Simultaneous high-pressure liquid chromatographic analysis of ampicillin and cloxacillin in serum and urine.

A rapid, specific and sensitive high pressure liquid chromatographic (HPLC) assay for the simultaneous determination of ampicillin and cloxacillin in serum and urine is developed. Ampicillin, cloxacillin and cephalexin (internal standard) were eluted from a 6.5 mu Synchropack RPP reversed phase column at ambient temperature using a mobile phase comprised of methanol:water (3/7v/v) and containing 0.011 M sodium-n-octane sulphonate, 0.005 M NaH2PO4 and 1.3% v/v of 0.5 M HCl (pH 2.7). The analysis time required no longer than 11 min. Equations are presented for the linear relationships between the peak height ratios of ampicillin/cephalexin and cloxacillin/cephalexin over the range 10-80 micrograms/ml (ampicillin) and 5-25 micrograms/ml (cloxacillin), respectively. The sensitivity limits for ampicillin and cloxacillin in serum and urine were 5 micrograms/ml and 1 microgram/ml, respectively. Quality criteria such as accuracy, precision and specificity were studied extensively. We investigated the applicability of the HPLC assay for the developed simultaneous determination of the cumulative amounts of ampicillin and cloxacillin, excreted unchanged in urine after an oral dose containing 500 mg ampicillin and 500 mg cloxacillin to a human volunteer.