Reversed-phase high-performance liquid chromatographic determination of cocaine in plasma and human hair with direct fluorimetric detection

Abstract 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°C and extracted with a commercial liquid-liquid method; the dried residue reconstituted with 500 μl of 0.05 M NaH 2 PO 4 (pH 5.2) was injected. Blood plasma samples (200 μl) were mixed with 150 μl of 0.1 M Na 2 HPO 4 (pH 8.9) and extracted with 5 ml of chloroform-2-propanol (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 × 4.6 mm I.D.) packed with spherical 5-μm 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.