Simultaneous Determination of Losartan

Potassium, Atenolol and Hydrochlorothiazide

in Pharmaceutical Preparations

by Stability-Indicating UPLC

D. Durga Rao1,2,&, N. V. Satyanarayana1, S. S. Sait1, Y. Ramakoti Reddy2, K. Mukkanti2

1 Dr. Reddys Laboratories Ltd., IPDO, Bachupally, Hyderabad, AP 500072, India;E-Mail: dantu_durga@yahoo.com; dantudr@drreddys.com

2 Department of Chemistry, J.N.T. University, Kukatpally, Hyderabad, AP 500072, India

Received: 27 November 2008 / Revised: 15 April 2009 / Accepted: 22 April 2009Online publication: 24 July 2009

Abstract

A stability-indicating UPLC method was developed for the simultaneous quantitative deter-mination of losartan potassium, atenolol, and hydrochlorothiazide in pharmaceutical dosageforms in the presence of degradation products. The separation was achieved on a simpleisocratic method (water: acetonitrile: triethyl amine: ortho phosphoric acid (60:40:0.1:0.1,v/v) at 0.7 mL min-1, a detection wavelength of 225 nm). The retention times of losartanpotassium, atenolol, and hydrochlorothiazide were 2.3, 0.6 and 0.9 min. The total runtimewas 3 min. Losartan potassium, atenolol, and hydrochlorothiazide were subjected to dif-ferent ICH prescribed stress conditions. The method was validated with respect to linearity,accuracy, precision, robustness and ruggedness.

Keywords

Column liquid chromatographyStability-indicatingLosartan potassium, atenolol and hydrochlorothiazide

Introduction

The aim of the present study was to

establish the inherent stability of losartan

potassium, atenolol, and hydrochlorothi-

azide through stress studies under a variety

of ICH recommended test conditions [13]

and to develop a stability-indicating assay

[46]. Losartan (L) (2-butyl-4-chloro-1-

[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imid-

azole-5-methanol mono potassium salt) is

an angiotensin II receptor (type AT1)

antagonist. Hydrochlorothiazide (H) (6-

chloro-3,4-dihydro-2H-1,2,4-benzothia-

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

thiazide diuretic. Atenolol (A) is a blocker

described as 4-[2-hydroxy-3-[(1-methyl-

ethyl)amino]propoxy] benzeneacetamide.

Literature survey reveals that a vari-

ety of spectrophotometric and chro-

matographic methods, and a stability

indicating LC method, have been

reported for determination of L in phar-

maceutical preparations in combination

with other drugs [712]. Spectrophoto-

metric and chromatographic methods

have been reported for determination of

A, in combination with other drugs, in

bulk and pharmaceutical preparations

[1316]. A variety of methods has been

used for determination of H [1723]. An

HPTLC method has been reported for

simultaneous quantitative determination

of the three drugs in tablets [24]. Whereas

no LC method has been reported for

simultaneous quantitative determination

of these in the combined dosage form.

Hence a UPLCmethod was developed

for simultaneous quantitative deter-

mination of the three drugs in combined

pharmaceutical dosage forms in the pres-

ence of degradation products.

Experimental

Chemicals

Standards were supplied by Dr. Reddys

Laboratories, Hyderabad, India. Com-

mercially available Repalol H tablets

(50 mg of L, 50 mg of A, 12.5 mg of H)

were purchased from Sun Pharmaceutical

Industries, India. LC grade acetonitrile,

2009, 70, 647651

DOI: 10.1365/s10337-009-1221-x0009-5893/09/08 2009 Vieweg+Teubner | GWV Fachverlage GmbH

Limited Short Communication Chromatographia 2009, 70, August (No. 3/4) 647

analytical grade triethyl amine and ortho

phosphoric acid were purchased from

Merck, Darmstadt, Germany; water was

prepared by Millipore MilliQ Plus water

purication system.

Equipment

The used Waters UPLC Acquity system

consisted of a binary solvent manager, a

sample manager and a UV detector. The

output signal was monitored and pro-

cessed by Empower software, a water

baths equipped with MV controller

(Julabo, Seelbach, Germany) was used

for hydrolysis studies. Photo stability

studies were carried out in a photo sta-

bility chamber (Sanyo, Leicestershire,

UK). Thermal stability studies were

performed in a dry air oven (MACK

Pharmatech, Hyderabad, India).

Chromatographic Conditions

The chromatographic column was a

Zorbax C-18, 50 mm 9 4.6 mm i.d with

1.8 lm particles. Mobile phase containeda mixture of water: acetonitrile: triethyl

amine: ortho phosphoric acid (60:40:

0.1:0.1, v/v). The ow rate of mobile phase

was 0.7 mL min-1 and the detection was

monitored at a wavelength of 225 nm. The

column temperature was maintained at

25 C and injection volume was 5 lL.

Preparation of Stock Solutions

A stock solution of L, A and H stan-

dard and sample (0.5 mg mL-1 of L,

0.5 mg mL-1 of A, 0.125 mg mL-1 of

H) was in acetonitrile: water (40:60, v/v).

Working solutions 0.05 mg mL-1 of L,

0.05 mg mL-1 of A, 0.0125 mg mL-1 of

H were prepared from above stock

solution in the mobile phase for assay

determination.

Preparation of SampleSolution

Twenty Repalol H tablets were weighed

and grinded in a mortar. Then an

equivalent to 50 mg of L (50 mg of

losartan potassium, 50 mg of atenolol,

12.5 mg of hydrochlorothiazide) was

transferred to a 100 mL volumetric ask,

70 mL of acetonitrile: water (40:60, v/v)

added and sonicated for 15 min and di-

luted to 100 mL (0.5 mg mL-1 of L,

0.5 mg mL-1 of A, 0.125 mg mL-1 of

H). About 5 mL of supernant solution

was diluted to 50 mL with the mobile

phase. This was ltered using a 0.45 l(Nylon 66-membrane) lter.

System Suitability SolutionCriteria

The system suitability was assessed by

ve replicate analyses of the drugs at

concentrations of 0.5 mg mL-1 of L or

A, 0.125 mg mL-1 of H.

Stress Studies

All stress decomposition studies were

performed at an initial drug concentra-

tion 0.05 mg mL-1 of L or A, 0.0125

mg mL-1 of H. Acid hydrolysis was

performed in 0.1 N HCl at 70 C for 4 h.The study in basic solution was carried

out in 0.1 N NaOH at 70 C for 4 h. Forstudy in neutral solution, drugs dissolved

in water were heated at 70 C for 3 h.Oxidation studies were carried out at

room temperature in 5% hydrogen per-

oxide for 4 h. Photo degradation stud-

ies were carried out at according to

Option 2 of Q1B in ICH guidelines [3].

Samples were exposed to light for an

overall illumination of 1.2 million lux

h-1 and an integrated near ultraviolet

energy of 200 W hm2. The drug product

was exposed to dry heat at 80 C for24 h. Samples were subjected to UPLC

analysis after suitable dilution (0.05

mg mL-1 of L or A, 0.0125 mg mL-1

of H).

Method Validation

Method validation was performed as per

ICH guidance for simultaneous deter-

mination of L, A and H in the formu-

lations.

Limit of Detection and Limitof Quantification

The LOD and LOQ for L, A and H

were estimated at a signal-to-noise ratio

of 3:1 and 10:1 by injecting a series of

dilute solutions with known concentra-

tion.

Precision

Precision was investigated using the

sample preparation procedure for six

real samples of commercial tablets

(Repalol H).

Repeatability (Intra-Day) The preci-sion of the assay method was evaluated

by carrying out six independent assays of

L, A and H (0.05 mg mL-1 of L or A,

0.0125 mg mL-1 of H) test samples

against qualied reference standard (%

RSD).

Intermediate Precision (Inter-Day)A dierent analyst on a dierent day in

the same laboratory evaluated the inter-

mediate precision % RSD of the meth-

od. Six samples of L, A and H tablets

against were assayed reference standard.

Linearity

Linearity solutions were prepared from

stock solution at ve concentration lev-

els from 50 to 150% of analyte concen-

trations (25 to 75 lg mL-1 for L, A and6.25 to 18.75 lg mL-1 for H). The slope,Y-intercept and correlation coecient

were calculated.

Accuracy

The accuracy of the method was eval-

uated in triplicate at three concentra-

tion levels, 50, 100 and 150% of the

target test concentration (0.5 mg mL-1

of L or A, 0.125 mg mL-1 of H).

The percentage of recoveries were

calculated.

648 Chromatographia 2009, 70, August (No. 3/4) Limited Short Communication

Robustness

The relative standard deviation for rep-

licate injections of L, A and H peaks and

the USP resolution factor between L, A

and H peaks were evaluated.

The mobile phase ow rate was

0.7 mL min-1. This was changed by

0.1 units to 0.6 and 0.8 mL min-1. The

eect of column temperature was studied

at 20 and 30 C instead of 25 C. Theeect of mobile phase composition was

studied by use of water: acetonitrile:

triethyl amine: orthophosphoric acid

62:38:0.1:0.1 and 58:42: 0.1:0.1 (v/v).

Specificity and Selectivity

The specicity of the method was

established through study of resolution

factors of the drug peaks from nearest

resolving peak, and among all other

peaks.

Solution Stability and MobilePhase Stability

The solution stability of L, A and H was

carried out by leaving the test solution in

tightly capped volumetric asks at room

temperature for 24 h and assayed at 6 h

interval, against the freshly prepared

standard solution. The mobile phase

stability was carried out by assaying the

freshly prepared standard solution at

24 h interval up to 48 h. The percentage

of RSD of assay of L, A and H was

calculated for the study period during

mobile phase and solution stability

experiments.

Results and Discussion

Force Degradation Studies

Degradation was not observed in L, A

and H under stress conditions like pho-

to, thermal degradation and water

hydrolysis. Degradation was observed at

RRT 1.26 with respect atenolol in 0.1 N

NaOH at 70 C for 4 h. The impuritywhich was formed at RRT 1.26 in

base degradation was the impurity of H

(4-amino-6-chlorobenzene-1,3-disulph-

onamide). This was conrmed with

spiking analysis with qualied reference

standard and also with spectral matching

and with mass spectroscopy.

Major degradation was observed

after 4 h in 5% hydrogen peroxide for L,

A and H. Peak purity test results con-

rmed that the drug peaks are homoge-

neous and pure in all the analyzed stress

samples.

Mass balance (% assay + % degra-

dents + % impurities) was calculated

for stress sample. The mass balance of

stressed sample was > to 99.0% for all

three drugs.

Method Development andOptimization of StabilityIndicating Assay Method

The main target of this study was the

separation of the degradation product

at 1.26 RRT (with respect to A). The

degradation samples were run using

dierent stationary phases like C18, C8,

Cyano and mobile phases containing

buers like phosphate, sulphate and

acetate at dierent pH (27) and using

organic modiers like acetonitrile and

methanol in the mobile phase. The

separation was satisfactory under the

isocratic conditions with 40% acetoni-

trile as organic modier in the mobile

phase.

The assay results (n = 3) for com-

mercially dosage from products, were

99.0, 99.3, 98.9% of L, 98.5, 98.8, 98.6%

of A and 99.2, 99.5, 99.4% of H.

Analyses were also performed

(n = 3) separately for losartan potas-

sium tablets and losartan potassium

and hydrochlorothiazide tablets. The

assay results for losartan potassium

were 99.1, 99.3, 99.5%. The assay re-

sults for losartan potassium and

hydrochlorothiazide were, 98.8, 99.0,

98.6% for losartan potassium and 99.0,

99.2, 99.0% for hydrochlorothiazide.

This shows that, adopted UPLC meth-

od can also be used separately for assay

estimation of Losartan potassium tab-

lets, and simultaneous estimation of

losartan potassium and hydrochloro-

thiazide tablets.

Validation of DevelopedStability-Indicating method

The relative standard deviation for

replicate injections of L, A and H was

0.2, 0.5 and 0.2% for L, A and H. The

USP resolution factor between A and H

was 5.7 and that between H and L was

19.8 indicating the suitability of the sys-

tem.

The limit of quantication was 0.39,

0.40 and 0.11 lg mL-1 and the limit ofdetection was 0.13, 0.15 and

0.04 lg mL-1 for L, A and H for 5 lLinjection volume.

The percentage RSD values for the

precision study were 0.4, 0.5, 0.4% (in-

ter-day precision) and 0.8, 0.8, 1.0%

(intra-day precision) for L, A and H

conrming a good precision.

The linear calibration plot for this

method was obtained over the calibra-

tion ranges 25.0875.24 lg mL-1 for L,25.0575.15 lg mL-1 for A, 6.2618.77 lg mL-1 for H. The correlationcoecient was greater than 0.999 for all

three drugs. The mean regressions

equations were found as A = 26538

C - 13476 (r2 = 0.9993, n = 5), A =

12541 C - 4094 (r2 = 0.9990, n = 5)

and A = 43100 C - 14825 (r2 = 0.9999,

n = 5) for L, A and H. A = aC + b,

where A is the peak area ratio of

the drugs, a is the slope, b is the inter-

cept and C is the concentration

(lg mL-1). The results show an excellentcorrelation existed between the peak

area and the concentration of the analyte

(Fig. 1).

The percentage recovery of L, A and

H in pharmaceutical dosage forms ran-

ged from 99.1 to 100.5% for L, 99.0 to

100.8% for A, 99.1 to 100.5% for H.

Excellent recoveries were made at each

added concentration.

For the robustness study, all changes

of conditions the system suitability

parameters were evaluatedone condi-

tion tested, the other conditions at the

optimum values. Relative standard

deviation for replicate injections of L, A

and H for all deliberate changes of con-

ditions was within 0.20.6%, 0.10.4%

and 0.20.8%. The USP resolution fac-

tor between A and H was within 4.86.5

and the USP resolution factor between

Limited Short Communication Chromatographia 2009, 70, August (No. 3/4) 649

H and K was within 18.120.3, thus

proven that the method is robust.

The solution stability and mobile

phase stability experiment data con-

rmed that sample solutions and mobile

phase used during the assay were stable

up to 48 h.

Figure 2 shows the specics. The

resolution factor for the drug peaks was

>1.5 from the nearest resolving peak

(RRT * 1.26).Intermediate precision conrmed

that separation between L, A and H was

satisfactory. The resolution factor be-

tween the A and H peaks was >4.0 and

between H and L was >15.0 indicat-

ing that the method remained selective

for all components under tested condi-

tions.

Conclusions

The novel isocratic UPLC method

proved to be simple, linear, precise,

accurate, robust, rugged and specic.

The total runtime was 3 min within

which three drugs and their degradation

products were separated. The method

was validated showing satisfactory data

for all validation parameters tested. The

developed method was stability indicat-

ing and could be used for simultaneous

quantitative determination of the drugs

in the presence of degradation products.

The method can also be useful for the

estimation of losartan potassium tablets,

and the simultaneous estimation of lo-

sartan potassium and hydrochlorothia-

zide tablets.

Acknowledgments

The authors wish to thank the manage-

ment of Dr. Reddys Laboratories for

supporting this work. Cooperation from

colleagues and of Research & Develop-

ment and Analytical Research & Devel-

opment of Dr. Reddys Laboratories is

appreciated.

References

1. International Conference on Harmoniza-tion (2003) Stability testing of new drugsubstances and products Q1A (R2).International Conference on Harmoniza-tion, IFPMA, Geneva

2. Singh S, Bakshi M (2000) Pharm TechOnline 24:114

3. International Conference on Harmoniza-tion (1996) Photo stability testing of newdrug substance and products Q1B. Inter-national Conference on Harmonization,IFPMA, Geneva

4. Bakshi M, Singh S (2002) J Pharm Bio-med Anal 28:10111040. doi:10.1016/S0731-7085(02)00047-X

5. SinghS, SinghB,BahugunaR,WadhwaL,Saxena R (2006) J Pharm Biomed Anal41:10371040. doi:10.1016/j.jpba.2006.01.030

Fig. 1. A typical UPLC chromatogram of L, A and H drug product

Fig. 2. A typical UPLC chromatogram of (a, b) stressed L, A and H drug product. (a) NaOHstressed L, A and H drug product. (b) Hydrogen peroxide stressed L, A and H drug product

650 Chromatographia 2009, 70, August (No. 3/4) Limited Short Communication

6. Carstensen JT, Rhodes CT (eds) (2000)Drug stability principles and practices,3rd edn

7. Lastra OC, Lemus IG, Sanchez HJ, PerezRF (2003) J Pharm Biomed Anal 33:175180. doi:10.1016/S0731-7085(03)00347-9

8. Shankar MB, Metha FA, Bhatt KK,Metha RS, Geetha M (2003) Indian JPharm Sci 65:167170

9. Prabhakar AH, Giridhar R (2002) JPharm Biomed Anal 27:861866. doi:10.1016/S0731-7085(01)00536-2

10. Erk N (2001) J Pharm Biomed Anal24:603611. doi:10.1016/S0731-7085(00)00434-9

11. Carlucci G, Palumbo G, Mazzeo P,Quaglia MG (2000) J Pharm BiomedAnal 23:185189. doi:10.1016/S0731-7085(00)00268-5

12. Hertzog DL, McCAerty JF, Fang X,Tyrrell RJ, Reed RA (2002) J Pharm

Biomed Anal 30:747760. doi:10.1016/S0731-7085(02)00385-0

13. Prasad CVN, Parihar C, Sunil K, Pari-moo P (1998) J Pharm Biomed Anal17:877884. doi:10.1016/S0731-7085(97)00241-0

14. Al-Ghannam SM (2006) J Pharm BiomedAnal 40:151156. doi:10.1016/j.jpba.2001.12.001

15. Agrekar AP, Powar SG (2000) J PharmBiomed Anal 21:11371142. doi:10.1016/S0731-7085(99)00210-1

16. Lamprecht G, Kraushofer T, StoschitzkyK, Linder W (2000) J Chromatogr B740:219226. doi:10.1016/S0378-4347(00)00080-3

17. Taomin H, Zhang H, Bei Y, Luping S,Xiaowei Z, Gengli D (2004) J PharmBiomed Anal 41:644648. doi:10.1016/j.jpba.2005.12.007

18. Razak OA (2006) J Pharm Biomed Anal34:433440. doi:10.1016/S0731-7085(03)00497-7

19. Lusina M, Cindric T, Tomaic J, Peko M,Poziac L, Musulin N (2005) Int J Pharm291:127137. doi:10.1016/ijpharm.2004.07.050

20. Sidiko E, Sevil MC, Sedef A (2003) JPharm Biomed Anal 33:505511. doi:10.1016/S0731-7085(03)00252-8

21. Murillo Pulgarin JA, Molina AA, Prez-Olivares Nieto G (2004) Anal Chim Acta518:3743. doi:10.1016/j.aca.2004.05.034

22. Takubo T, Okada H, Ishii M, Hara K,Ishii Y (2004) J Chromatogr B 806:199203. doi:10.1016/j.jchromb.2004.03.060

23. Erk N (2003) J Chromatogr B 784:195201. doi:10.1016/S1570-0232(02)00759-6

24. Sathe SR, Bari SB (2007) ACTA Chro-matogr 19:270278

Limited Short Communication Chromatographia 2009, 70, August (No. 3/4) 651

Simultaneous Determination of Losartan Potassium, Atenolol and Hydrochlorothiazide in Pharmaceutical Preparations by Stability-Indicating UPLCAbstractIntroductionExperimentalChemicalsEquipmentChromatographic ConditionsPreparation of Stock SolutionsPreparation of Sample SolutionSystem Suitability Solution CriteriaStress StudiesMethod ValidationLimit of Detection and Limit of QuantificationPrecisionLinearityAccuracyRobustnessSpecificity and SelectivitySolution Stability and Mobile Phase StabilityResults and DiscussionForce Degradation StudiesMethod Development and Optimization of Stability Indicating Assay MethodValidation of Developed Stability-Indicating methodConclusionsFig1Fig2

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