title: High-throughput simultaneous analysis of buprenorphine, methadone, cocaine, opiates, nicotine, and metabolites in oral fluid by liquid chromatography tandem mass spectrometry.
abstract: 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 μL) and OF (250 μL) 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 μg/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 μg/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 °C, for 72 h at 4 °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 Salivette® OF collection device from an opioid-dependent BUP-maintained pregnant woman. BUP ranged from 0 to 7,400 μg/L, NBUP from 0 to 71 μg/L, methadone from 0 to 3 μg/L, nicotine from 32 to 5,020 μg/L, cotinine from 125 to 508 μg/L, OH-cotinine from 11 to 51 μg/L, cocaine from 0 to 419 μg/L, BE from 0 to 351 μg/L, EME from 0 to 286 μg/L, AEME from 0 to 7 μg/L, morphine from 0 to 22 μg/L, codeine from 0 to 1 μg/L, 6AM from 0 to 4 μg/L, and heroin from 0 to 2 μg/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.
authors.affiliation: Servicio de Toxicología Forense, Dpto. Anatomía Patológica y Ciencias Forenses, Facultad de Medicina, Universidad de Santiago de Compostela, C/San Francisco s/n, 15782, Santiago de Compostela (A Coruña), Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Concheiro
authors.forename: Teresa R
authors.initials: TR
authors.lastname: Gray
authors.forename: Diaa M
authors.initials: DM
authors.lastname: Shakleya
authors.forename: Marilyn A
authors.initials: MA
authors.lastname: Huestis
mesh_terms: D000779:Anesthetics, Local / Q000032:analysis / Q000378:metabolism; D002047:Buprenorphine / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D003042:Cocaine / Q000032:analysis* / Q000378:metabolism; D005260:Female; D005731:Ganglionic Stimulants / Q000032:analysis / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D008691:Methadone / Q000032:analysis* / Q000378:metabolism; D009294:Narcotics / Q000032:analysis* / Q000378:metabolism; D009538:Nicotine / Q000032:analysis* / Q000378:metabolism; D011247:Pregnancy; D012463:Saliva / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000779:Anesthetics, Local; D005731:Ganglionic Stimulants; D009294:Narcotics; D002047:Buprenorphine; D009538:Nicotine; D003042:Cocaine; D008691:Methadone