International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491

Vol 2, Issue 4, 2010

ResearchArticle

HPLCMETHODDEVELOPMENTANDVALIDATIONFORSIMULTANEOUSESTIMATIONOF
ARTEMETHERANDLUMEFANTRINEINPHARMACEUTICALDOSAGEFORMS

J.SUNIL1*,M.SANJITHNATH1,U.SAMBAMOORTHY2
1DepartmentofPharmacy,KrishnaUniversity,Machilipatnam,Andrapradesh,India,2SwamiRamanandaTirthaInstituteof
PharmaceuticalSciences,Nalgonda,AndhraPradesh,India.Email:sunilpharma49@gmail.com

Received:06April2010,RevisedandAccepted:26May2010
ABSTRACT
ASimpleandpreciseHPLCmethodwasdevelopedfortheestimationofArtemetherandLumefantrineinpureandpharmaceuticaldosageforms.
ThequantificationwascarriedoutusingsymmetryC18,250x4.6mm,i.d,5mparticlesizeinisocraticmode,withmobilephasecompressingof
bufferandacetonitrileintheratioof40:60(v/v),pH30.5.Theflowratewas1.5ml/minandthedetectionwascarriedoutbyUVdetectordual
i.e,210and303nm.Theretentiontimeswere13.887and7.218minsforArtemetherandLumefantrine,respectively.Thepercentagerecoverywas
foundtobe98.87and99.78%forArtemetherandLumefantrine;respectively.Themethodwasvalidatedbyevaluationofdifferentparameters.
Keywords:HPLC,Artemether,Lumefantrine,UVdetector

INTRODUCTION15
Artemether (figure1) chemically know as 3R, 5aS, 6R, 8aS, 9R,
10S, 12R, 12aR)Decahydro10Methoxy3, 6, 9trimethyl3, 12
epoxy12Hpyrano [4, 3j] 1, 2benzodioxepin. Artemether is a
compound with a molecular formula C16H26O5 and molecular
weight of 298.4 g mol1 and is a white crystalline powder.
Artemether is practically insoluble in water, very soluble in
dichloromethane&acetoneandfreelysolubleinethylacetateand
dehydratedethanol.

different pharmaceutical dosage forms. There is no method has

been reported for estimation of Artemether and Lumefantrine,
thus an attempt was made to estimate Artemether and
LumefantrinebyusingHPLC.

Lumefantrine (figure2) chemically (1R, S)2Dibutylamino1{2,

7dichloro9[(Z)
(4chlorobenzylidene)9Hfluoren4yl}
ethanol, Lumefantrine is a compound with molecular formula
C30H32Cl3NOandmolecularweightof528.9gmol1,andisayellow
crystalline powder. Lumefantrine practically insoluble in water
andaqueousacids.

PharmacologyofArtemetherandLumefantrine:
Fig.1:Artemether

Both artemether and lumefantrine act as blood schizontocides.

Artemether is concentrated in the food vacuole. Itthen 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
polymerisation process,a critical detoxicifying pathway for the
malaria parasite. Both Artemether and Lumefantrine have a
secondary actioninvolvinginhibition of nucleicacid and protein
synthesis within the malarial parasite. An 8 aminoquinoline
derivativesuchasprimaquineshouldbegivensequentiallyafter
thecombinationincasesofmixedinfectionsofP.falciparumand
P. vivax to achieve hypnozoites eradication. The combination is
alsoassociatedwithrapidgametocyteclearance.
Branded Drugs:Laritherinjection3x1ml, Larither capsules
Stripof6capsules.Lumeraxtablets,Riamet,Coartem.
The literature survey617 indicates that Artemether and
Lumefantrine were estimation by UV, TLC, HPTLC and HPLC in

Fig.2:Lumefantrine
EXPERIMENTAL
All chemicals and reagents used were of AR/HPLC grade. Pure
standards of Artemether and Lumefantrine were obtained as gift
sample from Hetero Drugs, Hyderabad. The purities of these

Suniletal.
IntJPharmandPharmSci,Vol2,Issue4,9396
standardswere99.85and99.76%,respectively.Hydrochloricacid,
tween80,sodiumlaurylsulphate, sodiumacetate trihydrate,glacial
acetic acid, monobasic potassium phosphate, sodium hydroxide,
acetonitrile, potassium hydrogen phosphate, potassium hydroxide,
orthophosphoricacidandwaterusedwereofHPLCgrade.
Preparationofbuffer
A 1.36 gm of potassium dihydrogen orthophosphate was dissolved
in 900 ml of MilliQ water. Then the pH was adjusted to 3.0 with
ortho phosphoric acid. Then the volume was make up to 1000 ml
and was filtered through 0.45m nylon membrane filter and
degassed.
Preparationofmobilephase
A degassed mixture of Buffer and Acetonitrile in the ratio of 40:60
(v/v) was prepared and the mixture was filtered through 0.45
membranefiltersanditwasdegassed.
StandardPreparation
A 24.0 mg of Lumefantrine working Standard was weighed and
transferredaccuratelyintoa100mlcleananddryvolumetricflask;
itwasdissolvedin60mldiluent.Then5mlofArtemetherstandard
stock solution was added and diluted to volume with diluent and
mixedwell.
Samplepreparation
Not less than ten tablets of Lumefantrine was weighed and
powdered. A tablet powder equivalent to 24 mg of Lumefantrine
wasaccuratelyweighedandwastransferredinto100mlvolumetric
flask, Then 60ml of acetonitrile was added and sonicated for 30
minuteswithintermediateshaking.Makeupthenvolumewasmake
upwithbuffer(40ml).Aportionofthesolutionwasfilteredthrough
0.45m membrane filter and first few ml of the filtrate was
discarded.
Chromatographicconditions
Freshly prepared Buffer and acetonitrile 40:60 (v/v) mobile phase
andadjustpHto3werefilteredthrough0.45membranefilterand
sonicatedbeforeuse.FlowrateofMobilephasewasmaintainedat
1.5 ml/min. The column temperature maintained was ambient
temperature. The detection was carried out dual i.e, 210 and 303
nm, Injectionvolume 20l andtotalrun timewas20 min.Column
wasSymmetryC18,250x4.6mm,5particlesize.

Systemsuitabilityparameters
System suitability tests are an integral part of chromatographic
method.Toascertainitseffectiveness,systemsuitabilitytestswere
carried out on freshly prepared standard stock solution of
ArtemetherandLumefantrine.Inadditiontothisstandarddeviation
of Artemether and Lumefantrine standards were evaluated by
injecting a mixed standard of both Artemether and Lumefantrine
(800 and 240 ppm) as internal standard for five times at 20 min
intervalandthevalueswererecorded.Alltheaboveparametersare
shownintable1.
Artemetherstandardstockpreparation
A 40.0 mg of Artemether working standard was weighed and
transferredintoa50mlcleananddryvolumetricflask.Thenitwas
dissolvedanddilutedtovolumewithdiluent.
Assayprocedure
A 20 l of diluent, placebo, standard preparation (5 times) and
sample preparation were separately injected into the
chromatographic system. Then the chromatograms and the peak
responsesweremeasured.
Theplacebochromatogramwasexaminedforanyextraneouspeaks
that were observed in the chromatogram of sample preparation.
Chromatogram of the standard preparation was recorded and the
peak responses were measured. The tailing factor for the principal
peak should not more than 4.0 and the number of the theoretical
plates should not less than 5000. The % RSD (Relative Standard
Deviation) should not be more then 2.0. A 20 L of standard
preparation and assay preparation was separately injected in the
chromatogram, the chromatograms were recorded and the
responsesforthemajorpeaksweremeasured.
RESULTSANDDISCUSSION
In order to develop simultaneous estimation of two components
underisocraticconditions,themixtureofacetonitrilewithbufferin
different ratios were assayed as the mobile phase. A mixture of
buffer and acetonitrile in different ratios were also tried for the
assay of combined dosage forms. Finally a mixture of acetonitrile
potssiumdihydrogenorthophosphate(buffer)inratioof40:60v/v,
proved to be the effective mixture than the other mixture used for
theseparation.Thentheflowratesweretestedincludes0.5,0.8,1.0,
1.5and2.0ml,amongtheseflowrates1.5mLwasselectedforthe
assaybecausebetterresolutionofthepeakswereobserved.System
suitability test was applied to freshly prepared stock solution of
Artemether and Lumefantrine to check the parameters like Tailing
factors, Resolution, Theoretical plates, Relative standard deviation
asshownintable1.

Table1:Systemsuitabilityparameters
Parameters
Tailingfactors
Resolution
Theoreticalplates
Relativestandarddeviation

Lumefantrine
3.6
7.218
1992
0.10

Solubility
The solubility of Artemether in mg/ml in all medias can be
calculatedbythefollowingformula:
Solubility = Sample absorbance/standard absorbance std
dilution/sampledilutionPurity/100wtofsample
From the above observation, it was observed that Lumefantrine
showedacceptablesolubilityinallmediaexhibitinghighersolubility

Artemether
1.1
13.887
17863
1.08
in0.1NHClandTween80.
From the above observation it was observed that Artemether
showedacceptablesolubilityinallmediaexhibitinghighersolubility
in pH 4.5 Acetate buffer and 2% SLS Table2,3. The developed
methodwasstudiedforprecision.Theprecisionofthemethodwas
demonstrated by at least six determinations in method precision.
Standarddeviationandtheresultsaregivenintable4.Theaccuracy
of theproposed HPLC method wasexpressed intermsofrecovery.
The recovery studies was carried out and given in terms of
percentagerecoveryandgivenintable5
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Suniletal.
IntJPharmandPharmSci,Vol2,Issue4,9396

Table2:Solubilityresultsoflumefantrine
S.No.
1
2
3
4
5

Media
Purifiedwater
0.1NHCl
0.1NHCland1%Tween80
pH4.5Acetatebuffer
pH4.5Acetatebufferand2%of
Sodiumlaurylsulphate
pH6.8Phosphatebuffer
pH7.4Phosphatebuffer

6
7

Samplewt.inmg
25.39
70.81
400
150.40
170

Sampleabsorbance
0.671
0.516
0.482
0.513
0.401

0.627
0.678

25.0
25.0

Standardabsorbance
0.453
0.461
0.487
0.437
0.479

Solubility(mg/ml)
3.90
6.95
38.83
18.42
13.14

0.452
0.462

3.58
3.791

Table3:Solubilityresultsofartemether
S.No.

Media

Samplewt.

Sampleabsorbance

1
2
3
4
5

Purifiedwater
0.1NHCl
0.1NHCland1%Tween80
pH4.5Acetatebuffer
pH4.5Acetatebufferand2%
Sodiumlaurylsulphate
pH6.8Phosphatebuffer
pH7.4Phosphatebuffer

20.16
70.90
70.00
251.1
520
20.00
20.16

6
7

0.536
0.473
0.459
0.391
0.394

Standard
absorbance
0.325
0.391
0.345
0.309
0.325

Solubility
mg/ml
3.26
9.69
10.49
29.93
57.35

0.531
0.592

0.384
0.371

2.73
3.14

Table4:Precisionofmethod
Determinations
Lumefantrine
Artemether

1
98.9
99.6

2
99.6
97.8

3
97.8
100.3

4
100.1
99.8

5
98.8
98.9

6
99.5
99.2

Mean
99.11
99.26

Std.dev
0.73
0.79

%RSD
0.74
0.79

Table5:Recoverystudies
Drug

Lumefantrine
Artemether

Amountadded(g)
12.06
24.34
36.51
2.10
4.23
6.25

Amountrecovered(g)
12.02
24.28
36.49
2.08
4.17
6.18

Recovery(%)
99.66
99.75
99.94
99.1
98.6
98.9

mean

99.78
98.87

CONCLUSION
Theproposed methodwasfound to be simple,speciesspecificand
highlyaccurate,requiredlesstimeconsumptionforanalysisandthis
canbeemployedfortheroutineanalysis.
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