Chemical stability of rifampicin. The chemical stability of rifampicin was determined in the absence and presence of an anti-oxidant in aqueous solutions. Either 0.02 or 0.2 % w/v of ascorbic acid were added to a 1 mg/ml solution of rifampicin in PBS buffer (pH 7.4). The solutions were constantly stirred and at pre-determined time point (0, 6, 24, 48 and 72 h), samples were withdrawn from the solutions, diluted using mobile phase and analysed by HPLC. All samples were stored under light protected conditions at ambient temperature (23 ± 2oC) during the stability study (n = 3). The effect of rifampicin concentration and temperature on drug stability was also investigated by using two drug concentrations of rifampicin (1 mg/ml and 0.01 mg/ml) containing 0.02 % w/v ascorbic acid at two different temperatures, ambient temperature (23 ± 2oC) and 37oC. The hydrolysis of rifampicin was also investigated in PBS buffer (pH 7.4). For the co-analysis of rifampicin and RSV, a second HPLC method was developed. Analysis was performed using a 600 HPLC pump (Waters, UK) with a 717 auto- sampler (Waters, UK) and a 2487 dual λ absorbance detector (Waters, UK). The column used was a reversed-phase C18 (250 x 4.6 mm). UV-photodiode array detection was performed at 240 nm. The mobile phase consisted of acetonitrile: PBS buffer (36:64 % v/v) at a flow rate of 1.4 ml/min. Final pH of the mobile phase was adjusted to 7.5 by diluted hydrochloric acid. Column temperature was set to 28°C and 10 μl of samples was injected to the HPLC system. Calibration standards were produced by making up a stock solution in acetonitrile containing 1 mg/ml rifampicin and 1 mg/ml RSV (0.02% w/v butylhydroxytoluene (BHT) to avoid drug oxidation). Six serial dilutions were used to produce calibration standards in the range of 1-0.01 mg/ml. Calibration plots were constructed for rifampicin and RSV in standard solutions by plotting the concentrations of the compounds versus peak area response. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. Three calibration curves were run on a single day to determine the intra-day variance; this data was combined with two additional calibration curves on two further days to determine the inter-day variance. Precision and accuracy were calculated according to equations 2.1 and 2.2. Limits of detection (LOD) and quantification (LOQ) were calculated according to equations
Chemical stability of rifampicin. In the absence of the anti-oxidant, rifampicin (1 mg/ml) recovery at PBS buffer pH
Chemical stability of rifampicin. The chemical stability of rifampicin was determined by measuring the recovery of rifampicin from buffer solution. Briefly, solution of rifampicin in PBS buffer pH 7.4 was prepared and was constantly stirred at 37°C. At pre -determined time points, samples were withdrawn from the solution and analysed by HPLC and the recovery of rifampicin was evaluated.
