Drug Testing and Analysis最新文献

Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their acetates, HHC-O and HHCP-O, respectively, are emerging in Europe as alternatives to tetrahydrocannabinol (THC). This study aimed to elucidate the metabolic pathways of the semi-synthetic cannabinoids HHC, HHCP, HHC-O and HHCP-O from incubation with human hepatocytes. The metabolites of HHC were also identified in authentic urine samples. HHC, HHCP, HHC-O and HHCP-O were incubated with primary human hepatocytes for 1, 3 and 5 h. Authentic urine samples from cases screened positive for cannabis in blood using ELISA but confirmed negative were analysed both non-hydrolysed and hydrolysed for HHC metabolites. Potential metabolites were identified using ultra-high performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight mass spectrometer (QToF-MS). HHC and HHCP were primarily metabolised through monohydroxylation (monoOH), followed by oxidation to a carboxylic acid metabolite. HHC-O and HHCP-O were rapidly metabolised to HHC and HHCP, respectively. In authentic urine samples, 18 different metabolites were identified, and 99.3% of hydroxylated metabolites were glucuronidated. 11-OH-HHC, 5'OH-HHC and another metabolite with a monoOH on the side chain were the only metabolites present in all 16 urine samples. The metabolism of HHC and HHCP were similar, although the longer alkyl side chain of HHCP (heptyl) led to greater hydroxylation on the side chain than HHC (pentyl). The use of HHC and HHCP can be differentiated from the use of THC and other phytocannabinoids, but the use of the acetate analogues may not be differentiable from their non-acetate analogues.

The monitoring of endogenous steroids in urine has been an important component of the Athlete Biological Passport (ABP) for the last decade. Recently, the quantitation of endogenous steroids in blood has been incorporated into the ABP to increase sensitivity in circumstances where the excretion of urinary ABP biomarkers is low. Current ABP guidelines mandate the use of venous blood draws for blood steroid sample collections, however, recent efforts have focused on investigating the use of less invasive sample collection methods, such as capillary blood collected from the upper arm. The focus of this study was to compare the analytical results of venous and capillary blood collected weekly from 20 individuals, 10 males and 10 females, over six weeks. The two primary biomarkers of the blood steroid ABP module, testosterone (T) and the testosterone/androstenedione (T/A4) ratio, were compared, as well as luteinizing hormone (LH) and the T/LH ratio in male participants, two biomarkers known to be responsive to T use. All biomarkers showed excellent agreement between venous and capillary blood. Longitudinal stability between sample types within individuals was also comparable for all biomarkers. Finally, storage of simultaneously collected capillary samples at room temperature and frozen conditions was compared with evaluate the potential impact of non-cold chain shipping conditions. Most biomarkers showed excellent agreement between frozen and room temperature storage conditions. These results indicate capillary blood collections represent a promising alternative to venous blood collections for the blood steroid module of the ABP.

The instability of erythropoietin receptor agonists (ERAs, i.e., EPO) in urine presents a challenge to their detectability in doping control samples; however, this issue is not often seen in blood (serum) samples. With the anti-doping field beginning to transition into alternative blood collection technologies, it is important to understand recombinant EPO (rEPO) detectability in serum samples collected from one such capillary collection device, the Tasso+ SST. Twelve individuals were administered a single, 40 IU/kg dose of rEPO (epoetin alfa, EPOGEN®). Following administration, matched urine, venous serum, and capillary serum samples were concurrently collected. Urine aliquots were subject to various storage times and temperatures mimicking shipping conditions of doping control urine samples to assess EPO stability, while other urine aliquots, venous serum, and capillary serum aliquots were frozen until analysis to understand rEPO detectability across all three matrices. EPO and rEPO instability was identified in urine collected from 8 of 12 participants, especially in aliquots stored at room temperature and 37°C. In some of these unstable samples, rEPO was still detectable, while in others, no recombinant nor endogenous EPO was detectable and would have resulted in negative sample reports. Analyzing the concurrently collected urine, venous, and capillary serum samples, rEPO detectability was identical across the three matrices. In most cases, rEPO was detectable for at least 168 h post-administration. Noting greater stability in blood compared with urine, it is recommended that anti-doping authorities utilize this novel capillary serum collection technology to improve overall ERA detectability in doping control samples.

Synthetic opioids have been associated globally with adverse effects in drug users. The nitazene group of drugs is a relatively new addition to the synthetic opioid class emerging in Europe in 2019. Some nitazenes have been shown to be more potent than fentanyl. Overdose clusters in heroin users in Dublin (57 cases) and Cork (20 cases), Ireland, in November and December 2023, respectively, prompted a rapid response from a number of Irish laboratories to identify the substance(s) of concern. Light brown (tan) powders were obtained from cases associated with overdoses, and the results from these analyses by collaboration of four laboratories are reported here. The samples were found to contain N-pyrrolidino protonitazene (protonitazepyne), caffeine, paracetamol, benzoic acid and mannitol.

Accurate determination of carboxy-hemoglobin (COHb%) is essential for the assessment of hemoglobin mass (Hbmass) by CO-rebreathing. To analyze blood samples for a certain period of time after blood collection, it is necessary to know the stability of the COHb% during storage. The aim of the study was to determine the stability of COHb% at different storage temperatures over a period of up to 3 months. Twenty-five milliliters of cubital venous blood was taken from five volunteers (three females and two males) before and after inhalation of 0.8/1.0 mL/kg carbon monoxide and stored at +20°C and +4°C for 6 days and at -70°C for 12 weeks. Within the first 6 days, the blood was analyzed daily, then weekly for 12 weeks. Additionally, Hbmass was determined in 13 endurance athletes immediately after blood collection and after storage for 3 days (eight cyclists) and 7 days (five swimmers) at +20°C or +4°C. COHb% before and after CO inhalation was 1.56 ± 0.48 and 5.86 ± 1.12%, respectively, and remained unchanged over 6 days, with no difference between storage at different temperatures. The standard deviation (STD) over time was between 0.07% and 0.12%. Similarly, storage at -70°C for 12 weeks did not change COHb%, whereas STD was 0.07%. Hbmass determined immediately and, after 3 or 7 days of storage, differed by 10 ± 7 g and 15 ± 11 g corresponding to a typical error of 0.8% and 1.1%. Blood storage at +20°C and +4°C for 6 days and at -70°C for 12 weeks does not affect COHb% and has, therefore, no influence on Hbmass assessment.

Villocarine A is a bioactive indole alkaloid isolated from the Uncaria genus. It has demonstrated vasorelaxation activity and potential to protect the central nervous system. To identify the pharmacokinetic properties of villocarine A, a series of in vitro and in vivo studies have been performed. Villocarine A was found to be highly permeable (15.6 ± 1.6*10^-6 cm/s) across human colorectal adenocarcinoma cell monolayer with high protein binding (>91%) in both rat and human plasma. Hepatic extraction ratio of villocarine A was 0.1 in pooled rat liver and 0.2 in human liver microsomes and was found stable in rat plasma at 37°C. Due to the high permeability and low rate of metabolism properties, villocarine A was initially considered suitable for preclinical development and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification (linearity: 1-150 ng/ml) in rat plasma was developed and validated for in vivo studies. Essential pharmacokinetic parameters included the volume of distribution and clearance of villocarine A, which were found to be 100.3 ± 15.6 L/kg and 8.2 ± 1.1 L/h/kg, respectively, after intravenous administration in rats. Following oral dosing, villocarine A exhibited rapid absorption as the maximum plasma concentration (53.2 ± 10.4 ng/ml) occurred at 0.3 ± 0.1 h, post-dose. The absolute oral bioavailability of villocarine A was 16.8 ± 0.1%. To our knowledge, this was the first pharmacokinetic study of villocarine A, which demonstrated the essential pharmacokinetic properties of villocarine A: large volume distribution, high clearance, and low oral bioavailability in rats.

Doping offenses involve the use or attempted use of any prohibited method or substance as well as substituting samples. Consequently, it has been recommended that short tandem repeat (STR) analysis be used to determine if the doping control samples are from the same athlete. However, it has been recognized that it may be difficult to obtain full STR analysis using negligible amounts of DNA samples. Mitochondrial DNA (mtDNA) is characterized by its stability and high cellular copy number. Therefore, mtDNA testing in urine is expected to be used to analyze samples that cannot be analyzed using STR analysis. The objective of this study was to compare mtDNA testing with STR analysis by conducting sensitivity, concordance (whole blood, dried blood spot, and urine), and case-type studies. In sensitivity studies, mtDNA testing exhibited greater sensitivity compared with STR analysis. Concordance studies indicated that all samples were consistent with the mtDNA sequences and STR profiles. Allelic dropout occurred in some urine samples that were examined for STR analysis. Case-type sample studies demonstrated that mtDNA testing could be used to obtain DNA profiles of all the samples tested, including blood, dried blood spots, urine, blood residues on needles, and blood stains. In conclusion, mtDNA testing is valuable for analyzing highly degraded DNA samples, such as urine samples, compared with STR analysis. Urine testing should be performed for the initial testing procedure, because mtDNA is inherited maternally. In situations where the DNA match is detrimental to the athlete, additional blood STR analysis may be required.

Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is a type of high-resolution mass spectrometry that uses data-independent acquisition. Compared with more targeted acquisition schemes, the power behind this data-independent acquisition technique comes from its ability to mitigate interferences via the use of SWATH acquisition windows (Q1 quadrupole isolation windows) while still obtaining all accurate mass information. However, consistent with high-resolution mass spectrometry techniques, its routine and high throughput implementation in forensic toxicology is limited due to the complex processing power required to effectively manage the large amount of acquired data. It is therefore pivotal to create an efficient and validated identification criterion that confidently reports suspected positive detections as a confirmational technique for final reporting. This review examines all publications that implemented SWATH in a forensic toxicological framework with suggestive best practices and commonly used criteria. Seventeen publications were reviewed for extraction, liquid chromatography and mass spectrometry parameters, and more specifically for all SWATH applicable characteristics including spray voltages, collision energies and spreads, mass error, isotopic ratio difference, retention time error, and library score thresholds. Notwithstanding the challenges SWATH implementation faces for a laboratory, the technique demonstrates its potential to be utilized in routine forensic toxicology testing regimes and aids in the detection of both common and emerging novel drugs simultaneously.