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This article details the rapid extraction of amphetamines from oral fluid using low specimen volume, low sorbent bed mass, and fast gas chromatography with mass selective detection. The collection of a known amount of sample volume coupled with high percentage recovery of drug from the collection pad has allowed oral fluid to be increasingly employed as the specimen of choice for the detection of drug use in various applications. Additionally, low specimen volume for confirmation is required, so that adequate test volume remains for second analysis in case of batch failure or for testing at a different laboratory facility. The extracts were prepared using low bed mass sorbent, so less conditioning, washing, and elution solvent further reduced the overall cost of sample preparation. The limits of quantitation for the assay were 25 ng/mL; the intraday precision of the assays (n = 5) ranged from 0.3 to 3.99%; interday precision ranged from 0.72 to 4.6% for the amphetamine class. The percentage recovery of the drugs from the collection pads ranged from 85.4 to 89.1% (n = 6). The process lends itself to widely available automated processing instrumentation and significantly increases the efficiency of laboratories providing high-volume oral fluid analysis for drugs of abuse. Introduction Oral fluid is becoming increasingly popular in many areas of drug testing as a diagnostic fluid, partly due to the ease and non-invasiveness of collection (1). Applications for the detection of amphetamines in oral fluid analysis have been reported in clinical areas (2,3) as well as several drugs of abuse in epidemiological studies (4) and in roadside situations (5,6). The presence of measurable drug levels has enhanced the number of laboratories engaged in the analysis of oral fluid, and as the overall number of analyses increase, the rapid, reproducible extraction of drugs from the collection buffer is an essential component. However, because a limited sample volume is generally collected when oral fluid is to be tested, procedures for the use of reduced sample volume along with improved analytical capability are necessary. Most importantly for our application, the amount of specimen required for extraction is significantly reduced. Other advantages of the assays include excellent precision, accuracy, recovery from the collection pad, decreased sample preparation time, and reduced solvent volumes for conditioning, washing, and elution. The analytical procedure employed a gas chromatograph (GC) equipped with fast oven capability coupled to a mass selective detector (MS) with an inert source operating in electron impact mode. In 2004, the Substance Abuse and Mental Health Service Administration (SAMHSA) proposed the inclusion of oral fluid as a specimen for workplace drug testing (7). The proposed confirmatory cut-off concentration for amphetamine (AMP), methamphetamine (METH), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), and methylenedioxyethylamphetamine (MDEA) was 50 ng/mL. The objective of our study was twofold: to determine the efficiency of recovery of amphetamines from the oral fluid collection system and to achieve proposed regulatory "cut-off concentrations" for amphetamines using 62.5 L of neat oral fluid (0.25 mL of total specimen). Materials and Methods Quantisal™ oral fluid collection devices were obtained from Immunalysis (Pomona, CA). Amphetamine, methamphetamine, MDMA, MDA, MDEA, and their penta-deuterated analogues were obtained from Cerilliant (Round Rock, TX). Mixed-mode solid-phase extraction columns (HCX 130 mg/1 mL capacity; Part # 902-0013-A) were obtained from Biotage (Charlottesville, VA). All reagents were of high-performance liquid chromatography (HPLC) grade, and chemicals were of ACS grade or better. The derivatizing agent, heptafluorobutyric anhydride (HFBA) was obtained from Pierce Chemical (Rockford, IL). Achieving Proposed Federal Concentrations using Reduced Specimen Volume for the Extraction of Amphetamines from Oral Fluid Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. Journal of Analytical Toxicology, Vol. 31, October 2007 * Author to whom correspondence should be addressed. E-mail: cmoore@immunalysis.com. Christine Moore, Cynthia Coulter, and Katherine Crompton Immunalysis Corporation, 829 Towne Center Drive, Pomona, California 91767 Abstrac