Drug‐drug interaction and doping,part 1: An in vitro study on the effect of non‐prohibited drugs on the phase I metabolic profile of toremifene

The present study was designed to provide preliminary information on the potential impact of metabolic drug‐drug interaction on the effectiveness of doping control strategies currently followed by the anti‐doping laboratories to detect the intake of banned agents. In vitro assays based on the use of human liver microsomes and recombinant CYP isoforms were designed and performed to characterize the phase I metabolic profile of the prohibited agent toremifene, selected as a prototype drug of the class of selective oestrogen receptor modulators, both in the absence and in the presence of medicaments (fluconazole, ketoconazole, itraconazole, miconazole, cimetidine, ranitidine, fluoxetine, paroxetine, nefazodone) not included in the World Anti‐Doping Agency list of prohibited substances and methods and frequently administered to athletes. The results show that the in vitro model developed in this study was adequate to simulate the in vivo metabolism of toremifene, confirming the results obtained in previous studies. Furthermore, our data also show that ketoconazole, itraconazole, miconazole and nefazodone cause a marked modification in the production of the metabolic products (i.e. hydroxylated and carboxylated metabolites) normally selected by the anti‐doping laboratories as target analytes to detect toremifene intake; moderate variations were registered in the presence of fluconazole, paroxetine and fluoxetine; while no significant modifications were measured in the presence of ranitidine and cimetidine. This evidence imposes that the potential effect of drug‐drug interactions is duly taken into account in anti‐doping analysis, also for a broader significance of the analytical results. Copyright © 2014 John Wiley & Sons, Ltd.     

Drug‐drug interaction and doping,part 2: An in vitro study on the effect of non‐prohibited drugs on the phase I metabolic profile of stanozolol

The present study was designed to provide preliminary information on the potential impact of metabolic drug‐drug interaction on the effectiveness of doping control strategies currently followed by the anti‐doping laboratories to detect the intake of prohibited agents. In vitro assays based on the use of human liver microsomes and recombinant cytochrome P450 isoforms were developed and applied to characterize the phase I metabolic profile of the prohibited agent stanozolol, both in the absence and in the presence of substances (ketoconazole, itraconazole, miconazole, cimetidine, ranitidine, and nefazodone) not included in the World Anti‐Doping Agency (WADA) list of prohibited substances and methods and frequently administered to athletes. The results show that the in vitro model utilized in this study is adequate to simulate the in vivo metabolism of stanozolol. Furthermore, our data showed that ketoconazole, itraconazole, miconazole, and nefazodone caused a marked modification in the production of the metabolic products (3’‐hydroxy‐stanozolol, 4β‐hydroxy‐stanozolol and 16β‐hydroxy‐stanozolol) normally selected by the anti‐doping laboratories as target analytes to detect stanozolol intake. On the contrary, moderate variations were registered in the presence of cimetidine and no significant modifications were measured in the presence of ranitidine. This evidence confirms that the potential effect of drug‐drug interactions is duly taken into account also in anti‐doping analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro evaluation of the effects of anti‐fungals,benzodiazepines and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone: implications on doping control analysis

We have studied whether the phase II metabolism of 19‐norandrosterone, the most representative metabolite of 19‐nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti‐Doping Agency. In detail, we have evaluated the effect of non‐prohibited drugs belonging to the classes of anti‐fungals, benzodiazepines, and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl‐transferase were designed and performed to monitor the formation of 19‐norandrosterone glucuronide from 19‐norandrosterone. Determination of 19‐norandrosterone (free and conjugated fraction) was performed by gas chromatography – mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert‐butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed‐phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non‐prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19‐norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti‐fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non‐steroidal anti‐inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of non‐steroidal anti‐inflammatory drugs on non‐melanoma skin cancer incidence in the SKICAP‐AK trial

Recent studies link the prostaglandin metabolic pathway to skin carcinogenesis expanding possibilities that cyclooxygenase (COX) inhibitors may be utilized in non‐melanoma skin cancer (NMSC) chemoprevention. Using data from a study of the efficacy of retinol supplementation on incidence of NMSC, we sought to determine the role of non‐steroidal anti‐inflammatory drugs (NSAIDs) in NMSC development. Cox proportional hazards models describe the relationship between NSAID use and time to first squamous cell carcinoma (SCC) or basal cell carcinoma (BCC) among participants categorized by use pattern: continuous users (use for length of study duration), new users (use for less than study duration), and non‐users. For SCC and BCC, there was a statistically significant protective effect for participants who reported use for less than the study duration (HR = 0.49, 95%CI 0.28–0.87 and HR = 0.43, 95%CI 0.25–0.73, respectively). Categorical examination of NSAIDs (aspirin (ASA) vs. non‐ASA NSAIDs) showed significant effects for BCC among those using non‐ASA NSAIDs for less than the study duration (HR = 0.33, 95%CI 0.13–0.80). For SCC and BCC, NSAID use of shorter duration and potentially more recent, was more protective than longer duration of use. These results are counter to the idea that longer duration of NSAID use is more protective. Additional investigations are needed into the role NSAIDs play in the chemoprevention of NMSC. Copyright © 2009 John Wiley & Sons, Ltd.     

Anti‐doping analytes in serum: A comparison of SST and SST‐II Advance blood collection tubes

Serum insulin‐like growth factor‐1 (IGF‐1), procollagen type III N‐terminal peptide (PIIINP), and human growth hormone (hGH) isoforms were analyzed in identical serum samples collected into BD Vacutainer^® SST and BD Vacutainer^® SST‐II Advance serum separator tubes. Comparing the serum collected into each tube, measurement correlation was high (R² > 0.83) and percent bias was minimal (<|3.2%|) for all analytes measured using World Anti‐Doping Agency (WADA)‐approved tests. As such, it is recommended that both SST and SST‐II Advance tubes can be used interchangeably for anti‐doping purposes.     

Studies on the minor metabolite 6a‐hydroxy‐androstenedione for doping control purposes and its contribution to the steroid profile

Recent publications have shown that the concentrations of minor metabolites such as formestane and 6a‐hydroxy‐androstenedione (6aOHADION) are import parameters, capable of increasing the specificity and efficiency of steroid abuse screening. The importance of such minor metabolites has been recognized for some time, but setting up concentration thresholds is not that straightforward with a single quadrupole gas chromatograph mass spectrometer (GC‐MS) because of the low concentrations; this is especially the case for 6aOH‐ADION. The main aim of this study was to propose a concentration threshold above which the detected 6aOH‐ADION is considered suspicious and isotope ratio mass spectrometry (IRMS) is recommended. Routine doping control samples (2128) from athletes that entered our lab and were not found suspicious for the intake of any doping substance were used to determine the baseline concentrations of 6a‐OH‐ADION. For this purpose, the more sensitive gas chromatography‐tandem mass spectrometry (GC‐MS/MS) was used, capable of quantifying these low concentrations with high reliability. A urinary concentration threshold of 5 ng/mL was set. Concentrations above this threshold are considered suspicious and are forwarded to IRMS for confirmation in routine practice. In addition, an IRMS method was developed, capable of determining the 13C value of 6aOH‐ADION. If a urine sample has an elevated 6aOH‐ADION concentration and normal 13C values for the traditional IRMS target compounds, we are still able to check the 13C value of 6aOH‐ADION. Six excretion studies were executed to stress the applicability of the threshold by visualizing the concentration and δ13C value time profiles of 6aOH‐ADION. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification and validation of nine nonsteroidal anti‐inflammatory drugs in equine urine using gas chromatography–mass spectrometry for doping control

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine in accordance with the International Screening Limits (ISL) regulated by International Federation of Horseracing Authorities. After basic hydrolysis, samples were extracted with a C18 cartridge using automated solid‐phase extraction. Several derivatization reagents were investigated, and trimethylphenylammonium hydroxide/methanol (20/80, v/v) was selected. Analyses were carried out using gas chromatography–mass spectrometry with selected ion monitoring mode, but the method can be applied to a large number of analytes. The within‐laboratory reproducibility was not more than 12.8% (≤15%), and mean relative recoveries ranged from 91.1% to 104.1% for inter‐day and intra‐day precision. The decision limits (CCα) and detection capabilities (CCβ) were evaluated at concentrations near the ISL for each therapeutic substance. The validation results demonstrated that the method is highly reproducible, easily applicable, and suitable for the analysis of some NSAIDs in equine urine that have not been previously published. Finally, the method was also applied to known positive samples.     

Liposomes as potential masking agents in sport doping. Part 1: analysis of phospholipids and sphingomyelins in drugs and biological fluids by aqueous normal‐phase liquid chromatography‐tandem mass spectrometry

In the present work, aqueous normal‐phase liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS), in different acquisition modes, was employed for the direct analysis and profiling of nine phospholipid classes (phosphatidic acids, phosphatidylserines, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins) in biological and pharmaceutical matrices. After chromatographic separation by a diol column, detection and elucidation of phospholipid and sphingomyelin classes and molecular species were performed by different scan acquisition modes. For screening analysis, molecular ions [M + H]⁺ were detected in positive precursor ion scan of m /z 184 for the classes of phosphatidylcholines, lyso‐phosphatidylcholines and sphingomyelins; while phosphatidylethanolamines and lyso‐phosphatidylethanolamines were detected monitoring neutral loss scan of 141 Da; and phosphatidylserines detected using neutral loss scan of 184 Da. Molecular ions [M‐H]^‐ were instead acquired in negative precursor ion scan of m /z 153 for the classes of phosphatidic acids and phosphatidylglycerols; and of m /z 241 for the phosphatidylinositols. For the identification of the single molecular species, product ion scan mass spectra of the [M + HCOO]^‐ ions for phosphatidylcholines and [M + H]⁺ ions for the other phospholipids considered were determined for each class and compared with the fragmentation pattern of model phospholipid reference standard. By this approach, nearly 100 phospholipids and sphingomyelins were detected and identified. The optimized method was then used to characterize the phospholipid and sphingomyelin profiles in human plasma and urine samples and in two phospholipid‐based pharmaceutical formulations, proving that it also allows to discriminate compounds of endogenous origin from those resulting from the intake of pharmaceutical products containing phospholipidic liposomes. Copyright © 2016 John Wiley & Sons, Ltd.     

A rapid screening LC‐MS/MS method based on conventional HPLC pumps for the analysis of low molecular weight xenobiotics: application to doping control analysis

This study presents a fast multi‐analyte screening method specifically developed for the detection of xenobiotics in urine. The proposed method allows the screening of several classes of substance in a single chromatographic method with a run‐time of 11 min, inclusive of post‐run and reconditioning times. Chromatographic separation is achieved in 7.2 min using a reversed‐phase 2.7 µm fused‐core particle column, generating a back‐pressure not exceeding 400 bar and therefore enabling the use of traditional high performance liquid chromatography (HPLC) instruments. The effectiveness of this approach was evaluated, by liquid‐chromatography tandem mass spectrometry (LC‐MS/MS) in positive electrospray ionization, using 20 blank urine samples spiked with 45 compounds prohibited in sport: 11 diuretics, 16 glucocorticoids, 9 stimulants, 5 anti‐oestrogens, as well as formoterol, carboxy‐finasteride (previously prohibited by the World Anti‐Doping Agency (WADA) in 2008), gestrinone and tetrahydrogestrinone. Qualitative validation shows the proposed method to be specific with no significant interference. All of the analytes considered in this study were clearly distinguishable in urine, with limits of detection ranging from 5 ng/mL to 350 ng/mL, significantly below the Minimum Required Performance Levels (MRPL) set by WADA for the accredited sports anti‐doping laboratories. All compounds of interest were separated, including synthetic and endogenous glucocorticoids with similar retention times and fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.     

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

Sulfate metabolites have been described as long‐term metabolites for some anabolic androgenic steroids (AAS). 4‐chlorometandienone (4Cl‐MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl‐MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl‐MTD metabolism by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to establish potential long‐term metabolites valuable for doping control purposes. 4Cl‐MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and gas chromatography‐tandem mass spectrometry (GC‐MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.     

DNA typing for personal identification of urine after long‐term preservation for testing in doping control

When the tampering of a urine sample is suspected in doping control, personal identification of the sample needs to be determined by short tandem repeat (STR) analysis using DNA. We established a method for extracting DNA from urine samples stored at −20 °C without using any additives or procedures, which is consistent with how samples are required to be managed for doping control. The method, using the Puregene® Blood Core kit followed by NucleoSpin® gDNA Clean‐up or NucleoSpin® gDNA Clean‐up XS kit, does not need any special instrument and can provide a purified extract with high‐quality DNA from up to 40 mL of urine suitable for STR analysis using an AmpFl STR® Identifiler® PCR amplification kit. Storing urine at −20 °C is detrimental to the stability of DNA. The DNA concentration of preserved urine could not be predicted by specific gravity or creatinine level at the time of urine collection. The DNA concentration of a purified extract (10 μL) was required to be >0.06 ng/μL to ensure a successful STR analysis. Thus, the required extraction volumes of urine preserved for 3–7 years at −20 °C were estimated to be 30 mL and 20 mL to succeed in at least 86% of men and 91% of women, respectively. Considering the long half‐life of DNA during long‐term preservation, our extraction method is applicable to urine samples stored even for 10 years, which is currently the storage duration allowed (increased from 8 years) before re‐examination in doping control. Copyright © 2016 John Wiley & Sons, Ltd.     

Issues for sustainability of non‐government organisations in the alcohol and other drugs sector

Introduction and Aims. The study aimed to identify issues for sustainability of non‐government organisations in the alcohol and other drugs sector (AODS) in Australia within the current neoliberal context. Design and Methods. Six key‐informant group consultations were conducted in Sydney, Melbourne, Darwin (Australia). Participants were professionals working in the AODS from non‐government AOD organisations, government departments, philanthropic organisations and academic institutions (n = 40). Qualitative data were collected according to a discussion guide. Results. Issues related to problems with workforce capacity, independence, governance and funding. Factors contributing to these issues related to the competitive tendering funding model, for example: accountability requirements and tied funding. Discussion and Conclusions. Issues identified by the AODS were mostly similar to those identified by the broader non‐government organisations sector. There is much to learn from outside the AODS. Strategies to improve sustainability will include workforce development, improving governance and developing relationships within the sector and with government. These require a commitment to innovation and will entail movement of resources from service provision in the short term to organisational development for the long term.[Spooner C, Dadich A. Issues for sustainability of non‐government organisations in the alcohol and other drugs sector. Drug Alcohol Rev 2009]     

A rapid non‐target screening method for determining prohibited substances in human urine using liquid chromatography/high‐resolution tandem mass spectrometry

Target analysis using liquid chromatography–tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non‐target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid‐phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high‐resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%–131%), matrix effects (35%–237%), limites of detection (0.0002–100 ng/mL), and intra‐ and inter‐day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non‐target screening method is efficient for conducting anti‐doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.     

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.     

Direct injection horse‐urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3‐methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Levodopa and dopamine have been abused as performance‐altering substances in horse racing. Urinary 3‐methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 µg mL^?1 (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3‐methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3‐methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3‐methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion, while a second run at high collision energy was performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1–20 µg mL^?1. The relative standard deviations of intra‐ and inter‐ assay analysis of 3‐methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd.