do

Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS University, S.S. Nagar, Mysore 570015, India

a r t i c l e i n f o

Article history:

Received 23 February 2013

Hydrochlorothiazide (HCT), 6-chloro-3,4-dihydro-2H-1,2,4-

liquid chromatographic5e8 polarographic techniques.9

Amlodipine (AMLO)chemically, 2-[(2-aminoethoxy)methyl]-

4-(2-chlorophenyl)-1, 4- dihydro-6 methyl-3, 5-pyridine dicar-

boxylic acid 3-ethyl, 5-methyl ester, is an antihypertensive and

an antianginal agent in the form of the besylate salt. Its activity

review reveals various methods for estimation of amlodipine

angiotensin II receptor type 1 (AT1) receptor antagonist. Los-

artan administration results in a decrease in total peripheral

resistance and cardiac venous return. However, several

methods have been described for the determination of los-

artan potassium drug substance in tablets. Various methods

* Corresponding author. Tel.: 91 (0) 8212548353, 91 (0) 9886658520 (mobile); fax: 91 (0) 8212548359.ffmail.com (A.R. Tengli).

Available online at www.sciencedirect.com

.e

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 3e3 8E-mail addresses: anandrtengli@gmail.com, anandrtengli@redibenzothiadiazine-7- sulphonamide-1,1- dioxide is the potent

orally diuretic and antihypertensive agent related to chloro-

thiazide. This inhibits active chloride reabsorption and thus

increases the excretion of sodium chloride and water. There

aremany published determinationmethods for HCT in tablets

using spectrophotometric,1e3 fluorodensistometric,4 gas and

alone and its combination are by liquid chromatography

coupled with UV,10,11 voltametric,12 mass spectrophoto-

metric,13,14 HPLC,15e19 HPTLC.20

Losartan potassium is monopotassium salt of 4-butyl-4-

chloro-1- [[2-(1H-tetrazole-5-yl)[1,1-biphenyl]-4-yl]methyl]-

1H-imidazole-5-methanol. It is a selective, competitive1. Introduction resides mainly in the () isomer, that inhibits transmembraneinflux of calcium ions into vascular smooth muscle. LiteratureAccepted 8 March 2013

Available online 29 March 2013

Keywords:

HPLC

Hydrochlorothiazide

Amlodipine

Losartan and telmisartan

Simultaneous estimation0976-1209/$ e see front matter Copyright http://dx.doi.org/10.1016/j.ijcas.2013.03.003a b s t r a c t

A simple, sensitive and specific liquid chromatographic method with UV detection (230 nm)

was developed for the simultaneous estimation of hydrochlorothiazide, amlodipine and

losartan in tablet dosage form and telmisartan as an internal standard. Separation was

achievedwith a phenomenex luna 5mCN100R, 250 4.60mm5micron size column, ambienttemperature with a low pressure gradient mode with mobile phase containing acetonitrile,

water and 0.4% of potassium dihydrogen phosphate buffer pH 2.7 adjusted with ortho-

phosphoric acid (45:35:20). The flow rate was 1 mL min1 and eluent was monitored at

230 nm. The selected chromatographic conditions were found to effectively separate hy-

drochlorothiazide, amlodipine and losartan with retention time of 3.9, 4.9 and 5.8 min

respectively. The linearity range of hydrochlorothiazide, amlodipine and losartan found in

the range of 12.5e62.5 mg ml1, 2.5e12.5 mg ml1 and 50e250 mg ml1 respectively. The pro-

posedmethodwas found to be accurate, precise, reproducible and specific and it can also be

used for routine quality-control analysis of these drugs in combination tablets.

Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rightsreserved.Anandkumar R. Tengli*, B.M. Gurupadayya, Neeraj SoniOriginal Article

Simultaneous estimation of hyand losartan in tablet dosage f

journal homepage: www2013, JPR Solutions; Publirochlorothiazide, amlodipine,rm by RP-HPLC

l sevier .com/locate/ i jcasshed by Reed Elsevier India Pvt. Ltd. All rights reserved.

developed are HPLC,21e24 spectrophotometric,25e27 capillary

electrophoresis (CE),28 voltamatric,29 capillary zone electro-

phoresis HPTLC and liquid chromatography electrospray

ionization tandem mass spectrometry.

A new combination dosage form of HCT, AMLO and LOSAT

is indicated for the treatment and management of hyperten-

sion. The focus of present study was to develop and validate

simple precise.

Aristo Pharmaceutical Ltd. Mumbai, India. Potassium dihy-

system (Kyoto Japan), provided with high speed auto sampler,

stock solutions were stored at 4 C protected from light.

2.4. Preparation of calibration plot

Standard solutions were freshly prepared from the stock solu-

tion by diluting with mobile phase as 2, 4, 6, 8, 10 mg mL1 Hy-drochlorothiazide,5,10, 15, 20, 25mgmL1 amlodipineand3,6, 9,12, 15 mg mL1 losartan and IS (internal standard), respectively.Each solution was injected in triplicate and chromatographed

under the chromatographic conditions specified above. Telmi-

sartan (8 mg mL1) was used as internal standard for determi-

ters (Millipore, Milford, MA, USA). From the filtered solution,

rea

379

304

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 3e3 834Table 1 e Results of system suitability study.

Hydrochlorothiazide Amlodipine

Retentiontime (min)

Peak area Retentiontime (min)

Peak a

Mean 3.99 2,439,961 4.86 2,788,

SD 0.016 1,308,330 0.040 1,293,column oven, degasser and UV detector to provide a compact

and convenient for LC with LC solution soft ware chromato-

graphic analysis were performed on phenomenex luna 5m CN

100R, 250 4.60 mm 5 micron size column. The flow rate was1 mL min1 and the injection volume 20 ml, UV detection wasperformed at 230 nm. Peak identity was confirmed by both

retention time comparison and comparison spectra obtained

from the UV detector.

2.3. Standard preparation

Separate stock solutions of 1000 mg mL1 of Hydrochlorothia-zide, amlodipine and losartan were prepared in Acetonitrile,

then 1 mL of stock solution into a 10 mL of standard volu-

metric flask and diluted with mobile phase. The prepareddrogen phosphate, Orthophosphoric acid AR grade purchased

from Merck. HPLC solvents like, Acetonitrile, Methanol and

water from Ranchem, Mumbai. The pharmaceutical dosage

form containing 12.5 mg HCT, 2.5 mg AMLO and 50mg LOSAT,

Nusar-AMH 20 tablets (Emcure Pharmaceuticals Ltd.) pur-

chased from a local drug store. Telmisartan which was

employed as an internal standard (IS) was obtained from

Ranbaxy Laboratories, New Delhi.

2.2. Instrumentation

The development and validation of the assay was performed

on a Shimadzu LC2010 integrator a 4-liquid gradient HPLC2. Experimental

2.1. Chemicals and reagents

Working standards, amlodipine and losartan were from Aur-

bindo Laboratories, Hyderabad, India. HCT and TEL were fromRSD 0.408 53.620 0.827 46.381aliquots of appropriate volume were transferred to 10 mL

volumetric flasks and diluted to volume with mobile phase to

furnish the concentration range listed in Table 1

3. Method validation

3.1. Linear range

The linearity of the method was evaluated by analyzing

different concentration of the drugs. According to ICH rec-

ommendations,35 at least five concentrationsmust be used. In

this study five concentrations were chosen, in the ranges

12.5e62.5, 2.5e12.5 and 50e250 mg mL1 hydrochlorothiazide,amlodipine and losartan, respectively.

3.2. Accuracy and precision

The accuracy of the method was determined by recovery ex-

periments using the standard addition method. Each solution

was injected in triplicate and percentage recovery was

Losartan Telmisartan (IS)

Retentiontime (min)

Peak area Retentiontime (min)

Peak area

5.83 3,363,027 7.24 6,519,518

0.031 1,637,458 0.0609 15910nation of mixtures of Hydrochlorothiazide, amlodipine and

losartan with Telmisartan. Linear relationships were obtained

when drug to- internal standard peak area ratios were plotted

against the corresponding concentrations for each drug.

2.5. Sample preparation

Average weight was calculated by weighing 20 tablets. The

tablets were crushed into homogenous powder. A quantity of

powder equivalent to one tablet containing 12.5 mg of hy-

drochlorothiazide, 2.5 mg of amlodipine and 50mg of losartan

was transferred into a 100 mL volumetric flask. To this flask,

50 mL of Methanol were added, and the solution was soni-

cated for 25 min with intermittent shaking. The solution was

cooled to ambient temperature. Then the volume was made

up with Acetonitrile and centrifuged at 10,000 rpm for 10 min.

The centrifuged solution filtered through a 0.45 mm Nylon fil-0.537 48.689 0.853 0.24403

The selectivity of the method was evaluated by assessingACE inhibitor contain weak benzene chromophores and are

4.1.1. System suitabilityTheRSDvaluesofpeakareaand retention time for drugsand IS

are within 2% indicating the suitability of the system (Table 2).

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 3e3 8 35whether excipients present in the pharmaceutical formula-

tions interfered with the analysis. A placebo for each tablet

was prepared by mixing the respective excipients, and solu-

tions were prepared by following the procedure described in

the section on sample preparation. The commonly used tablet

excipients did not interfere with the method.

3.5. Robustness

Robustness is a measure of capacity of analytical methods to

remain unaffected by small but deliberate variation of the

operating conditions. This was tested by studying the effect of

changingmobile phase pH by0.2, the amount of buffer in themobile phase by 2%, and detector wavelength by 2 nm.

4. Results and discussion

To establish and validate an efficient method for analysis of

these drugs in pharmaceutical formulations, preliminary tests

were performed with the objective of selecting optimum

chromatographic conditions. The separation was tried using

either columns described previously in the literature or alter-

native stationary phases. The main problems encountered

during these investigations were lack of resolution between

hydrochlorothiazide, Amlodipine, Losartan and IS. To solve

these problems, three columns, C18, C8, and CN,were used for

simultaneous determination of the drugs. The best resolution

and peak shape, without excessive tailing, were obtained by

use of the CN column. The effect of both mobile phase

composition, flow rate and pH were also studied. The best

resolution with reasonable retention time was obtained with

mobile phase containing Acetonitrile, water and 0.4% of po-

tassium dihydrogen phosphate buffer pH 2.7 adjusted with

orthophosphoric acid (45:35:20) with 1.0 mL min1 flow rate.To avoid multiple peaks of peptides on reversed phase col-calculated. The precision of the method was assessed by

studying intra-day and inter-day variation. In the intra-day

studies, standard and sample solutions were analyzed in

triplicate on the same day and percentage RSDwas calculated.

In the inter-day studies, standard and sample solutions were

analysed in triplicate on three consecutive days and per-

centage RSD were calculated.

3.3. Limits of detection (LOD) and limit quantitation(LOQ)

In accordance with ICH recommendations, the approach

based on the standard deviation of the response and the slope

of the calibration plots was used to determine detection and

quantification limits. LOD and LOQ values were estimated as

[(standard deviation of repeatability)/(Slope of the regression

equation)] by multiplying with 3.3 and 10 respectively. The

values obtained are given in Table 1.

3.4. Selectivityumns, the pH must be controlled with buffers, for example

potassium dihydrogen phosphate. A major reason for using acharacterized by low molar absorptivity, so the detector was

set at 230 nm to increase the sensitivity of the method. The

specificity of the method is illustrated in (Figs. 1 & 2), which

indicates separation of the compoundswas complete. Average

retention times standard deviation for IS, HCT, AMLO andLOSAT were 3.9 0.016, 4.9 0.040, 5.8 0.031, and7.2 0.060 min, respectively, for six replicate analyses. Indetermination of accuracy and precision, recovery was

100 2%, which indicates the method is accurate, and intra-day and inter-day variation, as RSD, were no more than

1.25%, indicating the method is precise. In determination of

the robustness of themethod, slight variation of mobile phase

pH, amount of buffer, in the mobile phase, and detector

wavelength had no significant effect on chromatographic

resolution.

4.1. Method validationconcentration of 0.5 mMwas to achieve maximum sensitivity

of UV detection at low wavelengths. Members of this class of

Fig. 1 e HPLC Chromatogram of pure drug.Fig. 2 e HPLC Chromatogram of tablet formulation.

Table 2 e Intra-day and inter-day precision and accuracy of HCT, RPL and TEL.

Name ofthe drug

Actualconcentration

(mg mL1)

Intra-day Inter-day

Found concentration(mg mL1) SD

RSD (%) RME (%) Found concentration(mg mL1) SD

RSD (%) RME (%)

HCT 13 12.77 0.119 0.933 0.417 12.788 0.083 0.648 0.29025.5 25.39 0.097 0.385 0.172 25.562 0.241 0.945 0.42337.5 37.42 0.063 0.168 0.075 37.444 0.044 0.117 0.052

AMLO 2.5 2.458 0.038 1.560 0.698 2.482 0.008 0.337 0.1515 4.876 0.031 0.642 0.287 4.876 0.078 1.599 0.7157.5 7.452 0.043 0.580 0.260 7.442 0.071 0.952 0.426

LOSAT 50 49.598 0.377 0.760 0.340 49.584 0.650 1.310 0.586100 99.678 0.231 0.232 0.104 99.792 0.139 0.140 0.062150 149.698 0.191 0.128 0.057 149.574 0.375 0.251 0.112

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 3e3 8364.1.2. LinearityThe calibration curves were prepared by plotting the peak

areas of the drug to IS which were linear in the range of

12.5e62.5, 2.5e12.5 and 50e250 mg mL1 HCT, AMLO andLOSAT, respectively. Peak area ratios and concentrationswere

subjected to least square linear regressionanalysis to calculate

the calibration equations and correlation coefficients. The

mean regression equations were found as y 0.125x 0.126(R2 0.990, n 6), y 0.132x 0.106 (R2 0.990, n 6) andy 0.163x 0.143 (R2 0.991, n 6) for HCT, AMLO and LOSAT,respectively. y ax bwhere y is thepeakarea ratio of drugs,a is the slope, b is the intercept and x is the concentration

of the measured solution in mg mL1. The result shows thatthere is an excellent correlation between the peak area ratios

and the concentrations of drugs in the range tested.

4.1.3. LOD and LOQThe LODwas 0.03 mgmL1 for HCT, 0.03 mgmL1 for AMLO and0.108 mg mL1 for LOSAT at a signal to noise ratio of 3.1. The

limit of quantification was determined as 0.1 HCT, 0.1 mgmL1

for AMLO and 0.228 mgmL1 for LOSAT at a signal to noise ratioof 10:1.

4.1.4. PrecisionIntra-day precision was performed by relative standard devi-

ation of five repeated assayes of samples at the threeTable 3 e Results of recovery studies by standard addition me

Name of the drug Amount of drugin tablet (mg)a

Amount of puredrug added (mg)

HCT 37.5 37

37.5 37.5

37.5 38

AMLO 7.5 7

7.5 7.5

7.5 8

LOSAT 150 145

150 150

150 155

a Nusar-AMH tablet (12.5 mg of HCT, 2.5 mg AMLO, 50 mg LOSAT).

b Five independent analyses.

c Standard deviation.concentration levels. Inter-day precision was determined by

analyzing the same set of samples of five different days. The

RSD values were found to be 0.117e0.945% for HCT,

0.337e1.599% for AMLO and 0.128e1.310% for LOSAT respec-

tively, indicating good precision (Table 2).

4.1.5. RecoveryTo examine the accuracy of the method, recovery studies

were carried out by standard addition method. The percent

recovery of the added standard to the assay samples was

calculated from:

Recovery % C1 Cu=Ca 100Were C1 is the total concentration of analyte found; Cu is the

concentration analyte present in the formulation; and Ca is

the concentration added to the formulation. The average

percent recoveries recoveries obtained as 99.03e99.90% indi-

cate good accuracy of the method (Table 3)

4.1.6. SpecificityThe specificity of the RP-HPLCmethod was determined by the

complete separation of HCT, AMLO, LOSAT and IS as show in

(Figs. 1 & 2) with parameters like retention time (tR), resolu-

tion (Rs) and tailing factor (T ). The peaks obtained for HCT,

AMLO, LOSAT and IS were sharp and have a clear baseline

separation.thod.

Total found(mg)b (mean SDc)

RSD (%) Recovery of puredrug added (%)

74.39 0.045 0.061 99.7074.948 0.046 0.061 98.3775.45 0.0391 0.051 99.8814.45 0.026 0.179 99.3114.92 0.049 0.326 98.9615.45 0.023 0.148 99.47294.98 0.132 0.045 99.89299.9 0.079 0.026 99.93304.92 0.061 0.020 99.94

Acknowledgement

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Mysore, for providing all facilities to complete this research

work.Conflicts of interest

All authors have none to declare.4.1.7. RobustnessTo ensure the insensitivity of the HPLC method to minor

changes in the experimental conditions it is important to

demonstrate robustness of the method. None of the modifi-

cations caused a significant change in the resolution between

the drugs and IS, peak area RSD, USP tailing factor, peak width

or theoretical plates.

HPLC Chromatograms.

5. Conclusion

A simple, rapid, and reliable LC method has been established

for simultaneous determination of HCT, AMLO and LOSAT

either alone or in their ternary formulations. The method has

several advantages, including rapid analysis, a simple mobile

phase, simple sample preparation, and improved sensitivity.

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i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 3e3 838

Simultaneous estimation of hydrochlorothiazide, amlodipine, and losartan in tablet dosage form by RP-HPLC1. Introduction2. Experimental2.1. Chemicals and reagents2.2. Instrumentation2.3. Standard preparation2.4. Preparation of calibration plot2.5. Sample preparation

3. Method validation3.1. Linear range3.2. Accuracy and precision3.3. Limits of detection (LOD) and limit quantitation (LOQ)3.4. Selectivity3.5. Robustness

4. Results and discussion4.1. Method validation4.1.1. System suitability4.1.2. Linearity4.1.3. LOD and LOQ4.1.4. Precision4.1.5. Recovery4.1.6. Specificity4.1.7. Robustness

5. ConclusionConflicts of interestAcknowledgementReferences