POST MARKETING SURVEILLANCE OF ACTIVE PHARMACEUTICAL INGREDIENTS (API) IN ANTIMALARIAL DRUGS USED IN MALAWI THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL CHEMISTRY DEGREE BY IBRAHIM CHIKOWE, BSc (Ed). (10361119) DEPARTMENT OF CHEMISTRY UNIVERSITY OF GHANA, LEGON JULY 2013 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I, Ibrahim Chikowe , declare that the work contained in this thesis has been undertaken at the University of Ghana-Legon campus in the Chemistry Department under supervision and has never been presented for any other degree elsewhere. ………………………………………. Ibrah i m Chikowe (Student) ………………………….. ……………………………... Prof. Ivan Addae-Mensah Dr. (Mrs.) Dorcas Osei-Safo (Supervisor) (Supervisor) University of Ghana http://ugspace.ug.edu.gh ii ACKNOWLEDGEMENT This research project has been made possible with the support of the Almighty God through countless populace. Specifically, a vote of thanks is given to my supervisors; Prof. Ivan Addae- Mensah, Dr. (Mrs.) Dorcas Osei-Safo and Mr. J.J.E.K. Harrison. Other members of staff are equally thanked for their bountiful and precious help and support, although special thanks are poured to; the HoD, Prof. R. Kingsford-Adaboh, Dr. I.V. Oppong, Prof Akpabli, Prof. Asomaning, Dr. Mary Anti Chama, Dr. Klake and the department secretary, for the support and advice they gave me during my study period. I extend my vote of thanks to Deedat Chikowe a.k.a. Dida, Calista Chabwera Chikowe, Daniel Yeboah Konadu, Mr Bob Essien, Leornard Peter Masanja (Tanzanian), Prisca Mai-Mai Kanjere (Malawian) and my classmates Kojo Sekyi-Acquah (My Ghanaian Brother), Alhaji Mahama Appiah, Anita Adjowa Oppong, Godwin Akpeko Dziwornu, Cephas Ziwu, Eric Asiedu, Horatio Egbo Akpoviri (Nigerian), MacDonald Quansah, Samuel Offei-Dwamena and Mercy Ami Addoh for their hand in my work whenever need arose for their assistance. My distinguished acknowledgements go to the Department for International Development (DFID) / Wellcome Trust / National Commission for Science and Technology of Malawi (NCST) for funding my studies and the project. Finally, I express my love and gratitude to my Beloved family members for their prayers, help and best wishes in the course of my work and studies. To my treasured sister and second mother, Farida Saplana Bunaya Chikowe Nkhoma, Only Almighty God knows how best to reward you. May Almighty God Bless You All. University of Ghana http://ugspace.ug.edu.gh iii DEDICATION This project is dedicated to Me, My Father and My Mother. University of Ghana http://ugspace.ug.edu.gh iv TABLE OF CONTENTS DECLARATION ........................................................................................................................ i ACKNOWLEDGEMENT .......................................................................................................... ii DEDICATION .......................................................................................................................... iii LIST OF TABLES .................................................................................................................... ix LIST OF FIGURES ................................................................................................................ xiii LIST OF ABBREVIATIONS.................................................................................................... xv CHAPTER ONE .........................................................................................................................1 1 INTRODUCTION ............................................................................................................1 1.1 WHAT IS MALARIA? .............................................................................................1 1.2 OVERVIEW OF THE MALARIA SITUATION .......................................................2 1.3 MALARIA IN MALAWI ..........................................................................................3 1.4 PREVENTION OF ANTIMALARIAL DRUG RESISTANCE .................................5 1.5 STATEMENT OF THE PROBLEM .............................................................................5 1.6 AIM AND OBJECTIVES OF THE PROJECT .............................................................6 CHAPTER TWO ........................................................................................................................7 2 LITERATURE REVIEW .....................................................................................................7 2.1 LIFE CYCLE OF THE MALARIA PARASITE - PLASMODIUM ............................7 2.2 BRIEF HISTORY OF ANTIMALARIAL DRUGS ...................................................8 2.3 FORMS OF ANTIMALARIAL DRUGS ................................................................ 15 2.4 MEDICINAL CHEMISTRY OF ANTIMALARIAL DRUGS ................................. 15 2.5 PLASMODIA RESISTANCE TO ANTIMALARIAL DRUGS ............................... 23 2.6 POOR QUALITY DRUGS...................................................................................... 25 2.7 QUALITY CONTROL PARAMETERS IN POST-MARKETING SURVEILLANCE (PMS)...……………………………………………………………………………………..29 2.8 CASE STUDIES ON QUALITY CONTROL.......................................................... 30 2.9 QUALITY ASSESSMENT METHODS .................................................................. 41 CHAPTER THREE ................................................................................................................... 54 3 CURRENT INVESTIGATION .......................................................................................... 54 3.1 SAMPLING ................................................................................................................ 54 University of Ghana http://ugspace.ug.edu.gh v 3.1.1 STUDY AREA .................................................................................................... 54 3.1.2 SAMPLE SIZE .................................................................................................... 54 3.1.3 SAMPLING STRATEGY .................................................................................... 57 3.2 PRE-LABORATORY ANALYSIS ............................................................................. 58 3.2.1 REGISTRATION STATUS OF SAMPLES ......................................................... 58 3.2.2 ORIGIN OF THE COLLECTED DRUG SAMPLES ........................................... 59 3.2.3 VISUAL INSPECTION OF DOSAGE FORM AND PACKAGING .................... 59 3.3 LABORATORY ANALYSIS ..................................................................................... 60 3.3.1 QUALITATIVE COLOUR REACTIONS ........................................................... 60 3.3.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ASSAYS ... 75 3.4 DISCUSSION ............................................................................................................. 82 3.4.1 SQ-TLC AND HPLC RESULTS ......................................................................... 82 3.4.2 COMBINED HPLC AND SQ-TLC RESULTS .................................................. 100 3.4.3 REGISTRATION STATUS AND QUALITY OF THE SAMPLES ................... 103 3.4.4 SAMPLES WITH THE SAME BATCH NUMBER........................................... 104 3.4.5 OVERALL RESULTS ....................................................................................... 105 3.4.6 COMPARISON OF RESULTS WITH OTHER AFRICAN COUNTRIES ........ 109 3.4.7 CONCLUSIONS ............................................................................................... 110 CHAPTER FOUR ................................................................................................................... 113 4 EXPERIMENTAL ........................................................................................................... 113 4.1 THIN LAYER CHROMATOGRAPHY .................................................................... 113 4.1.1 EXPERIMENTAL CONDITIONS .................................................................... 113 4.1.2 PREPARATION OF DOSAGE FORM SOLUTIONS ....................................... 114 4.2 HPLC METHODS FOR THE ASSAY OF THE SELECTED ANTIMALARIALS .. 115 4.2.1 HPLC ASSAY OF ARTESUNATE ................................................................... 115 4.2.2 HPLC ASSAY OF ARTEMETHER / LUMEFANTRINE FDC FORMULATIONS ……………………………………………………………………………………118 4.2.3 HPLC ASSAY OF DIHYDROARTEMISININ ................................................. 122 4.2.4 HPLC ASSAY OF QUININE ............................................................................ 124 4.2.5 HPLC ASSAY OF SULPHADOXINE/SULPHAMETHOXYPYRIDAZINE/ ... 128 University of Ghana http://ugspace.ug.edu.gh vi PYRIMETHAMINE FORMULATIONS ......................................................................... 128 5 REFERENCES ................................................................................................................ 132 APPENDICES ....................................................................................................................... 148 University of Ghana http://ugspace.ug.edu.gh vii ABSTRACT The use of poor quality antimalarials causes low bioavailability of the active pharmaceutical ingredients (APIs) to the drug targets resulting in treatment ineffectiveness or failure and parasite resistance over a short period of drug usage. Resistance has rendered the hitherto cheap and effective drugs like chloroquine and sulphadoxine/pyrimethamine ineffective, resulting in their replacement with the more expensive artemisinin-based combination therapy (ACT) for malaria treatment. The high cost of the ACTs has made them attractive to counterfeiters leading to the proliferation of poor quality antimalarials on the drug markets. One hundred and twelve (112) samples of antimalarial drugs were purchased from licensed and unlicensed markets in all parts of Malawi. Samples were subjected to visual inspection of dosage form and packaging, registration verification with the Pharmacy, Medicines and Poisons Board of Malawi, basic tests and semi-quantitative thin layer chromatography (SQ-TLC) and HPLC tests to quantify the APIs in the samples and compare with pharmacopoeial specifications and the manufacturers’ claims. The results showed an 85 % registration status with all samples purported to be imported and 100% compliance with visual inspection requirements and basic tests confirming the presence of requisite APIs. The results of the SQ-TLC showed that 4.88% of artemether/lumefantrine (Atm/Lum) FDC samples were compliant with pharmacopoeial specifications, 2.44% were borderline compliant and a further 92.68% were non-compliant (48.15% of Atm component was overdose and 51.85% under dose; 57.14% of Lum component was overdose and the remaining 42.86% under dose). The HPLC results confirmed this result with 4.88% found to be compliant and 95.12% non-compliant (48.15% overdose and 51.85% under dose for Atm component; 50% overdose and 50% under dose for Lum component). SQ-TLC tests on artesunate/sulphadoxine/pyrimethamine (Ats/SP) samples showed that 77.78% were non- University of Ghana http://ugspace.ug.edu.gh viii compliant all of which were under dose, while 22.22% were borderline compliant. However, the HPLC results showed a compliant percentage of 11.10% and 88.90% non-compliance (under dose). The dihydroartemisinin/piperaquine phosphate (Dha/Pp) samples recorded SQ-TLC percentage of 42.86% being compliant, 14.29% borderline compliant and 42.86% non-compliant with all the non-compliant quantities of the components found under dose. With HPLC, they were found to be 28.57% compliant, 7.14% borderline compliant and 64.29% non-compliant (under dosed components). Dihydroartemisinin/sulphadoxine/pyrimethamine (Dha/SP) samples were found to be 100% non-compliant for both SQ-TLC and HPLC tests. The quantities of the APIs in the non-compliant Dha/S/P samples fell within the range of 51% and 84%. Samples containing sulphadoxine/pyrimethamine (SP) were found to be 4.35% compliant, 8.70% borderline compliant and 86.95% non-compliant using SQ-TLC method (25.00% of the non- compliant pyrimethamine component overdose, 75.00% under dose and 100.00% of sulphadoxine under dose). With HPLC, half (50.00%) of the samples that were borderline compliant were found to be non-compliant with the remaining half being compliant giving rise to the following results: 8.70% and 91.30% compliant and non-compliant respectively with all non- compliant sulphadoxine and 72.73% of pyrimethamine being under dose and 27.27% of the quantities of pyrimethamine being overdose. None of the quinine samples were compliant with SQ-TLC (28.57% borderline compliant, 71.43% non-compliant and overdose). HPLC analysis found 30.77%, 15.38% and 53.85% to be compliant, borderline compliant and non-compliant (85.71% overdosed) respectively. Generally, 85.71% failure rate was found arising from Atm/Lum (95.12%), Dha/P (64.30%), Dha/SP (100.00%), SP (91.30%), Ats/SP (88.90%) and quinine (53.80%) failure rates indicating wide spread circulation of poor quality antimalarial drugs in Malawi. University of Ghana http://ugspace.ug.edu.gh ix LIST OF TABLES TABLE 1: EXAMPLES OF ACTS AND NON-ACTS .......................................................................... 15 TABLE 2: COMBINED COUNTRY RESULTS: PERCENTAGE FAILURE OF SAMPLES .............................. 37 TABLE 3: KINDS OF CHROMATOGRAPHIC COLUMNS AND THEIR MODES OF SEPARATION ................ 51 TABLE 4: COLUMNS AND THEIR RESPECTIVE STATIONARY AND MOBILE PHASES AS FUNCTIONS OF POLARITY ........................................................................................................................... 52 TABLE 5: NUMBER OF SAMPLES BY ZONE OF COLLECTION............................................................ 56 TABLE 6: CATEGORIES OF COLLECTED ANTIMALARIAL DRUG SAMPLES ........................................ 57 TABLE 7: NUMBER OF SAMPLES COLLECTED IN EACH ZONE AND THEIR REGISTRATION STATUS ..... 58 TABLE 8: RESULTS FOR BASIC TESTS OF ARTESUNATE CONTAINING DRUG SAMPLES ..................... 64 TABLE 9: RESULTS FOR BASIC TESTS OF ARTEMETHER CONTAINING DRUG SAMPLES ..................... 66 TABLE 10: RESULTS FOR BASIC TESTS OF DIHYDROARTEMISININ CONTAINING DRUG SAMPLES ...... 68 TABLE 11: RESULTS FOR BASIC TESTS OF QUININE CONTAINING DRUG SAMPLES ........................... 69 TABLE 12: RESULTS FOR BASIC TESTS OF SULPHADOXINE AND PYRIMETHAMINE DRUG SAMPLES. . 70 TABLE 13: SOLVENT SYSTEMS FOR ARTEMISININ DERIVED ACTIVE PHARMACEUTICAL INGREDIENTS .......................................................................................................................................... 71 TABLE 14: SOLVENT SYSTEMS FOR NON-ARTEMISININ DERIVED ACTIVE PHARMACEUTICAL INGREDIENTS ...................................................................................................................... 72 TABLE 15 : COMPARATIVE STUDY OF DRUG API COMPLIANCE/NON-COMPLIANCE WITH REGARD TO COUNTRY OF ORIGIN (BASED ON HPLC) .............................................................................. 93 TABLE 16: COMPARATIVE STUDY OF COMPLIANCE/NON-COMPLIANCE OF DRUGS AS A WHOLE WITH REGARD TO COUNTRY OF ORIGIN.......................................................................................... 93 TABLE 17: OVERALL RESULTS OF THE STUDY ............................................................................ 105 University of Ghana http://ugspace.ug.edu.gh x TABLE 18: DISSOLUTION SOLVENTS OF APIS FOR TLC TESTS .................................................... 113 TABLE 19: CHROMATOGRAPHIC AND INSTRUMENTAL CONDITIONS FOR ARTESUNATE ASSAY ...... 115 TABLE 20: CONCENTRATIONS OF ARTESUNATE RS SOLUTIONS AND THEIR CORRESPONDING AUCS ........................................................................................................................................ 117 TABLE 21: CHROMATOGRAPHIC AND INSTRUMENTAL CONDITIONS OF ARTEMETHER/LUMEFANTRINE ASSAY .............................................................................................................................. 118 TABLE 22: CONCENTRATIONS OF ARTEMETHER RS SOLUTIONS AND THEIR AUCS ...................... 120 TABLE 23: CONCENTRATIONS OF LUMEFANTRINE RS SOLUTIONS AND THEIR AUCS ................... 121 TABLE 24: CHROMATOGRAPHIC AND INSTRUMENTAL CONDITIONS OF DIHYDROARTEMISININ ASSAY ........................................................................................................................................ 122 TABLE 25: CONCENTRATIONS OF DIHYDROARTEMISININ RS SOLUTIONS AND THE CORRESPONDING AUCS............................................................................................................................... 124 TABLE 26: CHROMATOGRAPHIC AND INSTRUMENTAL CONDITIONS FOR QUININE ASSAY ............. 125 TABLE 27: CONCENTRATIONS OF QUININE RS SOLUTIONS AND THEIR CORRESPONDING AUCS ... 127 TABLE 28: CHROMATOGRAPHIC CONDITIONS OF SULPHADOXINE / PYRIMETHAMINE ASSAY ........ 128 TABLE 29: CONCENTRATIONS OF SULPHADOXINE RS SOLUTIONS AND THEIR CORRESPONDING AUCS............................................................................................................................... 130 TABLE 30: CONCENTRATIONS OF PYRIMETHAMINE RS SOLUTIONS AND THEIR CORRESPONDING AUCS............................................................................................................................... 131 TABLE 31: LIST OF ARTEMISININ-BASED AND NON-ARTEMISININ BASED ANTIMALARIAL DRUGS PURCHASED FROM VARIOUS ZONES IN MALAWI .................................................................. 148 TABLE 32: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF ARTESUNATE ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS University of Ghana http://ugspace.ug.edu.gh xi WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. ARTESUNATE TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF ARTESUNATE ON THE PACK. .......................................................................................... 161 TABLE 33: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF ARTEMETHER ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. ARTEMETHER TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF ARTEMETHER ON THE PACK........................................................................................... 162 TABLE 34: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF LUMEFANTRINE ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. LUMEFANTRINE TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF LUMEFANTRINE ON THE PACK ....................................................................................... 165 TABLE 35: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF DIHYDROARTEMISININ (ARTENIMOL) ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. DIHYDROARTEMISININ TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF DIHYDROARTEMISININ ON THE PACK ............. 168 TABLE 36: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF SULPHADOXINE ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. SULPHADOXINE TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF SULPHADOXINE ON THE PACK ....................................................................................... 170 University of Ghana http://ugspace.ug.edu.gh xii TABLE 37: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF PYRIMETHAMINE ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. PYRIMETHAMINE TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF PYRIMETHAMINE ON THE PACK ..................................................................................... 173 TABLE 38: PERCENTAGE (%) AND MASS (MG) QUANTITIES OF RESULTS OF QUININE ACTIVE PHARMACEUTICAL INGREDIENT (API) BY TLC AND HPLC METHODS AND THEIR COMPARISONS WITH THE MANUFACTURER’S CLAIM AND PHARMACOPOEIAL REQUIREMENTS. QUININE TABLETS MUST CONTAIN AT LEAST 90.0% AND AT MOST 110.0% OF THE LABELLED AMOUNT OF QUININE ON THE PACK .................................................................................................. 176 TABLE 39: SAMPLES WITH THE SAME BATCH NUMBER AND THEIR RESPECTIVE MASSES (MG) ...... 178 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF FIGURES FIGURE 1: LIFE CYCLE OF PLASMODIUM .........................................................................................7 FIGURE 2: STRUCTURES OF THE FIRST PURE COMPOUNDS FOR MALARIA TREATMENT ......................9 FIGURE 3: DEVELOPMENT OF VARIOUS ANTIMALARIAL DRUGS FROM METHYLENE BLUE. .............. 11 FIGURE 4: CONVERSION OF ARTEMISININ TO ITS DERIVATIVES ..................................................... 14 FIGURE 5: CHEMICAL STRUCTURES OF SOME QUINOLINES ............................................................ 16 FIGURE 6: CHEMICAL STRUCTURES OF SOME SELECTED EXAMPLES OF ANTIFOLATES .................... 19 FIGURE 7: PROPOSED MECHANISM OF ARTEMISININ DEGRADATION BY IRON TO FORM TWO RADICALS ........................................................................................................................... 21 FIGURE 8: REACTION MECHANISM OF THE FERROUS HAEME WITH THE ENDOPEROXIDE BRIDGE ..... 22 FIGURE 9: BASIC TEST PROPOSED REACTION OF ARTEMISININ DECOMPOSITION PRODUCTS WITH VANILLIN ............................................................................................................................ 47 FIGURE 10. REACTION OF HYDROGEN PEROXIDE WITH POTASSIUM IODIDE ................................... 47 FIGURE 11: BASIC TEST PROPOSED REACTION FOR LUMEFANTRINE............................................... 48 FIGURE 13: MAP OF MALAWI SHOWING SAMPLING SITES ............................................................. 55 FIGURE 13: SOURCES OF THE COLLECTED DRUG SAMPLES ............................................................ 59 FIGURE 14: A SAMPLE OF DEVELOPED TLC PLATE OF AN ARTEMETHER CONTAINING DRUG .......... 73 FIGURE 15: CHROMATOGRAM OF A SAMPLE SOLUTION CONTAINING ARTESUNATE........................ 76 FIGURE 16: CHROMATOGRAM OF A SAMPLE SOLUTION CONTAINING ARTEMETHER AND LUMEFANTRINE ................................................................................................................... 77 FIGURE 17: CHROMATOGRAM OF A SAMPLE SOLUTION CONTAINING DIHYDROARTEMISININ .......... 78 FIGURE 18: CHROMATOGRAM OF A SAMPLE CONTAINING SULPHADOXINE AND PYRIMETHAMINE .. 80 FIGURE 19: CHROMATOGRAM OF A SAMPLE SOLUTION CONTAINING QUININE ............................... 82 University of Ghana http://ugspace.ug.edu.gh xiv FIGURE 20: SQ-TLC RESULTS OF INDIVIDUAL APIS IN THE ANTIMALARIAL DRUG SAMPLES ......... 84 FIGURE 21: SQ-TLC RESULTS OF DRUG SAMPLES WHEN ALL COMPONENTS ARE CONSIDERED ....... 88 FIGURE 22: HPLC RESULTS OF INDIVIDUAL APIS OF THE ANTIMALARIAL DRUG SAMPLES ............ 89 FIGURE 23: HPLC RESULTS OF DRUG SAMPLES WHEN ALL COMPONENTS ARE CONSIDERED .......... 92 FIGURE 24: COMPARISON OF HPLC AND SQ-TLC RESULTS ........................................................ 98 FIGURE 25: CORRELATION CURVE OF HPLC AND SQ-TLC RESULTS ............................................ 99 FIGURE 26: A COMPARISON OF FAILURE RATES FOR REGISTERED AND UNREGISTERED SAMPLES .. 103 FIGURE 27: FAILURE RATES OF THE DRUG SAMPLES BY ZONE .................................................... 106 FIGURE 28: CALIBRATION CURVE FOR ARTESUNATE RS CONC. AS A FUNCTION OF AUC ............ 117 FIGURE 29: CALIBRATION CURVE FOR ARTEMETHER RS CONC. AS A FUNCTION OF AUC ............ 121 FIGURE 30: CALIBRATION CURVE FOR LUMEFANTRINE RS CONC. AS A FUNCTION OF AUC ......... 122 FIGURE 31: CALIBRATION CURVE FOR DIHYDROARTEMISININ RS CONC. AS A FUNCTION OF AUC ........................................................................................................................................ 124 FIGURE 32: CALIBRATION CURVE FOR QUININE RS CONC. AS A FUNCTION OF AUC .................... 127 FIGURE 33: CALIBRATION CURVE FOR SULPHADOXINE RS CONC. AS A FUNCTION OF AUC ......... 130 FIGURE 34: CALIBRATION CURVE FOR PYRIMETHAMINE RS CONC. AS A FUNCTION OF AUC ....... 131 University of Ghana http://ugspace.ug.edu.gh xv LIST OF ABBREVIATIONS AA Artesunate-Amodiaquine ABC Artemisinin-Based Combination ACTs Artemisinin-Based Combination Therapies AD Anno Domini API Active Pharmaceutical Ingredient ATM Artemether ATS Artesunate CHAM Christian Health Association of Malawi DESI Desorption Electro Spray Ionization DHA Dihydroartemisinin 2, 4-DNPH 2, 4-Dinitrophenylhydrazine EDCTP European and Developing Countries Clinical Trials Partnership FDC Fixed dose combination GMP Good Manufacturing Practice GPHF Global Pharma Health Fund HCl Hydrochloric acid HIV Human Immunodeficiency Virus HoD Head of Department HPLC High Pressure Liquid Chromatography INN International Non-proprietary Name IPTp Intermittent Preventive Treatment of Pregnant women IRS Indoor Residual Spraying University of Ghana http://ugspace.ug.edu.gh xvi ITN Insecticide Treated Net KI Potassium Iodide LC-MS Liquid Chromatography-Mass Spectroscopy LUM Lumefantrine NABC Non-Artemisinin -Based Combination NMCP National Malaria Control Programme Ph. Int. International Pharmacopoeia PPQ/PP Piperaquine QAMSA Quality of selected Antimalarial Medicines circulating in Six countries of Sub-saharan Africa Rf Retardation Factor RS Reference Standard SP Sulphadoxine-Pyrimethamine SQ-TLC Semi-Quantitative Thin-Layer Chromatography TLC Thin-Layer Chromatography TS Test Solution USAID United States Agency for International Development USP United States Pharmacopoeia UV Ultraviolet VS Volumetric Solution WHO World Health Organization University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1 INTRODUCTION 1.1 WHAT IS MALARIA? Malaria was alleged to be caused by bad air (mal’aria in Italian) from which its name was derived. This allegation was held until the 1880s and 1890s when its cause was associated with a parasite and its transmission linked to mosquitoes by Alphonse Laveran, Battista, Ronald Ross and others [1]. Malaria is an infectious disease caused by a protozoon of the subphylum sporozoa of the genus Plasmodium and the species falciparum, malariae, vivax and ovale [2]. Molecular evidence has further shown two subspecies in P. ovale and one of them, P. knowlesi, is also capable of infecting humans through zoonosis, but it is mostly misdiagnosed as P. malariae [3, 4]. The Plasmodia are spread by an infected female anopheles mosquito that bites late at night and early morning [4]. Nevertheless, most malaria associated deaths are caused by P. falciparum and P. vivax, with the former accounting for 1 million deaths per annum; about 90% of all malarial deaths and approximately 250 million morbidity rates per year [3]. Malaria infection has several devastating physiological effects like anaemia and jaundice. P. falciparum infection causes many dangerous malaria cases and may further cause kidney failure, acidosis, cerebral malaria, kidney damage, multifunction failure, severe anaemia, spleen rupture, coma and death if not treated quickly and effectively [4, 5]. University of Ghana http://ugspace.ug.edu.gh 2 1.2 OVERVIEW OF THE MALARIA SITUATION Malaria is a disease that is still claiming millions of lives worldwide. Approximately 3.3 billion people were affected by the disease worldwide in 2010. Out of 216 million clinical episodes due to malaria reported in that year, 655,000 resulted in deaths. More deaths were recorded in Africa (~91%) compared to South–East Asia region (6%) and the East Mediterranean region (3%). Children accounted for up to 86% of these deaths globally, most probably due to their relatively low level of immunity which makes them easily overwhelmed by the infection if not promptly treated [3, 6]. These results indicate that the disease remains the main killer in Africa and its children are the most vulnerable [4]. This stunning death rate has been attributed mainly to the rapid development of resistance by malaria parasites to the conventional antimalarial drugs such as chloroquine, quinine and sulphadoxine/pyrimethamine FDC [7]. Currently, a new drug regimen called the artemisinin- based antimalarials has been developed, introduced and deployed to counterattack this resistance. That has seen the subsequent introduction of the new, more potent and prevalent artemisinin- based combination therapies (ACTs) in which an artemisinin-based compound is formulated in combination with a non-artemisinin antimalarial. These newly WHO-recommended first line therapy antimalarial drugs for uncomplicated and unconfirmed malaria have given such a good hope to the fight against malaria due to their ability to affect the Plasmodia at several stages of their life cycle, which affects their gametocyte presence and infection [8]. Since the ACTs are mainly aimed at uncomplicated malaria, the other drug types are still used for other serious and special cases of malaria [6]. However, the effective use of these newly introduced drugs is mainly hampered by the presence and use of substandard and fake drugs such that even this therapeutic University of Ghana http://ugspace.ug.edu.gh 3 line is equally threatened by parasite resistance. It is disappointing and frightening to note that some of these substandard drugs are produced even by the certified companies believed to be committed to good manufacturing practices (GMP) [9]. 1.3 MALARIA IN MALAWI 1.3.1 Malaria Situat ion In the Malawi Ministry of Health World Malaria Day communiqué of 2012, it was reported that for every 1000 people, 325 have a medical condition of malaria [10]. An estimated 30% of the outpatients treated at health facilities have malaria-related infections and it is estimated that 40% of children die of diseases related to malaria [11, 12]. Malaria suspected cases in Malawi have been on the increase since 2005. As a matter of illustration, 3.7 million cases were registered in 2005 and the number almost doubled to 6.1 million cases in 2009 of which 50% of these cases were reported in children alone [13]. In a 2010 Malawi National Malaria Indicator Survey in which Slide microscopy was used, a national parasite occurrence rate of 43.3% was found with vulnerability to parasite being found to be increasing with aging. In addition, severe anaemia study conducted simultaneously showed that it was prevalent in 12.3% of children under 5 years with an average HB concentration of less than 8g/dL, with vulnerability to severe anaemia found to be diminishing with aging. In addition, the study found that children who did not sleep under the ITN were more prone to severe anaemia and malaria parasite, with the poor contributing much to the parasite and anaemia prevalence [14]. In a related study, Harms and Feldmeier in Kabuluzi et al. reported that 66% of pregnant women were found anaemic and this condition was mainly attributed to malaria. University of Ghana http://ugspace.ug.edu.gh 4 Furthermore, the results showed that malaria parasitaemia contributed significantly to the rampant vitamin A deficiency in 44% and 70% of pregnant women and pre-school children respectively (MOH/CDC/UNICEF, 2001 in Kabuluzi et al., 2004) [15]. Malaria in Malawi is controlled by chemotherapy for infection prevention and treatment, with artemether-lumefantrine being the first line treatment for uncomplicated and unconfirmed cases since 2007 after replacing SP that also replaced chloroquine due to parasite resistance. However, antimalarial drugs like quinine, SP and others are still being used for special cases. Sleeping under insecticide treated net (ITN) or long lasting insecticide-treated nets (LLITN) and indoor residual spraying (IRS) are used for vector control [13, 16]. However, failure to significantly adhere to treatment and policies has been an area of concern in an effort to implement malaria interventions effectively [14]. 1.3.2 Distrib u t ion of Malaria Inhabitants are infected throughout the year in all parts of the country with highest transmission cases recorded in the low-lying areas during the rainy season. The areas with repeated high infection rates are those with high temperatures notably the lower Shire valley and lakeshore areas except mountainous parts of the North and South [13]. Malaria is amongst the diseases that are commonest amongst the poor people [14], who constitute 54% of the population and 65% of the population living on less than a dollar per day [13] and this makes most Malawians not able to access prompt malaria treatment within 24 hours of observing the infection symptoms as reported by UNICEF, which worsens the burden of this preventable and treatable disease [10]. University of Ghana http://ugspace.ug.edu.gh 5 1.4 PREVENTION OF ANTIMALARIAL DRUG RESISTANCE After a thorough understanding of available malaria treatment, most of the drugs have faced significant rates of resistance and the only hope for future malaria treatment currently rests on artemisinins even though other regimens are still in use. Therefore, it is imperative to protect these drugs from any impending drug resistance and this can be achieved by among others, proper use of good and effective quality ACTs and a relentless combat against the use of poor quality drugs as well as strict patient compliance to treatment regimen [17]. 1.5 STATEMENT OF THE PROBLEM No other disease has killed more human kind than malaria [18]. With the widespread development of resistant malarial parasites to widely used antimalarial drugs, various efforts have been suggested by WHO and its partners for malaria control. These recommendations have been duly adopted by governments and one such measure is the use of artemisinin-based combination drugs as first-line prescription for the treatment of malaria. However, most countries including Malawi import most of these drugs. With the high demand, cost of production and prices of these drugs, a significant number of fake and sub-standard drugs have been imported and/or produced locally. This has called for routine quality control activities to check this malpractice. However, national drug regulatory authorities in most developing countries like Malawi have not been able to conduct these crucial routine quality assessment activities to curb this rapid inflow of these unwanted drugs, due to the lack of the necessary state-of-the-art tools and qualified personnel. The National Malaria Control Programme (NMCP) points out that the quality control system for laboratories in Malawi is weak and it is an area of concern that needs a prompt University of Ghana http://ugspace.ug.edu.gh 6 attention. Therefore, this research was aimed at fulfilling and supplementing the drug regulatory efforts as outlined by the WHO Expert Committee on Quality Assurance of Medicines [13]. 1.6 AIM AND OBJECTIVES OF THE PROJECT The research was aimed at finding out if the antimalarial drugs used in Malawi comply with the WHO accepted standards for active pharmaceutical ingredients (APIs) in ACTs and Non-ACTs. To achieve this aim, the following specific objectives were set: i. To find out the registration status of the antimalarial drugs available on the markets. ii. To visually inspect dosage forms and packaging using the guidelines outlined in the WHO pharmacopoeia and the literature or otherwise. iii. To carry out a qualitative determination to establish the presence or otherwise of the active pharmaceutical ingredients using the authenticated rapid tests outlined in WHO publications. iv. To carry out a quantitative determination of the API content using SQ-TLC and HPLC. v. To further validate the newly developed methods and their adoption for analyzing antimalarial drugs used in Malawi. University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2 LITERATURE REVIEW 2.1 LIFE CYCLE OF THE MALARIA PARASITE - PL AS MODIUM The life cycle of malaria parasites is hosted by female anopheles mosquito (primary host) that harbours the sexual cycle, and humans (secondary host) that host the asexual cycle (Figure 1) [19]. Source: European Vaccine Initiative (EVI) [20] Figure 1 : Life cycle of plasm odium Malaria infection in humans starts with an exo-erythrocytic stage where an infected adult female anopheles mosquito bites in search of food and in the process, inoculates some sporozoites into the bloodstream of humans and some other mammals [2]. The sporozoites then invade, penetrate University of Ghana http://ugspace.ug.edu.gh 8 and localize in the liver tissues and subsequently infect the liver cells. The sporozoites start growing in the liver followed by asexual reproduction, budding into schizonts (tissue schizonts) that contain merozoites. Upon maturity, the schizonts burst open releasing the matured merozoites [19]. Some merozoites enter the bloodstream to start the erythrocytic stage while others may re-infect the liver initiating the secondary tissue stage [2], where they evolve to dormant hypnozoites in the liver especially in infections by P. vivax and P. ovale species leading to long incubations and late relapses [3, 4]. The merozoites in the bloodstream attack the erythrocytes (red blood cells) to consume some of the haemoglobin and develop into immature trophozoites. When the trophozoites mature, they form blood schizonts within which merozoites develop through asexual reproduction. A burst of the schizonts releases more merozoites into the bloodstream repeating the infection and multiplication cycle. Recurrence of the cycle persists unless it is sorted out by either the immune system or an antimalarial drug [21] and this is responsible for the malarial symptoms like fever and chills [2, 3]. This process also accounts for anaemia and HB concentration decrease. While some merozoites repeat the erythrocyte invasions, others differentiate to male and female gametocytes. When a female anopheles mosquito bites, it is infected with the gametocytes from the bloodstream that evolve into sporozoites which discharge into the salivary glands of the mosquito; a subsequent bite by the mosquito re-starts the cycle [2, 4]. 2.2 BRIEF HISTORY OF ANTIMALARIAL DRUGS Cinchona bark and Wormwood (Qinghao) had been used effectively as herbs in treating fevers several hundred years prior to the discovery of the mosquito cycle in the early 20th century. The University of Ghana http://ugspace.ug.edu.gh 9 mosquito cycle breakthrough brought novel methods in vector control. Later, pure chemical compounds and subsequent synthetic drugs overshadowed herbal materials. These two ancient natural treasures still live today as quinine and artemisinin extracted from cinchona bark and qinghao respectively and are still the prime antimalarials in use today [1]. 2.2.1 Quinin e and Its Derivat ives Figure 2 : Structures of the fi rst pure compou nds fo r ma la ria treat men t The alkaloids quinine and cinchonine were isolated from Cinchona bark in 1820 by two French chemists, Pierre Pelletier and Joseph Caventou [1]. The name quinine was derived from quina- quina (bark of barks), an Inca word [3]. French physicians started administering pure quinine for intermittent fever patients, which turned out successful, thus putting malaria amongst the first diseases to be cured by a pure chemical compound. University of Ghana http://ugspace.ug.edu.gh 10 William Henry Perkins, an English chemist aged 18 had the first but unsuccessful attempt to synthesize quinine and the successful synthesis of quinine was eventually achieved in 1944, though not commercially viable. Perkins’ failed attempt to synthesize quinine led to yet another important product, the first artificial textile dye called Mauveine (Methylene blue) that could not be washed by water, which later promoted the development of medicine. It had research application in microbiology and enabled microbiologists see microbial pathogens under microscope after staining them with the dye, a phenomenon that was not possible prior to the dye discovery [1]. Later, a German scientist, Paul Ehrlich found that malaria parasites were also successfully stained by the dye, also known as methylene blue and reasoned that it would eventually poison the parasites in vivo. In 1891, Ehrlich used synthetic drugs in humans for the first time in history when he used methylene blue to cure two patients of malaria based on his reasoning, making malaria the first disease to be treated by a synthetic compound. Methylene blue was later used as a prototype for the development of novel synthetic antimalarial drugs [1]. Mepacrine was produced for P. falciparum in 1932 by the Germans for their combat soldiers who were affected by malaria in earlier battles in Europe. Later, mepacrine (atebrine) manufacturing was extended to the USA, with chemical intermediates from Germany [18]. In 1925, the first 8-aminoquinoline plasmoquine (pamaquine) was developed for the prevention of P. vivax malaria relapses [1]. In 1934, chloroquine synthesis began indirectly by H. Andersag and by 1946, US clinical trials proved the superiority of chloroquine to atebrine (mepacrine). Eventually, chloroquine was recognized as a powerful antimalarial drug and was extensively used worldwide. In the 1950s and 1960s, WHO recommended chloroquine as the main choice for its Global Eradication University of Ghana http://ugspace.ug.edu.gh 11 Programme. Below is Figure 3 illustrating the development of various antimalarial drugs from the prototype methylene blue [22]. Figure 3 : Develop men t of va riou s ant ima la ria l drugs from m et h ylen e blu e. Antifolates were developed after the 1940s, following detailed studies of bacterial systems and this breakthrough helped in the development of drugs such as proguanil, a prodrug that metabolizes to active cycloguanil in vivo, pyrimethamine, sulphonamides such as sulphadoxine and sulphones like dapsone [3]. In the 1970s, pyrimethamine was combined with sulphadoxine University of Ghana http://ugspace.ug.edu.gh 12 for synergistic purpose, commercially available as Fansidar and it is still in use today in some parts of Africa [1]. Post-war efforts by American scientists have led to improvement of plasmoquine to the more effective primaquine as a standard drug for P. vivax malaria relapse prevention. Some drugs have been used as prototypes, such as SN10275 for mefloquine and 2-hydroxynaphthoquinones for atovaquone. Other drugs include WR 238605 and tafenoquine [1]. Since 1980, several drugs have been developed, while others are still underway all for purposes of malaria control and most notable ones are dapsone, mefloquine, halofantrine, doxycycline, primaquine and the artemisinins [23]. 2.2.2 Arte misin in and Derivat ives The Chinese herbal medicine practitioners originally used Artemisia annua (Qinghao) for treating haemorrhoid over at least 2000 years [1]. Qinghao therapy dates back to two prominent ancient physicians; Ge Hong and Li Shizhen who used it separately within 283-343 AD and 1518-1593 AD periods respectively [24, 25]. Ge Hong assembled and studied various herbal prescriptions and went further to author a book titled Zhou Hou Bei Ji Fang (Handy Therapies for Emergencies) where his prescriptions were coded. Li Shizhen also authored a book that had monographs of each herb or medicinal matter called Bencao Gangmu (Great Compendium of Herbs) that was published after his death [24]. In 1967, the Mao-led Chinese government decided to professionalize traditional medicine in a multi-disciplinary programme called Project 523. In the early 1970s, colourless pure crystals that University of Ghana http://ugspace.ug.edu.gh 13 were active against plasmodia in animal models were extracted from A. annua. The extract was called Qinghaosu or artemisinin [18]. Later, clinical trials of artemisinin showed that artemisinin was effective against chloroquine-resistant P. falciparum coupled with quick action and low toxicity. Structure elucidation of artemisinin revealed that it was a sesquiterpene lactone with the formula C15H23O5 and the chemical structure was found to have features uncommon to natural products, i.e. three fused rings; one ring having seven atoms and one ring spanned by a peroxide bridge (-O-O-) that could be broken easily (Figure 4) [24]. Artemisinin has rapid action, short half-life (1-3hrs), low toxicity and effective against antifolate and quinoline-resistant P. falciparum [24]. It is this rapid action that causes a prompt treatment for severe infections [26]. However, it has low solubility in water and oil coupled with poor availability and these properties limit its therapeutic value along with effectiveness [24]. Thus, artemisinin extract has poor pharmacological properties and this is improved by modifying the sesquiterpene lactone endoperoxide, which increases cost of production [3]. In this process, artemisinin is chemically converted to dihydroartemisinin, followed by partly synthetic derivatives that are more effective such as artesunate, artemether and arteether, also known as first generation derivatives of artemisinin [24]. Firstly, artemisinin is reduced with sodium borohydride (NaBH4) in the presence of methanol to produce dihydroartemisinin. Secondly, the derivatives are produced by other reactions as illustrated in Figure 4 below. University of Ghana http://ugspace.ug.edu.gh 14 Figure 4 : Conversion of art e misin in to it s derivat ives Currently, the discovery, design and development of antimalarials have stalled due to the dwindling interest in antimalarial drug development by the pharmaceutical industry because of University of Ghana http://ugspace.ug.edu.gh 15 the high risk and low investment returns associated with antimalarials. Any persistence in the current P. falciparum resistance development will see malaria incurable in some parts of the malarious areas [27]. Hence, there is a great need to safeguard the present drugs against resistance through proper drug use, handling and checking against poor quality drugs. 2.3 FORMS OF ANTIMALARIAL DRUG COMBINATIONS There are two forms of antimalarial drugs: artemisinin-based combination therapies (ACTs) and Non-artemisinin based combination therapies [28]. Table 1 below shows few examples of antimalarial drug combinations. Table 1 : Examp le s of ACTs and non -ACTs Artemi s i ni n - b ased comb i nat io n t he r apy (ACTs) Non - ACTS Artesunate-amodiaquine Sulphadoxine-pyrimethamine (SP) Artesunate-mefloquine SP-chloroquine Artemether-lumefantrine SP-amodiaquine Artesunate-sulphadoxine-pyrimethamine SP-mefloquine Dihydroartemisinin-piperaquine+derivatives Quinine-tetracycline-doxycycline Dihydroartemisinin-sulphadoxine-pyrimethamine Artesunate-sulphamethoxypyridazine- pyrimethamine 2.4 MEDICINAL CHEMISTRY OF ANTIMALARIAL DRUGS 2.4.1 Quinolin es and Relat ed Compou nds These compounds have a basic quinoline ring system and several compounds such as chloroquine, amodiaquine, primaquine, piperaquine, quinine, quinidine and mefloquine have been synthesized from the basic quinoline moiety of quinine (see Figure 5 for chemical University of Ghana http://ugspace.ug.edu.gh 16 structures of some quinolines). Related to the under listed drugs are halofantrine derived from phenanthrene ring system and lumefantrine from the fluorene ring [3]. Figure 5 : Chemical st ructures of so me quin olin es University of Ghana http://ugspace.ug.edu.gh 17 2. 4. 1. 1 Ch loroqu in e It is a synthetic derivative of 4-aminoquinoline, a blood schizonticide and one of the longest serving synthetic antimalarials. The exact mode of action of chloroquine is not known, but several mechanisms have been proposed. Plasmodium survives on amino acids obtained from the digestion of haemoglobin and this process produces β-hematin dimer haeme that is toxic to the parasite. So a bio-mineralization process is conducted by the parasite to form a non-toxic complex called Hemozoin. Chloroquine and other quinolines are used to block this haeme elimination process by producing complexes with the haeme, thus depriving the parasites of dimerization and crystallization processes for detoxification, thereby causing accumulation of toxic haeme within them and subsequently poisoning to death. In addition, the creation of drug- haeme complex hampers the formation of peptides and this also reduces necessary amino acid supply to the parasites, threatening their capability [3]. Moreover, chloroquine interferes with the biosynthesis of nucleic acids thereby inhibiting the DNA and the RNA of the parasite [29]. However, chloroquine has faced widespread P. falciparum parasite resistance due to the mutation of its transporter (PfCRT), with only North Africa, Carribean region and Central America still using it. This resistance dates back to 1957 when it was first discovered along the Venezuelan-Colombian border and spread across sub-saharan Africa in the late 1970s and 1980s [3]. In Africa, the resistance started in the Eastern African region (e.g. Kenya, Tanzania, Uganda, Rwanda), but took quite a long time to spread to the West Coast. Chloroquine resistance in West Africa therefore became a major problem only during the latter years of the 20th century [30, 31, 32]. University of Ghana http://ugspace.ug.edu.gh 18 2.4.2 Antifolat es These are antimalarial chemotherapeutic agents that act by competing with a natural substrate to suppress unpleasant body chemical reactions and they are called competitive (reversible) inhibitors. They are mostly structural analogues of their substrates. The Figure 6 below shows structures of some of these drugs. As competitive inhibitors, pyrimethamine and chloroguanil inhibit dihydrofolate reductase (DHFR) responsible for critical activities of bifunctional DHFR- thymidylate synthatase (TS) protein. This averts the biosynthesis of purines plus pyrimidines and eventually DNA synthesis, cell division as well as reproduction [3]. Sulphonamides like sulphadoxine and sulphamethoxypyridazine as well as sulpha drugs inhibit dihydropteroate synthase (DHPS) that is involved in the folate synthesis pathway and it is also crucial for the synthetic pathway of amino acids needed for parasite growth, thus preventing p- aminobenzoic acid (PABA) from converting to dihydrofolic acid and eventually tetrahydrofolate synthesis. For optimum efficacy against susceptible strains of malaria, sulphadoxine is combined with pyrimethamine in a product called Fansidar and many others for synergistic effect. However, the effectiveness of antifolates has been compromised by the emergence of the point mutation of the essential genes of the parasites; a form of mutation that occurs when a single base nucleotide is replaced by another nucleotide of a genetic matter, RNA or DNA [3]. University of Ghana http://ugspace.ug.edu.gh 19 Figure 6 : Chemical st ructures of so me se lected exa mp les of ant ifo lat es 2.4.3 Mechanism of Action of Artemisin in s A clear mechanism of action of these compounds is yet to be fully determined. However, various studies have proposed that the activity comes from the endoperoxide bridge through a two-step process of haemoglobin iron activation and alkylation [3, 33]. University of Ghana http://ugspace.ug.edu.gh 20 The haemoglobin iron activation starts when the haeme moiety containing a reduced iron, ferrous iron (Fe2+) (derived from the food vacuole’s haemoglobin digestion) interacts with the endoperoxide bridge [3]. The ferrous iron (Fe2+) is oxidised to ferric iron (Fe3+), releasing an electron to the endoperoxide bridge of the artemisinins, causing the drug activation; but the Fe2+ cation acts as a catalyst because it is regenerated after the process. The peroxide bond breaks forming carbon-centred alkoxyl radicals, which are believed to be the source of the drug activity, because all the derivatives that cannot produce this radical (those without endoperoxide bridge like deoxyartemisinin) are devoid of biological activity [34]. This proposed mechanism of artemisinin degradation by iron is shown in Figure 7 below. Apart from acting as a catalyst for artemisinin breakdown, haeme also forms complexes with artemisinin producing specific radicals that are neither in artemisinin nor haemin products alone as shown in Figure 7 below, which are believed to act at a specific target that has not yet been discovered [36]. Furthermore, the artemisinin derivatives are alleged to act by alkylating particular proteins comprising iron-sulphur protein, transporters and translationally controlled tumour protein (TCTP) homologue [3]. Figure 8 shows a proposed reaction between the generated radicals and the bio molecules and it also shows a ferrous haeme (Fe (II) protoporphyrin IX) activating an artemisinin to form a seco C-4 radical ( XXVI ) and alkylated adduct ( XXX) through pathway a (second row). University of Ghana http://ugspace.ug.edu.gh 21 Source: Scafat et al. [35] Figure 7 : Proposed mechanism of a rt e misin in degradation by iron to form two radicals Another possibility in which the radical exerts its activity is its loss of presumed water molecule from the haeme ( XXXI ) to alkylate the necessary parasite protein site in pathway b (first row) [37]. In contrast to other alkylating agents, artemisinin does not react with the DNA [38]. Therefore, the two processes of iron activation and alkylation deprive the parasite of its normal activities as follows: parasites consume part of the haeme of an erythrocyte they have invaded for their metalloenzymes and the rest is converted to hemozoin because the haeme is toxic to them [39, 40]. Alkylation prevents haemoglobin digestion by inhibiting proteases that facilitate the digestion. The process, therefore, deprives the parasite of vital amino acids. Alkylation also inactivates the University of Ghana http://ugspace.ug.edu.gh 22 histidine-rich protein involved in the polymerization of haeme to hemozoin and the poisonous haeme molecules accumulate, eventually destroying the parasite [41]. Source: Krishnaa et al. [37] Figure 8 : Reaction mechanism of the ferrou s ha em e wit h the endopero xide bridge These presumed actions of inhibition of histidine-rich protein (HRP) catalyst for hemozoin formation are believed to be caused by haeme-artemisinin adduct and C-4 radical alkylation respectively. Both actions lead to the subsequent accumulation of toxic haeme-FeIV. This toxic haeme-iron complex is formed when Fe2+ is changing to Fe3+ as it goes through a stage where University of Ghana http://ugspace.ug.edu.gh 23 FeIV is formed. This paralyses parasite activities through interaction with its proteins of the food vacuole and pro-oxidant activities [37]. However, evidence-based results have shown some traces of resistance reported to have been caused by the ability of the parasite to resist artemisinin, refuting earlier suggestions that it might be due to the host and pharmacokinetic factors [3]. 2.5 PLASMODIA RESISTANCE TO ANTIMALARIAL DRUGS Drug resistance of a parasite is confirmed when the effectiveness of a drug declines in either disease cure or symptoms recovery in a patient. The word ‘drug resistance’ is suitably used when referring to pathogen-caused diseases, whereby the drug is intended to destroy the pathogens. However, a drug is not always aimed at destroying or inhibiting pathogens; in such other case, drug ineffectiveness (resistance) is suitably called drug tolerance or dosage failure. Some pathogens are tagged as multidrug resistant when they can defend themselves against more than one type of drug and/or approaches concomitantly administered [42]. Bruce-Chwatt et al. ( in Bloland, 2001: 12) define resistance to antimalarials as the “ability of a parasite to survive and/or multiply despite administration and absorption of a drug given in doses equal to or higher than those usually recommended, but within tolerance of the subject. The drug in question must gain access to the parasite or the infected red blood cell for the duration of the time necessary for its normal action.” Recent studies have established that resistance is caused by host and parasite factors. The host factors include prophylaxis drugs over use, use of poor quality drugs and non-compliance as well as haphazard use of drugs by patients leading to unfinished therapeutic treatments, which in turn, cause a decrease in drug build up to a recommended level necessary for an effective physiological response . The parasite factors University of Ghana http://ugspace.ug.edu.gh 24 include change-flexibility in target genes and metabolic pathway, increase in concentration of target, abnormal rates of reproduction, sexual reproduction (in mosquitoes); which in turn cause the multiplication of resistant genes and drug deactivation [3, 43]. 2.5.1 Remedy fo r Drug Resist ance There are basically three ways of dealing with drug resistance and these are; prevention of drug resistance emergence, containment of the emerged resistance and drug efficacy scrutiny. Firstly, drug resistance can be prevented by controlling the parasite transmission, use of good quality antimalarials and adoption of combination therapy. Transmission of parasite can be prevented by the use of vaccines, vector control and decrease in infection reservoir; which has been blamed for spread of resistance and hence its decrease can be achieved by early diagnosis, efficient treatment and using gametocytocidal drugs. Furthermore, drug resistance emergence can be prevented by the use of combination therapy like ACTs and non-ACTs [17]. Besides, good quality ACTs are predictive of a successful parasite clearance as well as fight against resistance and this can be reinforced by easy access to ACTs, improved disease diagnosis, correct use of drugs especially in the private sector. This can be achieved by proper and updated education for the practitioners, increased compliance and supervising drug administration. Good quality drugs administration can also be achieved by routine quality assessment of drugs to fish out poor quality drugs from the market. Secondly, recommended drugs in the treatment guidelines are supposed to be routinely monitored for efficacy to detect any occurrence of resistance of the parasite quickly. This would help in establishing the extent of University of Ghana http://ugspace.ug.edu.gh 25 resistance and call for a prompt change of drug policy if necessary to prevent multi-drug resistance [17]. 2.5.2 EFFECTS OF ANTIMALARIAL DRUG RESISTANCE Parasite resistance to malaria treatment has had several devastating effects in the fight against malaria disease. This has led to an increase in morbidity and mortality rate (inclusive of anaemia and low birth weight), parasite transmission, rate and severity of pandemic, change in malaria distribution. In turn, this has caused pressure on the economy (government and individuals) due to increase in health services cost arising from prevalent treatment failures and death. These effects have made people resort to the informal private sector, thereby exposing themselves to poor quality drugs mostly blamed for escalation of drug resistance [17]. Specifically, some pockets of parasite resistance to artemisinin resistance have been attributed to the sub-therapeutic doses derived from fake and substandard drugs [8]. 2.6 POOR QUALITY DRUGS These are drugs that have either wrong ingredient, incorrectly formulated APIs, insufficient amount of APIs or dosage forms contaminated with other exogenous substances that may render the drugs harmful, ineffective or cause death. Poor quality drugs are classified into three types: counterfeit, substandard and degraded [44]. WHO classifies monotherapy of artemisinins as substandard even when they have enough requisite API [45]. Drugs are classified as counterfeit if they are fraudulently and deliberately mislabelled to depict one ingredient when they contain another (harmless or toxic). The ingredient might be either University of Ghana http://ugspace.ug.edu.gh 26 active or inactive against the disease they are intended for or contain the correct requisite API, but manufacturer or packaging is faked or mislabelled. The drug might also contain insufficient active ingredients, or without active ingredient. These drugs might be generic products, prescription medicines, over the counter medication or traditional remedies. In addition, it includes drugs that contain misleading information with respect to name, composition, strength, and manufacturer, country of manufacturing, and country of origin, marketing authorization holder or steps of distribution [46]. Small amounts of active ingredients are added sometimes aimed at just passing the rapid tests at entry points [47]. Substandard drugs are those drugs that are manufactured by well-known and established licensed companies with all the requirements, but deviate significantly from the accepted limits of API and other chemical additives’ contents as well as falling outside the recommended range of dissolution times. This is attributed to poor manufacturing practices, lack of technical know-how and unsatisfactory infrastructure. Degraded drugs on the other hand are those that contain inadequate amount of API and other unwanted compounds resulting from decomposition of the well manufactured drugs that have been exposed to adverse conditions such as humidity, light and heat. Nevertheless, it is challenging to know whether a drug falls outside accepted limits due to poor manufacturing practices or exposure to adverse effects. Besides, it is also challenging to decide between counterfeit and substandard drugs as the latter might be due to poor manufacturing practices or deliberate move by a manufacturer [48]. However, Bate et al. state that determination of a drug as counterfeit or substandard requires a forensic examination of the trademarks, product designs and holograms [45]. University of Ghana http://ugspace.ug.edu.gh 27 Research has shown that poor quality drugs are more widely found in Asia than Africa but the situation is rapidly deteriorating in the latter. This has been attributed to poor regulatory capabilities for manufacturing and importation activities [45]. It is estimated that 15% of the drugs used globally are counterfeit and 50% of the drugs used in some parts of Africa as well as Asia are counterfeit [49]. The fake drugs look almost the same as the original ones in both the packing materials and the barcodes, to a layman [50]. This situation has increased rapidly recently especially in Asia because most of these new drugs are very expensive as compared to the old ones. Market prices have shown that the current cost of ACTs is 10 to 20 times higher than the cost of SP and chloroquine. However, there is an expectation that the prices will decrease when the forces of demand and supply play their part [44]. An estimated US$20 billion, representing 7% of the pharmaceutical industry’s total revenue, is lost to counterfeiters annually [51]. Most counterfeit drugs have been found to contain incorrect ingredients and this can cause adverse effects especially on HIV treated patients due to a possible drug-drug interaction [47]. It is also alleged that since other ingredients in the counterfeited drugs are sometimes not known, they create a toxicity threat due to unilateral activity, by-products effects or combined effects with other body contents [52]. In addition, some of these drugs contain zero active ingredients and others contain insufficient doses of the active ingredients that are too low to eradicate the available pathogens, while others contain too much of active ingredients that might have various adverse effects especially for the drugs that have low therapeutic indices [48]. These poor quality drugs result in low bioavailability of drugs; hence sub-therapeutic blood concentrations [48]. This, in turn, can cause preferential selection for the drug sensitive parasites, University of Ghana http://ugspace.ug.edu.gh 28 leaving the drug resistant parasites unaffected. When using a long half-life drug that stays in the body for a long time like SP, it is later encountered with new infections that are then exposed to sub-therapeutic residual drug that is below the least required inhibitory concentration [53, 54]. The result of these sub-therapeutic concentrations is cross-resistance; a situation in which microorganisms are conditioned to tolerate toxic levels of a drug due to the previous experience to similar medicine or mode of action. This can arise by either deoxyribonucleic acid (DNA) shift or spontaneous metamorphosis [42]. These factors contribute greatly to drug resistance either collectively or autonomously [48]. Serious cases lead to treatment failure and death especially for vulnerable groups like children who have a rapid evolution of malaria from mild to severe infection [49]. Therefore, artemisinin-based treatment is equally threatened by the proliferation of resistant strains and no other hope is foreseen to surpass this remedy in case of full resistance in the near future. It is, therefore, recommended that the best way is to safeguard good manufacturing practices to minimize or eradicate the inflow of substandard drugs by strengthening the pharmacovigilance tools specifically in this case, post-marketing surveillance of the drugs already on the market to flush out such dangerous drugs and perpetrators [55]. There is also a need for careful implementation of universal medicine availability whilst safeguarding the quality of the drugs at the same time, because any compromise means resistance and subsequent change of treatment regimen to newer and more costly drugs [49], which are not yet even available. Thus, it is recommended that regulatory authorities in various countries should invest in modern facilities to enable them carry out their routine quality control activities well so that people can University of Ghana http://ugspace.ug.edu.gh 29 access best quality drugs. This would help in fighting against the inflow of these unwanted drugs that are already threatening to escalate the spread of resistance in Africa. This is very important as any ability of this resistance to infiltrate Africa would roll back malarial control initiative to zero [47]. In view of this widespread distribution of poor quality drugs most of which are counterfeit and/or substandard and the consequent growing public health crisis, an official World Health Organisation (WHO) body called the International Medical Products Anti-Counterfeiting Taskforce (IMPACT) was set up in 2006 by WHO and other stakeholders to coordinate the fight against the production and distribution of counterfeit drugs worldwide [56]. 2.7 QUALITY CONTROL PARAMETERS IN POST-MARKETING SURVEILLANC E (PMS) A drug product is mostly tested for safety, efficacy and quality to qualify it as suitable for human treatment or consumption. Quality control is the oldest parameter that has been in place for assessment of drug products, while safety and efficacy came into force towards the end of the 1950s after the Thalidomide tragedy. Drug product quality continues to be a very important assessment criterion and the procedures are performed in a wide range of infrastructure from simple to state-of-the-art facilities. For antimalarials, quality control involves physical methods done on liquids, solids and semi-solids. It entails chemical methods analyzing the API content, excipients and impurities, in vitro disintegration and dissolution tests as well as in vivo bioavailability tests [49]. University of Ghana http://ugspace.ug.edu.gh 30 Post marketing surveillance (PMS) involves the assessment of drug products when they are ready for market or at the market. Lucas et al. explain that a drug quality may involve assessment of the drug for stability and shelf life under specific moisture and temperature conditions. A resolution was passed by the International Conference on Harmonization (ICH), to standardize methods for medicine stability tests. In addition, stability of drugs was divided into three operational classes of long term, intermediate and accelerated stability studies [57]. Other quality control tests reported in the literature are residue analysis, excipients and binding materials status, degradation products and unidentifiable materials analysis, expiry status and content analysis, uniformity of weight, friability, tablet hardness, product shelf life under specific conditions, bioavailability and bioequivalence for generic drugs especially those with limited solubility and development of new analytical procedures that are cost effective and rapid [49]. 2.8 CASE STUDIES ON QUALITY CONTROL Several studies have been conducted in malaria endemic areas of the world on poor quality drugs. Such poor quality drugs have contributed to the abandonment of once effective antimalarial drugs and such poor quality drugs still pose a threat to the new artemisinin-based combination therapies that have been recommended and widely used as first line treatment for uncomplicated Malaria cases. Various studies have confirmed the widespread existence of these substandard and counterfeit antimalarial drugs. A few of these case studies are discussed. 2.8.1 Report s from Outside Africa In eastern Burma, following the death of a male patient diagnosed of uncomplicated hyperparasitaemic P. falciparum malaria and treated with an artesunate drug, quality analysis University of Ghana http://ugspace.ug.edu.gh 31 was conducted on the purported artesunate antimalarial drugs. The drugs were labelled as manufactured in China by Guilin pharmaceuticals. Tests (or analyses) showed that the drugs contained only 10mg out of the required 50 mg indicated on the packaging material and mainly contained paracetamol. The paracetamol addition may have been aimed at fever reduction to give a false impression of the drug’s efficacy, to cover up its failure to cure malaria [58]. In the Mekong region covering Cambodia, Burma (Myanmar), Laos, Thailand and Vietnam, a study conducted in 2001 to assess the quality of drugs using 104 samples collected from shops showed that 38% of the samples had zero artesunate. In 2004, a follow up study assessed 188 artesunate and 44 mefloquine tablet samples and results demonstrated a deteriorating situation as 53% had zero artesunate. In addition, out of the 44 mefloquine samples, 9% had amounts less than 10% of the claimed amount labelled on the pack [59]. Abdo-Rabbo et al. reported a study conducted in Yemen. Samples of antimalarials containing chloroquine and SP were investigated for content and dissolution compliance using validated methods (HPLC) in the pharmacopoeia. Chloroquine syrup samples registered a 6.7% failure rate, while tablets had 20% failure rate on content analysis. For dissolution analysis, 8% of the chloroquine tablets failed. In addition, 100% of the SP tablets passed the content analysis, but 80% failed the dissolution tests, with pyrimethamine having extremely poor results [60]. In 2007, a study carried out in Vientiane, Laos using visual and chemical analysis showed that 53% of the samples were falsified. Most of the fake artesunate pills had a deceptive visual look for an ordinary eye due to their sophiscated packaging, holograms and logos that could be University of Ghana http://ugspace.ug.edu.gh 32 detected as fake only when a UV light was used. Some of the samples were found with inappropriate materials such as flour, starch, chalk, acetaminophen (paracetamol) or chloroquine as well as fatal sulpha drugs for those allergic to them. Some samples also had a trace of artemisinin that showed positive result for Fast-Red dye test, yet insufficient to cure malaria [61]. Newton et al. analyzed 391 artesunate samples collected from the Thai-Myanmar border (16), Cambodia (48), Vietnam (75), Lao PDR (115) and Myanmar (137). Results showed that there were 16 different fake holograms within the samples. In addition, some samples had wrong and banned ingredients, carcinogens and raw materials of the narcotic drug, Ecstasy. Overall, 195 of 391 drugs had little or no artesunate representing 49.9% failure rate; a genuine drug was supposed to have 50mg of the API, but instead, majority of the samples had only 12mg. Those samples that were counterfeit were also found with some pollen, calcite as well as charcoal and further investigations traced these face medicines to South-East China [62]. Nayyar et al. reviewed published and unpublished study data from 7 countries of Southeast Asia and sub-Saharan African regions to establish the extent of distribution of poor quality antimalarials between 1999 and 2010. Data for five classes of antimalarial drugs consisting of artemether, artesunate, chloroquine, mefloquine, quinine, sulphadoxine–pyrimethamine and tetracycline were collected from Southeast Asia. For southeast Asia, multinational surveys of seven countries showed that while 497 out of 1437 (35%) of the drug samples had failed chemical tests, 46% (423/919) of the samples had failed packaging analyses and assessments, while 36% (450/1260) were found to be counterfeit. However, due to the lack of enough data, they could not establish the frequency of substandard antimalarials [63]. University of Ghana http://ugspace.ug.edu.gh 33 2.8.2 Report s from Africa In Tanzania, a sample of an antimalarial drug purported to contain dihydroartemisinin (60 mg/tablet, cotecxin) was found on analysis using both TLC and HPLC to contain none of the active ingredients [64]. In another study also conducted in the same country in 2005, 1080 samples consisting of 679 antifolates (SP, 394 and sulphamethoxypyrazine/pyrimethamine, 285), 260 amodiaquine, 63 quinine and 51 artemisinin derivatives were collected from retail shops in 21 districts. Out of the total number of the samples, 304 samples were selected for the laboratory analysis during which US pharmacopoeia monographs were used when available for dissolution and HPLC tests. The HPLC and dissolution test results showed that 13% of the antifolate samples had poor quality, while 23.8% and 7.5% of quinine and amodiaquine samples respectively were found to be of poor quality, with all the artemisinin derivative samples being found compliant [65]. In Cameroun, a study conducted in 2004 to investigate the quality of 284 samples comprising chloroquine, quinine and sulphadoxine-pyrimethamine components collected from 132 different unauthorised retailers and distributors of the rural and urban areas, in which simple colour reaction tests and SQ-TLC methods were used reported that 38% (50/133), 74% (52/70) and 12% (10/81) of the drug tablets had failed the quality tests due to the presence of either unsatisfactory, wrong, unknown or inactive pharmaceutical ingredient(s) [66]. In Ghana, a study was conducted by Osei-Safo et al. in 2009 on various samples of antimalarial drugs collected from selected parts of Accra. The results showed that out of 49 samples comprising single tablet artemisinin-based fixed-dose combination drugs, separately formulated University of Ghana http://ugspace.ug.edu.gh 34 artemisinin-based combination drugs packaged on the same blister to be taken concomitantly (ACTs) and artemisinin-based monotherapy formulations, only 28.6% (14/49) fulfilled the pharmacopoeia standards based on SQ-TLC. When these samples were further subjected to HPLC test, only 8.0% (4/49) of the samples complied with pharmacopoeia standards [67]. In 2012, a related study was conducted in Ghana by Addae-Mensah et al. as part of a joint Ghana- Togo study. 86 antimalarial drug samples consisting of 50 ACTs, 8 artemisinin monotherapy and 28 non-ACTs were collected from the major cities and border towns. The samples were analysed using basic tests, SQ-TLC and HPLC tests. The results showed that there was a widespread circulation of poor quality drugs, whereby 6 out of 7 zones of the collection exercise had samples that failed the tests, with one zone recording an 85% failure rate and more than 60% failure rate by the others. Out of the 50 ACTs, 8 (17%) were compliant with the Ph. Int. requirements, with 39 (83%) being non-compliant. In addition, out of the 8 artemisinin monotherapy samples, 1 (12.5%) was compliant, whereas a further 1 (12.5%) and 6 (75%) were marginally compliant and non-compliant respectively. While 12 (43%) of the non-ACTs passed the tests, 16 (57%) failed the tests. Majority of the samples failed due to the inadequacy of API amounts [68] In Kenya and DR Congo, a survey on the quality of artemisinin-based drugs conducted on 24 samples collected and analyzed using European Pharmacopoeia guidelines showed that 9 did not comply with the quantity requirement of 95-105% of the labelled content, but all the failed samples had some traces of the API [69]. Separately, another study was conducted in East DR Congo using European Pharmacopoeia guidelines on chloroquine syrup and injection batches, quinine injection batches, SP tablet batches and proguanil batches. Outcomes showed that samples from one batch out of the two chloroquine injection batches investigated using UV- University of Ghana http://ugspace.ug.edu.gh 35 spectrophotometry and HPLC-UV were overdose by 14% and samples from one out of the four selected quinine injection batches were overdose by 8%, whereas chloroquine syrup was compliant with the European Pharmacopoeia limits of 95-105%. Samples containing sulphadoxine-pyrimethamine components that were analysed using HPLC-UV only showed that sulphadoxine component of the samples were under dosed (91-94%) with respect to European Pharmacopoeia (95-105%), but within the compliance limits according to the United States Pharmacopoeia (90-110%) requirements in all the batches. However, samples from two of the sulphadoxine-pyrimethamine batches had pyrimethamine components that were overdose by 106% and 108% respectively and one batch had its samples found without any of the requisite ingredients. In addition, proguanil also passed after registering 98.7% of the label claim [70]. In Nigeria, a survey was conducted in 2001, in which 581 drug samples were collected from pharmacies. Two hundred and eighty-six (286) of the drug samples were antimalarials while the rest were antibacterial, antifungal and antituberculosis drugs. They collected drug samples that were on the WHO model list of essential drugs only. The samples were analyzed for API content using a corroborated HPLC method in accordance with British Pharmacopoeia (BP) specifications. The validated HPLC methods were used in this survey and the results compared with pharmacopoeia requirements. Out of the 286 antimalarial drug samples were 82 chloroquine phosphate (29 capsules, 20 syrups, 18 tablets and 15 injections), 31 chloroquine sulphate (11 syrups, 19 tablets and 1 capsule), 19 proguanil hydrochloride tablets, 10 quinine hydrochloride injection, 18 quinine sulphate (1 syrup and 17 tablets) and 126 sulphadoxine-pyrimethamine (SP) (100 SP tablets and 26 syrups). The results of chloroquine phosphate showed that 70% (20), 100% (20), 94% (17) and 93% (14) of the capsules, syrups, tablets and injections respectively University of Ghana http://ugspace.ug.edu.gh 36 fell outside the BP limits, while the failure rates in chloroquine sulphate samples were as follows; syrups (73%; 8/11), tablets (79%; 15/19) and 0% for the capsules. Out of the 17 quinine sulphate tablets, 24% (4) failed the tests. Of the 100 SP tablets and the 26 syrup samples, 13% (13) and 30.79% (8) respectively failed the tests. All proguanil hydrochloride, quinine hydrochloride injections and quinine sulphate syrup registered compliance with the BP specifications [71]. In 2003, WHO carried out a pilot study on samples of SP, chloroquine tablets and syrups collected from Ghana, Sudan, Gabon, Zimbabwe, Mali, Mozambique, Kenya and Tanzania. The samples were analyzed for quality in terms of API content and dissolution. Table 2 below shows the combined percentage results from all countries with the results of the samples failing pharmacopoeial standards out of the total number of samples analysed. Samples from Tanzania were not analyzed because their collection was delayed and samples from Gabon were not analysed for dissolution tests because they were too few. Therefore, the results showed a widespread circulation of poor quality antimalarial drugs throughout the study countries. The report also showed that most samples failed the tests due to insufficient amount of the APIs for chloroquine samples, but for SP samples, dissolution rather than API content accounted for an unusually high failure rate. Such poor results were attributed to poverty which in turn gave rise to poorly equipped laboratories, under-funded regulatory bodies and poor handling and manufacturing practices [72, 73]. However, the undesirable quality of the drugs might also have been caused by greed on the part of certain manufacturers. University of Ghana http://ugspace.ug.edu.gh 37 In Uganda, a market survey in May, 2007 identified a quinine BP 300mg tablet as fake. The general public was then notified of this drug by the National Drug Authority. The drugs were identified as having batch number 0908, manufactured in 2006 with 2009 as expiry date. It was found that the drugs were not manufactured by the sole licensed pharmaceutical producer of quinine in Uganda, the Kampala Pharmaceutical Industries Ltd as claimed on the package [74]. Table 2: Combin ed count ry re su lt s: pe rcent age failu re of sa mp les Count ry Chlo roq ui ne syr ups Chlo roq ui ne t ab lets Sulp hadoxine/ pyr i met ha mi ne Tablets API Conte nt (%) API Conte nt (%) Dissol ut io n (%) API Conte nt (%) Dissol ut io n (%) Gabon 0, (0/8) 29.0, (5/17) 14.29, (1/7) 0, (0/10) - Ghana 5.0, (1/20) 66.7, (12/18) 20.0, (3/15) 37.5, (3/8) 75.0, (3/4) Mali 66.7, (4/6) 47.3, (9/19) 5.2, (1/19) 0, (0/7) 100, (7/7) Kenya 25.0, (2/8) 42.8, (3/7) 28.6, (2/7) 0, (0/12) 91.7, (11/12) Mozambique 25.0, (3/12) 20.0, (3/15) 6.7, (1/15) 5.5, (1/18) 100, (18/18) Sudan 26.6, (4/15) 5.2, (1/19) 12.5, (2/16) 0, (0/20) 80.0, (12/15) Zimbabwe 13.3, (2/15) 57.1, (8/14) 7.1, (1/14) 10.0, (1/10) 100, (10/10) Average failure 23.0 38.3 12.2 7.6 91.1, (n= 6) Range 0-66.7% 20-66.7% 5.2-28.6% 0-37.5% 75-100%,(n= 6) In Kumasi (Ghana), a study was also conducted on artesunate containing samples to assess the content of the API and the uniformity test on tablets in accordance with European Pharmacopoeia and International Pharmacopoeia specifications. Results showed that content analysis gave API content range of 47.9-99.9% for all samples. Specifically, 11 (47.1%) failed the content uniformity test, while 6 (35.3%) passed the tests with respect to International Pharmacopoeia specifications. Additionally, 3 samples (17.6%) conformed to European Pharmacopoeia conditions for API content [75]. In 2009, fake Coartem tablets were reported to be University of Ghana http://ugspace.ug.edu.gh 38 circulating on the Ghanaian market. Analysis of the samples found them to contain none of the requisite APIs labelled on the pack [76]. In 2008, another study was carried out in 6 countries from the most malarious regions of Africa and the selected countries were Kenya, Rwanda, Uganda, Tanzania, Ghana and Nigeria. A total of 210 antimalarial drugs consisting of artemether-lumefantrine, artesunate, artemether, dihydroartemisinin, mefloquine and amodiaquine were selected and the semi-quantitative thin layer chromatography method coupled with dissolution tests were used for analysis. It was reported that dihydroartemisinin had the highest failure rate of 55% followed by amodiaquine (48%), SP (38%), artesunate (31%), artemether (27%), mefloquine (24%) and artemether- lumefantrine (19%). Furthermore, the country drug failure rates were Ghana (35%), Kenya (38%), Nigeria (32%), Rwanda (33%), Tanzania (32%) and Uganda (35%) [45]. In South East Nigeria, Onwujekwe et al. (2009) conducted a survey on artesunate, dihydroartemisinin, SP, quinine and chloroquine to assess their dissolution efficiency and content in some 4 artesunate samples as well as some 24 dihydroartemisinin samples using an uncertified HPLC method. All the Artesunate samples passed the tests, while 46% of the quinine as well as 39% of SP failed and were found to be substandard. Generally, 37% of the samples did not comply with the pharmacopoeia requirements [77]. In Tanzania, following the detection of a fake Metakelfin by the Tanzania Food and Drug Authority (TFDA) in 2009 on the markets through an inspection of some 40 pharmacies, the Drugs Board suspended the drugs’ importation, distribution, sale and use. The study revealed University of Ghana http://ugspace.ug.edu.gh 39 that the fake drugs had batch numbers that were not approved at the time of importation and some had low API content inconsistent with the 90-110% pharmacopoeia requirement [78]. A separate survey in the same country found some wheat flour instead of the labelled APIs [79]. Between 2001 and 2005, Thoithi et al., sampled 41 packs of antimalarials in Kenya comprising Artemisinin derivatives, SP, Quinine and Amodiaquine to assess them for content and dissolution compliance using pharmacopoeia guidelines. Many SP drugs failed especially the dissolution tests. In addition, half (50%) of the 20 SP samples and one-third (33.33%) of artemisinin-based drugs did not comply with the pharmacopoeial requirements [80]. A WHO (2011) report outlined the status of the quality for antimalarials in six sub-saharan countries. The six selected countries were Cameroon, Tanzania, Ghana, Kenya, Nigeria and Ethiopia and the samples collected were ACTs and SP. Out of the six countries, Nigeria had the highest failure rate of 64%, followed by Ghana (39%), Cameroon (37%), Tanzania (11%), Kenya (5%), and Ethiopia (0%). However, Ethiopia registered 41% of non-registered drugs, making the country vulnerable to fake drugs. Most of the poor drug results were attributed to low API content, degradation products and poor dissolution. For example, 62.5% of ACTs failed due to the low API content and dissolution failure. Samples containing SP also had a failure rate of 66.7%. The overall assessment showed that 33% of the drugs were non-compliant. Most of the countries with high failure rates had their antimalarial products sourced from many different manufacturers. Imported drugs had similar failure rate results for both registered and non- registered. The results analysis led to the speculation that the non-compliance might have come from poor manufacturing practices [55]. University of Ghana http://ugspace.ug.edu.gh 40 In Malawi, a national survey was conducted in 2007 to assess the quality of antimalarial drugs upon request from USAID-funded Strengthening Pharmaceutical System (SPS) programme, after being neglected in several WHO QAMSA multinational surveys. A total of 199 antimalarial drug samples consisting of 57.8% and 42.2% of ACTs and SP respectively were collected throughout the country from all the pharmaceutical outlets. After rejecting 26 (13%) monotherapies the remaining 173 samples were analysed. The samples were analyzed using the Minilab physical and basic tests in Malawi and confirmed later in Kenya. Results showed that all samples tested positive for disintegration and basic tests and 4% (8) failed the physical examination. In addition, 16% (8/50) of the samples failed confirmatory tests and out of the 8 samples that failed the confirmatory tests, 37.5% (3/8) were SP and 62.5% were ACTs (branded and generic); 12.5% (1/8) was not compliant with uniformity tests and a further 87.5% (7/8) failed dissolution test. This study helped the country to have an insight into establishing a national antimalarial drug monitoring system [81]. Several programmes and studies have been conducted in Malawi and some are still ongoing to kick out malaria. The options being employed include treatment modalities and diagnostic techniques, epidemiology, community and public health education, pharmacological and clinical efficacy and genetic diversity, intermittent preventive treatment of pregnant women (IPTp), insecticide treated net (ITNs) and indoor residual spraying (IRS) [82], with little or no attention being paid to quality control studies. In addition, reported cases of the surveillance of drug quality by the national drug regulatory authority on the required capacity, scale and frequency or their occurrence is not well documented in the published literature. This declaration of the capacity of the regulatory body is made following an ACT Consortium report by Staedke in 2009 University of Ghana http://ugspace.ug.edu.gh 41 [83] which recommended the establishment of drug safety register and systematic surveillance to monitor the quality, influx of poor quality drugs, their sources and the extent of the effects of transportation and storage on the quality of genuine drugs in Malawi. In addition, SPS in its 2011 report following a survey also categorised Malawi as one of the countries that had no or minimal capacity for pharmacovigilance (PV) due to the lack of legal or structural frameworks for PV systems, no coordinated passive/active surveillance and lack of national coordination in any ongoing PV activity [84]. Unconfirmed reports from the regulatory authority in 2012 revealed that such activities are being undertaken with specific activities and extent not mentioned. However, the Ministry of Health and the Drug Regulatory Board issued a statement that all drugs that are distributed in government and Christian Health Association of Malawi (CHAM) health centres are tested for efficacy, safety and quality [85], with no mention of drugs accessed from the private drug stores or pharmacies as well as the parameters assessed. In a nutshell, there has been widespread circulation of poor quality drugs in some parts of Asia and Africa. Most of them contain sub-therapeutic amounts of the APIs or no API at all or even toxic compounds and if this is not checked, it can lead to emergence of drug resistance. 2.9 QUALITY ASSESSMENT METHODS There is ample evidence that circulation of poor quality drugs in Africa is increasing at an alarming rate due to poor regulatory capabilities in most regulatory authorities in terms of manufacturing and import regulations [45]. As one of the ways of moving towards a counterfeit drugs-free society, the WHO Roll Back Malaria Initiative urges all those that have adopted its University of Ghana http://ugspace.ug.edu.gh 42 initiative into malaria programmes to implement one of its goals that stipulates that at least 80% of antimalarial products in a country should satisfy acceptable international quality standards [49]. So many mitigating factors have been employed to solve this problem of substandard and bogus drugs inflow. These include consistent monitoring of drugs quality to ensure the purchase, supply, import, manufacture and distribution of quality drugs. Guidelines and methodologies for the quality assurance surveillance are outlined in the pharmacopoeias for this exercise [86]. 2.9.1 Pharmacopoeia Methods There are several pharmacopoeias by individual countries, territories, regions and many more. However, few have been adopted, then widely used and these are United States Pharmacopoeia (USP), British Pharmacopoeia (BP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (Ph. Eur.), Pharmacopoeia of the People's Republic of China (PPRC) and International Pharmacopoeia (Ph. Int.). These pharmacopoeias have common features outlining recommended methods from published monographs with information such as procedures for analysis, determination of active pharmaceutical ingredients (APIs), excipients and dosage forms. Most of the pharmacopoeia guidelines are very expensive and difficult to organize for many programmes in developing countries. Therefore, to minimize this cost burden, the Directorate of Quality Assurance and Safety: Medicines of the WHO introduced a programme aimed at developing cost effective reliable methods of chemical analysis to be adopted by national drug regulatory authorities in standard noncompliance screening prior to drug’s use. Therefore, cost effective methods have been developed suitable for the screening of artesunate, artemether and University of Ghana http://ugspace.ug.edu.gh 43 other derivatives’ quality [87]. Some of these innovated methods are used together with those pharmacopoeia methods that are cheaper and easy to use. Indeed, the WHO developed the International Pharmacopoeia as a means of providing simple inexpensive but accurate methods that can be used in any less well-endowed analytical laboratory. 2.9.2 Case Studies on Method Develop men t s Green et al. developed and validated colorimetric [ARTS-Fast red TR (FRTR)] method. It was initially developed for the detection of fake artesunate, which was later extended to artemisinin, dihydroartemisinin and other derivatives. For artesunate analysis, artesunate is decomposed to an alkaline product which reacts with fast red TR salt giving a yellow colour. The absorbance of the yellow colour is then measured. Furthermore, Green et al. went on to find out the efficiency of using colorimetric and refractometric methods alone and together by comparing them with the HPLC assay results. Effectiveness of refractometry and colorimetry combined was observed in the assessment of artesunate, chloroquine injection, quinine and sulphadoxine with an accuracy of 0.96-1.00, while the accuracy was lower for enteric-coated chloroquine with an accuracy of 0.78 [88]. In 2004, German pharmaceutical companies involved in research set up a charitable trust called German Pharma Health Fund (GPHF), which developed a mini-laboratory tool called GPHF- Minilab containing consistent, cheap and simple methods for the prompt verification of drug quality for tuberculosis, malaria and HIV/AIDS, antibiotics and many more important drugs in low income or developing countries. This was aimed at detecting fake and substandard drugs [89]. University of Ghana http://ugspace.ug.edu.gh 44 An easy thin-layer chromatography (TLC) method was also developed and reported by Ioset and Kaur in 2009. The method was developed for artemisinin and its derivatives detection in antimalarial drugs, using 2,4-dinitrophenylhydrazine or 4-benzoylamino-2, 5- dimethoxybenzenediazonium chloride hemi (zinc chloride) salt as a spraying agent. The presence of artemisinin and derivatives is shown by a pink colour for the former reagent and blue colour for the latter reagent. However, the method cannot detect artemisinin and derivatives of contents 10% or less [90]. At the request of the WHO, Addae-Mensah and Osei-Safo in 2006 also developed and validated a user-friendly semi-quantitative thin layer chromatography method for artemisinin and non- artemisinin antimalarials with two suitable solvent systems for each API and anisaldehyde/methanol as a spraying reagent for the artemisinins. This method gives a valid estimation of the API in a drug which can then be more accurately verified by an HPLC method. The method is useful for rapid field estimation of the quality of all antimalarial drugs [91]. This method has subsequently been successfully used in two major World Health Organisation (WHO) and West African Health Organisation (WAHO)-sponsored surveys [67, 68, 92]. This method has also been used in the current study and the procedure has been articulated in the subsequent chapters. 2.9.3 Other Analyt ica l Methods Most of the methods developed above are mostly for rapid tests in areas that are economically challenged and they are partially accurate, although some accurate but expensive methods have also been developed the same way. Therefore, it is recommended that confirmatory tests using University of Ghana http://ugspace.ug.edu.gh 45 other more accurate, specific and precise though expensive expertise-technology dependent methods such as High Performance Liquid Chromatography (HPLC) and Dissolution tests should be used to complement the TLC and basic disintegration results respectively. TLC and basic disintegration tests are tentative or approximate hence can be easily challenged [48, 55]. Other tools that have been used in this course include GPHF Minilab, HPLC (coupled with either of the following; fluorescence, single wavelength ultraviolet/visible absorbance, photodiode array (PDA), electrochemical or refractive index), HPLC-Mass spectroscopy hyphenated method (LC- MS), Open air ionization-mass spectroscopy, desorption electro spray ionization (DESI)-mass spectroscopy and many more [48]. 2.9.4 Selected Qualit y Cont ro l Tools fo r the Presen t Study Following the recommendations by WHO and other players reported in the literature for post- market surveillance of antimalarial drugs, the following methods have been selected for the current study: 2. 9. 4. 1 Visu al Inspecti on A drug sample can be subjected to visual inspection which among others involves the verification of tablets’ mass uniformity, size, colour, crimping, weight, printing, bar codes and holograms in comparison to the authentic genuine drugs. However, this method has suffered a lot due to an overwhelming response by counterfeiting entities such that counter-mechanisms have been applied to beat this trap. The features used under this method are also difficult to determine, i.e. difficult to differentiate between a genuine and counterfeit drug. However, it is still suitable and widely used in surveillance in resource constrained poor developing countries [52]. University of Ghana http://ugspace.ug.edu.gh 46 2. 9. 4. 2 Basic Tests These are simple and readily applicable methods used to confirm the identity of APIs [93]. Due to the success and challenges the visual inspection method has met, there are some methods that are supposed to be done in addition to it such as basic or colorimetric tests. The literature and International pharmacopeia (Ph. Int.) have reported several methods of basic tests that are mainly based on the functional group of a drug [93, 94, 95, 96]. For example, artemisinins have been analyzed as follows: heating of a drug sample extract with potassium iodide producing a yellow colour; reaction of potassium iodide (KI) / starch with sample extract to produce a violet colour; reaction of Hydroxylamine HCl with a sample producing reddish violet colour. The colour change in each case indicates the presence of an API. The Figures 9 and 10 below show the reaction mechanisms of artemisinins reacting with KI / starch and vanillin / sulphuric acid (H2SO4) as examples [94]. Firstly, Figure 9 below shows an artemisinin derivative reacting at room temperature in the presence of glacial acetic acid and concentrated sulphuric acid mixture (10:1), producing a combination of α, β-unsaturated ketones (ketone-lactones). These products, therefore, react with an aromatic aldehydic vanillin whose product gives the observed corresponding pink colour in artemisinin derivatives basic tests [94]. Secondly, Figure 10 shows an alternative path a reaction can take; after an artemisinin derivative has reacted in the presence of an acid to produce many products, amongst them hydrogen peroxide (H2O2), i.e. from step XXXIII to XXX IV in Figure 9; the peroxide can be involved in a chemical reaction instead. Addition of KI at that stage results in the oxidation of iodide to iodine. Addition of starch reagent gives a colour reaction [94]. University of Ghana http://ugspace.ug.edu.gh 47 Figure 9 : Basic test prop osed reaction of a rt emi sin in decomposit ion products wit h v an illin Figure 10. Reaction of hydrogen pero xide wit h pot assiu m iodide Furthermore, alkaloidal antimalarials also have their basic tests outlined in the WHO literature and International Pharmacopoeia. These alkaloidal antimalarials react similarly with suitable reagents and chemicals using their distinctive chromophores in preferably colour and precipitate University of Ghana http://ugspace.ug.edu.gh 48 producing reactions. For example, lumefantrine tablets and suppositories are analyzed using such a test as shown in a proposed reaction mechanism shown in Figure 11 below. Figure 11 : Basic test prop osed reaction fo r lum efan t rin e. In this reaction, lumefantrine having allylic alcohol functional group is oxidised with manganese dioxide (MnO2) to an analogous ketone. The ketone then reacts with 2, 4-DNPH to produce an orange coloured precipitate of a hydrazone [92]. University of Ghana http://ugspace.ug.edu.gh 49 Basic tests are simple and readily available methods to confirm the presence of API using easily available reagents. It would also serve to inform if significant degradation has occurred in substances under unfavourable conditions [95]. However, this method cannot give quantity of an API and compounds with functional group similar to the required API can react similarly giving a false impression. For example, any compound with a similar allylic alcohol in Figure 11 can react similarly [92]. 2. 9. 4. 3 S em i- Quan ti tat i ve Thin Layer Chrom atograph y (SQ - T L C ) There has been a wide call to use semi quantitative TLC, and basic disintegration test in addition to the basic tests in pharmaceutical analysis, whereby the latter serves as a basic predictor of the time taken for the drug to break up for body absorption [49]. A TLC method, apart from its primary purpose, can also expose the impurities and substances resulting from degradation if they are in significant proportions [48]. This method is relatively easy, inexpensive, selective and cost effective. Results based on this method can be used to qualify a product as satisfying label specifications and legal for use [97]. In addition, it uses inexpensive and affordable technology, hence suitable for resource-limited communities and institutions [98]. Nevertheless, this method cannot detect the counterfeits containing incorrect inactive or active ingredients if they cannot be eluted by the solvent system and visually seen by the reagents employed for the correct API [97]. 2. 9. 4. 4 Hi gh Perf orm an ce Liqu id Chrom atogr aph y (HPL C) It is a chromatographic technique that separates a mixture of compounds for further activities such as identification, purification or quantification of compounds in a mixture. It is used for medical, legal, research and manufacturing purposes [99]. For example, it is generally used for the University of Ghana http://ugspace.ug.edu.gh 50 analysis of impurities of volatile and non-volatile compounds. It is also used for isolation, separation and purification of compounds, determination of ionic, zwitterionic and neutral molecules, preparative and process-scale separation ultra-tracing as well as qualitative and quantitative purposes [100]. It is a non-destructive method and can separate closely related compounds and a large variety of compounds such as organic, inorganic, biological, chiral and thermally labile compounds, polymers, small ions as well as macromolecules. HPLC has had a wide variety of applications such as measuring levels of active drugs, synthetic by-products, degradation products in medicines, hazardous compounds (like pesticides) as well as some compounds (amino acids, nucleic acids, and proteins) in physiological samples. In addition, it is used for monitoring environmental samples, purification of compounds, separation of polymers and determination of their weight distribution in mixtures, tracking synthetic reactions and quality control [100]. It works based on the basic principles of chromatography, where the components carried by a mobile phase interact with a stationary phase (sorbent) subsequently causing separation [100, 101]. The principle is the same as TLC, with the difference being the use of a pump in HPLC to pass the mobile phase and sample mixture with an operational pressure through a column, as opposed to TLC which depends on gravitational force for the mobile phase mobility. In addition, the sorbent materials in HPLC are smaller (average 2-5 micrometer), giving it a better resolving power. The typical solvents used are water, acetonitrile and/or methanol. The separation process is controlled by the temperature and composition of mobile phase, which influence the interaction between sample components and sorbent, which could be in the form of dipole- dipole, ionic and dispersive (hydrophobic) forces [99]. University of Ghana http://ugspace.ug.edu.gh 51 A typical HPLC system has a mobile phase reservoir, degasser, pump (50-350 bars), injector, column compartment, detector, data processor and waste collector. Using an HPLC requires suitable column, mobile phase solvents/reagents, standard solvent, analytical instruments (pH meter and balance), reference standard (for quantification), glassware and many more [102]. There are different kinds of detectors such as UV/Vis absorbance detector, fluorescence detector, electrochemical detector, conductivity detector, refractive index detectors, mass spectrometer [100], and photo diode array (PDA) [99]. There are different kinds of columns with different modes of separation as shown in Table 3 below [101]. Table 3: Kinds of chromat og rap h ic column s and their modes of sep a rat ion Type of Column Mode o f Separ at io n Normal / reversed phase Hydrophobicity (polarity) differences Gel filtration Size (molecular weight) differences Ion exchange Charge difference at particular pH (Bio-) affinity Interaction difference with ligand HPLC is widely used for pharmaceutical analysis in drug discovery, development, manufacturing and post-marketing quality control routine tests due to its sensitivity and accuracy. In pharmaceutical analysis, the widely used columns are reversed and normal phase columns [102], with the former preferentially used often [101]. The two columns differ due to the polarity of mobile and stationary phases as illustrated in Table 4 below. University of Ghana http://ugspace.ug.edu.gh 52 Table 4: Column s and their resp ective st at ion a ry and mobile phases as function s of polarit y Column Stat io na ry Phase Mobile Phase Normal phase Polar Non-polar Reversed phase Non-polar Polar In a normal phase chromatography, non-polar molecules are eluted first because the polar molecules associate with the polar stationary phase retaining them in the process. When polarity of the mobile phase is increased, elution time of the polar molecules increases, at the same time increasing the rate at which non-polar molecules are eluted. Due to the lack of reproducibility, it became unpopular until the development of the Hydrophilic Interaction Liquid Chromatography (HILIC) bonded phase, when it became useful again. In reversed phase chromatography, the polar silica gel in the stationary phase is covered with a non-polar layer of alkanes, after reaction of silica gel with long chain hydrocarbon-substituted Silane. This process reverses the polarity phase. Here, polar molecules are eluted first because it is the non-polar molecules that associate with the non-polar stationary phase and get retained. This allows polar molecules to elute first and quickly. Hence, polar compounds are used as mobile phases and water and acetonitrile are commonly used [103]. HPLC analysis can be run using either Isocratic or Gradient elution. Isocratic elution involves the use of a mobile phase with constant composition while in gradient elution, the solvent strength is adjusted during the separation and this is suitable for samples that are complex in nature [103]. Detection is usually done by UV method and/or mass spectrometer for analytes that University of Ghana http://ugspace.ug.edu.gh 53 do not show UV absorption. Therefore, to get a desirable well resolved peak, detector, column type and mobile phase are manipulated [103]. When used for quantification, the amount/concentration of the analyte is equivalent to the peak area of the chromatogram or area under the curve (AUC). The first step involves the creation of a calibration curve that is produced by generating various chromatographs from different known concentrations of a reference standard. The peak or curve areas are calculated and equated to their respective known concentrations and this data is used to plot a graph of area under the curve/peak (AUC) against the known concentration of the reference standard, to obtain a calibration curve. The second step involves the determination of the unknown analyte concentration. A measured amount of the sample of unknown concentration is injected into the HPLC system and the peak area of its chromatograph is calculated and its concentration is interpolated from the calibration curve automatically by a computer connected to the HPLC system. Like most analytical methods, HPLC also has some limitations like difficult resolution for complex samples, one sample analysis at a time, training needed for optimum separation, relatively long analysis times, sample preparation and need for complementary techniques like MS, NMR and IRS sometimes [100] as well as the high cost of columns, reference samples and the HPLC grade solvents. University of Ghana http://ugspace.ug.edu.gh 54 CHAPTER THREE 3 CURRENT INVESTIGATION 3.1 SAMPLING 3.1.1 Study Area The selection of study areas was based on the conventional random sampling in all the country’s 3 regions; north, central and south. The country was divided into 4 zones with each zone consisting of 5-7 districts based on the National Malaria Control Programme (NMCP) strategy partitions; giving the south west (1), south east (2), central (3) and north (4) zones. In each zone, the chosen districts were randomly selected based on the prevalence of Malaria, geographical position and economic activities. These are factors that are deemed to influence the demand and inflow of drugs. Therefore, the districts that were selected for the survey were Lilongwe, Blantyre, Zomba, Mzuzu (in Mzimba), Karonga, Dedza, Ntcheu, Mangochi, Mulanje, Thyolo, Mwanza, Kasungu, Mchinji and Nkhota-kota out of the 28 districts. Figure 12 below is a map of Malawi showing the zones and the districts in which sampling took place and Table 5 shows the number of samples that were collected in each zone. 3.1.2 Sample Size A total of 112 samples was purchased in all the zones based on their availability regardless of size, company name, brand, product name, dosage form and strength, though not more than one sample of the same name, batch number and characteristics was bought at one outlet. Some strategically designated areas were left out due to the lack of antimalarial drug outlets. University of Ghana http://ugspace.ug.edu.gh 55 Figure 12 : Map of Malawi showin g samp lin g sit es Zonal boundaries Zonal towns: where sample collections were made. University of Ghana http://ugspace.ug.edu.gh 56 Table 5 : Numbe r of samp le s by zon e of collection Zo ne o f Collectio n Place o f Collectio n Number o f Collected Samp l es 1 South west 41 2 South east 17 3 Central 25 4 North 29 Total 112 The samples consisted of 76 ACTs and 36 non-ACTs representing 67.86% and 32.14% of the total samples respectively. The ACTs were both in fixed dose and single dose co-packed on the same blister combinations, whereas all non-ACTs were fixed dose combinations. The large percentage of ACTs over the non-ACTs suggests that the implementation of ACT use in the country has been on the right track. The largest component of the ACT samples was the artemether-lumefantrine formulation (53.95%) and this might be attributed to the fact that it is a first line treatment for malaria in Malawi and has been found to be more tolerated with minimal side effects. Amongst the non-ACTs, sulphadoxine-pyrimethamine formulation had the largest quantity (63.89%) and this might also be attributed to the fact that it was the first-line treatment prior to the introduction of the artemether-lumefantrine formulation and that people are still using it because it is still being prescribed for special cases of malaria. However, the number of artemether-lumefantrine formulations is larger than sulphadoxine-pyrimethamine. Table 6 below summarizes the categories of drugs that were collected. The production, sale and use of blister co-packed ACTs was reported to be encouraging artemisinin monotherapy as people preferred using them alone, leaving the non-artemisinin drugs due to either bitterness or saving for another time [104]. University of Ghana http://ugspace.ug.edu.gh 57 Table 6: Categories of collected ant imala rial drug sa mp le s Fixed dose no n - ACTs Doses co - packed o n o ne blis ter Fixed dose ACTs API No. API No. API No. Quinine sulphate 6 Ats co-packed with SP 4 Atm/Lum 41 Quinine hydrochloride 3 Dha/Pp 14 Quinine bisulphate 4 Dha/SP 12 SP 23 Ats/SmP 5 TOTAL 36 4 72 Atm ; artemether, Ats ; artesunate, Lum ; lumefantrine, Dha ; dihydroartemisinin, S; sulphadoxine, P; pyrimethamine, Pp ; piperaquine phosphate, Sm ; sulphamethoxypyridazine. Hence, it was recommended that the co-packed blisters should be phased out [104]. However, the results show that there were 4 samples of single dose co-packed on the same blister drugs, a sign that this category of drugs is still in circulation. Considering the fact that the mostly affected people in Malawi are poor and coincidentally also illiterate, there is a high possibility of such occurrence of drug abuse. 3.1.3 Samplin g Strat egy Sampling was conducted between December and January, within the rainy season in Malawi. Malawi records peak malarial transmission in the rainy season that is experienced mostly between November and April. This agrees with the fact that during this period, there are so many stagnant water points, which are favourable breeding grounds for the malaria vector, the mosquito. The antimalarial drugs were purchased from every available outlet due to limited number of drug outlets in designated places. Included were both licensed and unlicensed markets `such as private pharmacies and hospitals, street vendors and shops. The samples were in the form of tablets, suspensions, injections and mixtures. Additionally, the samples were composed of APIs such as sulphadoxine, pyrimethamine, quinine, lumefantrine, piperaquine, sulphamethoxypyridazine, artemether, artesunate and dihydroartemisinin. The samples were University of Ghana http://ugspace.ug.edu.gh 58 labelled for identification as well as recording and kept in containers that protected them from light, moisture, crushing, heat and mechanical shock. 3.2 PRE-LABORATORY ANALYSIS 3.2.1 Regist rat ion Status of Samples The companies and individuals that manufacture, prepare, circulate and process drug products as well as biological products for human and veterinary use are required by law to register the products with a regulatory body to validate their safety, efficacy and quality. Therefore, the drugs were subjected to registration verification with the drug regulatory authority - the Pharmacy, Medicine and Poisons Board of Malawi as soon as the collection exercise was completed. There were 25 different brands of drugs purchased in all the regions and all of them were presumed to be imported as there were no samples labelled to have been manufactured or packed in Malawi. As of 31st December, 2011, 92% (23/25) of the brands and 86.61% (97/112) of the collected samples as well as their formulation types were registered with the regulatory board, according to their annual registration publication. Table 7 below demonstrates the drug registration status with respect to the zones of collection. Table 7 : Numbe r of samp le s collected in each zone and their regi st rat ion st at u s Zo ne o f Collectio n Number o f Samp l es per Zo ne Regist ered Samp l es per Z o ne Unreg iste red samp les per Zo ne 1 41 35 6 2 17 17 0 3 25 20 5 4 29 25 4 Table 7 demonstrates that zone 2 (central) had best registration standing with 100% (17/17) registration rate, followed by zone 4 with 86.21% (25/29), zone 1 with 85.57% (35/41) and zone University of Ghana http://ugspace.ug.edu.gh 59 3 with 80% (20/25). On the whole, the registration status of antimalarials in Malawi was found to be quite satisfactory, compared to countries such as Ghana and Togo. The most recent studies indicated 55% and 78% unregistered antimalarials in Ghana in 2008 and 2012 respectively and 17% unregistered antimalarials in Togo in 2012 [67, 68] 3.2.2 Origin of the Collected Drug Samp les None of the drug samples was manufactured locally and an inventory confirmed that they were imported from Asia, Africa and North America. Figure 13 below shows the countries and regions where the drugs were manufactured and imported into Malawi. Figure 13 : Sources o f the collected drug samp le s 3.2.3 Visual Inspection of Dosage Form a nd Packagi n g This was done with respect to guidelines outlined in the WHO International Pharmacopoeia (2006, Version 2). It is stated that “Every pharmaceutical preparation must comply with the 1.79% 11.61% 17.86% 8.04% 60.71% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% USA Tanzania Kenya China India America Africa Asia Areas of drug origin University of Ghana http://ugspace.ug.edu.gh 60 labelling requirements established under Good Manufacturing Practice.” Below are the requirements as stipulated in the pharmacopoeia: “(1) the name of the pharmaceutical product; (2) the name(s) of the active ingredient(s); International Non-proprietary Names (INN) should be used wherever possible; (3) the amount of the active ingredient(s) in each tablet and the number of tablets in a container; (4) the batch (lot) number assigned by the manufacturer; (5) the expiry date and, when required, the date of manufacture; (6) any special storage conditions or handling precautions that may be necessary; (7) directions for use, warnings, and precautions that may be necessary; and (8) the name and address of the manufacturer or the person responsible for placing the product on the market.” A sample was deemed to have passed the visual inspection if it contained all the pharmaceutical product information outlined above and the results showed that the manufacturers of all the collected drug samples complied with the packaging material labelling requirements. 3.3 LABORATORY ANALYSIS 3.3.1 Qualit at ive Colour Reaction s Drugs consist of active pharmaceutical ingredients (APIs) that are responsible for pharmacological effect and excipients, which are inactive pharmaceutical ingredients added together with the APIs for the former’s biopharmaceutical, stability and technical purposes. These are basically chemical compounds and they have some distinctive functional groups, University of Ghana http://ugspace.ug.edu.gh 61 which are mostly identical to a specific drug API or group of APIs. Reactions and suitable reagents are chosen to distinguish a particular API from the rest. The reactions that exhibit a colour change or produce a precipitate are preferable; a colour change or precipitate formation depicts the presence of the compound in question. This method has been widely used in API identification because it is rapid, cheap, simple and easily applicable; hence suitable for the poorly equipped laboratories in less privileged communities, especially in developing countries. It is mostly the first chemical test for drug products verification of the labelled ingredients after visual inspection. For each API, at least two methods of identification were used because there are some compounds that can mimic API reactions and produce false results [95]. The present drug samples had different APIs, formulations, excipients as well as dosage forms and each API had its distinct methodology and apparatus as specified in pharmacopoeias and the literature. Therefore, all samples were subjected to colorimetric tests to determine if they contained the APIs claimed by the manufacturers and below are the details of the methods and procedures that were used. 3. 3. 1. 1 Gen eral Procedu re Drug samples in the form of tablets were prepared for analysis by weighing them using analytical balance individually and all together. The individual and total masses were recorded and later averages derived from them, then compared for consistency. The tablets were ground using a pestle and a mortar to powder. The powdered samples were treated according to the procedures below. University of Ghana http://ugspace.ug.edu.gh 62 3. 3. 1. 2 S pecif ic Procedu re 3. 3. 1. 2. 1 Artesu n ate con tai n in g dru g sam ples Description of the artesunate containing samples:  Tablets of artesunate co-packed on the same blister with sulphadoxine/pyrimethamine combination tablets; artesunate/sulphadoxine/pyrimethamine: 100mg / 500mg / 25mg  Fixed dose combination tablets of artesunate/sulphamethoxypyridazine/pyrimethamine: 200mg / 500mg / 25mg and 100mg / 500mg / 12.5mg formulations 3. 3. 1. 2. 1. 1 Artesu n ate Colou r and other reaction s [96, 105] a) A quantity of the powdered tablets equivalent to 100mg of artesunate was weighed into a clean dry beaker and 40ml of dehydrated ethanol added. The mixture was shaken together to dissolve the active ingredients. This was then filtered and the filtrate divided into approximately 2 equal parts, 20ml each. To one half of the filtrate was added 0.5ml of hydroxylamine hydrochloride TS 2 and 0.25ml of 2M sodium hydroxide solution. This mixture was then heated on a water bath to boiling. The solution was then allowed to cool and two drops of 2M HCl solution added followed by 2 drops of iron (III) chloride (50 g/l). Expected observat ion : production of a light-red violet colour b) The other half of the filtrate from (a) above was evaporated on a water bath to a volume of about 5 ml. A few drops of the mixture were placed on a white porcelain dish and one drop of vanillin/sulphuric acid TS1 added. Expected observat ion : Production of a reddish-brown colour. University of Ghana http://ugspace.ug.edu.gh 63 3. 3. 1. 2. 1. 2 S u lph adoxin e: Colou r and other reaction s [105] a) A quantity of the pulverized tablet equivalent to 50mg of the fixed-dose combination was dissolved in 3ml of sodium hydroxide (0.1 mol/l) VS and heated gently. The mixture was then cooled and 1.0 ml of copper (II) sulphate (80 g/l) TS was added. Expected observat ion : production of a greenish yellow precipitate, the colour of which changes gradually to blue. b) A quantity of the powdered tablets equivalent to 50mg of the fixed-dose combination was dissolved in 2ml hydrochloric acid (~70 g/l) TS. The resulting solution was cooled in ice, treated with 4ml of sodium nitrite (10 g/l) TS and poured into 2ml of 2-naphthol TS1 containing 1g of sodium acetate R. Expected observat ion : production of an orange-red precipitate. 3.3.1.2.1.3 P yrim eth am in e : Colou r and other reaction s [93] a) A quantity of the powdered tablet equivalent to 0.25g was shaken and dissolved in 50ml of ethanol (~750g/L) TS and heated to 60 oC. The solution was then filtered and heated to dryness with constant weight (test substance). 0.05g of the test substance was then dissolved in 5ml of sulphuric acid (~100g/L) TS. Freshly prepared potassio-mercuric iodide TS was then added to the solution. Expected observat ion : Formation of a creamy white precipitate. b) A mixture of 5mL of water and 2mL of ethyl acetate R was added to 1 mL of methyl orange/ethanol TS and shaken; ethyl acetate remained colourless. A solution prepared by University of Ghana http://ugspace.ug.edu.gh 64 dissolving 5mg of test substance in 5ml of sulphuric acid (~5g/L) TS was added, shaken well and allowed to settle (about 30 minutes). Expected observat ion : Formation of a yellow colour in the ethyl acetate layer. 3.3.1.2.1.4 S u lph am eth oxypyri d azin e : Colou r and other reaction s [96, 105] a) A quantity of the powdered tablet equivalent to 20mg of sulphamethoxypyridazine was added to 10ml of sulphuric acid (100 g/l) TS. This was then shaken to dissolve and 0.1ml of potassium bromate (50 g/l) TS was added to the mixture. Expected obse rvat ion : Production of a yellow colour that changes to amber and brown precipitate gradually forms. b) A quantity of the powdered tablets equivalent to 50mg of the fixed-dose combination was dissolved in 2ml hydrochloric acid (~70 g/l) TS. The resulting solution was cooled in ice, treated with 4ml of sodium nitrite (10 g/l) TS and poured into 2ml of 2-naphthol TS1 containing 1g of sodium acetate R. Expected observat ion : Production of a bright orange-red precipitate. 3.3.1.2.1.5 Result s fo r the basic test s Table 8 : Result s fo r basic test s of a rt esu n at e cont ain in g drug samp les Identit y Artesunate Sulphadoxine Pyrimethamine Sulphamethoxypyridazine Test a Test b Test a Test b Test a Test b Test a Test b *24Y13 + + + + + + *4Y13 + + + + + + *34Y13 + + + + + + *32Y13 + + + + + + 44Y12 + + + + + + 42Y12 + + + + + + 43Y12 + + + + + + University of Ghana http://ugspace.ug.edu.gh 65 41Y12 + + + + + + 31Y12 + + + + + + * stands for samples co-packed on the same blister, + means API present. 3. 3. 1. 2. 2 Artem eth er con tai n in g dru g sam ples Description of the artemether containing drug samples:  Fixed dose combination tablets and suspension powder of artemether / lumefantrine: 80mg/480mg; 20mg/120mg; 40mg/240mg; 180mg/1080mg formulations. 3.3.1.2.2.1 Artem eth er : Colou r and other reaction s fo r a rt e met h e r tab le t s [96, 105] a) To a quantity of the powdered tablets equivalent to about 80 mg of Artemether was added 40 ml of dehydrated ethanol. The solution was shaken well to dissolve, and filtered. Half of the filtrate was evaporated to about 1 ml and 100 mg of potassium iodide was added and heated on a water bath for about five minutes. Expected observat ion : Production of a yellow colour. b) The remaining filtrate from test (a) above was evaporated to about 5 ml. A few drops of this solution was placed on a white porcelain dish and 1 drop of vanillin/sulphuric acid TS1 added. Expected observat ion : Production of a pink colour. 3.3.1.2.2.2 L um ef an trin e : Colou r and other reaction s [92] a) To a quantity of the powdered tablets equivalent to 10mg of lumefantrine was added 5ml of ethanol and shaken well to dissolve the active ingredient. 20 mg of MnO2 was added to the University of Ghana http://ugspace.ug.edu.gh 66 solution and boiled on a water bath for about a minute. The solution was filtered and a few drops of 2, 4-dinitrophenylhydrazine (2, 4-DNPH) solution were added and shaken. Expected observat ion : Appearance of an orange precipitate of hydrazone within a minute. b) To a quantity of the powdered tablets equivalent to 10 mg Lumefantrine in a test tube was added 5 ml of ethyl acetate. A few drops of 1M HCl solution were added. The solution was stirred, warmed and filtered. To a portion of the test solution was added a few drops of Dragendorff’s reagent. Expected observat ion : formation of a brown to orange precipitate within 5 minutes. 3.3.1.2.2.3 Result s fo r the basic test s Table 9 : Result s fo r basic test s of art e met h e r cont ain in g drug samp les Ident ity Arte me t her Lume fant r i ne Test a Test b Test a Test b 2X15 + + + + 4X20 + + + + 11X1 + + + + 11X11 + + + + 11X14 + + + + 11X17 + + + + 11X18 + + + + 11X20 + + + + 12X1 + + + + 12X11 + + + + 12X14 + + + + 13X1 + + + + 13X11 + + + + 14X1 + + + + 14X11 + + + + 15X1 + + + + 15X11 + + + + 16X1 + + + + 17X1 + + + + 18X1 + + + + 19X1 + + + + 21X1 + + + + University of Ghana http://ugspace.ug.edu.gh 67 22X1 + + + + 23X1 + + + + 24X1 + + + + 25X1 + + + + 31X1 + + + + 32X1 + + + + 31X11 + + + + 33X1 + + + + 34X1 + + + + 36X1 + + + + 41X11 + + + + 42X11 + + + + 43X1 + + + + 44X1 + + + + 45X12 + + + + 110X1 + + + + 16X11 + + + + 112X1 + + + + 113X1 + + + + 3.3.1.2.3 Dih ydroartem isin in (artenim ol) con tain in g dru g sam ples : Description of the dihydroartemisinin containing samples:  Fixed dose combination tablets of artenimol / piperaquine phosphate : 40mg / 320mg  Fixed dose combination tablets of dihydroartemisinin / sulphadoxine / pyrimethamine: 60mg / 500mg / 25mg. 3. 3. 1. 2. 3. 1 Dih ydroartem isin in (Artenim ol) Colou r and other reaction s [96, 105] a) To a quantity of the powdered tablets equivalent to 10mg of artenimol, 20ml of dehydrated ethanol was added, shaken to dissolve, filtered and evaporated to dryness. The following reagents were added to half of the residue: 0.1ml of dehydrated ethanol, 1ml of potassium iodide (80g/l) TS, 2.5ml of sulfuric acid (~100g/l) TS, and 4 drops of starch TS. Expected obse rvat ion : production of a violet colour University of Ghana http://ugspace.ug.edu.gh 68 b) The remaining residue from test (a) above was dissolved in 0.5ml of dehydrated ethanol R, and about 0.5ml of hydroxylamine hydrochloride TS2 and 0.25ml of sodium hydroxide (~80 g/l) TS were added. The mixture was heated in a water-bath to boiling and cooled. Two drops of hydrochloric acid (~70g/1) TS and 2 drops of ferric chloride (50g/l) TS were added. Expected obse rvat ion : immediate production of a deep violet colour. 3.3.1.2.3.2 P iperaqu in e : Colou r and other reaction s fo r pip e raq u in e [92] To a quantity of the powdered tablets equivalent to 10 mg piperaquine in a test tube was added 5cm3 of distilled water. A few drops of 1M HCl solution were added. The solution was stirred, warmed and filtered. To the filtrate was added a few drops of Dragendorff’s reagent. Expected observat ion : Formation of a copious brownish red precipitate. 3.3.1.2.3.3 Result s fo r the basic test s Table 10 : Result s fo r basic test s of dihydroa rt emisin in cont ain in g drug samp les Identity Dihydroartemisinin Sulphadoxine Pyrimethamine Piperaquine Test a Test b Test a Test b Test a Test b Test 11Z3 + + + 12Z3 + + + 14Z3 + + + 16Z1 + + + 17Z1 + + + 26Z1 + + + 27Z1 + + + 32Z3 + + + 33Z3 + + + 37Z1 + + + 41Z3 + + + 42Z5 + + + 43Z3 + + + 44Z3 + + + 15Z1 + + + + + + 13Z1 + + + + + + University of Ghana http://ugspace.ug.edu.gh 69 12Z1 + + + + + + 14Z1 + + + + + + 21Z1 + + + + + + 23Z1 + + + + + + 24Z1 + + + + + + 25Z1 + + + + + + 34Z1 + + + + + + 35Z1 + + + + + + 45Z1 + + + + + + 46Z1 + + + + + + 3.3.1.2.4 Qui n in e con tai nin g dru g sam ples : Description of the quinine containing samples:  Quinine injection containing 300mg of quinine dihydrochloride  Quinine suspension containing 150mg of quinine sulphate  Quinine mixture containing 50mg/5mL of quinine bisulphate BP 3.3.1.2.4.1 Colou r and other reaction s fo r quin in e in j ection and other dosage form s [105] a) A quantity of quinine containing samples equivalent to 5mg was dissolved in 10mL of water. 1 drop of sulphuric acid (~100 g/l) TS was added to the solution. Expected observat ion : Production of a strong blue fluorescence b) To another 10 ml of the solution prepared for test 1, was added 0.15 ml of bromine TS1 and 1 ml of ammonia (100 g/l) TS. Expected observat ion : Production of an emerald-green colour. 3.3.1.2.4.2 Result fo r the basic test s Table 11: Result s fo r basic test s of quin in e cont ain in g drug samp les Identity Test a Test b 11V5 + + University of Ghana http://ugspace.ug.edu.gh 70 12V5 + + 13V5 + + 41Q6 + + 42Q6 + + 43Q6 + + 4V5 + + 41R8 + + 42R4 + + 43R4 + + 31Q6 + + 32Q6 + + 33Q6 + + 3. 3. 1. 2. 5 S u lph adoxin e and Pyrim eth am in e dru g sam ples Description of the sulphadoxine and pyrimethamine drug samples:  Fixed dose combination tablets of sulphadoxine / pyrimethamine: 500mg / 25mg. Tests as described in sections 3.3.1.2.1.2 and 3.3.1.2.1.3 3.3.1.2.5.1 Result s fo r the basic test s Table 12 : Result s fo r basic test s of sulp h adoxin e and pyrimet h amin e drug samp le s. Identity Sulphadoxine Pyrimethamine Test a Test b Test a Test b 11P10 + + + + 11P2 + + + + 12P2 + + + + 14P10 + + + + 14P2 + + + + 16P10 + + + + 21P15 + + + + 21P2 + + + + 22P2 + + + + 23P2 + + + + 31P10 + + + + 31P2 + + + + 32P10 + + + + 32P2 + + + + 33P10 + + + + 35P2 + + + + 37P15 + + + + University of Ghana http://ugspace.ug.edu.gh 71 38P15 + + + + 41P2 + + + + 42P2 + + + + 44P2 + + + + 45P2 + + + + 48P5 + + + + *24Y13 + + + + *4Y13 + + + + *34Y13 + + + + *32Y13 + + + + The results of the colorimetric tests demonstrated that all the samples contained the requisite APIs claimed by the manufacturers. The next phase of the work involved the SQ-TLC and HPLC assays to quantify the APIs in the samples and compare with pharmacopoeial specifications and manufacturers’ claims. 3.3.2 Semi-Quant it at ive Thin Layer Chromat og rap h y (Sq-Tlc) SQ-TLC is a tool that has been used to verify identity and quantity of the active pharmaceutical ingredients (APIs) in drugs. This is a semi-quantitative tool because it gives the estimated amounts of the active ingredients in the drugs. This technique has two designated solvent systems for each drug API in a formulation [92] and the Tables 13 and 14 below summarise the drug APIs, their respective solvent systems and spraying reagents. Table 13 : Solven t syst em s fo r a rt emisin in deriv ed active pharmaceut ical ingredient s API Solve nt sys te m 1 (S1) Solve nt sys te m 2 (S2) Spray i ng re age nt Colo ur Artesunate Ethanol : Ammonia 100 : 0.5 Ethanol: Toluene: Ammonia 70 : 30 : 1.5 Anisaldehyde/ methanol Purple Artemether Petrol: Ethyl acetate 70 : 30 Toluene: Ethyl acetate 70 : 30 Anisaldehyde/ methanol Purple Artenimol Toluene: Ethyl acetate 60 : 40 Toluene: Ethyl acetate 70 : 30 Anisaldehyde/ methanol Purple University of Ghana http://ugspace.ug.edu.gh 72 Table 14 : Solven t syst em s fo r non - art emi sin in derived active pharmaceut ical ing redient s API Solve nt sys te m 1 (S1) Solve nt sys te m 2 (S2) Spray i ng re ag e nt Colo ur Chloroquine Methanol: ammonia 100:1.5 Ethyl acetate: acetic acid: water 60:20:20 I2-KI Brown Quinine Methanol: ammonia 100:1.5 Ethyl acetate: acetic acid: water 60:20:20 I2 –KI Brown Amodiaquine Ethanol: ammonia 100:1.5 Ethanol: Toluene: Ammonia 70:30:1.5 Cobaltous NO3 saturated with NaCl Green Pyrimethamine Ethyl acetate: methanol: ammonia 80:15:5 Ethyl acetate: acetic acid: water 60:20:20 I2-KI Brown Sulphadoxine Ethyl acetate: methanol: ammonia 80:15:5 Ethyl acetate: Methanol: acetic acid 75:25:1 I2-KI Brown Lumefantrine Ethyl acetate: acetic acid: toluene 4:2:18 Ethyl acetate: acetic acid 10:5 I2-KI Brown 3. 3. 2. 1 S pott in g, Developm en t and Detecti on o f Chrom atogram An origin line was marked at 1.5 cm from and parallel to the bottom end of the chromatoplate. Different amounts of a fixed 1mg/mL concentration of RS in µL (1.0-2.0) corresponding to the amounts in µg and a fixed amount of the drug sample (2µg) were spotted onto the plate. Solvent systems were placed in the TLC tanks lined with filter paper and allowed to stay for homogenous mixing and saturation of the solvents before the TLC plates were placed in them. The chromatoplates were left until the solvent front reached the upper limit line; then removed and left to dry. For alkaloidal drugs, drying was followed by spraying the appropriate reagent and then scanned onto a computer. For artemisinins, drying was followed by spraying with anisaldehyde and heated in an oven at 120 oC. The coloured (purple) spots (Figure 15) were scanned and saved on a computer. University of Ghana http://ugspace.ug.edu.gh 73 3. 3. 2. 2 Data Captu rin g and Analysis Using Microsoft Office Picture Manager, the brightness of the spots on the developed chromatograms was changed continuously and the rates at which the RS and test solution spots faded were compared simultaneously. The principle of the method is that the RS and the sample test solution spots on the chromatogram that had the same concentration would fade at the same time with the increase in the brightness of the computer screen. For example, if a spot of a sample test solution is presumed to contain 2 µg (µL), it must fade at the same time as 2 µg of the RS. Figure 14 below shows a sample of a developed TLC plate of an artemether containing antimalarial drug. Figure 14 : A sample of deve lop ed TLC plat e of an art emet h e r cont ain in g drug Solvent system Artemether sample spot Artemether RS spots of different quantities University of Ghana http://ugspace.ug.edu.gh 74 The concentration of a test solution is estimated by taking note of the RS spot that begins to fade simultaneously with the spot of the sample test solution and one that fades completely after the spot of the test solution. This gives the range in which the amount of the API test solution falls. For example, if the spot of the test solution (2µL) of a drug containing 200mg of an API expected to fade at the same time as the 2µL reference standard spot fades between 1.4µL and 1.6 µL spots of the RS, then; 1.4µL = lower limit 1.6µL = upper limit of the API content of the test solution In percentage equivalent: Lower limit = 1.4/2.0 100 = 70.0% Upper limit = 1.6/2.0 100 = 80.0% Converting percentage to mass: Lower limit = 1.4/2.0 200 mg = 140 mg Upper limit = 1.6/2.0 200 mg = 160 mg This implies that the API quantity is within 70 - 80% (140 – 160) mg, out of the 200 mg claimed by the manufacturer on the pack and it is also not compliant with the pharmacopoeial requirement that stipulates that each tablet must contain not less than 90% and not more than 110% of the manufacturers label claim. So this was done for all the 12 replicates, 6 from each of the two solvent systems. The average for the upper and lower limits was calculated for each drug sample. The data were transferred to Microsoft Windows Excel 2007 for further analysis. University of Ghana http://ugspace.ug.edu.gh 75 3.3.3 High Perfo rmance Liquid Chromat og rap h y (HPLC) Assay s This method was used as a validation tool for the results of the SQ-TLC method. There were different kinds of drug formulations whereby some drugs had 2 APIs while others had 3 APIs combined. A simultaneous assay was used where possible and single API analysis where the former was not feasible. The analysis was done with reference to pharmacopoeias and published validated assays subject to some adjustments in some cases where the available conditions and prescribed methods gave unrealistic results. The adjustments made have been outlined thoroughly in the experimental section. In all cases, an appropriate calibration curve using pure reference samples of the requisite API was plotted. These were used in calculating the API contents of the various dosage forms. Details are given in the experimental section. 3. 3. 3. 1 Detai ls of Variou s Dru g Assays 3. 3. 3. 1. 1 HP L C Assay of Artesu n ate The experimental conditions of the assay for the HPLC analysis of artesunate were formulated from the modification of a validated method by Ranher et al. [106]. Outlined below are the conditions of the adapted assay:  Column measurements: Discovery C-18 bonded, 5µm, 25cm x 4mm.  Mobile phase: 70: 30 v/v, 1% triethylamine (TEA) in methanol: buffer (10mM KH2PO4/ 85% H3PO4 , pH=2.5)  retention time (average): 5.1 minutes  detection wavelength: 216nm  Flow rate: 1.2 mL/ min.  volume of injection: 20 µL University of Ghana http://ugspace.ug.edu.gh 76 The adapted assay was successfully implemented and produced well resolved chromatograms. Figure 15 below is a typical example of the chromatograms obtained. Figure 15 : Chromat og ram o f a samp le solu t ion cont ain in g art esu n at e 3. 3. 3. 1. 2 HP L C Assay of Artem eth er/Lum ef an trin e FDC Form u lat ion The assay of artemether/lumefantrine was carried out using a modified method of Arun and Smith (2011) [107] details of which are given below.  Column measurements: Hyperprep PEP 300A C4, 8µm, 25cm x 4.6mm.  Mobile phase: 70: 30 v/v, acetonitrile: 10mM buffer consisting of KH2PO4 mixed with 1 mL of triethylamine (TEA) per liter and pH changed to 2.5 using 85% H3PO4 mixture.  Retention time (average): artemether appeared at 2.5 minutes and lumefantrine at 3 minutes. Artesunate peak Other drug substance University of Ghana http://ugspace.ug.edu.gh 77  Detection wavelength: 216nm  Flow rate: 1.5mL/min.  Injection volume: 20 µL A sample of the chromatograms produced from the modified experimental conditions is depicted in Figure 16 below. Figure 16 : Chromat og ram o f a samp le solu t ion cont ain in g art emet h er and lume fan t rin e 3. 3. 3. 1. 3 HP L C Assay of Dih ydroartem isin in (Artenim ol) The assay of dihydroartemisinin was adopted from the Ph. Int. [105] with few modifications and the following experimental conditions were produced and used after the adaptations: Artemether peak Lumefantrine peak Blank University of Ghana http://ugspace.ug.edu.gh 78  Column measurements: Kramasil C8, 5 µm, 25cm x 4.6mm  Mobile phase: 50: 50 v/v, water: acetonitrile  Retention time (average): 5.2 minutes  Flow rate: 1.5mL/min.  Detection wavelength: 210nm  Volume of injection: 10µL Using these conditions, dihydroartemisinin was unequivocally analyzed and well resolved chromatograms were generated (Figure 17). Figure 17 : Chromat og ram o f a samp le solu t ion cont ain in g dihydroart e misin in Dihydroartemisinin peak Piperaquine peak Related substance University of Ghana http://ugspace.ug.edu.gh 79 3. 3. 3. 1. 4 HP L C Assay o f Sulph adoxin e/P yrim eth am in e WHO expert committee, 2011 adopted a method for the simultaneous assay of sulphadoxine and pyrimethamine due to the dearth of the assay in the previous editions of the Ph. Int., which was intended to be included in the subsequent edition (2010 edition) [108]. In this study, the method conditions were adapted as follows:  Column measurements: Ascentis C-18 column, 5µm, 15cm x 4.60mm.  Mobile phase: 65: 10: 25 v/v, 20mM buffer (KH2PO4/Na2HPO4 of pH 5.6: methanol: acetonitrile.  retention time (average): 3.9 minutes for sulphadoxine and 8.7 minutes for pyrimethamine  Flow rate: 1mL/min  Detection wavelength: 240nm  Volume of injection: 10µL Figure 18 below is an illustration of a sample chromatogram that was produced from the application of the conditions of the modified assay. 3. 3. 3. 1. 5 HP L C Assay of Quin in e Quantitative determination of quinine described in the USP 24 [109] was modified and used in the HPLC assay of quinine preparations and the following conditions were generated after the changes:  Column measurements: Discovery C-18 bonded, 5 µm, 25cm x 4mm  Mobile phase: 80: 16: 2: 2 v/v, water: acetonitrile: methanesulfonic acid: TEA (pH 2.6)  Average Retention time: 4.7 minutes. University of Ghana http://ugspace.ug.edu.gh 80  Flow rate: 1.2mL/min.  Wavelength for detection: 235nm  Injection volume: 20 µL Figure 19 below is a demonstration of one of the chromatograms produced from the assay conditions outlined above. Figure 18 : Chromat o g ram o f a samp le cont ain in g sulp h adoxin e and pyrimet h amin e 3. 3. 3. 2 AP I Con tent from t he HPL C Assay The calculation of the API content in the HPLC analyzed samples is demonstrated below using sample, 31X1: Label claim of API content per tablet = ATM/LUM: 20mg /120mg (calculation of quantity of the artemether component only) Sulphadoxine peak Pyrimethamine peak University of Ghana http://ugspace.ug.edu.gh 81 Weight of 10 tablets = 2.9390g Average weight of each tablet = 0.2939g = 293.9mg Weight of tablets corresponding to 4 mg of artemether used for analysis is found as follows: = 4mg/20mg x 293.9mg = 58.78mg equivalent of artemether API From the calibration curve; AUC = 176.08 C + 74.287 and C = (AUC – 74.287)/176.08; But AUC= 139.301 C = (139.301-74.287)/176.08 = 0.3692mg/mL Sample solution prepared for the assay = 10mL Quantity of artemether in 10mL = 0.3692mg/mL x 10mL = 3.692mg Percentage of the actual amount to the expected amount = 3.692mg/4mg x 100 = 92.3% Therefore, the sample contains: 92.3/100 x 20mg = 18.46mg of artemether per tablet. Since pharmacopoeial specifications require the drug to contain not less than 90% and not more than 110% of the manufacturer’s claim, sample 31X1 will be deemed to have complied with pharmacopoeial specifications. University of Ghana http://ugspace.ug.edu.gh 82 Figure 19 : Chromat og ram o f a samp le solu t ion cont ain in g quin in e 3.4 DISCUSSION 3. 4. 1 S Q- T L C and HP L C Resu lt s There were three classes of results; identified as compliant (C), non-compliant (NC) and borderline compliant (BLC). A requisite API in a drug was classified as “compliant” if its quantity fell within acceptable limits of the Ph.Int.; “non-compliant” if the quantity was more (overdose) or less (under dose) than the acceptable limits and “borderline compliant” if the amount was marginally compliant with Ph. Int. limits by ±5 % fo r SQ- TLC and ±2 % fo r HPLC. In addition, a drug was graded as compliant if the entire component APIs in it were Quinine peak Related substances University of Ghana http://ugspace.ug.edu.gh 83 compliant; non-compliant if at least one of the API components was non-compliant and borderline compliant if all components are separately borderline compliant; one component or more is borderline amongst other compliant components of a drug. 3. 4. 1. 1 S Q - T L C Meth od Resu lt s 3. 4. 1. 1. 1 An alysis of Dru g AP I Com pon en ts Figure 20 below shows the quality status of the individual APIs when the SQ-TLC method was used to estimate their quantities. The samples were analysed for artemether, artesunate, dihydroartemisinin, sulphadoxine, pyrimethamine, lumefantrine and quinine active pharmaceutical ingredients (APIs). For artesunate, there were 33.33% (3/9) of samples that were borderline compliant, whereas 44.44% (4/9) were compliant and 22.22% (2/9) non-compliant (under dose). 9.76% (4/41) of artemether samples were found to be borderline compliant, with 24.39% (10/41) and 65.85% (27/41) being compliant and non-compliant respectively, with the non-compliance arising due to 13 (48.15%) samples being overdose and 14 (51.85%) being under dose. The samples containing lumefantrine posted borderline qualification of 17.07% (7/41) and 31.71% (13/41) were compliant, with the remaining 51.22% (21/41) being non-compliant; whereby twelve (12) (57.14%) samples of the non-compliant samples (21) were overdose and 9 (42.86%) of them under dose. For dihydroartemisinin, 7.69% (2/26) were found to be borderline compliant, 23.08% (6/26) compliant and 69.29% (18/26) non-compliant (under dose). University of Ghana http://ugspace.ug.edu.gh 84 Figure 20 : SQ-TLC result s o f i ndividual APIs in the ant imalaria l drug samp les Out of the 39 samples with sulphadoxine component, 7.69% (3/39) were found to be borderline compliant, 10.26% (4/39) compliant with a further 82.05% (32/39) being non-compliant (under 24.39% 65.85% 9.76% Artemet h er Compliant Noncompliant Borderline compliant 31.71% 51.22% 17.07% Lumefan t rin e Compliant Noncompliant Borderline compliant 23.08% 69.29% 7.69% Dihydroart emisin in Compliant Noncompliant Borderline compliant 27.27% 38.63% 34.09% Pyrimet h amin e Compliant Noncompliant Borderline compliant 44.44% 22.22% 33.33% Artesun at e Compliant Noncompliant Borderline compliant 10.26% 82.05% 7.69% Sulphadoxin e Compliant Noncompliant Borderline compliant 71.43% 28.57% Quinin e Noncompliant Borderline compliant University of Ghana http://ugspace.ug.edu.gh 85 dose). The quantity of pyrimethamine samples found to be borderline compliant were 34.09% (15/44) with 27.27% (12/44) and 38.63% (17/44) being compliant and non-compliant respectively, with 5 out of the non-compliant samples (17) being overdosed. The analysis of the quinine samples showed that while 28.57% (2/7) were borderline compliant, 71.43% (5/7) were non-compliant, with all of the 5 (100%) non-compliant samples being overdose. Some quinine containing samples were not analyzed for the quinine API due to the insufficiency of the RS material. Therefore, the results demonstrated the widespread occurrence of substandard API quantities, with sulphadoxine being the most compromised. 3. 4. 1. 1. 2 An alysis of Dru g Sam ples as a Whole Based on SQ - T L C Resu lt s Classification of a drug as being compliant or otherwise in the case of fixed dose combination (FDC) drugs depends on compliance of all APIs present. 3. 4. 1. 1. 2. 1 Artesu n ate/S u lph adoxin e/S u lph am eth oxypyridazin e/P yrim eth ami n e Con tai n in g Sam ples There were 9 samples of this API combination constituting 8.04% of the total samples (112). Out of the 9 samples, 5 samples had artesunate/sulphamethoxypyridazine/pyrimethamine while the rest (4) had artesunate/sulphadoxine/pyrimethamine API combination. Analysis of artesunate/sulphamethoxypyridazine/pyrimethamine samples showed that 40% (2/5) samples had been found to be non-compliant (under dose) for pyrimethamine component, whereas 20% (1/5) and 40% (2/5) samples were found to be compliant and borderline compliant respectively. In terms of artesunate component, 40% (2/5) were non-compliant while the other 40% (2/5) were compliant and the remaining 20% (1/5) of the samples was borderline compliant. University of Ghana http://ugspace.ug.edu.gh 86 Sulphamethoxypyridazine component was not successfully analyzed using SQ-TLC due to the procedural limitations of the analytical method, that is, although the spots of the sulphamethoxypyridazine on the TLC plate had the same retention time as the sulphadoxine spots, they could not be stained well with the spraying agent Iodine-Potassium iodide solution (I2/KI). Therefore, these samples were analyzed as a whole using the other available components. On the other hand, results of the components in the artesunate/sulphadoxine/pyrimethamine samples were as follows: samples with artesunate component being compliant were 2 (50%) and borderline compliant samples were also 2 (50%). For sulphadoxine component, all the samples were found to be non-compliant (under dose) and the pyrimethamine component registered 3 (75%) samples as being non-compliant all of which were also under dose and 1 (25%) sample being borderline compliant. Thus, on the whole, SQ-TLC tests showed that 22.22% (2/9) and 77.78% of these samples were borderline compliant (Ats/SmP) and non-compliant respectively. 3. 4. 1. 1. 2. 2 Artem eth er / Lum ef an trin e Con tain in g Sam ples Out of the 41 artemether/lumefantrine samples, 27 artemether and 21 lumefantrine samples were non-compliant, with 13 (48.15%) and 12 (57.14%) of the non-compliant artemether and lumefantrine components respectively being overdose. Therefore, overall SQ-TLC results of the artemether/lumefantrine components showed that 2.44% (1/41) samples were borderline compliant, whereas 4.88% (2/41) were compliant and 92.68% (38/41) non-compliant. University of Ghana http://ugspace.ug.edu.gh 87 3. 4. 1. 1. 2. 3 Dih ydroartem isin in /P iperaqu in e Phosph ate Con tain in g Sam ples These samples were analyzed for the dihydroartemisinin component only due to the lack of a piperaquine reference standard. Therefore, they were treated like a monotherapy drug in the analysis for dihydroartemisinin. Approximately 14.29% (2/14) of the samples were borderline compliant, with 42.86% (6/14) and 42.86% (6/14) being compliant and non-compliant correspondingly. All non-compliant components were under dose. 3. 4. 1. 1. 2. 4 Dih ydroartem isin in /S u lph adoxin e/P yrim eth am ine Con tai n in g Sam ples For the samples of this combination, 83.33% (10/12) of the samples had sulphadoxine component non-compliance (under dose), with 8.30% (1/12) of samples being compliant and another 8.30% (1/12) being borderline compliant. For the pyrimethamine component, none of the samples was found non-compliant, with 50% (6/12) being found borderline compliant and the other 50% (6/12) found to be compliant. All the samples had 100% non-compliance (under dose) for the dihydroartemisinin component. Thus, out of the 12 samples that contained dihydroartemisinin/sulphadoxine/ pyrimethamine, none was found to be compliant, with the failure rate mainly due to the failure of the dihydroartemisinin and sulphadoxine components. 3. 4. 1. 1. 2. 5 S u lph adoxin e/P yrim eth am in e Con tai n in g Sam ples Out of the 23 samples, 78.26% (18/23) had sulphadoxine component being non-compliant, with 13.04% (3/23) being compliant and 8.70% (2/23) being borderline compliant. For the pyrimethamine component, 52.17% (12/23) samples were non-compliant (3 overdose, 9 under dose), with 21.74% (5/23) found to be compliant and 26.09% (6/23) being borderline compliant. Therefore, on the whole, of the 23 samples that contained the combined sulphadoxine and University of Ghana http://ugspace.ug.edu.gh 88 pyrimethamine only, 8.70% (2/23) were found to be borderline compliant, 4.35% (1/23) being compliant and 86.96% (20/23) non-compliant. 3. 4. 1. 1. 2. 6 Qui n in e Con tai n in g Sam ples There were 13 samples consisting of quinine sulphate, quinine bisulphate and quinine hydrochloride and 7 samples were selected for SQ-TLC due to inadequate reference standard (RS). Out of this, 28.57% (2/7) of these samples were borderline compliant and 71.43% (5/7) being non-compliant, with 100% (5/5) of the non-compliance arising from overdosed API quantity. Figure 21 below summaries the statuses of the drugs as a whole based on SQ-TLC only. Figure 21 : SQ-TLC result s o f drug sa mp le s when all compon en t s a re considered ATM/LUM ATS/SP DHA/PP SP DHA/SP QN BLC 2.44% 22.22% 14.29% 8.70% 0% 28.57% C 4.88% 0.00% 42.86% 4.35% 0% 0% NC 92.68% 77.78% 42.86% 86.96% 100% 71.43% 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% 120.00% P er ce n ta ge (% ) Categories of drug samples and their API components SQ-TLC results of drug samples University of Ghana http://ugspace.ug.edu.gh 89 3. 4. 1. 2 HP L C Meth od Resu lt s 3. 4. 1. 2. 1 S umm ary of Resu lt s of the HP L C Assay Figure 22 : HPLC resu lt s of i ndividual APIs of the ant imala rial drug samp le s 34.15% 65.85% Artemet h er Compliant Noncompliant 66.67% 33.33% Artesun at e Compliant Noncompliant 26.83% 68.29% 4.88% Lumefan t rin e Compliant Noncompliant Borderline compliant 15.38% 80.77% 3.85% Dihydroart emisin in Compliant Noncompliant Borderline compliant 10.26% 84.62% 5.12% Sulfadoxin e Compliant Noncompliant Borderline compliant 63.64% 36.36% Pyrimet h amin e Compliant Noncompliant 20.00% 80.00% Sulfamet h oxyp yrazin e Compliant Noncompliant 30.77% 53.85% 15.38% Quinin e Compliant Noncompliant Borderline compliant University of Ghana http://ugspace.ug.edu.gh 90 Figure 22 above demonstrates a summary of the results obtained from the HPLC analysis of the APIs. With the HPLC method, the samples were analysed for artemether, artesunate, dihydroartemisinin, sulphadoxine, sulphamethoxypyridazine, pyrimethamine, lumefantrine and quinine active pharmaceutical ingredients (APIs). For artesunate component, 66.67% (6/9) of the samples were compliant and the remaining 33.33% (3/9) were non-compliant (under dose). Artemether component of the samples posted 34.15% (14/41) and 65.85% (27/41) compliant and non-compliant results respectively, with 48.15% (13/27) of the non-compliant samples being overdose. About 4.88% (2/41) of the lumefantrine component were found to be borderline compliant, whereas 26.83% (11/41) and 68.29% (28/41) were compliant and non-compliant respectively, and 50% (14/28) of the non-compliant samples were overdose. Of the samples that were analyzed for dihydroartemisinin, 3.85% (1/26) was found to be borderline compliant, with 15.38% (4/26) being found compliant and 80.77% (21/26) found non-compliant (under dose). In the case of sulphadoxine and pyrimethamine components, the HPLC results showed a corresponding 5.12% (2/39) and 10.26% (4/39) for borderline compliant and compliant with respect to sulphadoxine component and the remaining 84.62% (33/39) were non-compliant (under dose). For pyrimethamine, 63.64% (28/44) and 36.36% (16/44) of the samples were compliant and non-compliant respectively, of which 6/16 (37.50%) of the non-compliant samples were overdose. Sulphamethoxypyridazine component had approximately 20% (1/5) of the samples being certified as compliant, with 80% (4/5) being declared non-compliant all of which were under dose. Finally, 15.38% (2/13) of the quinine samples were borderline University of Ghana http://ugspace.ug.edu.gh 91 compliant, while 30.77% (4/13) were endorsed as compliant and 53.85% (7/13) turned out to be non-compliant; 85.71% (6/7) of the non-compliant samples were found overdose (Figure 22). These HPLC results were as a result of some SQ-TLC borderline samples in the numbers 3, 4, 1, 1, 13 and 1 for artesunate, artemether, lumefantrine, sulphadoxine, pyrimethamine and quinine respectively being found compliant using the HPLC method (indicated as *BLC in the table of results appendix II). Almost all the values for the latter method were also within the range of the TLC method estimation. In addition, borderline compliant samples for lumefantrine (2) and dihydroartemisinin (1) remained unchanged while the rest of the borderline samples were found non-compliant. Out of the total 139 non-compliant components in all the samples, 28.06% (39/139) were found to be overdose. Therefore, the HPLC results confirmed the widespread existence of substandard API component quantities in all the drug samples. Therefore, non- compliance was accounted for by both over dosage and under dosage of some of the components, both in significant proportions. 3. 4. 1. 2. 2 An alysis of AP Is of the Dru g Sam ples wit h Respe ct to their Cou n try of Origi n The samples were also analysed for API quantity with respect to their country of origin (source) to find out a source that produced antimalarial drugs with good quality or better drug compliance rate. In essence, a recommendation would be made as to the private traders as to whom they should import from for quality drugs. Only the HPLC results were used based on individual APIs and the drug as a whole. All the samples in this study were purported to be manufactured and imported from India (68), Tanzania (13), Kenya (20), China (9) and the USA (2). Based on HPLC results, the samples were comparatively analysed for compliance/non-compliance. University of Ghana http://ugspace.ug.edu.gh 92 Table 15 shows an outline of the sample APIs compliance status with respect to their sources and Table 16 shows the compliance status of the samples as a whole with respect to the same parameter. The results showed that failure rates for the individual APIs and the drug samples as a whole were similar, such that there were similar trends in all the samples imported from the different countries found to be of poor quality with very high failure rates recorded for all the samples and the sources. It was surprising to note that samples purported to be imported from both developing countries and well developed countries failed similarly. 3. 4. 1. 2. 3 An alysis of Dru g Sam ples as a Whole (HPL C Resu lt s) The samples were also analyzed in terms of a drug as a whole whereby a drug was qualified based on the collective qualification status of the individual APIs as well. Figure 23 below represents the overall drug’s qualification status based on HPLC. Figure 23 : HPLC resu lt s of drug samp le s when all compon en t s are considered ATM/LUM ATS/SP DHA/PP SP DHA/SP QN BLC 0% 0% 7.14% 0% 0% 15.38% C 4.88% 11.10% 28.57% 8.70% 0% 30.77% NC 95.12% 88.90% 64.29% 91.30% 100% 53.85% 0% 20% 40% 60% 80% 100% 120% P er ce n ta ge (% ) Categories of drug samples and their API components HPLC results of drug samples University of Ghana http://ugspace.ug.edu.gh 93 Table 15 : Comparat ive st udy of drug API compliance/n on -compliance wit h rega rd to count ry of origi n (Based on HPLC) Table 16: Comparat ive st udy of compliance/n on -compliance of drugs as a whole wit h rega rd to count ry of origi n APIs India Tanzani a Keny a China USA Non - co mp l i ance Non - co mp l i ant Non - co mp l i ant Non - co mp l i ant Non - co mp li ant Atm/Lum 91.18% (31/34) - 100.00% (5/5) 100.00% (3/3) 100% (2/2) Dha/Pp 20.00% (1/5) - - 88.89% (8/9) - Dha/SP 100.00% (12/12) - - - - SP 100.00% (6/6) 92.31% (12/13) 50.00% (2/4) - - Ats/S/SmP 75.00% (3/4) - 100.00% (5/5) - - Qn 57.14% (4/7) - 80.00% (4/5) - - Total 83.82% (57/68) 92.31% (12/13) 84.21% (16/19) 91.67% (11/12) 100% (2/2) APIs India Tanzani a Keny a China USA C NC BLC C NC BLC C NC BLC C NC BLC C NC BLC OD UD OD UD OD UD OD UD OD UD Ats 4 - - - - - - - 2 - 3 - - - - - - - - - Atm 11 11 12 - - - - - 1 1 3 - - - - - 1 1 - - Lum 9 10 13 2 - - - - 2 2 1 - - - - - - 2 - - Dha 3 - 13 1 - - - - - - - - 1 - 8 - - - - - S 1 - 20 1 2 - 10 1 1 - 3 - - - - - - - - - Sm - - - - - - - - 1 - 4 - - - - - - - - - P 18 - 4 - 6 3 4 - 4 3 2 - - - - - - - - - Qn 3 2 1 1 - - - - 1 4 - 1 - - - - - - - - Total 49 23 63 5 8 3 14 1 12 10 16 1 1 - 8 - 1 3 - - University of Ghana http://ugspace.ug.edu.gh 94 3. 4. 1. 2. 3. 1 Artesu n ate/S u lph adoxi n e/S u lph am eth oxypyridazin e/P yrim eth ami n e Con tai n in g Sam ples With the HPLC, all the sample components were successfully analyzed. These samples consisted of 5 artesunate/sulphamethoxypyridazine/pyrimethamine (Ats/SmP) samples and 4 artesunate/sulphadoxine/pyrimethamine (Ats/SP) samples. In Ats/SP samples, 100% (4/4) of the artesunate components were compliant. For sulphadoxine component, 25% (1/4) was borderline compliant with the rest (3/4) being non-compliant due to under dosage and 50% (2/4) of the pyrimethamine component was compliant, while the remaining 2 were non-compliant (under dose). In Ats/SmP samples, 40% (2/5) of the artesunate component was compliant whereas the remaining 3 components were non-compliant and under dose. For sulphamethoxypyridazine component, 20% (1/5) was borderline compliant with the rest being non-compliant and under dose, whereas 40.00% (2/5) of pyrimethamine component were borderline compliant and 60% (3/5) were found to be non-compliant and overdose. Overall, the results showed that approximately 11.10% (1/9) and 88.90% (8/9) of the total sample were compliant and non- compliant respectively. 3. 4. 1. 2. 3. 2 Artem eth er/L um ef an trin e Con tain in g Sam ples Out of the 36.6% (41/112) samples of artemether/lumefantrine analyzed, the HPLC results showed that 4.88% (2/41) samples were compliant and 95.12% (34/41) non-compliant. The non- compliant samples here had the quantities within the range 35-89% for under dosed and 110- 178% for over dosed (see section 3.4.1.2.1 for details). The HPLC tests found the SQ-TLC designated borderline compliant sample to be non-compliant in addition to those that were already non-compliant. University of Ghana http://ugspace.ug.edu.gh 95 3. 4. 1. 2. 3. 3 Dih ydroartem isin in /P iperaqu in e Phosph ate Con tain in g Sam ples With the HPLC as well, these samples were analyzed for the dihydroartemisinin component only due to the lack of the piperaquine RS material. Therefore, it was treated like a monotherapy drug in the analysis for dihydroartemisinin. Approximately 28.57% (4/14) of the samples were found compliant, whereas 7.14% (1/14) turned out to be borderline compliant, with 64.29% (9/14) being non-compliant due to under dosage. Using the HPLC, one of the two SQ-TLC borderline compliant samples was found to be non-compliant and the other one remained the same. 3. 4. 1. 2. 3. 4 Dih ydroartem isin in /S u lph adoxin e/P yrim eth am ine Con tai n in g Sam ples 12 samples contained dihydroartemisinin/sulphadoxine/pyrimethamine APIs and all the samples were non-compliant due to the high failure rate in the dihydroartemisinin and sulphadoxine components. For example, while 100% (12/12) of the pyrimethamine component was compliant, 100% of the dihydroartemisinin was non-compliant and 8.33% (1/12) of the sulphadoxine was compliant; with the further 91.67% (11/12) being non-compliant. All the non-compliant samples in both dihydroartemisinin and sulphadoxine components failed because they were under dosed. 3. 4. 1. 2. 3. 5 S u lph adoxin e/P yrim eth am in e Con tai n in g Sam ples Out of the 23 samples that contained sulphadoxine and pyrimethamine only, 8.70% (2/23) were found to be compliant and the remaining 91.30% (21/23) were non-compliant. The failure rate was overwhelming due to the high non-compliant status of the sulphadoxine component unlike the pyrimethamine. As a matter of illustration, 13.04% (3/23) of the sulphadoxine alone was confirmed compliant, while 4.35% (1/23) was borderline compliant and 82.61% (19/23) non- compliant. On the other hand, the quantity of pyrimethamine that turned out to be compliant and University of Ghana http://ugspace.ug.edu.gh 96 non-compliant was 52.17% (12/23) and 47.83% (11/23) respectively. All the non-compliant components fell within the 47-84% range, except 27.27% (3/11) of the non-compliant pyrimethamine component that were overdosed. One (1) out of the 2 SQ-TLC borderline compliant samples turned out to be non-compliant whereas the other one was found to be compliant when the HPLC method was used. 3. 4. 1. 2. 3. 6 Qui n in e Con tai n in g Sam ples The quinine samples were in different forms of quinine sulphate, quinine bisulphate and quinine hydrochloride. All the samples were duly analysed using HPLC. Approximately 30.77% (4/13) of these samples were compliant, with 15.38% (2/13) and 53.85% (7/13) being borderline compliant and non-compliant respectively; 85.71% (6/7) of the non-compliant quinine samples were found overdosed. The HPLC tests of the SQ-TLC borderline compliant samples showed that 1 sample turned out to be compliant, whereas one sample became non-compliant and one SQ-TLC non-compliant sample turned out to be borderline compliant. Therefore, majority of the samples have been found non-compliant with the Ph. Int. [96, 105] requirements. Some of the non-compliant samples had more than the requisite API amount, a situation which is abnormal for most substandard drugs. This might be attributed to either wrong calibration in a normal/licensed manufacturing company or counterfeiting, whereby production of the drugs takes place in substandard infrastructure using substandard materials and machinery by mostly unqualified personnel. University of Ghana http://ugspace.ug.edu.gh 97 The current study results are comparable to those found in recent studies conducted in Ghana by Osei-Safo et al. [92] and Konadu [68] as well as several other surveys in both Africa and Asia in which significant proportions of the samples were also found to be overdosed, with serious cases also reported in quinine samples as well. The results therefore show that whereas a given FDC drug may have certain individual components complying with pharmacopoeial specifications, other components may render the drug as a whole non-compliant. If therefore a component complies and is in sufficient quantities to effect reduction of parasitaemia, the drug as a whole may produce some treatment success, giving a false impression of total efficacy. The danger, however is that the absence or insufficient quantities of the other component(s) of the fixed dose combination drug to do what it/ is/are supposed to do in the overall cure or reduction in parasitaemia, could lead to rapid development of drug resistance. 3. 4. 1. 3 Com parison of SQ- TL C and HPL C Resu lt s After analyzing the results with respect to SQ-TLC and HPLC methods separately, the results were compared as well to find out if they were comparable. Figure 24 below illustrates the comparison of the results derived from the two analytical methods. University of Ghana http://ugspace.ug.edu.gh 98 Figure 24 : Comparison of HPLC and SQ-TLC resu lt s The API quantity of the samples was analyzed using HPLC and SQ-TLC assays. The results for the two methods were analogous such that similar decisions were made from them in a significant majority of the results and they were comparable for the case where drugs were analyzed as a whole (inclusive of all APIs) except for DHA/PP and QN (Figure 24). For individual APIs, looking at the artemether, artesunate, dihydroartemisinin, sulphadoxine and pyrimethamine as well with non-compliant results as an example, the HPLC results of 65.85% (27), 33.33% (3), 80.77% (21), 84.62% (33) and 36.36% (16) non-compliance were very closely related and comparable to the SQ-TLC non-compliance results of 65.85% (27), 22.22% (2), 92.68% 77.78% 42.86% 86.96% 100% 71.43% 95.12% 88.90% 64.29% 91.30% 100% 53.85% 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% 120.00% ATM/LUM ATS/SP DHA/PP SP DHA/SP QN ATM/LUM ATS/SP DHA/PP SP DHA/SP QN TL C H P LC Percentage (%) A n al yt ic al m e th o d a n d c at e go ri e s o f d ru g sa m p le s Comparison of HPLC and TLC results NC C BLC University of Ghana http://ugspace.ug.edu.gh 99 69.29% (18), 82.05% (32) and 38.63% (17) respectively. However, the HPLC and SQ-TLC results of lumefantrine and quinine APIs were slightly wider in difference. These slight and wide differences can be attributed to some of the borderline SQ-TLC compliant cases being found to be either fully compliant or non-compliant by HPLC. The HPLC assay was used to confirm and specify the estimated TLC results. Hence, the difference between the two methods was found to be insignificant because majority of the HPLC results fell within the TLC results ranges, whether compliant or non-compliant. For example, artesunate (3), artemether (4), lumefantrine (1), sulphadoxine (1), pyrimethamine (13) and quinine (1) samples changed from borderline compliant to compliant with HPLC assay (indicated as *BLC in the table of results, appendix II) and were still within the TLC range which made the TLC results statistics more closer to those of the HPLC results. Therefore, the HPLC and TLC results were comparable and equally important for the analysis (refer to appendix II). A correlation curve for each API in Figure 25 below further illustrates the comparability of the HPLC and SQ-TLC results; with a correlation coefficient of r = 0.949, showing a very strong similarity in the results. Figure 25 : Corre lat ion curve of HPLC and SQ-TLC resu lt s 0 50 100 150 200 0 20 40 60 80 100 120 140 160 H P LC A P I R es u lt s SQ-TLC API Results Correlation Curve of SQ-TLC vs HPLC for Each API University of Ghana http://ugspace.ug.edu.gh 100 3.4.2 Combin ed HPLC and SQ-TLC Result s 3. 4. 2. 1 Artesu n ate/S u lph adoxin e/S u lph am eth oxypyrazin e/P yrim eth am in e Con tai n in g Sam ples There were 9 samples constituting 8.04% of the total samples. 33.33% (3/9) of these samples had artesunate component failure, 77.78% (7/9) had sulphadoxine component failure and 55.56% (5/9) had a pyrimethamine component failure. The overall results for the drug samples with all the components analyzed showed that 1 out of 9 samples were fully compliant in all respects representing an overall 88.89% failure rate. 3. 4. 2. 2 Artem eth er/L um ef an trin e Con tain in g Sam ples 41 samples containing artemether/lumefantrine were analyzed representing 36.6% of the total sample size. The results of the artemether component showed a 65.85% (27/41) failure rate and lumefantrine component was found to be 68.85% (28) non-compliant. On overall, 39 out of 41 samples were not compliant representing a 95.12% failure rate. Being the first line treatment, this is a devastating situation to the efforts against Malaria in a country burdened with acute, frequent and persistent drug shortages in public hospitals, thus making people resort to private hospitals and pharmacies for prompt and easily accessible treatment. 3. 4. 2. 3 Dih ydroartem isin in /P iperaqu in e Phosph ate Con tain in g Sam ples These samples were analyzed for the dihydroartemisinin component only due to the lack of a piperaquine reference standard. Therefore, it was treated like a monotherapy drug in the analysis. 35.71% (5/14) of the samples were compliant. It is quite possible that a few more non- compliance cases would have been recorded if the piperaquine had also been analysed. University of Ghana http://ugspace.ug.edu.gh 101 3. 4. 2. 4 Dih ydroartem isin in /S u lph adoxin e/P yrim eth am ine Con tai n in g Sam ples There were 12 samples containing this formulation. The components dihydroartemisinin, sulphadoxine and pyrimethamine recorded 100%, 91.7% and 0% failure rates respectively. Generally, all the samples were non-compliant with the Ph. Int. requirements, even though all the samples had 100% compliance for pyrimethamine. This situation where one component of a multi component formulation complies while the other does not, was found to be common in all the samples analysed. The implication is that the synergism effect that is aimed at preventing development of parasite resistance against the drug over a short period of its usage is compromised. Hence, the drug only reduces the malaria causing parasite, which eliminates the signs/symptoms and once the parasites re-group and one falls sick again, it is unlikely that s/he would get cured as the parasites might have developed resistance against the drug. In cases where the API is overdosed, one is likely to get cured with serious or unexpected side effects and/or organ damages even unintended self-poisoning that can cause death. 3. 4. 2. 5 S u lph adoxin e / Pyrim e th am in e Con tai n in g Sam ples There were 23 samples that contained sulphadoxine and pyrimethamine only and 82.61% (19/23) of the sulphadoxine component were non-compliant, 4.35% (1/23) were borderline compliant and 13.04% (3/23) were compliant. In addition, 52.17% (12/23) of the pyrimethamine component were non-compliant and the rest were compliant. On overall, 91.30% (21/23) of the samples were non-compliant. This is also a worrisome development because sulphadoxine / pyrimethamine is still administered in Malawian hospitals especially to the vulnerable group of pregnant women, due to the lack of comprehensive studies on the possible adverse effects of ACTs on the developing foetus. Hence, administering of poor quality SP drugs could worsen the University of Ghana http://ugspace.ug.edu.gh 102 already fragile situation this group encounters and aggravate the parasite resistance the regimen has faced. 3. 4. 2. 6 Qui n in e Con tai n in g Sam ples There were 13 quinine containing samples consisting of quinine sulphate, quinine bisulphate and quinine hydrochloride. All the samples were analysed using HPLC, while some could not be analysed using SQ-TLC method because the extraction process was not efficient enough such that the results were not comparable to their HPLC counterparts like all other samples (see appendix II for details for such samples). The results showed that 53.85% of the samples were non-compliant. The high failure rate of the drugs and their individual components is worrisome and poses a serious parasite resistance (for under dosed) and toxicity (for overdosed) threats to the use of quinine for the treatment of more complicated cases of malaria. In addition, most of the malaria cases in Malawi are diagnosed without microscopic determination. In most cases, most types of fever are presumed to be malaria first, and treated as such. If indeed, such an ad hoc diagnosis is also treated with substandard or poor quality antimalarials, this could lead to treatment failure and/or fast development of resistance. This inference comes with the background reported by Bate et al.[45] that resistance development of chloroquine and sulphadoxine in Africa in the 1990s and the devastating impact of malaria on the people was partly due to the use of substandard drugs. University of Ghana http://ugspace.ug.edu.gh 103 3.4.3 Regist rat ion Status and Qualit y of the Samples The results of the registered and unregistered samples were compared to determine whether the registration status necessarily had an influence on the quality of the drugs. Below is Figure 26 summarizing the relationship between drug registration and compliance. Figure 26 : A comparison of fai lu re rat es fo r reg ist ered and unregist ered samp les Out of the 97 registered samples, 15.46% (15) of the samples were compliant, while 6.67% (1) of the 15 unregistered samples were compliant. Although the registered samples had a better compliance compared to the unregistered ones, the overall results show that better registration status does not necessarily always guarantee the quality a drug. These results are similar to the observations made in the 2008/2009 Ghana study [67]. This implies that a registered drug does not mean good quality drug and the fact that a drug is registered with the national regulatory authority does not make it good. This might be caused by lack of adherence of a supplier to registration guidelines, whereby they register a particular batch number of drug products and duplicate lots of that batch number, which are not checked by authorities. registered Unregistered compliant 15.46% 6.67% non-compliant 84.54% 93.33% 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% P er ce n ta ge (% ) Registered vs Unregistered samples University of Ghana http://ugspace.ug.edu.gh 104 In addition, this might also be caused by a supplier registering a particular product name or formulation and more products similar to it subsequently supplied without being checked. Furthermore, this problem might be attributed to lack of rigorous testing and assessment of the new products information required and presented for registration due to either lack of instruments for thorough testing or lack of qualified personnel. Additionally, this problem is also caused by supplier presenting a well manufactured drug product for the registration application process, which after passing the scrutiny, is diluted and subsequent products are prepared without regard to GMP. Finally, due to the lack of forensic testing of the drug products, some counterfeit products get their way into the market by mimicking the registered products, compromising the quality of the drug market as a whole. There is also the possibility of an importer simply not subjecting products for registration with all the tools involved, simply because the regulatory authorities are not vigilant enough in surveillance and application of appropriate sanctions. 3.4.4 Sample s wit h the Same Batch Number Such samples were expected to have the same quantities of APIs or negligible differences. On the contrary, majority of the samples had varying quantities within a pool of a batch number (see appendix III). This might be attributed to counterfeiting, lack of adherence to good manufacturing and laboratory practices, inconsistent quantities of excipients that are, among others, used to minimize the effect of adverse conditions (e.g. heat, humidity) on APIs and exposure to different environmental conditions that affect the stability of APIs significantly. University of Ghana http://ugspace.ug.edu.gh 105 3.4.5 Overa ll Resu lt s The detailed results on each of the samples analysed by both SQ-TLC and HPLC are as shown in appendix II. It has been shown that there was a 100 % compliance of the samples for the visual inspection of dosage form and packaging material as well as qualitative determination of active pharmaceutical ingredients (API). There was a good relationship between the former and the latter because the labelled contents on the packaging materials were correct. Therefore, there was an excellent correlation between the two results as expected. However, it has been reported earlier that with the advancement of counterfeiting today, visual inspection and basic tests could not qualify a drug as genuine despite chemical and physical similarities. Bate et al. state that determination of a drug as counterfeit or substandard requires a forensic examination of the trademarks, product designs and holograms [45]. Generally, using both HPLC and SQ-TLC results and the fact that if a component fails then the sample as a whole fails, there was an overall 85.71% (96) failure rate out of the 112 samples that were tested. The Table 17 and Figure 27 below illustrate the overall results. Table 17 : Overa ll resu lt s of the st udy APIs North Centr al South East South West Total Total C NC Total C NC Total C N C Total C NC Total C NC Atm/Lum 6 0 6 6 1 5 6 0 6 23 1 22 41 2 39 Dha/pp 4 0 4 3 1 2 2 2 0 5 2 3 14 5 9 Dha/sp 2 0 2 2 0 2 4 0 4 4 0 4 12 0 12 Sp 5 0 5 8 2 6 4 0 4 6 0 6 23 2 21 Ats/sp 5 1 4 3 0 3 1 0 1 0 0 0 9 1 8 Qn 7 3 4 3 2 1 0 0 0 3 1 2 13 6 7 Total 29 4 25 25 6 19 17 2 14 41 4 37 112 16 96 University of Ghana http://ugspace.ug.edu.gh 106 The failure rates were more serious for dihydroartemisinin/sulphadoxine/pyrimethamine combination 100% (12/12), followed by artemether/lumefantrine 95.12% (39/41), sulphadoxine/pyrimethamine 91.3% (21/23), artesunate/sulphadoxine/pyrimethamine 88.9% (8/9), dihydroartemisinin 64.29% (9/14) and quinine 53.8% (7/13) as shown in Table 17 above. This means that in multi component FDC drugs, manufacturers have to rigorously apply good manufacturing practices (GMP) and ensure that every component meets the requisite pharmacopoeial specifications. Compliance in such circumstances is therefore more difficult to achieve. The results have also shown that the failure rates were worst in the south west zone with 90.20% failure rate, followed by the north zone with 86.20%, then south east with 82.40% and the central with 76% as shown in the Figure 27 below. Figure 27 : Failu re rat e s of the drug samp les by Zon e 90.20% 82.40% 76% 86.20% 65.00% 70.00% 75.00% 80.00% 85.00% 90.00% 95.00% South West South East Central North Zonal drug failure rates University of Ghana http://ugspace.ug.edu.gh 107 The results suggest that there is a widespread circulation of poor quality antimalarial drugs with the south west zone being the worst affected. This has been greatly attributed to the presence of more economic activity places in south west region such as the Mulanje border, Mwanza border and Blantyre city that attract more imports and influence the import of poor quality drugs. In addition, this region has higher economic activities with higher likelihood of population increase, which provides a good market for goods. The north has two places of high economic activities such as Karonga border and Mzuzu and a large number of imports pass through this border from Tanzania and Kenya just like the Mwanza border in the south west zone. However, the other zones are equally centres of high economic activities because they also have cities and border posts like Zomba city and Mangochi district border for south east and Lilongwe city as well as Mchinji and Dedza border posts for the central zone, only that the activities are slightly lower than the former two zones with high failure rates. In addition, the borders are porous and despite the regulatory body carrying out some testing activities, some poor quality drugs are bound to be smuggled into the country; worse still the tests done at the border posts are not rigorous enough to detect a fake, substandard or degraded drug, other than just certifying that the sample contains the labelled APIs, which is not sufficient; that is, if at all they take place as per requirement because their occurrence is not well documented in the published literature. The high circulation of poor quality drugs implies that many people are using antimalarial drugs with insufficient or too much amount of the requisite APIs, hence such drugs would either not be effective because they cannot eradicate all the parasites from the body and those that survive can easily develop and transmit resistance (for under dose) or cause some toxicity concerns (for overdose). These effects might lead to worsening of an acute to severe malaria case, high cost of University of Ghana http://ugspace.ug.edu.gh 108 treatment to get cured, loss of public trust in the treatment regimen and unreliable studies on medical drug efficacy resulting in high mortality rate. Despite the effort put in by the Malawi government and its partners to minimize the impact of Malaria over the years, Malaria remains the country’s biggest health challenge and the most risky part is that almost the whole population is affected. Several programmes have been rolled out and phased out, others are still in progress, but the problem seems to be getting worse and there have been cases of resistance against antimalarials as well. Nevertheless, the trend is similar to the areas where resistance and high treatment failures have been attributed to poor quality drugs. Some of these suggestions are supported by an article in which the Malawi government is quoted to have ordered the removal of some antimalarial drugs such as sulphadoxine/pyrimethamine from the shops’ shelves. This particular antimalarial drug was suspected to have had its sensitivity to malaria parasites compromised, and has since been replaced with artemether/lumefantrine [110]. The post-marketing surveillance and pharmacovigilance system in Malawi has been under development since 2009 and therefore is still faced with severe limitations. This problem is also shared by other African countries like Tanzania and Kenya which are also sources of antimalarial drugs imported into Malawi. Unless this problem is solved with urgency, all efforts to kick out malaria will be in vain. Moreover, if there is development of resistance, this could be disastrous because currently there does not appear to be any new efficacious antimalarial drug in the pipeline to replace the current regimen. University of Ghana http://ugspace.ug.edu.gh 109 3.4.6 Compa rison of Resu lt s wit h Recent Result s fro m ot h er African Count rie s The results of the current Malawi study were compared with the results of other African countries from a recent survey reported in the WHO QAMSA report of 2011, where the participating countries Ethiopia, Kenya, Tanzania, Cameroon, Ghana and Nigeria had 0%, 5%, 11%, 37%, 39% and 64% failure rates respectively [55]. Another recent study report by Nayyar et al. (2012) was selected. In this survey, the authors reviewed several survey reports of poor quality antimalarial drugs from mostly Uganda, Cameroon, Kenya, Democratic Republic of Congo, Burkina Faso, Madagascar, Senegal, Ghana, Nigeria, Tanzania, Ethiopia, Gabon, Mali, Mozambique, Zimbabwe, Sudan, Burundi, Angola, Congo, Chad and Rwanda for the period between 1999 and 2011 [63]. The review report showed that the failure rates for all the surveys conducted in these countries within the stated period were in the range 12-69%, with the exception of one of the studies conducted in Ghana which reported an 82% failure rate in 2008 [63]. Therefore, the Malawian failure rate of 85.71% is very alarming. This problem might be attributed to lack of routine rigorous drug testing by international and local organizations as most international surveys have rarely included samples from Malawi and such activities are also rarely done internally if they take place at all because efforts to locate any such activity taking place at the required level have proved futile. In addition, Caudron et al., 2008 reported the results of a quality audit done by Medicin Sans Frontiers (MSF) pharmacists over a period of 4 years in 180 sites they visited, which showed that most regulated manufacturers bypassed their GMP compliance and set the standards of their drug University of Ghana http://ugspace.ug.edu.gh 110 products based on the recipient countries status with regard to the level of regulation capability and level of income as well as lack of prequalified standards by most developing countries to their suppliers [111]. So Malawi being one of the poorest countries apart from just being a developing country, with poor regulatory capability, without a pharmacovigilance system and lack of expertise in such areas, there is a possibility that these factors might be amongst the causes of the high failure rates, because most of the drugs circulating in these countries are manufactured by almost the same manufacturers. 3.4.7 Conclusion s The results have shown an alarming widespread use of poor quality drugs in Malawi, with the south west and the north zones appearing to be the hub of the circulation of substandard antimalarial drugs and the central zone having relatively high quality antimalarial drugs. In addition, the results have also shown that the failure rate is much higher compared to other African countries. The registration, visual and qualitative analysis of the drugs alone would not serve a purpose of certifying a drug as worth prescription and the fact that a drug is registered with the national regulatory authority does not guarantee its quality. As such, there is always a need to carry out thorough tests on the drugs if the purpose of such activities is to flush out the poor quality drugs. Some forms of drugs that are supposed to be phased out at the time of this study were found to be still in circulation. It has also been established that SQ-TLC method can be used if the HPLC method is beyond the capability of the community since the results of the two methods are comparable. University of Ghana http://ugspace.ug.edu.gh 111 Therefore, the results have shown that while the existence of a poor regulatory system is not a conclusion that can be drawn directly from the present study, it could be responsible for the high failure rate of medicines imported into the country. Hence, the regulatory system needs urgent attention by the authorities for improvement to be able to carry out thorough routine tests of drugs. This will result in better quality health care delivery through malaria and other diseases control, patients’ safety, fight and eradication of parasite resistance. 3.4.8 Recommendation s Well-planned and implemented surveys need to be conducted to give a true reflection of the nature of the poor antimalarial drugs as to whether those drugs found to be non-compliant are degraded due to exposure to adverse conditions, counterfeited or substandard as a result of poor manufacturing practices. From the fact that some substandard drugs might not be necessarily counterfeit, it is recommended that analysis be carried out to determine the factors that contributed to the current state of being substandard. This is so because a substandard drug can result during production for a number of reasons like deliberate deviation from standards to maximise profits, non-compliance with standard operating procedures (SOPs) and good manufacturing practices (GMPs), equipment error or malfunctioning, degradation and/or mishandling at any part of medicine continuum. Knowledge generated from the study would help in choosing appropriate action to address the situation. The studies should also include dissolution tests, which is also another important parameter that defines the quality of a drug plus many other important parameters. Such studies should also be extended to public hospitals and pharmacies. University of Ghana http://ugspace.ug.edu.gh 112 Good procurement practices amongst the public and private institutions must be promoted and appropriate measures taken to prevent the illegal importing and smuggling of drugs into the country. The drug regulatory authority’s capacity should be strengthened in terms of infrastructure and working personnel so that they will be able to conduct thorough initial quality and routine tests on new supplies, drug manufacturing and packaging companies and suppliers inspection to ensure strict adherence to the GMPs. Appropriate sensitisation of the public and drug handling personnel in charge of the supply chain is needed to let them know the appropriate quality control measures, proper drug storage and distribution. A properly functioning and sustainable pharmacovigilance system should be developed. This could partly be achieved by incorporating the pharmacovigilance topics in the various training disciplines such as medicine, pharmacy, nursing colleges, public health schools or even secondary schools at an introductory level. University of Ghana http://ugspace.ug.edu.gh 113 CHAPTER FOUR 4 EXPERIMENTAL 4.1 THIN LAYER CHROMATOGRAPHY 4.1.1 Expe ri men t a l Condition s The TLC tests were carried out at room temperature under the following conditions: a. Chromatograms were run in TLC tanks (22.5 cm x 10 cm) with filter papers lined on the inside walls, saturated with the mobile phase. b. Silica gel 60 F254 (Merck) pre-coated on aluminium foil or Silica gel 60 (Fluka) pre- coated on aluminium foil were used as stationary phase. c. Each drug API had two proposed solvent systems selected as mobile phase. d. Test solutions: 1mg/mL solutions of the authentic Reference Standards (RS) of the active pharmaceutical ingredient (API) and the dosage forms were prepared daily and stored at 4 0C. The RS were obtained from the European Directorate for the Quality of Medicines and Healthcare (EDQM), France. These were prepared by dissolving 10 mg of RS in 10 mL of their respective solvents as shown in Table 18 below. Table 18: Dissolu t ion solven t s of APIs fo r TLC test s Activ e Phar maceut ical Ingredie nt (API) Dissol ut io n Solve nt Quinine Methanol Dihydroartemisinin Methanol Sulphadoxine Methanol Pyrimethamine Methanol artemether Ethyl acetate Lumefantrine Ethyl acetate Artesunate 80% methanol University of Ghana http://ugspace.ug.edu.gh 114 e. Application: different volumes of a 1mg/mL concentration reference standard (RS) in the range 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4μL equivalent to the corresponding quantities in µg were applied to the plate at 1cm apart. The quantity of the dosage form was however kept constant; 2.0μL of a 1mg/mL solution equivalent to 2.0μg of each drug was applied. f. Detection: each active ingredient had its specific detection agent as specified in the pharmacopoeia and published literature and these have been outlined (Tables 13 and 14). 4.1.2 Prepa rat ion of Dosage Form Solut ion s An amount of the ground dosage form equivalent to 10mg of a given API was weighed into a clean dry beaker. 5 mL of a suitable dissolution solvent was added to the beaker, then the mixture shaken for at least 5 minutes and filtered. Another 3 mL of the solvent was added to the filtration residue, shaken gently, then allowed to settle for at least 5 minutes and filtered again. To check the effectiveness of the extraction, another 2 mL of the solvent was added to the residue and a TLC spot developed from residue solution alongside the RS. Absence of the spot from the residue meant complete API extraction. The filtrate was subsequently added to the other filtrates to make up 10 mL of the dosage form solution. So in effect, a 1mg/mL solution was prepared. For each of the FDC formulations, each API component was assayed independent of the other API constituents. For example, if artemether/lumefantrine is assayed for artemether, a quantity that will give out 10mg of artemether is weighed, irrespective of what the amount of lumefantrine will be given. Likewise when lumefantrine is assayed, an amount that will give 10mg of lumefantrine is weighed irrespective of the amount of artemether. Therefore, in University of Ghana http://ugspace.ug.edu.gh 115 extracting an API of interest, a solvent is selected such that what will dissolve it will dissolve only the analysed API, and leave the others intact, so that there is a preferential extraction. 4.2 HPLC METHODS FOR THE ASSAY OF THE SELECTED ANTIMALARIALS In addition to the SQ-TLC assay, the drug API contents were also assayed using HPLC procedures suitable for each particular API, adopted from the pharmacopoeias and the literature to authenticate the results of the SQ-TLC. 4.2.1 HPLC Assay o f Arte su n at e The assay for the HPLC analysis of artesunate was adopted from Ranher et al. validated HPLC method [106] with some few modifications to suit the current circumstances as shown in Table 19 below: Table 19: Chromat ograp h ic and inst ru men t a l condition s for art e su n at e assay Chro matogr ap hic par a met ers Prescrib ed met hod Adapt ed met hod Column measurements HiQ-Sil C8, 25cm x 4.6mm i.d. Discovery C-18 bonded, 5µm, 25cm x 4mm. Mobile phase (70:30 v/v) acetonitrile: 1M sodium acetate buffer (pH 3 adjusted with o-phosphoric acid) 70: 30 1% triethylamine(TEA) methanol: 10mM KH2PO4/ 85% H3PO4 (pH=2.5) Injection volume 20 µL 20 µL Flow rate 1.0mL/min 1.2 mL/ min. Retention time 4.883min. 5.1 minutes Detection Wavelength(UV/Vis) 220nm 216nm Internal standard artemether None The prescribed conditions of the assay using an HiQ-Sil C8 column gives some tailing problems, hence modifications leading to the adapted conditions above which gave well resolved peaks. University of Ghana http://ugspace.ug.edu.gh 116 The modifying agent, 1% triethylamine (TEA) was added to methanol and the pH of the 0.01 M buffer potassium dihydrogen phosphate was adjusted to 2.5 with 10mM of phosphoric acid in the 70/30 v/v mobile phase. 4. 2. 1. 1 P reparati on and Assay of Artesu n ate RS Solu tion f or Cali brati on Curve Artesunate concentrations comprising 0.706 mg/mL, 0.807 mg/mL, 0.908 mg/mL, 1.009 mg/mL, 1.211mg/mL, 1.312mg/mL and 1.413mg/mL were prepared through dilution of pipette volumes of 0.7mL, 0.8mL, 0.9mL, 1.0mL, 1.2mL, 1.3mL and 1.4mL respectively, from a 10.1mg/mL stock solution of the artesunate reference material using 2mL of mobile phase (70:30 methanol: buffer) and 8mL of HPLC grade water to produce 10 mL of working solution. The area under the curve (AUC) for each concentration was determined with six replicates each and an average AUC resulting from them. This data was used to plot a graph of average AUC against concentration (C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient (r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. The area under the curve (AUC) is the average area under a chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 20 and Figure 28 below). 4. 2. 1. 2 P reparati on and Assay of Soluti on s o f Artesu n ate Con tai n in g Tablets A quantity of powdered dosage form equivalent to 10 mg of artesunate was weighed into a clean dry 10 mL volumetric flask. 5mL of the mobile phase (70:30 v/v, 1% triethylamine in methanol: buffer) was then added and the mixture shaken for 15 minutes on an ultrasonic sonicator. Then, more mobile phase was added to the mark and the solution filtered through a 0.45 µm filter. University of Ghana http://ugspace.ug.edu.gh 117 Table 20 : Concent rat ion s of art e su n at e RS solut ion s and their corre sp onding AUCs ID Actual vol ume pipet ted (ml ) Conc. (mg/ ml) Peak Area ( AUC) Aver age AUC SD RSD % 1 2 3 4 5 6 1 0.7 0.706 403.4 403.3 402.9 403.5 403.5 403.5 403.3897 0.22 0.055 2 0.8 0.807 455.9 455.8 454.9 455.9 455.0 455.9 455.6458 0.47 0.103 3 0.9 0.908 515.8 515.4 515.2 515.4 515.2 515.2 515.3963 0.21 0.041 4 1.0 1.009 570.2 569.9 570.2 570.3 570.3 569.9 570.1942 0.17 0.031 5 1.2 1.210 640.0 640.0 640.1 640.0 639.9 640.0 640.0629 0.05 0.008 6 1.3 1.311 714.2 714.2 714.1 714.2 714.2 713.9 714.1686 0.09 0.012 7 1.4 1.412 767.3 767.5 767.3 767.2 768.0 767.3 767.4849 0.26 0.034 Figure 28 : Calib rat ion curve fo r a rt e su n at e RS Conc. as a function of AUC y = 502.02x + 52.657 R² = 0.994 403 453 503 553 603 653 703 753 803 0.7000 0.8000 0.9000 1.0000 1.1000 1.2000 1.3000 1.4000 1.5000 A rea und er the curve, A U C ( mg/L ) Concent rat ion, C (mg/mL) The calib rat ion curve for art esun at e University of Ghana http://ugspace.ug.edu.gh 118 4.2.2 HPLC Assay o f Arte met h e r / Lumefan t rin e FDC Formu lat ion s Artemether/lumefantrine drugs were analyzed using assay conditions derived from modifications of a method developed by Arun and Smith [107], to improve the excess retention times of the methods developed earlier by Sridhar et al. and Sunil et. al.; with retention times of 13.8 and 13.9 minutes respectively, which were deemed to be unfavourable for routine analysis. Table 21 below summarises the prescribed and adapted method conditions. Table 21 : Chro mat ograp h ic and inst ru men t a l condition s of art emet h e r/lu me fan t rin e as say Chro matogr ap hic par a met ers Prescrib ed met hod Adapt ed met hod Column measurements Hypersil (BDS) C18, 25cm x 4.6mm, 5µm. Hyperprep PEP 300A C4, 25cm x 4.6mm, 8µm. Mobile phase (70: 30 v/v) acetonitrile: 0.01M Potassium dihydrogen orthophosphate buffer at pH 4.0. (70: 30 v/v) acetonitrile: 10mM buffer consisting of KH2PO4 mixed with 1 mL of triethylamine per liter and pH changed to 2.5 using 85% H3PO4 mixture. Injection volume 20 µL 20 µL Flow rate 1mL/min. 1.5mL/min. Retention time Artemether = 2.5 minutes, lumefantrine = 3.0 minutes Detection Wavelength(UV/Vis) 253nm 216nm The counter-ion modifying agent triethylamine was added as well to get enhanced peak symmetry and minimized tailing. Additionally, due to the large difference in the ratio of artemether to lumefantrine of 1: 6, efforts were made to add an artemether amount detectable by using higher concentrations, whilst as well avoiding use of very high concentration of lumefantrine that would have been too much for the column or given peaks that shot over the height of the recording device. University of Ghana http://ugspace.ug.edu.gh 119 4. 2. 2. 1 P reparati on and Assay of Artem eth er RS Solu ti on f or Cali brati on Curve Artemether concentrations comprising 0.2797mg/mL, 0.3168mg/mL, 0.3564mg/mL, 0.3960mg/mL, 0.4356mg/mL, 0.4752mg/mL and 0.5148mg/mL were prepared through dilution of 0.28mL, 0.32mL, 0.36mL, 0.40mL, 0.44mL, 0.48mL and 0.52mL pipette volumes of 9.9 mg/mL stock solution of the artemether reference material respectively and topped up to 10 mL using mobile phase (70:30 v/v acetonitrile: buffer). Area under the curve (AUC) for each concentration was determined with six replicates for each and an average AUC derived from them. The data was used to plot a graph of average AUC against concentration (C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient (r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. The area under the curve (AUC) is the average area under a chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 22 and Figure 29). 4.2.2.2 Prepa rat ion and Assay of Lumefan t rin e RS Solut ion for Calib rat ion Curve Similarly, lumefantrine concentrations comprising 1.6782mg/mL, 1.9008mg/mL, 2.1384mg/mL, 2.3760mg/mL, 2.6136mg/mL, 2.8512mg/mL and 3.0888mg/mL were prepared through dilution of 0.28mL, 0.32mL, 0.36mL, 0.4mL, 0.44mL, 0.48mL and 0.52mL pipette volumes of 9.9mg/mL stock solution of the lumefantrine reference material for each concentration and top up to 10mL using mobile phase (70:30 acetonitrile: buffer). Area under the curve (AUC) for each concentration was determined with six replicates each and an average AUC derived from them. The data was used to plot a graph of average AUC against concentration (C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient(r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. The area under University of Ghana http://ugspace.ug.edu.gh 120 the curve (AUC) is the average area under a chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 23 and Figure 30). 4. 2. 2. 3 P reparati on and Assay of Soluti on s o f Artem eth er/ Lum ef an trin e Tablets A quantity of powdered dosage form equivalent to 4 mg was accurately weighed into a clean dry beaker. 1mL of acetic acid was added, allowed to react for a few minutes after which 5 mL of the mobile phase (70:30 v/v acetonitrile: buffer) was added. The mixture was then shaken for 15 minutes on an ultrasonic sonicator. Then, the solution was filtered into a 10 mL volumetric flask through a 0.45 µm filter, then the beaker was washed with the mobile phase and the residue added to the mark through the filter and kept well in readiness for the analysis. Table 22 : Concent rat ion s of art e met h e r RS solu t ion s and their AUCs ID Actual vol ume pipet ted (ml ) Conc. (mg/ ml) Peak Area ( AUC) Aver age AUC SD RSD 1 2 3 4 5 6 1 0.28 0.2797 121.0 120.5 130.2 122.5 120.5 120.2 122.8 3.88 3.16 2 0.32 0.3168 134.4 133.8 135.3 130.5 129 130.8 131.9 2.56 1.94 3 0.36 0.3564 136.0 138.7 138 142.9 140.0 131 138.1 4.04 2.92 4 0.40 0.3960 145.2 143.8 146.1 144.3 145.8 144.3 144.9 0.93 0.64 5 0.44 0.4356 142.0 155.2 151.6 150.1 150.2 150.9 150 4.33 2.88 6 0.48 0.4752 158.4 158.6 159.1 157.4 158.5 158.3 158.4 0.55 0.34 7 0.52 0.5148 166.6 167.2 165.3 167.6 166.2 169.6 167.2 1.45 0.88 University of Ghana http://ugspace.ug.edu.gh 121 Table 23 : Concent rat ion s of lu mefan t rin e RS solut ion s and their AUCs ID Actual vol ume pipet ted (ml ) Conc. mg/ ml Peak Area ( AUC) Aver age AUC SD % SD 1 2 3 4 5 6 1 0.28 1.6782 28344 28337 28093 28315 28297 28090 28226 121 0.4 2 0.32 1.9008 30243 30245 30685 30544 30345 30262 30416 185 0.6 3 0.36 2.1384 32159 32131 30909 32160 32122 32091 31883 500 1.6 4 0.40 2.3760 33503 33500 32033 33493 33502 33541 33214 602 1.8 5 0.44 2.6136 35452 35404 33385 35462 35391 35460 35020 836 2.4 6 0.48 2.8512 36537 36533 34161 36512 36334 36537 36015 954 2.7 7 0.52 3.0888 37822 37848 36892 37805 37843 37867 37651 386 1.0 Figure 29 : Calib rat ion curve fo r a rt e met h e r RS Conc. as a function of AUC y = 176.08x + 74.287 R² = 0.9941 122.78807 127.78807 132.78807 137.78807 142.78807 147.78807 152.78807 157.78807 162.78807 167.78807 172.78807 0.2804 0.3304 0.3804 0.4304 0.4804 0.5304 A rea und er the curve, A U C ( mg/L ) Concentrat io n, C (mg/mL) The calib ra tio n curve f o r artem eth er University of Ghana http://ugspace.ug.edu.gh 122 Figure 30 : Calib ra t ion curve fo r lum efan t rin e RS Conc. as a function of AUC 4.2.3 HPLC Assay o f Dihydroa rt emi sin in A method outlined in the Ph. Int. [105] was modified to suit the present study as shown in Table 24 below, with both the old method conditions and the new conditions outlined. Table 24 : Chro mat ograp h ic and inst ru men t a l condition s of dihydroa rt emisin in assay Chromatographic parameters Prescribed technique Adapted method Column measurements C-18 column, 10cm x 4.6mm, 3 µm Kramasil C8, 25cm x 4.6mm, 5µm Mobile phase 60:40 v/v, acetonitrile: water with gradient elution 50:50 v/v, water: acetonitrile Injection volume 20 µL 10µL Flow rate 0.6mL/min. 1.5mL/min. Retention time Total runtime of 30 min; no retention time given 5.2 minutes Detection wavelength (UV/Vis) 216nm 210nm y = 6320.7x + 17985 R² = 0.9934 28226.02 30226.02 32226.02 34226.02 36226.02 38226.02 40226.02 1.6854 1.8854 2.0854 2.2854 2.4854 2.6854 2.8854 3.0854 3.2854 A rea und er the curve, A U C (mg/L ) Concentrat io n, C (mg/mL) The calib ra tio n curve f o r lu mef a n trin e University of Ghana http://ugspace.ug.edu.gh 123 4. 2. 3. 1 P reparati on and Assay o f Dih ydroartem isin i n RS Solu ti on f or Cali brati on Curve Dihydroartemisinin concentrations comprising 0.7014mg/mL, 0.8016mg/mL, 0.9018mg/mL, 1.0020mg/mL, 1.1022mg/mL, 1.2024mg/mL and 1.3026mg/mL were prepared through dilution of pipetted volumes of 1.4mL, 1.6mL, 1.8mL, 2.0mL, 2.2mL, 2.4mL and 2.6mL respectively, from a stock solution of 12.5mg/mL dihydroartemisinin reference material and mobile phase (60:40 v/v acetonitrile: phosphate buffer) was added to make a 25 mL solution. Area under the curve (AUC) for each concentration was determined with six replicates each and an average AUC derived from them. The data was used to plot a graph of average AUC against concentration (C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient (r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. The area under the curve (AUC) is the average area under a chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 25 and Figure 31 below). 4. 2. 3. 2 P reparati on and Assay of Soluti on s o f Dih ydroartem isin in Con tai n in g Tablets A quantity of powder equivalent to 10 mg of dosage form was accurately weighed into a clean dry beaker. 5 mL of the mobile phase (60:40 v/v acetonitrile: phosphate buffer) was added as a diluent and sonicated for 15 minutes on an ultrasonic sonicator. The solution was then filtered through a 0.45 µm filter into a 10 mL volumetric flask and more diluent was used to wash the beaker and the residue was added to the mark through the filter and the solution kept well in readiness for the analysis. University of Ghana http://ugspace.ug.edu.gh 124 Table 25 : Concent rat ion s of dihydroa rt emi sin in RS solut ion s and the corre sp onding AUCs ID Actual Volume pipet ted (mL) Conc. (mg/ mL ) Peak Area ( AUC) Aver age AUC SD RSD 1 2 3 4 5 6 1 1.4 0.7014 328.6 328.6 326.6 328.6 329.1 325.6 327.8271 1.40 0.428 2 1.6 0.8016 355.2 355.2 358.2 357.2 358.2 354.2 356.3465 1.72 0.484 3 1.8 0.9018 393.3 391.5 394.3 390.0 390.3 390.8 391.7017 1.74 0.445 4 2.0 1.0020 440.1 439.1 441.1 440.1 441.1 439.1 440.0619 0.89 0.201 5 2.2 1.1022 486.6 485.6 488.0 485.5 485.6 484.6 486.0071 1.17 0.240 6 2.4 1.2024 530.8 531.0 530.9 531.9 529.9 530.9 530.8868 0.63 0.119 7 2.6 1.3026 564.6 561 563.6 563.5 564.6 564.6 563.6343 1.39 0.246 Figure 31 : Calib rat ion curve fo r dih ydroa rt emi sin in RS Conc. as a function of AUC 4.2.4 HPLC Assay o f Quinin e An USP 24 HPLC assay [109] was modified and used in the analysis of quinine preparations and the changes brought out the analytical conditions shown under the adapted method in Table 26. y = 410.18x + 31.35 R² = 0.995 320 370 420 470 520 570 620 0.7000 0.8000 0.9000 1.0000 1.1000 1.2000 1.3000 1.4000 A rea und er the curve, A U C ( mg/L ) Concentrat io n, C (mg/mL) The calib ra tio n curve f o r dihydroa rtem isin in University of Ghana http://ugspace.ug.edu.gh 125 Table 26 : Chro mat ograp h ic and inst ru men t a l condition s for quin in e assay Chro matogr ap hic par a met ers Prescrib ed techniq ue Adapt ed met hod Column measurements C-18 Phenomenex luna, 15cm x 4.66mm, 5 µm Discovery C-18 bonded , 25cm x 4.0 mm, 5 µm Mobile phase 81:15:2:2 v/v, water: acetonitrile: methanesulfonic acid solution: triethylamine (TEA), pH 2.6 80:16:2:2 v/v, water: acetonitrile: methanesulfonic acid: TEA, pH 2.6 Injection volume 10 µL 20 µL Flow rate 1.2mL/min. 1.2mL/min. Retention time 3.6minutes 4.7 minutes Detection Wavelength (UV/Vis) 235nm 235nm 4. 2. 4. 1 Mobil e Phase Preparati on Methanesu lfon ic acid solu t ion :  3.5 mL of methanesulfonic acid  2.0 mL of glacial acetic acid  Dilute with water to 50 mL of solution Triethyla min e solu t ion :  10.0 mL of triethylamine  Dilute with water to 100.0 mL of solution Final compo sit ion :  Water – acetonitrile – methanesulfonic acid solution – triethylamine solution  (80:16:2:2 v/v) adjusted to pH 2.6 4. 2. 4. 2 P reparati on and Assay of Qui n in e RS Solu ti on f or Cali brati on Curve 50.6mg of Quinine reference standard was weighed into a 50mL volumetric flask to give a resultant solution of concentration 1.012mg/mL. Concentrations comprising 0.3542mg/mL, University of Ghana http://ugspace.ug.edu.gh 126 0.4048mg/mL, 0.4554mg/mL, 0.5060mg/mL, 0.5566mg/mL, 0.6072mg/mL and 0.6578mg/mL were prepared by diluting pipetted volumes of 3.5mL, 4.0mL, 4.5mL, 5.0mL, 5.5mL, 6.0mL and 6.5mL respectively, from a stock solution of the quinine reference material into a 10mL volumetric flask. Area under the curve (AUC) for each concentration was determined with six replicates each and an average AUC derived from them. The data was used to plot a graph of average AUC against concentration(C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient(r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. Area under the curve is the average area under chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 27 and Figure 32). 4. 2. 4. 3 P reparati on and Assay of Solu ti on s of Quin in e Con tai n in g Dru g Sampl es 4. 2. 4. 3. 1 Qui n in e Suspen sion s / Mixt u res /In jecti on s A certain amount of the quinine suspension was sonicated for about 15 minutes and a quantity equivalent to 5mg was pipetted into a 10 mL volumetric flask. 8 mL of methanol was then added to the content of the flask and made up to the mark with the mobile phase. Where necessary, the solution was also filtered. For the injections, a 10mL solution was prepared in a volumetric flask after 5 ampoules of quinine injections were mixed together and 20 µL aliquot was measured from the stock quinine solution using a microlitre syringe. The prepared solution was diluted with 8mL of methanol and mobile phase was added to the 10mL mark. University of Ghana http://ugspace.ug.edu.gh 127 Table 27 : Concent rat ion s of quin in e RS solut ion s and their corresp onding AUCs ID Actual v ol ume pipet ted (mL) Conc. mg/ mL Peak ar e a ( AUC) Aver age AUC SD RSD 1 2 3 4 5 6 1 3.5 0.3542 7183 7178 7173 7173 7178 7178 7177 3.95 0.06 2 4 0.4048 8375 8416 8369 8445 8445 8374 8404 36.2 0.43 3 4.5 0.4554 9453 9433 9390 9445 9333 9463 9413 49.3 0.52 4 5 0.5060 10301 10471 10257 10309 10335 10470 10368 97.4 0.94 5 5.5 0.5566 11665 11450 11665 11664 11507 11445 11546 110.8 0.96 6 6 0.6072 12185 12348 12185 12329 12479 12312 12331 104.7 0.85 7 6.5 0.6578 13346 13364 13202 13439 13489 13398 13378 109.1 0.82 Figure 32 : Calib rat ion curve fo r quin in e RS Conc. as a function of AUC y = 20180x + 162.91 R² = 0.9979 7083.373 8083.373 9083.373 10083.373 11083.373 12083.373 13083.373 14083.373 0.3500 0.4000 0.4500 0.5000 0.5500 0.6000 0.6500 0.7000 A rea und er the curve, A U C ( mg/L ) Concentrat io n, C ( mg/mL) The calib ra tio n curve f o r qu in in e University of Ghana http://ugspace.ug.edu.gh 128 4.2.5 HPLC Assay o f Sulphadoxin e/Sulph am et h oxyp yridazin e/Pyri met h amin e Formu lat ion s Table 28 below shows experimental conditions that were used in the analysis of sulphadoxine / pyrimethamine containing drug samples, produced after modifying those of the WHO adopted monograph for inclusion into the Ph. Int. in 2010 [108]. Table 28 : Chromat og rap h ic condition s of sulp h adoxin e / pyrimet h amin e assay Chro matogr ap hic par a met ers Prescrib ed techniq ue Adapt ed met hod Column measurements Phenomenex Luna®, 25 cm x 4.6 mm, 5 μm. Ascentis C-18, 15 cm x 4.60 mm, 5 µm. Mobile phase 80:20 v/v, 10 mL of acetic acid R and 0.5 mL of TEA R dissolved in 800 mL of H20 R, diluted to 1000 mL and pH adjusted to 4.2 using NaOH (~400 g/L) TS: acetonitrile R. 65:10:25 v/v, 20 mM buffer (KH2PO4/Na2HPO4 of pH 5.6; methanol; acetonitrile. Injection volume 20 µL 10 µL Flow rate 2mL/min. 1 mL/min Retention time Run time, at least 25 minutes sulphadoxine = 3.9 minutes, pyrimethamine = 8.7 minutes Detection Wavelength(UV/Vis) 227 nm 240 nm Like in artemether/lumefantrine, the ratio of sulphadoxine to pyrimethamine of 20: 1 made the detection of pyrimethamine difficult and this was overcome by using higher concentrations of pyrimethamine whilst still maintaining appropriate concentration of sulphadoxine for the column. 4. 2. 5. 1 P reparati on and Assay of Sulph ad oxin e RS Solu tion f or Cali brati on Curve Different concentrations of sulphadoxine RS were prepared using mobile phase (65:10:25 v/v University of Ghana http://ugspace.ug.edu.gh 129 buffer: methanol: acetonitrile) as shown in Table 29 and Figure 33 below. Area under the curve (AUC) for each concentration was determined with six replicates each and an average AUC derived from them. The data was used to plot a graph of average AUC against concentration (C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient (r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. The area under the curve (AUC) is the average area under a chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 29 and Figure 33). 4. 2. 5. 2 P reparati on and Assay of Pyrim eth am in e RS Solu ti on for Cali brati on Curve Similarly, different concentrations of pyrimethamine RS were prepared using mobile phase (65:10:25 v/v buffer: methanol: acetonitrile) as shown in the Table 30 and Figure 33 below. Area under the curve (AUC) for each concentration was determined with six replicates each and an average AUC derived from them. The data was used to plot a graph of average AUC against concentration (C) using Microsoft Excel and the slope of the graph, intercept, correlation coefficient (r2) as well as equation of the straight line, AUC = mC + b were deduced and calculated from it. The area under the curve (AUC) is the average area under a chromatographic peak, m = slope of the straight line and b = AUC intercept (Table 30 and Figure 34). 4. 2. 5. 3 P reparati on and As say of Solu ti on of Sulph adoxin e/S u lph am eth oxypyridazin e/ Pyrim eth am in e Con tai n in g Tablets 100mg of sulphadoxine/sulphamethoxypyridazine and 5mg of pyrimethamine equivalents of the dosage form were weighed simultaneously into a clean dry beaker and the APIs extracted three University of Ghana http://ugspace.ug.edu.gh 130 times for completeness using acetonitrile and the mobile phase (65:10:25 v/v Buffer: methanol: acetonitrile) in a 50mL volumetric flask. Table 29 : Concent rat ion s of sulp h adoxin e RS solut ion s and their corre sp onding AUCs ID Actual vol ume pipet ted (mL) Conc. (mg/ mL) Peak Area (AUC) Aver age AUC SD SD % 1 2 3 4 5 6 1 1.6 0.7968 18728 18765 18756 18753 18785 18720 18751 24.2 0.13 2 1.8 0.8964 19558 19566 19530 19585 19569 19533 19557 21.6 0.11 3 2.2 1.0956 23670 23660 23657 23650 23653 23650 23656 7.71 0.03 4 3.8 1.8924 36670 36665 36666 36663 36664 36662 36665 2.9 0.01 5 4.2 2.0916 40433 40426 40430 40442 40449 40427 40434 9.3 0.02 6 5 2.4900 47100 47113 47112 47112 47110 47111 47111 2.6 0.01 Figure 33 : Calib rat ion curve fo r sulp h adoxin e RS Conc. as a function of AUC y = 16929x + 4894 R² = 0.9993 18751.15 23751.15 28751.15 33751.15 38751.15 43751.15 48751.15 0.7968 1.2968 1.7968 2.2968 2.7968 The calibratio n curve for sulphadoxine A rea und er the curve, A U C ( mg/L ) Concentrat io n , C (mg/mL ) University of Ghana http://ugspace.ug.edu.gh 131 Table 30 : Concent rat ion s of pyri met h amin e RS solut ion s and their corresp onding AUCs ID Actual vol ume pipet ted (mL) Conc. (mg/ mL ) Peak Area (AUC) Aver age AUC SD RSD 1 2 3 4 5 6 1 0.08 0.0402 1624 1624 1624 1624 1624 1624 1625 0.00 0.00 2 0.1 0.0503 1777 1777 1777 1777 1777 1727 1769 20.4 1.15 3 0.11 0.0553 1868 1868 1868 1868 1868 1868 1868 0.00 0.00 4 0.12 0.0604 1960 1960 1960 1960 1960 1960 1960 0.00 0.00 5 0.13 0.0654 2024 2024 2024 2024 2024 2024 2024 0.00 0.00 6 0.15 0.0743 2185 2185 2185 2185 2185 2185 2185 0.00 0.00 Figure 34 : Calib rat ion curve fo r py ri met h amin e RS Conc. as a function of AUC The list of antimalarial drugs purchased is as in appendix I. In addition, the detailed results of each of the samples analysed by both SQ-TLC and HPLC are given in appendix II. 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Retrieved from http://apps.who.int/phint/en/p/about/. 97. Sherma, J. (2007). Analysis of Counterfeit Drugs by Thin Layer Chromatography. Acta Chromatographica, No.19, 5-20. Retrieved from http://www.us.edu.pl/uniwersytet/jednostki/wydzialy/chemia/acta/ac19/zrodla/01_AC19.pdf. University of Ghana http://ugspace.ug.edu.gh 146 98. Risha, P., Layloff, T., Msuya, Z., & Ndomondo-Sigonda, M. (2006). Proficiency Testing as a Tool to Assess the Performance of Visual TLC Quantitation Estimates. Management Sciences for Health (MSH): Strategies for Enhancing Access to Medicines (SEAM). Retrieved from www.researchgate.net/. 99. Pharma Tips. (2013, January 19). UHPLC+ Focused: Ultra High Performance Liquid Chromatograph. Pharma Tips. Retrieved from http://pharmatips.doyouknow.in/Articles/Pharmaceutical-Equipment/UHPLC-Focused- Ultra-High-Performance-Liquid-Chromatograph.aspx. 100. Settle, F.A. (1997). Handbook of Instrumental Techniques for Analytical Chemistry. Prentice Hall PTR, Upper Saddle River, NJ 07458. 101. Koninklijke Philips Electronics N.V. ( 2008). High pressure liquid chromatography. Mi Plaza. Retrieved from http://www.innovationservices.philips.com/sites/default/files/materials-analysis-hplc.pdf. 102. Oona, M. (2009). An introduction to HPLC pharmaceutical analysis; Excerpt. Mourne Training Services (MTS). Retrieved from http://www.mournetrainingservices.co.uk/Preview_book_introduction_HPLC.pdf. 103. HPLC Basics. Fundamentals of liquid chromatography (HPLC). Courtesy of Agilent Technologies Inc. Retrieved from http://polymer.ustc.edu.cn/xwxx_20/xw/201109/P020110906263097048536.pdf. 104. Africa Fighting Malaria, AFM (2009, April 9). Antimalarial drugs-Challenges and suggestions for the way forward. March of Washingtons. Washington DC. Retrieved from http://fightingmalaria.org/pdfs/WMD09.pdf. University of Ghana http://ugspace.ug.edu.gh 147 105. World Health Organization, WHO. The International Pharmacopoeia (Ph. Int.), 4th Edition, Version 2; CD-ROM. (2006). Geneva. 106. Ranher, S.S., Gandhi, S.V., Kadukar, S.S., & Ranjane, P.N. (2010). A validated HPLC method for determination of artesunate in bulk and tablet formulation. Journal of Analytical Chemistry, 65(5), 507-510. Retrieved from http://link.springer.com/static- content/lookinside/510/art%253A10.1134%252FS1061934810050126/000.png. 107. Arun, A., & Smith, A.A. (2011). Simultaneous HPLC-UV method for the estimation of artemether and lumefantrine in tablet dosage form. International Journal of Pharmaceutical and Biomedical Research, 2(3), 201-205. ISSN No.: 0976-0350. 108. World Health Organization, WHO expert committee (2011). Sulphadoxine and pyrimethamine tablets. Adopted text for addition to The International Pharmacopoeia. Working document QAS/07.218/FINAL. Retrieved from http://www.who.int/medicines/publications/pharmacopoeia/Sulfadox-Pyrimeth-tab-QAS07-218 109. The United States Pharmacopoeia USP NF 24. Monographs of quinine sulphate and quinine sulphate tablets. United States Pharmacopoeia Convention: Rockville. p. 1458-1460. 110. Somanje, C. (2011, ). Malawi Orders Malaria Drugs off Shop Shelves. Eyes on Malaria Online; An AMMREN magazine . 7th Edition . Retrieved from http://trial.eyesonmalaria.org/content/malawi-orders-malaria-drugs-shelves-0. 111. Caudron, J.M., Ford, N., Henkens, M., Macé, C., Kiddle-Monroe, R., & Pinel, J. (2008). Substandard medicines in resource-poor settings: a problem that can no longer be ignored. Trop. Med. Int. Health, 13, 1062–1072. DOI: 10.1111/j.1365-156.2008.02106.x. University of Ghana http://ugspace.ug.edu.gh 148 APPENDICES APPENDIX I LIST AND DETAILS OF ANTIMALARIAL DRUGS PURCHASED Table 31: List of art e misin in - b ased and non - art emisin in based ant imala ria l drugs pu rchased from variou s zon e s in Malawi No. Code Name of drug Dosage for m Active ingredient Batch no. Manufactur e r Man –Exp Date ZONE 1: SOUTH WEST NON- ARTEMISININ- BASED DRUGS 1 *14P10 Malacure Tablet Sulphadoxine USP/ Pyrimethamine USP 500mg/ 25mg fixed dose S-60 S Kant India 07/2010- 06/2013 2 *16P10 Malacure Tablet Sulphadoxine USP/ Pyrimethamine USP 500mg/ 25mg fixed dose S-60 S Kant India 07/2010- 06/2013 3 *11P10 Malacure Tablet Sulphadoxine USP/ Pyrimethamine USP 500mg/ 25mg fixed dose S-60 S Kant India 07/2010- 06/2013 4 11P2 Sulphadar Tablet Sulphadoxine USP/ Pyrimethamine USP 500mg/ 25mg fixed dose 10011 Shellys Tanzania 07/2010- 06/2014 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 149 5 12P2 Sulphadar Tablet Sulphadoxine USP/ Pyrimethamine USP 500mg/ 25mg fixed dose 10008 Shellys Tanzania 05/2010- 04/2014 6 14P2 Sulphadar Tablet Sulphadoxine USP/ Pyrimethamine USP 500mg/ 25mg fixed dose 10011 Shellys Tanzania 07/2010- 06/2014 7 13V5 Quinaquin 100ml Mixture Quinine Bisulphate BP 50mg/ml OK 157 Elys Chemical Kenya 11/2010- 10/2012 8 12V5 Quinaquin 100ml Mixture Quinine Bisulphate BP 50mg/ml OK 159 Elys Chemical Kenya 11/2010- 10/2010 9 11V5 Quinaquin 100ml Mixture Quinine Bisulphate BP 50mg/ml OL 119 Elys Chemical 12/2010- 11/2012 ARTEMISININ- BASED DRUGS 10 17X1 Lonart-DS Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-266 Bliss GVS India 09/2011- 08/2013 11 19X1 Lonart-DS Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-259 Bliss GVS India 08/2011- 07/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 150 12 113X1 Lonart-DS Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-259 Bliss GVS India 08/2011- 07/2013 13 110X1 Lonart-DS Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-259 Bliss GVS India 08/2011- 07/2013 14 112X1 Lonart-DS Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-266 Bliss GVS India 09/2011- 08/2013 15 15X1 Lonart-DS Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-266 Bliss GVS India 09/2011- 08/2013 16 16X11 Lonart Suspension Artemether/ Lumefantrine 180mg/1080mg fixed dose LO-210 Bliss GVS India 08/2011- 07/2013 17 14X1 Lonart Forte Tablet Artemether/ Lumefantrine 40mg/240mg fixed dose LF-249 Bliss GVS India 08/2011- 07/2013 18 16X1 Lonart Forte Tablet Artemether/ Lumefantrine 40mg/240mg fixed dose LF-249 Bliss GVS India 08/2011- 07/2013 19 13X1 Lonart Forte Tablet Artemether/ Lumefantrine 40mg/240mg fixed dose LF-239 Bliss GVS India 05/2011- 04/2013 20 18X1 Lonart Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LN-450 Bliss GVS India 09/2011- 08/2013 21 12X1 Lonart-Dispersible Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LS-39 Bliss GVS India 09/2011- 08/2013 22 11X1 Lonart Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LN-449 Bliss GVS India 09/2011- 08/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 151 23 12X11 Artefan Suspension Artemether/ Lumefantrine 180mg/1080mg fixed dose SB0100I Ajanta India 09/2010- 08/2012 24 11X11 Artefan Suspension Artemether/ Lumefantrine 180mg/1080mg fixed dose SB0100I Ajanta India 09/2010- 08/2012 25 14X11 Artefan Suspension Artemether/ Lumefantrine 40mg/240mg fixed dose C0490J Ajanta India 10/2010- 09/2012 26 13X11 Artefan Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose C0520J Ajanta India 10/2010- 09/2012 27 15X11 Artefan Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose C0480J Ajanta India 10/2010- 09/2012 28 11X20 Coartem- Dispersible Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose F0493 Norvatis USA 08/2011- 07/2013 29 11X17 Fantem-Forte Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose M110371 Medinomics India 05/2011- 04/2013 30 12X14 Lum-Artem Tablet β-Artemether/ Lumefantrine 20mg/120mg fixed dose 1105062 Dawa Ltd Kenya 05/2011- 04/2014 31 11X14 Lum-Artem Tablet β-Artemether/ Lumefantrine 20mg/120mg fixed dose 1105062 Dawa Ltd Kenya 05/2011- 04/2014 32 11X18 LA-DS Tablet Artemether/ Lumefantrine 40mg/240mg fixed dose 1117 Global Pharma India 04/2010- 03/2013 33 17Z1 P-Alaxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40mg/320mg fixed dose PX-161 Bliss GVS India 08/2011- 07/2014 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 152 34 16Z1 P-Alaxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40mg/320mg fixed dose PX-161 Bliss GVS India 08/2011- 07/2014 35 15Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-18 Bliss GVS India 08/2011- 07/2014 36 14Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-17 Bliss GVS India 03/2011- 02/2014 37 13Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-18 Bliss GVS India 08/2011- 02/2014 38 12Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-18 Bliss GVS India 08/2011- 02/2014 39 *14Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40mg/320mg fixed dose 110123 Zhejiang Holley Nanhu China 01/2011- 01/2013 40 *12Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40mg/320mg fixed dose 110123 Zhejiang Holley Nanhu China 01/2011- 01/2013 41 *11Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40mg/320mg fixed dose 110123 Zhejiang Holley Nanhu China 01/2011- 01/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 153 ZONE 2: SOUTH EAST ARTEMISININ- BASED DRUGS 42 23X1 Lonart Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LN-262 Bliss GVS India 02/2010- 01/2012 43 21X1 Lonart Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LN-450 Bliss GVS India 09/2011- 08/2013 44 22X1 Lonart Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose LD-259 Bliss GVS India 08/2011- 07/2013 45 24X14 Lum-Artem Tablet β-Artemether/ Lumefantrine 20mg/120mg fixed dose 1105062 Dawa Ltd Kenya 05/2011- 04/2014 46 2X15 Co-Max Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose 021367 Universal Kenya 08/2010- 07/2012 47 25X11 Artefan Tablet Artemether/ Lumefantrine 80mg/480mg fixed dose C0601A Ajanta India 01/2011- 12/2012 48 *24Y13 Spafil Tablet Artesunate/Sulphadoxine/Pyrimetha mine 100/500/25mg co-packed on the same blister T0458 Fourrts 02/2011- 01/2014 49 25Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-18 Bliss GVS India 08/2011- 07/2014 50 24Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-16 Bliss GVS India 02/2011- 01/2014 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 154 51 21Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-18 Bliss GVS India 08/2011- 07/2014 52 23Z1 Alaxin Tablet Dihydroartemisinin/Suphfadoxine BP/Pyrimethamine BP 60mg/500mg/25mg fixed dose AP-16 Bliss GVS India 02/2011- 01/2014 53 26Z1 P-Alaxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg PX-161 Bliss GVS India 08/2011- 07/2014 54 27Z1 P-Alaxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg PX-116 Bliss GVS India 02/2011- 01/2014 NON- ARTEMISININ BASED DRUGS 55 21P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/ 25mg fixed dose 10011 Shellys Tanzania 07/2010- 06/2014 56 22P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/ 25mg fixed dose 10012 Shellys Tanzania 07/2010- 06/2014 57 23P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/ 25mg fixed dose 10012 Shellys Tanzania 07/2010- 06/2014 58 21P15 Methomine S Tablet Sulphadoxine BP/Pyrimethamine BP 500mg/25mg fixed dose 021350 Universal Kenya 08/2010- 07/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 155 ZONE 3 : CENTRAL ARTEMISININ- BASED DRUGS 59 31X11 Artefan Tablet Artemether/Lumefantrine 80mg/480mg fixed dose C0520J Ajanta India 10/2010- 09/2012 60 31X1 Lonart-Dispersible Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LS-39 Bliss GVS India. 09/2011- 08/2013 61 32X1 Lonart-Dispersible Tablet Artemether/ Lumefantrine 20mg/120mg fixed dose LS-39 Bliss GVS India. 09/2011- 08/2013 62 33X1 Lonart Forte Tablet Artemether/Lumefantrine 40mg/240mg fixed dose LF-249 Bliss GVS India 08/2011- 07/2013 63 34X1 Lonart-DS Tablet Artemether/Lumefantrine 800mg/480mg fixed dose LD-227 Bliss GVS India 05/2011- 04/2013 64 36X1 Lonart-DS Tablet Artemether/Lumefantrine 80mg/480mg fixed dose LD-227 Bliss GVS India 05/2011- 04/2013 65 31Y12 Co-Arinate FDC(Adult) Tablet Artesunate/Sulphamethoxypyridazin e/Pyrimethamine 200/500/25mg fixed dose 081 Dafra Kenya 02/2011- 02/2013 66 *32Y13 Spafil(Adults) Tablet Artesunate/Sulphadoxine/Pyrimetha mine 100/500/25mg co-packed on the same blister TR0458 Fourrts India 02/2011- 01/2014 67 *34Y13 Spafil(Adults) Tablet Artesunate/Sulphadoxine/Pyrimetha mine 100/500/25mg co-packed on the same blister TR0458 Fourrts India 02/2011- 01/2014 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 156 68 *32Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose 210610 Zhejiang Holley Nanhu China 06/2010- 06/2012 69 *33Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose 110123 Zhejiang Holley Nanhu 01/2011- 01/2013 70 34Z1 Alaxin +(plus) Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60/500/25mg fixed dose AP-18 Bliss GVS India 08/2011- 07/2014 71 35Z1 Alaxin +(plus) Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60/500/25mg fixed dose AP-16 Bliss GVS India 02/2011- 01/2014 72 37Z1 P-Alaxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose PX-146 Bliss GVS India 06/2011- 05/2014 NON- ARTEMISININ BASED DRUGS 73 *31P10 Malacure Tablet Sulphadoxine/Pyrimethamine 500mg/25mg fixed dose S-60 S Kant Mumbai 07/2010- 06/2013 74 *32P10 Malacure Tablet Sulphadoxine/Pyrimethamine 500mg/25mg fixed dose S-60 S Kant Mumbai 07/2010- 06/2013 75 *33P10 Malacure Tablet Sulphadoxine/Pyrimethamine 500mg/25mg fixed dose S-60 S Kant Mumbai 07/2010- 06/2013 76 31P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10008 Shellys Tanzania 05/2010- 04/2014 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 157 77 32P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10012 Shellys Tanzania 07/2010- 06/2014 78 35P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10011 Shellys Tanzania 07/2010- 06/2014 79 37P15 Methomine S Tablet Sulphadoxine BP/Pyrimethamine BP 500mg/25mg fixed dose 021350 Universal Kenya 08/2010- 07/2013 80 38P15 Methomine S Tablet Sulphadoxine BP/Pyrimethamine BP 500mg/25mg fixed dose 021350 Universal Kenya 08/2010- 07/2013 81 31Q6 Quinine Sulphate Suspension (QSM) Suspension Quinine Sulphate 150mg L-491 Lebene Laboratories India 06/2011- 05/2013 82 32Q6 Quinine Sulphate Suspension (QSM) Suspension Quinine Sulphate 150mg L-1180 Lebene Laboratories India 08/2010- 07/2012 83 33Q6 Quinine Sulphate Suspension (QSM) Suspension Quinine Sulphate 150mg L-491 Lebene Laboratories India 06/2011- 05/2013 ZONE 4 : NORTH NON- ARTEMISININ- BASED DRUGS 84 4V5 Quinaquin Mixture Quinine bisulphate BP 50mg/5ml OK 159 Elys Chemical Kenya 11/2010- 10/2012 85 41R8 Kwinil Injection Quinine di-HCl L01224 Intas Pharmaceuticals India 02/2010- 01/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 158 86 41Q6 Quinine Sulphate Suspension (QSM) Suspension Quinine sulphate 150mg/5ml L-491 Lebene Laboratories India 06/2011- 05/2013 87 42Q6 Quinine Sulphate Suspension (QSM) Suspension Quinine sulphate 150mg/5ml L-491 Lebene Laboratories India 06/2011- 05/2013 88 43Q6 Quinine Sulphate Suspension (QSM) Suspension Quinine sulphate 150mg/5ml L-2100 Lebene Laboratories India 12/2010- 11/2012 89 42R4 Curaquin Quinine syrup Quinine HCl BP 100mg/5ml 110433 Regal Pharmaceuticals Kenya 04/2011- 03/2014 90 43R4 Curaquin Quinine syrup Quinine HCl BP 100mg/5ml 110433 Regal Pharmaceuticals Kenya 04/2011- 03/2014 91 41P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10013 Shellys Tanzania 07/2010- 06/2014 92 44P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10008 Shellys Tanzania 05/2010- 04/2014 93 42P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10012 Shellys Tanzania 07/2010- 06/2014 94 45P2 Sulphadar Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose 10012 Shellys Tanzania 07/2010- 06/2014 95 48P5 Ekelfin Tablet Sulphadoxine USP/Pyrimethamine USP 500mg/25mg fixed dose OA 92 Elys Chemical Kenya 01/2010- 12/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 159 ARTEMISININ- BASED DRUGS 96 45X12 Co-Artesiane Suspension Artemether/Lumefantrine 180mg:60ml/1080mg:60ml 24243 Dafra Pharma Kenya 06/2010- 06/2012 97 4X20 Coartem- Dispersible (Children) Tablet Artemether/Lumefantrine 20/120mg fixed dose F0442 Novartis USA 05/2011- 04/2013 98 44X1 Lonart-DS Tablet Artemether/Lumefantrine 80/480mg fixed dose LD- 259 Bliss Gvs India 08/2011- 07/2013 99 43X1 Lonart Forte Tablet Artemether + lumefantrine 80/480mg fixed dose LF-249 Bliss Gvs India 08/2011- 07/2013 100 42X11 Artefan Tablet Artemether/Lumefantrine 20/120mg fixed dose C0411G Ajanta India 07/2011- 06/2013 101 41X11 Artefan Tablet Artemether/Lumefantrine 20/120mg fixed dose P0761G Ajanta India 07/2011- 06/2013 102 *4Y13 Spafil Tablet Artesunate/Sulphadoxine/Pyrimetha mine 100/500/25mg co-packed on the same blister TR0324 Fourrts India 05/2010- 04/2013 103 43Y12 Co-Arinate PDC Tablet Artesunate/Sulphamethoxypyridazin e/Pyrimethamine 100/250/12.5mg fixed dose 079 Dafra Pharma Kenya 03/2011- 03/2013 104 41Y12 Co-Arinate PDC Tablet Artesunate/Sulphamethoxypyridazin e/Pyrimethamine 200/500/25mg fixed dose 081 Dafra Pharma Kenya 02/2011- 02/2013 105 42Y12 Co-Arinate PDC Tablet Artesunate/Sulphamethoxypyridazin e/Pyrimethamine 200/500/25mg 081 Dafra Pharma Kenya 02/2011- 02/2013 106 44Y12 C0-Arinate PDC Tablet Artesunate/Sulphamethoxypyridazin e/Pyrimethamine 100/250/12.5mg 079 Dafra Pharma Kenya 03/2011- 03/2013 107 *44Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose 110123 Zhejiang Holley Nanhu China 01/2011- 01/2013 * Unregistered samples University of Ghana http://ugspace.ug.edu.gh 160 108 *43Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose 110123 Zhejiang Holley Nanhu China 01/2011- 01/2013 109 *42Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose 110620 Zhejiang Holley Nanhu china 06/2011- 06/2013 110 *41Z3 Duo-Cotecxin Tablet Dihydroartemisinin/Piperaquine Phosphate 40/320mg fixed dose 110123 Zhejiang Holley Nanhu China 01/2011- 01/2013 111 45Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60/500/25mg AP-18 Bliss GVS India 08/2011- 07/2014 112 46Z1 Alaxin Tablet Dihydroartemisinin/Sulphadoxine BP/Pyrimethamine BP 60/500/25mg AP-11 Bliss GVS India 01/2010- 01/2013 * Unregistered samples Total nu mb er o f unreg ister ed sa mp les * = 15 University of Ghana http://ugspace.ug.edu.gh 161 APPENDIX II HPLC AND TLC RESULT Table 32: Percent age (%) and ma ss (mg) quan t it ies of re su lt s of a rt e su n at e active pharmaceu t ical i ng redient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and pharmacopoeial requirements. Artesunate t ab let s must cont ain at least 90.0% and at most 110.0% of the lab elled amou n t of art esu n at e on the pack. Code Manufactur er’s Label Claim (mg) Semi - quanti tati ve TLC esti mati on of composi ti on in % and mg quanti ti e s of dosage for ms of ar te sunate compared to the manufacturer’s label claim r ange of dosage for ms (n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of ar te sunate dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Ethanol : Ammonia 100 : 0.5 Solve nt syste m 2 Ethanol: Toluene: Ammonia 70 : 30 : 1.5 % range ± rsd Quantity (mg) % range ± rsd Quantity (mg) % ± rsd Quantity (mg) *24Y13 ATS/SDX/PYR:100/500/25 87-97±5 87-97 82-92 ± 4 82-92 *BLC 97.57 ± 0.01 98 C *4Y13 ATS/SDX/PYR:100/500/25 90-100± 5 90-100 92-102 ± 4 92-102 C 94.8 ± 0.1 95 C *34Y13 ATS/SDX/PYR:100/500/25 100-110 ± 5 100-110 89-99 ± 5 89-99 C 97.65 ± 0.08 98 C *32Y13 ATS/SDX/PYR:100/500/25 90-100 ± 5 90-100 87-97 ± 5 87-97 *BLC 98.11 ± 0.02 98 C 44Y12 ATS/SM/PYR:100/250/12 90-100 ± 10 90-100 95-105 ± 10 95-105 C 90.4 ± 0.2 90 C 42Y12 ATS/SM/PYR:200/500/25 87-97 ± 5 174-194 85-95 ± 5 170-190 *BLC 92.77 ± 0.02 186 C 43Y12 ATS/SM/PYR:100/250/12.5 65-75 ± 10 65-75 70-80 ± 10 70-80 NC 78.22 ± 0.04 78 NC University of Ghana http://ugspace.ug.edu.gh 162 CODE: ZONESampleAPIManufacturer = Fixed dose combination * ZONESampleAPIManufacturer = Single dose of ATS + single dose of SP on the same blister Table 33: Percent age (%) and ma ss (mg) quan t it ies of re su lt s o f a rt e met h e r active pha rmaceut ical i ng redient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and pharmacopoeial requirements. Artemether t ab let s must cont ain at least 90.0% and at most 110.0% of the lab elled amou n t of art e met h e r on the pack 41Y12 ATS/SM/PYR:200/500/25 90-100 ± 5 180-200 89-99 ± 5 178-198 C 86.9 ± 0.2 174 NC 31Y12 ATS/SM/PYR:200/500/25 65-75 ± 10 130-150 77-87 ± 5 154-174 NC 88.96 ± 0.05 178 NC Code Manufacturer’s Label Claim (mg) Semi - quanti tati ve TLC esti mati on of comp osi ti on in % and mg quanti ti e s of dosage for ms of ar te me the r compared to the manufacturer’s label claim ( n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of ar te me the r dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Petrol: Ethyl acetate 70 : 30 Solve nt syste m 2 Petrol: Ethyl acetate 60 : 40 % range ± rsd Quantity (mg) % range ± rsd Quantity (mg) % ± rsd Quantity (mg) 2X15 ATM/LUM:20/120 127-137 ± 5 25-27 134-144 ± 5 27-29 NC overdose 144.1 ± 0.7 29 NC overdose 4X20 ATM/LUM:20/120 95-105 ± 5 19-21 92-102 ± 4 18-20 C 100 ± 2 20 C 11X1 ATM/LUM:20/120 59-69 ± 4 12-14 55-65 ± 5 11-13 NC 58.9 ± 0.6 12 NC 11X11 ATM/LUM:180 /1080 97-107 ± 5 175-193 99-109 ± 4 178-196 C 93 ± 2 167 C 11X14 ATM/LUM:20/120 79-89 ± 4 16-18 78-88 ± 4 16-18 NC 75.3 ± 0.9 15 NC 11X17 ATM/LUM: 80 /480 115-125 ± 15 92-100 125-135± 15 100-108 NC overdose 177.8 ± 0.3 142 NC overdose University of Ghana http://ugspace.ug.edu.gh 163 11X18 ATM/LUM: 40 /240 30-40 ± 5 12-16 29-39 ± 4 12-16 NC 26.5 ± 0.8 11 NC 11X20 ATM/LUM:20/120 132-142 ± 4 26-28 134-144 ± 5 27-29 NC overdose 135 ± 4 27 NC overdose 12X1 ATM/LUM:20/120 119-129 ± 4 24-26 115-125 ± 5 23-25 NC overdose 119.7 ± 0.8 24 NC overdose 12X11 ATM/LUM:180 /1080 105-115 ± 5 189-207 100-110 ± 5 180-198 *BLC 103.3 ± 0.8 186 C 12X14 ATM/LUM:20/120 30-40 ± 5 6-8 30-40 ± 5 6-8 NC 35 ± 2 7 NC 13X1 ATM/LUM: 40 /240 92-102 ± 4 37-41 94-104 ± 5 38-42 C 96.8 ± 0.2 39 C 13X11 ATM/LUM: 80 /480 35-45 ± 5 28-36 35-45 ± 5 28-36 NC 39.6 ± 0.5 32 NC 14X1 ATM/LUM: 40 /240 64-74 ± 5 26-30 67-77 ± 5 27-31 NC 69 ± 2 28 NC 14X11 ATM/LUM: 40 /240 75-85 ± 5 30-34 72-82 ± 4 29-33 NC 82.7 ± 0.3 33 NC 15X1 ATM/LUM: 80 /480 65-75 ± 5 52-60 62-72 ± 4 50-58 NC 67.3 ± 0.3 54 NC 15X11 ATM/LUM:20/120 40-50 ± 0 8-10 40-50 ± 0 8-10 NC 45 ± 1 9 NC 16X1 ATM/LUM: 40 /240 125-135 ± 10 50-54 120-130 ± 5 48-52 NC overdose 163.2 ± 0.4 65 NC overdose 17X1 ATM/LUM: 80 /480 84-94 ± 5 67-75 84-94 ± 5 67-75 NC 81.205±0 65 NC 18X1 ATM/LUM:20/120 120-130 ± 5 24-26 120-130 ± 5 24-26 NC overdose 162.7 ± 0.3 33 NC overdose 19X1 ATM/LUM: 80 /480 105-115 ± 5 84-92 105-115 ± 5 84-92 *BLC 103.4 ± 0.4 83 C 21X1 ATM/LUM:20/120 97-107± 5 19-21 97-107 ± 5 19-21 C 97 ± 1 19 C 22X1 ATM/LUM: 80 /480 120-130 ± 5 96-104 115-125± 15 92-100 NC overdose 131.5 ± 0.2 105 NC overdose 23X1 ATM/LUM:20/120 135-145 ± 5 27-29 129-139 ± 4 26-28 NC overdose 149 ± 1 30 NC overdose University of Ghana http://ugspace.ug.edu.gh 164 CODE: ZONESampleAPIManufacturer = Fixed dose combination 24X14 ATM/LUM:20/120 52-62 ± 4 10-12 50-60 ± 0 10-12 NC 53.2 ± 0.3 11 NC 25X11 ATM/LUM: 80 /480 105-115 ± 10 84-92 107-117 ± 5 86-94 NC overdose 113 ± 4 90 NC overdose 31X1 ATM/LUM:20/120 85-95 ± 10 17-19 84-94 ± 5 17-19 *BLC 92.4 ± 0.6 18 C 32X1 ATM/LUM:20/120 92-102 ± 4 18-20 95-105 ± 5 19-21 C 97 ± 1 19 C 31X11 ATM/LUM: 80 /480 32-42 ± 4 26-34 34-44 ± 5 27-35 NC 39 ± 1 31 NC 33X1 ATM/LUM: 40 /240 74-84 ± 5 30-34 77-87 ± 5 31-35 NC 81.0 ± 0.3 32 NC 34X1 ATM/LUM: 80 /480 95-105 ± 5 76-84 95-105 ± 5 76-84 C 102.1 ± 0.7 82 C 36X1 ATM/LUM: 80 /480 99-109 ± 4 79-87 95-105 ± 5 76-84 C 104.8 ± 0.3 84 C 41X11 ATM/LUM:20/120 105-115 ± 5 21-23 100-110± 10 20-22 *BLC 106.2 ± 0.4 21 C 42X11 ATM/LUM:20/120 24-34 ± 5 5-7 25-35 ± 5 5-7 NC 28 ± 1 6 NC 43X1 ATM/LUM: 40 /240 132-142 ± 4 53-57 130-140 ± 0 52-56 NC overdose 173.8 ± 0.3 70 NC overdose 44X1 ATM/LUM: 80 /480 135-145 ± 5 108-116 135-145± 10 108-116 NC overdose 142.6 ± 0.7 114 NC overdose 45X12 ATM/LUM:180 /1080 90-100 ± 5 162-180 90-100 ± 5 162-180 C 96.0 ± 0.9 173 C 110X1 ATM/LUM: 80 /480 120-130 ± 5 96-104 124-134 ± 5 99-107 NC overdose 124.2 ± 0.5 99 NC overdose 16X11 ATM/LUM:180 /1080 100-110 ± 5 180-198 100-110 ± 5 180-198 C 99.3 ± 0.9 179 C 112X1 ATM/LUM: 80 /480 92-102 74-82 90-100 ± 5 72-80 C 100.9 ± 0 81 C 113X1 ATM/LUM: 80 /480 112-122 ± 4 90-98 115-125 ± 5 92-100 NC overdose 140.0 ± 0.5 112 NC overdose University of Ghana http://ugspace.ug.edu.gh 165 Table 34: Percent age (%) and mas s (mg) quan t it ies of re su lt s of lu mefan t rin e active pha rmaceut ical i ngredient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and pharmacopoeial requirements. Lumefantrine t ab let s must cont ain at least 90.0% and at most 110.0% of the lab elled amou n t of Lume fan t rin e on the pack Code Manufacturer’s Label Claim (mg) Semi - quanti tati ve TLC esti mati on of comp osi ti on in % and mg quanti ti e s of dosage for ms of lume fantr i ne compar ed to the manufacturer’s label claim ( n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of lume fantr i ne dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Ethyl acetate: acetic acid: toluene 4:2:18 Solve nt syste m 2 Ethyl acetate: acetic acid 10:5 % range ± rsd Quantity (mg) % range ± rsd Quantity (mg) % ± rsd Quantity (mg) 2X15 ATM/LUM:20/120 95-105 ± 5 114-126 95-105 ± 5 114-126 C 97 ± 2 116 C 4X20 ATM/LUM:20/120 112-122± 4 134-146 113-123±5 136-148 NC overdose 119.5 ± 0.4 143 NC overdose 11X1 ATM/LUM:20/120 104-114± 5 125-137 95-105 ± 5 114-126 C 112.5 ± 0.5 135 NC overdose 11X11 ATM/LUM:180/1080 117-127± 5 1264-1372 120-130 ± 0 1296-1404 NC overdose 127 ± 1 1372 NC overdose 11X14 ATM/LUM:20/120 60-70 ± 0 72-84 60-70 ± 0 72-84 NC 69.3 ± 0.3 83 NC 11X17 ATM/LUM:80/ 480 94-104 ± 5 451-499 98-108 ± 5 470-518 C 101.0 ± 0.4 485 C 11X18 ATM/LUM:40/240 95-105 ± 5 228-252 90-100 ± 0 216-240 C 92.9 ± 0.3 223 C 11X20 ATM/LUM:20/120 109-119± 4 131-143 110-120 ± 0 132-144 NC overdose 114.5 ± 0.5 137 NC overdose 12X1 ATM/LUM:20/120 100-110 ± 5 120-132 94-104 ± 5 113-125 C 113.0 ± 0.4 136 NC 12X11 ATM/LUM:180/1080 122-132± 4 1318-1426 119-129 ± 4 1285-1393 NC overdose 123 ± 2 1328 NC overdose University of Ghana http://ugspace.ug.edu.gh 166 12X14 ATM/LUM:20/120 92-102 ± 4 110-122 90-100 ± 5 108-120 C 95.3 ± 0.2 114 C 13X1 ATM/LUM:40/240 77-87 ± 5 185-209 74-84 ± 5 178-202 NC 81.1 ± 0.3 195 NC 13X11 ATM/LUM:80/ 480 74-84 ± 5 355-403 75-85 ± 5 360-408 NC 85 ± 1 408 NC 14X1 ATM/LUM:40/240 100-110 ± 5 240-264 109-119 ± 4 262-286 BLC 115.0 ± 0.9 276 NC 14X11 ATM/LUM:40/240 100-110 ± 5 240-264 105-115 ± 5 252-276 BLC 114.0 ± 0.4 274 NC 15X1 ATM/LUM:80/ 480 99-109 ± 4 475-523 103-113 ± 5 494-542 BLC 110.9 ± 0.2 532 BLC 15X11 ATM/LUM:20/120 80-90 ± 10 96-108 90-100 ± 5 108-120 *BLC 90.5 ± 0.3 109 C 16X1 ATM/LUM:40/240 122-132± 4 293-317 120-130 ± 10 288-312 NC overdose 127 ± 1 305 NC overdose 17X1 ATM/LUM:80/ 480 57-67 ± 5 274-322 59-69 ± 4 283-331 NC 59 ± 1 283 NC 18X1 ATM/LUM:20/120 70-80 ± 0 84-96 72-82 ± 4 86-98 NC 79.19 ± 0.0 95 NC 19X1 ATM/LUM:80/ 480 107-117± 5 514-562 109-119 ± 4 523-571 NC overdose 113.5 ± 0.4 545 NC overdose 21X1 ATM/LUM:20/120 105-115 ± 5 126-138 109-119 ± 4 131-143 NC overdose 113 ± 4 136 NC 22X1 ATM/LUM:80/ 480 57-67 ± 5 274-322 60-70 ± 0 288-336 NC 63 ± 2 302 NC 23X1 ATM/LUM:20/120 97-107 ± 5 116-128 99-109 ± 4 119-131 C 103.3 ± 0.3 124 C 24X14 ATM/LUM:20/120 112-122± 4 134-146 115-125 ± 5 138-150 NC overdose 117.5 ± 0.8 141 NC overdose 25X11 ATM/LUM:80/ 480 90-100 ± 10 432-480 100-110 ± 5 480-528 C 112.2 ± 0.6 539 NC 31X1 ATM/LUM:20/120 115-125 ± 5 138-150 119-129 ± 4 143-155 NC overdose 116.9 ± 0.5 140 NC overdose 32X1 ATM/LUM:20/120 112-122± 4 134-146 112-122 ± 4 134-146 NC ove rdose 117 ± 2 140 NC overdose University of Ghana http://ugspace.ug.edu.gh 167 ` CODE: ZONE SampleAPIManufacturer = Fixed dose combination 31X11 ATM/LUM:80/ 480 100-110 ± 10 480-528 105-115 ± 10 504-552 BLC 118.6 ± 0.6 569 NC overdose 33X1 ATM/LUM:40/240 95-105 ± 5 228-252 95-105 ± 5 228-252 C 108.0 ± 0.2 259 C 34X1 ATM/LUM:80/ 480 100-110 ± 10 480-528 102-112 ± 4 490-538 BLC 111.6 ± 0.3 536 NC overdose 36X1 ATM/LUM:80/ 480 90-100 ± 5 432-480 95-105 ± 5 456-504 C 103.3 ± 0.1 496 C 41X11 ATM/LUM:20/120 45-55 ± 5 54-66 40-50 ± 0 48-60 NC 50 ± 6 60 NC 42X11 ATM/LUM:20/120 87-97 ± 5 104-116 90-100 ± 5 108-120 BLC 88 ± 2 106 BLC 43X1 ATM/LUM:40/240 72-82 ± 4 173-197 75-85 ± 5 180-204 NC 81.8 ± 0.3 196 NC 44X1 ATM/LUM:80/ 480 100-110 ± 5 480-528 95-105 ± 5 456-504 C 104.0 ± 0.2 499 C 45X12 ATM/LUM:180/1080 125-135 ± 5 1350-1458 125-135 ± 5 1350-1458 NC overdose 129 ± 2 1393 NC overdose 110X1 ATM/LUM:80/ 480 62-72 ± 4 298-346 60-70 ± 0 288-336 NC 63 ± 1 302 NC 16X11 ATM/LUM:180/1080 107-117± 5 1156-1264 108-118 ± 5 1166-1274 NC overdose 119.3 ± 0.1 1288 NC overdose 112X1 ATM/LUM:80/ 480 100-110 ± 5 480-528 94-104 ± 5 451-499 C 102.5 ± 0 492 C 113X1 ATM/LUM:80/ 480 90-100 ± 5 432-480 95-105 ± 5 456-504 C 109.96±0.0 528 C University of Ghana http://ugspace.ug.edu.gh 168 Table 35: Percent age (%) and mass (mg) quan t it ies of resu lt s of dihydroa rt e misin in (Art enimol) active pharmaceut ical i ngredient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and pharmacopoeial req u i re men t s. Dihydroa rt e misin in tab let s mu st cont ain at least 90.0% and at most 110.0% of the lab e lled amou n t of Dihydroa rt emi sin in on the pack Code Manufacturer’s Label Claim (mg) Semi - quanti tati ve TLC esti mati on of comp osi ti on in % and mg q uanti ti e s of dosage for ms of dihydroar te mi si ni n compar ed to the manufacturer’s label claim (n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of dihydroar te mi si ni n dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Toluene: Ethyl acetate 60 : 40 Solve nt syste m 2 Toluene: Ethyl acetate 70 : 30 % range ± rsd Quantity (mg) % range ± rsd Quantity (mg) % ± rsd Quantity (mg) 11Z3 DHA/PPQ: 40 /320 100-110 ± 5 40-44 95-105 ± 5 38-42 C 98.7 ± 0.2 39 C 12Z3 DHA/PPQ: 40 /320 84-94± 5 34-38 84-94± 5 34-38 NC 86 ± 2 34 NC 14Z3 DHA/PPQ: 40 /320 70-80 ± 5 28-32 70-80 ± 5 28-32 NC 70 ± 1 28 NC 16Z1 DHA/PPQ: 40 /320 90-100 ± 10 36-40 94-104± 5 38-42 C 97.5 ± 0.9 39 C 17Z1 DHA/PPQ: 40 /320 85-95 ± 10 34-38 85-95 ± 10 34-38 BLC 85 ± 1 34 NC 26Z1 DHA/PPQ: 40 /320 95-105 ± 10 38-42 95-105 ± 10 38-42 C 103 ± 1 41 C 27Z1 DHA/PPQ: 40 /320 102-112± 4 41-45 100-110 ± 5 40-44 BLC 88 ± 5 35 BLC 32Z3 DHA/PPQ: 40 /320 67-77 ± 5 27-31 66-76 ± 5 26-30 NC 73 ± 2 29 NC 33Z3 DHA/PPQ: 40 /320 62-72 ± 4 25-29 64-74 ± 5 26-30 NC 71 ± 1 28 NC University of Ghana http://ugspace.ug.edu.gh 169 37Z1 DHA/PPQ: 40 /320 90-100 ± 5 36-40 95-105 ± 5 38-42 C 101 ± 6 40 C 41Z3 DHA/PPQ: 40 /320 69-79 ± 5 28-32 67-77 ± 5 27-31 NC 74 ± 3 30 NC 42Z3 DHA/PPQ: 40 /320 90-100 ± 10 36-40 95-105 ± 5 38-42 C 87 ± 1 35 NC 43Z3 DHA/PPQ: 40 /320 70-80 ± 5 28-32 70-80 ± 10 28-32 NC 71 ± 3 28 NC 44Z3 DHA/PPQ: 40 /320 90-100 ± 5 36-40 90-100 ± 5 36-40 C 88 ± 1 35 NC 15Z1 DHA/SDX/PYR:60/500/25 47-57 ± 5 28-34 45-55 ± 5 27-33 NC 51.4 ± 0.2 31 NC 13Z1 DHA/SDX/PYR:60/500/25 56-66 ± 4 34-40 60-70 ± 5 36-42 NC 56.8 ± 0.2 34 NC 12Z1 DHA/SDX/PYR:60/500/25 57-67 ± 5 34-40 57-67 ± 5 34-40 NC 52.5 ± 0.1 32 NC 14Z1 DHA/SDX/PYR:60/500/25 60-70 ± 5 36-42 55-65 ± 5 33-39 NC 61.6 ± 0.1 37 NC 21Z1 DHA/SDX/PYR:60/500/25 60-70 ± 5 36-42 60-70 ± 5 36-42 NC 56.48±0.09 34 NC 23Z1 DHA/SDX/PYR:60/500/25 45-55 ± 5 27-33 50-60 ± 5 30-36 NC 54.4 ± 0.2 33 N C 24Z1 DHA/SDX/PYR:60/500/25 54-64 ± 5 32-38 52-62 ± 4 31-37 NC 52.5 ± 0.1 32 NC 25Z1 DHA/SDX/PYR:60/500/25 55-65 ± 5 33-39 57-67 ± 5 34-40 NC 51.25±0.06 31 NC 34Z1 DHA/SDX/PYR:60/500/25 49-59 ± 4 29-35 49-59 ± 4 29-35 NC 51.4 ± 0.1 31 NC 35Z1 DHA/SDX/PYR:60/500/25 56-66 ± 5 34-40 52-62 ± 4 31-37 NC 51.8 ± 0.1 31 NC 45Z1 DHA/SDX/PYR:60/500/25 57-67 ±5 34-40 57-67±5 34-40 NC 51.67±0.05 31 NC 46Z1 DHA/SDX/PYR:60/500/25 57-67± 5 34-40 55-65 ± 10 33-39 NC 51.92±0.09 31 NC CODE: ZONESampleAPIManufacturer = Fixed dose combination University of Ghana http://ugspace.ug.edu.gh 170 Table 36: Percent age (%) and Mass (mg) quan t it ies of resu lt s of su lp h adoxin e active pha rmaceut ical i ng redient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and p h a rmacopoei al req u i re men t s. Sulph adoxin e tab let s must cont ain at least 90.0% and at most 110.0% of the lab elled amou n t of Sulph adoxin e on the p ack Code Manufacturer’s Label Claim (mg) Semi - quanti tati ve TLC esti mati on of composi ti on in % and mg qu anti ti e s of dosage f or ms of sul ph adoxine compared to the manufacturer’s label claim n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of s ul ph adoxi ne dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Ethyl acetate/methanol/ Ammonia 80:15:5 Solve nt syste m 2 Ethyl acetate/acetic acid/ water 60:20:20 % range ± rsd Quantity (mg) % range ± rsd Quantity (mg) % ± rsd Quantity (mg) 11P10 SDX /PYR: 500 /25 80-90 ± 5 400-450 77-87±5 385-435 NC 78 ± 2 390 NC 11P2 SDX /PYR: 500 /25 82-92±4 410-460 80-90 ± 0 400-450 NC 87 ± 2 435 NC 12P2 SDX /PYR: 500 /25 70-80 ± 5 350-400 70-80 ± 5 350-400 NC 67 ± 4 335 NC 14P10 SDX /PYR: 500 /25 74-84± 5 370-420 77-87 ± 5 385-435 NC 85 ± 2 425 NC 14P2 SDX /PYR: 500 /25 79-89± 4 395-445 78-88 ± 5 390-440 NC 82 ± 2 410 NC 16P10 SDX /PYR: 500 /25 82-92± 4 410-460 80-90 ± 5 400-450 NC 83 ± 3 415 NC 21P15 SDX /PYR: 500 /25 77-87± 5 385-435 78-88 ± 4 390-440 NC 82 ± 3 410 NC 21P2 SDX /PYR: 500 /25 87-97± 5 435-485 80-90 ± 0 400-450 NC 77 ± 1 385 NC 22P2 SDX /PYR: 500 /25 85-95 ± 5 425-475 83-93 ± 5 415-465 NC 83 ± 2 415 NC 23P2 SDX /PYR: 500 /25 90-100± 5 450-500 90-100 ± 0 450-500 C 96 ± 2 480 C University of Ghana http://ugspace.ug.edu.gh 171 31P10 SDX /PYR: 500 /25 94-104±5 470-520 85-95 ± 5 425-475 C 83 ± 2 415 NC 31P2 SDX /PYR: 500 /25 94-104± 5 470-520 90-100± 10 450-500 C 97 ± 3 485 C 32P10 SDX /PYR: 500 /25 85-95 ± 5 425-475 80-90 ± 0 400-450 NC 81 ± 2 405 NC 32P2 SDX /PYR: 500 /25 82-92 ± 4 410-460 82-92± 4 410-460 NC 85 ± 2 425 NC 33P10 SDX /PYR: 500 /25 70-80 ± 5 350-400 69-79 ± 4 345-395 NC 82 ± 2 410 NC 35P2 SDX /PYR: 500 /25 50-60 ± 0 250-300 50-60 ± 0 250-300 NC 53 ± 3 265 NC 37P15 SDX /PYR: 500 /25 70-80 ± 5 350-400 75-85 ± 5 375-425 NC 71 ± 2 355 NC 38P15 SDX /PYR: 500 /25 85-95± 10 425-475 85-95 ± 5 425-475 * BLC 91.7 ± 0.2 459 C 41P2 SDX /PYR: 500 /25 85-95 ± 5 425-475 80-90 ± 5 400-450 NC 89 ± 3 445 BLC 42P2 SDX /PYR: 500 /25 55-65 ± 5 275-325 52-62 ± 4 260-310 NC 47 ± 2 235 NC 44P2 SDX /PYR: 500 /25 72-82 ± 4 360-410 75-85 ± 10 375-425 NC 85.17 ± 0.06 426 NC 45P2 SDX /PYR: 500 /25 82-92 ± 4 410-460 85-95 ± 5 425-475 NC 85.1 ± 0.2 426 NC 48P5 SDX /PYR: 500 /25 87-97 ± 5 435-485 90-100± 10 450-500 BLC 85 ± 2 425 NC *24Y13 ATS/SDX /PYR:100/ 500 /25 79-89 ± 4 395-445 73-83 ± 5 365-415 NC 72 ± 2 360 NC *4Y13 ATS/SDX /PYR:100/ 500 /25 77-87 ± 5 385-435 77-87 ± 5 385-435 NC 89 ± 2 445 BLC *34Y13 ATS/SDX /PYR:100/ 500 /25 65-75 ± 5 325-375 63-73 ± 5 315-365 NC 74 ± 4 370 NC *32Y13 ATS/SDX /PYR:100/ 500 /25 64-74 ± 5 320-370 63-73 ± 5 315-365 NC 68 ± 5 340 NC 15Z1 DHA/SDX /PYR:60/ 500 /25 84-94 ± 5 420-470 88-98 ± 4 440-490 BLC 85.694 ± 0 428 NC University of Ghana http://ugspace.ug.edu.gh 172 Sm = sulphamethoxypyridazine. With TLC, it gave a spot with the same retention time as sulphadoxine, but it failed to stain well with I2/KI for the SQ-TLC. However, HPLC analysis was successful. CODE: ZONESampleAPIManufacturer = Fixed dose combination * ZONESampleAPIManufacturer = Single dose of ATS + single dose of S/P on the same blister 13Z1 DHA/SDX /PYR:60/ 500 /25 84-94± 5 420-470 82-92 ± 4 410-460 NC 86 ± 2 430 NC 12Z1 DHA/SDX /PYR:60/ 500 /25 72-82 ± 4 360-410 70-80 ± 10 350-400 NC 82.7 ± 0.9 414 NC 14Z1 DHA/SDX /PYR:60/ 500 /25 65-75± 10 325-375 70-80 ± 5 350-400 NC 79 ± 2 395 NC 21Z1 DHA/SDX /PYR:60/ 500 /25 72-82 ± 4 360-410 78-88 ± 5 390-440 NC 77 ± 2 385 NC 23Z1 DHA/SDX /PYR:60/ 500 /25 90-100± 5 450-500 90-100 ± 5 450-500 C 90 ± 2 450 C 24Z1 DHA/SDX /PYR:60/ 500 /25 70-80± 10 350-400 75-85 ± 10 375-425 NC 80 ± 2 400 NC 25Z1 DHA/SDX /PYR:60/ 500 /25 79-89± 4 395-445 75-85 ± 5 375-425 NC 86.7 ± 0.6 434 NC 34Z1 DHA/SDX /PYR:60/ 500 /25 80-90 ± 5 400-450 75-85 ± 5 375-425 NC 85.4 ± 0.2 427 NC 35Z1 DHA/SDX /PYR:60/ 500 /25 87-97 ± 5 435-485 80-90 ± 0 400-450 NC 84.9 ± 0.1 425 NC 45Z1 DHA/SDX /PYR:60/ 500 /25 74-84 ± 5 370-420 75-85 ± 5 375-425 NC 87.5 ± 0.8 438 NC 46Z1 DHA/SDX /PYR:60/ 500 /25 74-84 ± 5 370-420 80-90 ± 5 400-450 NC 75.2 ± 0.1 376 NC 44Y12 ATS/SM/PYR:100/250 /12.5 - - - - 90 ± 3 225 C 42Y12 ATS/SM/PYR:200/ 500 /25 - - - - 88.6 ± 0.2 443 NC 43Y12 ATS/SM/PYR:100/250 /12.5 - - - - 87 ± 2 218 NC 41Y12 ATS/SM/PYR:200/ 500 /25 - - - - 87.9 ± 0.5 440 NC 31Y12 ATS/SM/PYR:100/250 /12.5 - - - - 86.6 ± 0.2 217 NC University of Ghana http://ugspace.ug.edu.gh 173 Table 37: Percent age (%) and Mass (mg) quan t it ies of re su lt s of py ri met h amin e active pharmaceut ical i ng redient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and pharmacopoeial requirements. Pyrimet h a min e tab let s must cont ain at least 90.0% and at most 110.0% of the lab elled amou n t of Pyrim et h amin e on the pack Code Manufacturer’s Label Claim (mg) Semi - quanti tati ve TLC esti mati on of comp osi ti on in % and mg quanti ti e s of dosage for ms of pyrimethamine compared to the manufacturer’s l abe l clai m n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of pyr i me thami ne dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Ethyl Acetate/methanol/ammoni a 85:10:5 Solve nt syste m 2 Ethyl acetate/acetic acid/water 60:20:20 % range ± rsd Quantity( mg) % range ± rsd Quantity (mg) % ± rsd Quantity (mg) 11P10 SDX/PYR:500/25 80-90 ± 0 20-23 90-100 ± 0 23-25 * BLC 92±0 23 C 11P2 SDX/PYR:500/25 105-115± 10 26-29 110-120 ± 5 28-30 NC overdose 114±0 29 NC overdose 12P2 SDX/PYR:500/25 69-79 ± 4 17-20 69-79± 4 17-20 NC 76±0 19 NC 14P10 SDX/PYR:500/25 92-102 ± 4 23-26 94-104 ± 5 24-26 C 110±0 28 C 14P2 SDX/PYR:500/25 87-97 ± 5 22-24 89-99 ± 5 22-25 *BLC 93±0 23 C 16P10 SDX/PYR:500/25 87-97 ± 5 22-24 92-102 ± 4 23-26 C 92±0 23 C 21P15 SDX/PYR:500/25 85-95 ± 5 21-24 85-95 ± 5 21-24 * BLC 93±0 23 C 21P2 SDX/PYR:500/25 87-97 ± 5 22-24 87-97 ± 5 22-24 BLC 89±0 22 NC 22P2 SDX/PYR:500/25 60-70 ± 10 15-18 57-67 ± 5 14-17 NC 64±0 16 NC University of Ghana http://ugspace.ug.edu.gh 174 23P2 SDX/PYR:500/25 107-117± 5 27-29 107-117± 5 27-29 NC overdose 122±0 31 NC overdose 31P10 SDX/PYR:500/25 80-90 ± 10 20-23 85-95 ± 5 21-24 NC 83±0 21 NC 31P2 SDX/PYR:500/25 92-102 ± 4 23-26 94-104± 5 24-26 C 97±0 24 C 32P10 SDX/PYR:500/25 85-95 ± 5 21-24 84-94 ± 4 21-24 *BLC 94±0 24 C 32P2 SDX/PYR:500/25 79-89 ± 4 20-22 80-90 ± 5 20-23 NC 96±0 24 C 33P10 SDX/PYR:500/25 84-94 ± 5 21-24 82-92± 4 21-23 NC 87±0 22 NC 35P2 SDX/PYR:500/25 40-50 ± 0 10-13 40-50 ± 0 10-13 NC 48±0 12 NC 37P15 SDX/PYR:500/25 80-90 ± 5 20-23 70-80 ± 0 18-20 NC 78±0 20 NC 38P15 SDX/PYR:500/25 99-109 ± 4 25-27 95-105 ± 5 24-26 C 103±0 26 C 41P2 SDX/PYR:500/25 110-120 ± 5 28-30 110-120 ± 5 28-30 NC overdose 113±0 28 NC overdose 42P2 SDX/PYR:500/25 60-70 ± 5 15-18 50-60 ± 0 13-15 NC 51±0 13 NC 44P2 SDX/PYR:500/25 80-90 ± 15 20-23 75-85 ± 10 19-21 NC 102±0 26 C 45P2 SDX/PYR:500/25 95-105 ± 5 24-26 99-109 ± 5 25-27 C 108±0 27 C 48P5 SDX/PYR:500/25 104-114± 5 26-29 102-112± 4 26-28 * BLC 110±0 28 C *24Y13 ATS/SDX/PYR:100/500/25 85-95 ± 5 21-24 90-100 ± 0 23-25 * BLC 91±0 23 C *4Y13 ATS/SDX/PYR:100/500/25 80-90 ± 5 20-23 85-95 ± 5 21-24 NC 98±0 25 C *34Y13 ATS/SDX/PYR:100/500/25 60-70 ± 0 15-18 60-70 ± 0 15-18 NC 70±0 18 NC *32Y13 ATS/SDX/PYR:100/500/25 75-85 ± 5 19-21 72-82 ± 4 18-21 NC 78±0 20 NC University of Ghana http://ugspace.ug.edu.gh 175 CODE: ZONESampleAPIManufacturer = Fixed dose combination * ZONESampleAPIManufacturer = Single dose of Ats + single dose of SP on the same blister *BLC= Samples that changed to C with HPLC analysis. 15Z1 DHA/SDX/PYR:60/500/25 90-100 ± 0 23-25 87-97 ± 5 22-24 * BLC 94±0 24 C 13Z1 DHA/SDX/PYR:60/500/25 87-97 ± 5 22-24 88-98 ± 4 22-25 * BLC 91±0 23 C 12Z1 DHA/SDX/PYR:60/500/25 90-100 ± 5 23-25 88-98 ± 4 22-25 * BLC 98±0 25 C 14Z1 DHA/SDX/PYR:60/500/25 95-105 ± 5 24-26 95-105 ± 5 24-26 C 106±0 27 C 21Z1 DHA/SDX/PYR:60/500/25 89-99 ± 4 22-25 88-98 ± 5 22-25 * BLC 105±0 26 C 23Z1 DHA/SDX/PYR:60/500/25 95-105 ± 5 24-26 90-100 ± 0 23-25 C 100±0 25 C 24Z1 DHA/SDX/PYR:60/500/25 95-105 ± 5 24-26 90-100 ± 10 23-25 C 95±0 24 C 25Z1 DHA/SDX/PYR:60/500/25 84-94 ± 5 21-24 90-100 ± 5 23-25 * BLC 90±0 23 C 34Z1 DHA/SDX/PYR:60/500/25 90-100 ± 0 23-25 92-102 ± 4 23-26 C 98±0 25 C 35Z1 DHA/SDX/PYR:60/500/25 104-114± 5 26-29 100-110 ± 0 25-28 * BLC 98±0 25 C 45Z1 DHA/SDX/PYR:60/500/25 97-107 ± 5 24-27 100-110 ± 0 25-28 C 103±0. 26 C 46Z1 DHA/SDX/PYR:60/500/25 94-104 ± 5 24-26 94-104 ± 5 24-26 C 99±0 25 C 44Y12 ATS/SM/PYR:100/250/12.5 104-114± 5 13-14 107-117± 5 13-15 NC overdose 119±0 15 NC overdose 42Y12 ATS/SM/PYR:200/500/25 95-105 ± 5 24-26 107-117± 5 27-29 BLC 116±0 29 NC overdose 43Y12 ATS/SM/PYR:100/250/12.5 115-125 ± 5 14-16 105-115 ± 10 13-14 NC overdose 113±0 14 NC overdose 41Y12 ATS/SM/PYR:200/500/25 90-100 ± 5 23-25 85-95 ± 5 21-24 * BLC 101.73±0 25 C 31Y12 ATS/SM/PYR:100/250/12.5 99-109 ± 4 12-14 97-107 ± 5 12-13 C 110±0 14 C University of Ghana http://ugspace.ug.edu.gh 176 Table 38: Percent age (%) and Mass (mg) quan t it ies of resu lt s o f quin in e active pharmaceut ical i ng redient (API) by TLC and HPLC methods and their comparisons with the manufacturer’s claim and pharmacopoeial requirements. Quinine tablets mu st cont ain at least 90.0% and at most 110.0% of the lab e lled amou n t of Quinin e on the pack Code Manufacturer’s Label Claim Semi - quanti tati ve TLC esti mati on of comp osi ti on in % and mg q uanti ti e s of dosage for ms of q ui ni ne compar ed to the manufacturer’s label claim n = 6 for each sol ve nt syste m, total n = 12 ) Remar k s based on TLC resul ts HPLC deter mi nati on of comp osi ti on of q ui ni ne dosage for ms in % and mg quanti ti e s (n = 6) Remar k s based on HPLC resul ts Solve nt syste m 1 Methanol: ammonia 100:1.5 Solve nt syste m 2 Ethyl acetate: acetic acid: water 60:20:20 % range ±rsd Quantity (mg) % range ±rsd Quantity (mg) % ±rsd Quantity (mg) 11V5 QUN:50mg/ 5ml 102-112 ± 4 51-56 102-112 ± 4 51-56 *BLC 109.7 ± 0.3 55 C 12V5 QUN:50mg/ 5ml 110-120 ± 5 55-60 103-113 ± 10 52-57 NC overdose 120 ± 1 60 NC overdose 13V5 QUN:50mg/ 5ml 104-114 ± 5 52-57 104-114 ± 5 52-57 BLC 112 ± 1 56 NC overdose 41Q6 QUN:50mg/ 5ml - - - - - 102 ± 4 51 C 42Q6 QUN:50mg/ 5ml - - - - - 56 ± 7 28 NC 43Q6 QUN:50mg/ 5ml - - - - - 97 ± 4 49 C 4V5 QUN:50mg/ 5ml 107-117± 5 54-59 107-117± 5 54-59 NC overdose 112 ± 2 56 BLC 41R8 QUN:100mg/5ml 130-140 ± 10 130-140 135-145 ± 5 135-145 NC overdose 291 ± 1 291 NC overdose 42R4 QUN:100mg/5ml 125-135 ± 5 125-135 125-135 ± 5 125-135 NC overdose 151 ± 1 151 NC overdose 43R4 QUN:100mg/5ml 117-127± 5 117-127 120-130 ± 0 120-130 NC overdose 156 ± 2 156 NC overdose University of Ghana http://ugspace.ug.edu.gh 177 31Q6 QUN:150mg - - - - - 110.4 ± 0.1 166 C 32Q6 QUN:150mg - - - - - 110.5 ± 0.1 166 BLC 33Q6 QUN:150mg - - - - - 122.1 ± 0.8 183 NC overdose CODE: ZONESampleAPIManufacturer = Fixed dose combination * ZONESampleAPIManufacturer = Single dose of Ats + single dose of SP on the same blister *BLC= Samples that changed to C with HPLC analysis. University of Ghana http://ugspace.ug.edu.gh 178 APPENDIX III Table 39: Sample s wit h the same batch numb er and their re sp ective mas ses (mg) Batch No. Sampl e s wi th the same batch numbe r and the ir masse s S-60 14P10 16P10 11P10 31P10 32P10 33P10 85mg/110mg 83mg/92mg 78mg/92mg 83mg/83mg 81mg/94mg 82mg/87mg 10011 11P2 14P2 21P2 35P2 87mg/114mg 82mg/93mg 77mg/89mg 53mg/48mg 10008 31P2 44P2 12P2 97mg/97mg 85mg/102mg 67mg/76mg OK 159 12V5 4V5 120mg 112mg LD-266 112X1 15X1 17X1 101mg/103mg 67mg/111mg 81mg/59mg LD-259 19X1 113X1 110X1 22X1 44X1 103mg/114mg 140mg/110mg 124mg/63mg 131mg/63mg 143mg/104mg LF-249 14X1 16X1 33X1 43X1 69mg/115mg 163mg/127mg 81mg/108mg 174mg/82mg University of Ghana http://ugspace.ug.edu.gh 179 LN-450 18X1 21X1 163mg/79mg 97mg/113mg LS-39 12X1 31X1 32X1 120mg/113mg 92mg/117mg 97mg/117mg SB0100I 12X11 11X11 103mg/123mg 93mg/127mg C0520J 31X11 13X11 39mg/119mg 40mg/85mg 1105062 12X14 11X14 24X14 35mg/95mg 75mg/69mg 53mg/118mg PX-161 17Z1 16Z1 26Z1 85mg/- 98mg/- 103mg/- AP-18 15Z1 13Z1 12Z1 25Z1 21Z1 34Z1 45Z1 51mg/86mg/94mg 57mg/86mg/91mg 53mg/83mg/98mg 51mg/87mg/90mg 56mg/77mg/105mg 51mg/85mg/98mg (52/88/105)mg 110123 14Z3 12Z3 11Z3 33Z3 44Z3 43Z3 41Z3 70mg/- 86mg/- 99mg/- 71mg/- 88mg/- 71mg/- 74mg/- AP-16 24Z1 23Z1 35Z1 University of Ghana http://ugspace.ug.edu.gh 180 53mg/80mg/95mg 54mg/90mg/100mg 52mg/85mg/98mg 10012 22P2 23P2 32P2 42P2 45P2 83mg/64mg 96mg/122mg 85mg/96mg 47mg/51mg 85mg/108mg 021350 21P15 37P15 38P15 82mg/93mg 71mg/78mg 91mg/103mg LD-227 34X1 36X1 102mg/112mg 105mg/103mg 081 31Y12 41Y12 42Y12 89mg/87mg/110mg 87mg/88mg/102mg 93mg/89mg/116mg TR0458 32Y13 34Y13 98mg/68mg/78mg 98mg/74mg/70mg L-491 31Q6 33Q6 41Q6 42Q6 166mg 183mg 102mg 56mg 110433 42R4 43R4 151mg 156mg 079 43Y12 44Y12 78mg/87mg/111mg 90mg/90mg/119mg University of Ghana http://ugspace.ug.edu.gh 181 APPENDIX IV SAMPLES OF TLC PLATES ARTESUNATE SAMPLE ARTEMETHER SAMPLE Ethanol: Toluene: Ammonia Ethanol: Ammonia Pet. Ether: Ethyl Acetate Toluene: Ethyl acetate (70:30:1.5) (100:0.5) (70:30) (70:30) University of Ghana http://ugspace.ug.edu.gh 182 LUMEFANTRINE SAMPLE DIHYDROARTEMISININ SAMPLE Ethyl Acetate: Acetic acid:Toluene Ethyl acetate: Acetic acid Toluene: Ethyl acetate Toluene: Ethyl acetate (4:2:18) (10:5) (60:40) (70:30) University of Ghana http://ugspace.ug.edu.gh 183 QUININE SAMPLE SULPHADOXINE SAMPLE Methanol: Ammonia Ethyl acetate: Acetic acid: Water Ethyl acetate: Methanol: Ethyl acetate: Methanol: Ammonia Acetic acid (100:1.5) (60:20:20) (80:15:5) (75:25:1) University of Ghana http://ugspace.ug.edu.gh 184 PYRIMETHAMINE Ethyl acetate: Methanol: Ammonia (80:15:5) Ethyl acetate: Acetic acid: Water (60:20:20) University of Ghana http://ugspace.ug.edu.gh 185 APPENDIX IV SAMPLES OF API HPLC CHROMATOGRAMS Sample s of ch romat og ra ms of si x rep licate inj ection s of a 0.7mg/mL test art esu n at e RS p repa ra t ion. 1st Injection 2nd Injection University of Ghana http://ugspace.ug.edu.gh 186 3rd Injection 4th Injection University of Ghana http://ugspace.ug.edu.gh 187 5th Injection 6th Injection University of Ghana http://ugspace.ug.edu.gh 188 Sample s of ch romat og ra ms of si x rep licate inj ection s of a 0.7mg/mL test dihydroa rt emisin in RS prepa rat ion. 1st Injection 2nd Injection University of Ghana http://ugspace.ug.edu.gh 189 3rd Injection 4 th Injection University of Ghana http://ugspace.ug.edu.gh 190 5th Injection 6th Injection University of Ghana http://ugspace.ug.edu.gh 191 Sample s of ch romat og ra ms of si x rep licate inj ection s of a 0.4mg/mL test quin in e RS preparat io n. 1st Injection 2nd Injection University of Ghana http://ugspace.ug.edu.gh 192 3rd Injection 4th Injection University of Ghana http://ugspace.ug.edu.gh 193 5th Injection 6th Injection University of Ghana http://ugspace.ug.edu.gh 194 Sample s o f ch ro mat ogra m s of si x rep licate i nj ectio n s of a 0.8mg/ mL and 0.04mg/ mL te st su lp h adoxin e and py rimet h amin e RS solut ion re sp ective ly 1st Injection 2 nd Injection University of Ghana http://ugspace.ug.edu.gh 195 3rd Injection 4th Injection University of Ghana http://ugspace.ug.edu.gh 196 5th Injection 6th Injection University of Ghana http://ugspace.ug.edu.gh 197 Sample s of ch ro mat og ra m s of si x rep licate i nj ection s of a 0.3mg/mL and 1.7mg/mL test art emet h er and lu me fan t rin e RS solut ion re sp ective ly. 1st Injection 2nd Injection University of Ghana http://ugspace.ug.edu.gh 198 3rd Injection 4th Injection University of Ghana http://ugspace.ug.edu.gh 199 5th Injection 6th Injection University of Ghana http://ugspace.ug.edu.gh