Drug Testing Programs Research Articles

Cannabinoid Therapy in Athletics: A Review of Current Cannabis Research to Evaluate Potential Real-World Cannabinoid Applications in Sport.

The increasing legalization of Cannabis sativa plant products has sparked growing interest in their therapeutic applications. Prohibition laws established in 1937 hindered formal research on cannabis, a plant with cultural and medicinal roots dating back to 2700 BC in Chinese history. Despite regulatory hurdles, published research on cannabis has emerged; yet elite athletes remain an underrepresented population in these studies. Athletes, known for exploring diverse substances to optimize performance, are drawn to the potential benefits of cannabinoid therapy, with anecdotal reports suggesting positive effects on issues ranging from anxiety to brain injuries. This review aims to evaluate empirical published cannabis research with a specific focus on its potential applications in athletics. The changing legal landscape, especially the removal of cannabis from drug testing programs in leagues such as the National Basketball Association (NBA), and endorsements by Major League Baseball (MLB) for cannabinoid products and the National Football League (NFL) for cannabis research, reflects a shift in the acceptability of such substances in sports. However, stigma, confusion, and a lack of education persist, hindering a cohesive understanding among sports organizations, including business professionals, policymakers, coaches, and medical/training staff, in addition to athletes themselves. Adding to the confusion is the lack of consistency with cannabinoid regulations from sport to sport, within or out of competition, and with cannabis bioactive compounds. The need for this review is underscored by the evolving attitudes toward cannabinoids in professional sports and the potential therapeutic benefits or harms they may offer. By synthesizing current cannabis research, this review aims to provide a comprehensive understanding of the applications and implications of cannabinoid use in the realm of athletics.

Investigations into the Concentrations and Metabolite Profiles of Doping Agents and Antidepressants in Human Seminal Fluid Using Liquid Chromatography-Mass Spectrometry.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called adverse analytical finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e., samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. SIGNIFICANCE STATEMENT: This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

A comprehensive gas chromatography electron ionization high resolution mass spectrometry study of a new steroidal selective androgen receptor modulator (SARM) compound S42.

The synthetic 20-keto-steroid S42 (1) demonstrated selective androgen receptor modulator (SARM) properties in preclinical studies and, consequently, received growing attention also in the context of sports drug testing programs. Fundamental understanding of the behavior of S42 (1) and of relevant derivatives in gas chromatography-electron ionization MS experiments at high resolution (GC-EI-HRMS) is indispensable to develop a reliable qualitative and quantitative doping control method for S42 (1) and its metabolites in body fluid matrices. We present important fundamental mechanistic data on the EI fragmentation behavior of S42 (1) and of silyl ether derivatives as well as of stable isotope-labelled reference material.

Re-examining mandatory drug testing in Australian prisons.

Mandatory drug testing is commonly used in Australian prisons to detect and deter drug use. In this commentary, we review the limited evidence for mandatory drug testing programs, highlight potential harms associated with their implementation and provide recommendations for drug surveillance in prisons concordant with a harm minimisation framework.

Complementary information concerning the suspected interindividual transmission of GW1516, a substance prohibited in sport, through intimate contact: a case report

PurposeInadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed.MethodsTo probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography–tandem mass spectrometry. Further, drug-residue testing using patients’ ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry.ResultsIn one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete’s doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid.ConclusionsThe combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments.

Chiral analysis of ostarine in commercially available products and doping control urine samples

Various substances classified as prohibited in sport by the World Anti-Doping Agency (WADA) comprise one or more chiral centers. One such substance is enobosarm ((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2‑hydroxy-2-methylpropanamide, S-ostarine, S-22, MK-2866, GTx-024, VERU-024). Although ostarine is not an approved drug, it has been readily available via Internet-based suppliers, and to date it is unknown to what extent R-ostarine, S-ostarine, or a mixture of both is used in these products. Likewise, such information is not available with regards to urine samples that returned an adverse analytical finding in the context of sports drug testing programs and, hence, this pilot-study aimed at establishing a test method to provide first insights into the presence of R- and S-ostarine in commercially available products and in authentic doping control urine samples.Here, a chiral liquid chromatographic - mass spectrometric method, employing a teicoplanin-containing (chiral) stationary phase that allows the differentiation of R- and S-enantiomers of ostarine, is presented. Ostarine products were extracted and diluted prior to analysis, while urine samples were prepared for analysis by enzymatic hydrolysis followed by liquid-liquid extraction.Seven products declaring to contain ostarine were investigated, five of which were ordered via the Internet and two were confiscated and made available by a customs authority. Only 2 of the 5 purchased products contained ostarine. In these two products and the confiscated products, only the ostarine S-enantiomer was found. Further, five authentic doping control urine samples containing the target analyte were subjected to chiral analysis, also confirming the sole presence of the S-ostarine enantiomer.The developed approach proved suitable for the separation of ostarine enantiomers, which adds further information e.g. in the context of illicit drug production, trafficking, and doping controls, especially when additional details are desirable that might assist in determining the origin of a drug or drug residue.

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.

Prevalence of carboxy-Δ8-tetrahydrocannabiniol in antidoping samples.

Δ9-Tetrahydrocannabinol (Δ9-THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8-tetrahydrocannabinol (Δ8-THC), have become available for sale. Δ8-THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8-THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9-THC (Δ9-THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8-THC, which metabolizes to Δ8-THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8-THC. This study aims to estimate the prevalence of Δ8-THC use in the athlete community by monitoring for Δ8-THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9-THC-COOH. One thousand samples with a presumptive Δ9-THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8-THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing.

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 h. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.

Insulin-mimetic peptides in sports drug testing.

Because of its influence on carbohydrate metabolism and, at the same time, anti-catabolic effects, the misuse of the peptide hormone insulin and its synthetic analogs is prohibited in sports at all times according to the regulations of the World Anti-Doping Agency (WADA). The biological effects of insulin and its analogs are mediated through binding to the insulin receptor, which was also found to be activated by different peptides structurally largely unrelated to insulin. Such insulin-mimetic peptides or selective-insulin receptor modulators (SIRMs) represent a novel class of potential performance-enhancing agents, which is currently not explicitly mentioned on the WADA Prohibited List. Within this research project, advanced solid-phase extraction (SPE) and liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a fast, reliable, and specific assay for the detection of the insulin-mimetic peptides S597 and S519 from plasma. Method validation demonstrated a detection limit of 0.5ng/mL and successfully illustrated the applicability of the approach to routine sports drug testing programs. Moreover, sophisticated and comprehensive in vitro metabolism experiments were conducted, and several metabolic degradation products were identified, which will enhance the information generated from future analyses of doping control samples.

Implementation of the First Comprehensive State Oral Fluid Drug Testing Program for Roadside Screening and Laboratory Testing in DUID Cases - A Five-Year Review.

Oral fluid (OF) is a valuable specimen for Driving under the influence of drugs (DUID) applications. This study demonstrates the implementation of the first comprehensive OF drug testing program in the United States, including approved roadside screening OF devices for law enforcement and validated LC-MS-MS confirmation methods. Three roadside OF screening devices were evaluated: the Dräger DrugTest® 5000, Abbott SoToxa®, and Randox Evidence MultiSTAT™. Two qualitative LC-MS-MS confirmation methods were validated per ASB Standard 036. The first method utilized an automated Dispersive Pipette Extraction (DPX) extraction using Integra and Hamilton STARlet platforms for drugs of abuse. The second method used a liquid-liquid extraction to detect cannabinoids. The prevalence of drugs in blood and OF was monitored over five years of casework. Calibration curves were analyzed with each batch to monitor OF concentrations for research purposes. Three roadside OF screening devices were deemed fit for purpose. Devices demonstrated appropriate sensitivity, specificity, positive and negative predictive values, and accuracy above 80% for targeted drugs except for benzodiazepines (DrugTest® 5000) and amphetamine (SoToxa®). The validated LC-MS-MS OF confirmation methods met the National Safety Council recommended cutoffs for 18/21 (86%) of the targets. Over five years of casework, THC and cocaine were detected at a positivity rate of 90% and 97% in OF versus 75% and 44% in blood, respectively. OF: blood ratios exceeded unity for parent drugs. Median concentrations of THC in OF and blood were 31 and 3.5 ng/mL, respectively. OF is a viable alternative or supplemental specimen for DUID investigations. Collecting OF close to the driving event increases the opportunity to identify pharmacologically active substances, and when combined with blood analysis results, an elevated OF: blood ratio provides valuable information for DUID investigation purposes.

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a β2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10ng/mL in urine. In consideration of cmax values (7.8ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

General Review on Opioids

Opioids are widely utilized by the public as both legal and illegal substances. Opiates, particularly heroin, are among the most widely used illicit narcotics and still account for the majority of seizures globally, despite the development of Novel Psychoactive Substances (NPS), particularly new fentanyl derivatives. The article gives a general review of opiates' pharmacology, analgesic action, and classification. It discussed how they might be abused by the general public and in the event of tampering. Because of the hazards of accidents linked to opiate use and dependence, opiates are furthermore closely monitored in workplace drug testing programs around the world. Also it covered analytical techniques for opiate detection in biological fluids. In the modern era, substance abuse has emerged as a global epidemic. Opium and its derivatives are second only to cannabis in terms of global consumption among illegal narcotics.

Insights from Drug Checking Programs: Practicing Bootstrap Public Health Whilst Tailoring to Local Drug User Needs.

The year 2021 was the most deadly year for overdose deaths in the USA and Canada. The stress and social isolation stemming from the COVID-19 pandemic coupled with a flood of fentanyl into local drug markets created conditions in which people who use drugs were more susceptible to accidental overdose. Within territorial, state, and local policy communities, there have been longstanding efforts to reduce morbidity and mortality within this population; however, the current overdose crisis clearly indicates an urgent need for additional, easily accessible, and innovative services. Street-based drug testing programs allow individuals to learn the composition of their substances prior to use, averting unintended overdoses while also creating low threshold opportunities for individuals to connect to other harm reduction services, including substance use treatment programs. We sought to capture perspectives from service providers to document best practices around fielding community-based drug testing programs, including optimizing their position within a constellation of other harm reduction services to best serve local communities. We conducted 11 in-depth interviews from June to November 2022 via Zoom with harm reduction service providers to explore barriers and facilitators around the implementation of drug checking programs, the potential for integration with other health promotion services, and best practices for sustaining these programs, taking the local community and policy landscape into account. Interviews lasted 45-60 min and were recorded and transcribed. Thematic analysis was used to reduce the data, and transcripts were discussed by a team of trained analysts. Several key themes emerged from our interviews: (1) the instability of drug markets amid an inconsistent and dangerous drug supply; (2) implementing drug checking services in dynamic environments in response to the rapidly changing needs of local communities; (3) training and ongoing capacity building needed to create sustainable programs; and (4) the potential for integrating drug checking programs into other services. There are opportunities for this service to make a difference in overdose deaths as the contours of the drug market itself have changed over time, but a number of challenges remain to implement them effectively and sustain the service over time. Drug checking itself represents a paradox within the larger policy context, putting the sustainability of these programs at risk and challenging the potential to scale these programs as the overdose epidemic worsens.

Mass spectrometry in sports drug testing-Analytical approaches and the athletes' exposome.

Test methods in anti-doping, most of which rely on the most modern mass spectrometric instrumentation, undergo continuous optimization in order to accommodate growing demands as to comprehensiveness, sensitivity, retrospectivity, cost-effectiveness, turnaround times, etc. While developing and improving analytical approaches is vital for appropriate sports drug testing programs, the combination of today's excellent analytical potential and the inevitable exposure of humans to complex environmental factors, specifically chemicals and drugs at the lowest levels, has necessitated dedicated research, particularly into the elite athlete's exposome. Being subjected to routine doping controls, athletes frequently undergo blood and/or urine tests for a plethora of drugs, chemicals, corresponding metabolic products, and various biomarkers. Due to the applicable anti-doping regulations, the presence of prohibited substances in an athlete's organism can constitute an anti-doping rule violation with severe consequences for the individual's career (in contrast to the general population), and frequently the question of whether the analytical data can assist in differentiating scenarios of 'doping' from 'contamination through inadvertent exposure' is raised. Hence, investigations into the athlete's exposome and how to distinguish between deliberate drug use and potential exposure scenarios have become a central topic of anti-doping research, aiming at supporting and consolidating the balance between essential analytical performance characteristics of doping control test methods and the mandate of protecting the clean athlete by exploiting new strategies in sampling and analyzing specimens for sports drug-testing purposes.

Annual banned-substance review-Analytical approaches in human sports drug testing 2021/2022.

Also in 2021/2022, considerable efforts were invested into advancing human sports drug testing programs, recognizing and taking into account existing as well as emerging challenges in anti-doping, especially with regard to substances and methods of doping specified in the World Anti-Doping Agency's 2022 Prohibited List. In this edition of the annual banned-substance review, literature on recent developments published between October 2021 and September 2022 is summarized and discussed. Focus is put particularly on enhanced analytical approaches and complementary testing options in human doping controls, appreciating the exigence and mission in anti-doping and, equally, the contemporary "new normal" considering, for example, the athlete's exposome versus analytical sensitivity and applicable anti-doping regulations for result interpretation and management.

Will tetrahydrocannabinol be formed from cannabidiol in gastric fluid? An in vivo experiment.

Cannabidiol (CBD) products have ascribed an uprising trend for their health-promoting effects worldwide. In contrast to Δ9-tetrahydrocannabinol (THC), CBD exhibits no state of euphoria. Since conversion of CBD into THC in an acidic environment has been reported, it has not been proved whether this degradation will also occur in human gastric fluid. A total of 9 subjects ingested 400mg CBD as a water-soluble liquid together with lecithin as an emulsifier and ethanol as a solubilizer. Blood samples were taken up to 4h, and urine samples were submitted up to 48h. THC, 11-hydroxy-Δ9-THC (THC-OH), 11-nor-9-carboxy-Δ9-THC (THC-COOH), CBD, 7-hydroxy cannabidiol (7-OH-CBD), and 7-carboxy cannabidiol (7-CBD-COOH) were determined in blood and THC-COOH and 7-CBD-COOH in urine by LC-MS/MS. Neither THC, THC-OH, nor THC-COOH were detectable in any serum specimen. Only two urine samples revealed THC-COOH values slightly above the threshold of 10ng/ml, which could also be caused by trace amounts of THC being present in the CBD liquid. It can be concluded that negative consequences for participants of a drug testing program due to a conversion of CBD into THC in human gastric fluid appear unlikely, especially considering a single intake of dosages of less than 400mg. Nevertheless, there is a reasonable risk for consumers of CBD products being tested positive for THC or THC metabolites. However, this is probably not caused by CBD cyclization into THC in human gastric fluid but is most likely due to THC being present as an impurity of CBD products.

Rapid semi-automated simplified liquid extraction (SLE) and data processing for 43 drugs of abuse in oral fluid

Oral fluid (OF) is commonly used as a non-invasive biological specimen to screen for the presence of drugs. Since 2004 in Victoria, OF has been used by law enforcement to randomly screen drivers on the roadside for the presence of methylamphetamine, MDMA and tetrahydrocannabinol. Over time, the drug-testing program has expanded to 150,000 random tests per year and produces approximately 12–14,000 positive roadside detections for laboratory confirmation. This sample load is a significant burden for manual sample preparation techniques. The need for high-throughput detection methods to maintain strict turnaround times has driven procurement and development of a cost-effective benchtop liquid handing system (LHS) for a rapid and robust sample preparation protocol at a fraction of traditional LHS expense. Rapid automated sample preparation was performed using the Integra Assist PLUS variable-channel width pipetting system. This system allowed for the development of a modular, programmable simplified liquid extraction (SLE) process using Biotage Isolute+ 96-well plates (400 μL). In addition to the three target drugs in these OF specimens, a further 40 drugs of abuse were targeted for detection and quantitation (1–50 ng/mL), including classes of amphetamines, benzodiazepines, cannabinoids, stimulants, and opioids. For extraction, 100 uL OF (analysed in duplicate) was added to a 2 mL polypropylene tube and placed on the LHS. The custom designed automated program then added 200 uL ammonium carbonate buffer (2 M, pH9), mixed the samples and transferred to a 96-well SLE plate. Positive pressure nitrogen gas was applied to ensure samples transferred into the SLE sorbent before a second LHS program added 900 μL chloroform:isopropanol (9:1) to each well. Eluate was collected into 1 mL glass inserts containing 50 μL 0.1 M hydrochloric acid and evaporated to dryness. A final LHS program added 100 μL methanol to each well for reconstitution. The 96-well plate was then placed on a Shimadzu Nexera X2 liquid chromatography system and 1 μL injected onto an analytical column [Kinetex C18, 4.6 × 50 mm, 2.6 μm, mobile phase of 50 mM ammonium formate buffer (pH 3.5)/0.1% formic acid in acetonitrile, 5 minute gradient separation]. Analytes were detected using a SCIEX 4500 Triple Quadrupole mass spectrometer in positive electrospray MRM mode with the use of carbon-13 isotopes to assist in decreasing detector response and aid MRM ratio confirmation. Data was processed using MultiQuant software utilising custom queries for rapid data review, assessment of duplicate concentrations and report generation. The method was fully validated in accordance with international guidelines for OF collected in StatSure and Quantisal buffers. Matrix effects and extraction efficiencies were acceptable with most analytes showing > 80% response and low variation (less than 5% RSD). Precision, accuracy and repeatability were typically within 6% RSD for all analytes, except for EDDP (methadone metabolite) and norbuprenorphine which showed poor precision in StatSure buffer, while the 7 aminobenzodiazepine metabolites showed poor precision in Quantisal buffer. The Integra Assist PLUS automated pipettor was sourced for approximately one tenth the price of a full LHS system, and only occupies a fraction of the bench space. Further, the reliable automated process reduces the risk of sample well errors or spills. The modular set-up of the technique required minimal manual handling and was able to prepare a complete 96-well plate for analysis within 90 minutes. Further, the use of a 3D printer allowed for design and implementation of custom equipment within the workflow. Finally, automated custom queries used for assessment of results ensured tight turnaround times are maintained. The developed fast and reliable extraction technique combined with rapid LC-MS/MS analysis of a broad range of drugs in OF allows for the opportunity of greater throughput, decreased turnaround times and lower risk from manual handling processes.

Caution in workplace drug testing: Controlled drugs

Increasing substance abuse in the world, affects many areas of life, and this situation in workplaces causes exceptionally low work performance and even occupational accidents, resulting in problems that can create high costs for employers. In this context, workplace drug testing (WDT) programs are designed to detect the status of alcohol, illegal substances, or certain abused prescription drugs. These drug tests are a deterrent and preventive application of substance use in the workplace. The increase in the non-medical use and misuse of prescription drugs such as pregabalin (PGB) is a serious public issue in most countries such as Turkey. PGB was included controlled drug list in 2019. In the routine practice of WDT, classical substances are analyzed and PGB is not. In this study it's aimed to Evaluate the toxicological data obtained via a comprehensive retrospective investigation of urine samples of cases from a medico-legal aspect by creating a wide perspective for investigating psychoactive substances susceptible to abuse in Turkey. Furthermore, the study aimed to raise awareness on the subject by conducting a more comprehensive retrospective scan to determine the status of abused drugs, particularly the status of prescription/controlled ones. Forty-seven cases were admitted to Ege University Addiction Toxicology Laboratory between 2017 and 2021 due to special WDT requests. Thirty-one samples of the cases, of which there was a witness sample, were included in the study. The cases consisted of current employees or the ones who applied to work in companies from different transportation sectors and those who were asked for a drug test to be able to travel abroad. During the pre-interview of the cases by the physician, sociodemographic information, data on drug use, and urine samples were collected following the safety chain procedures. Urine samples were first subjected to screening analysis by immunoassay (Biochip). Confirmation analysis was performed with GC-MS of positive samples. All samples were subjected to a general toxicological screening in terms of 147 parameters with LC-MS/MS. Among the cases, 61.3% ( n = 19) were male, and the ages varied between 22 and 56. There were cases declaring drug use in the last 72 hours for the treatment of analgesic, cardiac, hypertension, osteoporosis problems etc. In case No. 1, cannabis (THC) and ATS were detected in the screening analysis and confirmed by GC-MS. In the analysis with LC-MS/MS, PGB, which was not included in the analysis scale in GC-MS, was detected in this case. He stated no drug/substance use. Case No. 1 was also reported to have attempted to forge the identity card upon identifying the personnel performing identity verification during the workplace drug test. PGB was found positive for case No. 29 with LC-MS/MS, while he also was noted to have been referred for toxicological analysis as the employer noticed the decrease in work performance. Employer-reported data overlapped with clinical signs of PGB use and a positive laboratory PGB result. Additionally, there was no drug containing PGB among the cold medicine and analgesic drugs (Voltaren) declared. The increase in the non-medical use and misuse of prescription drugs is a serious public issue in most countries such as Turkey. Most of the abusers have perceptions that the prescribed drugs are more reliable, and healthier than illegal substances, in addition, it is much more easy access. It is remarkable that we detected pregabalin, one of the controlled/prescription drugs in 2 cases in our study. In addition to conventional substance screening included in the international WDT guideline, the study's findings demonstrated, from a medico-legal perspective, the importance of meticulous investigation with clinical awareness of the prescripted substances susceptible to abuse such as PGB due to their increasing abuse potential.

Cut-off dilemma: Differences from the forensic and clinic perspective

Opioids are a major concern in many countries due to the serious health consequences associated with their use. The United Nations Office on Drugs and Crime (UNODC) opioid crisis strategy report states that the number of people using opioids for non-medical purposes around the world has nearly doubled in the last decade and the prevalence of opioid use has increased by 76% in the same period. The same report stated that opioid use is responsible for approximately two-thirds of deaths attributed to substance use disorders worldwide. Opiates are one of the five classes of abused drugs mandated for testing in the Drug Testing Program of the Substance Abuse and Mental Health Services Administration (SAMHSA). SAMHSA regulated opiate cut-off value from 300 ng/mL to 2000 ng/mL for clinical cases only in 2010. In our country, similar legislation was made in 2016 and unfortunately, the cut-off value was increased to 2000 ng/mL not only for clinical but also for forensic cases. In this study, it is aimed to examine the effects of the opiate cut-off values of 300 and 2000 ng/mL on the toxicological analysis results. Opiate results were evaluated in terms of 300 and 2000 ng/mL cut-off values in the urine samples of the cases who applied to Ege University BATI Institute, Addiction Toxicology Laboratory between 2014–2021 with illegal substance analysis. Urine samples were accepted to the laboratory and urine integrity test was performed before analysis. Urine samples were first subjected to pre-screening analysis by immunoassay method. Confirmation analysis was performed with gas chromatography-mass spectrometry (GC-MS, Agilent 7890B) of the samples that were positive after the pre-scan analysis. A total of 11,348 cases were screened in terms of opiates and the mean age of the cases is 25.4. Of the total cases, 3.45% ( n = 392) were opiate positive. Of the total cases, 83.7% ( n = 328) were male and 16.1% ( n = 64) of them were female. When the cases were classified according to the admitted reasons for the analysis request; alcohol, substance and drug use reason were 67,3%, psychiatric disorders 20.9% and 1.5% due to war & injury and falling, 7,4% for other reasons. According to the cut-off value of 300 ng/mL, the rate of opiate positive cases between 300 < n < 2000 were 39.8%. The cut-off value of 2000 ng/mL, the rate of 2000 < n opiate positive cases were 60.2%. According to the results, if the cases between 2014 and 2021 were evaluated according to the 300 ng/mL cut-off value, treatment and/or forensic proceedings would have been applied to 156 people because they were opiate positive. However, the presence of morphine in the urine samples of forensic toxicological cases, which may cause opiate positivity can be caused by legal prescription drugs, oral use of poppy seeds, or drug use. As the intake of some codeine-containing drugs even at therapeutic amounts and ingestion of food products containing poppy seeds can give a positive opiate result with the 300 ng/mL screening cut-off value, the most appropriate thresholds should extensively studied. The cut-off values used in forensic toxicological analyzes should be paid special attention in clinical and forensic analyzes. Since the cut-off values used in clinical and forensic cases are the same, awareness is the responsibility of forensic toxicologists and the physician who evaluates them medico-legally. Even in the presence of heroin and morphine in the urine matrix, too many true positive cases can be overlooked, since the threshold value used for forensic purposes is the same as that used for clinical purposes and remains below this value.