Stability indicating Method Development and Validation for simultaneous estimation of Hydrochlorothiazide, Amlodipine and Olmesartan in tablet dosage form by using RP-HPLC

S. Ashutosh Kumar¹*, Manidipa Debnath², Dr. J.V.L.N. Seshagiri Rao³, Dr. D. Gowri Sankar⁴

¹Department of Pharmaceutical Analysis and Quality Assurance, AKRG College of Pharmacy, Nallajerla, West Godavari, 534112, A.P, India

²Department of Pharmaceutics, AKRG College of Pharmacy, Nallajerla, West Godavari, 534112, A.P, India

³Prof. Pharmaceutical Analysis, Yalamarty College of Pharmacy, Tarluwada Visakhapatnam, 530052, A.P, India

⁴Department of Pharmaceutical Analysis and Quality Assurance, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, 530003, A.P

*Corresponding Author E-mail: ashu.mpharm2007@gmail.com

ABSTRACT:

Objective: The present work was undertaken with the aim to develop and validate a rapid and consistent RP-HPLC method in which the peaks will be appear with short period of time as per ICH Guidelines. The proposed method was simple, fast, accurate and precise method for the Quantification of drug in the dosage form, bulk drug as well as for routine analysis in Quality control. Method: RP-HPLC method was developed and validated for simultaneous estimation of Hydrochlorothiazide, Amlodipine and Olmesartan in bulk drug and in combined dosage forms. The HPLC separation was achieved on a Symmetry C18 (4.6 X 150mm, 5m, Make: XTerra) or equivalent in an Isocratic Mode. The mobile phase was composed of TEA Buffer (40%) whose pH was adjusted to 3.5 by using Ortho Phosphoric Acid and Acetonitrile (60%) [HPLC Grade] The flow rate was monitored at 0.8ml per min. The wavelength was selected for the detection was 230 nm. The run time was 9min. Result: The retention time found for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan were 3.034 min., 4.062 min. and 5.165 min. respectively. The % recovery was found to be 99.3% - 101.7% for the drug Hydrochlorothiazide. The % recovery was found to be 98.3% - 99.3% for the drug Amlodipine. The % recovery was found to be 98.3% - 100.7% for the drug Olmesartan. The linearity was established in the range of 25 to 62.5ppm for the drug Hydrochlorothiazide and 10 to25ppm for the drug Amlodipine and 10 to100 ppm for the drug Olmesartan. The LOD for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan were found to be 0.009µg/ml, 0.06µg/ml and 0.06µg/ml respectively. The LOQ for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan were found to be 0.03µg/ml, 0.2µg/ml and 0.2µg/ml respectively. Conclusion: The proposed method was adequate sensitive, reproducible, and specific for the determination of Hydrochlorothiazide, Amlodipine and Olmesartan in bulk as well as in Tablet dosage form. The validation of method was carried out utilizing ICH-guidelines. The described RP-HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form. Overall the proposed method was found to be suitable and accurate for the Quantitative determination of the drug in Tablet dosage form. The method was simple, precise, accurate and sensitive and applicable for the simultaneous determination of Hydrochlorothiazide, Amlodipine and Olmesartan in bulk drug and in combined dosage forms.

KEYWORDS: Hydrochlorothiazide, Amlodipine, Olmesartan, ICH Guideline, RP-HPLC, LOD, LOQ.

In the literature, several analytical methods have been reported for the determination of OLME in biological fluids and pharmaceutical formulations, including liquid chromatography coupled with mass spectrometry, high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC) and Spectrophotometric estimation [2–5]. In these methods, OLME was analyzed either alone or in combination with other drugs. Amlodipine besylate (AMLO), chemically (RS)-3-ethyl 5-methyl 2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (6), is a long-acting calcium channel blocker that is used as an antihypertensive agent (7–9) (Figure 1). For AMLO, alone or in combination, several analytical methods such as HPLC and HPTLC have been reported for its estimation in biological fluids and pharmaceutical formulations [10–14]. Hydrochlorothiazide (HCTZ), chemically 6-chloro-1, 1-dioxo-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide, is a diuretic [15] (Figure 1). Various analytical methods have been reported for the analysis of HCTZ alone and in combination in biological fluids and pharmaceutical preparations [16–18]. A literature survey revealed that no method has been reported on these drugs in combined pharmaceutical dosage forms. Therefore, in the present study, an attempt was made to develop a simple, precise, accurate and robust HPLC method on the analysis of OLME, AMLO and HCTZ in bulk and pharmaceutical formulation.

MATERIALS AND METHOD ^[16-21]:

Chemicals and Reagents Used:

The following chemicals were procured for the process: Water [HPLC Grade], Acetonitrile [HPLC Grade], Methanol [HPLC Grade], Hydrochlorothiazide, Amlodipine and Olmesartan [Working standards], Orthophosphoric Acid and TEAall the chemicals were procured from STANDARD SOLUTIONS and the tablets were collected from the Local market.

Apparatus and Chromatographic Conditions:

Equipment: High performance liquid chromatography equipped with Auto Sampler and DAD or UV detector.

UV/VIS spectrophotometer: LAB INDIA UV 3000⁺

pH meter: Adwa – AD 1020

Weighing machine: Afcoset ER-200A

Temperature: Ambient

Column: Symmetry C₁₈ (4.6 X 150mm, 5mm, Make: XTerra) or equivalent

Phosphate Buffer: 0.1ml of TEA in 1000 ml Water [HPLC Grade] pH adjusted with Orthophosphoric Acid.

pH: 3.5

Mobile phase: Buffer: Acetonitrile (40: 60v/v)

Flow rate: 0.8 ml per min

Wavelength: 230 nm

Injection volume: 20ml

Run time: 9min.

Preparation of buffer:

The buffer solution was prepared by dissolving accurately weighed 0.1ml of TEAand transferred into a clean and dry 1000ml volumetric flask, dissolved and diluted with 1000ml water [HPLC Grade]. The final pH of the buffer was adjusted to 3.5 by using Ortho Phosphoric Acid.

Preparation of mobile phase:

The Mobile Phase was prepared by mixing 400 ml (40%) of the above buffer and 600 ml of Acetonitrile [HPLC Grade] (60%) and degassed in an ultrasonic water bath for 10 minutes. Then the resultant solution was filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation: The Mobile phase was used as Diluent.

Preparation of the Hydrochlorothiazide, Amlodipine and Olmesartan Standard and Sample Solution:

Preparation of Stock solution:

The stock solution was prepared by weighing accurately 12.5mg Hydrochlorothiazide, 5.0mg Amlodipine and 20.0mg Olmesartan and transferred into clean and dry 10ml volumetric flask. Initially About 7ml of diluent was added to the flask respectively and sonicated. The volume was made upto the mark with the same diluent. From the above prepared Stock solution pipette out 0.4ml of Hydrochlorothiazide, Amlodipine and Olmesartan solution and transferred into a clean and dry 10ml volumetric flask, the diluent was added upto the mark to get final concentration.

Preparation of Sample Solution:

The sample solution was prepared by weighing equivalently 190mg of Hydrochlorothiazide, Amlodipine and Olmesartan and transferred into a 10ml clean and dry volumetric flask and about 7ml of diluent was added and sonicated to dissolve it completely and the volume made up to the mark with the same solvent. From above prepared stock solution pipette out 0.4ml of solution and transferred into a clean and dry 10 ml volumetric flask, the diluent was added upto the mark to get final concentration. The standard and sample solutions were injected five times and the peak areas were recorded. The mean and percentage relative standard deviation were calculated from the peak areas.

System Suitability:

The Tailing factor for the peaks due to Hydrochlorothiazide, Amlodipine and Olmesartan in Standard solution should not be more than 2.0. The Theoretical plates for the Hydrochlorothiazide, Amlodipine and Olmesartan peaks in Standard solution should not be less than 2000. The system suitability of the method was checked by injecting five different preparations of the Hydrochlorothiazide, Amlodipine and Olmesartan standard. The parameters of system suitability were checked.

Assay calculation for Hydrochlorothiazide, Amlodipine and Olmesartan:

Assay % =

Where

AT = average area counts of sample preparation.

AS = average area counts of standard preparation.

WS = Weight of working standard taken in mg.

WT =Weight of test taken in mg.

DS =Dilution of standard solution

DT =Dilution of sample solution

P = Percentage purity of working standard

System Suitability Results for Hydrochlorothiazide:

1) The Tailing factor obtained from the standard injection was 1.1.

2) The Theoretical Plates obtained from the standard injection was 2876.9.

Assay Result for Hydrochlorothiazide:

System Suitability Results for Amlodipine:

1) The Tailing factor obtained from the standard injection was 1.2.

2) The Theoretical Plates obtained from the standard injection was 3741.4.

Assay Result for Amlodipine:

System Suitability Results for Olmesartan:

1) The Tailing factor obtained from the standard injection was 1.1.

2) The Theoretical Plates obtained from the standard injection was 3831.1.

Assay Result for Olmesartan:

VALIDATION DEVELOPMENT [22]

1. PRECISION:

It is a measure of degree of repeatability of an analytical method under normal operation and it is normally expressed as % of relative standard deviation (% RSD). The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. (Table no. 1)

Table no.1: Precision result for the drug Hydrochlorothiazide, Amlodipine and Olmesartan

Injection	Area for Hydrochloro- thiazide	Area for Amlodipine	Area for Olmesartan
Injection-I	7001650	944170	4286742
Injection-II	7000180	944201	4287137
Injection-III	7001711	944153	4286253
Injection-IV	7001861	944108	4285164
Injection-V	7001234	944067	4284208
Average	7001327	944139	4285901
Standard Deviation	682.12	52.7	1200.8
%RSD	0.009	0.005	0.02

Acceptance Criteria: The %RSD for the area of all the five injections should not be more than 2%.

2. INTERMEDIATE PRECISION/RUGGEDNESS:

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day by using different make column of same dimensions. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. (Table no. 2)

Table no.2: Ruggedness result for the drug Hydrochlorothiazide, Amlodipine and Olmesartan

Injection	Area for Hydrochloro-thiazide	Area for Amlodipine	Area for Olmesartan
Injection-I	6998931	939860	4275891
Injection-II	6991563	939762	4276186
Injection-III	6992013	939718	4274398
Injection-IV	6991894	939693	4273406
Injection-V	6990863	939815	4277811
Average	6993053	939769.6	4275537
Standard Deviation	3316.3	68.5	1699.2
%RSD	0.04	0.007	0.039

Acceptance Criteria: The %RSD for the area of all the five injections should not be more than 2%.

3. ACCURACY:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and value found. The standard solution with Accuracy -50%, Accuracy -100% and Accuracy -150% were injected into chromatographic system and calculated the amount found and amount added for Hydrochlorothiazide, Amlodipine and Olmesartan and further calculated the individual recovery and mean recovery values. (Table no. 3)

Acceptance Criteria: The %Recovery for each level should be between 98.0 to 102.0%.

4. LINEARITY:

It is the ability of the method to elicit test result that is directly proportional to analytic concentration within a given range. It is generally reported as variance of slope or regression line. It is determined by series of three to six injections of five of more standards. Different levels of solution were prepared and injected to the chromatographic system and the peak area was measured. Plotted a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient. The calibration curve was represented in fig. no. 2, 3 and 4. (Table no. 4)

Acceptance Criteria: The correlation coefficient should not be less than 0.99

Table No. 3. Accuracy result for the drug Hydrochlorothiazide, Amlodipine and Olmesartan

Drug	% Concentration	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	% Mean Recovery
Hydrochlorothiazide	50%	3583524	6.30	6.40	101.6%	100.9%
	100%	7001563	12.6	12.5	99.3%
	150%	10078298	17.7	18.0	101.7%
Amlodipine	50%	465931	2.5	2.47	99.07%	98.9%
	100%	935201	5.0	4.96	99.3%
	150%	1387547	7.5	7.37	98.3%
Olmesartan	50%	2182012	10.0	10.0	100.7%	98.9%
	100%	4285508	20.0	19.7	98.9%
	150%	6112231	28.7	28.2	98.3%

Table No. 4. Linearity Curve for the drug Hydrochlorothiazide, Amlodipine and Olmesartan

Linearity Level	Hydrochlorothiazide		Amlodipine		Olmesartan
	Conc.	Area	Conc.	Area	Conc.	Area
I	25ppm	3610874	10ppm	454409	40ppm	2162681
II	37.5ppm	5354435	15ppm	698979	60ppm	3207767
III	50ppm	6963745	20ppm	913122	80ppm	4296375
IV	62.5ppm	8709208	25ppm	1165154	100ppm	5198765
V	75ppm	10063272	30ppm	1353907	120ppm	6098386
Correlation Coefficient	0.9996		0.999		0.9992

5. LIMIT OF DETECTION:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value.

Limit of Detection for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan:

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio. Limit of detection is the lowest concentration of the substance that can be detected, not necessarily quantified by the method. (Regression statistics) The minimum concentration at which the analyte can be detected is determined from the linearity curve by applying the following formula.

Limit of detection (LOD) = 3.3

Where S – slope of the calibration curve

σ – Residual standard deviation

Calculation of S/N Ratio for Hydrochlorothiazide:

Average Baseline Noise obtained from Blank: 48 µV

Signal Obtained from LOD solution (0.26% of target assay concentration): 144 µV

S/N = 144/48 = 3.0

Calculation of S/N Ratio for Amlodipine:

Average Baseline Noise obtained from Blank: 48 µV

Signal Obtained from LOD solution (0.62% of target assay concentration): 145 µV

S/N = 145/48 = 3.02

Calculation of S/N Ratio for Olmesartan:

Average Baseline Noise obtained from Blank : 48 µV

Signal Obtained from LOD solution (0.62% of target assay concentration): 145 µV

S/N = 145/48 = 3.02

Acceptance Criteria: The S/N Ratio value should be 3 for LOD solution.

6. LIMIT OF QUANTIFICATION:

It is defined as lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and reliability by a given method under stated experimental conditions. LOQ is expressed as a concentration at a specified signal to noise ratio.

Limit of Quantification for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan:

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio. Limit of Quantification is the lowest concentration of the substance that can be estimated quantitatively. It can be determined from linearity curve by applying the following formula

Limit of Quantification (LOQ) = 10

Where S – slope of the calibration curve

σ – Residual standard deviation

Calculation of S/N Ratio for Hydrochlorothiazide:

Average Baseline Noise obtained from Blank : 48 µV

Signal Obtained from LOD solution (0.62% of target assay concentration) : 480 µV

S/N = 480/48 = 10.0

Calculation of S/N Ratio for Amlodipine:

Average Baseline Noise obtained from Blank : 48 µV

Signal Obtained from LOQ solution (2.0% of target assay concentration) : 481µV

S/N = 481/48= 10.02

Calculation of S/N Ratio for Olmesartan:

Average Baseline Noise obtained from Blank : 48 µV

Signal Obtained from LOQ solution (2.0% of target assay concentration) : 481µV

S/N = 481/48= 10.02

Acceptance Criteria: The S/N Ratio value should be 10 for LOQ solution.

7. ROBUSTNESS:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method. The standard and samples of Hydrochlorothiazide, Amlodipine and Olmesartan were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

a. The flow rate was varied at 0.6 ml/min to 1.0ml/min.:

The Standard solution of Hydrochlorothiazide, Amlodipine and Olmesartan was prepared and analysed using the varied flow rates along with method developed flow rate. On evaluation of the above results, it was concluded that the variation in flow rate does not affected the method significantly. Hence it was indicated that the method was robust even by change in the flow rate. (Table No. 5).

Table No. 5 System Suitability Results for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan (Change in Flow Rate)

Name of the drug	Flow Rate (ml/min.)	System Suitability Results
Name of the drug	Flow Rate (ml/min.)	USP Plate Count	USP Tailing
Hydrochlorothiazide	0.6	2112.6	1.0
	0.8	2876.9	1.1
	1.0	2056.4	1.0
Amlodipine	0.6	3334.9	1.1
	0.8	3741.4	1.2
	1.0	3315.8	1.1
Olmesartan	0.6	3427.1	1.1
	0.8	3831.1	1.1
	1.0	3325.0	1.1

b. The Organic composition in the Mobile phase was varied from 70% to 80%.:

The Standard solution for the drug Hydrochlorothiazide, Amlodipine and Olmesartan was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition. On evaluation of the above results, it was concluded that the variation in 10% Organic composition in the mobile phase does not affected the method significantly. Hence it was indicated that the method was robust even by change in the Mobile phase ±10. (Table no. 6)

Table No. 6 System Suitability Results for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan (Change % composition in Organic Phase)

Name of the drug	Change in Organic composition in the Mobile Phase	System Suitability Results
Name of the drug	Change in Organic composition in the Mobile Phase	USP Plate Count	USP Tailing
Hydrochlorothiazide	10% Less	2128.0	1.0
	Actual	2876.9	1.1
	10% More	2117.8	1.0
Amlodipine	10% Less	3348.1	1.1
	Actual	3741.4	1.2
	10% More	3330.9	1.1
Olmesartan	10% Less	3469.1	1.1
	Actual	3831.1	1. 1
	10% More	3450.9	1.1

8. STABILITY INDICATING STUDIES [23]:

The International Conference on Harmonization (ICH) guideline entitled stability testing of new drug substances and products requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. The aim of this work was to perform the stress degradation studies on the Hydrochlorothiazide, Amlodipine and Olmesartan using the proposed method. The aim of work was to perform the stress degradation studies on the Hydrochlorothiazide, Amlodipine and Olmesartan using the proposed method. Drug product and placebo were subjected to forced degradation at various stressed conditions like Hydrolytic degradation under acidic condition, Hydrolytic degradation under alkaline condition, Thermal induced degradation, Oxidative degradation and Photolytic degradation. All the samples were analyzed for purity peak of Hydrochlorothiazide, Amlodipine and Olmesartan. In all the samples, Peak purity meets the acceptance limits. (Purity angle should be less than purity threshold. Hydrochlorothiazide, Amlodipine and Olmesartan peak should not have any flag in purity results table (which was analyzed by Waters with Empower-2 software).

a. Hydrolytic degradation under acidic condition:

0.4ml of Hydrochlorothiazide, Amlodipine and Olmesartan was took from stock solution was prepared and taken in clean, dry 10ml volumetric flask in which 3 ml of 0.1N HCl was added. Then the volumetric flask was kept at normal condition for 90 minutes and further it was neutralized with 0.1 N NaOH and the volume was made upto the mark [10ml] with the diluent. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

b. Hydrolytic degradation under alkaline condition:

0.4ml of Hydrochlorothiazide, Amlodipine and Olmesartan was took from stock solution was prepared and taken in a clean, dry 10ml volumetric flask in which 3ml of 0.1N NaOH was added. Then the volumetric flask was kept at normal condition for 90 minutes and further it was neutralized with 0.1 N HCL and the volume was made upto the mark [10ml] with the diluent. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

c. Thermal induced degradation:

0.4ml of Hydrochlorothiazide, Amlodipine and Olmesartan was took from stock solution was prepared and taken in a clean, dry 10ml volumetric flask in which 3 ml of the diluent was added. Then the volumetric flask was kept at reflex condition for 60 minutes and the volume was made upto the mark [10ml] with the same diluent. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

d. Oxidative degradation:

0.4ml of Hydrochlorothiazide, Amlodipine and Olmesartan was took from stock solution was prepared and taken in a 10ml clean, dry volumetric flask in which 1 ml of 3 % w/v of hydrogen peroxide solution was added and the volume was made up to the mark with the diluent. Then the volumetric flask was kept at room temperature for 15 min. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

RESULTS AND DISCUSSION:

The present work was undertaken with the aim to develop and validate a rapid and consistent RP-HPLC method development in which the peaks will be appear with short period of time as per ICH Guidelines. The proposed method was simple, fast, accurate and precise method for the Quantification of drug in the Pharmaceutical dosage form, bulk drug as well as for routine analysis in Quality control. Overall the proposed method was found to be suitable and accurate for the Quantitative determination of the drug in tablet dosage form. The method was simple, precise, accurate and sensitive and applicable for the simultaneous determination Hydrochlorothiazide, Amlodipine and Olmesartan in bulk drug and in combined dosage forms. The High performance liquid chromatography (HPLC) methods was developed and validated for simultaneous estimation of Hydrochlorothiazide, Amlodipine and Olmesartan in bulk drug and in combined dosage forms. The HPLC separation was achieved on a Symmetry C₁₈ (4.6 x 150mm, 5mm, Make: XTerra) or equivalent in an Isocratic Mode. The mobile phase was composed of TEA Buffer (40%) whose pH was adjusted to 3.5 by using Ortho Phosphoric Acid and Acetonitrile (60%) [HPLC Grade] The flow rate was monitored at 0.8 ml per min. The wavelength was selected for the detection was 230 nm. The run time was 9min. The retention time found for the drugs Hydrochlorothiazide, Amlodipine and Olmesartan were 3.034 min., 4.062 min. and 5.165 min. respectively. The chromatogram was represented in fig. no.2.

CONCLUSION:

Development of new analytical methods for the determination of drugs in pharmaceutical dosage is important in pharmacokinetic, toxicological biological studies. Pharmaceutical analysis occupies a pivotal role in statuary certification of drugs and their formulations either by the industry or by the regulatory authorities. In industry, the quality assurance and quality control departments play major role in bringing out a safe and effective drug or dosage form.

The current good manufacturing practices (CGMP) and the Food Drug Administration (FDA) guidelines insist for adoption of sound methods of analysis with greater sensitivity and reproducibility. Therefore, the complexity of problems encountered in pharmaceutical analysis with the importance of achieving the selectivity, speed, low cost, simplicity, sensitivity, specificity, precision and accuracy in estimation of drugs. It was concluded that the proposed new RP-HPLC method developed for the quantitative determination of Hydrochlorothiazide, Amlodipine and Olmesartan in bulk as well as in its formulations was simple, selective, sensitive, accurate, precise and rapid. The method was proved to be superior to most of the reported methods. The mobile phases were simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence the method can be easily adopted as an alternative method to report routine determination of Hydrochlorothiazide, Amlodipine and Olmesartan depending upon the availability of chemicals and nature of other ingredients present in the sample. The method also finds use in clinical, biological and pharmacokinetic studies for the drug Hydrochlorothiazide, Amlodipine and Olmesartan. The method was validated as per ICH guidelines, and validation acceptance criteria were met in all cases.

FUTURE ASPECT:

The proposed method can be use in future for the clinical, biological and pharmacokinetic studies of Hydrochlorothiazide, Amlodipine and Olmesartan.

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Received on 10.03.2014 Modified on 30.03.2014

Asian J. Research Chem. 7(5): May 2014; Page 538-548

INTRODUCTION: