Investigation of urinary excretion of hydroxyethyl starch and dextran by uhplc-hrms in different acquisition modes.

Plasma volume expanders (PVEs) such as hydroxyethyl starch (HES) and dextran are misused in sports because they can prevent dehydration and reduce haematocrit values to mask erythropoietin abuse. Endogenous hydrolysis generates multiple HES and dextran oligosaccharides which are excreted in urine. Composition of the urinary metabolic profiles of PVEs varies depending on post-administration time and can have an impact on their detectability. In this work, different mass spectrometry data acquisition modes (full scan with and without in-source collision-induced dissociation) were used to study urinary excretion profiles of HES and dextran, particularly by investigating time-dependent detectability of HES and dextran urinary oligosaccharide metabolites in post-administration samples. In-source fragmentation yielded the best results in terms of limit of detection (LOD) and detection times, whereas detection of HES and dextran metabolites in full scan mode with no in-source fragmentation is related to recent administration (< 24 hours). Urinary excretion studies showed detection windows for HES and dextran respectively of 72 and 48 hours after administration. Dextran concentrations were above the previously proposed threshold of 500 µg · mL(-1) for 12 hours. A "dilute-and-shoot" method for the detection of HES and dextran in human urine by ultra-high-pressure liquid chromatography-electrospray ionization-high resolution Orbitrap™ mass spectrometry was developed for this study. Validation of the method showed an LOD in the range of 10-500 µg · mL(-1) for the most significant HES and dextran metabolites in the different modes. The method allows retrospective data analysis and can be implemented in existing high-resolution mass spectrometry-based doping control screening analysis.

Prevalence of legal and illegal stimulating agents in sports.

This paper reviews the prevalence of legal and illegal stimulants in relation to doping-control analysis. Stimulants are among the oldest classes of doping agents, having been used since ancient times. Despite the ease with which they can be detected and the availability of sensitive detection methods, stimulants are still popular among athletes. Indeed, they remain one of the top three most popular classes of prohibited substances. Because the list of legal and illegal stimulants is extensive only a selection is discussed in detail. The compounds selected are caffeine, ephedrines, amphetamine and related compounds, methylphenidate, cocaine, strychnine, modafinil, adrafinil, 4-methyl-2-hexaneamine, and sibutramine. These compounds are mainly prevalent in sport or are of therapeutic importance. Because stimulants are the oldest doping class the first detection methods were for this group. Several early detection techniques including GC-NPD, GC-ECD, and TLC are highlighted. The more novel detection techniques GC-MS and LC-MS are also discussed in detail. In particular, the last technique has been shown to enable successful detection of stimulants difficult to detect by GC-MS or for stimulants previously undetectable. Because stimulants are also regularly detected in nutritional (food) supplements a section on this topic is also included.

Direct quantification of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis.

An accurate and precise method for the quantification of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for doping analysis purposes has been developed. The method involves the use of only 200 microL of urine and the use of D(9)-THCA as internal standard. No extraction procedure is used. The urine samples are hydrolysed using sodium hydroxide and diluted with a mixture of methanol/glacial acetic acid (1:1). Chromatographic separation is achieved using a C8 column with gradient elution. All MS and MS/MS parameters were optimised in both positive and negative electrospray ionisation modes. For the identification and the quantification of THCA three product ions are monitored in both ionisation modes. The method is linear over the studied range (5-40 ng/mL), with satisfactory intra-and inter-assay precision, and the relative standard deviations (RSDs) are lower than 15%. Good accuracy is achieved with bias less than 10% at all levels tested. No significant matrix effects are observed. The selectivity and specificity are satisfactory, and no interferences are detected. The LC/MS/MS method was applied for the analysis of 48 real urine samples previously analysed with a routine gas chromatography/mass spectrometry (GC/MS) method. A good correlation between the two methods was obtained (r(2) > 0.98) with a slope close to 1.

Development and validation of an LC-MS/MS method for the quantification of ephedrines in urine.

The objective of this study was to develop a simple and robust LC-MS/MS method for the quantification of ephedrine type substances in urine. Sample preparation consisted of a 10-fold dilution step of the samples into the internal standard solution (ephedrine-d(3), 4 microg/mL in water). Baseline separation of the diastereoisomers norpseudoephedrine-norephedrine and ephedrine-pseudoephedrine was performed on a C8-column using isocratic conditions followed by positive electrospray ionisation and tandem mass spectrometric detection. The mobile phase consisted of 98/2 (H(2)O/ACN) containing 0.1% HAc and 0.01% TFA. Calibration curves were constructed between 2.5 and 10 microg/mL for norephedrine and norpseudoephedrine and 5 and 20 microg/mL for ephedrine, pseudoephedrine and methylephedrine. The bias ranged from -5.5 to 12% for norephedrine, -4.1 to 8.0 % for norpseudoephedrine, 0.3 to 2.1 % for ephedrine, 1.6 to 2.6 % for pseudoephedrine and 2.9 to 5.0 % for methylephedrine. Precision of the method varied between 2.8 and 10.4% for all compounds and the matrix effect was less than 15%. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC-NPD method. Results show a good correlation between both methods with correlation coefficients higher than 0.95 for all analytes.

Stability of selected chlorinated thiazide diuretics.

In sports, diuretics are used for two main reasons: to flush previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. A common property observed for thiazides is hydrolysis in aqueous media resulting in the formation of the degradation product aminobenzenedisulphonamide. This degradation product can be observed for several thiazides. Because there is limited information regarding the effect of pH, temperature and light on the stability of thiazides, these parameters were investigated for chlorothiaizide, hydrochlorothiazide and altizide. For all three compounds the degradation product could be detected after incubation at pH 9.5 for 48h at 60 degrees C. At lower pH and temperature the degradation product could not be detected for all compounds. When samples were exposed to UV-light altizide and hydrochlorothiazide were photodegraded to chlorothiazide. When the degradation rate between the different compounds was compared for a given temperature and pH, altizide is the most unstable compound. This study confirms that thiazide degradation products can be formed in urine during transport. Hence doping control laboratories shall include them into their routine testing methods as required by WADA.

Implementation of gas chromatography combined with simultaneously selected ion monitoring and full scan mass spectrometry in doping analysis.

A comprehensive screening method for the detection of prohibited substances in doping control is described and validated. This method is capable of detecting over 150 components mentioned on the list of the World Anti-Doping Agency including anabolic androgenic steroids, stimulants and all narcotic agents that are currently analysed using different analytical methods. The analytes are extracted from urine by a combined extraction procedure using freshly distilled diethyl ether and tert-butyl methyl ether as extraction solvents at pH 9.5 and 14 respectively. Prior to GC-MS analysis the residues are combined and derivatised using a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, NH(4)I and ethanethiol. The mass spectrometer is simultaneously operated in the full scan mode (mass range varies along with GC-oven temperature program) and in the selected ion monitoring mode. The obtained limits of detection are in compliance with the requirements set by the World Anti-Doping Agency. Besides narcotics, stimulants and anabolic androgenic agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. This comprehensive screening method reduces the amount of urine needed and increases the sample throughput without a loss in sensitivity and selectivity.

Development and validation of a quantitative gas chromatography-mass spectrometry method for the detection of endogenous androgens in mouse urine.

A quantitative method based on gas chromatography-mass spectrometry (GC-MS) has been developed for the detection of 16 endogenous androgens in the urine of mice. The substances are extracted from 100 microL urine with freshly distilled diethyl ether after alkalinisation. The substances are derivatised with a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide/NH(4)I/ethanethiol (383/1/2, v/w/v) and detected by GC-MS in the selected ion monitoring mode. The results of the method validation indicate good linearity, accuracy and precision, making the method suitable for the quantification of endogenous androgens in mouse urine. The selectivity of the method showed that no interfering peaks were observed at the retention times of the analytes. The method allows for the direct quantification and identification of testosterone and 15 other endogenous androgens at low concentrations (ng/mL) in mouse urine. The applicability of the method is shown by the analysis of a mouse urine. Several endogenous steroids could be detected.

Validation of an extended method for the detection of the misuse of endogenous steroids in sports, including new hydroxylated metabolites.

Endogenous steroids are amongst the most misused doping agents in sports. Their presence poses a major challenge for doping control laboratories. Current threshold levels do not allow for the detection of all endogenous steroid misuse due to great interindividual variations in urinary steroid concentrations. A method has been developed and validated to screen for traditionally monitored endogenous steroids in doping control as well as specific hydroxylated/oxygenated metabolites in order to enhance the detection capabilities for the misuse of endogenous steroids.

Comprehensive analysis by liquid chromatography Q-Orbitrap mass spectrometry: Fast screening of peptides and organic molecules.

The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS2 . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS2 enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1 ng mL-1 ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.

Comprehensive screening method for the qualitative detection of narcotics and stimulants using single step derivatisation.

A selective and sensitive screening method for the detection of prohibited narcotic and stimulating agents in doping control is described and validated. This method is suitable for the detection of all narcotic agents mentioned on the World Anti-Doping Agency (WADA) doping list in addition to numerous stimulants. The analytes are extracted from urine by a combined extraction procedure using CH(2)Cl(2)/MeOH (9/1, v/v) and t-butylmethyl ether as extraction solvents at pH 9.5 and 14, respectively. Prior to GC-MS analysis the obtained residues are combined and derivatised with MSTFA. The mass spectrometer is operated in the full scan mode in the range between m/z 40 and 550. The obtained limits of detection (LOD) for all components included in this extensive screening method are in the range 20-500 ng/ml, which is in compliance with the requirements set by WADA. Besides narcotic and stimulating agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. As an example, a positive identification of hydroxyl-methoxy-tamoxyfen is shown.

Results of stability studies with doping agents in urine.

For a correct interpretation of analytical results in doping control, knowledge on the stability of prohibited substances in the urinary matrix is a prerequisite. So far, limited data is available on the stability of prohibited substances in unaltered urine because most of the studies investigating the stability of drugs have used stabilized, sterilized, or filtered urine. In this work, the long-term stability of ephedrine, methylephedrine, cathine, 19-norandrosterone glucuronide, and a wide range of diuretics was determined over a period of 9 months at -20 degrees C, 4 degrees C, 22 degrees C, and 37 degrees C. Short-term stability, including the influence of 6 freeze-thaw cycles and 15 h storage at 60 degrees C, was also investigated. Often, a tolerance limit of 15%, similar to what is commonly used in the evaluation of precision data during method validation, is used to evaluate stability. This paper describes an alternative approach, using measurement uncertainty data to evaluate long-term stability with a probability of 95%, and proposes a simple alternative for investigating the stability for non-threshold substances. The results indicate that all the investigated substances are stable (alpha=0.05) when stored at -20 degrees C and 4 degrees C, but that at higher temperatures significant degradation effects can occur. The study also shows that degradation can be dependent on the urinary matrix and that the results from stability studies using stabilized, filtered, or sterilized urine can underestimate degradation effects.

Detection of hydroxyethylstarch (HES) in human urine by liquid chromatography-mass spectrometry.

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC-MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS(2). The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.

Nutritional supplements: prevalence of use and contamination with doping agents.

Based upon recent sales numbers, nutritional supplements play a key role in the lifestyle of a substantial proportion of the population. As well as products such as vitamins or minerals, several precursors of anabolic steroids are marketed as nutritional supplements. Another group of commercially available supplements are products for weight loss based upon herbal formulations originating from Ephedra species. Apart from supplements indicating the presence of these active compounds, numerous non-hormonal nutritional supplements were found that were contaminated with non-labelled anabolic steroids. Stimulating agents other than naturally occurring analogues of ephedrine were detected. A major group using dietary supplements are sportsmen, ranging from amateur level to elite athletes. Besides the possible health risks associated with the use of dietary supplements, athletes should take care not to violate the rules of the World Anti-Doping Agency because athletes remain responsible for substances detected in their biofluids, irrespective of their origin. Several analytical methods have been developed to determine the presence of doping agents as contaminants. The present review attempts to address the issues concerning the use of nutritional supplements and the detection of doping agents as contaminants in dietary supplements.

Detection of budesonide in human urine after inhalation by liquid chromatography-mass spectrometry.

Budesonide, a corticosteroid frequently used in the treatment of asthma, is most often administered via inhalation. Its use in sports is allowed when medically necessary. A fast, sensitive and accurate LC-MS method was developed and validated for the quantification of budesonide and its major metabolite 16alpha-hydroxyprednisolone in urine samples after inhalation of a metered dose (Pulmicort-Turbohaler 200). Sample preparation consists of an alkaline liquid-liquid extraction with ethyl acetate. Analysis was performed using liquid chromatography-tandem mass spectrometry with electrospray ionization (ESI). The method was linear in the range of 5-100 and 0.5-10ng/mL for 16alpha-hydroxyprednisolone and budesonide, respectively. The limits of quantification were 5ng/ml for 16alpha-hydroxyprednisolone and 0.5ng/mL for budesonide. The accuracy ranged from 2.2 to 3.5% for 16alpha-hydroxyprednisolone and from 0.8 to 16.4% for budesonide. After administration of 200microg of budesonide to five healthy volunteers budesonide could not be detected in any urine sample whereas 16alpha-hydroxyprednisolone was detectable up to 12h post-administration.

Quantitative LC-MS determination of strychnine in urine after ingestion of a Strychnos nux-vomica preparation and its consequences in doping control.

A simple, fast and sensitive method for the quantitative determination of strychnine residues in urine has been developed and validated. The method consists of a liquid-liquid extraction step with ethyl acetate at pH 9.2, followed by LC-MS/MS in positive atmospheric pressure chemical ionization (APCI)-mode. The method is linear in the range of 1-100 ng/mL and allows for the determination of strychnine at sub-toxicological concentrations. The accuracy of the method ranged from 1.3% to 4.4%. The method was used to determine the excretion profile of strychnine after the ingestion of an over-the-counter herbal preparation of Strychnos nux-vomica. Each volunteer ingested a dose equivalent to 380 micro g of strychnine. This dose is lower than the prescription dose but results in the detection of strychnine for over 24-h post administration. Maximum detected urinary concentrations ranged from 22.6 to 176 ng/mL. The results of this study show that the use of this type of preparation by athletes can lead to a positive doping case.

Quantitative analysis of androst-4-ene-3,6,17-trione and metabolites in human urine after the administration of a food supplement by liquid chromatography/ion trap-mass spectrometry.

6-OXO, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6alpha-ol-3,17-dione and androst-4-ene-6alpha,17beta-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6alpha-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6alpha,17beta-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6alpha-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6alpha,17beta-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.

Endogenous origin of norandrosterone in female urine: indirect evidence for the production of 19-norsteroids as by-products in the conversion from androgen to estrogen.

Recently the use of high resolution mass spectrometry or tandem mass spectrometry has enabled the detection of low amounts of anabolic steroids. As a consequence, the post-administration detection time of these drugs has been extended. Recent investigations have shown that norandrosterone, previously unequivocally regarded as evidence of nandrolone administration, might be an endogenous steroid present in small amounts in urine of humans. In this study, very low concentrations (<1 ng/ml) of norandrosterone in urine of a female athlete were detected using tandem mass spectrometry. The presence of norandrosterone was strongly correlated with high plasma 17beta-estradiol levels during the menstrual cycle. Analysis of urine samples from pregnant women supports the hypothesis of formation of precursors for urinary 19-norandrosterone during aromatization of androgens to estrogens. The detection of low urinary concentrations of norandrosterone (0.2-0.5 ng/ml) in samples after strenuous exercise could be regarded as an additional evidence for the existence of such a pathway.

Urinary concentrations of morphine after the administration of herbal teas containing Papaveris fructus in relation to doping analysis.

A quantitative method for the analysis of morphine in human urine in the concentration range between 0.25 and 2 microg/ml is described and validated. Morphine was determined after enzymatic hydrolysis of the urine. After liquid-liquid extraction with dichloromethane-methanol (9:1) at pH 9.5, morphine was derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and analyzed with GC-MS (full scan). The limit of quantification of the method was 0.25 microg/ml. Two Papaveris fructus containing herbal teas were administered to five male volunteers and urine samples were taken quantitatively during the first 12 h after the administration. The morphine concentration in the tea was 10.4 and 31.5 microg/ml, respectively. Morphine was detected in the urine of all volunteers by 1 h after drinking the tea. Maximum morphine concentrations, 4.3 and 7.4 microg/ml, respectively, were obtained 4-6 h after administration. Doping positive urine samples were delivered for 1-9 h.

Detection and disposition of tolmetin in the horse.

Non-steroidal anti-inflammatory drugs (NSAIDs) are prohibited by the International Federation of Horse Racing Authorities but are commonly used in veterinary practice. Plasma and urinary concentrations of the NSAID tolmetin were determined by a high-performance liquid chromatographic procedure with UV detection following oral administration of a dose of 1 g to six fasted untrained standard bred mares. With a limit of quantitation (LOQ) of 0.05 microg/ml tolmetin was present in plasma for 9-12 h post-administration. Maximum concentrations of 2.1+/-0.89 microg/ml were found after 0.7+/-0.25 h. The elimination half-life was 2+/-1.25 h. Plasma protein binding at concentrations of 0.25 and 2.5 microg/ml was 92+/-4.9 and 84+/-4.2%, respectively. As early as 1 h after dosage, tolmetin could be detected in unhydrolysed urine and remained detectable up to 48 h (LOQ=0.5 microg/ml). The maximum concentrations occurred 1.8+/-0.4 h after administration. The percentage of the dose excreted as unchanged tolmetin within 12 h was 58+/-7.9%. Neither conjugates nor metabolites could be detected under the experimental conditions studied. For confirmatory analysis in doping control, an LC-MS method was developed. Analysis was performed on an ion trap LC-MS system equipped with an ESI probe in positive MS(2) mode.

Detection and determination of anabolic steroids in nutritional supplements.

A method is described for the determination of anabolic steroids including testosterone, 19-nor-4-androstene-3,17-dione, 4-androstene-3,17-dione and nandrolone in food supplements. Initial clean-up is done by HPLC followed by determination with GC/MS. A 'contaminated' food supplement was analysed and appeared to contain 19-nor-4-androstene-3,17-dione and 4-androstene-3,17-dione. One capsule of this nutritional supplement was ingested by five male volunteers. Urine samples were collected and analysed by GC/MS and GC/MS-MS. Neither the ratio testosterone/epitestosterone, nor the ratio androstenedione/epitestosterone increased significantly. Concentrations above 2 ng/ml for norandrosterone, the major metabolite of nandrolone, were detected until 48-144 h after ingestion of the food supplement.