Hsiu-Chuan Liu

Direct Injection LC–MS-MS Analysis of Opiates, Methamphetamine, Buprenorphine, Methadone and Their Metabolites in Oral Fluid from Substitution Therapy Patients

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed, validated and applied to simultaneous analysis of oral fluid samples for the following 10 analytes: methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, norbuprenorphine, morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, amphetamine, and methamphetamine. The oral fluid sample was briefly centrifuged and the supernatant was directly injected into the LC-MS-MS system operated under reverse-phase chromatography and electrospray ionization (ESI). Deuterated analogs of the analytes were adopted as the internal standards and found to be effective (except for buprenorphine) to compensate for potential matrix effects. Each analytical run took <10 min. Linearity range (r(2) > 0.99) established for buprenorphine and the other nine analytes were 5-100 and 1-100 ng/mL. Intra- and interday precision (% CV) ranges for the 10 analytes were 0.87-12.2% and 1.27-12.8%, while the corresponding accuracy (%) ranges were 91.8-113% and 91.9-111%. Limits of detection and quantitation established for these 10 analytes were in the ranges of 0.1-1.0 and 0.25-1.0 ng/mL (5 ng/mL for buprenorphine). The method was successfully applied to the analysis of 62 oral fluid specimens collected from patients participating in methadone and buprenorphine substitution therapy programs. Analytical results of methadone and buprenorphine were compared with data derived from GC-MS analysis and found to be compatible. Overall, the direct injection LC-MS-MS method performed well, permitting rapid analysis of oral fluid samples for simultaneous quantification of methadone, buprenorphine, opiate and amphetamine drug categories without extensive sample preparation steps.

Simultaneous Quantitation of Amphetamines and Opiates in Human Hair by Liquid Chromatography–Tandem Mass Spectrometry

In this study, an incubation, solid-phase extraction (SPE) and LC-MS-MS procedure was developed, validated and used for simultaneous analysis of amphetamine (AP), methamphetamine (MA), morphine (MOR), codeine (COD), 6-acetylmorphine (6-AM) and 6-acetylcodeine (6-AC) in hair. Hair samples were initially cut into sections, washed with dichloromethane, then sonicated in a methanol-trifluoroacetic acid mixture. The resulting solutions were processed with a SPE procedure before undergoing LC-MS-MS analysis. Mass spectrometric analysis was performed in positive-ion, multiple reactions monitoring (MRM) mode, using appropriate collision energy for each selected precursor ion. The overall protocol, when applied to the analysis of hair (50 mg) samples fortified with 100-10,000 pg/mg of the analytes, was found to achieve 55.5-74.6% recovery of the six analytes with the following analytical parameters: (i) intra- and interday precision/accuracy data for the six analytes in the 1.6-7.6%/-6.0-12.8% and 1.3-6.6%/-6.9-9.3% ranges, respectively; (ii) r(2) > 0.998 for all six analytes and (iii) LOD 2 pg/mg for AP and MA, and 8 pg/mg for MOR, COD, 6-AM and 6-AC; LOQ 10 pg/mg for all six analytes. This method was then utilized to (i) analyze hair samples collected from 86 self-reported drug users and (ii) evaluate the deposition pattern of drugs in head hairs from four female MA and heroin users in a rehabilitation facility. This relatively simple protocol was found superior over the GC-MS methods we have previously developed and utilized in our laboratory for the analysis of these six analytes.

The Quantitative Determination of Opiates and Amphetamines in Oral Fluid by Direct Injection Liquid Chromatography-Tandem Mass Spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of opiates and amphetamines in oral fluid was developed and validated. A simple sample preparation of oral fluid involving centrifugation and injection of 10 μL of the supernatant substantially reduced total analysis time. Owing to the basic nature of the drugs and their metabolites, LCMS/ MS analysis was performed by reverse-phase chromatography and electrospray ionization (ESI) which is a rugged and efficient ionization technique for basic drugs. Stable isotope-labeled internal standards were used in the analysis and the calibration to compensate for any matrix effects. The method was fully validated with the linearity of the developed method established in the concentration range of 1 to 500 ng/mL and the limits of detection and quantitation were 1 ng/mL for amphetamine, methamphetamine, morphine, codeine, 6-acetylmorphine and 6-acetylcodeine. The precision and accuracy were determined by spiking oral fluid samples at six concentration levels. For all analytes, the relative standard deviations of intra- and inter-day precision were 0.9 to 6.4% and 0.9 to 12.9%, respectively. The method was also successfully applied to the analyses of 34 oral fluid specimens collected from patients participating in the substitution therapy program.