Chiral separation of the enantiomers of ephedrine, amphetamine, methamphetamine, MDA, MDMA, and phentermine in blood using LC-MS/MS
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Amphetamine-type stimulants are widely abused due to their ability to stimulate the central nervous system and elicit feelings of confidence, wakefulness, mood elevation, and euphoria. After cannabis, amphetamines were the most abused group of illicit substances in 2016 according to the National Forensic Laboratory Information System Annual Report. Included in this group are ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-metheylenedioxymethamphetamine (MDMA). Structurally, each of these compounds contain a chiral center, causing them to have an R-(-) and S-(+)- enantiomer (also called levo- and dextro-, respectively). Despite their similarity, the R- and S- enantiomers display differing pharmacological effects, with the S-enantiomer producing a stronger, longer-lasting effect than the R-enantiomer. Because of this, R-methamphetamine, for example, has therapeutic uses and is the active ingredient in some over-the-counter nasal decongestant products (e.g. Vicks® vapor inhaler). S-methamphetamine, on the other hand, is generally found in illicit sources. As a result of these chiral centers, these compounds have differing legal statuses. The aim of this research was to develop and validate a method for the separation and analysis of eleven amphetamine-class compounds in blood for forensic casework. This was accomplished using a liquid-liquid extraction and analysis on a liquid chromatography-tandem mass spectrometer (LC-MS/MS) (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was achieved using a Phenomenex Lux® AMP chiral column (Phenomenex, Torrance, CA, USA), under gradient aqueous and organic mobile phase conditions, with a total run time of just over 17 minutes. The target analytes included 1S,2R-ephedrine, 1R,2S-ephedrine, R-amphetamine, S-amphetamine, R-methamphetamine, S-methamphetamine, phentermine, R-MDMA, S-MDMA, R-MDA, and S-MDA with 1S,2R-ephedrine-d3 and MDMA-d5 as the internal standards (Cerilliant, Round Rock, TX, USA). The analytical method was validated according to the Scientific Working Group for Forensic Toxicology guidelines (now a subcommittee of the Organization of Scientific Area Committees for Forensic Science), including the assessment of its linearity, limits of detection and quantitation, bias, precision, interferences, matrix effects, carryover, and processed sample stability . The limit of detection (LOD) was 2 µg/L for all compounds except MDMA and MDA, which had LODs of 10 µg/L. The lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) was 10 µg/L and 1000 µg/L for all compounds, respectively. The precision was within 15% for all analytes, with the bias extending outside the ±20% range for at least one set of samples for all analytes except 1S,2R-ephedrine and both MDA enantiomers. Matrix effect studies showed average ion enhancement (140%-361%), extraction efficiencies (60%-123%), and process efficiencies (105%-432%) across all analytes. No interferences were detected from isotope internal standards, postmortem blood, antemortem blood, or 85 commonly seen drugs in forensic casework. No carryover was observed following injections of analytes at the ULOQ (1000 µg/L). To demonstrate applicability in authentic casework, the method was applied to 28 cases that had previously been analyzed using a non-chiral method. By selectively identifying R- and S- enantiomers, this method may be used in forensics laboratories where the question of the licit or illicit use of amphetamines is of importance.