Validation of an ultra performance liquid chromatography tandem mass spectrometry (UPLC™/MS/MS) method for forensic toxicological analysis : confirmation and quantitation of lysergic acid diethylamide (LSD) and its congeners in forensic samples

Abstract

The Royal Canadian Mounted Police (RCMP) Forensic Laboratory Services (FLS) needed a method to confirm positive lysergic acid diethylamide (LSD) immunoassay screening results. As a result, an ultra performance liquid chromatography tandem mass spectrometry (UPLC¢â/MS/MS) method was validated for the confirmation and quantitation of LSD, iso-LSD, N-demethyl-LSD (nor-LSD), and 2-oxo-3-hydroxy-LSD (O-H-LSD). The method was validated in urine and whole blood, where linearity, accuracy, precision, sensitivity, stability, selectivity, recovery, matrix effects, and reproducibility were evaluated. The method involved a liquid-liquid extraction (LLE) of the analytes and the deuterated internal standard from 1 mL of urine or whole blood with dichloromethane:isopropyl alcohol after being basified. The average recovery for all analytes was ¡Ã 62%, and the matrix effect was found to be insignificant. MS/MS analysis was conducted with a triple quadrupole mass spectrometer by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The lowest limit of quantitation (LLOQ) was 20 pg/mL for LSD and iso-LSD, and 50 pg/mL for nor-LSD and O-H-LSD. The method was linear, accurate, precise, selective, and reproducible from 20 to 2000 pg/mL for LSD and iso-LSD, and from 50 to 2000 pg/mL for nor-LSD and O-H-LSD with an r2 ¡Ã 0.99. The refrigerated and frozen long term stability was investigated for 90 days. LSD was stable at all temperatures for 90 days. Iso-LSD in blood was also stable at all temperatures for 90 days, but iso-LSD in urine showed an initial decrease followed by a gradual increase back to day 0 concentrations. Nor-LSD was stable at all temperatures up to day 14, with >43% decrease by day 30, with no additional decrease for the next 60 days. O-H-LSD in urine was stable at all temperatures for 90 days, but by day 90 O-H-LSD in whole blood stored refrigerated decreased in concentration by >37%. Additionally, a case sample that was stored at -50¡ÆC for ten years was found to still contain measurable amounts of each compound. The method was applied to blind samples and a case that screened positive with immunoassay. Retention time, relative retention time, and ion ratios were used as identification parameters and found to correctly identify the analytes 100% of the time with no false positives. The case sample showed that the concentration of O-H-LSD was 4 times greater than LSD in urine. Furthermore, both the detection of O-H-LSD in a blood case sample, and LSD in a vitreous humor case sample were the first to be documented.