Methylation of phase II metabolites of endogenous anabolic androgenic steroids to improve analytical performance.

The study of intact phase II metabolites of endogenous anabolic androgenic steroids (EAAS) gives important information about metabolism and has the potential to improve the detection of doping with testosterone. For analysis with liquid chromatography-mass spectrometry (LC-MS), chemical derivatization at the steroid moiety is a technique to improve the positive ionization efficiency of glucuronidated/sulfated EAAS under collision-induced dissociation (CID) conditions. However, regarding the chromatographic performance, there are still challenges to address, for example, poor peak shape, which is mainly caused by nondefined adsorption in the chromatographic system. Here, we show a novel derivatization technique for the analysis of selected phase II metabolites of EAAS, where the acidic moiety of the glucuronide/sulfate is methylated with different methylation reagents to reduce nondefined adsorption. The methylation reagent trimethylsilyl-diazomethane (TMSD) was preferred over the other tested reagents methyl iodide (MeI) and dimethyl sulfate (DMS). Glucuronidated and sulfated testosterone and epitestosterone were methylated, and their chromatographic performance and CID ion mass spectra obtained in positive ionization mode were investigated. The peak width and peak height were significantly improved for all substances. Methylated testosterone sulfate showed the best results with a 3.5 times narrower peak and 14 times increased intensity compared with underivatized testosterone sulfate. Furthermore, CID ion mass spectra obtained in positive ionization mode showed product ions characteristically for the steroidal backbone for all substances. This preliminary study shows the potential of methylation as a supplementary derivatization technique, which can assist in the development of more sensitive methods due to the improvements in method performance.

Use of Selective Serotonin Reuptake Inhibitors - Validity of Self-Report versus Plasma Concentrations and Pharmacy Dispensations - A Cross-Sectional Analysis of the Norwegian Women and Cancer Study.

Purpose: To validate self-reported current use of selective serotonin reuptake inhibitors (SSRI) in the Norwegian Women and Cancer study (NOWAC) and to identify factors associated with discordance between data sources. Material and Methods: This is a cross-sectional record-linkage study comparing SSRI-use derived from four data sources: 1) Specific SSRI questions in the main NOWAC questionnaire, 2) Open questions on medication use in the small questionnaire following blood samples, 3) plasma concentration measurements for a subsample, and 4) pharmacy dispensations from the Norwegian prescription database (NorPD) where current use of SSRI was defined by Legend Time Duration (LTD). Among 105 855 women, aged 46 to 64 years and randomly selected from the general population, 70,191 had data on SSRI-use from both NOWAC and NorPD. Plasma concentration was measured for 93 pairs of self-reported SSRI-users and non-users, with dispensation data available for 68 pairs. Validity was assessed by sensitivity and specificity; agreement was assessed by Cohen's kappa. Factors associated with discordance between information sources were analyzed by multiple binary logistic regression. Results: We found high sensitivity (89.5%) and specificity (98.7%) for the specific questions in the main questionnaire compared with pharmacy dispensations. Measured against plasma concentrations, current SSRI-use defined by open questions and pharmacy dispensations both had high sensitivity (100% and 92.5%, respectively) and specificity (98.6% both). Agreements (kappa) were similarly high for all comparisons (≥0.80). The factors associated with discordance between data sources included poor health, comorbidity, being single and not being in full time work. Education was inversely associated with discordance. Conclusion: Self-reported current use of SSRI from the NOWAC questionnaires is highly valid and, according to plasma concentrations, perhaps even more so than pharmacy dispensations. Factors associated with discordance between information sources should be taken into account in the interpretation of future analyses which include SSRI-use in the NOWAC study.

Detection of DHCMT long-term metabolite glucuronides with LC-MSMS as an alternative approach to conventional GC-MSMS analysis.

Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic-androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17ß-hydroxymethyl-17α-methyl-5ß-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1'N-glucuronide and 17-epistanozolol-1'N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given.

Development and validation of a simple online-SPE method coupled to high-resolution mass spectrometry for the analysis of stanozolol-N-glucuronides in urine samples.

Stanozolol is still the most commonly used illicit anabolic-androgenic steroid (AAS) in professional sports. Therefore, accurate and fast analysis and long detection windows are of great interest in the field of antidoping analysis. In this work, a very simple, fast, and highly sensitive online solid-phase extraction method coupled with liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMSMS) for the analysis of stanozolol-N-glucuronides was developed. This fully validated procedure is characterized by only a few manual steps (dilution and addition of internal standard) in the sample preparation. A limit of identification (LOI) of 75 pg/mL, high accuracy (87.1%-102.1%), precision (3.1%-7.8%), and sensitivity was achieved. Furthermore, good linearity (> 0.99) and robustness, as well as no carry-over effects, could be observed. In addition to excellent confirmation analysis performance, this method shows sufficient potential for the identification and characterization of unknown metabolites. Using this method, it was possible to unambiguously confirm the presence of 1'N- and 2'N-stanozolol-glucuronide in human urine for the first time due to the access to reference material.

Quantification of endogenous steroid sulfates and glucuronides in human urine after intramuscular administration of testosterone esters.

For an effective detection of doping with pseudo-endogenous anabolic steroids, the urinary steroid profile is of high value. In this work, the aim was to investigate steroid metabolism disruption after exogenous intramuscular administration of different testosterone esters. The investigation focused on both sulfo - and glucoro conjugated androgens. A single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido®) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanone®), was given to six healthy volunteers. Urine was collected throughout a testing period of 60 days. A LC-MS method was developed and validated for the analysis of eight conjugated steroids in their intact form. The results show that urinary changes in both sulfo - and glucuro conjugated steroid levels are prominent after the injection of testosterone esters. A promising potential marker for the intake of exogenous testosterone is the combined ratio of epitestosterone sulfate/epitestosterone glucuronide to testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) as a complementary biomarker for testosterone abuse. This represents a new piece of evidence to detect testosterone doping, representing a new approach and being independent from the metabolic connections of the markers in the steroid passport.

Mucosal Metabolomic Profiling and Pathway Analysis Reveal the Metabolic Signature of Ulcerative Colitis.

The onset of ulcerative colitis (UC) is characterized by a dysregulated mucosal immune response triggered by several genetic and environmental factors in the context of host-microbe interaction. This complexity makes UC ideal for metabolomic studies to unravel the disease pathobiology and to improve the patient stratification strategies. This study aims to explore the mucosal metabolomic profile in UC patients, and to define the UC metabolic signature. Treatment- naïve UC patients (n = 18), UC patients in deep remission (n = 10), and healthy volunteers (n = 14) were recruited. Mucosa biopsies were collected during colonoscopies. Metabolomic analysis was performed by combined gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). In total, 177 metabolites from 50 metabolic pathways were identified. The most prominent metabolome changes among the study groups were in lysophosphatidylcholine, acyl carnitine, and amino acid profiles. Several pathways were found perturbed according to the integrated pathway analysis. These pathways ranged from amino acid metabolism (such as tryptophan metabolism) to fatty acid metabolism, namely linoleic and butyrate. These metabolic changes during UC reflect the homeostatic disturbance in the gut, and highlight the importance of system biology approaches to identify key drivers of pathogenesis which prerequisite personalized medicine.

Lipidomics in Ulcerative Colitis Reveal Alteration in Mucosal Lipid Composition Associated With the Disease State.

BACKGROUND: The onset of ulcerative colitis (UC) is associated with alterations in lipid metabolism and a disruption of the balance between pro- and anti-inflammatory molecules. Only a few studies describe the mucosal lipid biosignatures during active UC. Moreover, the dynamics of lipid metabolism in the remission state is poorly defined. Therefore, this study aims to characterize mucosal lipid profiles in treatment-naïve UC patients and deep remission UC patients compared with healthy subjects. METHODS: Treatment-naïve UC patients (n = 21), UC patients in deep remission (n = 12), and healthy volunteers (n = 14) were recruited. The state of deep remission was defined by histological and immunological remission defined by a normalized TNF-α gene expression. Mucosa biopsies were collected by colonoscopy. Lipid analysis was performed by means of ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS-MS). In total, 220 lipids from 11 lipid classes were identified. RESULTS: The relative concentration of 122 and 36 lipids was altered in UC treatment-naïve patients and UC remission patients, respectively, compared with healthy controls. The highest number of significant variations was in the phosphatidylcholine (PC), ceramide (Cer), and sphingomyelin (SM) composition. Multivariate analysis revealed discrimination among the study groups based on the lipid profile. Furthermore, changes in phosphatidylethanolamine(38:3), Cer(d18:1/24:0), and Cer(d18:1/24:2) were most distinctive between the groups. CONCLUSION: This study revealed a discriminant mucosal lipid composition pattern between treatment-naïve UC patients, deep remission UC patients, and healthy controls. We report several distinctive lipids, which might be involved in the inflammatory response in UC, and could reflect the disease state.

A Quantitative Analysis of Colonic Mucosal Oxylipins and Endocannabinoids in Treatment-Naïve and Deep Remission Ulcerative Colitis Patients and the Potential Link With Cytokine Gene Expression.

BACKGROUND: The bioactive metabolites of omega 3 and omega 6 polyunsaturated fatty acids (ω-3 and ω-6) are known as oxylipins and endocannabinoids (eCBs). These lipid metabolites are involved in prompting and resolving the inflammatory response that leads to the onset of inflammatory bowel disease (IBD). This study aims to quantify these bioactive lipids in the colonic mucosa and to evaluate the potential link to cytokine gene expression during inflammatory events in ulcerative colitis (UC). METHODS: Colon biopsies were taken from 15 treatment-naïve UC patients, 5 deep remission UC patients, and 10 healthy controls. Thirty-five oxylipins and 11 eCBs were quantified by means of ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. Levels of mRNA for 10 cytokines were measured by reverse transcription polymerase chain reaction. RESULTS: Levels of ω-6-related oxylipins were significantly elevated in treatment-naïve patients with respect to controls, whereas the levels of ω-3 eCBs were lower. 15S-Hydroxy-eicosatrienoic acid (15S-HETrE) was significantly upregulated in UC deep remission patients compared with controls. All investigated cytokines had significantly higher mRNA levels in the inflamed mucosa of treatment-naïve UC patients. Cytokine gene expression was positively correlated with several ω-6 arachidonic acid-related oxylipins, whereas negative correlation was found with lipoxin, prostacyclin, and the eCBs. CONCLUSIONS: Increased levels of ω-6-related oxylipins and decreased levels of ω-3-related eCBs are associated with the debut of UC. This highlights the altered balance between pro- and anti-inflammatory lipid mediators in IBD and suggests potential targets for intervention.

Analytical Quality by Design-based development and validation of ultra pressure liquid chromatography/MS/MS method for glycopeptide antibiotics determination in human plasma.

AIM: An ultra pressure liquid chromatography (UPLC)/MS/MS method for vancomycin and teicoplanin determination in human plasma was developed in accordance with analytical quality by design (AQbD) concept and fully validated. MATERIALS & METHODS: Chromatographic separation was performed on ACQUITY UPLC C18 charge surface hybrid (CSH) column (2.1 mm × 50 mm, 1.7 µm particle size) in gradient mode and the mobile phase consisted of 0.1% formic acid in water and pure acetonitrile. The experimental design methodology was used for the definition of optimal chromatographic and protein precipitation conditions. RESULTS: The linearity ranges were 0.05-10 µg ml-1 for vancomycin and 0.5-200 µg ml-1 for total teicoplanin. The relative standard deviations for precision estimation were below 15% and the accuracy was within 85-115% for all quality control levels. CONCLUSION: The method was utilized for glycopeptide antibiotics bioanalysis.

Urinary excretion studies of meldonium after multidose parenteral application.

Meldonium is a drug exhibiting cardioprotective and anti-ischemic effects. Due to its potential performance-enhancing benefit in sports, meldonium was added to the World Anti-Doping Agency list of prohibited substances in 2016. Since then, a high number of adverse analytical findings reported on meldonium has questioned meldonium`s detection time in urine. Hence, the objective of the current study was to characterize the pharmacokinetic urinary excretion pattern of meldonium when administered as multiple intravenous injections. Three injections of 250 mg meldonium were given over a time period of five days to six healthy volunteers and urine samples were collected for eight months after the last injection of the drug. For the quantification of meldonium in urine, a liquid chromatography-tandem mass spectrometry method was fully validated according to the World Anti-Doping Agency guidelines in terms of specificity, matrix interferences, intra- and inter-day precision, accuracy, carry-over, robustness, linearity, limit of detection, and limit of quantification. The assay was successfully applied to the pharmacokinetic study. A three-compartment model was found to best describe the pharmacokinetics of meldonium with average alpha, beta, and gamma half-lives of 1.4 h, 9.4 h, and 655 h, respectively. The detection time in urine varied between 94 and 162 days.

Unambiguous identification and characterization of a long-term human metabolite of dehydrochloromethyltestosterone.

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long-term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long-term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α-methyl-17ß-hydroxymethyl - structures were synthesized and compared to a major DHCMT long-term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α-chloro-18-nor-17ß-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol.

Detection of testosterone esters in blood.

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma. Furthermore, the suitability of different types of blood collection devices was evaluated. Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested. A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®)) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon(®)). Blood was collected throughout a testing period of 60 days. The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All investigated testosterone esters could be detected in post-administration blood samples. The detection time depended on the type of ester administered. Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied. The testosterone ester detection window, however, was comparable.

Use of dried blood spots in doping control analysis of anabolic steroid esters.

Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness, robustness against manipulation and fastness DBS sampling recommends itself as an advantageous technique in doping control analysis. The present approach highlights the development of a screening assay for the analysis of eight anabolic steroid esters (nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters allows an unequivocal proof of the administration of conjugates of exogenous testosterone and its derivatives. Precise, specific and linear conditions were obtained by means of liquid chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb range was accomplished by the preparation of the methyloxime derivatives of the target compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-nandrolone undecanoate) were applied to compensate for the broad range in chain length of the esters. The assay presented here outlines the application of DBS for the analysis of anabolic steroid esters in doping controls for the first time providing great potential to simplify the proof of exogenous administration of testosterone.

Carbon isotope ratio determination and investigation of seized testosterone preparations.

In the present study, the content of a number of black market testosterone products collected in Austria has been analyzed. Additionally, (13) C/(12) C ratios were measured for testosterone in the products after cleavage of the testosterone ester. The aim was to determine whether some of these products had similar (13) C/(12) C ratios to those normally found for endogenous testosterone, which could prevent a positive isotopic ratio mass spectrometric (IRMS) finding in doping control. Moreover, it was investigated to what extent the preparations contained the masking agent epitestosterone, in order to lower the testosterone/epitestosterone (T/E) ratio in urinary steroid profiles. Out of 30 analyzed products, the declared ingredients differed from the actual content in 10 cases. Epitestosterone, however, could not be found in any of the products. The products displayed δ(13)C(VPDB) values between -23.6 and -29.4‰. For more than half of these products, the values were within a range reported for endogenous urinary steroids.

Synthesis and analytics of 2,2,3,4,4-d5-19-nor-5alpha-androsterone--an internal standard in doping analysis.

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.

Investigation of the silylation of ephedrines using N-methyl-N-trimethylsilyl-trifluoroacetamide.

This paper describes a gas chromatography (GC)--mass spectrometry (MS) method for the simultaneous quantification of ephedrine, pseudoephedrine, cathine, norephedrine and methylephedrine in urine. The sample preparation step includes solid phase extraction and derivatisation with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). An evaluation of various silylation conditions compatible with screening methods in doping control analysis is presented. The method was found to be well suited for quantification of ephedrines in doping control.

Quantification and stability of salbutamol in human urine.

A sensitive method for the quantification of free salbutamol in human urine is described. Sample clean up is performed using SPE on a mixed phase extraction column. Derivatisation is performed with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and the extract is analysed by GC-MS. The method was found to be suitable for use in the doping field, where a cut-off limit of 1 microg salbutamol/mL urine is set by the International Olympic Committee (IOC) and approved by the World Anti-Doping Agency (WADA). Above that value a doping violation occurs. In addition, the stability of salbutamol in human urine has been evaluated.