Trace-level determination of nifedipine in human serum by reversed phase high performance liquid chromatography

A rapid, simple, specific and sensitive high performance liquid chromatographic method has been developed for the determination of nifedipine (adalate) in human serum using diazepam (valium) as an internal standard (I.S.). The method utilized a 5 microm nonpolar C18 reversed phase Hypersil (ODS) column (250 x 3 mm i.d.). The mobile phase consisted of 60% v/v, acetonitrile in water, adjusted to pH 3.7 with glacial acetic acid and ammonium acetate, and pumped at a flow rate of 2 ml/min. The effluent was monitored by UV detection at 340 nm and a sensitivity fixed at 0.02 aufs. Each analysis required no longer than 4 min. The minimum detectable amount of nifedipine in serum was 3 ng/ml, and the mean absolute recovery was 93.5%. The within and between-day coefficients of variation at three different concentrations from 15-160 ng/ml ranged from 2.07 to 7.76%, and from 3.15 to 7.98% respectively. Calibration graphs for 10 to 200 ng/ml were linear with a mean correlation coefficient, r (n = 36) of 0.9991. The method was validated for accuracy, sensitivity, selectivity and reproducibility and finally was utilized and proved to be suitable in a bioavailability study of two products of nifedipine following oral administration to healthy male subjects.