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Abstract: A simple, rapid, precise and accurate reversed phase high performance liquid chromatographic method
has been developed for the simultaneous determination of Artemether in combination with Lumefantrine. This
method uses a Hypersil ODS C18 (250mm4.6mm5 particle Size) analytical column, a mobile phase of
methanol: 0.05 % trifluroacetic acid with triethylamine buffer pH 2.8 adjusted with orthophosphoric acid in ratio
(80:20 v/v). The instrumental settings are a flow rate of 1.5 ml/min and PDA detector wavelength at 210 nm. The
retention times for Artemether and Lumefantrine are 6.15 min and 11.31min, respectively. The method is validated
and shown to be linear. The linearity range for Artemether and Lumefantrine are 20-120 & 120-720 g/ml
respectively. The Percentage recovery for Artemether and Lumefantrine are ranged between 99.50101.16 and
99.78101.21 respectively. The correlation coefficients of Artemether and Lumefantrine are 0.999, and 0.999,
respectively. The relative standard deviation for six replicates is always less than 2%. The Statistical analysis
proves that the method is suitable for analysis of Artemether and Lumefantrine as a bulk drug and in
pharmaceutical formulation without any interference from the excipients.
Key words - Artemether, Lumefantrine, Validation and RP-HPLC.
Introduction
Artemether
is
chemically
(3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-10-methoxy3,6,9- trimethyl- 3,12-epoxy-12H-pyrano [4,3-j]-1,2benzodioxepin1 and is used as antimalarial agent.
Lumefantrine is chemically 2, 7-Dichloro-9-[(4chlorophenyl) methylene]--[(dibutylamino) methyl]9H-fluorene-4-methanol2 and is used in the treatment
of uncomplicated falciparum malaria. Both of these
drugs available in combined tablet dosage form with
B
Figure 1.: Structures of Antimalarial Drugs:
A- Artemether and B- Lumefantrine
Experimental
Instrumentation
The LC system was from Perkin Elmer Quaternary
pump Series 200 and was comprised of auto sampler
injector; and an Intelligence PDA detector connected
to the Total Chrome Navigator version 6.3. For
controlling the instrumentation as well as processing
the data generated was used.
Material and reagents
Artemether API and Lumefantrine API were obtained
as gift sample from Ajantha Pharmaceutical Ltd
(Mumbai, Maharashtra, India). Acetonitrile (HPLC
grade), triethylamine (AR grade), methanol (HPLC
grade),
trifluroacetic
Acid
(AR
grade),
orthophosphoric acid (AR grade) were obtained from
Rankem Pvt. Ltd. Delhi, India. The 0.45 m
membrane filter was used throughout the experiment.
The tablets of ART in combination with LUM
(Lumerax) were purchased from Local market. Double
distilled water was used throughout the experiment.
Other chemicals used in the experiment were of
analytical or HPLC grade.
Chromatographic conditions
The isocratic mobile phase consists of methanol: 0.05
% trifluroacetic acid with triethylamine buffer pH 2.8
adjusted with orthophosphoric acid in ratio 80:20 v/v,
flowing through the column at a constant flow rate of
1.5 ml/min. A Hypersil ODS C18 column (250mm
1723
1724
% Label claim
%RSD
100.05 0.657
99.67 0.247
99.22 0.204
100.16 0.115
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 II.
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 ART and LUM
1725
LUM
11.31
1.45
61407.69
% drug Found
ART
LUM
100.09
100.06
99.94
100.01
100.05
99.97
% drug Found
ART
LUM
100.21
100.88
99.94
100.01
99.97
99.42
% drug Found
ART
LUM
100.20
99.97
99.94
100.01
100.04
100.27
% drug Found
ART
LUM
99.67
99.91
99.94
100.01
99.89
99.90
1726
Conclusion
The proposed RPHPLC method enables simultaneous
determination of ART & LUM enabling good
separation and resolution of the chromatographic
peaks. This is the first reported method for
References
1. Martindale: The complete drug reference 36th
edition, Pharmaceutical press, Lambeth High
Street, London, 2009, 598-599.
2. Martindale: The complete drug reference 36th
edition, Pharmaceutical press, Lambeth High
Street, London, 2009, 605.
3. Navratnam V., Mansor, L.K., Chin, M. Asokan,
Nair N.K., Determination of Artemether and
dihydroartemisinin in blood plasma by hplc for
application in pharmacological studies, Journal
of chromatography B. 669, 1995, 289-294.
4. Karbwang J., Na-Bangchang K., Molunto P.,
Banmairuroi
V.,
Congpuong
K.,
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