World Journal of Pharmaceutical ReseaRch Siddardha et al ...

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Siddardha et al. World Journal of Pharmaceutical Research

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR

THE SIMULTANEOUS ESTIMATION OF IRBESARTAN AND

HYDROCHLOROTHIAZIDE IN PHARMACEUTICAL DOSAGE

FORM BY RP-HPLC

M.Siddardha*, K.Mangamma And G.Manikumar

Department of Pharmaceutical Analysis, University College of Pharmacy, JNTU, Kakinada,

E.G.Dist, A.P, India.

ABSTRACT

A simple, specific, accurate and precise reverse phase high pressure

liquid chromatographic method has been developed for the

simultaneous determination of Irbesartan and Hydrochlorothiazide in

tablet dosage form by using Hypersil BDS C18, 250mm X 4.6 mm, 5µm

column. The samples were analyzed by using Methanol: water (80: 20)

v/v as a mobile phase at the flow rate of 1.0 ml/min in isocratic mode

and detection wavelength is 257 nm. Both the drugs were eluted within

5 minutes and gave sharp peaks with high theoretical plate count and

low tailing factor. The retention time for Irbesartan and

Hydrochlorothiazide was found to be 2.872 and 3.943 min

respectively. The validation was carried according to ICH guidelines.

Calibration curve was linear with correlation coefficient of 0.997 and

0.999 over a concentration range of 60µg/mL-240µg/mL and 5-20µg/ml for Irbesartan and

Hydrochlorothiazide respectively. The percent recovery was 99.80 for Irbesartan and 99.80

for Hydrochlorothiazide indicating accuracy and reliability of method. So the method can be

used for estimation of combination of these drugs in tablet dosage form.

Key words: Irbesartan, Hydrochlorothiazide, RP-HPLC

1. INTRODUCTION

Irbesartan is a non-peptide compound, chemically described as a 2-butyl-3-[p-(o-1H-tetrazol-

5ylphenyl) benzyl]-1,3-diazaspiro[4.4]non-1-en-4-one. Its empirical formula is C25H28N6O,

World Journal of Pharmaceutical ReseaRch

Volume 3, Issue 1, 1060-1075. Research Article ISSN 2277 – 7105

Article Received on 21 October2013 Revised on 23 November 2013, Accepted on 26 December 2013

*Correspondence for

Author:

M.Siddardha,

Department of Pharmaceutical

Analysis, University College of

Pharmacy, JNTU, Kakinada,

E.G.Dist, A.P, India.

[email protected]

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and its structural formula is given in Fig.1. Hydrochlorothiazide is 6-chloro-3, 4-dihydro-2H-

1, 2, 4-benzothiadiazine-7-sulfonamide 1,1dioxide. Its empirical formula is

C7H8ClN3O4S2 and its structural formula is given in Fig.2. Irbesartan is a type of medicine

called an angiotensin II receptor antagonist. It works by preventing the action of a hormone in

the body called angiotensin II. Irbesartan blocks the receptors that angiotensin II acts on, and

so prevents its actions. The main result of this is that the peripheral blood vessels are allowed

to widen, which means that there is more space and less resistance in these blood vessels.

This lowers the pressure inside the blood vessels.

Hydrochlorothiazide is a type of medicine known as a thiazide diuretic. Thiazide diuretics act

in the kidneys, where they increase the production of urine. They work by causing the

kidneys to increase the amount of salts, such as potassium and sodium that are filtered out of

the blood and into the urine. When these salts are filtered out of the blood by the kidneys,

water is also drawn alongside. As hydrochlorothiazide increases the removal of salts from the

blood, it also causes more water to be drawn out of the blood and into the urine.

A combination of 3000mg of Irbesartan and 12.5mg Hydrochlorothiazide is available

commercially as tablets. This combination is used in the treatment of Hypertension.

Literature survey revealed that UV spectrophotometric, HPLC methods, HPTLC and

Capillary electrophoresis methods are available for estimation of MET and FENO

individually and also in combination with other drugs [6-15]. Few reports are also available

on the simultaneous estimation of Irbesartan and Hydrochlorothiazide using techniques like

Spectrophotometric and RP-HPLC but they are suffering from their own drawbacks like high

retention time or poor resolution or use of expensive solvents etc.

The aim of present work is to develop and validate a new, simple, better and economical

method for the simultaneous estimation of Irbesartan and Hydrochlorothiazide in tablet

dosage form by RP-HPLC with improved conditions and parameters for routine use in the

laboratories. The chemical structures of the assayed compounds are given below.

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Fig-2 Structure of Irbesartan

Fig-3 Structure of Hydrochlorothiazide

2. Reagents and materials

Pure drugs samples of Irbesartan and Hydrochlorothiazide were obtained as a gift samples

from Aurobindo Pharma Ltd, Hyderabad, Andhrapradesh, India. HPLC grade methanol,

acetonitrile were procured from Merck. In-house Millipore water was used throughout the

study.

3. Instrumentation

The apparatus used in this study were Waters Alliance E2690 with empower 2 software and

UV-2489 detector with auto sampler HPLC.UV Double Beam spectrometer systronics UV

3000 was used to find the lambda maximum of the drugs. ADWA AD102 U pH meter,

ENERTECH SE60US ultrasonicator were also used.

Table-1 Chromatographic conditions

Parameters Optimised condition

Mobile Phase Methanol:water(80:20)

Column type Hypersil BDS C18, 250mm X 4.6 mm, 5µ

Flow rate 1.0ml/min

Detection wavelength UV at 257nm

Temperature Ambient

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Injection volume 20µl

Retention time Irbesartan-2.86min,Hydrochlorothiazide-3.94min

4. Determination of working wavelength (λ max)

Fig-3 Overlay spectra of both Irbesartan and Hydrochlorothiazide

To ascertain the maximum wavelength (λ max), the standard solutions were scanned between

200-400nm in UV spectrometer and the wavelength of 257nm is best suitable for both the

analytes (Irbesartan and Hydrochlorothiazide) and hence the experiment is carried out in the

wavelength of 257nm.

5. Preparation of solutions

5.1. Preparation of Mobile phase

800mL (80%) of methanol is mixed with the 200mL (20%) of nanopure water and filtered

through vacuum filtration using 0.45µm filter and degassed in a ultrasonic water bath for

10min.

5.2. Preparation of Diluent

Mobilephase was used as diluent.

5.3. Preparation of Standard stock solution

Standard stock solutions of Irbesartan and hydrochlorothiazide were prepared by dissolving

300mg of Irbesartan and25mg of hydrochlorothiazide were dissolved in 20 mL of diluent

separately and sonicated for 10min and made up the volume to 100mL with diluent and were

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labelled as standard stock solution-1(Irbesartan 3mg/mL) and standard stock solution-

2(hydrochlorothiazide 0.25mg/mL).

5.4. Preparation of mixed standard solution

0.4mL of standard stock solution-1, 0.4mL of standard stock solution-2 was transferred to 10

mL volumetric flask and made up the volume to 10mL with diluent.

5.5. Preparation of sample solution

Twenty tablets were weighed, ground in to a fine powder and mixed thoroughly. A quantity

of powder equivalent to 300mg of Irbesartan (25mg of hydrochlorothiazide) was weighed and

transferred in to a 100ml volumetric flask and was dissolved in the diluents. The volume was

made up to the mark with the same and the resulting solution was labelled as test stock

solution. A solution containing 120µg/ml of Irbesartan (10µg/ml of hydrochlorothiazide) was

prepared by appropriate dilution of the test stock solution. The solutions thus prepared were

filtered through 0.45µ membrane filter and the filtrate was sonicated for 10min.

6. Assay

20µL of test solution containing (120µg/mL of Irbesartan and 10µg/mL of

Hydrochlorothiazide) was injected three times at the optimised method conditions and the

chromatograms of three injections were recorded and the % Assay was calculated using the

following formula:

%Assay= Sample Area x Standard Wt x Tablet Avg Wt x 100

Standard Area x Sample Wt x Label Amt

7. Method validation

The developed method is validated by following the ICH guidelines with respect to

parameters such as system suitability, linearity, accuracy, precision, limit of detection, limit

of quantification, robustness and specificity.

7.1. System Suitability

To verify the working condition of analytical system whether it can give accurate and precise

results, 20µL of working standard solution containing 120µg/mL of Irbesartan and 10µg/mL

of hydrochlorothiazide was injected for 5 times and the chromatograms were taken for the

same and the results were tabulated.

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7.2. Linearity

The linearity of the Irbesartan and Hydrochlorothiazide was established over the range of

60µg/mL-240µg/mL and 5-20µg/mL respectively and the chromatograms were taken for the

same and calibration plot was drawn and regression analysis was performed and regression

coefficient was calculated.

7.3. Accuracy

The accuracy of the method was evaluated by recovery studies which were carried out by

standard addition method, where a known amount of sample was spiked with mixed standard

solution at three levels 50%,100% and 150%.At each levels recovery studies were performed

in triplicate and expressed as percentage recoveries.

7.3.1. Preparation of mixed standard solution

30mg of Irbesartan and 2.5mg oh hydrochlorothiazide were accurately weighed in to a 10 mL

volumetric flask and made up the volume with diluent. The resulting solution contains the

3000µg/mL of Irbesartan and 250µg/mL of hydrochlorothiazide.

7.3.2. Preparation of standard and test solutions

Mixed standard solutions containing 40, 80 and 120µg/mL of Irbesartan (5, 10 and 15µg/mL

of hydrochlorothiazide) respectively were prepared in triplicates, from the mixed standard

stock solution by appropriate dilutions. Sample solutions containing 40µg/mL of Irbesartan

(5µg/mL of hydrochlorothiazide) were prepared by appropriate dilution of sample stock

solution (containing 3000 µg/mL of Irbesartan and 250 µg/mL of hydrochlorothiazide).

7.3.3. Procedure for spiking

Spiking at 50%level was accomplished in triplicate, by adding 3ml of unfilterated sample

solution to 1.5mL of mixed standard stock solution in a test tube. The contents of the test tube

were shaken for some time and filtered through a whatmann filter in to a 10mL volumetric

flask and washed 2 times with diluent and made up the volume with the diluent and filtered

through 0.44 µm filter in to autoinjector vial.In same way the 100% and 150% spiking was

prepared by adding 3mL of unfiltered sample solution in to 3mL and 4.5mL of mixed

standard stock solution respectively in to a separate test tubes.

%Recovery = ((b-a)/c)*100

Where a=response of test solution

b=response of spiked solution

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c=response of standard solution

7.4. Precision

The precision was checked at levels of repeatability and also checked for both system

precision and method precision. In case of system precision only one preparation was done at

the level of 120µg/mL, injected on the same day for 5times and where as for interday

precision it was determined by injecting by preparing on different days. In case of method

precison five different preparations were done and both intra and inter day precison was

performed. The results of the repeatability and intermediate precision were calculated and

tabulated.

7.5. Limit of detection

7.2µg/mL of Irbesartan and 2µg/mL of hydrochlorothiazide was prepared by making

appropriate dilutions from standard stock solutions and injected in to HPLC system and

optimised chromatographic conditions; using signal to noise ratio, LOD was calculated.

LOD=3.3(σ/S)

σ = standard deviation

S=Slope of the calibration curve

7.6. Limit of quantification

18µg/mL of Irbesartan and 3.2µg/mL of hydrochlorothiazide was prepared by making

appropriate dilutions from standard stock solutions and injected in to HPLC system and

optimised chromatographic conditions, signal using signal to noise ratio, LOQ was calculated

LOD=10(σ/S)

σ = standard deviation

S=Slope of the calibration curve

7.7. Robustness

Robustness was performed by making deliberate changes in the chromatographic conditions

like

1) Change in flow rate (±0.2mL/min)

2) Change in detection wavelength (±2nm)

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7.8. Specificity

Specificity was performed by injecting placebo which was used for preparation of test

solution. This placebo was filtered through 0.44µm filter and injected in to HPLC system.

8. RESULTS AND DISCUSSION

To develop a precise, accurate and suitable RP-HPLC method for the estimation of Irbesartan

and Hydrochlorothiazide, different mobile phases were tried but the proposed

chromatographic conditions were found to be appropriate for quantitative determination.

Irbes

arta

n - 2

.869

Hyd

roch

loro

thia

zide

- 3.

942

AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Fig-4 Chromatogram of standard

Irbes

arta

n - 2

.869

Hyd

roch

loro

thia

zide

- 3.

942

AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Fig-5 Chromatogram of sample

AU

-0.008

-0.006

-0.004

-0.002

0.000

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Fig-6 Chromatogram of placebo

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8.1. Assay

The assay was performed on the tablet formulation and the % drug contents of Irbesartan and

hydrochlorothiazide were found to be 99.80 and 99.80% which were in acceptance limits

Table-2 Results of Assay

Parameters Irbesartan Hydrochlorothiazide

Sample Area 1246839 546612

Standard Area 1246748 546597

Standard Wt 300 25

Sample Wt 501 501

Average tablet wt 500 500

Label claim 300 25

%Assay 99.80 99.80

8.2. System suitability

The system suitability of the present method was accomplished from the resolution,

theoretical plates, assymetry factor of Irbesartan and hydrochlorothiazide at the optimised

conditions. The parameters were recorded and tabulated and were found to be within the

acceptance criteria.

Table-3 System suitability data of Irbesartan and hydrochlorothiazide

S.No Name Retention Time

Area Height USP Tailing

USP Plate count

1 Irbesartan 2.872 1246426 252167 1.12 8086.89

2 Hydrochlorothiazide 3.943 546725 91227 1.08 9873.71

8.3. Linearity

The linearity of the Irbesartan and Hydrochlorothiazide was established over the range of

60µg/mL-240µg/mL and 5-20µg/mL respectively and correlation coefficient of the respective

calibration curves were 0.997and 0.999 for Irbesartan and hydrochlorothiazide respectively

which were well within the acceptance criteria.

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Table-4 Linearity results for Irbesartan and Hydrochlorothiazide

Standard Irbesartan Hydrochlorothiazide

Concentration (µg/mL) Peak area Concentration

(µg/mL) Peak area

CC1 60 623030 5 273334

CC2 90 935298 7.5 410569

CC3 120 1246810 10 546585

CC4 150 1557240 12.5 683562

CC5 180 1870197 15 820392

CC6 210 2182190 17.5 947307

CC7 240 2394180 20 1094198

Statistica

l analysis

Slope -- 10036 -- 54427

Y-intercept -- 38667 -- 1943

Correlation

coefficient(r2) -- 0.997 -- 0.999

Fig-6Linearity curve of Irbesartan

Fig-7 Linearity curve of Hydrochlorothiazide

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8.4. Accuracy

The accuracy of the present method was evaluated from recovery studies, by standard

addition method which is performed at three levels of 50,100&150% levels. The mean

percentage recoveries at each level of Irbesartan and Hydrochlorothiazide were found to be

98.60 to 101.30 and 99.13 to 101.73 respectively.

Table-5 Accuracy data for Irbesartan

Spiked levels

Standard sample Spiked %Recovery

Mean % recovery Conc

(µg/mL) Peak area

Conc (40µg/mL) Peak area

Conc (µg/mL) Peak area

50 40

440111 430003

80

867895 97.98 98.66

441209 440034 872376 98.75

440093 440003 873540 99.27

100 80

841506

Mean

120

1285768 100.90 101.30

841517 1275768 99.71

841530 1305768 103.27

150 120

1251221

160

1690890 100.24

99.33 1262683 1687890 99.09

1271224 436680 1690890 98.66

Table-6 Accuracy data for Hydrochlorothiazide

Spiked levels

Standard Sample Spiked %Rec

overy

Mean %

recovery Conc (µg/mL)

Peak area

Conc (5µg/mL) Peak area

Conc (µg/mL) Peak area

50 5

273246 273557

10

547547 99.38

99.13 275465 275437 547980 98.74

276547 278965 550480 99.26

100 10

546474

Mean

15

836268 102.53

101.73 544568 822376 100.33

554365 843246 102.33

150 15

836578

20

1114915 100.28

99.68 862376 1134150 99.51

843246 275986 1112893 99.25

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8.5. Precision

The precision of the proposed method was established from the %RSDs of the percentage

assays of the drugs at the levels of repeatability (intra-day) and intermediate precision

(interday) and also at system and method precision. In system precision, the %RSDs of

Irbesartan and hydrochlorothiazide at intra-day precision were found to be 0.035% and

0.029% respectively, at inter-day precision were found to be 0.015 and 0.022% respectively.

In method precision, the %RSDs of Irbesartan and hydrochlorothiazide at intra-day precision

were found to be 0.067% and 0.041% respectively, at inter-day precision were found to be

0.012 and 0.036% respectively. As the %RSDs were found to be within the acceptance limit

(%RSD < 2%) at all the levels, the proposed method was said to be precise.

Table-7 Intra day and inter day precision data for System precision

Injection

No.

Intraday(peak area) Inter day(peak area)

Irbesartan Hydrochlorothiazide Irbesartan Hydrochlorothiazide

Injection-1 1246538 546435 1246650 546591

Injection-2 1246634 546876 1246974 546647

Injection-3 1246738 546647 1246556 546342

Injection-4 1246876 546576 1246889 546497

Injection-5 1247645 546675 1246997 546435

Average 1246886 546641.8 1246813 546650

Standard

deviation 442.349 160.5263 198.8535 121.510

%RSD 0.035 0.029 0.015 0.022

Table-8 Intra day and inter day precision data for Method Precision

Preparation

No.

Intraday(peak area) Inter day(peak area)

Irbesartan Hydrochlorothiazide Irbesartan Hydrochlorothiazide

Prep-1 1246982 546672 1246812 546342

Prep-2 1246567 546545 1246538 546687

Prep-3 1245345 546342 1246876 546456

Prep-4 1247657 546223 1246637 546785

Prep-5 1246545 546778 1246556 546778

Average 1246619 546512 1246684 546609.6

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Standard

deviation 842.6845 229.0557 152.608 200.1982

%RSD 0.067 0.041 0.012 0.036

8.6. Limit of detection and Quantification

8.6.1. Limit of detection for Irbesartan

The LOD is determined by the following formula

LOD=3.3 σ/S

=3.3(199.1213/10036)

=0.065µg/mL

8.6.2. Limit of Quantification for Irbesartan

The LOQ is determined by the following formula

LOQ=10 σ/S

=10(199.1213/10036)

=0.198 µg/mL

8.6.3. Limit of detection for Hydrochlorothiazide

The LOD is determined by the following formula

LOD=3.3 σ/S

=3.3(212.920/54427)

=0.013µg/mL

8.6.4. Limit of Quantification for Hydrochlorothiazide

The LOQ is determined by the following formula

LOQ=10 σ/S

=10(212.920/54427)

=0.039 µg/mL

The results were calculated and tabulated as follows

Table-9 Results of LOD and LOQ

Name of the Analyte LOD LOQ

Irbesartan 0.065µg/mL 0.198µg/mL

Hydrochlorothiazide 0.013µg/ml 0.039µg/mL

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8.7. Robustness

Robustness of the proposed method was done by making deliberate changes in the flow rate

and wavelength from the optimized conditions of the developed method and computing the

%RSD of the peak areas. The %RSD for Irbesartan and Hydrochlorothiazide at 0.8 ml/min

was found to be 0.87% and 0.67% respectively and at 1.2 ml/min they were found to be

0.67% and 0.23% respectively. At a wavelength of 255nm, the %RSDs for the drugs were

found to be 0.76%, 0.98% and at 259nm they were found to be 0.23% and 0.32%

respectively. As the %RSDs of peak areas and system suitability parameters were found to be

within the acceptance limit, the proposed method was said to be robust.

Table-10 Robustness data of Irbesartan and Hydrochlorothiazide

Parameter

Irbesartan Hydrochlorothiazide

Mean Rt

Mean Peak area

%RSD of peak areas

Mean Rt

Mean Peak area

%RSD of peak areas

Flow rate

(mL/min)

0.8 3.08 1286686 0.87 4.45 556789 0.67

1.0 2.86 1246767 0.71 3.94 546674 0.15

1.2 2.40 1278274 0.67 3.39 568798 0.23

Wavelength

(nm)

255 2.85 1244342 0.76 3.93 546323 0.98

257 2.86 1245127 0.65 3.94 546129 0.45

259 2.87 1247767 0.23 3.95 546189 0.32

As the peaks of analytes were well resolved and had no interference of excipients, it was

concluded that the proposed method was specific to the drugs under study. As all the

validation parameters studied, complied with the acceptance criteria, the proposed method

was said to be validated in accordance with ICH guidelines.

9. CONCLUSION

A simple and rapid reverse phase HPLC method was developed and validated according to

ICH guidelines for the simultaneous determination of Irbesartan and Hydrochlorothiazide in

tablet dosage form. The validation data signifies good specificity, accuracy, precision, and

reliability of the method. Because of its simplicity and low cost, the method can be used for

routine practices in the laboratories. The proposed method was found to be simple, precise,

accurate, rapid and specific for determination of Irbesartan and hydrochlorothiazide from

pure and its dosage forms. The mobile phase is simple to prepare and economical. The

sample recoveries in the formulation were in good agreement with their respective label

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claims and they suggested non-interference of formulation excipients in the estimation.

Hence, this method can be easily and conveniently adopted for routine analysis of Irbesartan

and hydrochlorothiazide in pure form and its dosage form and also can be used for

dissolution or similar studies.

10. REFERENCES

1) ICH Harmonized Tripartite guideline for validation of Analytical procedures Q2 (R1)

step 4 version Nov 2005.

2) David CL, Michael Webb. Pharmaceutical Analysis. London: Black well publishing;

1994. p. 2-4.

3) Gurdeep R. Chatwal, ShamK.Anand Instrumental methods of chemical analysis 5th

revised ed. Mumbai Himalaya publishing house 2009 p 2.570, 2.652.

4) B.K.Sharma. Instrumental methods of chemical analysis 25th Ed, Meerut, Goel

publishing house 2006 p 286-385.

5) Text book of Pharmaceutical Analysis, 7th edition by Asuthoshkar.

6) Hemamrutha, S.; Rambabu, R.; Vidhyadhara S. Simultaneous estimation and method

development of irbesartan and hydrochlorothiazide by RP HPLC .International Journal of

Pharmacy 2013; 3(2): 360.

7) Chabukswar, Aniruddha R.; Jagdale, Swati C.; Kuchekar, Bhanudas S.; Lokhande,

Pradeep D.; Shinde, Santosh N.; Ingale, Kunal D.; Kolsure, Anuja K. simultaneous

estimation of Hydrochlorothiazide and Irbesartan in tablet formulations by RP-HPLC-

PDA . Der Pharma Chemica2010; 2(4), 148

8) R. A. Mhaske .simultaneous determination of anti-hypertensive drugs Irbesartan,

Losartan, diuretics Hydrochlorothiazide and Chlorthalidone stability-indicating by HPLC

method. IJPSR 2012;3(4):1116-1123

9) Sane r. t. ; Francis m. ; Pawar s. ; et al. Determination of irbesartan in human plasma by

HPLC and a HPTLC method. Indian drugs coden indrba Source2003; 40(2): 104-110.

10) Tandogan, Ankara, Turkey; et al, assay for the antihypertensive drugs, irbesartan and

hydrochlorothiazide by high-performance liquid chromatographic method. Journal of

Chromatography B 02/2003; 784(1):195-201.

11) Lara F. Tutunji, Maha F. Tutunji, Mamoun I. Alzoubi, Manal H. Khabbas, Adi I. Arida .;

developed and validated for the simultaneous determination of irbesartan and

hydrochlorothiazide in human plasma by liquid chromatography/tandem mass

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spectrometric method. Journal of pharmaceutical and biomedical analysis Molecules2012,

17(3), 3461-3474.

12) Kishanta Kumar Pradhan .developed and validated for estimation of Irbesartan by

stability indicating UV-method. Asian Journal of Research in Chemistry 2010; 3(3):728-

731.

13) V.P. Rane ,K.R. Patil , J.N. Sangshetti , R.D. Yeole and D.B. Shinde, developed and

validated for the quantitative simultaneous estimation of Irbesartan (IRB) and

hydrochlorothiazide (HCTZ) in combined pharmaceutical dosage form by stability-

indicating liquid chromatography method. J ChromatogrSci (2010); 48 (7): 595-600.

14) A. Ramprasad Reddy, GVS. Kumar*, SB. Puranik, PeerlaGiriprasad and KA. Sridhar

developed as stability indicating reversed phase was developed and validated for

simultaneous estimation of irbesartan and hydrochlorothiazide in bulk and tablet

formulation. International journal of pharmaceutical, chemical and biological sciences 12,

2(4), 696-703.

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