RESEARCH
c
i
1,
i-m
population - are at risk of malaria. In 2013, about 198 mil- the population in Malawi lives in a region of high malaria
Chikowe et al. Malaria Journal  (2015) 14:127 
DOI 10.1186/s12936-015-0637-z(IPTP), long-lasting insecticide-treated nets (LLITNs),1Department of Chemistry, University of Ghana, Legon, Accra, Ghana
Full list of author information is available at the end of the articlelion cases were reported worldwide with an estimated
584,000 deaths. The WHO African Region recorded 90%
of these deaths, mostly among children under five years of
age [2]. Although, preventive and control measures intro-
duced since 2000 are yielding positive results, leading to
transmission. There is an estimated five million cases an-
nually; responsible for about 30% of the outpatients
treated at health facilities and about 40% of all hospitaliza-
tions of children under five years of age [3].
Malaria control strategic plans in Malawi comprise the
following four key interventions: prompt access to ACT,
intermittent preventive treatment during pregnancy
* Correspondence: dosafo@ug.edu.ghproximately 3.2 billion people - about half of the world’slargely by administration of sub-therapeutic doses derived from falsified and substandard medicines necessitates
regular monitoring of the quality of these medicines to avert any potential public health disaster. This study aimed
at determining the active pharmaceutical ingredient (API) content of anti-malarial medicines available in Malawi with
respect to the manufacturers’ label claim and pharmacopoeia specifications.
Methods: Samples of anti-malarial medicines (112) collected from both licensed and unlicensed markets throughout
Malawi were subjected to visual inspection of dosage form and packaging, and registration verification with the
regulatory body. Basic (colourimetric) tests were employed to establish the presence and identity of the requisite APIs.
Semi-quantitative thin layer chromatography (SQ-TLC) was employed as a quick assay for the verification of identity
and estimation of the API content while HPLC assays were used to quantify the APIs. The results were compared with
pharmacopoeia specifications and manufacturers’ label claims. For combination therapies, a sample was considered to
have failed if one or more of its component APIs did not meet pharmacopoeia specifications.
Results: There was 86.6% registration status and 100% compliance with visual inspection and basic tests confirming
the presence of requisite APIs. The identification test was confirmed by the SQ-TLC assay. API quantification by HPLC
assay however, showed that 88.4% (99/112) of the samples failed the quality tests due to the presence of either
insufficient or excessive API.
Conclusions: The results suggest the existence of substandard anti-malarial medicines in Malawi. The presence of both
excessive and insufficient artemisinin-based and non-artemisinin-based API, clearly points to poor adherence to GMP
and improper handling during storage or distribution. The country relies heavily on imported anti-malarial medicines
so there is an urgent need to carry out regular and thorough post-market surveillance of medicines to ensure better
quality health care delivery.
Keywords: Malawi, Anti-malarial medicines, Substandard, API, Post-marketing surveillance
Background
No other disease has killed more humans than malaria [1]
and it is still claiming millions of lives worldwide. Ap-
the reduction of mortality rates by 47% globally and 54%
in Africa, the disease continues to be a major public health
problem in most malaria-endemic countries [2]. As in
most of sub-Saharan Africa [2], one hundred percent ofPost-marketing surveillan
medicines used in Malaw
Ibrahim Chikowe1,2, Dorcas Osei-Safo1*, Jerry JEK Harrison
Abstract
Background: The growing concern over the extent of ant© 2015 Chikowe et al.; licensee BioMed Centr
Commons Attribution License (http://creativec
reproduction in any medium, provided the or
Dedication waiver (http://creativecommons.or
unless otherwise stated.Open Access
e of anti-malarial
Daniel Y Konadu1 and Ivan Addae-Mensah1
alarial medicine resistance in sub-Saharan Africa, drivenal. This is an Open Access article distributed under the terms of the Creative
ommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
iginal work is properly credited. The Creative Commons Public Domain
g/publicdomain/zero/1.0/) applies to the data made available in this article,
Chikowe et al. Malaria Journal  (2015) 14:127 Page 2 of 11and indoor spraying with residual insecticides [3]. Since
2007, artemether-lumefantrine has been adopted as the
first-line treatment for uncomplicated and unconfirmed
cases, after replacing sulphadoxine-pyrimethamine (SP),
which also replaced chloroquine in 1993 due to parasite
resistance. SP and other anti-malarial medicines, such as
quinine (QN), are still being used for special cases [2].
Plasmodium resistance to malaria treatment has sev-
eral devastating effects. It has led to an increase in mor-
bidity and mortality rate, parasite transmission, severity
of the pandemic and change in malaria distribution. As
a result, this has caused pressure on the economy due
to increase in cost of health services arising from preva-
lent treatment failures and deaths [4]. Thus, patients re-
sort to illegal medicines, exposing them to poor quality
medicines. Fake/spurious/substandard/degraded/coun-
terfeit medicines are mostly blamed for the escalation
of medicine resistance. For example, some pockets of
parasite resistance to artemisinin-based medicines have
been attributed to the sub-therapeutic doses derived
from falsified and substandard medicines [5]. Various
studies have reported the widespread circulation of poor
quality medicines in some parts of Asia and Africa. Most
of them have been shown to contain sub-therapeutic
amounts of the APIs or no API at all or even toxic
compounds [6-8].
Currently, the only hope for future malaria treatment
rests on artemisinin-based combination therapy (ACT).
However, with the high demand and cost of production
of these medicines, the poor regulatory systems that
exist in most endemic countries including Malawi allow
unscrupulous persons to easily infiltrate the weak chain
supply systems with both imported and/or domestic
poor quality medicines [9,10]. Therefore, it is imperative
to protect these medicines from any impending medi-
cine resistance through GMP, relentless combat against
the circulation of poor quality medicines and strict pa-
tient compliance to treatment regimen.
The WHO Expert Committee on Quality Assurance
of Medicines calls for routine quality control activities
to check this malpractice. Therefore, this study aimed
at evaluating the quality of the anti-malarial medicines
used in Malawi with respect to the active pharmaceut-
ical ingredient (API) content in both ACT and non-
ACT. To achieve this aim, the following specific objectives
were set: to find out the registration status of the anti-
malarial medicines available on the markets; to visually
inspect dosage forms and packaging using the guide-
lines outlined in the WHO pharmacopoeia and the
literature; to carry out a qualitative determination to es-
tablish the presence or otherwise of the APIs using the
authenticated rapid tests outlined in the WHO publica-
tions; to carry out a quantitative determination of the
API content.Methods
Sampling procedures
Samples were collected by the first author after seeking
permission from the regulatory boards; the Pharmacy,
Medicines and Poisons Board and the National Health
Sciences Research Committee (NHSRC) of Malawi. The
medicines were bought under the guise of a patient, but
in the case where pharmacy technicians refused to sell
without prescription or many brands of medicine were
being bought at once, it was explained that they were for
research purposes. In the situation where the investiga-
tor introduced himself as a researcher, he ensured that
he was sold the medicines from the open shelves to
avoid a tendency where vendors give out only authentic
goods to regulatory authorities or researchers.
The country was divided into four zones based on the
National Malaria Control Programme (NMCP) strategy
partitions designated as south west (1), south east (2),
central (3) and north (4) zones. Few districts from each
zone were selected based on malaria prevalence rates,
economic activities and geographical position (border
towns). A master list of pharmacies and private health
facilities in the districts of interest was compiled. The
pharmacies were considered based on proportionate
sampling, called probability proportionality to size (PPS),
i.e. more samples from districts with more pharmacies/
health facilities. The districts of Lilongwe, Mzuzu and
Blantyre were further divided into Enumeration areas
(EAs) as these had pharmacies in separate townships as
well, unlike the other districts that had few pharmacies/
private health facilities all clustered at one place. Each
township acted as an EA. The simple random sampling
(SRS) procedures were used to select the EAs because
the EAs were few per district. The random walk method
was used in the selection of pharmacies/health facilities
from the EAs while for the rest of the districts all
pharmacies were selected as there were few pharmacies.
Samples were collected between December and January
(within the rainy season of November to April) when
Malawi records peak malarial transmission due to abun-
dance of stagnant water points, which are favourable
breeding grounds for the mosquito vector. The anti-
malarial medicines were purchased from both licensed
and unlicensed markets such as private pharmacies and
hospitals, street vendors and shops. The samples were
purchased 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 were bought at one outlet. They
were labelled, recorded and kept in containers that pro-
tected them from extreme light, moisture, crushing,
heat and mechanical shock. For more details of the sam-
ples see Additional file 1. The sampling sites are shown
in Additional file 2.
Chikowe et al. Malaria Journal  (2015) 14:127 Page 3 of 11Reference standards
The Reference standards were purchased from the European
Directorate for the Quality of Medicines and Healthcare
(EDQM), France.
Medicine analysis
Registration verification and visual inspection
The samples were subjected to registration verification
with the medicine regulatory authority - the Pharmacy,
Medicines and Poisons Board of Malawi after the collec-
tion exercise. This was followed by visual inspection
with respect to technical regulatory information as out-
lined in the WHO International Pharmacopoeia [11,12].
Basic/Colourimetric tests
To determine if the samples contained the APIs claimed
by the manufacturers on the packaging materials, all of
them were subjected to colourimetric tests using the
suitable reactions and reagents outlined in the pharma-
copoeias and the literature [13-15].
Semi-quantitative thin layer chromatography (SQ-TLC) assay
SQ-TLC was employed as a quick assay for the verification
of identity and estimation of the API content in the medi-
cine samples according to published protocols [16,17].
HPLC assay
HPLC procedures suitable for determining the API in
each anti-malarial sample adopted from the pharmaco-
poeias and the literature [17-21] were employed. Calibra-
tion curves were prepared using varying concentrations of
the various Reference Standards (RS). The Area Under the
Curve (AUC) for each concentration was determined from
six replicates and an average AUC was obtained. This data
was used to generate calibration curves of a plot of aver-
age AUC against concentration (C) using Microsoft Excel
and the slope of the graph, intercept, correlation coeffi-
cient (r2) as well as equation of the straight line, AUC=
mC+ b were deduced and calculated. The quantities of
the APIs in the medicine samples then were calculated
from their corresponding calibration curves. Six replicates
were carried out for each API component and the mean
and standard deviations were calculated (see Additional
files 3 and 4).
Assay for artesunate in ATS/SP and ATS/SmP samples
The assay for ATS was adopted from Ranher et al. [18]
with a few modifications as follows: column measure-
ments: Discovery C-18 bonded, 5 μm, 25 cm x 4 mm;
mobile phase: 70: 30 v/v, 1% triethylamine (TEA) in
methanol: buffer (10 mM KH2PO4/ 85% H3PO4, pH =
2.5); retention time (average): 5.1 minutes; detection
wavelength: 216 nm; flow rate: 1.2 mL/min.; volume of
injection: 20 μL. For each ATS-containing sample, aquantity of the powdered dosage form equivalent to
10 mg of artesunate was weighed into a clean dry 10 mL
volumetric flask. 5 mL of the mobile phase 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.
Assay for artemether and lumefantrine in ATM/LUM samples
The assay protocol for ATM and LUM was derived from
modifications of a method developed by Arun and Smith
[19]. The adapted method is as follows: column mea-
surements: Hyperprep PEP 300A C4, 25 cm x 4.6 mm,
8 μm; mobile phase: 70: 30 v/v acetonitrile: 10 mM buf-
fer consisting of KH2PO4 mixed with 1 mL of triethyla-
mine per liter and pH changed to 2.5 using 85% H3PO4
mixture; retention time (average): ATM 2.5 minutes,
LUM 3.0 minutes; detection wavelength: 216 nm; flow
rate: 1.5 mL/min.; volume of injection: 20 μL. The
counter-ion modifying agent triethylamine was added to
obtain enhanced peak symmetry and minimize tailing.
Furthermore, due to the large difference in the ratio of
ATM to LUM (1:6), efforts were made to add a detect-
able amount of ATM without unnecessarily overloading
the column with high concentrations of LUM. Sample
solutions of the tablets were prepared by accurately
weighing 4 mg of the powdered dosage form weighed
into a clean dry beaker. 1 mL of acetic acid was added,
allowed to react for a few minutes after which 5 mL of
the mobile phase was added. The mixture was then
shaken for 15 minutes on an ultrasonic sonicator, fil-
tered into a 10 mL volumetric flask through a 0.45 μm
filter, and made up to the mark through the filter with
the mobile phase.
Assay for dihydroartemisinin in DHA/SP and DHA/Pp samples
A method outlined in the Ph. Int. [12] was modified as
follows: column measurements: Kramasil C8, 25 cm x
4.6 mm, 5 μm; mobile phase: 50:50 v/v, water: aceto-
nitrile; retention time (average): 5.2 minutes; detection
wavelength: 210 nm; flow rate: 1.5 mL/min.; volume of
injection: 10 μL. For each sample, a quantity of pow-
dered tablets equivalent to 10 mg of DHA was accur-
ately weighed into a clean dry beaker. This was extracted
four times with diethyl ether as SP and Pp are practically
insoluble in diethyl ether. This solution was evaporated
to dryness. The residue was re-dissolved in 5 mL of the
mobile phase, sonicated for 15 minutes, filtered through
a 0.45 μm filter into a 10 mL volumetric flask and made
up to the mark.
Assay for quinine in QN samples
QN was assayed according to a modified version in USP
24 [20]. Column measurements: Discovery C-18 bonded,
Chikowe et al. Malaria Journal  (2015) 14:127 Page 4 of 1125 cm x 4.0 mm, 5 μm; mobile phase: 80:16:2:2 v/v,
water: acetonitrile: methanesulfonic acid: TEA, pH 2.6;
retention time (average): 5.7 minutes; detection wave-
length: 235 nm; flow rate: 1.2 mL/min.; volume of injec-
tion: 20 μL. For the suspensions and mixtures, a certain
amount was sonicated for about 15 minutes and a quan-
tity equivalent to 5 mg was pipetted into a 10 ml volu-
metric flask. Methanol (8 ml) was added to the contents
of the flask and made up to the mark with the mobile
phase. Where necessary, the solution was also filtered.
For the injections, a 10 mL 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 8 mL of methanol
and the mobile phase was added to the 10 mL mark.
Assay for sulphadoxine/sulphamethoxypyridazine and
pyrimethamine
The experimental conditions employed in the analysis of
S, Sm and P samples, were modified after a WHO-
adopted monograph for inclusion into the Ph. Int. in 2010
[21]. Column measurements: Ascentis C-18, 15 cm x
4.60 mm, 5 μm; mobile phase: 65:10:25 v/v, 20 mM buffer
(KH2PO4/Na2HPO4 of pH 5.6; methanol; acetonitrile;
retention time (average): sulphadoxine/sulphamethoxypyr-
idazine 3.9 minutes, pyrimethamine 8.7 minutes; detection
wavelength: 240 nm; flow rate: 1 mL/min.; volume of in-
jection: 10 μL. Solutions of sulphadoxine/sulphamethoxy-
pyridazine and pyrimethamine containing tablets were
prepared as follows: a quantity of the powdered dosage
form equivalent to 100 mg of sulphadoxine/sulphamethox-
ypyridazine and 5 mg of pyrimethamine were weighed
simultaneously into a clean dry beaker. The APIs were ex-
tracted three times for completeness using acetonitrile and
finally made up to the mark with the mobile phase in a
50 mL volumetric flask.
Validation
Accuracy, precision, linearity and specificity parameters
were evaluated for all the various determinations. Accur-
acy of results of an analytical method can also be estab-
lished when validation parameters including precision
(RSD values), linearity (R2 values), accuracy (% recovery)
and specificity (retention times) were evaluated for all
the various determinations (n = 6).
Data interpretation
Poor quality medicines may be degraded, substandard or
counterfeit. According to the WHO, Spurious/Falsely-
Labelled/ Falsified/Counterfeit (SFFC) medicines (branded
or generic) can be classified as “any medicines or pharma-
ceutical products that are deliberately and fraudulently
mislabelled for identity and/or source” The definitionincludes products with correct or wrong ingredients, with-
out active ingredients, with insufficient active ingredients,
or with false packaging [22]. Substandard medicines, also
known as Out of Specification (OOS) products are those
that are genuine and legally produced but fall outside the
specifications or acceptance criteria established in product
dossiers, drug master files, pharmacopoeias or by the
manufacturer. A degraded medicine can be classified
along with substandard medicine. However, they differ in
that they might be originally of specification, but in the
course of time naturally or catalysed by external factors,
fall out of specification within its shelf-life [5].
In this study, a sample was considered to have failed
the quality evaluation if it did not meet any of the fol-
lowing criteria: 1) failure of visual inspection of dosage
form and packaging, 2) failure to produce the expected
colour reaction in the basic test and 3) failure to pro-
duce the expected spot colour or Rf on TLC compared
to the reference standard. With respect to API content,
a component API was classified as “compliant (C)” if
its quantity fell within the acceptable International
Pharmacopoeia limits of 90-110% of the amount of API
stated on the label claim; “non-compliant (NC)” if the
quantity was less than the lower (insufficient) or more
than upper (excessive) acceptable limits [11,12]. Thus an
ACT was considered compliant or to have passed the
API content test only when both or all of its component
APIs were compliant.
Results
Sample description
The anti-malarial samples analysed were 112, comprising
36 non-ACT, 4 ACT of single dose medicines co-packed on
the same blister to be taken concomitantly and 72 ACT of
fixed dose combinations. Artemether-lumefantrine (ATM/
LUM) tablets, being the main ACT and serving as the first-
line treatment for malaria in the country, formed the bulk
of samples (36.6%) while SP tablets represented 20.5%.
The formulations also included suspensions, injections
and mixtures containing other APIs such as quinine
(QN), piperaquine (Pp), sulphamethoxypyridazine (Sm),
artesunate (ATS) and dihydroartemisinin (DHA). A total
of 153 samples were collected, but the number analysed
was limited by available assays and reference standards
(RS). Table 1 shows a summary of the samples collected
and analysed.
Registration status of samples
The samples were subjected to registration verification
with the Pharmacy, Medicine and Poisons Board of
Malawi as soon as the collection exercise was completed.
As of 31st December, 2011, 86.6% (97/112) of the col-
lected samples as well as their formulation types were
registered with the regulatory board, according to their
annual registration publication. None of the samples was
manufactured locally; all of them were imported samples
with 60.7% manufactured in India and the rest originat-
ing from China, Kenya and Tanzania.
Visual inspection of dosage form and packaging
The visual inspection of the dosage forms and packaging
showed total (100%) compliance of the samples with the re-
quirements. Labelling information regarding dosage form,
brand name, active ingredient/strength, batch number,
manufacture and expiry dates were provided (see Add-
itional file 1). Tablets did not present with non-uniform col-
ouration or signs of breakage.
Basic tests
API content claims were confirmed by colourimetric
tests, which demonstrated that all the samples contained
the requisite APIs claimed by the manufacturers. How-
ever, it has been reported that with the advancement of
counterfeiting, visual inspection and basic tests alone
cannot qualify a medicine as genuine despite chemical
and physical similarities. According to Bate et al., the de-
termination of a medicine as counterfeit or substandard
requires a forensic examination of the trademarks; prod-
uct designs and holograms [23], but the present study
did not go as far as that.
HPLC assay
A major challenge in the simultaneous assay of ATM/LUM
tablets was the choice of a solvent that would not interfere
with the analyte peaks, dissolve both APIs long enough for
the analysis to be carried out and also give well-resolved
peaks. This was overcome by using acetic acid followed
Table 1 Categories of collected anti-malarial medicine
samples
Non-ACT ACT co-packed
on one blister
Fixed dose ACT
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.
Chikowe et al. Malaria Journal  (2015) 14:127 Page 5 of 11Figure 1 Chromatogram of a 0.3 mg/mL and 1.7 mg/mL of ATM and LUM RS solutions.
Chikowe et al. Malaria Journal  (2015) 14:127 Page 6 of 11by acetonitrile to extract the active ingredients. From
the chromatograms, acetic acid eluted first and did not
interfere with the analyte peaks. Acetonitrile, with its
low cut-off wavelength also did not interfere. The
Figure 2 Chromatogram for ATM/LUM tablet.addition of the modifying agent triethylamine greatly
enhanced peak symmetry and minimized tailing. A
small shoulder appeared in the LUM peak and was ob-
served in both the RS and the samples. Its presence did
not hinder the computation of the AUC and was
attributed to the column type, the mobile phase com-
position or both (Figures 1 and 2). Another challenge
encountered was the difficulty in detecting artemether
due to its low molar absorptivity and its low concentra-
tion (16.7%) in the fixed dose combination. Thus in the
preparation of the calibration curves, different LUM
Figure 3 Calibration curve for lumefantrine.concentrations were tried to obtain a range that would
allow for the suitable detection of ATM and at the same
time, not correspond to too high concentrations of
LUM. The curves obtained were linear with R2 values of
0.994 for ATM and 0.993 for LUM (Figures 3 and 4).
The assay of SP samples was also problematic with re-
spect to the high ratio of sulphadoxine to pyrimeth-
amine, 20:1 respectively. Suitably high concentrations of
sulfadoxine API were used to aid in the detection of
pyrimethamine. The calibration curves obtained were
linear with an R2 value of 0.999 and 0.998 for sulfadox-
ine and pyrimethamine respectively (Table 2). Details
and sample chromatograms of all samples assayed to-
gether with their corresponding calibration curves are
presented in Additional files 3 and 4.
Chikowe et al. Malaria Journal  (2015) 14:127 Page 7 of 11Validation
The accuracy of results of an analytical method can be
established when validation parameters such as preci-
sion, linearity and specificity are clearly demonstrated. A
summary of the method validation results is presented
in Table 2, with details in Additional file 4. The average
RSD values are ≤ 2%. The linearity values (R2) are also
above 0.95 demonstrating a very good correlation be-
tween the peak area (AUC) and the concentration of
the analyte APIs, linear across the 80-120% RS concen-
trations. The retention times of the analyte in the
sample and the RS are also comparable, demonstrating
high specificity.
Quality of anti-malarial medicines
Although all the samples passed the visual inspection
and qualitative determination tests, the HPLC assay re-
vealed that 88.4% (99/112) did not meet the require-
Figure 4 Calibration curve for artemether.ments for API content (Table 3). The main cause of
the failure was either the presence of insufficient API
(i.e. < 90%) or excessive API (>110%). See Additional
file 3 for detailed results.
The 112 anti-malarial samples consisted of 9 APIs in
various combinations with the exception of QN, which
occurred as a monotherapy. Pp could not be assayed due
Table 2 Results of analytical method validation
Validation parameter ATM ATS DHA
Precision (RSD) 0.836023149 0.088086277 1.1653302
Linearity(R2) 0.9940 0.9941 0.9950
Slope 176 502 410.1
Intercept 74.28 52.65 31.35
Specificity (Retention time)
Medicine Sample API 2.528 5.081 5.225
Reference Standard 2.548 5.091 5.225to unavailability of a reference standard. Out of the 4
ATS/SP samples, ATS was compliant in all, P was com-
pliant in 2 samples while S was compliant in none. The
total noncompliance of the S component thus resulted
in a 100% failure of all the ATS/SP samples.
ATS was compliant in only 2 out of the 5 ATS/SmP
samples while Sm and P were compliant in 1 and 2 sam-
ples respectively. Regardless of having at least one case
of compliance for each constituent API, this did not
occur in the same sample. Hence, there was not a single
sample in which all the individual APIs were compliant,
resulting in 100% failure. In the 23 SP samples, compli-
ance for S and P was in 3 and 12 samples respectively.
Compliance for both constituents occurred together in
only 2 samples resulting in 91.3% failure rate. There
were 12 DHA/SP samples and although API content test
for P was 100% compliant, 11 samples failed the S API
content test while DHA was 100% noncompliant. Hence,
no DHA/SP sample passed the quality test. Since Pp API
in DHA/Pp could not be assayed, based on DHA alone,
4 out of the 14 samples were compliant (71.4% failure
rate). In the case of the 41 ATM/LUM samples, only 2
samples had both APIs being compliant in the same
sample. Overall, ATM was compliant in 14 samples
while LUM was compliant in 11 samples. The failure
LUM QN S P
78 0.896337033 2.00605798 1.77262931 0.0030604
0.9934 0.9979 0.9993 0.9975
6320 20180 16929 16600
17985 162.9 4894 948.09
2.992 6.113 3.928 8.689
3.035 6.113 3.928 8.689
rate of the samples was 91.5%. In the 13 QN monother-
apy, 61.5% of the samples failed the quality test.
Failure rate of anti-malarial medicines versus registration status
Comparison of the failure rate of registered and unregis-
tered samples suggested that registration status did not
have significant influence on the quality of the anti-malarial
medicines. Out of the 97 registered samples, 15.5% (15) of
Samples with the same batch number
API content of samples within same batches was com-
pared to ascertain their uniformity. Some of the batch
numbers were unregistered while others were duly reg-
istered. The results (Table 4) revealed wide variations
in API content within batches, regardless of registra-
tion status.
Reporting of results
The findings of the study have not yet been made avail-
able to any of the regulatory bodies - the Pharmacy,
Medicines and Poisons Board, the National Health
Sciences Research Committee (NHSRC) of Malawi or
the WHO Rapid Alert System.
Discussion
The study showed a good correlation between both vis-
ual inspection of dosage form and packaging material on
one hand and qualitative determination (basic tests) of
API on the other because the labelled contents on the
packaging materials were found to be correct. Thus,
none of the samples can be considered as falsified with
Figure 5 A comparison of failure rates for registered and
unregistered samples.
fai
nt
Chikowe et al. Malaria Journal  (2015) 14:127 Page 8 of 11the samples were compliant, while 6.7% (1) of the 15 un-
registered samples were compliant. Although the registered
samples had a slight edge over the unregistered ones, the
overall results (Figure 5) show that 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 [16].
Table 3 Level of compliance of individual API content and
Anti-malarial
sample
Number of
samples
Level of compliance of API content
API compliant noncomplia
ATS/SP 4 ATS 4 0
S 0 4
P 2 2ATS/SmP 5 ATS 2 3
Sm 1 4
P 2 3
ATM/LUM 41 ATM 14 27
LUM 11 30
DHA/Pp 14 DHA 4 10
Pp - -
DHA/SP 12 DHA 0 12
S 1 11
P 12 0
SP 23 S 3 20
P 12 11
QN 13 QN 5 8
Total 112 73 145respect to visual inspection and qualitative determin-
ation. However, the quantitative assay revealed noncom-
pliance of API content in a significant majority of the
samples. There were 145 instances of the presence of a
noncompliant API in the various categories of the anti-
malarial medicines against 73 cases of compliance. In
the combination formulations, more often than not,
ling rates of anti-malarial samples
Remarks Number and rate
of failing samples
All the 4 samples were noncompliant 4 (100%)
Compliant APIs did not occur in the
same samples therefore all the samples
were noncompliant
5 (100%)
Only 2 samples had both APIs being
compliant in the same sample.
39 (95.1%)
Pp API could not be assayed. Based on DHA
alone, 4 out of 14 samples were compliant
10 (71.4%)
All the samples were noncompliant even
though P was 100% compliant
12 (100%)
Only 2 samples had both APIs being
compliant in the same sample
21 (91.3%)5 out 13 samples were compliant 8 (61.5%)
99 (88.4%)
Table 4 Samples with the same batch number and their respective API content
Anti-malarial sample showing API
and strength
Batch No. Samples with the same batch number and their respective API content with HPLC assay
SDX/PYR S-60 14P10* 16P10* 11P10* 31P10* 32P10* 33P10*
500/25 mg 425/28 mg 415/23 mg 390/23 mg 415/21 mg 405/24 mg 410/22 mg
SDX/PYR 1001 11P2 14P2 21P2 35P2
500/25 mg 1 435/29 mg 410/23 mg 385/22 mg 265/12 mg
SDX/PYR 1000 31P2 44P2 12P2
500/25 mg 8 485/24 mg 426/26 mg 335/19 mg
QUN Bi-SO4 OK 12 V5 4 V5
50 mg/5 ml 159 60 mg 56 mg
ATM/LUM LD- 112X1 15X1 17X1
80/480 mg 266 81/492 mg 54/532 mg 65/283 mg
ATM/LUM LD- 19X1 113X1 110X1 22X1 44X1
80/480 mg 259 83/545 mg 112/528 mg 99/302 mg 105/302 mg 114/499 mg
ATM/LUM LF- 14X1 16X1 33X1 43X1
40/240 mg 249 28/276 mg 65/305 mg 32/259 mg 70/196 mg
ATM/LUM LN- 18X1 21X1
20/120 mg 450 33/95 mg 19/136 mg
ATM/LUM LS- 12X1 31X1 32X1
20/120 mg 39 24/136 mg 18/140 mg 19/140 mg
ATM/LUM SB01 12X11 11X11
180/1080 mg 00I 186/1328 mg 167/1372 mg
ATM/LUM C052 31X11 13X11
80/480 mg 0 J 31/569 mg 32/408 mg
ATM/LUM 1105 12X14 11X14 24X14
20/120 mg 062 7/114 mg 15/83 mg 11/141 mg
DHA/Pp PX- 17Z1 16Z1 26Z1
40/320 mg 161 34/- 39/- 41/-
DHA/SDX/PYR AP-18 15Z1 13Z1 12Z1 25Z1 21Z1 34Z1 45Z1
60/500/25 mg 51/428/24 mg 34/430/23 mg 32/414/
25 mg
31/434/
23 mg
34/385/
26 mg
31/427/
25 mg
31/438/
26 mg
DHA/Pp 1101 14Z3* 12Z3* 11Z3* 33Z3* 44Z3* 43Z3* 41Z3*
40/320 mg 23 28 mg/- 34 mg/- 39 mg/- 28 mg/- 35 mg/- 28 mg/- 30 mg/-
DHA/SDX/PYR AP- 24Z1 23Z1 35Z1
16 32/400/24 mg 33/450/25 mg 31/425/
25 mg
60/500/25 mg
SDX/PYR 1001 22P2 23P2 32P2 42P2 45P2
500/25 mg 2 415/16 mg 480/31 mg 425/24 mg 235/13 mg 426/27 mg
SDX/PYR 0213 21P15 37P15 38P15
500/25 mg 50 410/23 mg 355/20 mg 459/26 mg
ATM/LUM LD- 34X1 36X1
80/480 mg 227 82/536 mg 84/496 mg
ATS/SMP/PYR 081 31Y12 41Y12 42Y12
200/500/25 mg 178/217/
14 mg
174/440/
25 mg
186/443/
29 mg
ATS/SDX/PYR TR0458 32Y13* 34Y13*
Chikowe et al. Malaria Journal  (2015) 14:127 Page 9 of 11
pe
37
6
m
4
m
12
22
Chikowe et al. Malaria Journal  (2015) 14:127 Page 10 of 11compliant APIs did not occur together in the same sam-
ple, resulting in the observed high failure rate in the
quality evaluation (88.4%). Although the presence of in-
sufficient API was the main cause of failing samples,
there were cases of the presence of excessive API. In two
previous studies on anti-malarial samples distributed in
Ghana, where low quantities of API was also identified
as a major contributory factor in failing rates, the
artemisinin-based components of ACT were the insuffi-
cient APIs. Thus it was deduced that manufacturers
could deliberately or otherwise be reducing quantities of
the more expensive API as a means of cutting down on
production cost. However, in the current quality evaluation,
the artemisinin-based components have been detected in
excessive quantities as well. This observation suggests poor
adherence to SOPs, GMPs and proper registration proce-
dures and is corroborated by the wide differences in API
quantities of samples within batches (Table 4). In either
case, there is the danger of sub-therapeutic doses of the
noncompliant component promoting resistance or in the
case where this component is present in excessive doses,
posing a risk of toxicity to patients.
Although the registration status of anti-malarials used
in Malawi was found to be quite satisfactory (13.4% un-
registered), compared to countries such as Ghana and
Togo where most recent studies indicated 55% and 78%
unregistered anti-malarials in Ghana in 2008 and 2012
[16,17] respectively and 17% unregistered anti-malarials
Table 4 Samples with the same batch number and their res
100/500/25 mg 98/340/20 mg 98/
QUN di-HCl L-491 31Q6 33Q
150 mg/5 ml 166 mg 183
QUN HCl 110433 42R4 43R
100 mg/5 ml 151 mg 156
ATS/SMP/PYR 079 43Y12 44Y
100/250/12.5 mg 78/218/14 mg 90/
* Unregistered samples.in Togo in 2012 [17], it has been established that regis-
tration of a medicine with the national regulatory au-
thority does not necessarily guarantee its quality. ATM/
LUM, SP and QN are the most commonly used medi-
cines against malaria in Malawi, a country burdened
with high transmission rate of malaria. Hence, the failure
rate of these important medicines - ATM/LUM (95.1%),
SP (91.3%) and QN (61.5%) - is alarming considering
the fact most of the malaria cases in Malawi are diag-
nosed without microscopic determination. Most types of
fever are presumed to be malaria first, and treated as
such. If indeed, such ad hoc diagnoses are also treated
with substandard anti-malarials, this could lead to treat-
ment failure and/or fast development of resistance. Thisinference is based on the report by Bate et al. that resist-
ance development of chloroquine and sulphadoxine in
Africa in the 1990s and the devastating impact of
malaria on the people were partly due to the use of sub-
standard medicines [23].
Comparison of current results with recent results from
other African countries
Recent surveys on the quality of medicines circulating in
many African countries have shown similar trends of
poor quality anti-malarial medicines [7,16,17,24]. Most
regulated manufacturers bypassed their GMP compli-
ance and set the standards of their medicine 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 [25]. Malawi, being a devel-
oping country and one of the poorest for that matter, is
bound to suffer from poor regulatory capability and lack
of expertise in routine rigorous medicine testing, consid-
ering the heavy reliance on imported anti-malarials.
Most international surveys have rarely included samples
from Malawi and efforts to locate any such internal ac-
tivity at the required level have so far proved futile.
Conclusions
The findings of the study suggest a widespread use of sub-
standard anti-malarial medicines throughout the country
ctive API content (Continued)
0/18 mg
41Q6 42Q6
g 153 mg 84 mg
g
5/15 mgwith respect to API content. The detection of indiscrimin-
ate cases of excessive as well as insufficient API in both
artemisinin-based and non-artemisinin-based components
can be attributed to improper GMP and lack of quality
control in the distribution chain. Therefore, there is an ur-
gent need for regular rigorous testing by the National
Medicines Regulatory Authority to deter importers from
flooding the markets with poor quality medicines. Despite
the effort put in place by the Government and its partners
to minimize the impact of malaria over the years, the dis-
ease remains the country’s biggest health challenge. The
post-marketing surveillance and pharmacovigilance sys-
tem has been under development since 2009 and yet, it is
still faced with severe limitations.
8. Maponga C, Ondari C. The quality of antimalarials; a study in selected
Chikowe et al. Malaria Journal  (2015) 14:127 Page 11 of 11Additional files
Additional file 1: List and details of anti-malarial medicines
purchased.
Additional file 2: Figure S1. Map of Malawi showing sampling sites.
Additional file 3: Results of HPLC assay for API content in
anti-malarial medicines.
Additional file 4: Detailed analytical results.
Abbreviations
API: Active pharmaceutical ingredients; ACT: Artemisinin-based combination
therapy; ATM: Artemether; ATS: Artesunate; C: Compliant;
DHA: Dihydroartemisinin; EA: Enumeration area; EDQM: European Directorate
for the Quality of Medicines and Healthcare; FDC: Fixed dose combination
therapy; GMP: Good manufacturing practice; HPLC: High pressure liquid
chromatography; LUM: Lumefantrine; NC: Non-compliant; NMCP: National
Malaria Control Programme; P: Pyrimethamine; Ph. Int.: International
pharmacopoeia; Pp: Piperaquine; PPS: Probability proportionality to size;
QN: Quinine; RS: Reference standard; S: Sulphadoxine;
SM: Sulphamethoxypyridazine; SOP: Standard operating procedure;
SP: Sulphadoxine-pyrimethamine; SQ-TLC: Semi-quantitative thin layer
chromatography; SRS: Simple random sampling; WHO: World Health
Organization..
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
IAM conceived the study and designed the experiment. IC carried out the
sampling and wrote the first draft. IC and DYK performed the experiments.
IAM, DOS and JJEK supervised the study. DOS produced the final manuscript.
All authors read and approved the final manuscript.
Acknowledgements
The study was carried out in the laboratories of the Department of
Chemistry, University of Ghana Legon. Funding was received from the
Department for International Development UK (DFID)/Wellcome Trust
through the National Commission for Science and Technology (NCST)
Malawi under the Health Research Capacity Strengthening Initiative (HRCSI).
We would like to thank Mr. Wilford Mathiya of the Pharmacy, Medicines and
Poisons Board (Malawi), Mr. Rage Majamanda of the National Health Sciences
Research Committee (NHSRC) (Malawi) and Dr Mathildah Chithila, Dr Happy
Phiri, Mr Andrew Mpesi and Mr George Ng’ambi of the HRCSI Secretariat
(Malawi) for their prompt assistance(s) during the sample collection exercise.
Author details
1Department of Chemistry, University of Ghana, Legon, Accra, Ghana.
2Ministry of Education, Science and Technology, Lilongwe, Malawi.
Received: 31 October 2014 Accepted: 3 March 2015
References
1. Butler AR, Khan S, Ferguson E. A brief history of malaria chemotherapy. J R
Coll Physicians Edinb. 2010;40:172–7.
2. WHO. World Malaria Report 2014. Geneva, Switzerland: World Health
Organization; 2014.
3. Programme NMC. NMCP: Malaria strategic plan 2011–2015; towards
universal access. Lilongwe, Malawi: Ministry of Health; 2011.
4. Ettling M, McFarland DA, Schultz LJ, Chitsulo L. Economic impact of malaria
in Malawian households. Trop Med Parasitol. 1994;45:74–9.
5. Dondorp AM, Yeung S, White L, Nguon C, Day NPJ, Socheat D, et al.
Artemisinin resistance: current status and scenarios for containment.
Nat Rev Microbiol. 2010;8:272–80.
6. Newton PN, White NJ, Rozendaal JA, Green MD. Murder by fake medicines.
BMJ. 2002;324:800–1.7. Nayyar GML, Breman JG, Newton PN, Herrington J. Poor-quality antimalarial,
medicines in southeast Asia and sub-Saharan Africa. Lancet Infect Dis.
2012;12:488–96.African countries. World Health Organization (WHO) Department of Essential
Medicines and Medicines. WHO/EDM/PAR/2003.4; 2003 [http://apps.who.int/
medicinedocs/pdf/s4901e/s4901e.pdf]
9. Staedke S: Medicine safety and quality. London School of Hygiene and
Tropical Medicine; Uganda Malaria Surveillance Project. ACT Consortium;
2009. [http://www.actconsortium.org/data/files/
actc_safety_and_quality__overview.pdf]
10. Strengthening Pharmaceutical Systems (SPS) Program: Safety of medicines in
sub-Saharan Africa: assessment of pharmacovigilance systems and their
performance. US Agency for International Development by the Strengthening
Pharmaceutical Systems (SPS) Program. Arlington, VA: Management Sciences
for Health; 2011. [http://apps.who.int/medicinedocs/en/d/Js19152en/]
11. World Health Organization: The International pharmacopoeia (including first,
second and third supplements). 4th Edition, Version 2; Online, Geneva; 2013.
[http://apps.who.int/phint/en/p/about/]
12. World Health Organization. The International pharmacopoeia 4th Edition,
Version 2; CD-ROM, Geneva. Antwerp, Belgium: Human Info NGO/WIT; 2006.
13. World Health Organization, BTD. Basic tests for medicines: pharmaceutical
substances, medicinal plant materials and dosage form; 1998. [http://
whqlibdoc.who.int/publications/1998/9241545135.pdf]
14. World Health Organization. New basic tests for antimalarials. CH-1211
Geneva 27, Switzerland: Quality Assurance and Safety: Medicines (QSM),
Department of Essential Medicines and Medicines Policy (EDM), World
Health Organization Working Document QAS/06.159; 2006.
15. WHO: Basic tests for pharmaceutical dosage forms. Geneva; World Health
Organization, 1991. [http://apps.who.int/medicinedocs/pdf/h1794e/h1794e.pdf]
16. Osei-Safo D, Harrison JJEK, Addae-Mensah I: Validation and application of
quality assurance methods developed for artemisinin-based antimalarial
medicines to assess the quality of a selection of such medicines distributed
in Accra, Ghana. African Journal of Pharmaceutical Sciences and Pharmacy
2010, 1: 1–25. Retrieved from [http://www.ajpspjournal.com/article/view/
6206]
17. Osei-Safo D, Agbonon A, Konadu DY, Harrison JJEK, Edoh M, Gordon A,
Gbeassor M, Addae-Mensah I: Evaluation of the quality of artemisinin-based
antimalarial medicines distributed in Ghana and Togo. Malar Res Treat 2014,
Article ID 806416, 12 pages. [http://www.hindawi.com/journals/mrt/2014/
806416/]
18. Ranher SS, Gandhi SV, Kadukar SS, Ranjane PN. A validated HPLC method
for determination of artesunate in bulk and tablet formulation. J Anal Chem.
2010;65:507–10.
19. Arun A, Smith AA. Simultaneous HPLC-UV method for the estimation of
artemether and lumefantrine in tablet dosage form. Int J Pharma Biomed
Res. 2011;2:201–5. ISSN No.: 0976–0350.
20. The United States Pharmacopoeia USP NF 24. Monographs of quinine
sulphate and quinine sulphate tablets. Rockville: United States
Pharmacopoeia Convention; 2006. p. 1458–60.
21. WHO expert committee. Sulphadoxine and pyrimethamine tablets. Adopted
text for addition to The International Pharmacopoeia. World Health
Organization Working document QAS/07.218/FINAL; 2011. [http://www.who.
int/medicines/publications/pharmacopoeia/Sulfadox-Pyrimeth-tab-QAS07-
218FINAL.pdf]
22. World Health Organization. Counterfeit drugs. Guidelines for the
development of measures to combat counterfeit drugs. Geneva: World
Health Organization; 1999. WHO/EDM/QSM/99.1.
23. Bate R, Coticelli P, Tren R, Attaran A. Antimalarial medicine quality in the
most severely malarious parts of Africa – A six country study. PLoS One.
2008;3:e2132.
24. WHO QAMSA. Survey of the quality of selected antimalarial medicines
circulating in six countries of sub-Saharan Africa. Geneva: World Health
Organization Quality Assurance and Safety; Essential Medicines and
pharmaceutical Policies, WHO/EMP/QSM/2011.1; 2011. [http://www.who.int/
medicines/publications/WHO_QAMSA_report.pdf]
25. Caudron JM, Ford N, Henkens M, Macé C, Kiddle-Monroe R, Pinel J.
Substandard medicines in resource-poor settings: a problem that can no
longer be ignored. Trop Med Int Health. 2008;13:1062–72.