Tetrahydrogestrinone: discovery, synthesis, and detection in urine

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.     

Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

Coupling HPLC to on-line, post-column (bio)chemical assays for high-resolution screening of bioactive compounds from complex mixtures

It is challenging to screen and identify bioactive compounds from complex mixtures. We review a recently developed technique that couples high-performance liquid chromatography (HPLC) to on-line, post-column (bio)chemical assays and parallel chemical analysis to screen and identify bioactive compounds from complex mixtures without the need for cumbersome purification and subsequent screening. In this system, HPLC separates complex mixtures and a post-column (bio)chemical assay determines the activity of the individual compounds present in the mixtures. Parallel chemical-detection methods (e.g., diode-array detection, mass spectrometry and nuclear magnetic resonance) identify and quantify the active compounds simultaneously. We focus on relatively widely used on-line, post-column assays for antioxidant screening and less widely used hyphenated systems involving assays based on enzymes and receptors. These strategies have proved to be very useful for rapid profiling and identification of individual active components in mixtures to provide a powerful method for natural product-based drug discovery.     

Screening for unknown synthetic steroids in human urine by liquid chromatography-tandem mass spectrometry

Chemically modified steroids (designer steroids), including tetrahydrogestrinone and norbolethone, pose a threat to the integrity of the sport community. These compounds have recently been detected in urine specimens from athletes, resulting in temporary or permanent suspension from amateur and/or professional competition. Triple quadrupole mass spectrometers enable doping control laboratories to screen for unknown, anabolic, androgenic steroids utilizing precursor ion scans. On the basis of common dissociation patterns of steroids with common structural features, characteristic product ions were selected to serve as diagnostic markers for previously unidentified drugs or drug metabolites in human urine samples. An assay was established to complement standard screening procedures. Urine specimens were enzymically hydrolyzed, partitioned into ether, concentrated, and analyzed by precursor ion scanning. Spectra from samples fortified with eight standard compounds (methyltestosterone, ethyltestosterone, 1-testosterone, gestrinone, dihydrogestrinone, tetrahydrogestrinone, norbolethone, and propyltrenbolone) and one deuterium-labeled analog (d(4)-tetrahydrogestrinone) at 50 ng/ml of urine, had precursor ion peaks other than those from common endogenous steroids. Subsequent product ion scan experiments on precursor ions of peaks of unknown origin provided structural identification of the unknown compounds.     

Another designer steroid: discovery, synthesis, and detection of 'madol' in urine

Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.     

Optimization of the Extraction and Analysis of Natural Androgen Steroids and Their Metabolites in Urine by GC/MS and GC/FID

A multiresidue method was developed for screening, quantification, and confirmation of nine natural androgen steroids and their metabolites in urine. Steroids were first extracted from urine by solid phase extraction, enzymatically deglucuronated, re-extracted using a liquid/liquid extraction for purification, and finally acetylated for GC/MS and GC/FID analysis. Each step of sample preparation, as well as analysis, was optimized: solid phase extraction, liquid/ liquid extraction, and derivatization reaction … Therefore, a rugged sample preparation procedure was developed leading to extracts of sufficient purity (recoveries >66% and few matrix compounds). The whole methodology allowed reliable detection and quantification of the nine steroids at low concentration levels. Linearity and repeatability were established and were found to be satisfactory (R² > 0.996, RSD < 11%). Finally, the method was applied to quantify compounds of interest in real samples collected from healthy volunteers and patients treated with 4-androstenedione or dehydroepiandrosterone.     

Identification and quantification of 34 drugs and toxic compounds in blood,urine, and gastric content using liquid chromatography with tandem mass spectrometry

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid‐phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02–20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01–10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65–117% and 67–121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05–20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.     

Liquid chromatography methodologies for the determination of steroid hormones in aquatic environmental systems

Steroid hormones are a diverse group of natural and synthetic compounds. Their wide use in human and veterinary medicine results in their continual introduction into the environment. In recent years, environmental concern over steroids that act as endocrine disruptors has increased because of their adverse effects on organisms or their progeny. Moreover, as these compounds are not totally removed from sewage in wastewater treatment plants, they can reach the aquatic environment and persist due to their physicochemical characteristics.For this reason, a major trend in analytical chemistry is the development of rapid and efficient procedures for the extraction, determination and quantification of steroid hormones in environmental samples. Over the past few decades, the significant expansion of liquid chromatography technology utilizing mass spectrometry detection has led to applications with increased selectivity and sensitivity. Optimized extraction and microextraction techniques combined to these liquid chromatography techniques have lowered detection and quantification limits to the ng L⁻¹–μg L⁻¹ range, which is the concentration of steroid hormones in liquid, solid and biota samples.In this paper, the state-of-the-art techniques for the analysis of steroid hormones focused mainly in based liquid chromatography methods in liquid and aquatic solid and biota samples are reviewed. Handling, storage, extraction and detection methodologies are reviewed and compared for all families of steroid hormones.     

Fast and sensitive supercritical fluid chromatography – tandem mass spectrometry multi-class screening method for the determination of doping agents in urine

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48–68% of the compounds and higher than 50% for 83–87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.     

Detection challenges in quantitative polymer analysis by liquid chromatography

Accurate quantification of polymer distributions is one of the main challenges in polymer analysis by liquid chromatography. The response of contemporary detectors is typically influenced by compositional features such as molecular weight, chain composition, end groups, and branching. This renders the accurate quantification of complex polymers of which there are no standards available, extremely challenging. Moreover, any (programmed) change in mobile‐phase composition may further limit the applicability of detection techniques. Current methods often rely on refractive index detection, which is not accurate when dealing with complex samples as the refractive‐index increment is often unknown. We review current and emerging detection methods in liquid chromatography with the aim of identifying detectors, which can be applied to the quantitative analysis of complex polymers.     

Flow-Based Methods with Chemiluminescence Detection for Food and Environmental Analysis: A Review

This paper presents an overview of flow-based methods in food and environmental analysis using chemiluminescence (CL) detection covering the period from 2005 to the present. The review discussses both automated flow methods of analysis [such as flow-injection analysis (FIA), sequential-injection analysis (SIA) and their variants] and separation techniques [liquid chromatography (LC) and capillary electrophoresis (CE) coupled to CL detection]. The most widely used CL reactions are presented together with representative applications in food and environmental analysis (determination of naturally occurring compounds, contaminants, additives as well as determination of inorganic and organic compounds).     

Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine

The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids - metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol - were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GC/MS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GC/MS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Simultaneous quantitation of 17 female sex hormones in illegal products by validated liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry methods

The consumption of food and drugs adulterated with female sex hormones can have an extremely adverse effect on human health. Therefore, developing appropriate monitoring methods for the identification of various exogenous female sex hormones is crucial for minimizing and eliminating the related health risks. Herein, 17 female hormones categorized into two groups: estrogen and progestin, were analyzed using reversed-phase liquid chromatography coupled to Orbitrap or triple quadrupole mass spectrometry. The fragmentation patterns for all compounds were discovered, and fragmented structures were also derived from them through liquid chromatography–high-resolution mass spectrometry followed by qualitative sample analysis. In addition, a quantitative analysis of 67 samples of illicit drugs and dietary supplements was performed using the validated liquid chromatography-tandem mass spectrometry method. Female hormone components were detected in two samples of an unauthorized injectable solution and a tablet-type drug. Medroxyprogesterone was detected in the samples in the range of 96.4–206 ng/g. Notably, eight components similar in structure to steroids were simultaneously detected as male sex hormones by confirming their fragmentation ion patterns using liquid chromatography–high-resolution mass spectrometry. The developed methods thus offer a dependable and practically applicable approach for the screening and detection of exogenous female sex hormones in real food and drug samples to ensure public health.     

Properties of the Adsorption Equilibrium Isotherms Used and Measured in RPLC

Monitoring anabolic steroids in meat-producing animals is a challenging task. It implies very specific and sensitive analytical methods able to detect and identify sub-μg kg^?1 residue levels in complex biological matrices such as meat, urine, or hair. Gas and liquid chromatography coupled to mass spectrometry are the most efficient means of achieving these objectives. In this paper we review how developments in mass spectrometry have been rapidly applied to this problem, how efficient analysis of anabolic steroids in urine, edible tissue, and hair has been achieved, and, later, how measurement of conjugate steroids and determination of the origin of natural steroid hormones has been achieved. The performance characteristics of different mass spectrometers (quadrupole, ion-trap, electromagnetic, isotope-ratio, tandem, and hybrid instruments), the efficiency of different acquisition techniques (LR-SIM, HR-SIM, MRM), and, finally, sample introduction (gas chromatography and liquid chromatography, with discussion of alternative interfaces) are discussed, with numerous applications.     

Rapid screening of polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography-tandem mass spectrometry

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.     

Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry

Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneous hydrolysis of methandrostenolone sulphate gave 17-epimethandrostenolone and several dehydration products. This reaction had a half-life of 16 min in equine urine at 27 degrees C. Mono- and dihydroxylated metabolites were also identified. Several screening methods were evaluated for detection and confirmation of methandrostenolone use including thin-layer chromatography and high-performance liquid chromatography. Coupled-column liquid chromatography was used for automated clean-up of analytes difficult to isolate by manual methods. The recovery of methandrostenolone was 101 +/- 3.3% (mean +/- S.D.) at 6.5 ng/ml and both methandrostenolone and 17-epimethandrostenolone were quantified in urine by ultraviolet detection up to six days after a 250-mg intramuscular dose to a horse. The utility of on-line tandem mass spectrometry for confirmation of suspected metabolites is also shown.     

Evaluation of micellar liquid chromatography and capillary zone electrophoresis for dope control in sport

Micellar liquid chromatography (MLC) and capillary zone electrophoresis (CZE) have been evaluated for the analysis of twelve banned drugs in sport including diuretics and -blockers. In MLC, a sodium dodecylsulphate aqueous solution has been used as mobile phase using an octadecylsilica column. In CZE, a pH 8 buffer solution and a silica capillary have been employed. Parameters of retention and efficiency have been compared. Limits of detection with UV detection at 254 nm and relative standard deviations for atenolol, furosemide, nadolol, spironolactone and triamterene were established and compared in both techniques. Examples of direct urine injection into the separation systems are presented. Drugs overlapping in MLC are well resolved in CZE, while the opposite is true for a limited number of drugs. Some interferences from urine may arise in CZE. The selectivity of analysis would be greatly enhanced by using both techniques, which require only filtration as pre-treatment.     

A cleanup method for perchlorate determination in urine

There is increasing concern about perchlorate exposure because of perchlorate's potential effects on organisms as a thyroid hormone disruptor, as well as its contamination of the environment being much more widespread than previously thought. Perchlorate is excreted primarily into urine, therefore, evaluating perchlorate residues in urine should be a reasonable approach for determining exposure and if successful could be used as an effective biomarker of perchlorate exposure. Since the presence of ions and other biomolecules in matrices like urine usually confounds accurate determination of perchlorate by ion chromatography, it is necessary to develop efficient methods for perchlorate determination in these matrices. We developed a method that reduces the background signal of urine, which is typically the problem with the analysis of biological fluids and tissues by ion chromatography. Relatively high recovery of perchlorate was shown. In cow urine samples spiked with perchlorate at 2.5, 10, and 100 μg/L, perchlorate recoveries were 67% ± 2.5, 77% ± 3.6, and 81% ± 1.7 (mean ± S.D.), respectively. In addition, the detection limit was as low as 12.6, 12.3, and 18.7 μg/L in cow, vole, and human urine samples, respectively.