REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF COCAINE IN PLASMA AND HUMAN HAIR WITH DIRECT FLUOROMETRIC DETECTION

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine with direct fluorimetric detection, particularly intended for the routine analysis of hair and blood samples, is described. Benzoylecgonine, eluting before cocaine in a completely resolved peak, is also detectable. Detection is based on the weak native fluorescence of cocaine and benzoylecgonine, depending on the benzene ring present in both molecules. Hair samples (20-200 mg) were incubated overnight in 2 ml of 0.25 M HCl at 45 degrees C and extracted with a commercial liquid-liquid method; the dried residue reconstituted with 500 mu l of 0.05 M NaH2PO4 (pH 5.2) was injected. Blood plasma samples (200 mu l) were mixed with 150 mu l of 0.1 M Na2HPO4 (pH 8.9) and extracted with 5 ml of chloroform-2-propano1 (9:1); the organic phase was evaporated and the residue dissolved and injected as above. Isocratic reversed-phase liquid chromatography was carried out on a column (150 x 4.6 mm I.D.) packed with spherical 5-mu m poly(styrene-divinylbenzene) particles; the mobile phase was 0.1 M potassium phosphate (pH 3)-methanol-tetrahydrofuran (70:25:5). The excitation and emission wavelengths were set at 230 and 315 nm, respectively. Under the described conditions, cocaine eluted in a symmetrical peak with a capacity factor of about 5. The limit of detection was about 1 ng/ml (0.2 ng injected), with a signal-to-noise ratio of 3. Using external standardization and partial loop filling, the intra-assay precision of the assay was characterized by R.S.D.s of 5.0 and 3.6% (n = 6) for cocaine concentrations of 10 and 100 ng/ml, respectively, and in inter-assay tests (n = 6) R.S.D.s of 7.5 and 5.2% were achieved for the same cocaine levels. The linearity of the method was fairly good in the concentration range 1.5-500 ng/ml (r(2) = 0.9998). Possible interferences from as many as 90 therapeutic and/or illicit drugs were excluded.