Simultaneous RP-LC Determinationof Losartan Potassium, Ramipril,and Hydrochlorothiazide in PharmaceuticalPreparations

M. M. Baing, V. V. Vaidya&, R. T. Sane, S. N. Menon, K. Dalvi

Department of Chemistry, Ramnarain Ruia College, Matunga, Mumbai 400 019, India; E-Mail: vaidya_vikas@yahoo.com

Received: 29 March 2006 / Revised: 4 May 2006 / Accepted: 29 May 2006Online publication: 19 August 2006

Abstract

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic methodhas been developed for simultaneous determination of losartan potassium, ramipril, andhydrochlorothiazide. The three drugs were separated on a 150 mm 4.6 mm i.d., 5 lmparticle, Cosmosil C18 column. The mobile phase was 0.025 M sodium perchlorateacetonitrile,62:38 (v/v), containing 0.1% heptanesulphonic acid, pH adjusted to 2.85 with orthophos-phoric acid, at a flow rate of 1.0 mL min)1. UV detection was performed at 215 nm.The method was validated for linearity, accuracy, precision, and limit of quantitation. Linear-ity, accuracy, and precision were acceptable in the ranges 3565 lg mL)1 for losartan,1.753.25 lg mL)1 for ramipril, and 8.7516.25 lg mL)1 for hydrochlorothiazide.

Keywords

Column liquid chromatographyPharmaceutical preparationsLosartan potassiumRamiprilHydrochlorothiazide

Introduction

Losartan potassium (2-butyl-4-chloro-1-

[(2-1H-tetrazole-5-yl)[1,1-biphenyl]-4-yl]methyl-1H-imidazole-5-methanol) is an

angiotensin II receptor antagonist used

for treatment of hypertension. Ramipril

([2S-{1(R*(R*)}2a,3ab,6ab]}-1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]aminol]-1-

oxopropyl]octahydrocyclopenta(b)pyrole-

2-carboxylic acid) is another angioten-

sion-converting enzyme-inhibiting anti-

hypertensive drug. Hydrochlorothiazide

(6-chloro-3,4-dihydro-2H-1,2,4-benzothi-

adiazine-7-sulfonamide 1,1-dioxide) is a

thiazide-class diuretic used for treatment

of hypertension [1]. The structures of

these three drugs are shown in Fig. 1. A

combination of 50 mg losartan potas-

sium, 2.5 mg ramipril, and 12.5 mg

hydrochlorothiazide is very widely used

for treatment of hypertension but a lit-

erature search revealed that no HPLC

method is available for simultaneous

HPLC determination of these three drugs

in such pharmaceutical preparations. An

HPLC method was therefore developed

for analysis of losartan potassium,

ramipril, and hydrochlorothiazide in

their combined dosage forms [27]. The

method described is simple, rapid, pre-

cise, and accurate.

Experimental

Chemicals and Reagents

Standards were obtained from Sun

pharmaceutical Industries, Mumbai,

India. Loram-H tablets, manufactured by

Unichem Laboratories, Mumbai, India,

were obtained commercially. Acetoni-

trile, methanol, and orthophosphoric

acid were from Qualigens and sodium

perchlorate and heptanesulphonic acid

were from S.D. Fine Chem. Double-

distilled water was used throughout the

work. All dilutions were performed in

standard volumetric asks.

Preparation of Stock andWorking Standard Solutions

A stock solution of losartan potassium

(1,000 lg mL)1) was prepared by dis-solving 25.05 mg losartan potassium

(99.44%) in methanol in a standard

25-mL volumetric ask (solution A). A

stock solution of ramipril (1,000 lgmL)1) was prepared by dissolving

25.00 mg ramipril (99.80%) in methanol

in a standard 25-mL volumetric ask

(solution B). A stock solution of hydro-

chlorothiazide (1,000 lg mL)1) wasprepared by dissolving 25.05 mg hydro-

DOI: 10.1365/s10337-006-0008-60009-5893/06/09 2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

2006, 64, 293296

Short Communication Chromatographia 2006, 64, September (No. 5/6) 293

chlorothiazide (99.60%) in methanol in a

standard 25-mL volumetric ask (solu-

tion C). Internal standard (IS; amlodipine

besylate) stock solution (100 lg mL)1)was prepared by dissolving 10.0 mg am-

lodipine besylate in methanol in a 100-

mL standard volumetric ask. Solutions

containing mixtures of losartan potas-

sium, ramipril, and hydrochlorothiazide

at seven different concentrations were

prepared in the mobile phase and am-

lodipine besylate was added to each as

internal standard. The concentration

ranges for each of the three drugs in the

working standard solutions were 3565

lg mL)1 for losartan, 1.753.25 lg mL)1

for ramipril, and 8.7516.25 lg mL)1 forhydrochlorothiazide.

Sample Preparation

Twenty tablets were weighed and their

average weight was calculated. The tab-

lets were crushed to a homogeneous

powder and a quantity equivalent to one

tablet (158.6 mg) was weighed in a

100-mL volumetric ask, dissolved in

methanol, and ltered through Whatman

no. 41 lter paper. The ltrate (1 mL)

was quantitatively transferred to a 10-mL

volumetric ask, 1 mL internal standard

solution was added, and solution was

diluted to volume with mobile phase.

Instrumentation andChromatographic Conditions

Chromatography was performed with a

Jasco PU 980 isocratic pump, a Jasco

AS-2057 autosampler, and a Jasco UV-

970 variable-wavelength UVvisible

detector. Chromatograms and data were

recorded by means of Borwin Chro-

matographic software version 1.21.

Compounds were separated on a

150 mm 4.6 mm i.d., 5 lm particle,Cosmosil C18 column. The mobile phase

was 0.025 M sodium perchlorateacetoni-

trile, 62:38 (v/v), containing 0.1% hep-

tanesulphonic acid (pH adjusted to 2.85

with orthophosphoric acid). The ow rate

was 1.0 mL min)1. Twenty microlitres of

sample was injected and the detection

wavelength was 215 nm for determination

of all three drugs. The overlain UV spec-

tra of losartan potassium, ramipril, and

hydrochlorothiazide are shown in Fig. 2

and typical HPLC chromatograms ob-

tained from simultaneous determination

of losartan potassium, ramipril, and

hydrochlorothiazide standards and from

a pharmaceutical formulation are shown

in Figs. 3 and 4, respectively.

Results and Discussion

Method Validation

The chromatographic conditions were

optimized to obtain good baseline sepa-

ration and peak shapes.

Linearity

Linearity was evaluated by analysis of

working standard solutions of losartan

potassium, ramipril, and hydrochloro-

thiazide of seven dierent concentrations

N

NCl

N

N NH

NCH2OHnC4H9

K+

HN

OO

H3C

O

CH3HN

HH

H CO2H

H

NH

NHS

OOS

Cl

OO

H2N

Fig. 1. The structures of losartan potassium (top), ramipril (middle), and hydrochlorothiazide(bottom)

Fig. 2. UV spectra of (a) losartan potassium, (b) hydrochlorothiazide, (c) amlodipine besylate (IS),and (d) ramipril, all in methanol

294 Chromatographia 2006, 64, September (No. 5/6) Short Communication

[8, 9]. The linear ranges were from 35 to

65 lg mL)1 for losartan potassium, 1.75to 3.25 lg mL)1 for ramipril, and 8.75 to16.25 lg mL)1 for hydrochlorothiazide.The drug-to-IS peak-area ratio and con-

centration of each drug were subjected to

regression analysis to calculate the cali-

bration equations and correlation coef-

cients. The regression data obtained for

the three pharmaceuticals are listed in

Table 1. The results show that within

these concentration ranges there was

excellent correlation between peakarea

ratio and concentration of each drug.

Limits of Detection and Quantitation

The limits of detection (LOD) and

quantitation (LOQ) were established at

signal-to-noise ratios of 3:1 and 10:1,

respectively [8, 9]. The LOD and LOQ of

losartan potassium, ramipril and hydro-

chlorothiazide were determined experi-

mentally by injecting each drug six times.

The LOD for losartan potassium, ram-

ipril and hydrochlorothiazide were 0.006,

0.030, and 0.006 lg mL)1, respectively.The LOQ for losartan potassium,

ramipril, and hydrochlorothiazide were

0.02, 0.08, and 0.02 lg mL)1, respectively.

Precision

Repeatability was studied by determina-

tion of system precision for six replicate

injections of the mixed standard solutions

[9]. The relative standard deviations were

less than 2% for the three drugs. Method

precision was determined from results

from seven independent determinations

at 100% of the test concentrations of lo-

sartan potassium, ramipril, and hydro-

chlorothiazide in the product. The RSD

were 0.62, 0.55, and 0.50, respectively.

System Suitability

System-suitability tests were performed in

accordance with USP 24/NF 19 to con-

rm the reproducibility of the equipment

was adequate for the analysis to be per-

formed. The test was performed by

injecting 20 lL of a standard solution oflosartan potassium, ramipril, and hydro-

chlorothiazide containing 50.00, 2.5, and

12.50 lg mL)1, respectively. This wasrepeated ve times. RSD values for lo-

sartan potassium, ramipril, and hydro-

chlorothiazide were 0.055, 0.03, and

0.001, respectively. These values met

the requirements of USP24/NF19 (RSD

Fig. 3. Typical HPLC chromatogram obtained from simultaneous determination of losartanpotassium, ramipril, hydrochlorothiazide, and amlodipine besylate (IS) in a standard solution

Fig. 4. Typical HPLC chromatogram obtained from simultaneous determination of losartanpotassium, ramipril, and hydrochlorothiazide in a pharmaceutical formulation, with amlodipinebesylate as internal standard

Table 1. Results from determination of linearity

Analyte Slope(mean)

Intercept(mean)

RSD ofslope(n = 3)

RSD ofintercept(n = 3)

Correlationcoecient(r2; n = 7)

Losartan potassium 0.2818 )0.2637 0.02 )0.07 0.9999Ramipril 0.0936 0.0011 0.035 1.82 0.9996Hydrochlorothiazide 0.3037 )0.044 0.019 )1.46 0.9997

Table 2. Results from determination of system suitability

Characteristic Hydrochlorothiazide Losartanpotassium

Ramipril Amlodipinebesylate (IS)

Resolution 19.13 2.90 5.31Tailing factor 0.55 0.66 0.45 0.62Theoretical plates 3,766 7,743 2,670 8,523

Short Communication Chromatographia 2006, 64, September (No. 5/6) 295

< 2%) and were therefore satisfactory.

Theoretical plates, resolution, and tailing

factor were also determined. The values

obtained are listed in Table 2.

Accuracy

The accuracy of the method was deter-

mined by measuring the recovery of the

drugs by the method of standard addi-

tions. Known amounts of each drug cor-

responding to 100, 110, 120, and 130% of

the label claim for each drugwere added to

a pre-analysed sample, to determine

whether the excipients present in the for-

mulation led to positive or negative inter-

ferences [9]. Each set of additions was

repeated seven times .The accuracy was

expressed as the percentage of the analytes

recovered by the assay; the results ob-

tained are listed in Table 3. The results

indicate the method enables highly accu-

rate simultaneous determination of the

three drugs.

Assay

The validated HPLC method was used

for simultaneous determination of losar-

tan potassium, ramipril, and hydrochlo-

rothiazide in their combined dosage

form. In the assay experiment seven

samples were weighed separately and

analysed. The mean assay results, ex-

pressed as a percentage of the label claim,

are listed in Table 4. The results indicate

that the amount of each drug in the tab-

lets is within the requirements of 90

110% of the label claim.

Conclusion

A simple, sensitive method has been

established for isocratic separation and

simultaneous determination of losartan

potassium, ramipril, and hydrochloro-

thiazide in their combined dosage form.

The linearity, precision, and accuracy of

the method prove it is highly reproducible

under quality-control conditions if the

procedures described above are followed

precisely.

References

1. Merck Index (2001) 13th edn. Merck &Co., inc., USA

2. Kanumula GV, Bhanu R (2000) IndianDrugs 37:3841

3. Zarapkar SS, Rane SH (2000) IndianDrugs 37:589593

4. Shah SA, Rathod IS, Suhagia BN, SalveSS, Patel JB (2001) J AOAC Int 84:17151723

5. Hertzog DL, McCafferty JF, Fang X,Tyrrell RJ, Reed RA (2002) J Pharm Bio-med Anal 30:747760

6. Gandhimathi M, Ravi TK, Ninan A,Varghese A (2004) Indian Drugs 41:3639

7. Valiyare GR, Chandra A, Apte SK, Ma-hadik AA (2005) Indian Drugs 42:309312

8. Snyder LR, Kirkland JJ, Glajch JL (1997)Practical HPLC method development, 2ndedn. Wiley, USA

9. ICH (1996) Validation of analytical pro-cedures: methodology, ICH harmonisedtripartite guidelines, adopted 6 Nov 1996

Table 3. Results from determination of the accuracy of the method

Analyte Initialconcentration(mg)

Concentrationadded (mg)

Totalconcentration(mg)

Concentrationfound (mg)

RSD(%)n = 7

Recovery(%)

Bias (%)

Losartan potassium 50 0 50 50.03 0.40 100.07 )0.0750 5 55 54.94 0.23 99.90 0.1050 10 60 60.04 0.27 100.08 )0.0850 15 65 64.97 0.22 99.95 0.05

Ramipril 2.50 0 2.50 2.49 0.44 99.93 0.072.50 0.25 2.75 2.74 0.36 99.91 0.092.50 0.50 3.00 2.99 0.20 99.91 0.0862.50 0.75 3.25 3.24 0.27 99.86 0.14

Hydrochlorothiazide 12.50 0 12.50 12.48 0.41 99.86 0.1312.50 1.25 13.75 13.74 0.40 99.94 0.0612.50 2.50 15.00 14.99 0.18 99.94 0.0612.50 3.75 16.25 16.23 0.34 99.90 0.094

Table 4. Results from assay experiment

Losartan potassium Ramipril Hydrochlorothiazide

Means amount ofdrug found (mg per tablet)

50.06 2.50 12.53

Mean recovery (%) 100.13 100.20 100.31RSD 0.62 0.30 0.45

The label claimed was 50 mg losartan potassium, 2.5 mg ramipril, and 12.5 mg hydrochlorothi-azide

296 Chromatographia 2006, 64, September (No. 5/6) Short Communication

Simultaneous RP-LC Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide in Pharmaceutical PreparationsAbstractIntroductionExperimentalChemicals and ReagentsPreparation of Stock and Working Standard SolutionsSample PreparationInstrumentation and Chromatographic ConditionsResults and DiscussionMethod ValidationLinearityFig1Fig2Limits of Detection and QuantitationPrecisionSystem SuitabilityFig3Fig4Tab1Tab2AccuracyAssayConclusionReferencesCR1CR2CR3CR4CR5CR6CR7CR8CR9Tab3Tab4

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 150 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/Description >>> setdistillerparams> setpagedevice