International Journal of Pharmaceutical Science and Health Care
Available online on http://www.rspublication.com/ijphc/index.html
Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
HPLC METHOD DEVELOPMENT AND VALIDATION FOR
SIMULTANEOUS ESTIMATION OF ANTIMALARIAL DRUGS
ARTEMETHER AND LUMEFANTRINE
SANJANA GAIKWAD1*, MADHUKAR TAJNE1, NARESH GAIKWAD1, ANWAR
DAUD2 ,DINESH LONARE3,MANISH LONARE4
1. University Department of Pharmaceutical Sciences, RTM Nagpur University, Nagpur (M.S.) India.
2.Zimz Laboratories, Kalmeshwar, Nagpur(M.S.),India
3.NEERI, Nagpur, India 4.Higher and Technical Education Department, Government of Maharashtra.
ABSTRACT
The present study describes the development and validation of a simultaneous HPLC-UV method for the estimation
of
antimalarial
drugs
Artemether
and
Lumefantrine
in
fixed-dose
combination.
A Simple and precise HPLC method was developed for the estimation of Artemether and Lumefantrine in pure and
pharmaceutical dosage forms. The quantification was carried out using symmetry C18, 100 x 3 mm, 1.9μm particle s
ize in gradient mode, with mobile phase compressing of following
Buffer: 5.65 gm of 1-Hexanesulphonic Acid and 2.75 gm sodium dihydrogen phosphate (Monohydrate) was
dissolved in 1000 mL of water, pH 2.3±0.05 was adjusted with diluted phosphoric acid.
Solution A: Filtered and degassed mixture of 245 mL of Buffer and 305 mL of Acetonitrile was prepared.
Solution B:
Filtered and degassed mixture of 135 mL of Buffer, 415 mL of Acetonitrile and 50 mL of 1–Propanol was prepared.
The percentage recovery was found to be 100.46
and
100.33
% for Artemether and Lumefantrine.
The method was validated by evaluation of different parameters. The method showed to be linear, precise, accurate,
specific and robust. Three batches of Artemether and Lumefantrine tablets were assayed by the validated method.
The Artemether contents in the tablets was satisfactory
KEY WORDS: Artemether, Lumefantrine, Antimalarial, Simultaneous determination, Method development,
Method validation
INTRODUCTION
Malaria is one of the oldest infirmities of humans and even today approximately 40% of world’s populations are at
risk of this disease. Antimalarial drugs have played a mainstream role in management and control of malaria in
human host [1]. For decades, malaria chemotherapy has been reliant mostly on relatively small number of
chemically related drugs with lack of structural multiplicity. These handful of drugs have their own precincts, of
which the acquirement and spread of parasite multidrug resistance has been the most damaging [2]. Artemisininbased combination therapy [ACT] is increasingly being prescribed as promising treatment. ACT is based on the use
of two drugs Artemether and Lumifantrine is an ACT widely used nowadays and consists of a registered fixed dose
combination [3]
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International Journal of Pharmaceutical Science and Health Care
Available online on http://www.rspublication.com/ijphc/index.html
Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
Artemisinin-based combination therapy (ACT) is increasingly being advocated as promising treatment. ACT is
based on the use of two drugs with different modes of action: an artemisinin-derivative that causes rapid and
effective reduction of parasite biomass and gametocyte carriage and a partner drug that has a longer duration of
action [4]. Artemether–Lumefantrine is an ACT widely used nowadays and consists of a registered fixed dose
combination of Artemether (20 mg) and Lumefantrine (120 mg).World Health Organization (WHO) recommends
this association as first line therapy for falciparum malaria in endemic areas [5].
The increasing use of Artemether–Lumefantrine association as an effective treatment for resistant malaria demands
the need of analytical methods to simultaneously quantify these drugs in tablets in order to evaluate its quality. Some
papers have described the analysis of Artemether in plasma, based on HPLC with electrochemical [6–8] or mass
spectrometry detection [9]. Few methods are available to assay Artemether in pharmaceutical products [10,11]. The
quantitative determination of Lumefantrine in plasma has been described using HPLC with UV detection [12–15].
However, there is no method reported regarding the simultaneous quantitation of Artemether and Lumefantrine.
Hence, the aim of this study was to develop and validate a HPLC method, using UV detection, to simultaneously
quantify Artemether and Lumefantrine in fixed dose combination tablets, thus an attempt was made to estimate
Artemether and Lumefantrine by using HPLC. Ultraviolet detection is not adequate for Artemether quantitation in a
biological matrix due to its low sensitivity and selectivity.
Artemether and Lumefantrine capsules are indicated for the treatment of P. falciparum malaria cases resistant to
both chloroquine and sulphadoxine, pyrimethamine combination. Artemether is a sesquiterpene lactone derived
from artemisinin. Artemisinin is a compound derived from the sweet wormwood plant and has been used for
centuries in traditional Chinese medicine to treat fever. Lumefantrine is a synthetic aryl-amino alcohol antimalarial
(quinine, mefloquine and halofantrine are members of the same group).
Artemether is chemically, (3R,5aS,-6R, 8aS,9R,10S,12R,12aR)-Decahydro-10-methoxy-3,6,9- trimethyl- 3,12epoxy-12H-pyrano [4,3-j]-1,2-benzodioxepin and is used as antimalarial agent
(Figure.1A). Lumefantrine is
chemically, 2, 7-Dichloro-9-[(4-chlorophenyl) methylene]- α-[(dibutylamino)methyl]-9H-fluorene-4-methanol and
is used in the treatment of uncomplicated falciparum malaria (Figure.1B).
FIGURES
Figure 1: Artemether
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Figure 2 : Lumefantrine
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International Journal of Pharmaceutical Science and Health Care
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Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
Pharmacology of Artemether and Lumefantrine:
Both Artemether and Lumefantrine act as blood schizontocides. Artemether is concentrated in the food vacuole. It
then splits its endoperoxide bridge as it interacts with haem, blocking conversion to haemozoin, destroying existing
haemozoin and releasing haem and a cluster of free radicals into the parasite. Lumefantrine is thought to interfere
with the haem polymerization process, a critical detoxicifying pathway for the malaria parasite. Both Artemether
and Lumefantrine have a secondary action involving inhibition of nucleic acid and protein synthesis within the
malarial parasite. An 8 ‐ amino‐quinoline derivative such as primaquine should be given sequentially after
combination in cases of mixed infections of P. falciparum and P. vivax to achieve hypnozoites eradication. The
combination is also associated with rapid gametocyte clearance.[16]
MATERIALS AND METHODS
Artemether and Lumefantrine reference standards were obtained from Zim Laboratories, Nagpur. Ultra-pure water
was obtained from a Millipore system (Bedford, MA, USA). Acetonitrile (HPLC grade) was obtained from E-Merck
(India) Ltd, Mumbai, India. All other chemicals used in the analysis were AR grade.
Assay
assay was carried by HPLC as given below.
Chromatographic conditions:

Column
: Packing L1 (100 X 3 mm X 1.9µ)
(ZODIAC100 C18 is suitable or equivalent)

Flow rate
:
0.5 mL/min.

Wavelength
:
210 nm & 380 nm

Injection volume:

Temperature
10 µL
:
30°C
Preparation of Buffer: 5.65 gm of 1-Hexanesulphonic Acid and 2.75 gm sodium dihydrogen phosphate
(Monohydrate) was dissolved in 1000 mL of water, pH 2.3±0.05 was adjusted with diluted phosphoric acid.
Diluent:
A mixture of 300 mL of Water, 270 mL of Acetonitrile, 100 mL of Buffer and 100 mL of 1–Propanol was prepared.
Solution A: a filtered and degassed mixture of 245 mL of Buffer and 305 mL of Acetonitrile was prepared.
Solution B: a filtered and degassed mixture of 135 mL of Buffer, 415 mL of Acetonitrile and 50 mL of 1–Propanol
was prepared and the gradient program for mobile phase is given in table 1
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International Journal of Pharmaceutical Science and Health Care
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Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
Table 1. Gradient program prescribed
Time (min.)
Solution A (%)
Solution B (%)
Wavelength (nm)
0.00
100
0
210
10.00
100
0
210
10.01
0
100
380
14.00
0
100
380
15.00
100
0
380
23.00
100
0
380
Preparation for standard solution:
Artemether working standard 80 mg and 480 mg of Lumefantrine working standard were weighed and transferred to
100.0 mL dry volumetric flask, 10.0 mL of 1-Propanol was added to it and mixture was sonicated to dissolve, 20.0
mL of Acetonitrile was added and sonicated until dissolved and 5.0 mL of 5% Phosphoric acid solution was added
and sonicated and the volume was made up to 100.0 mL with diluent. And it was filtered through a 0.2-µm pore size
nylon membrane (800 ppm of Artemether and 4800 ppm of Lumefantrine).
Preparation of Sample solution:
Three different batches of commercially available batches were analyzed using the validated method. For the
analysis, six replicates of each batch were assayed. The tablets were weighed and finely powdered containing eq. to
80 mg of Artemether was transferred in 100.0 mL dry volumetric flask 10.0 mL of 1-Propanol was added and
sonicated until dissolved, 20.0 mL of Acetonitrile was added and sonicated until dissolved and 5.0 mL of 5%
Phosphoric Acid solution was added and sonicated until dissolved and made up the volume 100.0 mL with diluent.
The sample was stirred for 1 hour by magnetic stirrer and centrifuged at 3000 RPM for 5 minutes. Sample was
filtered through a 0.2-µm pore size nylon membrane (800 ppm of Artemether and 4800 ppm of Lumifantrine). The
system was equilibrated before making each injection.
Procedure:
Filter through a 0.2-µm pore size nylon membrane and separately inject 10 µL of the standard preparation and
sample preparation into the chromatograph and record the chromatograms and measure the response for the major
peaks and calculate the result by comparison.
Standard preparation
Artemether
Accurately, about 20 mg of standard Artemether was weighed and transferred to separate 100 ml volumetric flasks.
10.0 mL of 1-Propanol was added to it and mixture was sonicated to dissolve, 20.0 mL of Acetonitrile was added
and sonicated until dissolved and 5.0 mL of 5% Phosphoric acid solution was added and sonicated and the volume
was made up to 100.0 mL with diluent to obtain standard stock solutions of each drug of concentration 200 μg/ml.
The stock solutions were filtered through a 0.45 μ membrane filter paper.
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International Journal of Pharmaceutical Science and Health Care
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Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
Lumefantrine
Accurately, about 100 mg of standard Lumefantrine was weighed and transferred to separate 100 ml volumetric
flasks. 10.0 mL of 1-Propanol was added to it and mixture was sonicated to dissolve, 20.0 mL of Acetonitrile was
added and sonicated until dissolved and 5.0 mL of 5% Phosphoric acid solution was added and sonicated and the
volume was made up to 100.0 mL with standard stock solutions of each drug of concentration 1000 μg/ml. The
stock solutions were filtered through a 0.45 μ membrane filter paper.
Calibration curve solutions
From the mentioned stock solutions of Artemether and Lumefantrine calibration curve solutions containing 20
μg/ml to 120 μg/ml of Artemether and 120 μg/ml to 720 μg/ml of Lumefantrine in each calibration level were
prepared.
Preparation of sample solutions
Twenty tablets were weighed and finely powdered. A quantity equivalent to one tablet containing 80 mg of
Artemether and 480 mg of Lumifantrine was transferred in a 100 mL volumetric flask and add 10.0 mL of 1Propanol was added to it and mixture was sonicated to dissolve, 20.0 mL of Acetonitrile was added and sonicated
until dissolved and 5.0 mL of 5% Phosphoric acid solution was added, sonicated and the volume was made up to
100.0 mL.
RESULTS AND DISCUSSION
Optimization of chromatographic conditions
The chromatographic method was optimized by different experiments were performed to achieve the adequate
retentions and resolution for the peaks of Artemether and Lumefantrine. To set the adequate retentions and
resolution, the effects of the mobile phase components, changes in ionic strength were studied, initially Acetonitrile
and water in different ratios were tried. But Artemether gave broad peak shape while Lumefantrine gave no peak, so
water was replaced by potassium dihydrogen buffer and mixture of methanol and potassium dihydrogen phosphate
buffer in different ratios were tried later 1-Hexanesulphonic Acid and 2.75 gm sodium dihydrogen phosphate were
tried. It was found that both peak shows broad peaks finally pH 2.3 adjusted with orthophosphoric acid in ratio gave
acceptable retention time and good resolution for Artemether and Lumefantrine
Validation of the method
Specificity
The peak purity test was used to check the specificity and the sample preparation done by PDA detector. The peak
purity for drugs was found to be 999. The result of the peak purity analysis shows that the peaks of the analytes were
pure and also the formation excipients were not interfering with the analytes peaks the results indicated that peaks
were pure.
Calibration and linearity
The standard solutions containing 20 μg/ml to 120 μg/ml of Artemether and 120 μg/ml to 720 μg/ml of
Lumefantrine in each linearity level were prepared. In the simultaneous determination, the calibration curves were
linear and the coefficient of correlation was found to be 0.997 and 0.998 for Artemether and Lumefantrine,
respectively and the solutions were injected in triplicate.
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Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
Precision (repeatability)
Concentration of The precision of the method was studied by determining the concentrations of each ingredient in
the tablets six times. In the precision study, % relative standard deviation of the Artemether and Lumefantrine were
found to be 0.657 and 0.247 respectively. The results of precision study indicate that the method is reproducible.
Accuracy (recovery test)
Recovery experiments were used to study accuracy by adding known amounts of the pure drug. The recovery was
done at three levels: 80%, 100%, and 120% of the label claim. Three samples were prepared for each recovery level.
The average of recovery of three levels for Artemether and Lumefantrine were found to be 100.46 and 100.33
respectively (Table 2).
Table 2. Results of recovery tests for Artemether (ART) and Lumefantrine (LUM)
Level of
Amount of drug
addition (%)
added(mg)
Recovery (%)
ART
LUM
ART
LUM
80
64
384
99.82±0.43
99.62±0.68
100
80
480
100.54±0.51
100.32±0.67
120
96
576
101.32±0.89
101.05±0.82
Average
ART
LUM
100.46
100.33
Intermediate precision
Intermediate precision of the method was done by analyzing the sample six times on different days, by different
chemists, using different analytical column of the make, and different HPLC systems. The percentage assay was
calculated using the calibration curve. The assay results are shown in Table 3.
Determination of the limits of detection and Quantitation
For determining the limits of detection (LOD) and quantitation (LOQ), the method based on the residual standard
deviation (SD) of a regression line and slope was adopted. To determine the LOD and LOQ, a specific calibration
curve was studied using samples containing the analytes in
the range of the detection and quantitation limits. The LOD for Artemether and Lumefantrine were 0.0019 and
0.00047 μg/ml and the LOQ were 0.0060 and 0.0014 μg/ml respectively.
Robustness
The robustness of the method was performed by deliberately changing the chromatographic conditions. The organic
strength and buffer pH were varied by ±2% and 0.2 units, respectively.
The robustness was determined by
analyzing the same sample under a variety of conditions. The factors consider being variations in the pH (0.2 units)
and strength of Acetonitrile (±2%).The results and the experimental range of the selected variables, together with the
optimized conditions.
There were no significant changes in the chromatography pattern when the above
modifications were made in the experimental conditions, showing that the method is robust.
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Issue 6, Vol. 3 (May-June 2016)
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Determination of active ingredients in tablets
The contents of two drugs in tablets were determined by the proposed method using a calibration curve. The
determinations were done in two sets, one for precision and the second for intermediate precision, and six samples
were prepared for each set. The results are shown in table III.
Table 3. Assay of Active Ingredients in Tablets
Set
Ingredients
Label claim(mg)
Found(mg)
% Label claim
Precision
ART
80
80.34
100.03±0.34
LUM
480
479.58
99.43±0.45
Intermediate
ART
80
80.75
100.43±43
precision
LUM
480
480.23
100.04±01
CONCLUSION
The proposed RP–HPLC method enables simultaneous determination of Artemether and Lumefantrine enabling
good separation and resolution of the chromatographic peaks. This is the useful method for simultaneous
quantitative analysis of Artemether and Lumefantrine, and is a significant advance in chromatographic analysis of
such pharmaceutical mixtures. The method is suitable for qualitative and quantitative analysis of these
pharmaceutical products. The results obtained are in a good agreement with the declared contents. Statistical
analysis showed the method is accurate and precise. There was no interference from excipients in the tablets.
ACKNOWLEDGEMENTS
The authors are thankful to Zim Laboratories for providing gift samples and also Head, University Department of
Pharmaceutical Sciences, RTM Nagpur University, India for successful conduct and completion of this research
work.
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International Journal of Pharmaceutical Science and Health Care
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Issue 6, Vol. 3 (May-June 2016)
ISSN 2249 – 5738
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FIGURES
Figure 1: Artemether
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Figure 2 : Lumefantrine
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