In vitro synthesis and characterisation of three fenoterol sulfoconjugates detected in fenoterol post-administration urine samples

Fenoterol, a fast-acting beta(2)-adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for beta(2)-agonists, which are prohibited in sports by the World Anti-Doping Agency, is commonly performed by liquid chromatography/mass spectrometry after hydrolysis of phase II metabolites. The continuing development of analytical procedures has led to direct injection of urine samples without sample preparation becoming a viable tool. For the detection of substances without sample preparation, including hydrolysis, detailed information of the phase II metabolism of the substances is essential. In this study, human S9 fractions of different tissues and two recombinant sulfotransferases were investigated for their potential to form fenoterol sulfoconjugates, which were characterised in detail. Two mono-sulfoconjugates and one bis-sulfoconjugate were synthesised and their structures confirmed by liquid chromatography-high-resolution/high-accuracy mass spectrometry. All of the metabolites were identified as esterified phenolic compounds. Excretion studies with orally and inhalatively administered fenoterol proved the occurrence of the sulfoconjugates in vivo. Inhalatively administered fenoterol resulted in the detection of the two mono-sulfoconjugates in low amounts in urine due to the lower inhalation dose of fenoterol compared to the oral dose. After oral uptake of fenoterol, the two mono-sulfoconjugates and a fenoterol bis-sulfoconjugate were detected in urine. This is the first report of the bis-sulfoconjugate

Probing for corticotropin-releasing hormone (CRH) in human blood for doping control purposes using immunoaffinity purification and LC-HRMS/MS

Corticotropin-releasing hormone (CRH), a peptide hormone whose secretion leads to adrenal cortisol release, is classified as prohibited substance by the World Anti-Doping Agency (WADA). In order to comprehensively enforce anti-doping regulations, a detection method for CRH in blood (serum and plasma) is required. In this study, two different immunoaffinity purification strategies were optimized and employed for sample preparation, followed by nano-ultra high performance liquid chromatography (UHPLC) coupled to high resolution/high accuracy tandem mass spectrometry (HRMS/MS). For that purpose, a CRH primary polyclonal antibody was immobilized to either IgG-covered paramagnetic particles or a monolithic protein A/G surface. The first approach using magnetic beads was fully validated in both human plasma and serum, while the Mass Spectrometric Immunoassay (MSIA (TM)) procedure was cross-validated for selected parameters. The resulting assays' LLODs were estimated at 200 pg mL(-1) (magnetic beads) and 500 pg mL(-1) (MSIA (TM)). In addition to human CRH, also animal analogs such as ovine and bovine CRH were found to be traceable using the established approach. For all target analytes comprising of 41 amino acids (approximately 4.7 kDa), high-resolution/high-accuracy product ion mass spectra were generated, and diagnostic y-and b-ions were identified. Proof-of-concept data were obtained by the analysis of plasma samples collected in the course of CRH stimulation blood tests. Specimens were collected from three patients 15 and 30 min after intravenous application of a 100 mu g single dose of human CRH, yielding plasma concentrations between 7.6 and 18.9 ng mL(-1)

Quantitative determination of adrenaline and noradrenaline in urine using liquid chromatography-tandem mass spectrometry

Thomas A, Geyer H, Mester HJ, Schaenzer W, Zimmermann E, Thevis M. Quantitative determination of adrenaline and noradrenaline in urine using liquid chromatography-tandem mass spectrometry. CHROMATOGRAPHIA. 2006;64(9-10):587-591.The quantitative determination of adrenaline (A) and noradrenaline (N) in human urine provides a helpful tool for sports sciences as well as clinical purposes. A rapid and highly specific procedure based on an off-line sample preparation using weak cation exchange solid phase extraction (CX-SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed that allowed the unambiguous identification of the target analytes at low (10 ng mL(-1) A and N), medium and high (500 ng mL(-1) A and N) physiological concentration levels. Recovery rates ranged from 104 to 111 %, precisions of better than 15% and a linear approximation in the aimed working range were obtained for both compounds. Thus, this method provides a rugged and highly specific alternative to the commonly used electrochemical or fluorescence detection strategies. In- and out-of-competition catecholamine concentrations in urine samples of six professional German first division soccer players were determined demonstrating an obvious decrease of the N/A-ratio post-competition, compared to pre-competition and out-of-competition values providing a parameter for the estimation of physiological and psychological stress

High-throughput screening for various classes of doping agents using a new dilute-and-shoot' liquid chromatography-tandem mass spectrometry multi-target approach

A new multi-target approach based on liquid chromatography electrospray ionization tandem mass spectrometry (LC-(ESI)-MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs for example, diuretics, beta2-agonists stimulants and narcotics are detectable at concentration levels far below the required limits

New potential markers for the detection of boldenone misuse

Boldenone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Boldenone misuse is commonly detected by the identification of the active drug and its main metabolite, 5 beta-androst-1-en-17 beta-ol-3-one (BM1), by gas chromatography-mass spectrometry (GC-MS), after previous hydrolysis with beta-glucuronidase enzymes, extraction and derivatization steps. However, some cases of endogenous boldenone and BM1 have been reported. Nowadays, when these compounds are detected in urine at low concentrations, isotope ratio mass spectrometry (IRMS) analysis is needed to confirm their exogenous origin. The aim of the present study was to identify boldenone metabolites conjugated with sulphate and to evaluate their potential to improve the detection of boldenone misuse in sports. Boldenone was administered to a healthy volunteer and urine samples were collected up to 56 h after administration. After a liquid-liquid extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using electrospray ionisation in negative mode by monitoring the transition of m/z 365-350, specific for boldenone sulphate. Boldenone sulphate was identified in the excretion study urine samples and, moreover, another peak with the same transition was observed. Based on the MS/MS behaviour the metabolite was identified as epiboldenone sulphate. The identity was confirmed by isolation of the LC peak, solvolysis and comparison of the retention time and MS/MS spectra with an epiboldenone standard. These sulphated metabolites have not been previously reported in humans and although they account for less than 1% of the administered dose, they were still present in urine when the concentrations of the major metabolites, boldenone and BM1, were at the level of endogenous origin. The sulphated metabolites were also detected in 10 urine samples tested positive to boldenone and BM1 by GC-MS. In order to verify the usefulness of these new metabolites to discriminate between endogenous and exogenous origin of boldenone, four samples containing endogenous boldenone and BM1, confirmed by IRMS, were analysed. In 3 of the 4 samples, neither boldenone sulphate nor epiboldenone sulphate were detected, confirming that these metabolites were mainly detected after exogenous administration of boldenone. In contrast, boldenone sulphate and, in some cases, epiboldenone sulphate were present in samples with low concentrations of exogenous boldenone and BM1. Thus, boldenone and epiboldenone sulphates are additional markers for the exogenous origin of boldenone and they can be used to reduce the number of samples to be analysed by IRMS. In samples with boldenone and BM1 at the concentrations suspicion for endogenous origin, only if boldenone and epiboldenone sulphates are present, further analysis by IRMS will be needed to confirm exogenous origin. (C) 2012 Elsevier Ltd. All rights reserved