HepaChip-MP - a twenty-four chamber microplate for a continuously perfused liver coculture model.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

Quantification of AICAR-ribotide concentrations in red blood cells by means of LC-MS/MS.

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) arguably provides performance-enhancing properties even in the absence of physical exercise and, therefore, the substance is banned in elite sports since 2009. Due to the natural presence of AICAR in human blood and urine, uncovering the misuse by direct qualitative analysis is not possible. Entering the circulation, the riboside is immediately incorporated into red blood cells (RBCs) and transformed into the corresponding ribotide (5'-monophosphate) form. Within the present study, an analytical method was developed to determine AICAR-ribotide concentrations in RBC concentrates by means of liquid chromatography-tandem mass spectrometry. The method was validated enabling quantitative result interpretation considering the parameters specificity, precision (intra- and interday), linearity, recovery, accuracy (LOD/LOQ), stability and ion suppression. By analysing 99 RBC samples of young athletes, normal physiological levels of AICAR-ribotide were determined (10-500 ng/mL), and individual levels were found to be stable for several days. Employing in vitro incubation experiments with AICAR riboside in fresh whole blood samples, the ribotide concentrations were observed to increase significantly within 30 min from baseline to 1-10 µg/mL. These levels are considered conserved for the lifetime of the erythrocyte and, thus, the results of the in vitro model strongly support the hypothesis that measuring abnormally high AICAR-ribotide concentrations in RBC of elite athletes has the potential to uncover the misuse of this substance for a long period of time.

Expanding sports drug testing assays: mass spectrometric characterization of the selective androgen receptor modulator drug candidates RAD140 and ACP-105.

RATIONALE: Anabolic agents have been top-ranked for many years among statistics of adverse analytical findings compiled by the World Anti-Doping Agency (WADA). Besides archetypical anabolic-androgenic steroids (AAS), alternative substances with similar effects concerning bone and muscle anabolism have been therapeutically pursued. A prominent emerging class of drugs is the chemically heterogeneous group of selective androgen receptor modulators (SARMs), some of which have been detected in doping control samples between 2009 and 2012 despite missing clinical approval. METHODS: In order to support the momentum of expanding the preventive and proactive measures among anti-doping laboratories, the analytical characterization of substances with misuse potential is of great importance. In the present study, the SARM drug candidates RAD140 (comprising a 5-phenyloxadiazole nucleus) and ACP-105 (bearing an N-substituted tropanol pharmacophore) were studied regarding their mass spectrometric behavior under ESI-MS(/MS) and EI-MS(/MS) conditions. Reference material was synthesized according to established protocols and dissociation pathways of RAD140 and ACP-105 were elucidated with liquid chromatography/electrospray ionization quadrupole/time-of-flight or iontrap/orbitrap and gas chromatography/electron ionization quadrupole/time-of-flight high resolution/high accuracy mass spectrometry. RESULTS: Fragmentation pathways to diagnostic product ions of RAD140 (e.g. m/z 223 and 205 using ESI-MS/MS and m/z 421 and 349 using EI-MS/MS) and ACP-105 (such as m/z 233 and 193 or 231 and 217 for ESI-MS/MS and EI-MS/MS measurements, respectively) were proposed as substantiated by determined elemental compositions and MS(n) experiments as well as comparison to spectra of a structural analog. Notably, for the formation of the characteristic fragment ion at m/z 421 of RAD140, the comparably seldom intramolecular migration of a trimethylsilyl residue triggered by electron ionization was suggested as corroborated by all of the above-mentioned analytical means. CONCLUSIONS: The obtained data will support future sports drug testing methods and facilitate and accelerate the implementation of this analyte and related compounds or metabolites in both GC/MS(/MS)- and LC/MS(/MS)-based routine doping control procedures.

Does the analysis of the enantiomeric composition of clenbuterol in human urine enable the differentiation of illicit clenbuterol administration from food contamination in sports drug testing?

RATIONALE: Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzyl alcohol) is approved for human and veterinary use primarily for the treatment of pulmonary afflictions. Despite the authorized administration in cases of medical indications, the misuse of clenbuterol in animal husbandry as well as elite and amateur sport has frequently been reported, arguably due to growth-promoting properties. Due to various recent incidences of doping control specimens containing clenbuterol, strategies towards the discrimination of a surreptitious application from unintended intake via animal-derived edibles or dietary supplements were required. METHODS: The enantiomeric compositions of clenbuterol in human urine samples derived from administration studies with therapeutic amounts of the ß(2)-agonist and authentic doping control specimens were determined. Due to the facts that therapeutic clenbuterol consists of a racemic mixture of (+)- and (-)-stereoisomers and that the first mentioned (dextrorotatory) stereoisomer is retained to a greater extent in edible animal tissue, the differentiation of a recent administration of therapeutic (and thus racemic) clenbuterol from food contamination (stereoisomerically depleted clenbuterol) was considered. Employing deuterated clenbuterol as internal standard, the target analytes were extracted from human urine by means of concerted liquid-liquid and solid-phase extractions and subjected to chiral liquid chromatography hyphenated to high resolution/high accuracy mass spectrometry with electrospray ionization. RESULTS: Both enantiomers of clenbuterol were baseline separated and relative abundances of corresponding labeled and unlabeled stereoisomers were determined, demonstrating that the therapeutic use of clenbuterol results in racemic mixtures in urine for at least 24 h while adverse analytical findings presumably originating from food contaminations can yield (-)-clenbuterol-depleted pairs of analytes. CONCLUSIONS: The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (-)-clenbuterol in edible animal tissue is time-dependent and thus results can still be inconclusive as to the inadvertent ingestion of clenbuterol when clenbuterol administration to animals was conducted until slaughter.

Traditional Chinese medicine and sports drug testing: identification of natural steroid administration in doping control urine samples resulting from musk (pod) extracts.

The administration of musk extract, that is, ingredients obtained by extraction of the liquid secreted from the preputial gland or resulting grains of the male musk deer (eg, Moschus moschiferus), has been recommended in Traditional Chinese Medicine (TCM) applications and was listed in the Japanese pharmacopoeia for various indications requiring cardiovascular stimulation, anti-inflammatory medication or androgenic hormone therapy. Numerous steroidal components including cholesterol, 5α-androstane-3,17-dione, 5ß-androstane-3,17-dione, androsterone, etiocholanolone, epiandrosterone, 3ß-hydroxy-androst-5-en-17-one, androst-4-ene-3,17-dione and the corresponding urea adduct 3α-ureido-androst-4-en-17-one were characterised as natural ingredients of musk over several decades, implicating an issue concerning doping controls if used for the treatment of elite athletes. In the present study, the impact of musk extract administration on sports drug testing results of five females competing in an international sporting event is reported. In the course of routine doping controls, adverse analytical findings concerning the athletes' steroid profile, corroborated by isotope-ratio mass spectrometry (IRMS) data, were obtained. The athletes' medical advisors admitted the prescription of TCM-based musk pod preparations and provided musk pod samples for comparison purposes to clarify the antidoping rule violation. Steroid profiles, IRMS results, literature data and a musk sample obtained from a living musk deer of a local zoo conclusively demonstrated the use of musk pod extracts in all cases which, however, represented a doping offence as prohibited anabolic-androgenic steroids were administered.

Synthesis, mass spectrometric characterization, and analysis of the PPARδ agonist GW1516 and its major human metabolites: targets in sports drug testing.

The elucidation of metabolic pathways and the detection of emerging therapeutics potentially enhancing athletic performance are of paramount importance to doping control authorities to protect the integrity of elite sports. A new drug candidate belonging to the family of the peroxisome proliferator-activated receptor-delta agonists termed GW1516 (also referred to as GW501516) has been prohibited by the World Anti-Doping Agency in 2009 due to its potential to artificially increase endurance. Consequently, sports drug testing laboratories need to establish detection methods enabling the identification of the intact substance and/or its metabolite(s) that unambiguously prove the presence or absence of the target substances in doping control specimens. Simulating human metabolic reactions using liver microsomal preparations, minute amounts of possible urinary metabolites were obtained that were characterized by mass spectrometry-based methods. Subsequently, the most abundant metabolic products were chemically synthesized and as well characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Finally, GW1516 and two oxidized metabolites were implemented in a routine doping control analytical assay based on liquid chromatography-(tandem) mass spectrometry (LC-MS/MS), which was tested for its -fitness-for-purpose using spiked urine samples.

Hypoxia-inducible factor stabilizers and other small-molecule erythropoiesis-stimulating agents in current and preventive doping analysis.

Increasing the blood's capacity for oxygen transport by erythropoiesis-stimulating agents (ESAs) constitutes a prohibited procedure of performance enhancement according to the World Anti-Doping Agency (WADA). The advent of orally bio-available small-molecule ESAs such as hypoxia-inducible factor (HIF) stabilizers in the development of novel anti-anaemia therapies expands the list of potential ESA doping techniques. Here, the erythropoiesis-stimulating properties and doping relevance of experimental HIF-stabilizers, such as cobaltous chloride, 3,4-dihydroxybenzoic acid or GSK360A, amongst others, are discussed. The stage of clinical trials is reviewed for the anti-anaemia drug candidates FG-2216, FG-4592, GSK1278863, AKB-6548, and BAY85-3934. Currently available methods and strategies for the determination of selected HIF stabilizers in sports drug testing are based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). For the support of further analytical assay development, patents claiming distinct compounds for the use in HIF-mediated therapies are evaluated and exemplary molecular structures of HIF stabilizers presented. Moreover, data concerning the erythropoiesis-enhancing effects of the GATA inhibitors K7174 and K11706 as well as the lipidic small-molecule ESA PBI-1402 are elucidated the context of doping analysis.

Structure elucidation of the diagnostic product ion at m/z 97 derived from androst-4-en-3-one-based steroids by ESI-CID and IRMPD spectroscopy.

Structure elucidation of steroids by mass spectrometry has been of great importance to various analytical arenas and numerous studies were conducted to provide evidence for the composition and origin of (tandem) mass spectrometry-derived product ions used to characterize and identify steroidal substances. The common product ion at m/z 97 generated from androst-4-ene-3-one analogs has been subject of various studies, including stable isotope-labeling and (high resolution/high accuracy) tandem mass spectrometry, but its gas-phase structure has never been confirmed. Using high resolution/high accuracy mass spectrometry and low resolution tandem mass spectrometry, density functional theory (DFT) calculation, and infrared multiple photon dissociation (IRMPD) spectroscopy employing a free electron laser, the structure of m/z 97 derived from testosterone was assigned to protonated 3-methyl-2-cyclopenten-1-one. This ion was identified in a set of six cyclic C(6)H(9)O(+) isomers as computed at the B3LYP/6-311++G(2d,2p) level of theory (protonated 3-methyl-2-cyclopenten-1-one, 2-methyl-2-cyclopenten-1-one and 2-cyclohexen-1-one). Product ions of m/z 97 obtained from MS(2) and MS(3) experiments of protonated 3-methyl-2-cyclopenten-1-one, 2-methyl-2-cyclopenten-1-one, 2-cyclohexen-1-one, and testosterone corroborated the suggested gas-phase ion structure, which was eventually substantiated by IRMPD spectroscopy yielding a spectrum that convincingly matched the predicted counterpart. Finally, the dissociation pathway of the protonated molecule of testosterone to m/z 97 was revisited and an alternative pathway was suggested that considers the exclusion of C-10 along with the inclusion of C-5, which was experimentally demonstrated with stable isotope labeling.

Determination of salbutamol and salbutamol glucuronide in human urine by means of liquid chromatography-tandem mass spectrometry.

The determination of salbutamol and its glucuronide in human urine following the inhalative and oral administration of therapeutic doses of salbutamol preparations was performed by means of direct urine injection utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and employing d(3)-salbutamol and d(3)-salbutamol glucuronide as internal standards. Unconjugated salbutamol was detected in all administration study urine samples. Salbutamol concentrations following inhalation were commonly (99%) below 1000 ng/ml whereas values after oral administration frequently (48%) exceeded this threshold. While salbutamol glucuronide was not detected in urine samples collected after inhalation of the drug, 26 out of 82 specimens obtained after oral application contained salbutamol glucuronide with a peak value of 63 ng/ml. The percentage of salbutamol glucuronide compared to unconjugated salbutamol was less than 3%. Authentic doping control urine samples indicating screening results for salbutamol less than 1000 ng/ml, showed salbutamol glucuronide concentrations between 2 and 6 ng/ml, whereas adverse analytical findings resulting from salbutamol levels higher than 1000 ng/ml, had salbutamol glucuronide values between 8 and 15 ng/ml. The approach enabled the rapid determination of salbutamol and its glucuronic acid conjugate in human urine and represents an alternative to existing procedures since time-consuming hydrolysis or derivatization steps were omitted. Moreover, the excretion of salbutamol glucuronide in human urine following the administration of salbutamol was proven.

Structure characterisation of urinary metabolites of the cannabimimetic JWH-018 using chemically synthesised reference material for the support of LC-MS/MS-based drug testing.

As recently reported, the synthetic cannabinoid JWH-018 is the subject of extensive phase I and II metabolic reactions in vivo. Since these studies were based on LC-MS/MS and/or GC-MS identification and characterisation of analytes, the explicit structural assignment of the metabolites was only of preliminary nature, if possible at all. Here, we report the chemical synthesis of five potential in vivo metabolites of JWH-018 derivatives featuring an alkylcarboxy (M1), a terminal alkylhydroxy (M2), a 5-indolehydroxy (M3), an N-dealkylated 5-indolehydroxy (M4) and a 2'-naphthylhydroxy (5) analogue, respectively, and their characterisation by nuclear magnetic resonance spectroscopy. The collision-induced dissociation (CID) patterns of the protonated compounds were studied by high-resolution/high-accuracy tandem mass spectrometry (MS( n )) applying an LTQ Orbitrap with direct infusion and electrospray ionisation of target analytes. An unusual dissociation behaviour including a reversible ion-molecule reaction between a naphthalene cation (m/z 127) and water in the gas phase of the MS was shown to be responsible for nominal neutral losses of 10 u in the course of the CID pathway. LC-MS/MS-supported comparison of synthesised reference standards with an authentic urine sample using an API 4000 QTrap mass spectrometer identified the synthetic JWH-018 analogues M1-M4 as true in vivo metabolites, presuming a chromatographic separation of potentially present regioisomeric analogues. Existing doping control methods were expanded and validated according to international guidelines in order to allow for the detection of the carboxy and the alkylhydroxy metabolites, respectively, as urinary markers for the illegal intake of the synthetic cannabinoid JWH-018. Both metabolites were quantified in authentic doping control urine samples that had been suspicious of JWH-018 abuse after routine screening procedures, and a stable isotope-labelled (13)C(8)-(15)N-carboxy metabolite was synthesised for future analytical applications.

Screening for benfluorex and its major urinary metabolites in routine doping controls.

Benfluorex [1-(m-trifluoromethylphenyl)-2-(ß-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as well as liquid chromatography-electrospray ionization-high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid-liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48-54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5-8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.

Development of liquid chromatography-tandem mass spectrometry-based analytical assays for the determination of HIF stabilizers in preventive doping research.

Hypoxia-inducible factor (HIF) stabilizers increase blood haemoglobin levels after oral administration and their use in sports was recently banned by the World Anti-Doping Agency. For the support of analytical assay development, the metabolic fate of two model HIF stabilizers, based on the isoquinoline-3-carboxamide scaffold of the lead drug candidate FG-2216, was assessed by in vitro methods. The analytes were identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative ionization mode using an API 4000 Qtrap as well as an exactive high resolution-high accuracy MS. The model HIF stabilizer N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-carbonyl)-amino]-acetic acid (1), was converted into 3 major phase I metabolites by hydroxylation, dealkylation, and dehydrogenation. The structures of the hydroxylated and the dealkylated metabolites were confirmed by LC-coupled nuclear magnetic resonance spectroscopy. Moreover, glucuronic acid conjugates of the active drug and one of the dealkylated phase I metabolite were identified. Hydroxylation of model compound 2 (N-[(1-chloro-4-hydroxy-isoquinolin-3-carbonyl)-amino]-acetic acid) yielded two metabolites, regioisomeric to the dealkylated product of 1. Mass spectral data of compounds 1 and 2, as well as a structure-related analogue were included into a multi-target analytical assay based on direct injection and LC-MS/MS analysis of human urine. The method was validated for quantitative purposes. In an approach of preventive doping research, more comprehensive screening methods applying precursor ion (m/z 166) and neutral loss (-10 Da) scans were developed, allowing for the detection of unknown metabolites and structurally analogous HIF stabilizers emerging from ongoing lead structure developments.

Characterization of in vitro generated metabolites of the selective androgen receptor modulators S-22 and S-23 and in vivo comparison to post-administration canine urine specimens.

Selective androgen receptor modulators (SARMs) have great therapeutic potential in various diseases including cancer cachexia, sarcopenia, and osteoporosis, and the number of drug candidates has been growing over the last decade. The SARM drug candidates S-22 and S-23 belong to one of the most advanced groups of androgen receptor modulators and are based on an arylpropionamide-derived core structure. Due to their anabolic effects, SARMs have been prohibited in elite sports and have been a subject of sports drug testing programmes since January 2008. Consequently, the structure of analytically useful urinary metabolites should be elucidated to provide targets for sensitive and retrospective analysis. In the present study, the phase-I and -II metabolism of S-22 and S-23 was simulated using hepatic human enzymes, and resulting metabolites were characterized by means of state-of-the-art mass spectrometric approaches employing high resolution/high accuracy Orbitrap mass spectrometry. Subsequently, the newly defined target compounds including the glucuronic acid conjugates of S-22 and S-23, their corresponding monohydroxylated and bishydroxylated analogs, as well as their B-ring depleted counterparts were implemented into an existing routine doping control procedure, which was examined for its specificity for the added substances. In order to obtain proof-of-concept data for authentic urine specimens, canine urine samples collected up to 72 h after oral administration of S-22 to dogs were analyzed using the established approach outlining the capability of the presented assay to detect the glucuronide of S-22 as well as the B-ring-depleted metabolite (M3) in all samples following therapeutic (31.4 µg/kg) dosing. Finally, M3 was chemically synthesized, characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry, and chosen as primary target for future doping control analyses.

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S-4) for routine doping control purposes.

Selective androgen receptor modulators (SARMs) are potent anabolic agents with tissue-selective properties. Due to their potential misuse in elite sport, the World Anti-Doping Agency (WADA) has prohibited SARMs since 2008, and although no representative drug candidate has yet received full clinical approval, recent findings of SARMs illegally sold via the internet have further supported the need to efficiently test for these compounds in doping controls. In the present communication, the mass spectrometric characterization of urinary metabolites of the SARM Andarine (also referred to as S-4) compared with earlier in vitro and animal studies is reported. Liquid chromatography interfaced to high-resolution/high-accuracy (tandem) mass spectrometry was used to identify phase I and II metabolites, confirming the predicted target analytes for sports drug testing purposes including the glucuronic acid conjugates of the active drug, its monohydroxylated and/or deacetylated product, the hydrolysis product resulting from the removal of the compound's B-ring, as well as the sulfate of the monohydroxylated and the deacetylated phase I metabolite. The obtained data will support future efforts to effectively screen for and confirm the misuse of the non-approved drug candidate Andarine.

Mass spectrometry-based characterization of new drugs and methods of performance manipulation in doping control analysis.

Efficient and comprehensive sports drug testing necessitates frequent updating and proactive, preventive anti-doping research, and the early implementation of new, emerging drugs into routine doping controls is an essential aspect. Several new drugs and drug candidates with potential for abuse, including so-called Rycals (ryanodine receptor calstabin complex stabilizers, for example, S-107), hypoxia-inducible factor (HIF) stabilizers, and peroxisome-proliferator-activated receptor (PPAR) delta agonists (for example, GW1516), were studied using different mass spectrometry- and ion mobility-based approaches, and their gas phase dissociation behaviors were elucidated. The detailed knowledge of fragmentation routes allows a more rapid identification of metabolites and structurally related, presumably "tailor-made", analogs potentially designed for doping purposes. The utility of product ion characterization is demonstrated in particular with GW1516, for which oxidation products were readily identified in urine samples by means of diagnostic fragment ions as measured using high resolution/high accuracy mass spectrometry and higher energy collision-induced dissociation (HCD).

Quantification of urinary AICAR concentrations as a matter of doping controls.

Influencing the endurance in elite sports is one of the key points in modern sports science. Recently, a new class of prohibited substances reached in the focus of doping control laboratories and their misuse was classified as gene doping. The adenosine monophosphate activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance even in sedentary mice after treatment. Due to endogenous production of AICAR in healthy humans, considerable amounts were present in the circulation and, thus, were excreted into urine. Considering these facts, the present study was initiated to fix reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 499 athletes were analysed, and AICAR concentrations in urine were determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of sport and type of sample collection (in competition/out of competition). The method was fully validated for quantitative purposes considering the parameters linearity, inter- (12%, 7% and 10%) and intraday precision (14%, 9% and 12%) at low, mid and high concentration, robustness, accuracy (approx. 100%), limit of quantification (100 ng/mL), stability and ion suppression effects, employing an in-house synthesised (13)C(5)-labelled AICAR as internal standard.

Metalloproteinase mediated occludin cleavage in the cerebral microcapillary endothelium under pathological conditions.

Reactive oxygen species (ROS) as well as matrix metalloproteinases (MMPs) induce modifications in the tight junction (TJ) protein occludin which is crucial for the blood-brain barrier (BBB) function. We investigated the role of ROS and MMPs in endothelial autoregulatory response on oxidative stress with respect to occludin and the BBB integrity. The ROS hydrogen peroxide (H(2)O(2)) was applied to our well-established BBB cell culture model based on primary porcine brain capillary endothelial cells (PBCEC). At low concentrations (2.5 mM), H(2)O(2)-induced barrier impairment correlated with an altered occludin phosphorylation. At high, cell toxic H(2)O(2) concentrations (>or=10 mM) occludin cleavage occurred and elevated levels of active MMP-2 were detected. Under those conditions intercellular gaps were formed within the monolayer visualizing the barrier breakdown also determined by impedance analysis. A primary structure sequence analysis revealed potential type IV collagenase sensitive motives in the first extracellular loop, thus providing evidence that occludin might be an MMP-2 substrate. MMP inhibition by the metalloproteinase inhibitor GM6001 prevented occludin degradation and reduced the intercellular gap formation. However, the barrier function quantified by impedance measurement could not be maintained despite MMP inhibition. When we applied an enzymatic activity level which caused occludin cleavage in injured PBCEC to intact PBCEC, neither occludin cleavage nor barrier impairment was observed. Thus, in our model occludin cleavage is not an autoregulatory mechanism of microcapillary endothelium in barrier modulation under oxidative stress, but only occurs upon endothelial damage.

Characterization of two major urinary metabolites of the PPARdelta-agonist GW1516 and implementation of the drug in routine doping controls.

Since January 2009, the list of prohibited substances and methods of doping as established by the World Anti-Doping Agency includes new therapeutics such as the peroxisome-proliferator-activated receptor (PPAR)-delta agonist GW1516, which is categorized as a gene doping substance. GW1516 has completed phase II and IV clinical trials regarding dyslipidemia and the regulation of the lipoprotein transport in metabolic syndrome conditions; however, its potential to also improve athletic performance due to the upregulation of genes associated with oxidative metabolism and a modified substrate preference that shifted from carbohydrate to lipid consumption has led to a ban of this compound in elite sport. In a recent report, two presumably mono-oxygenated and bisoxygenated urinary metabolites of GW1516 were presented, which could serve as target analytes for doping control purposes after full characterization. Hence, in the present study, phase I metabolism was simulated by in vitro assays employing human liver microsomal fractions yielding the same oxygenation products, followed by chemical synthesis of the assumed structures of the two abundant metabolic reaction products. These allowed the identification and characterization of mono-oxygenated and bisoxygenated metabolites (sulfoxide and sulfone, respectively) as supported by high-resolution/high-accuracy mass spectrometry with higher-energy collision-induced dissociation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Since urine samples have been the preferred matrix for doping control purposes, a method to detect the new target GW1516 in sports drug testing samples was developed in accordance to conventional screening procedures based on enzymatic hydrolysis and liquid-liquid extraction followed by liquid chromatography, electrospray ionization, and tandem mass spectrometry. Validation was performed for specificity, limit of detection (0.1 ng/ml), recovery (72%), intraday and interday precisions (7.7-15.1%), and ion suppression/enhancement effects (<10%).

Unusual mass spectrometric dissociation pathway of protonated isoquinoline-3-carboxamides due to multiple reversible water adduct formation in the gas phase.

The study of the collision-induced dissociation behavior of various substituted isoquinoline-3-carboxamides, which are amongst a group of drug candidates for the treatment of anemic disorders (e.g., FG-2216), allowed for the formulation of the general mechanisms underlying the unusual fragmentation behavior of this class of compounds. Characterization was achieved with high-resolution/high accuracy LTQ-Orbitrap tandem mass spectrometry of the protonated precursor ions. Presented data were substantiated by the synthesis and analysis of proposed gas-phase intermediate structures and stable isotope labeled analogues, as well as by density functional theory calculations. Exemplary, CID of protonated N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-yl)carbonyl]glycine gives rise to the isoquinoline-3-carboxy-methyleneamide product ion which nominally eliminates a fragment of 11 u. This was attributed to the loss of methyleneamine (-29 u) and a concomitant spontaneous and reversible water addition (+18 u) to the resulting acylium ion to yield the protonated isoquinoline-3-carboxylic acid. The same water addition pattern is observed after loss of propylene (-42 u). A further nominal loss of 10 u is explained by the elimination of carbon monoxide (-28 u) followed by another water adduct formation (+18 u) to yield the protonated 1-chloro-3,4,7-trihydroxy-isoquinoline. The source of the multiple gas-phase water addition remained unclear. This atypical fragmentation pattern proved to be highly characteristic for all studied and differentially substituted isoquinoline-3-carboxamides, and offers powerful analytical tools for the establishment of a LC/MS(/MS) based screening procedure for model HIF-stabilizers and their potential metabolites in clinical, forensic and sports drug testing.

Electron ionization mass spectrometry of the ryanodine receptor-based Ca(2+)-channel stabilizer S-107 and its implementation into routine doping control.

New insights into the biochemistry of cardiac arrhythmia and skeletal muscle fatigue have yielded new drug candidates to counteract these phenomena. Major biological targets have become ryanodine receptor (RyR)-based Ca(2+)-release channels, which tend to 'leak' under various circumstances including strenuous exercise and, thus, cause aberrant calcium sparks that entail impaired muscle function. Therapeutics, which are referred to as rycals, are currently being developed to treat cardiac arrhythmia via enhancement of calstabin-ryanodine affinities that causes a stabilization of the RyR. These therapeutics possess potential for misuse in sports, and an early implementation of target analytes such as the benzothiazepine derivatives S-107 and JTV-519 or putative metabolites into doping control screening procedures is recommended. Reference compounds, deuterated analogues, and a putative metabolic product were synthesized, and electron ionization mass spectra of these products were studied and dissociation pathways elucidated by means of tandem mass spectrometry (MS/MS) and accurate mass measurements. The characterized analytes were incorporated into existing sports drug testing assays based on liquid-liquid extraction and subsequent gas chromatography/mass spectrometry (GC/MS) analysis, and specificity, lower limit of detection (4-6 ng/mL), intraday and interday precision (1.5-17.2%), as well as recovery (63-66%) were determined. The established procedure proved suitable for routine doping control analysis to detect a potential misuse of the drug candidate S-107 in elite sport.