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.

Determination of amphetamine enantiomers in urine by conductive vial electromembrane extraction and ultra‐high performance supercritical fluid chromatography tandem mass spectrometry

“…Comparing the proposed EME method with sample preparation protocols reported in other chromatography‐based bioanalysis of amphetamine enantiomers, it is evident that the organic solvent consumption of the majority of reported methods is an order of magnitude higher than EME 40–44 . Notable exceptions are methods proposed by Chermá et al, 45 where urine was diluted with water, and by Hädener et al, 46 which employed on‐line column switching in combination with LC–MS.…”

“…Comparing the proposed EME method with sample preparation protocols reported in other chromatography-based bioanalysis of amphetamine enantiomers, it is evident that the organic solvent consumption of the majority of reported methods is an order of magnitude higher than EME. [40][41][42][43][44] Notable exceptions are methods proposed by Chermá et al, 45 where urine was diluted with water, and by Hädener et al, 46 (3:2 v/v) showed that carry-over was reduced in the latter injection solvent. In the present application, the total amphetamine concentration was known prior to the enantioselective analysis, and carry-over related issues could be circumvented by injection of blank samples.…”

Deciphering microbiota of acute upper respiratory infections: a comparative analysis of PCR and mNGS methods for lower respiratory trafficking potential

“…Much of respiratory medicine is reliant on timely and precise diagnostics for critical This is significant to respiratory medicine because this technology can be used to supplement current syndromic-based tests and data can quickly and effectively be phenotypically characterized for clinical (co)infection and comorbid consideration. This has significance for laboratory medicine because it demonstrated this approach can rapidly be interpreted with a user-friendly and reliable platform for collective intention without overburdening laboratory investments in technology and people [75,76]. Furthermore, this approach could be advanced into a pan-microbial diagnostic testing that utilizes a single workflow for all specimen types.…”

Quantifying 64 drugs, illicit substances, and D- and L- isomers in human oral fluid with liquid-liquid extraction

“…Oral fluid drug testing is increasingly emerging as an alternative biological matrix for detecting drugs and monitoring patient medication compliance [4][5][6][7][8]. Moreover, in certain clinical situations clinicians may find oral fluid more beneficial for detection of specific drugs over UDT [9].…”