Complementary information concerning the suspected interindividual transmission of GW1516, a substance prohibited in sport, through intimate contact: a case report.

PURPOSE: Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed. METHODS: To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography-tandem mass spectrometry. Further, drug-residue testing using patients' ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry. RESULTS: In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete's doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid. CONCLUSIONS: The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments.

DropWise: current role and future perspectives of dried blood spots (DBS), blood microsampling, and their analysis in sports drug testing.

For decades, blood testing has been an integral part of routine doping controls. The breadth of information contained in blood samples has become considerably more accessible for anti-doping purposes over the last 10 years through technological advancements regarding analytical instrumentation as well as enhanced sample collection systems. Particularly, microsampling of whole blood and serum, for instance as dried blood spots (DBS), has opened new avenues in sports drug testing and substantially increased the availability and cost-effectiveness of doping control specimens. Thus, microvolume blood specimens possess the potential to improve monitoring of blood hormone and drug levels, support evaluation of circulating drug concentrations in competition, and enhance the stability of labile markers and target analytes in blood passport analyses as well as peptide hormone and steroid ester detection. Further, the availability of the fraction of lysed erythrocytes for anti-doping purposes warrants additional investigation, considering the sequestering capability of red blood cells (RBCs) for certain substances, as a complementary approach in support of the clean sport.

Nickel in equine sports drug testing - pilot study results on urinary nickel concentrations.

RATIONALE: The issue of illicit performance enhancement spans human and animal sport in presumably equal measure, with prohibited substances and methods of doping conveying both ways. Due to the proven capability of unbound ionic cobalt (Co(2) (+) ) to stimulate erythropoiesis in humans, both human and equine anti-doping regulations have listed cobalt as a banned substance, and in particular in horse drug testing, thresholds for cobalt concentrations applying to plasma and urine have been suggested or established. Recent reports about the finding of substantial amounts of undeclared nickel in arguably licit performance- and recovery-supporting products raised the question whether the ionic species of this transition metal (Ni(2) (+) ), which exhibits similar prolyl hydroxylase inhibiting properties to Co(2) (+) , has been considered as a substitute for cobalt in doping regimens. METHODS: Therefore, a pilot study with 200 routine post-competition doping control horse urine samples collected from animals participating in equestrian, gallop, and trotting in Europe was conducted to provide a first dataset on equine urinary Ni(2) (+) concentrations. All specimens were analyzed by conventional inductively coupled plasma mass spectrometry (ICP-MS) to yield quantitative data for soluble nickel. RESULTS: Concentrations ranging from below the assay's limit of quantification (LOQ, 0.5 ng/mL) up to 33.4 ng/mL with a mean value (± standard deviation) of 6.1 (±5.1) ng/mL were determined for the total nickel content. CONCLUSIONS: In horses, nickel is considered a micronutrient and feed supplements containing nickel are available; hence, follow-up studies are deemed warranted to consolidate potential future threshold levels concerning urine and blood nickel concentrations in horses using larger sets of samples for both matrices and to provide in-depth insights by conducting elimination studies with soluble Ni(2) (+) -salt species. Copyright © 2016 John Wiley & Sons, Ltd.

Methotrexate reduces HbA1c concentration but does not produce chronic accumulation of ZMP in patients with rheumatoid or psoriatic arthritis.

OBJECTIVES: The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy. METHOD: MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (µU/mL)/22.5]. RESULTS: Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p < 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations. CONCLUSIONS: MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP.

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

Fenoterol, a fast-acting ß2-adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for ß2-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 chromatography­high-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 monosulfoconjugates 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.

Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing.

The discovery and implementation of the long-term metabolite of metandienone, namely 17ß-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites-17ß-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and 17α-hydroxymethyl-17ß-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one-using a fungus (Cunninghamella elegans) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete's urine, different analytical approaches were followed, such as liquid chromatography-tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography-tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R(2) > 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation <20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Identification and characterization of urinary prenylamine metabolites by means of liquid chromatography-tandem mass spectrometry.

Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin(®) 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%.

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, 5ß-androst-1-en-17ß-ol-3-one (BM1), by gas chromatography-mass spectrometry (GC-MS), after previous hydrolysis with ß-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 56h 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.

Clenbuterol - regional food contamination a possible source for inadvertent doping in sports.

The misuse of the sympathomimetic and anabolic agent clenbuterol has been frequently reported in professional sport and in the livestock industry. In 2010, a team of athletes returned from competition in China and regular doping control samples were taken within the next two days. All urine samples contained low amounts (pg/ml) of clenbuterol, drawing the attention to a well-known problem: the possibility of an unintended clenbuterol intake with food. A warning that Chinese meat is possibly contaminated with prohibited substances according to international anti-doping regulations was also given by Chinese officials just before the Bejing Olympic Games in 2008. To investigate if clenbuterol can be found in human urine, a study was initiated comprising 28 volunteers collecting urine samples after their return from China. For the quantification of clenbuterol at a low pg/ml level, a very sensitive and specific isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed using liquid/liquid re-extraction for clean-up with a limit of detection and quantification of 1 and 3 pg/ml, respectively. The method was validated demonstrating good precision (intra-day: 2.9-5.5 %; inter-day: 5.1-8.8%), accuracy (89.5-102.5%) and mean recovery (81.4%). Clenbuterol was detectable in 22 (79%) of the analyzed samples, indicating a general food contamination problem despite an official clenbuterol prohibition in China for livestock.

Di(2-ethylhexyl) phthalate metabolites as markers for blood transfusion in doping control: intra-individual variability of urinary concentrations.

To indicate homologous or autologous blood transfusion in sports drug testing, quantification of increased urinary concentrations of di(2-ethylhexyl) phthalate (DEHP) metabolites presents a promising approach; however, the possible intra-individual variation of the metabolite concentrations over time has not been well characterized. The aim of this study was to explore the intra-individual variability of urinary DEHP metabolites among seven volunteers without special occupational exposure to DEHP during one week (n = 253) in order to investigate the possibility of increased urinary concentrations of the metabolites caused by, for example, residential, dietary, or environmental exposure. Quantification of three DEHP metabolites--mono(2-ethylhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate--was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography-tandem mass spectrometry. Although urinary concentrations of DEHP metabolites showed considerable intra-individual variation, no increased values were observed comparable to the concentrations measured in urine specimens collected after blood transfusion.

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. Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time-consuming, stand-alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP-2), and 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2-ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion. The assay was fully validated for qualitative purposes considering the parameters specificity, intra- (3.2-16.6%) and inter-day precision (0.4-19.9%) at low, medium and high concentration, robustness, limit of detection (1-70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects. The analyses of post-administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high-throughput method that increases the efficiency of the labs daily work.

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes.

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.

Evaluation of commercially available assays for the measurement of equine insulin.

Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when the provided assay buffer was used with the Siemens Count-a-Coat Insulin radioimmunoassay (RIA) but not when charcoal-stripped equine plasma was used as the diluent. This modified RIA and the Mercodia Equine Insulin ELISA were evaluated further by directly examining accuracy by comparing their results for 18 equine plasma samples with values obtained using liquid chromatography and high-resolution/high-accuracy mass spectrometry (LC-MS). Compared with LC-MS measurements, the modified Siemens Insulin RIA rendered a moderate Lin's concordance coefficient (ρ(c)) of 0.41, whereas the Mercodia Equine Insulin ELISA rendered a very poor ρ(c) of 0.06. This suggests that the Siemens Insulin RIA is appropriate to use for routine evaluations when LC-MS is not available.

Investigation of the in vitro metabolism of the emerging drug candidate S107 for doping-preventive purposes.

The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance-enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography-mass spectrometry with electrospray ionisation in positive mode (LC-ESI-MS/MS). Their collision-induced dissociation behaviour was studied by high-resolution/high accuracy Orbitrap MS(n) analysis, supported by stable isotope labelling, H/D-exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N- and S-oxidation of the 1,4-benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N- and O-demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N-demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107-N(+)-glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with ß-glucuronidase (Escherichia coli). A comprehensive LC-ESI-MS(/MS)-based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future.

Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing.

Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n = 100), in hospitalized patients receiving blood transfusion (n = 10), and in athletes (n = 468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.

Different mechanical loading protocols influence serum cartilage oligomeric matrix protein levels in young healthy humans.

The purpose of the study was to investigate whether a relationship between the loading mode of physical activity and serum cartilage oligomeric matrix protein (COMP) concentration exists and whether the lymphatic system contributes to COMP release into the serum. Serum COMP levels were determined in healthy male subjects before, after and at 18 further time points within 7 h at four separate experimental days with four different loading interventions. The loading intervention included high impact running exercise, slow but deep knee bends, and lymphatic drainage of 30 min duration, respectively, and a resting protocol. The serum COMP levels were measured using a commercially available quantitative enzyme-linked immunosorbent assay. An increase (p < 0.001) in serum COMP concentration was detected immediately after 30 min running exercise. Slow but deep knee bends did not cause any significant changes in serum COMP levels. Lymphatic drainage also had no effect on the serum COMP concentration. After 30 min of complete rest the serum COMP level was significantly (p = 0.008) reduced. The elevation of COMP serum concentration seems to depend on the loading mode of the physical activity and to reflect the extrusion of COMP fragments from the impact loaded articular cartilage or synovial fluid.

Enzyme-assisted synthesis and structural characterization of pure benzodiazepine glucuronide epimers.

The three hydroxybenzodiazepines oxazepam, temazepam, and lorazepam used for their anxiolytic, sedative, and anticonvulsant properties are metabolized by glucuronidation, which is the predominant pathway in the clearance mechanism of exogenous and endogenous substances during phase II metabolism. The aim of this study was the synthesis of benzodiazepine-O-glucuronides as analytical reference substances. All benzodiazepines are prescribed clinically as racemic formulations. The resulting conjugates from the coupling reactions with glucuronic acid are epimeric pairs of glucuronides. Due to the importance of stereochemical factors in drug disposition it is necessary to separate the diastereomeric forms after synthesis. An enzyme-assisted synthesis was developed and optimized by using microsomal UGT from fresh swine liver to receive multimilligram amounts of the benzodiazepine glucuronides, which were not accessible by standard synthetic procedures, like the Koenigs-Knorr- and Williamson-ether-synthesis. Swine liver microsomes were prepared by homogenization and differential centrifugation of liver tissue. In the presence of liver microsomes the benzodiazepines and cofactor UDPGA were incubated for 24h. After incubation the microsomes were removed by protein precipitation and the residual benzodiazepines by liquid-liquid extraction (dichloromethane). The epimeric pairs of benzodiazepine glucuronides were separated by preparative high performance liquid chromatography (HPLC) followed by solid phase extraction (SPE) to obtain the pure benzodiazepine glucuronide epimers. The synthesis products were characterized by mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy.