These include methanol-potassium Selleckchem 5-Fluoracil dihydrogen phosphate, methanol-ammonium

acetate, acetonitrile-potassium dihydrogen phosphate, acetonitrile-ammonium acetate, methanol-water. The mobile phase consisting of acetonitrile, methanol, 1% phosphate buffer (pH-3) in ratio of 18:58:24 (v/v/v) that was set at a flow rate of 1 ml/min was found to be optimum and further optimized by adjusting pH 3–4 by adding orthophosphoric acid. The composition of acetonitrile, methanol, 1% phosphate buffer in ratio of 18:58:24 (v/v/v) with pH-3 gave the best results. In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 96 h at an interval of 24 h at room temperature.

The results show that for solutions, the retention Selleck Compound Library time and peak area of diazepam hydrochloride remained unchanged and no significant degradation within the indicated period, this indicates that both solutions were stable for 72 h. The sample solution was injected and a chromatogram was recorded. The injections were repeated six times and the peak areas were recorded. The amount of drug present in the pharmaceutical formulation was calculated using standard calibration curve (concentration in μg/ml was taken on X-axis and average peak area on Y-axis). Percentage of drug present in each tablet was found to be 100.2. A representative chromatogram has been given in Fig. 1. most Different concentrations in the range of 0.5–50 μg/ml

were prepared. Each of the levels of concentration was prepared in triplicate.11 20 μl of each of standard solutions were injected into the HPLC system to get the chromatograms. The retention time, average peak areas were recorded. Calibration curve was constructed by plotting average peak area against concentration and regression equation was computed. The linearity range was found to be 2–20 μg/ml. The results were shown in Table 1. The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range, regression graph is presented in Fig. 2. The precision of method was ascertained from the peak area response obtained by actual determination of six replicates of a fixed amount of drug. The percent relative standard deviations were calculated for diazepam and presented in the Table 2. The precision of the method was found to be 1.02. Accuracy of developed method was confirmed by doing recovery study as per ICH norms. A known quantity of the pure drug was added to the pre-analyzed sample formulation (10 μg/ml) at three different concentration levels 80%, 100% and 120% by replicate analysis (n = 3). From the recovery study it was clear that the method is very accurate for quantitative estimation of diazepam hydrochloride in tablet dosage form as all the statistical results were within the range of acceptance, 99.4–100.3%, which shows that there is no interference with excipients.