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

Letrozole (1-(bis-(4-cyanophenyl)methyl)-1,2,4-triazole) is used therapeutically as a non-steroidal aromatase inhibitor (Femara) to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes it may be used to counteract the adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase the testosterone concentration by stimulation of the testosterone biosynthesis. The use of aromatase inhibitors has been prohibited by IOC/WADA regulations for male and female athletes since September 2001 and January 2005, respectively. Spot urine samples from women suffering from metastatic breast cancer and being treated with letrozole were collected and analysed to develop/optimise the detection system for metabolites of letrozole to allow the identification of athletes who do not comply with the internationally prohibited use of this cancer drug. The assay was based on gas chromatography/mass spectrometry (GC/MS) and the main metabolite of letrozole (bis-4-cyanophenylmethanol) was identified by comparison of its mass spectrum and retention time with that of a bis-4-cyanophenylmethanol reference. The full-scan spectrum, diagnostic ions and a validation of the method for the analysis of bis-4-cyanophenylmethanol are presented.

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.

Mass spectrometry of steroid glucuronide conjugates. II-Electron impact fragmentation of 3-keto-4-en- and 3-keto-5alpha-steroid-17-O-beta glucuronides and 5alpha-steroid-3alpha,17beta-diol 3- and 17-glucuronides.

The steroid glucuronide conjugates of 16,16,17-d(3)-testosterone, epitestosterone, nandrolone (19-nortestosterone), 16,16,17-d(3)-nortestosterone, methyltestosterone, metenolone, mesterolone, 5alpha-androstane-3alpha,17beta-diol, 2,2,3,4,4-d(5)-5alpha-androstane-3alpha,17beta-diol, 19-nor-5alpha-androstane-3alpha,17beta-diol, 2,2,4,4-d(4)-19-nor-5alpha-androstane-3alpha,17beta-diol and 1alpha-methyl-5alpha-androstane-3alpha/beta,17beta-diol were synthesized by means of the Koenigs-Knorr reaction. Selective 3- or 17-O-conjugation of bis-hydroxylated steroids was performed either by glucuronidation of the corresponding steroid ketole and subsequent reduction of the keto group or via a four-step synthesis starting from a mono-hydroxylated steroid including (a) protection of the hydroxy group, (b) reduction of the keto group, (c) conjugation reaction and (d) removal of protecting groups. The mass spectra and fragmentation patterns of all glucuronide conjugates were compared with those of the commercially available testosterone glucuronide and their characterization was performed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. For mass spectrometry the substances were derivatized to methyl esters followed by trimethylsilylation of hydroxy groups and to pertrimethylsilylated products using labelled and unlabelled trimethylsilylating agents. The resulting electron ionization mass spectra obtained by GC/MS quadrupole and ion trap instruments, full scan and selected reaction monitoring experiments are discussed, common and individual fragment ions are described and their origins are proposed.

High speed determination of beta-receptor blocking agents in human urine by liquid chromatography/tandem mass spectrometry.

Beta-receptor blocking agents are present on the international market in a huge variety. The International Olympic Committee prohibits the use of these drugs in several sport sections and doping control laboratories analyse urine samples of high-performance athletes with different techniques. Therefore, fast and reliable methods are required to enable a sensitive detection of many drugs and a high throughput of samples. In the present study a screening procedure is described using high speed liquid chromatography and multiple reaction monitoring to identify 32 beta-receptor blocking agents extracted from human urine. Urine specimens (blank urine samples, spiked urine samples and specimens of excretion studies) were hydrolysed, extracted and analysed within 7 min. Quasi-molecular ions (M(+) + H) of the beta-blockers are generated by means of an atmospheric pressure chemical ionization interface followed by collision-induced dissociation in a triple quadrupole mass spectrometer and subsequent detection of daughter ions. Proposals for the origin of common and individual secondary ions are presented.

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.

Screening of beta-2 agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives.

A new method for a comprehensive screening and confirmation of beta-2 agonists in human urine is presented based on gas chromatography-low-resolution mass spectrometry (GC-MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with beta-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other beta-2 agonists remain unchanged. Liquid-liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.

Detection of the plasma volume expander hydroxyethyl starch in human urine.

The plasma volume expander hydroxyethyl starch (HES) is usually administered in cases of hypovolaemic shocks but in 1998 the press reported its misuse in endurance sports. Since January 2000, it has been put on the list of prohibited substances of the International Olympic Committee (IOC) and its misuse is to ban by doping controls. Therefore, a rapid method enabling the screening for HES in human urine was developed which can be easily adopted by IOC laboratories to analyse routine urine samples for this remedy. Excretion study urine samples obtained from patients treated with HES, blank urine specimen and reference standards, were hydrolysed with hydrochloric acid and without any further purification of the resulting monosaccharides their per-timethylsilylated derivatives were performed. By means of gas chromatography-mass spectrometry the products were separated and the alpha- and beta-isomers of glucose, 2-, 3- and 6-hydroxyethyl glucose derivatives were identified. Typical ion traces of 2- and 3-substituted glucose (m/z 248, m/z 261 and m/z 235, m/z 248, respectively) support the fast determination of the substances whose electron impact mass spectra are presented and 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.

Gas chromatography/mass spectometry identification of long-term excreted metabolites of the anabolic steroid 4-chloro-1,2-dehydro-17alpha-methyltestosterone in humans.

The misuse of anabolic steroids by athletes has been banned by sports organizations and is controlled by the analysis of urine samples obtained from athletes using gas chromatography/mass spectrometry (GC/MS). To extend the retrospectivity of the analytical methods, research is focused on long-term excreted metabolites. Preliminary results concerning the long-term detection of metabolites of the anabolic androgenic steroid 4-chloro-1,2-dehydro-17alpha-methyltestosterone I are presented. A new metabolite 4-chloro-3alpha, 6 beta, 17beta-trihydroxy-17alpha-methyl-5beta-androst-l-en-16-one was isolated by high performance liquid chromatography (HPLC) from urine following a single oral administration of 40 mg of I and characterized. Metabolite II was excreted into urine with a maximum excretion rate at approximately 48 h after administration and could be detected by gas chromatography/high resolution mass spectrometry (GC/HRMS) for up to 14 days. Two further partly characterized metabolites III and IV were confirmed for more than 9 days. The same three metabolites, II-IV, in varying amounts were also detected in urine samples from athletes who administered I.

17-Epimerization of 17 alpha-methyl anabolic steroids in humans: metabolism and synthesis of 17 alpha-hydroxy-17 beta-methyl steroids.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.

Metabolism of stanozolol: identification and synthesis of urinary metabolites.

Urinary metabolites of stanozolol (17 alpha-methyl-17 beta-hydroxy-5 alpha-androst-2-eno(3,2-c)-pyrazole) following oral administration were isolated by chromatography on XAD-2 and by preparative high-performance liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionisation. Stanozolol is excreted as a conjugate but is metabolized to a large extent. All identified metabolites are hydroxylated, namely at C-3' of the pyrazole ring and at C-4 beta, C-16 alpha and C-16 beta of the steroid. Less than 5% of the metabolites are found in the unconjugated urine fraction: 3'-hydroxy-stanozolol (II) and 3'-hydroxy-17-epistanozolol (III). Conjugated excreted metabolites are 3'-hydroxystanozolol (II), stanozolol (I), 4 beta-hydroxy-stanozolol (IV), 16 beta-hydroxystanozolol (V), 16 alpha-hydroxystanozolol (VI), two isomers of 3',16-dihydroxystanozolol (VII, VIII), two isomers of 4 beta, 16-dihydroxystanozolol (IX, X) and a 3',?-dihydroxystanozolol (XI). 3'-Hydroxystanozolol, 4 alpha-hydroxystanozolol, 4 beta-hydroxystanozolol, 16 alpha-hydroxy-, 16 alpha-hydroxy-17-epi- and 16 beta-hydroxystanozolol were synthesised to confirm the structural assignment of the main metabolites.