Quality Assessment of Artemether/Lumefantrine Tablets Sampled from Pharmacies in Accra, Using the MVHimagePCv8.exe Color Software

doi:10.4236/pp.2013.48081

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Pharmacology & Pharmacy, 2013, 4, 567-572

Published Online November 2013 (http://www.scirp.org/journal/pp)

http://dx.doi.org/10.4236/pp.2013.48081

Open Access PP

567

Quality Assessment of Artemether/Lumefantrine Tablets

Sampled from Pharmacies in Accra, Using the

MVHimagePCv8.exe Color Software

Ebenezer Adu Nyarko, Henry Nettey

Department of Pharmaceutics and Microbiology, School of Pharmacy, College of Health Sciences, University of Ghana, Legon,

Ghana.

Email: [email protected]

Received August 21st, 2013; revised September 25th, 2013; accepted October 5th, 2013

tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-

erly cited.

ABSTRACT

Background: Widespread resistance has been recorded with the use of mono-therapy in the management of malaria. In

2000, Ghana initiated the process of using Artemisinin-based combination therapy (ACT) following the World Health

Organization’s (WHO) recommendation. Globally and in Ghana, there stands a high risk of development of resistance

to the ACTs due to the act of counterfeiting or substandard drugs. In 2009, there was a report that fake Coartem, an

ACT had been found in Ghana by the Drug Quality and Information (DQI) Program; this is a serious national problem

that needs redress thus the need to conduct this study to check if there are any substandard or counterfeit Artemether/

Lumefantrine tablets on the Ghanaian market. Method: Using Representative sampling method, a total of nine different

brands or samples of artemether/lumefantrine tablets were sampled from nine different Pharmacies in Accra. The sam-

ples were analyzed using a validated MVHimagePCv8.exe colour software technology. Results: The International Con-

ference on Harmonization (ICH) and United States Pharmacopoeia (USP) recommend that for assay of tablets, the per-

centage concentration should fall within 80% - 120%. After the analysis, seven out of the nine samples passed the test

to varying degrees. Two samples (AL-S4 and AL-S6) however failed the test with AL-S4 recording artemether concen-

tration (126.07%) above and Lumefantrine concentration (78.38%) below the recommended figure while AL-S6’s

51.53% failed to meet the minimum allowable concentration for lumefantrine in a tablet. Conclusion: The results pre-

sented show that some Artemether/Lumefantrine tablets on the Ghanaian market still have issues with regards to quality

or level of active ingredients. There would therefore be the need for further studies to be conducted into these products

especially those that failed the test.

Keywords: Artemisinin-Based Combination Therapy; Resistance; Counterfeit or Substandard Drugs

1. Introduction

Malaria is a potentially fatal blood-borne disease caused

by a eukaryotic protoctist of the genus Plasmodium, a

parasite that is transmitted to human and animal hosts by

the female Anopheles mosquito.

There are four main species of the parasite (P. falci-

parum, vivax, ovale, and malariae) that are known to

affect man [1]. Malaria caused by P. falciparum is the

most deadly and prevalent (90% - 98%). It is the most

common in Africa, south of the Sahara, accounting in

large part for the extremely high mortality in this region

[2]. In Ghana, malaria has been reported to account for

about 33.45% of all outpatient attendances and 30.3% of

all hospital admissions [2].

Management of the disease in Ghana and other parts of

the continent has moved from single drug therapies like

chloroquine to the new artemisinin based combination

therapy (ACT) due to the issue of resistance to mono-

therapy. Fixed-combination and multiple-drug therapies

are used to exploit the synergistic and additive potential

of individual drugs. The aim is to improve efficacy and

to retard the development of resistance to the individual

components of the combination.

In Africa, countries which are currently deploying

Quality Assessment of Artemether/Lumefantrine Tablets Sampled from Pharmacies in Accra,

Using the MVHimagePCv8.exe Color Software

568

ACTs in the general health services include: Burundi,

Comoros, Ethiopia, Ghana, Liberia, Mozambique, Sao

Tome and Principe, Sierra Leone, South Africa, Sudan,

Zambia and Zanzibar. The first and second lines of treat-

ment are artesunate/Amodiaquine, Artemether/Lumefan-

trine and rarely artesunate/sulfadoxine-pyrimethamine

depending on the cost and the country in question [3]. In

Ghana, the first line of treatment is Artesunate/Amodia-

quine (AA) but due to widely reported cases of unbear-

able side effects, most prescribers and patients as such

are now opting for the relatively expensive Artemether/

Lumefantrine (AL). In view of this, the Ghanaian market

is dominated by this product (AL) with many generics

available [4].

In Ghana and other parts of Africa, there stands a high

risk of development of resistance to the ACTs due to the

act of non-compliance, counterfeiting or substandard drugs.

The global nature of counterfeiting has since 1982 been

documented by the World Health Organization [5]. For

example, study on the quality of antimalarial drugs sup-

plied by Nigerian pharmacies was conducted using vali-

dated HPLC methods against British Pharmacopoeia (BP)

specifications. Overall 48% (279/581) of drugs analyzed

failed to comply with BP specifications [6]. In 2009, there

was a report that fake Coartem had been found in Ghana

by the Drug Quality and Information (DQI) Program.

These reports and many others pose serious health

threats to the nation and therefore need the attention and

vigilance of every individual.

This study was therefore aimed at assessing the quality

of the various brands of Artemether/lumefantrine prod-

ucts available at Pharmacies in Accra using very simple

analytical tools easily accessible to all. In a quest to de-

termine the possible presence of counterfeit or substan-

dard products on the Ghanaian market, the simplicity of

the method can be adopted by all pharmacies or indi-

viduals and used as a first-line screening method to weed

out poor quality drugs.

2. Materials and Methods

There are many experimental methods available in lit-

erature for the assay of artemether/lumefantrine tablets.

High Performance Liquid Chromatography (HPLC) ana-

lysis has been widely used in such analysis due to its sen-

sitivity, accuracy and precision. However for rapid ana-

lysis of such tablets, simple, effective, and validated me-

thods like the one used in this work can be employed.

Sampling: A representative sampling procedure was

employed. In this technique, a list of all the samples

known randomly to be still available on the Ghanaian

market was made. Representative samples of each ge-

neric/brand were obtained from different pharmacies

within Accra. In all, a total of nine different samples of

artemether/lumefantrine products were obtained from

nine different pharmacies in Accra.

3. Assay

Preparation of artemether standard: Exactly 60 mg of

pure artemether and 360 mg of pure lumefantrine (Table

1) were weighed, put together and thoroughly mixed. A

quantity of the mixture containing 24 mg of artemether

was weighed and dissolved in 2 ml of methanol to make

a 12 mg/ml solution. Portions of this solution were taken

and serially diluted to obtain 10 mg/ml and 8 mg/ml stan-

dard artemether solutions. The colorimetric method of

drug analysis was validated by Green et al. [7].

Preparation of samples for assay of artemether: Six (6)

tablets of each artemether/lumefantrine sample were

weighed and the average weight determined. The tablets

were crushed with a pestle in a mortar into powder. An

amount of the powder containing 20 mg of artemether

was weighed into each of 3 separate test tubes and la-

beled A1, A2, and A3. Exactly 2 ml of methanol was

added to each test tube to make a 10 mg/ml concentra-

tion.

Assay of artemether content in samples: Exactly 0.2 ml

of each of the samples labeled A1, A2 and A3 was pipet-

ted into 3 separate wells of a 48-well plate taking note of

the sample placed in each well. Then 0.2 ml triplicates of

each concentration of the standards prepared were also

placed in their respective wells. 0.2 ml of 85% O-phos-

phoric acid was added to the samples and standards in

the wells and shaken gently and carefully on the bench to

ensure complete mixing. The mixture was allowed to

stand on the bench for 4 - 5 hours.

The measurement of color intensity through image

analysis was performed as outlined by Green et al. [8].

Briefly, the 48-well plate was placed on a battery-oper-

ated light box (Visual Plus SV-650, Taiwan). A plastic

hood with apertures in the upper and lower ends was

placed over the light box. A digital camera was placed

over the upper orifice of the hood and set for “macro”

and “no flash”. This setup gave a consistent background

illumination for each plate analyzed. The picture re-

corded was transferred to a computer for digital image

analysis using MVHimagePCv8.exe color software. The

picture was then analyzed with the red vs. blue pixels

Table 1. Properties of drug standards.

Standard Batch

number

Man.

date

Exp.

date

Percentage

purity (%)

Artemether 20111125 Nov.

2011

Nov.

2014 95.3

Lumefantrine L20100804Aug.

2010

Aug.

2013 98.6

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Quality Assessment of Artemether/Lumefantrine Tablets Sampled from Pharmacies in Accra,

Using the MVHimagePCv8.exe Color Software

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569

of the MVHimagePCv8.exe color software. A measure-

ment of this pixel intensity as a function of artemether

concentration gave the most sensitive concentration

curve.

0.05 ml triplicate of each concentration of the standards

prepared were also placed in their respective wells. A 1

ml aliquot of 1.1 M acetic acid was added to the mixture

and shaken gently to ensure complete mixing of the

mixture. Exactly 0.1 ml of Congo red was added to the

mixture in the wells. A picture of the resulting color was

taken after 5 minutes and analyzed with red pixels of the

MVHimagePCv8.exe color software.

Preparation of lumefantrine standard: From the same

mixture prepared earlier, an amount containing 48 mg of

lumefantrine was weighed and dissolved in 4 ml of ethyl

acetate to make a 12 mg/ml solution. Portions of this

solution were taken and serially diluted to obtain 10

mg/ml and 8 mg/ml standard lumefantrine solutions. 4. Results

Preparation of samples for lumefantrine analysis:

From the six tablets earlier crushed, an amount of the

powder containing 40 mg of lumefantrine was weighed

into each of 3 separate test tubes and labeled L1, L2, and

L3. Exactly 4 ml of ethyl acetate was added to each test

tube to make 10 mg/ml concentrations.

The percentage content of the samples was obtained

through calculations using the linear equation obtained

from the regression analysis of artemether and lumefan-

trine standards. Below are representative sample calcula-

tions for artemether and lumefantrine content of Coar-

tem™. The calibration curves for artemether and lume-

fantrine standards are shown in Figures 1 and 2 respec-

tively. The lower and upper concentration limits (80%

and 120% respectively) used in the standard preparation

are those set by the United States Pharmacopeia [9].

Assay of lumefantrine content in samples: Exactly 0.5

ml of methanol was pipetted into the wells of 24-well

plates. Exactly 0.05 ml of each of the samples labeled L1,

L2 and L3 was pipetted into their respective wells of the

plate, taking note of the sample placed in each well. Then

y=0.534x‐ 9.530

R²=0.927

020406080100 120 140

%CONCENTRATIONS

Figure 1. Graph of red pixels against standard artemether concentrations (%).

y=0.36x+28.28

R²=0.999

020406080100 120 140

%CONCENTRATIONS

Figure 2. Graph of red pixels against standard lumefantrine concentrations (%).

Quality Assessment of Artemether/Lumefantrine Tablets Sampled from Pharmacies in Accra,

Using the MVHimagePCv8.exe Color Software

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Artemether content in Coartem

Standard curve equation Artemether standard… Y =

0.5346X − 9.5306 (Y: pixels, X: %conc.).

Average of pixels for artemether in Coartem (Y) =

49.9.

Therefore the percentage concentration of artemether

in Coartem (X) = (49.9 + 9.5306)/0.5346 = 111.168%.

Lumefantrine content in Coartem

Standard curve equation for Lumefantrine standard…

Y = 0.36X + 28.283 (Y: pixels, X: % conc.).

Average of pixels for Lumefantrine in Coartem (Y) =

58.87.

Therefore the percentage concentration of Lumefan-

trine in Coartem (X) = (58.87 − 28.283)/0.36 = 84.964%.

In all, a total of 9 samples obtained from Pharmacies

in Accra were analyzed (Tables 2 and 3). The results

obtained showed that, two samples; AL-S4 and AL-S6

had their Artemether and Lumefantrine contents respec-

tively falling outside specification (Table 4).

AL-S4 recorded the highest artemether concentration

of 126.07% (above normal range) while AL-S1 had the

lowest but acceptable artemether concentration of 93.40%.

None of samples recorded the desired 100% content of

Table 2. Sample content of artemether and their pixel read-

ings.

Samples Red Pixels 1 Red Pixels 2 Red Pixels 3Average

AL-S1 39.6 41.2 46.1 40.40

AL-S2 38.8 44.0 44.2 42.33

AL-S3 41.4 39.5 44.8 41.90

AL-S4 59.9 55.8 57.9 57.87

AL-S5 61.9 50.0 51.6 54.50

AL-S6 45.2 50.3 53.8 49.77

AL-S7 43.0 52.8 53.8 49.87

AL-S8 39.4 49.9 49.9 49.90

AL-S9 35.7 40.8 45.8 40.77

Table 3. Sample content of Lumefantrine and their red

pixel readings.

Samples Red Pixels 1 Red Pixels 2 Red Pixels 3Average

AL-S1 55.9 62.1 56.5 58.17

AL-S2 56.3 57.5 57.9 57.23

AL-S3 63.5 63.8 64.7 64.70

AL-S4 52.9 53.1 63.5 56.50

AL-S5 58.2 59.9 59.3 59.13

AL-S6 47.6 46.8 46.1 46.83

AL-S7 58.6 65.9 66.5 63.67

AL-S8 60.4 57.5 58.7 58.87

AL-S9 60.4 64.6 62.1 62.37

Table 4. Percentage content of Artemether (Art) and Lume-

fantrine (Lum) in samples (n = 3). Per cent accuracy is de-

fined as the difference between the expected value and cal-

culated value divided by the expected value times one hun-

dred. Calculated amount of active ingredient (mg/tablet)

and per cent accuracy are shown here.

Samples Art Amt

(mg) %

Art

Accuracy

(Art)

Lum Amt

(mg) %

Lum

Accuracy

(Lum)

AL-S118.68 93.406.6 99.612 83.0116.99

AL-S219.40497.022.98 96.512 80.4219.58

AL-S319.24296.213.79 121.392 101.161.16

AL-S425.214 126.07 26.07 94.056

78.38 21.62

AL-S523.956119.7819.78 102.828 85.6914.31

AL-S622.184110.9210.92 61.836

51.53 48.47

AL-S722.222111.1111.11 117.948 98.291.71

AL-S822.234111.1711.17 101.94 84.9515.05

AL-S91.818 94.095.91 113.616 94.685.32

the artemether in the tablet; the closest was AL-S2 which

had 97.02%.

The trend observed in the case of lumefantrine content

in the samples was questionable. Seven out of the nine

samples had their contents within range with two prod-

ucts AL-S4 (78.38%) and AL-S6 (51.53%) falling below

the minimum acceptable concentration. With the excep-

tion of AL-S3 (101.16%), AL-S9 (94.67%) and AL-S7

(98.28%), all the other samples that passed the test, did

so with concentrations slightly above the minimum al-

lowable concentration of 80%.

Quantitative analysis using pixel measurements

showed the artemether content of all the samples ana-

lyzed to be within 15% of the expected values, except

AL-S4 and AL-S5 which showed greater deviation of

26.07% and 19.78% respectively (Table 4). Accuracy (%

deviation from expected value) for the lumefantrine con-

tent of the samples was much lower. Table 4 shows that

only 5 out of the 9 samples analyzed were within 15% of

the expected values. Four samples, AL-S1, AL-S2, AL-

S4 and AL-S6 showed deviations of 17%, 19.6%, 21.6%

and 45% respectively.

5. Discussion

Generally, counterfeit drugs are known to be drug prod-

ucts that have been criminally and intentionally misla-

beled with regards to their identity or source. Examples

include fake packaging, absence of active ingredients or

their presence at incongruous amounts [10]. On the other

hand, substandard drugs are genuine products that do not

meet the specified quality standards. Several factors

could contribute to products becoming substandard. The

most obvious cause is an ineffective quality operations

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571

system. Production plants with ineffective quality opera-

tions that do not operate according to Good Manufactur-

ing Practice (GMP) may end up manufacturing products

that would not meet specifications and thus considered

substandard. However it is important to state that a drug

or product may lose its quality after production if trans-

portation, storage and other post production protocols are

not effective or followed effectively.

For a product to pass quality test, analytical results

obtained in laboratory experiment or calculated from

experimental measurements are compared with stated

acceptance criteria in internationally accepted compendia

(e.g. USP). The acceptance criteria are mostly set to

make room for analytical errors, unavoidable variations

in manufacturing and compounding and for deterioration

to an extent considered acceptable under practical condi-

tions [9].

An official standard product shall be formulated with

the intent to provide 100% of the quantity of each active

ingredient stated on the label. Variations are however

possible and do occur no matter how carefully the proc-

essing was done. In view of this, the International Con-

ference of Harmonization (ICH) and the United States

Pharmacopoeia (USP) recommend that assay of a drug

substance or finished product shall range from 80% to

120% of the test concentration [11].

In reference to the above stated criteria, the analysis

employed in this study used the above mentioned range

to determine the acceptability or otherwise of both Arte-

mether and Lumefantrine in the tablet samples analyzed.

Thus any sample that had their percentage concentrations

falling within the range was said to be satisfactory. Sam-

ples that fell below or above the range were said to have

questionable states of quality.

AL-S4 failed artemether content, not because there

was not enough active drug ingredient, but because there

was more than the allowable amount. This could have

happened because of poor quality control measures at the

manufacturing facility, or a deliberate attempt to increase

the content of one ingredient (most likely the cheaper) to

make up for a lower amount of the second ingredient.

The lumefantrine content of AL-S6 is of grave concern.

Although it is difficult to ascertain the cause of the very

low lumefantrine content, drug degradation cannot be

used as a reason for this observation. The sample was

well within the expiry date and it is highly unlikely that

the active ingredient will degrade by almost 50%. It

could be attributed to poor manufacturing practice or a

deliberate attempt to decrease the amount of the Lume-

fantrine content.

Most of the samples analyzed showed a percent error

of 15% or less. Very few, however, showed very high

variability. A further modification of the method, for

example, use of a better solvent for both ingredients

might improve the product yield. The method used here

has been shown in other experiments in our lab (results

not shown here) to be reliable and robust.

6. Conclusions

The results showed a trend that looked like artemether

concentrations are being increased at the expense of

lumefantrine probably due to cost. However, this is an

unacceptable practice because the role of lumefantrine in

its right amount is important in this combination therapy

and cannot be compromised.

In addition to augmenting the effect of highly effective

artemether, lumefantrine also prevents the development

of resistance against the artemisinins, a group of antima-

larials that the world is trying hard to protect against re-

sistance to their use.

Malaria is a deadly disease that affects people of all

ages and it is a major cause of morbidity and mortality in

Ghana. Since its discovery many drugs have been used to

manage the disease with a lot of changes being made

over the years due to the development of resistance.

This problem has been largely attributed to non com-

pliance and the availability and use of substandard or

counterfeit products on the market resulting in treatment

failure and the subsequent development of resistance.

Methods for drug analysis have been confined to well

equip laboratories with expensive and sophisticated equip-

ments thus making it difficult to quickly and easily ana-

lyze and detect possible substandard drugs entering the

market.

With the development of such simple methods of drug

analysis, more drugs can be tested quickly and easily to

help do away with the substandard ones.

The MVHimagePCv8.ex colour technology for drug

analysis was used in this project to analyze Artemether/

Lumefantrine products obtained from Community Phar-

macies in Accra, Ghana. The results after the analysis

revealed that, two out of the 9 samples (AL-S4 and AL-

S6), failed the test. These results therefore show that

there may still be antimalarials on the market that are

substandard and thus need to be investigated and dealt

with.

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