Mass spectrometry of steroid glucuronide conjugates. I. Electron impact fragmentation of 5alpha-/5beta-androstan-3alpha-ol-17-one glucuronides, 5alpha-estran-3alpha-ol-17-one glucuronide and deuterium-labelled analogues

Owing to the developments of analytical instruments and interfaces (e.g. coupling high-performance liquid chromatography to mass spectrometry), there has been increased interest in new reference materials, for example in doping analysis with steroid glucuronide conjugates. The synthesized reference material has to pass several characterization steps including the use of gas chromatography/mass spectrometry (GC/MS) for its structure confirmation. In the present study, the fragmentation and mass spectrometric behaviour of several steroid glucuronide conjugates of endogenous and anabolic steroids after derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated products were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The mass spectra of the derivatives of androsterone glucuronide, d5-androsterone glucuronide, epiandrosterone glucuronide, etiocholanolone glucuronide, 11beta-hydroxy etiocholanolone glucuronide, 19-norandrosterone glucuronide, d4-19-norandrosterone glucuronide and 1alpha-methyl-5alpha-androstan-3alpha-ol-17-one glucuronide are presented and the origin of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on different derivatization techniques including derivatization with d18-bistrimethylsilylacetamide, methyl ester and trimethylsilyl ester derivatization and selected reaction monitoring. Typical fragmentation patterns which are related to the steroid structure are discussed.     

Mass spectrometry of partially methylated alditol acetates derived from hydroxyethyl starch

The degradation and derivatization of hydroxyethyl starch to partially methylated alditol acetates (PMAAs) allows its detection by gas chromatography/mass spectrometry. The derivatization was performed by permethylation of the carbohydrate, hydrolysis of the permethylated polysaccharide, reduction of the resulting monosaccharides to alditoles and finally acetylation. A close similarity in the fragmentation of the PMAAs obtained was observed in both electron ionization (EI) and chemical ionization (CI) mass spectra owing to the comparable structures of the derivatives. CI measurements permitted the recognition of introduced hydroxyethyl groups in the glucose residues by detection of [M(+)+1]-60 signals. Investigations concerning the EI fragmentation schemes allowed secure determinations of monohydroxyethyl monosaccharides and differentiations between the possible positions (C-2, C-3 and C-6) of the substituted hydroxyethyl groups. Proposed generations of the main fragment ions are presented.     

The influence of recycled material on the crystallization kinetics of thermoplastic polymers

The injection moulding of semi-crystalline thermoplastic polymers requires an exact knowledge of the thermodynamic data and of the crystallization kinetics. The behaviour of the polymer melt during rapid cooling in the mould determines, to a great extent, the quality and usability of a final product. Technical raw materials are often equipped with nucleating agents in order to obtain crystallization within the desired temperature range and at the required rate. The use of recycled material (regranulate) shows an analogous effect such as the addition of nucleating agents, i.e. crystallization begins at a higher temperature and a higher crystallization rate is detected compared to materials without added regranulate. Heat flux DSC was used to study the crystallization of polyamides, polyolefins and polyoxymethylene during cooling at various cooling rates. Although the temperature gradients and pressures which occur in the proceesing machine cannot be realised in DSC tests, the DSC results reproduce the direction of influence of the regranulate additive very clearly.     

Characterization of chemically modified steroids for doping control purposes by electrospray ionization tandem mass spectrometry

The discovery of the designer steroid tetrahydrogestrinone (THG) in elite athletes' doping control samples in 2003 demonstrated the availability of steroid derivatives prepared solely for doping purposes. Modern mass spectrometers utilizing electrospray ionization and collisionally activated dissociation (CAD) of analytes allow the structural characterization of steroids and their derivatization sites by the elucidation of fragmentation behaviors. A total of 21 steroids comprising either a 4,9,11-triene, a 3-keto-4-ene or a 3-keto-1-ene nucleus were investigated regarding their dissociation pathways, deuterated analogues were synthesized and fragmentation routes were postulated, permitting the identification of steroidal structures and modifications. Compounds based on a 4,9,11-triene steroid with an ethyl residue at C-13 (gestrinone analogues) generate abundant fragment ions at m/z 241 and 199, whereas the substitution of the C-13 ethyl group by a methyl residue (trenbolone analogues) results in a shift of m/z 241 to 227. Substances related to testosterone with a 3-keto-4-ene structure give rise to abundant fragment ions at m/z 109 and 97 whereas steroids with a 3-keto-1-ene nucleus eliminate the A-ring including the carbons C-1-C-4, in addition to C-19 that is proposed to migrate from C-10 to C-1 under CAD conditions.     

Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for beta2-agonists in human or equine urine

Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine specimens, acidic re-extraction of the analytes. In addition, a degradation product of formoterol caused by acidic hydrolysis during sample preparation could be identified and utilized as target compound in screening and also confirmation methods. The screening procedures cover 18 or 19beta(2)-agonists, the estimated limits of detection of which for equine and human urine samples vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively. A single LC/MS/MS analysis can be performed in 9 min.     

Modelling and optimisation of solid electrolyte sintering behaviour by thermokinetic analysis

Burnout of organic binder and sintering behaviour of yttria doped zirconia electrolyte green tapes were investigated by thermogravimetry and dilatometry. The burnout which precedes the actual sintering, as well as the sintering itself were described by a mathematical model based on formal kinetics. Thus, the measured data were reduced to a set of a few parameters which can be easily used to simulate the kinetic behaviour for an arbitrary temperature profile, so that good accordance between prediction and respective measurement was accomplished. Using rate controlled binder burnout and rate controlled sintering, optimisation of the temperature profile was achieved with respect to time and energy saving. These results were confirmed by SEM analysis of the microstructure and determination of the electrical conductivity.     

6alpha-Methylandrostenedione: gas chromatographic mass spectrometric detection in doping control

In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared on the sport supplement market. Scientific studies have proven aromatase inhibition and anabolic and mild androgenic properties; however, no preparation has been approved for medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified as a prohibited substance according to the regulations of the World Anti-Doping Agency (WADA). For the detection of its misuse the metabolism was studied following the administration of two preparations obtained from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxyprogesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be excreted as glucuronide and was still detectable in microg/mL amounts until urine collection was terminated (after 25 h). Additionally, samples from routine human sports doping control had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-3,17-dione. Screening analysis can be easily performed by the existing screening procedure for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is possible as the mono-TMS derivative.     

Determination of (13)C/(12)C ratios of endogenous urinary steroids excreted as sulpho conjugates

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry‐based method for the measurement of stable carbon isotopes of endogenous urinary steroids excreted as sulphates is presented. The key element in sample preparation is the consecutive cleanup with high‐performance liquid chromatography of underivatized and acetylated steroids, which allows the isolation of seven analytes (pregn‐5‐ene‐3β,17α,20α‐triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), androst‐5‐ene‐3β,17β‐diol and androst‐5‐ene‐3β,17α‐diol) from a single urine specimen. These steroids are of particular importance to doping controls as they should enable the sensitive and retrospective detection of DHEA abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers the items, repeatability and reproducibility. The specificity was further demonstrated by gas chromatography/mass spectrometry for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring ¹³C/¹²C ratios and urinary concentrations of all implemented steroids, a reference population of n = 67 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values. The applicability of the developed method was tested by means of a DHEA excretion study. Despite the fact that orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal clearance of sulphated steroids is slow, only the ¹³C/¹²C ratio of EpiA demonstrated the potential to prolong the detection time for DHEA misuse. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry

Anastrozole (2,2'-[5-(1H-1,2,4-triazol-1-ylmethyl)-1.3-phenylene]bis(2-methylpropionitrile)) and exemestane (6-methylenandrostan-1,4-diene-3,17-dione) are therapeutically used to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes they may be used to counteract adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase plasma testosterone concentrations. Excretion study urine samples and spot urine samples from women suffering from metastatic breast cancer, being treated with anastrozole or exemestane, were collected and analyzed to develop/optimize a detection system for anastrozole and exemestane to allow the identification of athletes who do not comply with the internationally prohibited use of these cancer drugs. The assay was based on liquid-liquid extraction after enzymatic hydrolysis following liquid chromatography/tandem mass spectrometry (LC/MS/MS). Anastrozole, exemestane and its main metabolite (17-dihydroexemestane) were identified in urine by comparison of mass spectra and retention times with respective reference substances. An assay validation for the analysis of anastrozole and exemestane was performed regarding lower limits of detection (anastrozole: 0.02 ng/mL; exemestane: 3.1 ng/mL; dihydroexemestane: 0.5 ng/mL), interday precisions (6.6-11.1%, 4.9-9.1% and 5.6-8.3% for low [10 ng/mL], medium [50 ng/mL] and high [100 ng/mL] concentration) and recoveries (ranged from 85-97%).     

Identification of the aromatase inhibitor aminoglutethimide in urine by gas chromatography/mass spectrometry

Aminoglutethimide is used therapeutically as an aromatase inhibitor in the treatment of metastatic breast cancer in post-menopausal women. For doping purposes, aminoglutethimide may be used for treatment of adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase the testosterone concentration and stimulation of testosterone biosynthesis. The use of aromatase inhibitors has been prohibited for male athletes since September 1, 2001. The purpose of this study was to develop methods for the identification of the parent compound or its main metabolite and the inclusion of this information into established screening procedures in doping analysis. An excretion study was conducted using oral application of one single therapeutic dose (500 mg) of Orimeten. The analysis was performed by gas chromatography/mass spectrometry (GC/MS). Aminoglutethimide is excreted almost totally as unconjugated parent compound and is detectable by different screening procedures for up to 165 h. Most suitable for the detection of aminoglutethimide is the screening procedure for heavy volatile nitrogen-containing drugs ('Screening 2'). However, since only competition samples are analysed in that screening procedure, the additional inclusion of aminoglutethimide in the screening procedure for anabolic androgenic agents ('Screening 4') is recommended. Full mass spectra and diagnostic ions for the analysis of aminoglutethimide are presented.