SENSITIVE AND SPECIFIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY WITH ULTRAVIOLET DETECTION FOR THE DETERMINATION OF COCAINE AND ITS METABOLITES IN RAT PLASMA

A sensitive, specific and precise HPLC-UV assay was developed to quant itate cocaine (COG) and its metabolites benzoylecgonine (BE), norcocai ne (NC) and cocaethylene (CE) in rat plasma. After adding 50 mu l of t he internal standard solution (bupivacaine, 8 mu g/ml) and 500 mu l of Sorensen's buffer (pH 6) to 100 mu l of rat plasma sample, the mixtur e was extracted with 10 ml of chloroform. The organic layer was transf erred to a clean test tube and was evaporated under nitrogen. The resi due was reconstituted in 100 mu l of mobile phase and 35 mu l was inje cted onto the HPLC column. The mobile phase consisted of methanol-acet onitrile-50 mM monobasic ammonium phosphate (5:7:63, v/v/v) and was ma intained at a flow-rate of 0.4 ml/min. Separation of COC and its metab olites was achieved using a Supelcosil ABZ + plus deactivated reversed -phase column (250 x 2.1 mm I.D., 5 mu m). Calibration curves were lin ear over the range of 25-5000 ng/ml for COC and its three metabolites. The absolute extraction efficiencies for BE, COG, NC, CE and bupivaca ine were 56.6%, 78.6%, 61.1%, 76.4% and 67.0%, respectively. COC and i ts metabolites were stable in mobile phase for 24 h at room temperatur e and in rat plasma for 2 weeks at -20 degrees C, The limits of detect ion for BE, COG, NC and CE were 20, 24, 15 and 12.9 ng/ml, respectivel y. These values correspond to 0.70, 0.84, 0.525 and 0.452 ng of the ac cording compound being injected on column. The within-day coefficient of variation for the four compounds ranged from 3.0% to 9.9% while the between-day precision varied from 3.6% to 14%. This method was used t o analyze rat plasma samples after administration of COC alone and in combination with alcohol. The pharmacokinetic profiles of COC and its metabolites in these rats are also described. (C) 1997 Elsevier Scienc e B.V.