Analysis of doping control samples using supercritical fluid chromatography-tandem mass spectrometry: Ready for routine use

Supercritical fluid chromatography is proving to be a good separation and sample preparation tool for various analytical applications and, as such, has gained the attention of the anti-doping community. Here, the applicability of supercritical fluid chromatography hyphenated to tandem mass spectrometry for routine doping control analysis was tested. A multi-analyte method was developed to cover 197 drugs and metabolites that are prohibited in sport. More than 1000 samples were analyzed by applying a “dilute and inject” approach after hydrolysis of glucuronide metabolites. Additionally, a comparison with routinely used liquid chromatography-mass spectrometry was performed with 250 of the 1000 samples and a number of past positive anti-doping samples. It revealed some features where supercritical fluid chromatography-tandem mass spectrometry was found to be complementary or advantageous to liquid chromatography-mass spectrometry for anti-doping purposes, such as better retention of analytes that are poorly retained in reversed-phase liquid chromatography. Our results suggest that supercritical fluid chromatography-tandem mass spectrometry is sensitive (limit of detection <50% relevant minimum required performance level required by the World Anti-Doping Agency for anti-doping analysis), reproducible, robust, precise (analytes of interest area coefficient of variation <5%; retention time difference coefficient of variation <1%) and complementary to existing techniques currently used for routine analysis in the World Anti-Doping Agency accredited laboratories

Automated Real-Time Tumor Pharmacokinetic Profiling in 3D Models: A Novel Approach for Personalized Medicine

Cancer treatment often lacks individual dose adaptation, contributing to insufficient efficacy and severe side effects. Thus, personalized approaches are highly desired. Although various analytical techniques are established to determine drug levels in preclinical models, they are limited in the automated real-time acquisition of pharmacokinetic profiles. Therefore, an online UHPLC-MS/MS system for quantitation of drug concentrations within 3D tumor oral mucosa models was generated. The integration of sampling ports into the 3D tumor models and their culture inside the autosampler allowed for real-time pharmacokinetic profiling without additional sample preparation. Docetaxel quantitation was validated according to EMA guidelines. The tumor models recapitulated the morphology of head-and-neck cancer and the dose-dependent tumor reduction following docetaxel treatment. The administration of four different docetaxel concentrations resulted in comparable courses of concentration versus time curves for 96 h. In conclusion, this proof-of-concept study demonstrated the feasibility of real-time monitoring of drug levels in 3D tumor models without any sample preparation. The inclusion of patient-derived tumor cells into our models may further optimize the pharmacotherapy of cancer patients by efficiently delivering personalized data of the target tissue

Single-gene resolution of diversity-driven overyielding in plant genotype mixtures

In plant communities, diversity often increases productivity and functioning, but the specific underlying drivers are difficult to identify. Most ecological theories attribute positive diversity effects to complementary niches occupied by different species or genotypes. However, the specific nature of niche complementarity often remains unclear, including how it is expressed in terms of trait differences between plants. Here, we use a gene-centred approach to study positive diversity effects in mixtures of natural Arabidopsis thaliana genotypes. Using two orthogonal genetic mapping approaches, we find that between-plant allelic differences at the AtSUC8 locus are strongly associated with mixture overyielding. AtSUC8 encodes a proton-sucrose symporter and is expressed in root tissues. Genetic variation in AtSUC8 affects the biochemical activities of protein variants and natural variation at this locus is associated with different sensitivities of root growth to changes in substrate pH. We thus speculate that - in the particular case studied here - evolutionary divergence along an edaphic gradient resulted in the niche complementarity between genotypes that now drives overyielding in mixtures. Identifying genes important for ecosystem functioning may ultimately allow linking ecological processes to evolutionary drivers, help identify traits underlying positive diversity effects, and facilitate the development of high-performance crop variety mixtures

Analysis of cobalt for human sports drug testing purposes using ICP- and LC-ICP-MS

Due to the current demands in the fight against manipulation of blood and blood components, commonly referred to as blood doping in sports drug testing, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. Similar to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport, aiming at improving an athlete's aerobic capacity and endurance performance. Amongst other strategies, the administration of ionic cobalt (Co2+) can increase the number of erythrocytes by stimulating the endogenous erythropoietin (EPO) biosynthesis. Conversely, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, an analytical differentiation of permitted and banned contributions to urinary Co-concentrations is desirable. An excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days. Urine, plasma, and concentrated red blood cells were analyzed for their cobalt content. The samples were collected starting 7 days before the administration until 7 days after. Total Co concentrations were analyzed by using inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake. Furthermore, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. The analytical approach offers the option to further develop detection methods of illegal Co2+ supplementation in sport

Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administration in Humans

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performance-enhancement in sports was demonstrated recently, and ecdysterone was consequently included in the Monitoring Program, to detect potential patterns of misuse in sport. Only few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, post-administration urine samples in twelve volunteers (single dose of 50 mg of ecdysterone) were analyzed using dilute-and-inject liquid-chromatography–tandem mass spectrometry. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14-deoxy-poststerone, was achieved. Ecdysterone was the most abundant analyte present in post-administration urine samples, detected for more than 2 days, with a maximum concentration (Cmax) in the 2.8–8.5 h urine (Cmax = 4.4–30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14-deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5–39.5 h (Cmax = 0.1–6.0 µg/mL) and 23.3–41.3 h (Cmax = 0.1–1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14-deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS

Microsensor for in situ flow measurements in benthic boundary layers at submillimeter resolution with extremely slow flow

The currents in the lowest few millimeters of the bottom boundary layer of lakes are highly important for the dissipation of kinetic energy and for chemical processes such as oxygen transfer into the sediment. So far, no high-resolution flow velocity profiles close to the sediment - water interface have been reported for such systems because a suitable flow meter was lacking. This article introduces a novel sensor for the measurement of extremely lowflow velocities. The sensor is based on a gas transducer surrounded by a gas reservoir. It measures the change in the partial pressure of a tracer gas outside the reservoir tip due to advective transport. The sensor is suitable for measurements of velocities smaller than 1 mm s(-1), with a spatial resolution of 100 to 250 mu m. The flow measurements prove to be insensitive to temperature changes between 5 and 15 C. The sensor is robust against relative pressure changes, and angular differences in the sensitivity can be calibrated. We present high- resolution in situ measurements at the bottom of a pre- alpine lake with shear velocities as low as 0.13 +/- 0.02 cm s(-1). The velocity profile nicely resolves the transition zone between the viscous and the logarithmic boundary layer