Simple, accurate, precise, reproducible, requiring no prior separation, and economical procedures for simultaneous estimation of benazepril hydrochloride (BEN) and Hydrochlorothiazide (HCT) in tablet dosage form have been developed. Simultaneous equation method employs for estimation of both drugs in methanol at 240 nm and 270 nm as two analytical wavelengths. BEN and HCT at their respective
Benazepril hydrochloride (BEN) is chemically 3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-acetic acid monohydrochloride (Figure
(a) Structure of hydrochlorothiazide. (b) Structure of benazepril hydrochloride.
Hydrochlorothiazide (HCT) is chemically 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide (Figure
BEN and HCT are official in the british Pharmacopeia and the Indian Pharmacopeia. Many methods have been reported in the literature for both determination of BEN, individually and with other drugs in combination [
A double-beam Shimadzu UV-visible spectrophotometer 1700 (Pharma spec), with wavelength accuracy of ±0.5 nm and a pair of 1 cm matched quartz cells, was used to measure absorbance of the resulting solution. All weighing was done on an electronic balance (Shimadzu BL-220H). All statistical calculations were carried out using Microsoft Excel 2007 analytical tool.
Analytically pure BEN and HCT were procured as gift sample from Dishman Pharmaceuticals Ltd. and Cadila Pharmaceutical Pvt. Ltd. (Ahmedabad, India). Methanol (E. Merck, Mumbai, India) analytical grade was used as a diluent. Tablet formulation (Lotensin HCT, Novartis Pharmaceutical Pvt. Ltd.) containing labelled amount of 10 mg of benazepril hydrochloride and 12.5 mg of hydrochlorothiazide was purchased from local market.
Accurately weighed 100 mg of BEN and HCT standards was transferred to separate 100 mL volumetric flasks and dissolved in 50 mL Methanol. The flasks were shaken, and the volume was made up to the mark with methanol to give solutions containing 1000
Selection of analytical wavelengths was done by taking pure samples of BEN and HCT, which were separately dissolved in methanol, to give two solutions of 10
Overlain spectrum of BEN and HCT in Methanol.
The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines [
Developed analytical method shows linearity response over the range of 2–12
The intraday and interday precision study of the proposed simultaneous equation spectrophotometric method was carried out by estimating responses three times on the same day and on three different days (first, second, and third days) for three different concentrations of BEN (6, 8, and 10
The accuracy of the method was determined by calculating recoveries of BEN and HCT by the method of standard additions. Known amount of BEN (50%, 100%, and 150%) and HCT (50%, 100%, and 150%) was added to a pre quantified sample solutions, and the amounts of BEN and HCT were estimated by measuring the response at the appropriate wavelengths. The recovery was verified by estimation of drugs in triplicate preparations at each specified concentration level.
Calibration curve was repeated 5 times, and the standard deviation (SD) of the intercepts was calculated. Then LOD and LOQ were measured as follows:
Solution stability of the method was studied by observing the stability of both drug solutions at
The pharmaceutical dosage form used in this study was Lotensin HCT tablets with a content of 10 mg BEN and 12.5 mg HCT (as per USP) per tablet. Twenty tablets of brand Lotencin HCT tablets were weighed and finely powdered. Accurately weighed tablet powder equivalent to 10 mg was taken in 100 mL volumetric flask. Few mL of methanol was added and sonicated for 5 min. The volume was made up to the mark with methanol. Aliquot portion of this solution was further diluted to achieve final concentration of 10
A simultaneous equation spectrophotometric method was successfully developed for the determination of BEN and HCT from their combined dosage form.
The proposed simultaneous equation method shows good linearity in the concentration range of 2–12
The %RSD values for BEN and HCT were found to be 1.32% and 1.96%, respectively. The low values of relative standard deviation (less than 2%) indicate that the proposed method is repeatable. The %RSD values for intraday study were found to be 0.47–0.74% and 0.13–0.24% for BEN and HCT, respectively. The %RSD values for interday study were found to be 0.30–0.81% and 0.18–0.40% for BEN and HCT, respectively. The low RSD value indicates that the proposed method is precise (Tables
Summary of validation parameters.
Parameters | BEN | HCT |
---|---|---|
Limit of detection | 0.103 | 0.025 |
Limit of quantitation | 0.312 | 0.075 |
Accuracy (%) | 100.0–100.6 | 99.8–100.2 |
Precision | ||
Intraday ( |
0.47–0.74% | 0.13–0.24% |
Interday ( |
0.30–0.81% | 0.18–0.40% |
Repeatability (RSD, |
1.32% | 1.96% |
Specificity | Specific | Specific |
Robustness | Robust | Robust |
Solvent suitability | Suitable for 24 hrs. | Suitable for 24 hrs. |
Statistical data of BEN and HCT.
Parameters | BEN at 240 nm | HCT at 270 nm |
---|---|---|
Linear range | 2–12 µg/mL | 4–14 µg/mL |
Slope | 0.0181 | 0.05 |
Intercept | 0.0129 | 0.035 |
Standard deviation of intercept | 0.0031 | 0.0010 |
Standard deviation of slope | 0.0005 | 0.0019 |
Regression coefficient ( |
0.9997 | 0.9988 |
Determination of precision for BEN.
BEN | Conc. ( |
Intraday mean ± SD ( |
%RSD | Interday mean ± SD ( |
%RSD |
---|---|---|---|---|---|
At 240 nm | 6 |
|
0.47 |
|
0.81 |
8 |
|
0.74 |
|
0.37 | |
10 |
|
0.51 |
|
0.30 | |
At 270 nm | 6 |
|
1.88 |
|
1.88 |
8 |
|
1.59 |
|
1.58 | |
10 |
|
1.32 |
|
1.21 |
Determination of precision for HCT.
HCT | Conc. ( |
Intraday mean ± SD ( |
%RSD | Interday mean ± SD ( |
%RSD |
---|---|---|---|---|---|
At 240 nm | 8 |
|
0.017 |
|
1.68 |
10 |
|
0.024 |
|
1.35 | |
12 |
|
0.18 |
|
1.06 | |
At 270 nm | 8 |
|
0.13 |
|
0.34 |
10 |
|
0.19 |
|
0.40 | |
12 |
|
0.24 |
|
0.18 |
The accuracy of the method was determined by calculating recoveries of BEN and HCT by the method of standard additions. The percent recovery was found to be 100.0%–100.6% for BEN and 99.8%–100.2% for HCT (Table
Determination of accuracy.
% level | Amount of drug added ( |
Recovered amount of drug ( |
% recovery | |||
---|---|---|---|---|---|---|
BEN ( |
HCT ( |
BEN ( |
HCT ( |
BEN | HCT | |
50 | 6 | 6 | 6.04 | 6.00 | 100.6 | 100.2 |
100 | 8 | 8 | 8.01 | 7.98 | 100.1 | 99.89 |
150 | 10 | 10 | 10.00 | 9.98 | 100.0 | 99.9 |
The proposed validated method was successfully applied to determine BEN and HCT in tablet dosage form. The results obtained for BEN and HCT were comparable with the corresponding labelled amounts (Table
Assay results of marketed formulation.
Formulation | Actual concentration ( |
Amount obtained ( |
% BEN | % HCT | ||
---|---|---|---|---|---|---|
BEN | HCT | BEN | HCT | |||
Tablet | 4.0 | 5.0 | 3.90 | 4.97 | 98.25% | 99.32% |
Sensitive, precise, and accurate simultaneous UV spectroscopic method was developed and validated. The proposed method is accurate, precise, reproducible, and economic and can be successfully used for routine analysis of simultaneous estimations of BEN and HCT. The method was validated as per the ICH guidelines in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and limits of quantification (LOQ), and robustness. The proposed method can be used for quality control assay of BEN and HCT in their pharmaceutical dosage form.
All authors do not have conflict of interests.
The authors are thankful to Dishman Pharmaceuticals Ltd. for providing BEN and Cadila Pharmaceutical Pvt. Ltd. (Ahmedabad, India) for providing HCT as gratis samples. The authors are also heartily thankful to Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar, Anand, for funding the entire project and providing the necessary facilities for research work.