Changes in blood parameters after intramuscular testosterone ester injections – Implications for anti‐doping

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the ‘haematological module’ of the athlete biological passport (ABP) used by the World Anti‐Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo‐controlleddouble‐blind design. Urine samples and blood samples were collected twice pre‐treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post‐injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®‐treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope‐ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF‐Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3–10 days after injection. From an anti‐doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow‐up testing and substantiating likely misuse of testosterone.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Bioanalysis for cocaine,opiates, methadone,and amphetamines exposure detection during pregnancy

Drug exposure during pregnancy constitutes a major legal issue and a public health concern. Drug and metabolite determination in biological matrices from mother and newborn is an objective indication of prenatal drug exposure. However, limited data are available regarding the interpretation of these analytical results in terms of window of detection and degree of exposure. We collected paired maternal hair, meconium, placenta, and umbilical cord from 727 mother‐newborn dyads. We analyzed these specimens by liquid chromatography‐tandem mass spectrometry for the determination of cocaine, opioids, methadone, and amphetamines, and compared the analytical results from the four different matrices. The cases were divided in non‐exposure, low, and frequent exposure, based on maternal hair concentrations and segmental analysis by trimesters. For cocaine, 62 cases tested positive in hair, 9 in meconium, 6 in placenta and 7 in umbilical cord. In the case of opioids, 14 maternal hair cases were positive, 11 meconium and umbilical cord and 9 placenta samples. For methadone, 11 cases were positive in hair, 9 in meconium and 6 in placenta and umbilical cord. For amphetamines, 18 cases were positive according to maternal hair, but all meconium, placenta, and umbilical cord tested negative. Maternal hair was the most sensitive specimen to detect drug exposure during pregnancy. Meconium, placenta, and umbilical cord tested positive if hair concentrations showed frequent drug use during the whole pregnancy, especially during the third trimester. Meconium, placenta, and umbilical cord also tested positive for morphine and metabolites, if this drug was administered during labour and delivery. Copyright © 2016 John Wiley & Sons, Ltd.     

Surveillance of drug abuse in Hong Kong by hair analysis using LC‐MS/MS

The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non‐governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6‐acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug‐positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug‐testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management.     

Mass spectrometric characterization of the hypoxia‐inducible factor (HIF) stabilizer drug candidate BAY 85‐3934 (molidustat) and its glucuronidated metabolite BAY‐348, and their implementation into routine doping controls

The development of new therapeutics potentially exhibiting performance‐enhancing properties implicates the risk of their misuse by athletes in amateur and elite sports. Such drugs necessitate preventive anti‐doping research for consideration in sports drug testing programmes. Hypoxia‐inducible factor (HIF) stabilizers represent an emerging class of therapeutics that allows for increasing erythropoiesis in patients. BAY 85‐3934 is a novel HIF stabilizer, which is currently undergoing phase‐2 clinical trials. Consequently, the comprehensive characterization of BAY 85‐3934 and human urinary metabolites as well as the implementation of these analytes into routine doping controls is of great importance. The mass spectrometric behaviour of the HIF stabilizer drug candidate BAY 85‐3934 and a glucuronidated metabolite (BAY‐348) were characterized by electrospray ionization‐(tandem) mass spectrometry (ESI‐MS(/MS)) and multiple‐stage mass spectrometry (MSⁿ). Subsequently, two different laboratories established different analytical approaches (one each) enabling urine sample analyses by employing either direct urine injection or solid‐phase extraction. The methods were cross‐validated for the metabolite BAY‐348 that is expected to represent an appropriate target analyte for human urine analysis. Two test methods allowing for the detection of BAY‐348 in human urine were applied and cross‐validated concerning the validation parameters specificity, linearity, lower limit of detection (LLOD; 1–5 ng/mL), ion suppression/enhancement (up to 78%), intra‐ and inter‐day precision (3–21%), recovery (29–48%), and carryover. By means of ten spiked test urine samples sent blinded to one of the participating laboratories, the fitness‐for‐purpose of both assays was provided as all specimens were correctly identified applying both testing methods. As no post‐administration study samples were available, analyses of authentic urine specimens remain desirable. Copyright © 2016 John Wiley & Sons, Ltd.     

Tyrosine kinase inhibitor (TKI)‐induced cardiotoxicity: approaches to narrow the gaps between preclinical safety evaluation and clinical outcome

Although therapies targeted to inhibit the activity of certain tyrosine kinases (TK) have helped advance cancer therapy in recent years, reports of cardiac toxicity following treatment with tyrosine kinase inhibitors (TKIs) were unexpected and not well predicted by preclinical studies. Such clinical findings exposed gaps in current preclinical drug testing for predicting the development of cardiac toxicities in humans. These gaps included a lack of a comprehensive TKI mechanism of action determination and appropriate cardiac functional evaluation. New preclinical approaches are suggested to address these issues. In addition to tyrosine kinase inhibition, other factors that may play a role in drug‐induced cardiac effects should be assessed, such as unintended secondary targets of TKIs, toxic drug metabolites and drug accumulation in the heart. Both on‐target and off‐target toxic effects of TKIs on cultured cardiac myocytes have now been shown to be detectable, providing a rationale for using cardiomyocytes as a screening tool to study potential TKI‐mediated cardiotoxicity. Incorporating isolated perfused heart methodology to chronic/subchronic rodent studies or including echocardiography in chronic large animal toxicity studies may improve the detection of changes in cardiac function over current methods, and they may eventually become a routine tool for screening drugs with suspected cardiotoxic potential. Further, assessing drug toxicity and efficacy together in an animal model of disease is highly informative for candidate drug selection, and should be encouraged to assess specific safety endpoints, such as cardiovascular function. Together, these approaches will help better close the gaps between preclinical testing and clinical outcomes. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

A simple extraction and LC‐MS/MS approach for the screening and identification of over 100 analytes in eight different matrices

In the present study, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach using one single work‐up approach in whole blood, plasma, serum, post‐mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post‐mortem blood, tissue (homogenized 1 + 4 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether‐ethyl acetate mixture (1:1). Separation and identification were performed using LC‐QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi‐reaction‐mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z‐drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in‐house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work‐up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists. Copyright © 2014 John Wiley & Sons, Ltd.     

Utility of thin layer chromatography for detection of opioids and benzodiazepines in a clinical setting

This study examined the utility of thin layer chromatography (TLC) for detection of recent use of opioids and benzodiazepines among drug addicts seeking treatment at the Drug Dependence Treatment Centre of All India Institute of Medical Sciences, New Delhi, India. Over a period of 5 years (1991-1995), 6,055 urine samples were analyzed for opioids (morphine, codeine, buprenorphine, dextropropoxyphene, pentazocine) and benzodiazepines (diazepam, nitrazepam) by TLC. Out of all the drug tests (n = 9,922) carried out, 24% of the drugs had been used during the past 72 hr. Averaged across all drugs, the detection rates corresponding to 24, 48, and 72 hr by TLC were 37%, 36%, and 31%, respectively. A high percentage of negative TLC results was observed in these samples. Moderate sensitivity of the TLC assay procedure, low consumption of drug, short time between drug use and urine collection, overreporting by the subjects, and drug use history of the subject obtained from multiple sources led to high negative results. These findings suggest that all the TLC negative results also need further confirmation by an alternative, more sensitive technique in a clinical setting. This will make the drug abuse testing program more meaningful.     

Determination of drugs in exhumed liver and brain tissue after over 9 years of burial by liquid chromatography–tandem mass spectrometry—Part 2: Benzodiazepines,opioids, and further drugs

In this publication, benzodiazepines, opioids, and further drugs were analyzed in exhumed brain and liver tissue samples in 116 cases (total) after 9.5–16.5 years of burial. Solid phase extraction followed by liquid chromatography–tandem mass spectrometry was applied. Data from literature is listed summarizing the detectability of the presented analytes after a certain time of burial. In our study, 60% of the analyzed benzodiazepines, 100% of the opioids, and 82% of further drugs were detectable. Only the benzodiazepines lorazepam, nitrazepam, flunitrazepam, and its metabolite norflunitrazepam, and the drugs butylscopolamine, metronidazole, and omeprazole were not detectable at all. Percentage of positive findings (total, and separately for brain and liver tissue) and postmortem period are listed for each analyte. Correlation of detectability depending on postmortem period and condition of tissue are presented exemplarily for midazolam. No substantial correlation was observed. Despite a long time of burial, most benzodiazepines, opioids, and further drugs were detectable in the examined tissue samples. Our results may be a good support for future exhumations in which toxicological analyses are relevant.     

Drug use and sport--a commentary on: Injury, pain and prescription opioid use among former National Football League football players by Cottler et al

The accompanying paper by Cottler et al. reports on findings from a telephone survey study that examined opioid analgesic use and misuse by U.S. professional football players. The study shows high rates of misuse of these medications, and provides an opportunity to consider the intersection between sports and drug use. While in recent years there has been increasing focus upon the use of performance enhancing drugs (e.g., steroids) in athletes, the present report provides valuable information about a relatively unexplored but important topic: opioid analgesic misuse by athletes. The data provided show that misuse of opioids in this population is cause for concern, suggest that study of other groups of athletes should be undertaken, and that further assessment of opioid use in football players is also needed. The study also provides an opportunity to conceptualize drug (and non-drug) use in athletes, as a means to either return athletic functioning to a previous level of performance, or to enhance functioning. Discussions of drug use in sports need to appreciate the complexity of such use, which can be indicated and appropriate under certain circumstances, but which can also be inappropriate and problematic under others-for example, for drugs such as opioid analgesics.     

基于液相色谱-二级质谱联用技术精神科药物中毒筛查平台的构建与临床应用

目的：建立精神科药物的中毒筛查平台,辅助临床精神科药物中毒病患的诊断与急救。方法：依托液相色谱串联质谱（LC-MS/MS）仪,建立目前常用精神科药物的检测方法;采集疑似精神科药物过量或中毒病患的胃液、尿液或血液,处理后采用LC-MS/MS进行快速筛查和定量,获得药物浓度,及时进行药物剂量调整及中毒抢救,并进行复查。结果：本中毒筛查平台共收到院内外急诊病例1 183例。药物过量或中毒病例中前四位氯氮平占34.09%,镇静催眠药占29.54%,喹硫平占15.91%,氨磺必利占11.36%。儿童药物中毒半数由药物误服引起,误服的药物排名前三位为：五氟利多、氟哌啶醇和氯氮平。以上患者经剂量调整或抢救结束后再次复查,血中药物降至安全范围或完全消除。结论：本平台的建立可以快速获取患者中毒药物信息,提高临床医生确诊率,减少患者在中毒剂量的暴露时间,缩短转归时间。     

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography–high‐resolution tandem mass spectrometry (LC–HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC–HRMS/MS system consisted of a YMC‐UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β‐glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono‐hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2–19‐fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.