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D. Jothieswari et al., Design and RP - HPLC Method for the Simultaneous determination of Valsartan and Hydrochlorothiazide in Bulk and in Pharmaceutical Formulation


Design and RP - HPLC Method for the Simultaneous determination of Valsartan and Hydrochlorothiazide in Bulk and in Pharmaceutical Formulation 1





Jothieswari.D *, Priya.D , Brito Raj.S , Mohanambal.E , Wasim Raja.S 1

* Department of Pharmaceutical Analysis, Sri Venkateswara College of Pharmacy, Chittoor ,AP, India. Department of Pharmaceutical Chemistry, Sri Venkateswara College of Pharmacy, Chittoor, AP, India. 3 .Department of Pharmaceutics, Sri Venkateswara College of Pharmacy, Chittoor, AP, India. 4 Department of Pharmacy Practice, Sri Venkateswara College of Pharmacy, Chittoor AP, India. 2

Received for publication, September 2, 2011, and in Revised form, September 9, 2011; Published online in, October 7, 2011

ABSTRACT A reverse phase high performance liquid chromatographic method has been developed for the simultaneous estimation of valsartan and hydrochlorothiazide in bulk and in pharmaceutical formulation using RP - C18 column. The mobile phase (acetonitrile: methanol: 50 mM phosphate buffer adjusted to pH 3 with orthophosphoric acid) was pumped at a flow rate of 1.0 ml/ min in the ratio of 20: 50: 30%v/v and the eluents were monitored at 250 nm. Linearity was obtained in the concentration range of 4 - 40 µg/ ml for valsartan and 1 – 10 µg/ ml for hydrochlorothiazide. The method was statistically validated and RSD was found to be less than 2% indicating high degree of accuracy and precision of the proposed HPLC method. Due to its simplicity, rapidness, high precision and accuracy, the proposed HPLC method can be applied for determining valsartan and hydrochlorothiazide in bulk and in pharmaceutical dosage form. KEY WORDS: Valsartan, Hydrochlorothiazide, RP – HPLC method, Validation.

INTRODUCTION Valsartan (VAL) chemically, N - (1 - oxopentyl) - N - [(2' - (1H - tetrazol - 5 - yl) (1, 1' - biphenyl) - 4 - yl) methyl] - L - valine, is a potent angiotensin receptor 1 2-4 5-7 8 blocker . Methods such as HPLC , LC – MS , in plasma , 9 Capillary electrophoresis and simultaneous UV 1011 spectrophotometric methods are reported for estimation of VAL alone or in combination with other drugs. Hydrochlorothiazide (HCT), 6 - chloro - 3, 4 dihydro - 7 - sulfamoyl - 2H - 1, 2, 4 - benzothia - diazine 12 1, 1 - dioxide, is a thiazide diuretic . It increases sodium and chloride excretion in distilled convoluted tubule. Many analytical methods were reported for the analysis of HCT alone and combination with other drugs by stability 13 14, 15 indicating method , RP - HPLC methods , and 16, 17 Spectrophotometric methods . We have developed a simple, precise, accurate and specific RP - HPLC method for the simultaneous determination of VAL and HCT in bulk and in pharmaceutical dosage forms. Because analytical methods


must be validated before use by the pharmaceutical industry, the proposed HPLC – UV detection method was validated in accordance with International Conference on 18, 19 Harmonization (ICH) guidelines , by assessing its selectivity, linearity, accuracy, precision, limit of detection and limit of quantization.

MATERIALS AND METHODS Analysis was performed with a Shimadzu (Japan) chromatograph equipped with an LC - 10 AT VP solvent delivery system, a universal loop injector (Rheodyne 7725 i, Rheodyne Inc., Cotati, CA, USA) of injection capacity of 20 µL, and an SPD - 10 AVP UV – Visible detector set at 250 nm. The equipment was controlled by a PC work station with Winchrome software. Compounds were separated on a To whom correspondence should be addressed: Mrs. D. Jothieswari,

E-mail: Phone: +9199899165610

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D. Jothieswari et al., Design and RP - HPLC Method for the Simultaneous determination of Valsartan and Hydrochlorothiazide in Bulk and in Pharmaceutical Formulation

(150 mm × 4.6 mm i.d., 5 - µm particle size) Phenomenex Luna C18 column under reversed phase partition conditions. The mobile phase was a 20: 50: 30% v/v mixture of Acetonitrile: Methanol: Phosphate buffer (50 mM, pH 3 ± 0.1, adjusted with orthophosphoric acid). The flow rate was 1.0 ml/ min and the run time was 10 min. Before analysis both the mobile phase and sample solutions were degassed by the use of a sonicator (Soltec, Soluzionitecnologiche, Luglio, Italy) and filtered through a 0.2 - µm filter (Gelman science, India). The identity of the compounds was established by comparing the retention times of compounds in the sample solution with those in standard solutions. Chromatography was performed in an ambient temperature maintained at 20 ± 1°C. The UV spectrum of VAL and HCT for selecting the working wavelength of detection was taken using a Shimadzu UV 1700, UV - Visible spectrophotometer (Shimadzu, Kyoto, Japan).

up to 50 ml with methanol. The solution was then centrifuged at 1000 rpm for 10 min and the clear supernatant was collected and filtered through 13 mm membrane syringe filter (pore size 0.2 µm). From the clear solution, further dilutions were made by diluting 1.0 ml into 25 ml with mobile phase; from that 4.0 ml into 10 ml with mobile phase to obtain 16 µg /ml of VAL which is also contains 2.5 µg /ml of HCT theoretically. Each sample solution was injected and the peak areas were measured for the determination of VAL and HCT in tablet formulation.

VALIDATION The objective of method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision (repeatability and intermediate precision), accuracy, selectivity and specificity. Accuracy was assessed by measuring recovery at three different levels.

REAGENTS AND CHEMICALS Pharmaceutically pure samples of VAL and HCT were obtained as a gift samples from Caplin point, Chennai, India and the commercial formulation Exforge HCT tablets: VAL - 160 mg and HCT - 25 mg was procured from US market (Novartis pharmaceutical corporation, Switzerland). HPLC grade methanol, acetonitrile, and water and potassium dihydrogen orthophosphate (A.R. grade) were obtained from Qualigens India Pvt. Limited, Mumbai, India. Preparation of stock solution of VAL and HCT About 100 mg of VAL and 25 mg of HCT were accurately weighed and transferred in to 50 ml volumetric flasks separately. It was dissolved in methanol and the solution was made up to the volume with same. From this standard stock solution, the mixed standard solution was prepared by pipetting both 1 ml of mother liquors into the same 25 ml volumetric flask and made up to the volume with mobile phase to contain 80 µg/ ml and 20 µg/ ml of VAL and HCT, respectively. Calibration standards of both analyte were prepared at the concentrations of 4, 8, 16, 24, 32 and 40 µg/ ml for VAL and 1, 2, 4, 6, 8 and 10 µg/ ml for HCT. Assay of tablet formulation The contents of twenty commercial tablets were weighed and their mean mass was determined. After grinding the tablets into a fine powder in a glass mortar, an accurately weighed quantity of the tablet powder equivalent to 50 mg of VAL was quantitatively transferred into a 50 ml volumetric flask with about 45ml of methanol. The contents were sonicated for 15 min, to ensure the complete solubility of the drug. The mixture was then made


RESULTS AND DISCUSSION This work was focused on optimization of the conditions for the simple and rapid as well as low cost effective analysis including a selection of the proper column or mobile phase to obtain satisfactory results. The optimum mobile phase containing acetonitrile: methanol: potassium dihydrogen orthophosphate buffer (50 mM; pH 3.0 ± 0.1) in the ratio of 20: 50: 30% v/v. Under these experimental conditions sharp peaks were obtained for VAL and HCT at the retention times 8.86 min and 3.34 min, respectively. A typical chromatogram of sample solution is shown in Fig.1. Fig 1. Optimized chromatogram for HCT and VAL (10 µg /ml)

METHOD VALIDATION The system suitability parameters like capacity factor, number of theoretical plates, and USP tailing factor

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D. Jothieswari et al., Design and RP - HPLC Method for the Simultaneous determination of Valsartan and Hydrochlorothiazide in Bulk and in Pharmaceutical Formulation

for both analytes were found to be within the limit indicating the suitability of the system (Table 1). Fig 2. Chromatogram obtained from HCT (2.5 µg /ml) and VAL (16 µg /ml) in tablets Linearity Linearity was tested in the concentration range 4 –

40 µg/ ml for VAL and 1 – 10 µg/ ml for HCT. The solutions were chromatographed six times, in accordance with the International Conference on Harmonization. Separate calibration plots for VAL and HCT were constructed by plotting peak area against the respective concentrations and the method was evaluated by determination of the correlation coefficient and intercept, calculated in the corresponding statistical study (ANOVA; P < 0.05), 2 correlation coefficient r values > 0.999 and intercepts very close to zero confirmed the good linearity of the method. The P values calculated for the calibration plots were greater than 0.05, indicating the variances were not significantly different (Table 2).

method was confirmed by the repeated analysis of formulation for six times (Table 3). Table 2. Results from study of linearity Parameters Detection wavelength Beer’s law limit (µ µg /ml) Correlation coefficient (r) Regression equation (y = mx + c) Slope (b) Intercept (a) LOD (µ µg /ml) LOQ (µ µg /ml) P value** F - value

VAL* 8.86

HCT* 3.34

Tailing factor Asymmetrical factor Number of Theoretical plates Capacity factor Resolution

1.31 1.49

1.48 1.84



4.98 1.15 Between VAL and HCT 5.52

*Mean of six determinations. Assay of tablet formulation The percentage label claim present in tablet formulation was found to be 100.27 ± 0.1708, 100.03 ± 0.0579 for VAL and HCT, respectively. Precision of the


HCT* 250 nm

4 - 40

1 - 10



Y = 950860.6502 x + 84887.9113 950860.6502 84887.9113 0.2077 0.6294 0.5108 14852.41

y = 264705.7201x + 953.7155 264705.7201 953.7155 0.1636 0.4959 0.8635 36931.44

z*Mean of six determinations; **P > 0.05. Precision Intraday and Inter day precision was determined by repeating assay three times on same day for intraday and on three different days for inter day precision (Table 4).

Table 3. Results from assay of tablet formulation Drug

Amount taken (µ µg/ ml)





Table 1. Results from system suitability study Parameters Retention time

VAL* 250 nm

Amount added (µ µg/ ml) 3.933 7.955 11.978 1.920 4.008 6.014

Recovery (% )

102.22 101.08 100.73 102.20 100.25 100.20


COV (%)





Accuracy To check the accuracy of the developed methods and to study the interference of formulation excipients, analytical recovery experiments were carried out as per ICH guidelines. The results of the recovery studies and its statistical validation data given in Table 5 indicate high accuracy of the proposed method. The percentage recovery was found to be in the range of 100.73 – 102.22% for VAL and 100.20 – 102.20% for HCT.

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D. Jothieswari et al., Design and RP - HPLC Method for the Simultaneous determination of Valsartan and Hydrochlorothiazide in Bulk and in Pharmaceutical Formulation

Table 4. Intraday and inter day precision of the method Drug

Label claim (mg/ tablet; n = 6)

Amount found (mg)

Drug Content (%)


COV (%)













0.170 3 0.057 8


S.D., Standard deviation; COV, coefficient of variance; S.E., standard error

for accuracy, precision, specificity, and linearity. The run time is relatively short (10 min), which enables rapid quantification of many samples in routine and quality control analysis of tablets. The method also uses a solvent system with the same composition as the mobile phase for dissolving and extracting drugs from the matrices, thus minimizing noise. Thus the proposed method is rapid, selective, requires a simple sample preparation procedure, Moreover, the lower solvent consumption leads to a cost effective and represents a good procedure of VAL and HCT determination in bulk and in pharmaceutical dosage forms.


Table 5. Results of recovery analysis Drug



Concentration (µ µg /ml)

8 24 40 2 6 10

Intraday precision (n = 6) Mean RSD (%) 8.027 0.75 24.053 0.48 40.048 0.54 20.023 1.70 6.047 1.59 10.035 1.03

Inter day precision (n = 6) Mean RSD (%) 8.007 1.15 24.056 0.58 40.083 0.52 2.008 1.76 6.038 1.38 10.041 1.39




S.D., Standard deviation; COV, coefficient of variance Robustness As defined by the ICH, the robustness of an analytical procedure describes to its capability to remain unaffected by small and deliberate variations in method parameters. Robustness was performed by small variation in the chromatographic conditions and found to be unaffected by small variations like ± 2% variation in volume of mobile phase composition, ± 0.1 ml/ min in flow rate of mobile phase, ± 0.1 variation in pH. Specificity The specificity of the HPLC method was ascertained by analyzing standard drug and sample solutions. The retention time of VAL and HCT was confirmed by comparing the retention time with that of the standard. Conclusions A simple isocratic RP - HPLC method with UV detection has been developed for simultaneous determination of VAL and HCT. The method was validated





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