High-throughput simultaneous analysis of buprenorphine, methadone, cocaine, opiates, nicotine, and metabolites in oral fluid by liquid chromatography tandem mass spectrometry

A method for simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), morphine, codeine, 6-acetylmorphine (6AM), heroin, 6-acetylcodeine (6AC), nicotine, cotinine, and trans-3'-hydroxycotinine (OH-cotinine) by liquid chromatography tandem mass spectrometry in oral fluid (OF) was developed and extensively validated. Acetonitrile (800 mu L) and OF (250 mu L) were added to a 96-well Isolute-PPT+protein precipitation plate. Reverse-phase separation was achieved in 16 min and quantification was performed by multiple reaction monitoring. The assay was linear from 0.5 or 1 to 500 mu g/L. Intraday, interday, and total imprecision were less than 13% (n = 20), analytical recovery was 92-114% (n = 20), extraction efficiencies were more than 77% (n = 5), and process efficiencies were more than 45% (n = 5). Although ion suppression was detected for EME, cocaine, morphine, 6AC, and heroin (less than 56%) and enhancement was detected for BE and nicotine (less than 316%), deuterated internal standards compensated for these effects. The method was sensitive (limit of detection 0.2-0.8 mu g/L) and specific (no interferences) except that 3-hydroxy-4-methoxyamphetamine interfered with AEME. No carryover was detected, and all analytes were stable for 24 h at 22 A degrees C, for 72 h at 4 A degrees C, and after three freeze-thaw cycles, except cocaine, 6AC, and heroin (22-97% loss). The method was applied to 41 OF specimens collected throughout pregnancy with a SalivetteA (R) OF collection device from an opioid-dependent BUP-maintained pregnant woman. BUP ranged from 0 to 7,400 mu g/L, NBUP from 0 to 71 mu g/L, methadone from 0 to 3 mu g/L, nicotine from 32 to 5,020 mu g/L, cotinine from 125 to 508 mu g/L, OH-cotinine from 11 to 51 mu g/L, cocaine from 0 to 419 mu g/L, BE from 0 to 351 mu g/L, EME from 0 to 286 mu g/L, AEME from 0 to 7 mu g/L, morphine from 0 to 22 mu g/L, codeine from 0 to 1 mu g/L, 6AM from 0 to 4 mu g/L, and heroin from 0 to 2 mu g/L. All specimens tested negative for EDDP and 6AC. This method permits a fast and simultaneous quantification of 16 drugs and metabolites in OF, with good selectivity and sensitivity.