Acoustic ejection tandem mass spectrometry for high-throughput screening of phencyclidine-type substances in urine, including authentic cases.

BACKGROUND: The abuse of the Phencyclidine-type substances, especially ketamine is a serious problem worldwide, and retrospective analysis are important for both the analysis and the identification of forms of drug abuse. The current major analytical methods, while all excellent in terms of accuracy, are time- and reagent-consuming. This depletion is made even more unfortunate by the fact that a large number of samples are negative in retrospective analyses. It is clear that a set of methods that can be analyzed both accurately and quickly need to be developed and applied to the screening and analysis of large quantities of samples. RESULTS: We described a urine test based on acoustic ejection mass spectrometry, which allows precise injection at very low volumes and near 1 ejection s-1 and data acquisition. The confidence in identification was increased by the characterization of the abundance ratio of the two pairs of ions. Urine samples could be diluted with water and loaded into a 384-well plate for sampling without complicated sample preparation. The sample in the transparent 384-well plate was pre-scanned by the laser, and then 20 nL droplets were ejected into the ion source for targeted analysis of 2 ion transitions per droplet totaling 9 targeted analytes in the sequence of acquisition methods. It took 90 min to screen 250 samples in this approach, yielding 10 ng mL-1 detection limits. Positive samples were further analyzed by UHPLC-MS/MS for confirmation and quantification of up to 36 analytes. SIGNIFICANCE: This was the first fast screening method for phencyclidine-type substances based on acoustic ejection mass spectrometry, which greatly reduces the analytical time, and can accomplish in 1.5 h what UHPLC-MS/MS needs 3 days to complete. And the samples can be analyzed without complicated sample preparation, and also can obtain good detectability. It was applied to a short-term retrospective analysis in Shanghai, and its accuracy was also extremely high.

Technical and health governance aspects of the external quality assessment system for classical and new psychoactive substances analysis testing in blood.

New psychoactive substances (NPS) are uncontrolled analogues of existing drugs or newly synthesized chemicals that exhibit psychopharmacological effects. Due to their diverse nature, composition, and increasing prevalence, they present significant challenges to the healthcare system and drug control policies. In response, healthcare system laboratories have developed analytical methods to detect NPS in biological samples. As a Regional Reference Centre, the Sicilian CRQ Laboratory (Regional Laboratory for Quality Control) developed and conducted an External Quality Assessment (EQA) study to assess, in collaboration with the Istituto Superiore di Sanità (ISS), the ability of different Italian laboratories to identify NPS and traditional drugs of abuse (DOA) in biological matrices. Two blood samples were spiked with substances from various drug classes, including synthetic cannabinoids, cathinones, synthetic opiates, and benzodiazepines, at concentrations ranging from 2 to 10 ng/mL. The blood samples were freeze-dried to ensure the stability of DOA and NPS. Twenty-two laboratories from the Italian healthcare system participated in this assessment. The information provided by the laboratories during the registration in an in-house platform included a general description of the laboratory, analytical technique, and the chosen panels of analytes. The same platform was employed to collect and statistically analyze the data and record laboratory feedback and comments. The evaluation of the results revealed that the participating laboratories employed three different techniques for analyzing the samples: GC-MS, LC-MS, and immunoenzymatic methods. Approximately 90 % of the laboratories utilized LC-MS techniques. Around 40 % of false negative results were obtained, with the worst results in the identification of 5-chloro AB PINACA. The results showed that laboratories that used LC-MS methods obtained better specificity and sensitivity compared to the laboratories using other techniques. The results obtained from this first assessment underscore the importance of external quality control schemes in identifying the most effective analytical techniques for detecting trace molecules in biological matrices. Since the judicial authorities have not yet established cut-off values for NPS, this EQA will enable participating laboratories to share their analytical methods and expertise, aiming to establish common criteria for NPS identification.

Validation and application of an automated multitarget LC-MS/MS method for drugs of abuse testing using exhaled breath as specimen.

Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel "collectors" that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography - mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake.

Evaluation of spatial and temporal changes in illicit drug use in the Taipei metropolitan area via wastewater-based epidemiology.

Taiwan, identified as pivotal in the Asian drug trafficking chain, has been experiencing a surge in illicit drug-related issues. Wastewater-based epidemiology (WBE) has emerged as a promising approach for comprehensive evaluation of actual illicit drug usage. This study presents the first WBE investigation of illicit drug consumption in Taiwan based on the analysis of wastewater from four wastewater treatment plants (WWTPs) in the Taipei metropolitan area. Additionally, it demonstrates a high correlation between the amounts of illicit drugs seized and influent concentrations over an extended period of time. The reliability of solid-phase extraction and analysis via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was validated for 16 illicit drugs (methamphetamine, ketamine, cocaine, codeine, methadone, morphine, meperidine, fentanyl, sufentanil, para-methoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), 3,4-methylenedioxymethamphetamine (MDMA), cathinone, methcathinone, mephedrone (MEPH), and 4-methylethcathinone (4-MEC)). Methamphetamine, ketamine, and 4-MEC were consistently detected in all wastewater samples, underscoring their prevalence in the Taipei metropolitan area. Biochemical oxygen demand (BOD) and ammonia nitrogen (ammonia N) were employed to reduce uncertainty in estimations of population size during back-calculation of illicit drug consumption. The results indicate that methamphetamine was the most consumed drug (175-740 mg day-1 1000 people-1), followed by ketamine (22-280 mg day-1 1000 people-1). In addition, urban-related WWTPs exhibited higher consumption of methamphetamine and ketamine than did the suburban-related WWTP, indicating distinct illicit drug usage patterns between suburban and urban regions. Moreover, an examination of temporal trends in wastewater from the Dihua WWTP revealed a persistent predominance of ketamine and methamphetamine, consistent with statistical data pertaining to seizure quantities and urine test results. The study provides encouraging insight into spatial and temporal variations in illicit drug usage in the Taipei metropolitan area, emphasizing the complementary role of WBE in understanding trends in illicit drug abuse.

Fiber Laser-Generated Silver-109 Nanoparticles for Laser Desorption/Ionization Mass Spectrometry of Illicit Drugs.

Cannabinoids and opioids are the most prominently used drugs in the world, with fentanyl being the main cause of drug overdose-related deaths. Monitoring drug use in groups as well as in individuals is an important forensic concern. Analytical methods, such as mass spectrometry (MS), have been found most useful for the identification of drug abuse on a small and large scale. Pulsed fiber laser 2D galvoscanner laser-generated nanomaterial (PFL 2D GS LGN) was obtained from monoisotopic silver-109. Nanomaterial was used for laser desorption/ionization mass spectrometry of selected illicit drug standards with standard high-resolution reflectron-based time-of-flight MALDI apparatus. Δ9-THC, 11-OH-THC, 11-COOH-THC, fentanyl, codeine, 6-monoacetylmorphine (6-MAM), heroin, tramadol, and methadone were chosen as test compounds. Illicit drugs were tested in a concentration range from 100 µg/mL to 10 pg/mL, equating to 50 µg to 50 fg per measurement spot. For all analyzed compounds, identification and quantification by silver-109-assisted laser desorption/ionization (LDI) MS was possible, with uncommon [M + 109Ag3]+ and [M - H]+ ions present for certain structures. The results of the quantitative analysis of drugs using silver-109 PFL 2D GS LGN for LDI MS are presented. Laser-generated NPs are proven to be useful for the analysis of selected drugs, with exceptionally good results for fentanyl monitoring in a broad range of concentrations.

Detecting novel psychoactive substances around the world.

PURPOSE OF REVIEW: The worldwide spread of novel psychoactive substances (NPS) in the illicit drug market and their continuous increase in number and type, for the purpose of bypassing controlled substance legislation, represents a continuing challenge for forensic scientists, clinicians and enforcement authorities. We aim to provide information regarding the most urgent harms related to NPS consumption in different world regions and the current state of the art for NPS analysis. RECENT FINDINGS: Unfortunately, the identification of NPS in biological samples is controversial, especially when samples are limited, or the drug is promptly and extensively metabolized. This causes a lack of information on their real diffusion in different parts of the world and in different populations. New technologies and instrumental detection of NPS in alternative samples are offering comprehensive information about NPS use. SUMMARY: The lack of detection and underreporting of NPS in biological samples makes it difficult to obtain complete qualitative and quantitative information about NPS prevalence. The most innovative strategies that have been proposed in the last 2 years to assist NPS analysis and possibly facilitate the understanding of the NPS diffusion around the world are presented.

Consumption of illicit drugs and benzodiazepines in six Spanish cities during different periods of the COVID-19 pandemic.

Wastewater-based epidemiology (WBE) can provide objective and real time information about the use of addictive substances. A national study was conducted by measuring the most consumed illicit drugs, other drugs whose consumption is not so widespread but has increased significantly in recent years, and benzodiazepines in untreated wastewater from seven wastewater treatment plants (WWTPs) in six Spanish cities. Raw composite wastewater samples were collected from December 2020 to December 2021, a period in which the Spanish and regional governments adopted different restriction measures to contain the spread of the COVID-19 pandemic. Samples were analyzed using a validated analytical methodology for the simultaneous determination of 18 substances, based on solid-phase extraction and liquid-chromatography tandem mass spectrometry. Except for heroin, fentanyl, 6-acetylmorphine and alprazolam, all the compounds were found in at least one city and 9 out of 18 compounds were found in all the samples. In general, the consumption of illicit drugs was particularly high in one of the cities monitored in December 2020, when the restrictions were more severe, especially for cannabis and cocaine with values up to 46 and 6.9 g/day/1000 inhabitants (g/day/1000 inh), respectively. The consumption of MDMA, methamphetamine and mephedrone was notably higher in June 2021, after the end of the state of alarm, in the biggest population investigated in this study. Regarding the use of benzodiazepines, the highest mass loads corresponded to lorazepam. This study demonstrates that WBE is suitable for complementing epidemiological studies about the prevalence of illicit drugs and benzodiazepines during the COVID-19 pandemic restrictions.

Target analysis of psychoactive drugs in oral fluid by QuEChERS extraction and LC-MS/MS.

This study aimed to validate a modified QuEChERS method, followed by liquid chromatography-tandem mass spectrometry, for the determination of 51 psychoactive substances and screening of 22 ones in oral fluid from electronic dance music party (EDM) attendees. Unstimulated oral fluid was collected in a polypropylene tube and stored in a glass vial at -20 ºC. The sample was extracted with acetonitrile:water and MgSO4/NaOAc, followed by cleanup with primary secondary amine and MgSO4. The effectiveness of the sample storage conditions was shown to be comparable to when the Quantisal™ buffer was used, with no substantial concentration loss (< 15%) for all the substances after up to 72 hours at -20º C. The method was satisfactorily validated, with limits of detection (LOD) and quantification (LOQ) ranging from 0.04 to 0.5 ng/mL and 0.1-1.5 ng/mL, respectively, and was applied to the analysis of 62 real samples. The main substances detected were 3,4-methylenedioxymethamphetamine (MDMA) (<0.5-829 ng/mL) and/or methylenedioxyamphetamine (MDA) (10.1 - 460.6 ng/mL), found in 27 samples, and cocaine (13.0-407.3 ng/mL) and its metabolites (benzoylecgonine 0.17-214.1 ng/mL; ecgonine methyl ester 1.8-150.1 ng/mL) in eight samples. Methamphetamine (11-439 ng/mL) was detected in eight samples, along with MDMA and MDA; eutylone was detected in two cases (4.7 and 24.1 ng/mL) reported as "ecstasy" ingestion. A comparison between self-reported drug use and results of oral fluid analysis indicated that the use of illicit substances is often underreported among EDM attendees, who are often unaware of the substances they consume.

Development of an innovative analytical method for forensic detection of cocaine, antidepressants, and metabolites in postmortem blood using magnetic nanoparticles.

Cocaine and antidepressants rank high globally in substance consumption, emphasizing their impact on public health. The determination of these compounds and related substances in biological samples is crucial for forensic toxicology. This study focused on developing an innovative analytical method for the determination of cocaine, antidepressants, and their related metabolites in postmortem blood samples, using unmodified commercial Fe3O4 nanoparticles as a sorbent for dispersive magnetic solid-phase extraction (m-d-SPE), coupled with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. An aliquot of 100 µL of whole blood and 5 µL of the internal standard pool were added to 30 mg of nanoparticles. The nanoparticles were separated from the sample using a neodymium magnet inserted into a 3D-printed microtube rack. The liquid was then discarded, followed by desorption with 300 µL of 1/1/1 acetonitrile/methanol/ethyl acetate. The sample was vortexed and separated, and 1.5 µL of the organic supernatant was injected into the LC-MS/MS. The method was acceptably validated and successfully applied to 263 postmortem blood samples. All samples evaluated in this study were positive for at least one substance. The most frequent analyte was benzoylecgonine, followed by cocaine and cocaethylene. The most common antidepressants encountered in the analyzed samples were citalopram and fluoxetine, followed by fluoxetine's metabolite norfluoxetine. This study describes the first report of this sorbent in postmortem blood analysis, demonstrating satisfactory results for linearity, precision, accuracy, and selectivity for all compounds. The method's applicability was confirmed, establishing it as an efficient and sustainable alternative to traditional techniques for forensic casework.

Screening anabolic androgenic steroids in human urine: an application of the state-of-the-art gas chromatography-Orbitrap high-resolution mass spectrometry.

Over the past few decades, anabolic androgenic steroids (AASs) have been abused in and out of competition for their performance-enhancing and muscle-building properties. Traditionally, AASs were commonly detected using gas chromatography-mass spectrometry in the initial testing procedure for doping control purposes. Gas chromatography-Orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) is a new technology that has many advantages in comparison with GC-MS (e.g., a maximum resolving power of 240,000 (FWHM at m/z 200), excellent sub-ppm mass accuracy, and retrospective data analysis after data acquisition). Anti-doping practitioners are encouraged to take full advantage of the updated techniques of chromatography-mass spectrometry to develop sensitive, specific, and rapid screening methods for AASs. A new method for screening a wide range of AASs in human urine using GC-Orbitrap-HRMS was developed and validated. The method can qualitatively determine 70 anabolic androgenic steroids according to the minimum required performance limit of the World Anti-Doping Agency. Moreover, the validated method was successfully applied to detect six metabolites in urine after the oral administration of metandienone, and their excretion curves in vivo were studied. Metandienone M6 (17ß-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one) has been identified as a long-term urinary metabolite which can be detected up to 7 weeks, thus providing a longer detection window compared with previous studies. This study provides a rationale for GC-Orbitrap-HRMS in drug metabolism and non-targeted screening.

Appearance of hexahydrocannabinols as recreational drugs and implications for cannabis drug testing - focus on HHC, HHC-P, HHC-O and HHC-H.

This study investigated the effects of hexahydrocannabinol (HHC) and other unclassified cannabinoids, which were recently introduced to the recreational drug market, on cannabis drug testing in urine and oral fluid samples. After the appearance of HHC in Sweden in 2022, the number of posts about HHC on an online drug discussion forum increased significantly in the spring of 2023, indicating increased interest and use. In parallel, the frequency of false positive screening tests for tetrahydrocannabinol (THC) in oral fluid, and for its carboxy metabolite (THC-COOH) in urine, rose from <2% to >10%. This suggested that HHC cross-reacted with the antibodies in the immunoassay screening, which was confirmed in spiking experiments with HHC, HHC-COOH, HHC acetate (HHC-O), hexahydrocannabihexol (HHC-H), hexahydrocannabiphorol (HHC-P), and THC-P. When HHC and HHC-P were classified as narcotics in Sweden on 11 July 2023, they disappeared from the online and street shops market and were replaced by other unregulated variants (e.g. HHC-O and THC-P). In urine samples submitted for routine cannabis drug testing, HHC-COOH concentrations up to 205 (mean 60, median 27) µg/L were observed. To conclude, cannabis drug testing cannot rely on results from immunoassay screening, as it cannot distinguish between different tetra- and hexahydrocannabinols, some being classified but others unregulated. The current trend for increased use of unregulated cannabinols will likely increase the proportion of positive cannabis screening results that need to be confirmed with mass spectrometric methods. However, the observed cross-reactivity also means a way to pick up use of new cannabinoids that otherwise risk going undetected.

The return of drugs courts: Some important considerations.

Drugs courts can be praised for the rehabilitative health-treatment response they deliver to people with drugs dependence problems. This is when contrasted with traditional courts that operate on adversarial and retributive justice principles and do little to alleviate cycles of repeat drugs offending. Whilst drugs courts have been met with success and embraced in several jurisdictions worldwide, fundamental points need to be raised on the model that is re-emerging in England and Wales. Indications are it will involve drugs testing and a 'graduated sanctions and incentives system' that comprises short custodial sentences for non-compliance. This essay discusses the newly emerging drugs court model as signposted within different legislative and public policy documents and raises questions relating to the precise model these courts will take; whether they prioritise harm reduction or if 'abstinence' goals will predominate. We question whether people with drugs dependence problems should be sanctioned to short custodial prison sentences if in breach of a drugs court order. And perhaps more fundamentally: do we need drugs courts in the English and Welsh justice system?

Doping yesterday, today, tomorrow: A challenge for the clinical laboratory.

This work highlights the role of the clinical laboratory, in the early detection of the use of substances prohibited for doping. This is because most people who practice sports today are non-professional athletes and amateurs, in particular young kids. These persons are not subjected to anti-doping controls but are at risk for their health. Endocrinologists and laboratory tests, by detecting evidence of such usage can help protect their health. Anti-doping testing require specific instruments for qualitative and quantitative chemistry, to meet regulations of official competitions but are impossible to be used in every person because of high cost. A particular role the clinical laboratory can acquire in the future is through its molecular biology sections, when genetic doping will probably be a reality and quantitative chemistry will be unable to detect it. A brief history of doping is provided to understand the reasons of its spread. Although doping has great resonance nowadays, it is not a recent problem. It was common among ancient Greek wrestlers and Romans, who used mixtures of herbs and stimulants. Ancient Greece started the Olympic Games and winners assumed great esteem, akin to demi-god status. Therefore, any attempt to improve athletic performance was a norm, also because the damage caused by the substances used was not known at that time. The use became so widespread that soldiers also used drugs to better combat during recent wars, and doping was practiced by athletes, actors and musicians in attempts to obtain better performance results. Today, doping has been refined so as not to be discovered and there is a continuous race between those who promote new substances and those who, like the World Anti-Doping Agency (WADA), were created to defend the health of athletes and comply with regulations of competitions. The clinical laboratory plays a fundamental role in identifying the use of prohibited substances, especially in competitions not classified as official, which are the majority and involve thousands of amateurs. In this paper a series of laboratory tests are proposed in this perspective, at low cost without the need of qualitative/quantitative chemical analyses required by the sport jurisdictions. Finally, a glance into genetic doping illustrates a likely future and imminent practice.

Point-of-care testing of methamphetamine and cocaine utilizing wearable sensors.

The imperative for the point-of-care testing of methamphetamine and cocaine in drug abuse prevention necessitates innovative solutions. To address this need, we have introduced a multi-channel wearable sensor harnessing CRISPR/Cas12a system. A CRISPR/Cas12a based system, integrated with aptamers specific to methamphetamine and cocaine, has been engineered. These aptamers function as signal-mediated intermediaries, converting methamphetamine and cocaine into nucleic acid signals, subsequently generating single-stranded DNA to activate the Cas12 protein. Additionally, we have integrated a microfluidic system and magnetic separation technology into the CRISPR system, enabling rapid and precise detection of cocaine and methamphetamine. The proposed sensing platform demonstrated exceptional sensitivity, achieving a detection limit as low as 0.1 ng/mL. This sensor is expected to be used for on-site drug detection in the future.

Automated extraction and LC-MS-MS analysis of 11-nor-9-carboxy-tetrahydrocannabinol isomers and prevalence in authentic urine specimens.

11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH) is the most frequently detected illicit drug metabolite in the military drug testing program. An increasing number of specimens containing unresolved Δ8-THCCOOH prompted the addition of this analyte to the Department of Defense drug testing panel. A method was developed and validated for the quantitative confirmation of the carboxylated metabolites of Δ8- and Δ9-THC in urine samples utilizing automated pipette tip dispersive solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Analytes were separated isocratically over an 8.5-min runtime and detected on an MS-MS equipped with an electrospray ionization source operated in negative mode. A single point calibrator (15 ng/mL) forced through zero demonstrated linearity from 3 to 1,000 ng/mL. Intra- and inter-day precision were ≤9.1%, and bias was within ±14.1% for Δ8-THCCOOH and Δ9-THCCOOH. No interferences were found after challenging the method with different over-the-counter drugs, prescription pharmaceuticals, drugs of abuse and several cannabinoids and cannabinoid metabolites, including Δ10-THCCOOH. Urine specimens presumptively positive by immunoassay (n = 2,939; 50 ng/mL Δ9-THCCOOH cutoff) were confirmed with this analytical method. Δ8-THCCOOH and Δ9-THCCOOH were present together above the 15 ng/mL cutoff in 33% of specimens. However, nearly one-third of the specimens analyzed were positive for Δ8-THCCOOH only. This manuscript describes the first validated automated extraction and confirmation method for Δ8- and Δ9-THCCOOH in urine that provides adequate analyte separation in urine specimens with extreme isomer abundance ratios.

Quantitation of hexahydrocannabinol (HHC) and metabolites in blood from DUID cases.

Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The aim of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross-reactivity. Blood samples from driving under the influence of drugs (DUID) cases screened positive for cannabis using enzyme-linked immunoadsorbent assay (ELISA) and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS-MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias and autosampler stability. Cross-reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5 and 200 ng/mL. The cross-reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LC-MS-MS method, 9R-HHC-COOH, 9S-HHC-COOH and 11-OH-9R-HHC showed matrix effects <25% at both concentrations, while 8-OH-9R-HHC, 9R-HHC and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and intra day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper LOQ was 20 and 200 ng/mL, respectively. Reanalysis of cases (n = 145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.

Novel dummy molecularly imprinted polymer for simultaneous solid-phase extraction of stanozolol metabolites from urine.

Stanozolol, a synthetic derivative of testosterone, is one of the common doping drugs among athletes and bodybuilders. It is metabolized to a large extent and metabolites are detected in urine for a longer duration than the parent compound. In this study, a novel dummy molecularly imprinted polymer (DMIP) is developed as a sorbent for solid-phase extraction of stanozolol metabolites from spiked human urine samples. The optimized DMIP is composed of stanozolol as the dummy template, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker in a ratio of 1:10:80. The extracted analytes were quantitively determined using a newly developed and validated ultrahigh-performance liquid chromatography tandem mass spectrometry method, where the limits of detection and quantitation were 0.91 and 1.81 ng mL-1, respectively, fulfilling the minimum required performance limit decided on by the World Anti-Doping Agency. The mean percentage extraction recoveries for 3'-hydroxystanozolol, 4ß-hydroxystanozolol, and 16ß-hydroxystanozolol are 97.80% ± 13.80, 83.16% ± 7.50, and 69.98% ± 2.02, respectively. As such, the developed DMISPE can serve as an efficient cost-effective tool for doping and regulatory agencies for simultaneous clean-up of the stanozolol metabolites prior to their quantification.

Evidence of ostarine cross-contamination via sweat in 2 athletes sharing the same neoprene hamstring sleeves. An original situation of drug transfer where the anti-doping rule violation was suspended by the sport authorities.

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete's sample constitutes one of the most frequent anti-doping rules violation. It is possible to challenge this violation but it is the athlete who has to demonstrate he / she is innocent. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete or his/her legal representative must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete about the fact that he / she did not knowingly take the prohibited substance, i.e. that the violation was not intentional. During a 2-weeks period, a male athlete tested two times positive for ostarine in urine (<0.1 ng/ml) and he challenged these results. His hair and nail tests returned negative (LOQ at 0.5 pg/mg). He admitted using two neoprene hamstring sleeves of another athlete who confessed abusing ostarine. This was confirmed in his hair (190 pg/mg), his fingernail clippings (780 pg/mg) and his toenail clippings (45 pg/mg). To document the presence of ostarine in the hamstring sleeves and therefore possible drug transfer, the hamstring sleeves were analysed. Ostarine was identified in 12 different selected pieces (about 1 g) of the sleeves at concentrations ranging from 3 to 142 pg/g. Sport authorities (USADA) agreed that the most likely source of contamination was the hamstring sleeves, thus confirming the scenario of drug transfer and gave the athlete a no fault.

Understand the FDA-cleared fentanyl testing: A clinical evaluation of the SEFRIA fentanyl immunoassay.

BACKGROUND: Screening for fentanyl has been adopted by many clinical laboratories to detect illicit drug use and monitor medication adherence. However, compared to other urine drug testing, fentanyl screening assays are relatively new and therefore their clinical performances are largely unknown. This study extensively evaluated the clinical performance, positive cutoff, and interference profile of SEFRIA fentanyl immunoassay in real patient settings. METHODS: The FDA-cleared cutoff of 1.0 was verified with 21 urine samples with low or undetectable levels of fentanyl. After assay implementation, all screened-positive samples were confirmed by liquid chromatography-tandem mass spectrometry. A new cutoff was derived from the numeric values of the false positive (FP) screening results. The FP rates before and after implementing the new cutoff were compared. Interferences were identified by an untargeted drug analysis and confirmed by spiking experiments. RESULTS: A total of 3951 screening results were reviewed in the first two months of the assay utilization, 410 were screened-positive, and 157 (38 %) were FP. After a new cutoff of 1.3 was implemented, the FP rate was reduced to 17 % based on 11119 screening results. Trazodone, labetalol, and haloperidol were identified as major interferents, accounting for 56 % of the FP results using the cutoff of 1.3. CONCLUSION: By applying the new cutoff and including an interference comment to positive screening results, the FP rate was reduced from initial 38 % to 7.5 % (17 % times 56 %).