Materials and instrumentation
Fimasartan Potassium Trihydrate was obtained from Ajanta Pharma Limited, Aurangabad. Cilnidipine was also provided by a reputed company. HPLC grades Methanol, Acetonitrile, and Milli Q water were used. All other chemicals were analytical reagent grade. Chromatographic analysis was carried out using an Agilent Technologies HPLC, DAD detector, and EZ Chrome software for data acquisition. A 0.45 μm Nylon filter was used for filtration.
Chromatographic condition
The Methanol, Acetonitrile, and Phosphate buffer pH 3.0 in the ratio of 60:05:35%v/v/v was used as a mobile phase. The flow rate was maintained at 1.0 mL/min. The injection volume is kept 20 μL and the detection was carried out at 240 nm.
Preparation of standard stock solution
A standard stock solution of 100 μg/mL for Fimasartan Potassium Trihydrate and Cilnidipine was prepared by using methanol as diluent.
Preparation of working solution of the mixture of Fimasartan Potassium Trihydrate and Cilnidipine
A working solution of 30 μg/mL for Fimasartan Potassium Trihydrate and 5 μg/mL for Cilnidipine were prepared by appropriate dilution.
Calibration standards
Six standard concentrations with 15, 30, 45, 60, 75 and 90 μg/mL were prepared from the stock solution of Fimasartan Potassium Trihydrate drug powder (pure form). Similarly, six standard strengths with 2.5, 5, 7.5, 10, 12.5 and 15 μg/mL were prepared from the stock solution of Cilnidipine drug powder (pure form).
Preparation of mobile phase
Prepare a mixture of Methanol, Acetonitrile and Potassium Dihydrogen Phosphate buffer in the volume ratio 60:05:35%v/v/v for method optimization. Mix well and sonicate to degas the mixture. Adjust pH 3.2 with Orthophosphoric acid.
Method validation
Analytical validation parameters for the analysis of the proposed method were determined according to ICH (Q2 R1) guideline. [21] The parameters such as linearity, range, accuracy, precision, specificity, robustness, LOD & LOQ were performed [21, 22].
System suitability
The typical values for evaluating system suitability of a chromatographic procedure include the RSD < 1%, Asymmetry factor < 2, and Theoretical plates > 2000. The determination of system suitability of the analytical method was accomplished by assaying three samples of the same strength as Fimasartan Potassium Trihydrate and Cilnidipine. The sample concentration of Fimasartan and Cilnidipine used in this analysis was 30 μg/mL and 5 μg/mL respectively. The retention time, peak area, theoretical plates, and asymmetry factor were evaluated for system suitability.
Linearity
The linearity response was determined by analysing 06 independent level of calibration curve in the range of 15–90 µg/mL (15, 30, 45, 60, 75 & 90 µg/mL) for FIK and 2.5–15 µg/mL (2.5, 5.0, 7.5, 10.0, 12.5 & 15.0 µg/mL) for CLN.
The plot of mean peak area against concentration was plotted. Correlation coefficient and regression line equations for FIK and CLN were calculated.
Accuracy and precision
The accuracy of the method was performed in triplicate at three different concentration levels of 50, 100 and 150% of the targeted concentration of drugs. The accuracy of the method was evaluated by calculating percentage recovery. Repeatability was performed under 6 replicates at the assay concentration of FIK and CLN. Intra-day and inter-day variations of FIK and CLN were performed in triplicate at three different concentration levels of 50, 100, and 150%. Results are expressed in the form of RSD.
Specificity
Specificity was performed by injecting diluent, placebo and sample solution to check the interference of excipients.
LOD and LOQ
The limit of detection (LOD) and limit of quantification (LOQ) were calculated by formula as given in ICH (Q2R1).
$$\begin{aligned} {\text{ LOD}} & = 3.3 \times {\text{Standard}}\,{\text{deviation}}/{\text{Slope}} \\ {\text{LOQ}} & = 10 \times {\text{Standard}}\,{\text{deviation}}/{\text{Slope}} \\ \end{aligned}$$
Robustness
The robustness of the method was established by introducing small deliberate change in experimental conditions like flow rate, pH of Mobile phase and Mobile phase composition. The changes made in flow rate ± 0.1 mL/min, for pH of Mobile phase ± 0.2 and mobile phase composition ± 2%.
Assay
The combination of Fimasartan Potassium Trihydrate (60 mg) and Cilnidipine (10 mg) (Film Coated Tablet) is approved by CDSCO for clinical Trial Phase-III trial. So, there is no any single marketed pharmaceutical formulation is available.
The sample (synthetic mixture) was analyzed for assay containing 60 µg/mL of FIK and 10 µg/mL of CLN by optimized HPLC method. (conc. or dose ratio is suggested by CDSCO) [23].