Validation of urine drug-of-abuse testing methods for ketobemidone using thin-layer chromatography and liquid chromatography–electrospray mass spectrometry

Abstract

High-performance thin-layer chromatography (TLC) with visual detection (post-chromatographic derivatization) was used in screening for the drug ketobemidone in human urine samples. High-performance liquid chromatography with electrospray mass spectrometry (LC–ESI–MS) was used for final confirmation of the result. The clean-up was performed by mixed-mode solid-phase extraction, and nalorphine was used as internal standard. A screening cut-off for TLC was established at 0.2 μg/ml. The mean recovery for LC–MS was 91% ( n=60) with coefficients of variation (C.V.) in the range of 7 to 16%. Qualifying fragment ions of ketobemidone ( m/z 190, 201 and 230) were generated by up front collision-induced dissociation (CID) on a single quadrupole instrument. Relative ion intensities were within ±15% deviation compared with standards in the same batch. The limit of detection for LC–MS was 0.025 μg/ml. Positive clinical samples from drug abusers ( n=10) had concentrations in the range 0.07 to 3.2 μg/ml, which could be determined by LC–MS without matrix interference. During screening of unknown clinical samples ( n=27) the results from TLC was in agreement with LC–MS data. After acid hydrolysis of conjugates in clinical samples the analyte response of ketobemidone and norketobemidone was increased by a factor of approximately two and twelve, respectively. A qualitative GC–MS technique was demonstrated for the detection of the spasmolyticum A29 (N, N-dimethyl-4,4-diphenyl-3-buten-2-amine), which can be found in a preparation combined with ketobemidone (Ketogan).