Quantification of illicit drugs in urine for forensic analysis in routine laboratories using ion trap LC-MSn

Abstract

Aim Analysis of illicit drugs has become a major task in forensic and clinical research toxicology, doping control, workplace drug testing or law enforcement laboratories. Xenobiotics in biological samples are generally screened using various qualitative methods, often followed by targeted quantitative confirmation methods. In the past decades, liquid chromatography coupled to mass spectrometry has evolved to become the standard analytical technique for confirmatory analysis. Recently, a fast, robust and easy-to-use LC-MSn system was introduced for routine drug screening. In this study, quantitative analysis of amphetamines in extracted urine samples was carried out to demonstrate the applicability of the same instrument configuration for illicit drug confirmatory analysis, which enables usage of a single bench-top LC-MSn platform for high-confident multi-purpose analyses. Methods Authentic reference standard solutions of amphetamine (AMP), methamphetamine (MET), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), methylbenzodioxolylbutanamine (MBDB) and their respective deuterated internal standards (IS) were used. The LC-MS analyses have been performed on an amaZon speed ion trap system with the ESI interface coupled to an UltiMate 3000 RSLC using an Acclaim RSLC 120 C18 column (2.2 μm; 2.1 × 100 mm) and a gradient system of acetonitrile/water/ammonium formate/formic acid as mobile phase. Data acquisition utilized SmartFrag and a novel smartMRM mode for high-sensitivity multi-target analysis. Data analysis combined library based unequivocal analyte identification and subsequent quantitation. Applicability of the method was tested with spiked urine matrix. The sample preparation was conducted by solid-phase extraction or by liquid-liquid extraction. Results The most common amphetamines and their respective internal standards were used to create a routine-ready quantitation method based on an actual LC-MSn screening platform. First, electrospray ionization conditions were optimized to achieve best selectivity and sensitivity for the target analytes. The chromatographic method was developed to efficiently separate the analytes while retaining the analysis time as short as possible. A novel targeted smartMRM mode enabled acquisition of full scan MS and MS2 spectra, and if necessary MS3, for multiple targeted experiments within defined retention time windows. Ideally, only the target analyte and its respective IS were present in a given time window: Minimal overlap of chromatographic peaks results in highest data acquisition rates for each target and thus yields highly reproducible and accurate values for a subsequent qualitative and quantitative analysis. Specificity, sensitivity, linearity, recovery and matrix effects were evaluated during method development with spiked urine samples extracted by LLE or SPE. Conclusion In contrast to LC triple quad instruments, MRM on an ion trap mass spectrometer provides full scan fragment information up to MSn which can be used for unambiguous identification and quantitation at the same time. In this way, primary analytical target identification was achieved by spectral library search based on full scan MSn spectra and retention time matching. Absolute quantitation, necessary to determine the actual analyte concentrations in a sample were achieved by generating MS2 extracted ion chromatograms of the main target ion transitions. According to the obtained results, the utilized LC ion trap system allows for qualitative library based screening but also enables quantitative target confirmation on the same instrument platform after thorough method development and validation.