A novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-D-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC-MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R2) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged from 52–88 % in plasma and 51–118 % in urine. The limit of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL respectively with the exception of cotinine-N-β-D-glucuronide which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14 % and ≤17 % respectively. Matrix effect (%) was sufficiently minimized to ≤19 % for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze thaw cycles, 24 hours at room temperature, 24 hours in the refrigerator (4 °C) and 1 week in the freezer (−20 °C). Reconstituted plasma and urine extracts were stable for at least 72 hours storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrolment) and on the pharmacokinetic study day.

An anti-diazepam, molecularly imprinted polymer (MIP) has been synthesized and used to extract diazepam and other benzodiazepines from hair samples via a molecularly imprinted solid-phase extraction (MISPE) protocol. Optimum retention of diazepam on the MIP columns was achieved using an apolar solvent, and the binding capacity of the polymer toward diazepam was found to be 110 ng of diazepam/mg of polymer. The recovery of a 50 ng diazepam standard spiked into blank hair was 93%, with good precision (RSD = 1.5%). The LOD and LOQ of diazepam in spiked hair samples were 0.09 and 0.14 ng/mg, respectively. The MISPE method was demonstrated to be applicable to the analysis of diazepam metabolites and other benzodiazepine drugs, in addition to diazepam itself. The application of the extraction method to postmortem hair samples yielded results that were in good agreement with the corresponding ELISA data (from blood samples) and data arising from the analysis of the same blood samples using a validated in-house SPE-LC-MS-MS method.

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A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates from hair using a single extraction method. As part of the method development, Gemini C18, Synergi Hydro RP, and Zorbax Stablebond-Phenyl LC columns were tested with three different mobile phases. Analyte recovery and limit of detection were evaluated for two different solid-phase extraction methods that used Bond Elut Certify and Clean Screen cartridges. Phosphate buffer (pH 5.0) was chosen as the optimum hair incubation medium because of the high stability of cocaine and 6-monoacetylmorphine using this method and faster sample preparation. The optimized method was fully validated. Linearity was established over the concentration range 0.2-10 ng/mg hair, and the correlation coefficients were all greater than 0.99. Total extraction recoveries were greater than 76%, detection limits were between 0.02 and 0.09 ng/mg, and the intra- and interday imprecisions were generally less than 20% in spiked hair. The intra- and interbatch imprecision of the method for a pooled authentic hair sample ranged from 1.4 to 23.4% relative standard deviation (RSD) and 8.3 to 25.4% RSD, respectively, for representative analytes from the different drug groups. The percent matrix effect ranged from 63.5 to 135.6%, with most analytes demonstrating ion suppression. Sixteen postmortem samples collected from suspected drug-related deaths were analyzed for the 17 drugs of abuse and metabolites included in the method. The method was sufficiently sensitive and specific for the analysis of drugs and metabolites in postmortem hair samples. There is scope for the inclusion of other target drugs and metabolites in the method.