Drug Testing and Analysis最新文献

Bisphosphonate drugs and myo-inositol trispyrophosphate are of concern to the racing industry and have been listed as prohibited substances in equine sports. The current bisphosphonate plasma screening analysis employed at the Australian Racing Forensic Laboratory involves the use of sequential solid-phase extraction procedures, passing the samples through a mixed mode cartridge, followed by a weak anion exchange cartridge. The eluates collected following the second extraction are then methylated and analysed by liquid chromatography-mass spectrometry. Under these extraction conditions, some bisphosphonates have shown poor recovery. To improve the extraction efficacy, the effects of cartridge chemistry were evaluated. In particular, the weak anion exchange cartridges used for screening were compared to an affinisep AttractSPE polymeric phase cartridge. The effectiveness of each extraction approach was assessed through both a visual comparison of signal to noise in extracted chromatograms and recovery measurements to determine the best approach for routine screening.

Bioanalysis, such as the quantification of drugs in different matrices, is of great importance in forensic toxicology. Nowadays, mainly so-called multianalyte approaches are used given their increased speed and effectiveness. However, such multianalyte procedures can be difficult to develop and maintain with sufficient robustness in the laboratory. One aspect of this is the tedious, manual preparation of spiking solutions containing such a great number of analytes. Therefore, the current study aimed to develop and validate a fast, simple, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 82 classic drugs and to evaluate an alternative autosampler-assisted automated approach for the preparation of spiking solutions. Simple protein precipitation of 200-μL whole blood was used followed by analysis by reversed-phase LC-MS/MS in advanced scheduled multiple reaction monitoring (MRM) mode. The method was fully validated according to international guidelines, including selectivity, recovery, matrix effects, linearity, bias/imprecision, processed-sample stability, and limits. Validation criteria were fulfilled for all analytes except for buprenorphine and five benzodiazepines. In the context of a multianalyte procedure, a (multipurpose) autosampler-assisted automatic preparation of calibrator spiking solutions proved comparable to manual preparation. Thus, automated preparation can overcome the frequently performed manual, time-consuming, and error-prone steps of multianalyte approaches and still allow for customized calibration ranges. Since its introduction, more than 8000 cases have been measured with the presented method, and 35 proficiency tests have been passed.

The applicability of urinary minimum reporting limits (MRLs) to determine in-competition use of prohibited substances is an evolving topic. Most stimulants are subject to a universal MRL, despite the wide range of commercially available dosages for commonly used stimulants. Further, it is unknown whether the urinary MRL is reflective of a pharmacological dose ingested after the start of the in-competition period. To evaluate whether urinary MRLs can distinguish between in-competition and out-of-competition use, a controlled administration study was performed with three commonly used stimulants-amphetamine, methylphenidate, and modafinil at relatively low but therapeutically relevant dosages. Four to six volunteers were administered a particular drug once per day for five consecutive days. Urine, serum, dried blood spots (DBS), and oral fluid (OF) were collected during the active administration period and for 48 h after cessation of use. For all participants, urinary concentrations for all target analytes exceeded the MRL even 48 h after cessation of use. In serum and DBS, most volunteers showed detectable amounts at 48 h post use. Peak concentrations were variable between target compounds even with similar administered dosages. Further, there was a reproducible difference between serum and DBS concentrations. Interpretation of results from OF measurements was challenging due to the inability to normalize for hydration status and OF viscosity. Analyte concentrations decreased steadily over the washout period but did not correlate across matrices for all target analytes. The study reiterates the challenges associated with determining in-competition use by relying on urinary concentrations.

Ethanol, a central nervous system depressant and banned substance in horseracing, has reportedly been administered to horses prior to competition to "calm a horse's nerves." In this study, the pharmacokinetics of two metabolites of ethanol were studied to better understand the behavior of this compound in the horse and provide a scientific basis for regulation of its administration. Six horses received a single intravenous (30 mL; 1200 mg) and oral (90 mL; 3600 mg) administration of ethanol (vodka, 40% ABV) in a balanced cross-over design. Blood and urine samples were collected at various times post administration for up to 24 h. Concentrations of ethyl glucuronide and ethyl sulfate were determined using liquid chromatography-tandem mass spectrometry and pharmacokinetic analysis performed. Behavioral, locomotor activity and effects on heart rate were assessed. The maximum concentration (mean ± SD) of ethyl glucuronide was 71.5 ± 42.7 and 105.0 ± 47.5 ng/mL at 0.88 h following IV and oral administration, respectively. The maximum concentrations for the ethyl sulfate metabolite following IV and oral administration were 1.61 ± 0.60 and 3.46 ± 1.68 ng/mL, respectively. Urine concentrations of both metabolites were non-detectable by 24 h post ethyl alcohol administration. No observable behavioral responses were noted following IV or oral administration. Significant decreases in heart rate were noted at various times starting at 10 min until 4 h post administration in the oral dose group. Both ethyl glucuronide and ethyl sulfate could be useful markers for detection of illicit administration of ethanol to horses.

Gamma-hydroxybutyrate (GHB), an endogenous compound related to the neurotransmitter gamma-aminobutyric acid (GABA), is used as a therapeutic and recreational drug and as a "weapon" in drug-facilitated crimes. The very short window of detection of GHB in conventional matrices (blood and urine) makes necessary the use of alternative matrices like hair. Hair has a long window of detection and the possibility to perform segmental analysis, which makes it very useful for proving GHB intake. In the present work, a method for quantification of GHB in hair was developed and validated. Hair (10 mg) was washed twice with dichloromethane and then incubated at room temperature with Milli-Q water in an ultrasound bath for 30 min. Analysis was performed by UPLC-MS/MS using a CORTECS UPLC HILIC (1.6 μm), 2.1 × 100-mm column, and a gradient with acetonitrile and ammonium acetate (10 mM) at pH 6.0, with a total run-time of 10 min. For detection, a triple quadrupole mass spectrometer in ESI negative mode was used. The method was validated, following the criteria established in the "AAFS Standard Practices for Method Validation in Forensic Toxicology" guideline, obtaining satisfactory results for linearity (0.5-50 ng/mg), accuracy (95.0%-103.2%), imprecision (< 10.2%), limit of detection (0.1 ng/mg) and quantification (0.5 ng/mg), exogenous selectivity (no interferences), matrix effect (less than -44.2%), extraction efficiency (> 86.4%), process efficiency (> 46.1%), and autosampler stability (< 4.3%). The method was used for the analysis of 26 authentic hair samples, 25 from non-drug users, obtaining values between < LOQ and 6.25 ng/mg of endogenous GHB and 1 from a former GHB chronic user to prove abstinence.

In September 2019, a 22-year-old man with a history of drug abuse presented to the hospital with altered mental status. Due to a suspected drug overdose, a blood sample taken on admission and a urine sample collected 30 h thereafter were submitted to our laboratory to test for illegal drugs, pharmaceutical substances, and designer drugs. During the routine toxicological analysis of the serum sample, morphine and phenobarbital were identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Additionally, two compounds showing identical accurate masses and isotope ratios as the designer benzodiazepine diclazepam and the benzodiazepine lormetazepam were found. However, retention times differed significantly from the expected values, and the acquired MS/MS spectra did not match the library entries of the two compounds, indicating the presence of two previously unknown substances. After further investigation, SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone), a methaqualone analog, which has recently emerged on the research chemical market, and its hydroxy metabolite were tentatively identified by accurate mass, isotope matching, and plausible fragmentation. However, for unequivocal confirmation and quantification, a reference standard is required. As no reference material was available by the end of 2019, SL-164 was obtained from an online shop, and its identity and purity (97.8%) were confirmed by nuclear magnetic resonance spectroscopy. The subsequent quantitative analysis revealed a concentration of 390 ng/mL SL-164 in serum. In the urine sample, the parent compound was not detected, but three suspected monohydroxylated metabolites were found. This example shows that LC-QTOF-MS is a powerful approach for the (tentative) identification of unknown compounds in biological matrices.

Synthetic cannabinoid receptor agonists (SCRAs) continue to show high prevalence on the new psychoactive substances drug market. Around 2019-2020, new SCRAs bearing a cumyl moiety emerged: Cumyl-CBMEGACLONE and Cumyl-NBMEGACLONE, carrying a cyclobutyl methyl (CBM) and a norbornyl methyl moiety (NBM) attached to the γ-carbolinone core. These were followed by Cumyl-NBMINACA, the indazole carboxamide analog of Cumyl-NBMEGACLONE. The study aimed at evaluating the human phase-I metabolism of these compounds and at identifying suitable urinary markers to prove their consumption. After enzymatic hydrolysis, 14 authentic urine samples (eight for Cumyl-CBMEGACLONE, four for Cumyl-NBMEGACLONE, and two for Cumyl-NBMINACA) were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Results were compared with in vitro metabolites generated by pooled human liver microsomes incubation. Fifteen human phase-I metabolites were identified for Cumyl-CBMEGACLONE, nine for Cumyl-NBMEGACLONE, and thirteen for Cumyl-NBMINACA. The main in vivo metabolites were built by monohydroxylation, dihydroxylation, or trihydroxylation. The following urinary biomarkers are suggested for detecting the consumption of the investigated SCRAs: products of monohydroxylation at the CBM and at the core for Cumyl-CBMEGACLONE; two products of monohydroxylation at the norbonyl methyl tail for Cumyl-NBMEGACLONE; and metabolites built by dihydroxylation at the NBM substructure and by an additional hydroxylation at the cumyl moiety for Cumyl-NBMINACA.

This paper aims to study the metabolism of thyroid hormones (TH) in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was applied to samples collected before and after the administration of sodium triiodothyronine (T3) and sodium levothyroxine (T4) to a euthyroid volunteer and to samples of athletes declaring and not declaring thyroid supplementation. Samples were analyzed by LC-MS/MS after enzymatic hydrolysis, liquid-liquid, and solid-phase extractions. Ratios between T3/thyronine and T4/3,3'-T2 may be used for the detection of the administration of exogenous T3 in urine. Meanwhile, 3-T1 concentrations may be used to detect exogenous T4 administration. Nevertheless, these markers may not work properly in hypothyroid population, as athletes seem to be. The levels of T3 and T4 of athletes were lower than those of a euthyroid state even when they are under administration of TH supplements. The HTP axis high efficiency does not allow observing differences between athletes who do not declare and those who declare having used TH supplementation by direct measurements of T3 and T4 in urine. The detection of TH administration in urine (triiodothyronine and levothyroxine) may work when dealing with euthyroid individuals. Nevertheless, in individuals with hypothyroidism where the tendency is toward the maintenance of homeostasis, and it may be not possible to detect their consumption by applying cut-off values.

Anabolic-androgenic steroids (AASs) are a subclassification of image performance enhancing drugs (IPEDs). While AAS use is most prevalent among people in athletics, there is also high lifetime prevalence of AAS use among prisoners. This study reports the qualitative detection of AASs in seized samples from the Scottish prisons from 2019-2023. Additionally, methods were developed for the quantitative analysis of AASs using gas chromatography-mass spectrometry (GC-MS) and applied to 61 samples of tablets or powders seized from Scottish prisons between July 2022 and July 2023. Since 2022, there has been an increase in AAS detections in the Scottish prisons. Oxymetholone was the most prevalent AAS, followed by metandienone (methandrostenolone, methandienone), methyltestosterone, oxandrolone, mestanolone (methylandrostanolone), stanozolol, and androstenedione. Multiple AASs were found in 21 samples and 10 samples contained other drugs, including amitriptyline, sertraline, zopiclone, mirtazapine, sildenafil, etizolam, Δ⁹-tetrahydrocannabinol, and the synthetic cannabinoid MDMB-INACA. Most AAS samples were tablets (77.0%), although they were also detected in powders, herbal material, e-cigarettes, and a fragmented soap bar-type sample. There was a large variation in the concentration of AASs in the tablets and powders seized from the Scottish prisons, demonstrating AASs are another highly variable component of the polydrug use situation in prisons, the effects of which need to be examined further.