A novel analytical strategy based on gas chromatography-Orbitrap high-resolution mass spectrometry combined with solid-phase extraction for the monitoring of stanozolol misuse in human urine.

RATIONALE: Stanozolol, an anabolic androgenic steroid listed in Part S1 of the World Anti-Doping Agency Prohibited List, exhibits a low response and significant matrix interference in urine samples when using liquid-liquid extraction-gas chromatography-mass spectrometry (GC-MS). Enhancing sample preparation techniques remains essential for the effective detection of stanozolol and its metabolites. METHODS: A method for determining stanozolol and its metabolites (3'-OH-stanozolol, 4ß-OH-stanozolol, and 16ß-OH-stanozolol) in human urine was developed and validated using GC-Orbitrap high-resolution MS combined with optimized mixed-mode solid-phase extraction (SPE). This method was applied to urine samples from two volunteers who orally administered a single dose of stanozolol, with samples collected over a 30-day period post-administration. RESULTS: The optimized mixed-mode SPE method reduced matrix interference and achieved satisfactory extraction efficiency and high sensitivity, enabling confident identification of all targets in human urine. Validation showed extraction recovery of 74% to 81% and limits of detection from 0.1 to 0.25 ng mL-1. The method was successfully applied to detect urinary excretion profiles of stanozolol and its metabolites in positive volunteer samples. CONCLUSION: This study presents a novel detection protocol for stanozolol and its metabolites, enhancing the monitoring of stanozolol abuse and contributing to the integrity of sports competitions. This protocol offers a robust tool for anti-doping laboratories, aiding in the accurate detection of stanozolol misuse and supporting the enforcement of fair play in athletics.

Facile fabrication of the immuno-MALDI-MS chip for the enrichment of abused drug in human urine integrated with MALDI-MS analysis.

BACKGROUND: Drug abuse can result in both physical and mental health issues for individuals, and can also contribute to broader societal problems. The number of drug abuse cases rose to 296 million in 2021. The sample pretreatment methods commonly employed typically require longer processing times and occasionally necessitate derivatization. Furthermore, with the increase in sample sizes, traditional chromatography-mass spectrometry methods for analyzing abused drugs were no longer sufficient to handle such numerous samples. In this study, immuno-MALDI-MS chip were fabricated for specific enrichment of illicit drugs, integrating with the rapid and accurate capabilities of MALDI-MS for high-throughput analysis of drug abuse. RESULTS: The immuno-MALDI-MS chip was successfully prepared by coating an aluminum chip with antibody-conjugated boronic acid-modified gold nanoparticles. Ketamine, a frequently abused illicit drug, served as the proof of concept for this study. The immuno-MALDI-MS chip was employed to selectively enrich ketamine in human urine samples, facilitating direct MALDI-MS analysis with the addition of α-CHCA matrix solution. The challenge of detecting abused drugs, exacerbated by interfering peaks in the low m/z region from salts and small molecules in human urine samples, was successfully overcome. The developed method exhibited a wide linear range of 10-5000 ng/mL with a limit of detection of 3.3 ng/mL for ketamine. Notably, the proposed method enabled high-throughput screening and accurate confirmation of ketamine concentrations in suspects' urine samples within few minutes, requiring a minimal sample volume of 1 µL. The obtained data were in complete agreement with the previous GC/MS analysis. SIGNIFICANCE: A straightforward, cost-effective and sensitive method for the selective enrichment and absolute quantification of abused drugs was developed using a homemade immuno-MALDI-MS chip integrated with MALDI-MS analysis. This method combines the advantages of immunoassay and mass spectrometry, offering both speed and accuracy. The reported method for the quantification of ketamine in human urine offers a practical approach and has the potential to analyze emerging new psychoactive substances in the future.

Fentanyl, Heroin, Methamphetamine, and Cocaine Analyte Concentrations in Urine Drug Testing Specimens.

Importance: The US is experiencing a protracted drug overdose crisis primarily associated with exposure to illicitly manufactured fentanyl (IMF), methamphetamine, and cocaine. Overdose risk and treatment responses may be directly affected by absolute drug exposure concentrations and drug use prevalence. Objective: To quantify changes in absolute drug exposure concentrations from 2013 to 2023. Design, Setting, and Participants: This cross-sectional study analyzed urine drug testing (UDT) results from urine specimens collected between January 1, 2013, and August 22, 2023, in 49 states and the District of Columbia. Urine specimens were obtained from patients aged 18 years or older who presented to substance use disorder treatment clinics. The UDT was ordered by clinicians based on medical necessity. Exposures: Urine specimens were analyzed for the following drugs or metabolites (analytes tested in parentheses): fentanyl (fentanyl), heroin (6-monoacetylmorphine), cocaine (benzoylecgonine), and methamphetamine (methamphetamine) using liquid chromatography with tandem mass spectrometry. Main Outcomes and Measures: Relative concentrations of fentanyl, heroin, cocaine, and methamphetamine. Creatinine-normalized drug concentration values were log-transformed prior to visualization and statistical analyses. The Mann-Kendall trend test was performed to examine trends over time. To estimate the geospatial and temporal patterns of drug concentration, a second series of models (1 for each drug) with an interaction effect for clinic location and collection year were fit. Results: A total of 921 931 unique UDT samples were collected from patients (549 042 males [59.6%]; median [IQR] age, 34 [27-44] years). The adjusted fentanyl concentration in urine specimens was 38.23 (95% CI, 35.93-40.67) ng/mg creatinine in 2023 and 4.61 (95% CI, 3.59-5.91) ng/mg creatinine in 2013. The adjusted methamphetamine concentration was 3461.59 (95% CI, 3271.88-3662.30) ng/mg creatinine in 2023 and 665.27 (95% CI, 608.51-727.32) ng/mg creatinine in 2013. The adjusted cocaine concentration was 1122.23 (95% CI, 1032.41-1219.87) ng/mg creatinine in 2023 and 559.71 (95% CI, 524.69-597.06) ng/mg creatinine in 2013. The adjusted heroin concentration was 58.36 (95% CI, 48.26-70.58) ng/mg creatinine in 2023 and 146.59 (95% CI, 136.06-157.92) ng/mg creatinine in 2013. Drug concentrations varied across US Census divisions. Conclusions and Relevance: This cross-sectional study found that absolute concentrations of fentanyl, methamphetamine, and cocaine in urine specimens increased from 2013 to 2023, with a decrease in heroin concentration during that period. The findings suggest that exposure to these substances, as well as the illicit drug supply, has fundamentally changed in many parts of the US, highlighting the need to reinforce surveillance initiatives and accelerate efforts to treat individuals with IMF and/or stimulant exposure.

Implementation of a community-based LC-UV drug checking service: promising preliminary findings on feasibility and validity.

BACKGROUND: The increasing diversity of psychoactive substances on the unregulated drug market poses significant health, psychological, and social risks to people who use drugs (PWUD). To address these risks, various harm reduction (HR) policies have been implemented, including drug checking services (DCS). Many analytical methods are used for DCS. While qualitative methods (e.g., thin layer chromatography, spectroscopy) are easier to implement, they are not as accurate as quantitative methods (e.g., LC-UV, LC-MS). Some HR programmes have implemented high-performance liquid chromatography coupled with UV detection (LC-UV). This article presents the cross-validation of this quantitative method with a reference liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) method. METHODS: Drug samples were provided by PWUD to a DCS called DrugLab in Marseille, France. The samples were weighed and prepared through dissolution in methanol, followed by ultrasonic bathing. Samples were analysed onsite using LC-UV analysis. They were then subsequently analysed with the reference LC-HRMS method. The LC-UV instrument in DrugLab was calibrated after being purchased; analysis of standard solutions was routinely performed once a month and after maintenance operations. For the LC-HRMS instrument, calibration and quality control procedures followed European Medicines Agency (EMA) guidelines. Statistical analyses were conducted including Spearman correlation tests using IBM® SPSS® Statistics version 20. RESULTS: A total of 102 samples representing different product classes and cutting agents were cross-validated. Differences between both analyses methods for each molecule analysed were ≤ 20%, with significant correlations between both methods' results for most substances. Notably, LC-HRMS provided lower concentration values for cocaine and acetaminophen, whereas it provided higher values for other substances. Correlations were significant for cocaine, ketamine, MDMA, heroin, amphetamine, caffeine, acetaminophen, and levamisole. CONCLUSIONS: This study demonstrates that the results provided by DrugLab were accurate and reliable, making LC-UV an adaptable, stable, and suitable analytical method for simple matrices like drugs in a DCS context. However, this cross validation does not guarantee accuracy over time. A proficiency test project in HR laboratories across France is currently under development in order to address potential drifts in LC-UV accuracy.

Inkjet Printing Patterned Plasmonic SERS Platform with Surface-Optimized Paper for Label-Free Detection of Illegal Drugs in Urine.

Rapid quantitative testing of illegal drugs is urgently needed for precisely cracking down on drug crimes. Herein, an optimized paper-based surface-enhanced Raman spectroscopy (SERS) platform with patterned printing of plasmonic nanoparticles was constructed for the on-site quick testing of illegal drugs in urine. The filter paper was first coated with a layer of positive-charged chitosan, so as to reduce its roughness by filling the holes of the cellulose matrix and enhance the adhesion of negative-charged silver ink. Subsequently, hydrophobic modification was performed based on the binary silylation reaction, which could obviously improve the sensitivity of the paper-based SERS substrate by concentrating the amount of analyte. Meanwhile, SERS-active silver ink was fabricated and further printed on the surface of the above modified paper with custom-designed pattern (3 × 6). The performance of this SERS platform was assessed by using crystal violet (CV) as a model tag, and the obtained results proved it possesses excellent sensitivity and reproducibility, in which the relative standard deviation (RSD) dropped remarkably. More importantly, as a proof of concept, rapid detection of standard methylamphetamine (MAMP), one of the most widely abused drugs, was achieved with a limit of detection (LOD) of 1.43 ppb using a portable Raman spectrometer. And it also had a good capability in human urine sample detection, with a correlation index (R2) up to 0.9927. This optimized paper-based SERS platform was easily manufactured, cheap, and portable, providing a new strategy for the on-site detection of illicit drugs.

The Evolution of Illicit-Drug Detection: From Conventional Approaches to Cutting-Edge Immunosensors-A Comprehensive Review.

The increasing use of illicit drugs has become a major global concern. Illicit drugs interact with the brain and the body altering an individual's mood and behavior. As the substance-of-abuse (SOA) crisis continues to spread across the world, in order to reduce trafficking and unlawful activity, it is important to use point-of-care devices like biosensors. Currently, there are certain conventional detection methods, which include gas chromatography (GC), mass spectrometry (MS), surface ionization, surface-enhanced Raman spectroscopy (SERS), surface plasmon resonance (SPR), electrochemiluminescence (ECL), high-performance liquid chromatography (HPLC), etc., for the detection of abused drugs. These methods have the advantage of high accuracy and sensitivity but are generally laborious, expensive, and require trained operators, along with high sample requirements, and they are not suitable for on-site drug detection scenarios. As a result, there is an urgent need for point-of-care technologies for a variety of drugs that can replace conventional techniques, such as a biosensor, specifically an immunosensor. An immunosensor is an analytical device that integrates an antibody-based recognition element with a transducer to detect specific molecules (antigens). In an immunosensor, the highly selective antigen-antibody interaction is used to identify and quantify the target analyte. The binding event between the antibody and antigen is converted by the transducer into a measurable signal, such as electrical, optical, or electrochemical, which corresponds to the presence and concentration of the analyte in the sample. This paper provides a comprehensive overview of various illicit drugs, the conventional methods employed for their detection, and the advantages of immunosensors over conventional techniques. It highlights the critical need for on-site detection and explores emerging point-of-care testing methods. The paper also outlines future research goals in this field, emphasizing the potential of advanced technologies to enhance the accuracy, efficiency, and convenience of drug detection.

Analysis of the polymeric mixture in hypoxen, using high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Hypoxen is a medication known for providing individuals with a "second wind," by lowering the threshold for muscle fatigue and enhancing the body's efficiency under challenging conditions. Athletes who have used this medication report enhanced training outcomes and increased physical endurance. It is crucial to emphasize that hypoxen is not categorized as a prohibited substance as yet and is thus assumed safe for use in competitive sports. However, the polymeric nature of hypoxen presents challenges in detection and identification. METHODS: To prevent the potential misuse of this substance as a doping agent, doping control laboratories must therefore develop a detection method. This study aimed to address this gap by developing a comprehensive detection method for the polymeric mixtures within hypoxen, employing liquid chromatography-electrospray ionization-mass spectrometry. RESULTS: Among the different columns tested, the Accucore and Syncronis HILIC columns demonstrated exceptional performance, yielding excellent separation with high-quality results. The study found that the hypoxen consisted of between 1 and 6 repetitions of 2,4-dihydroxyphenylene units. It was crucial to highlight that each unit contained multiple isomers due to the incorporation of the sulfonic acid group at both the -SH and -OH positions. CONCLUSIONS: The results of this study will significantly contribute to the precise identification of hypoxen use, thereby aiding in the scrutiny of its abuse in competitive sports.

Impaction-based exhaled breath sampling for substance monitoring: a prospective pilot study (Drugxhale).

For decades, intake monitoring of drugs using urine as the matrix of choice is the gold standard in drug treatment centers. A properly conducted urine drug test can identify recent use of prescribed, non-prescribed and illicit drugs. However, issues like adulteration, substitution and privacy issues have driven the search for alternative matrices. This prospective pilot study evaluates the use of an impaction-based breath sampling device, Breath Explor®, as an alternative to traditional urine-based drug monitoring. Breath samples were analyzed using a validated 32-component liquid chromatography-tandem mass spectrometry method. Recovery data represent the efficiency of extracting the analytes from the breath devices. Both automated and manual processing of the Breath Explor® devices showed mean recovery rates ranging from 39.5% to 55.4% for the 32 analytes. Despite the small number of subjects, breath analysis proved to be a convenient and easy-to-use methodology. An overall kappa-values of 0.5 indicated a moderate level of agreement with urine analysis, underscoring its potential as a complementary diagnostic tool. All participants tested positive in their breath sample for methadone (70% methadone and 100% EDDP), while a significant portion (90%) tested positive for 6-monoacetylmorphine. This innovative approach offers several advantages, including non-invasiveness, reduced risk of adulteration, and the ability to perfom repeated automated sampling and confirmation testing. These findings suggest that breath-based substance monitoring could complement or even replace traditional urine-based methods in clinical practice.

Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control.

Aim: The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated.Materials & methods: Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the elimination profiles of osilodrostat using a validated method involving efficient enzymatic hydrolysis followed by LC/ESI-HRMS analysis.Results: Applying the validated quantification method with an LLOQ of 0.05 ng/ml, hydrolyzed osilodrostat could be quantified in post-administration urine samples from 48 to 72 h post-administration; by contrast, both hydrolyzed osilodrostat and M1c were detected up to 2 weeks. In addition, confirmatory analysis identified the presence of hydrolyzed osilodrostat for up to 72 h post-administration.Conclusion: For doping control purposes, we recommend monitoring both hydrolyzed M1c and osilodrostat because of the greater detectability of M1c and the availability of a reference material of osilodrostat, which is essential for confirmatory analysis.

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Detection of ethanol, cannabinoids, benzodiazepines, and opioids in older adults evaluated for serious injuries from falls.

BACKGROUND: In 2020, there were 36.7 million reported falls among older adults (65+) in the United States. Ethanol and other sedating substances may increase fall risk among older adults due to their effect on cognitive and physical function. We estimate the prevalence of these substances in blood specimens of older adults presenting with a fall injury at selected trauma centers. METHODS: The initial study collected blood specimens from May 2020 through July 2021 from adults undergoing a trauma team evaluation at selected United States Level 1 trauma centers. We limited our study to older adults evaluated after a fall (n = 1,365) and selected a random sample (n = 300) based on age, sex, and trauma-center quotas. Medical health records and blood specimens obtained at trauma center presentation were analyzed. We estimated the prevalence of ethanol, benzodiazepines, cannabinoids, and opioids in the blood specimens. Two-sample tests of binomial proportions and Chi-square two-tailed tests were used to compare prevalence estimates of substances by demographic characteristics. RESULTS: At least one substance was detected among 31.3% of samples analyzed. Prevalences of specific substances detected were 9.3% (95% CI: 6.0-12.6%) for benzodiazepines, 4.3% (95% CI: 2.0-6.7%) for cannabinoids, 8.0% (95% CI: 5.2-11.7%) for ethanol, and 15.0% (95% CI: 10.9-19.1%) for opioids. There were 18 deaths (6%; 95% CI: 3.6-9.3%). One-third of decedents had at least one substance detected in their blood. DISCUSSION: Opioids were the most frequently detected substance, followed by benzodiazepines, ethanol, and cannabinoids. Substance use prevalence was not uniform across demographics, with differences observed by sex and age. CONCLUSIONS: This study provides insight into the frequency of the presence of substances that may contribute to fall risk and serious injury among older adults. Screening older adults for substances that impair cognitive and physical function can enhance clinical fall prevention efforts.

Evaluating fentanyl test strips as a harm reduction strategy in rural and urban counties: study protocol for a randomized controlled trial.

BACKGROUND: Opioid-related fatalities are a leading cause of death in Ohio and nationally, with an increasing number of overdoses attributable to fentanyl. Rapid fentanyl test strips can identify fentanyl and some fentanyl analogs in urine samples and are increasingly being used to check illicit drugs for fentanyl before they are used. Fentanyl test strips are a promising harm reduction strategy; however, little is known about the real-world acceptability and impact of fentanyl test strip use. This study investigates fentanyl test strip distribution and education as a harm reduction strategy to prevent overdoses among people who use drugs. METHODS: The research team will recruit 2400 individuals ≥ 18 years with self-reported use of illicit drugs or drugs purchased on the street within the past 6 months. Recruitment will occur at opioid overdose education and naloxone distribution programs in 16 urban and 12 rural Ohio counties. Participating sites will be randomized at the county level to the intervention or non-intervention study arm. A brief fentanyl test strip educational intervention and fentanyl test strips will be provided to participants recruited from sites in the intervention arm. These participants will be eligible to receive additional fentanyl test strips for 2 years post-enrollment. Participants recruited from sites in the non-intervention arm will not receive fentanyl test strip education or fentanyl test strips. All participants will be followed for 2 years post-enrollment using biweekly, quarterly, and 6-month surveys. Primary outcomes include (1) identification of perceived barriers and facilitating factors associated with incorporating fentanyl test strip education and distribution into opioid overdose education and naloxone distribution programs; (2) differences in knowledge and self-efficacy regarding how to test drugs for fentanyl and strategies for reducing overdose risk between the intervention and non-intervention groups; and (3) differences in non-fatal and fatal overdose rates between the intervention and non-intervention groups. DISCUSSION: Findings from this cluster randomized controlled trial will contribute valuable information about the feasibility, acceptability, and impact of integrating fentanyl test strip drug checking in rural and urban communities in Ohio and help guide future overdose prevention interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT05463341. Registered on July 19, 2022. https://clinicaltrials.gov/study/NCT05463341.

Differences in toxicology reports and hospital emergency care for patients suspected of experiencing drug-facilitated crimes: an analysis according to gender. / Atención de pacientes con sospecha de sumisión química en un servicio de urgencias hospitalario y resultados del análisis toxicológico: diferencias según el sexo.

OBJECTIVE: To analyze gender-related differences in patient and care characteristics and in toxicology findings in suspected cases of drug facilitated crime (DFC). METHODS: Observational cross-sectional study of all patients in suspected DFC cases attended in the emergency department of Hospital Clínico San Carlos and of their blood or urine samples analyzed by the National institute of Toxicology and Forensics in Madrid between March 1, 2015, and March 1, 2023. We analyzed variables from patient records and the toxicology reports according to gender. RESULTS: A total of 514 suspected DFC episodes were studied; 101 (19.6%) were proactive crimes, 61 (11.9%) opportunistic, and 352 (68.5%) mixed. The median (interquartile range) age was 25 years (21-34 years), and 370 (72%) were women. Eighty-three percent of the patients had amnesia, and 48% of the cases involved sexual assault or robbery. Toxicology identified substances in 78% of the patients (alcohol, 53%; street drugs, 37%; and/or psychopharmaceuticals or opioids, 23%). Independent variables associated with female gender in the multivariate analysis, according to adjusted odds ratio (aORs) were age less than 25 years (aOR, 2.73; 95% CI, 1.75 4.24; P < .001); physician-referred emergency (aOR, 1.77; 95% CI, 1.12-2.80; P = .03); robbery (aOR, 0.25; 95% CI, 0.15-0.41; P < .001); alcohol-positive test result (aOR, 1.91; 95% CI, 1.21-3.00; P = .01); and a drug-positive result (aOR, 0.43; 95% CI, 0.28-0.64; P < .001). Police and a forensic physician intervened in 13% of the cases, and in such cases the victim was more likely to be female (aOR, 3.97; 95% CI, 1.41-11.13; P < .001). Toxicology identified the presence of an unknown substance in 39%, and a woman was less likely to be involved in such cases (aOR, 0.43; 95% CI, 0.28-0.67; P < .001). CONCLUSIONS: The majority of victims of DFCs were female, and the crimes were mixed, involving involve alcohol, psychopharmaceuticals or street drugs. Female victims were more likely to be under the age of 25 years, be referred to the emergency service by a physician, be attended by a forensic physician for sexual assault, and have an alcoholpositive toxicology report. Women were also less likely to report a robbery or have a toxicology report identifying drugs or an unknown substance.

OBJETIVO: Analizar las diferencias en las características de los pacientes atendidos por sospecha de sumisión química (SQ) y en los resultados del análisis toxicológico (AT) en función del sexo. METODO: Estudio observacional transversal retrospectivo que incluyó a todos los casos con SQ atendidos en el servicio de urgencias del Hospital Clínico San Carlos y las muestras (sangre o orina) para el AT en el Instituto Nacional de Toxicología y Ciencias Forenses de Madrid entre el 1 de marzo de 2015 y el 1 de marzo de 2023. Se analizan variables de la historia clínica y del AT según el sexo. RESULTADOS: Se incluyeron 514 episodios con sospecha de SQ [101 (19,6%) proactiva, 61 (11,9%) oportunista y 352 (68,5%) mixta] en pacientes con una mediana de 25 años (RIC: 21-34), 370 (72%) de sexo femenino. El 83% presentó amnesia y el 48% asoció agresión sexual o robo. En el 78% se identificó alguna sustancia en el AT (53% alcohol etílico, 37% drogas y/o 23% psicofármaco u opiáceos). En el análisis multivariado las variables que se asociaron de manera independiente con el sexo femenino fueron la edad menor de 25 años con ORa de 2,73 (IC 95%: 1,75-4,24; p < 0,001), con médico deriva a urgencias con ORa de 1,77 (IC 95%: 1,12-2,80; p = 0,03), delito de robo con de ORa 0,25 (IC 95%: 0,15-0,41; p < 0,001), alcohol etílico en el AT con ORa 1,91 (IC 95%: 1,21-3,00; p = 0,01) y alguna droga en el AT con ORa 0,43 (IC 95%: 0,28-0,64; p < 0,001). En el 13% de casos hubo intervención policial y médico-forense y fue más probable que fuera a una mujer, con ORa 3,97 (IC 95%: 1,41-11,13; p < 0,001). En el 39% de AT se identificó alguna sustancia desconocida y fue menos probable que fuera mujer, con ORa de 0,43 (IC 95%: 0,28-0,67; p < 0,001). CONCLUSIONES: La mayoría de casos registrados fueron mujeres con sospecha de SQ mixta por alcohol, psicofármacos o drogas de abuso. Las mujeres presentaron mayor probabilidad de tener menos de 25 años, ser derivada a urgencias por un médico, de intervención médico-forense por agresión sexual y encontrar alcohol etílico en el AT.

Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of AICAR.

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation. METHOD: In this study, an automated 2D-HPLC separation technique was used to separate and purify AICAR and endogenous reference substances in urine samples. Then, AICAR was derivatized with 3-TMS as the main derivative product, while the endogenous reference compounds remained in their original form. Subsequently, the developed GC/C/IRMS method was utilized for the detection of the target and reference substances. Followed, we evaluated the applicability of this method using urine samples from two Asian males administered a low dose of AICAR (3 grams). RESULTS: The advantages of this study include: 1) reduced sample pretreatment time: the established 2D-HPLC separation method can separate the target and endogenous reference substances in one step; 2) low interference: the isotope chromatograms have low background interference, and the separation of endogenous reference substances is purer; 3) more accurate result calculations: this method only requires derivatization and result correction for AICAR, with the endogenous reference substances measured in their original form, reducing biases from corrections of multiple substances. The detection method performed well, with a concentriton limit of 2500 ng/mL, meeting the needs of routine detection concentrations. The CIR results from volunteer samples indicated that samples collected within 16 hours post-administration exceeded the threshold set in the literature. CONCLUSION: This study successfully established a 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing the internal and external sources of AICAR. After administering a low dose of AICAR to the Asian population, exogenous drug characteristics were manifested within 16 hours. This observation, when compared to the 40-hour detection window cited in the literature, suggests that the length of the detection window is positively correlated with the dosage of the test drug.

Impact of reduced sampling frequency of illicit drug wastewater monitoring in the Netherlands.

Drug consumption estimates are traditionally based on surveys or information from police seizures. Alternatively, residues of illicit drugs in untreated wastewater (influent) can be used to calculate mass loads and subsequently estimate drug consumption in the community throughout the week. For this purpose, wastewater is commonly sampled for seven consecutive days within the Sewage analysis CORe group Europe (SCORE), while other sampling schemes may be implemented in long-term studies outside this consortium. The current study demonstrates how sampling frequency of illicit drug residues in the influent of wastewater treatment plants (WWTPs) affects the derived weekly average. Thirty WWTPs were sampled over the course of 12 years and influents were analyzed for five drugs (metabolites): 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, amphetamine, benzoylecgonine (a metabolite of cocaine), and 11-nor-9-Carboxy tetrahydrocannabinol (THC-COOH). Subsequently, small and large WWTPs were grouped with a threshold of 100,000 inhabitants. After data curation, standardized loads were calculated (mg/d per 1000 inhabitants). Weekly averages of loads of the drug residues were calculated based on six scenarios (sampling one to six weekdays) and compared to the weekly average in the control situation (sampling seven weekdays) in a Monte Carlo simulation. Results indicate that drug residues with more dynamic loads over a week require more frequent sampling. The analysis illustrates that a decreased sampling frequency (4 or 5 days per week) still leads to a representative weekly average for all drugs tested when a deviation up to a factor of 1.25 is deemed acceptable. However, knowledge on typical levels is necessary to define outliers. We therefore recommend to study dynamics in drug residue loads for WWTPs before reducing sampling frequency in long term monitoring programs.

On the Value of Measuring Recent Drug Use by Self-Reports and Urinalysis in Clinical Trials.

Background: Valid measurement of drug use in patients enrolled in clinical trials that treat substance use disorder is vital to determine the trial's outcome. Self-reports are often used but their validity has been studied with mixed results. Urinalysis may sometimes be employed as an alternative or supplement to self-reports. Objectives: This study examined how estimating drug use by either method would affect the results from a randomized clinical trial conducted in a methadone treatment program. At the initial Baseline interview and four follow-up interviews, participants were asked about their drug use history and provided a urine specimen for drug testing. Results: In most cases, the urinalyses detected more drugs than the patients had reported using. A major exception was heroin, whose use was an eligibility criterion for enrollment in the study and methadone treatment. Conclusions: The patients' self-reports would have led us to conclude that the use of heroin and fentanyl had declined from the initial Baseline interview to the final follow-up interview, while the urinalysis results indicated no change in exposure to heroin and an increase in exposure to fentanyl. Clinical trials would be well served to employ the use of biological tests in addition to self-reports to measure recent drug use and to accurately estimate the efficacy of the experimental protocols and patients' exposure to drugs.

How do perceptions of procedural justice, police legitimacy, and legitimacy of laws influence intentions to drug drive?

INTRODUCTION: There is a need for improved drug driving enforcement to promote greater driver compliance with drug driving laws. In Australia, Roadside Drug Testing (RDT) suffers from operational challenges that undermine its effectiveness in reducing drug driving. OBJECTIVE: To identify potential improvements to RDT, this study investigated the extent to which drivers perceive RDT to be procedurally just and that the policing of drug driving and the associated laws are legitimate. These perceptions were then compared with those applying to Random Breath Testing (RBT) and examined in relation to their respective influence on intentions to drug and drink drive in the future. METHOD: A sample of 1,483 licensed drivers from three Australian states completed an online survey. RESULTS: Those participants who reported engaging in drug driving perceived RDT to be less procedurally just than non-drug drivers. Similarly, drug drivers perceived the police and associated drug driving laws to be less legitimate than non-drug drivers. Furthermore, drug drivers who had been tested at an RDT operation in the past perceived RDT to be less procedurally just and considered drug driving policing and laws to be less legitimate, compared with the corresponding perceptions of drink drivers who had been tested at an RBT operation. A regression analysis indicated that stronger intentions to drug drive in the future were associated with lower perceptions of police legitimacy and the legitimacy of drug driving laws, but not with the elements of procedural justice. However, follow-up analyses indicated that the influence of procedural justice on intentions was mediated by the two legitimacy variables, thus weakening its direct impact on intentions. PRACTICAL APPLICATIONS: The results highlight the need for road safety authorities to enhance the perceived legitimacy of drug driving enforcement and associated laws. Changes to current police practices and/or drug-driving laws may also be needed to enhance the effectiveness of RDT.

Exploring methandienone metabolites generated via homogenized camel liver: Advancements for anti-doping applications through High Resolution-Liquid Chromatography Mass Spectrometry analysis.

RATIONALE: Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. METHODS: This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. RESULTS: The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. CONCLUSIONS: These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.

Prevalence and correlates of fentanyl test strip use among people who use drugs in Rhode Island.

BACKGROUND: Illicitly manufactured fentanyl accounts for a majority of overdose fatalities in the US. Research has demonstrated that fentanyl test strips (FTS) help people who use drugs (PWUD) avoid unintended exposure to fentanyl and overdose. This study assesses characteristics associated with FTS use among PWUD in Rhode Island. Such findings may shed light on whether there are subgroups of PWUD who are less likely to be using FTS and therefore may benefit from their use. METHODS: From September 2020 - February 2023, participants were recruited to participate in RAPIDS, a clinical trial assessing whether FTS provision can reduce overdose rates. Baseline data were used to assess correlates of lifetime FTS use through bivariable and multivariable analyses. We also examined drug testing patterns relating to FTS use in the past month. RESULTS: Of 509 people enrolled, 376 (73.9 %) had heard of FTS before enrollment. Among this group, 189 (50.3 %) reported lifetime FTS use and 98 (26.1 %) reported use in the last month. In bivariable analyses, lifetime injection drug use, responding to an overdose, and drug selling were associated with FTS use. Solitary drug use was not associated with FTS uptake. In the multivariable analysis, gender and lifetime naloxone administration were associated with FTS use. Of those who used FTS in the past month, 76.5 % had at least one test that was positive for fentanyl. CONCLUSIONS: We found high uptake of FTS use among PWUD in Rhode Island. Our results also suggest a need for targeted outreach to increase FTS uptake among sub-groups of PWUD. CLINICAL TRIAL REGISTRATION: The Rhode Island Prescription and Illicit Drug Study is a registered clinical trial, NCT043722838.

Trends in polysubstance use among patients in methadone maintenance treatment in Ireland: Evidence from urine drug testing 2010-2020.

INTRODUCTION: The benefits of methadone maintenance treatment (MMT) may be compromised by the continued use of other substances during treatment. Polysubstance use has been identified as a major contributing factor to treatment discontinuation, a known risk factor for drug overdose. We examined trends in immunoassay drug positivity rates for amphetamines, benzodiazepines, cannabis, cocaine and opioids, and (2) trends in polysubstance positivity rates for drug combinations associated with increased risk of drug overdose among patients attending the national drug treatment centre in Ireland for MMT between 2010 and 2020. METHODS: Repeated cross-sectional study of patients attending the national drug treatment centre (NDTC) for MMT (total N = 1942) between 2010 and 2020, focused on urine drug samples provided for testing to the NDTC clinical testing laboratory (n = 221,564). Samples were analysed using immunoassay during the study period. Mixed-effects logistic regression models evaluate time trends in drug positivity. A random intercept accounts for repeat testing of individual patients. The study reports Adjusted Odds Ratios (AOR) for time (per year) with 95 % Confidence Intervals (95 % CI). RESULTS: Drug positivity rates increased over time for benzodiazepines (AOR 1.02, 95 % CI 1.01-1.03, p < .0001), cannabis (AOR 1.06, 95 % CI 1.05-1.08, p < .0001) and cocaine (AOR 1.28, 95 % CI 1.27-1.29, p < .0001), with decreasing trends for opioids (AOR 0.91, 95 % CI 0.91-0.92, p < .0001). Methadone and benzodiazepines were co-detected in over two-thirds of all samples during the study period. Co-detection of methadone and benzodiazepines with cocaine was also found to be increasing (AOR 1.24, 95 % CI 1.23-1.25, p < .0001), with weighted polysubstance positivity rates reaching 29.2 % in 2020. The co-detection of methadone and benzodiazepines with opioids decreased over the study period (AOR 0.92, 95 % CI 0.91-0.92, p < .0001), ranging from 36.7 % in 2010 to 26.9 % in 2020. CONCLUSION: Interventions are needed to target the persistently high use of benzodiazepines among patients in receipt of methadone due to their synergistic effects with opioids on respiratory depression, enhancing the risk of overdose. The growing use of cocaine among people in MMT also needs to be addressed.

The sensitive detection of low molecular mass peptide drugs in dried blood spots by solid-phase extraction and LC-HRMS.

Dried blood spot (DBS) technique has become a new popular topic in anti-doping field in recent years due to its advantages of sample stability and easy operation. It can be employed as a supplementary method to routine urine analysis. However, the small volume of DBS samples (usually 10-20 µL) significantly reduces the application value of this technique. Therefore, the development of sensitive detection methods for the analysis of prohibited substances in DBS is particularly important. In this study, based on the characteristics of low molecular mass peptide (LMMP) drugs, systematic optimization strategies were utilized for the first time to establish a sensitive detection method for LMMPs in DBS. Without using DMSO to enhance mass spectrometry ionization efficiency of peptides, the limits of detection (LOD) ranged between 0.05 and 3.74 ng/mL, significantly better than the previously reported method (0.5-20 ng/mL). This method was validated according to the guidelines of the World Anti-Doping Agency (WADA), and corresponding post-administration study was conducted, demonstrating that the method could be applied to routine analysis of LMMP drugs in DBS. Moreover, since DMSO is not involved, this method also has the potential to simultaneously detect both LMMP and small molecular drugs.