Materials
PER, IND, and AML were procured from the reputed pharmaceutical industry, Gujarat, India. Solvents and reagent like methanol (HPLC grade)—SRL Chemicals Pvt. Ltd., Ahmedabad, India; ACN (AR grade)—Astron chemicals Pvt Ltd., Ahmedabad, India; and 1.0% ortho-phosphoric acid (OPA) (AR grade)—Astron chemicals, Ahmedabad, India, were purchased. Triplixam tablet (Serdia Pharmaceutical Ltd.) was used having label claim: Each tablet contains perindopril erbumine 4 mg, indapamide 1.25, and amlodipine besylate 5 mg.
Instrument
Analytical balance making a model of Mettler Toledo electronic balance (ME204, Mettler Toledo Group, Mumbai, India) was used. HPLC System of Waters HPLC system consisted of a binary pump (model Waters 515 HPLC pump), loop injector, and PDA detector (Waters 2998) were connected with empower - version 2 software. Phenomenex C-18 column (250 mm × 4.6 mm, 5.0 μ) was used as a stationary phase for the better separation compound.
Chromatographic conditions
The Phenomenex C-18 column (250 mm × 4.6 mm, 5.0 μ) equilibrated with mobile phase acetonitrile: methanol: water (30:20:50, v/v/v) was further adjusted to pH 3.0 with the help of 1.0% ortho-phosphoric acid. The flow rate was kept to be 1 ml/min, eluents were supervised using a PDA detector at 215.0 nm with the fixed volume of injection that was 20 μl, and the stop time optimized was 11 min.
Preparation of standard stock solution
Weight accurately PER (4.0 mg), IND (1.25 mg), and AML (5.0 mg) and transferred to three different 10 ml volumetric flasks containing a minute amount of methanol (5.0 ml). Volumes were to be adjusted up to the mark with methanol to yield the strength of solution containing 400 μg/ml of PER, 125 μg/ml of IND, and 500 μg/ml of AML. Pipette out 1.0 ml from each of the above solutions and mix into 10 ml volumetric flask, make up to the mark with the mobile phase. The working standard solutions of 40 μg/ml of PER, 12.5 μg/ml of IND, and 50 μg/ml of AML were prepared.
Validation of the developed RP-HPLC method
Validation of the proposed developed RP-HPLC method was performed according to ICH Q2 (R1) for the accuracy, precision, robustness, and various other typical parameters.
Linearity and range
Appropriate aliquots of PER, IND, and AML working standard solution of 7 different concentration was taken in different 10-ml volumetric flasks and diluted up to the mark with a mobile phase to obtain a final concentration of 0.40, 1.60, 2.80, 4.00, 5.20, 6.40, and 7.60 μg/ml of PER; 0.125, 0.500, 0.875, 1.25, 1.625, 2.0, and 2.375 μg/ml of IND; and 0.50, 2.00, 3.50, 5.00, 6.50, 8.00, and 9.50 μg/ml of AML. The solution was injected using a 20-μl fixed loop system and the chromatogram was recorded. The calibration curve constructed by plotting the average peak area against concentrations and regression equation was computed (n = 5).
Precision
Intraday and interday as intermediate precision were performed as a part of precision. Working standard solutions of 40 μg/ml of PER, 12.5 μg/ml of IND, and 50 μg/ml of AML were prepared and analyzed as a part of the precision study. Intraday precision was performed for the solutions of PER, IND, and AML mixture at 3 levels covering respectively of lower (0.400, 0.125, 0.500 μg/ml), medium (4.0, 1.25, 5.0 μg/ml), and higher (7.60, 2.75, 9.50 μg/ml) concentration of the calibration curve analyzed three times on the same day. Interday precision was determined by analyzing the sample solution of PER, IND, and AML mixture at three levels covering lower, medium, and higher concentration periods for three different days. Mean and %RSD values were calculated by using the obtained peak area.
The repeatability was studied by injecting six times of the middle concentration of the calibration range and estimating the area of each injection and determines the %RSD.
Accuracy
The accuracy of the method was determined by calculating the recovery of PER, IND, and AML by the method of standard addition. The known amount of PER, IND, and AML at three levels of 80, 100, and 120% were spiked to prequantified sample solution, and the amount of 2.24, 2.80, and 3.36 μg/ml of PER; 0.70, 0.88, and 1.05 μg/ml of IND; and 2.80, 3.50, and 4.20 μg/ml of AML was added to prequantified sample solution. By putting the values of area to the calibration curve, the percentage (%) recoveries of PER, IND, and AML were estimated and regression analysis was carried out.
Detection limit and quantitation limit
The lowest concentration of drug which can be reliably detected and differentiate from the background is known as a limit of detection (LOD) and which can be quantified at the lowest concentration is known as LOQ, i.e., the limit of quantification. LOD and LOQ were calculated using the following equation as per ICH guidelines.
LOD = 3.3 × σ/SD
LOQ = 10 × σ/SD
where σ is the standard deviation of y-intercept of the regression line and S is the slope of the calibration curve.
Specificity
The specificity studies have been carried out by spiking commonly used excipients into a preweighed quantity of active pharmaceutical ingredients. The chromatogram was recorded for the appropriate dilution.
Robustness
Robustness of the method was studied by providing deliberate changes in the experimenting condition like mobile phase, temperature, and wavelength and flow rate. The mean and %RSD of peak retention time was calculated.
Solution stability
The stock standard solutions of the mixture were stored at normal room temperature for 48 h and were estimated using RP–HPLC at a specific time interval of 0, 8, 24, 36, and 48 h. The standard stock solution of 40 μg/ml of PER, 12.5 μg/ml of IND, and 50 μg/ml of AML was prepared. From the above, the stock solution withdrawn was 1.0 ml and was transferred into another 10.0 ml volumetric flask and make up the volume up to the mark with a mobile phase to obtain the final concentration 4.0 μg/ml for PER, 1.25 μg/ml for IND, and 5.0 μg/ml for AML and used for the solution stability studies. The solution stability study of the solution was studied by applying and analyzing it three times in the middle concentration of the calibration range.
System suitability
A system suitability test was an integral part of the process development to verify that the system is appropriate for the analysis of PER, IND, and AML to be carried out. The system suitability test for the proposed chromatographic system was performed following the validation run. Six replicate injections of a standard and one injection of a sample were made and evaluated the efficiency of the system by verification of different parameters like Rt, resolution, asymmetric factor, and theoretical plate.
Analysis of marketed formulations
Finely powdered and accurately weighted twenty tablets were prepared by using a mortar pestle. The powder equivalent to 4.0 mg of PER, 1.25 mg of IND, and 5.0 mg of AML was accurately weighed and transferred to a 10-ml volumetric flask containing minute ml (5.0 ml) of methanol and flask was sonicated for 5.0 min. The solution was filtered into using the Whatman filter paper (No. 42), the residues were washed twice with a few ml of methanol, and both filtrate and washing were combined in the same volumetric flask. The volume was adjusted to the mark with the methanol. From the above solution, pipetted out 1.0 ml of aliquots was transferred into another 10 ml volumetric flask and the volume was made up to the mark with methanol to obtain the concentration of stock solutions 40 μg/ml of PER, 12.5 μg/ml of IND, and 50 μg/ml AML. 1.0 ml above stock solution was pipetted out into another 10 ml volumetric flask and make up the volume up to the mark with a mobile phase to obtain a final concentration 4.0 μg/ml of PER, 1.25 μg/ml of IND, and 5.0 μg/ml of AML, respectively. 100 μl Hamilton syringe was used to inject the middle concentration of the calibration range of these solutions, and all the three drugs were simultaneously estimated by the proposed chromatographic method. The percentage (%) amount of PER, IND, and AML was noted by using the regression equation.