A Human Oral Fluid Assay for D- and L- Isomer Detection of Amphetamine and Methamphetamine Using Liquid-Liquid Extraction

Abstract

Medical providers are increasingly confronted with clinical decision-making that involves (meth)amphetamines. And clinical laboratories need a sensitive, efficient assay for routine assessment of D- and L-isomers to determine the probable source of these potentially illicit analytes. This paper presents a validated method of D- and L-isomer detection in human oral fluid from an extract used for determination of a large oral fluid assay (63 analytes) on an older AB SCIEX 4000 instrument. Taken from the positive extract, D- and L-analytes were added. The method for extraction included addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. The samples were suspended in 50% MeOH in water, diluted with mobile phase, with separation and detection accomplished using LC-MS/MS to determine analyte concentration. Once samples were confirmed positive for (meth)amphetamine from the large oral fluid assay, they were further examined for the enantiomeric forms with 50 μl aliquots of the standards and samples of interest combined with 450 μl of D- and L-assay mobile phase, then analyzed using chiral column separation, and LC-MS/MS detection with standard curve spanning the range from 2.5 to 1000 ng/mL. The result is a sensitive and accurate detection of D- and L-isomers of amphetamine and methamphetamine in human oral fluid performed on an older model mass spectrometer (AB SCIEX 4000). The novelty of this assay is twofold (a) the 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples, and (b) its adoption characteristics as a reflex test from a large ODT panel without the need to invest in newer or expensive LC-MS/MS instruments. Finally, this assay also has potential to add a valuable option to high-throughput laboratories seeking a D- and L-testing alternative to urine drug testing methods.