JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2005; 40: 739–753 Published online 1 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.846 Validated toxicological determination of 30 drugs of abuse as optimized derivatives in oral fluid by long column fast gas chromatography/electron impact mass spectrometry Teemu Gunnar, 1 * Kari Ariniemi 1 and Pirjo Lillsunde 1,2 1 National Public Health Institute, Drug Research Unit, Mannerheimintie 166, 00300 Helsinki, Finland 2 Helsinki University of Technology, Laboratory of Inorganic and Analytical Chemistry, 02015 Espoo, Finland Received 30 December 2004; Accepted 9 February 2005 An analytical procedure was developed for the simultaneous sensitive identification, screening and quantitation of 30 drugs of abuse using 250 μl of human oral fluid. The method employs sequential mixed-mode solid-phase extraction (SPE), optimized derivative formation and long-column fast gas chromatography/electron impact mass spectrometry (GC/EI-MS). After sequential SPE elution, the most sensitive and stable derivatives were formed by taking careful account of the characteristics of the active functional groups and possible steric hindrances affecting derivatization chemistry. Amphetamine- type stimulant drugs were acylated with heptafluorobutyric anhydride, benzodiazepines and 1 9 - tetrahydrocannabinol were silylated with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and benzoylecgonine, codeine, ethylmorphine, 6-monoacetylmorphine, morphine, pholcodine, buprenorphine and norbuprenorphine with N-methyl-N-(trimethylsilyl)trifluoroacetamide. In addition, the following analytes were included: methadone, cocaine, alprazolam, midazolam, fentanyl and zolpidem. In GC separation, fast temperature ramping and high carrier gas flow-rate combined with long 30 m columns of i.d. 0.32 mm offered a reduction in analysis time and sharp peak shapes while still maintaining sufficient resolution and high sample capacity. Validated parameters including selectivity, linearity, accuracy, intra- and inter-day precision, extraction efficiency and limit of quantitation were all within required limits. In contrast to previously published methods, this single procedure is suitable for the simultaneous toxicological determination of the most common illicit drugs and benzodiazepines, and also zolpidem, in a small amount of oral fluid. Copyright  2005 John Wiley & Sons, Ltd. KEYWORDS: oral fluid; drugs of abuse; fast gas chromatography/mass spectrometry; quantitative determination; toxicological analysis INTRODUCTION Oral fluid (OF) is a complex biological matrix consist- ing of the secretory products of salivary glands (saliva) mixed with other fluids, substances and cellular debris that are present in the oral cavity. Over 90% of saliva is produced mainly by the three major salivary glands (parotid, submandibular and sublingual), whereas the numerous labial, buccal and palatal glands make a smaller contribution. 1,2 The primary transportation mechanism of drug molecules from plasma to saliva is passive diffusion across the concentration gradient through lipid membranes, although other mechanisms such as active transportation L Correspondence to: Teemu Gunnar, National Public Health Institute, Drug Research Unit, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: teemu.gunnar@ktl.fi Contract/grant sponsor: Ministry of the Interior. Contract/grant sponsor: Ministry of Transport and Communications. Contract/grant sponsor: European Union. and ultrafiltration through pores in the membrane might also have a role. 1,3,4 The main factors increasing the pas- sive diffusion process are lipophilia, neutrality and non- bonding to plasma proteins of a certain drug molecule. 1,4,5 Therefore, low concentrations of certain drug molecules (e.g. benzodiazepines) can be at least partly explained by high plasma protein binding, which prevents passive dif- fusion through membranes, and parent compounds rather than more hydrophilic metabolites are generally the pri- mary target analytes in OF analyses. Furthermore, if the salivary flow is increased, for example by mechanical (chew- ing) or chemical stimulation (citric acid, sour candy), the bicarbonate electrolyte concentration rises, resulting in a higher pH value for saliva. 1,6,7 This decreases the saliva- to-plasma ratio (S/P) of weakly basic drug molecules (e.g. cocaine, opiates) with pK a values close to the pH of saliva (6.0–7.8). The saliva collection method used should, there- fore, be taken into account when quantitative data are being interpreted. Copyright  2005 John Wiley & Sons, Ltd.