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Caffeine is an ergogenic substance that is consumed globally in many forms. The use of buccally absorbable formulations instead of gastrointestinal uptake has become increasingly popular over the years, especially when accelerated absorption with minimal gastrointestinal stress is desired. This study investigated the impact of five different formulations and administration routes of caffeine on the whole blood concentrations of caffeine, paraxanthine, and theobromine: caffeinated capsules, tablets, shots, pouches, and chewing gums. A uniform dose of caffeine (200 mg) was administered to 16 healthy recreational athletes (26.0 ± 2.1 years) using a randomized crossover design. Samples were taken in the form of dried blood spots at 16 different time points in a 2-hr timeframe after drug administration. The samples were analyzed using a validated liquid chromatography–tandem mass spectrometry method. The results for caffeine showed no significant differences in the overall bioavailability (area under the concentration–time curve), maximal concentration, and time to maximum concentration. However, when analyzing the bioavailability of caffeine in the first 5, 10, and 15 min, the liquid caffeine formulation was superior to other administered forms (p <.05). This indicates that caffeine solubility has a major influence on its absorption rate. In sports, the rate of caffeine absorption must be considered, not only when ingesting anhydrous caffeine, but also when choosing buccal absorption. These findings imply that general guidelines for ergogenic caffeine use should consider the formulation used and, accordingly, the corresponding route of absorption. [ABSTRACT FROM AUTHOR]

Peptides with a molecular mass between 2 and 10 kDa that are prohibited in elite sports usually require dedicated sample preparation and mass spectrometric detection that commonly cannot be combined with other (lower molecular mass) substances. In most instances, the physicochemical differences are too significant to allow for a generic analytical procedure. A simplification of established and comparably complex analytical approaches is therefore desirable and has been accomplished in the context of this study. With urine samples representing still the most frequently collected doping control specimens, efficient extraction of peptidic analytes from this matrix was a major goal of this method, as demonstrated for the included compounds such as insulins (human, lispro, aspart, glulisine, tresiba, glargine metabolite, bovine insulin, porcine insulin), growth hormone‐releasing hormones (sermorelin, CJC‐1295, tesamorelin) incl. their respective metabolites, insulin‐like‐growth factors (long‐R3‐IGF‐I, R3‐IGF‐I, des1–3‐IGF‐I), synacthen, gonadorelin and mechano growth factors (human MGF, MGF‐Goldspink). Sample preparation and detection are controlled by five internal standards, covering all five included peptide drug categories. Nearly all requirements of the recent technical documents from the World Anti‐Doping Agency (WADA) considering their minimum required performance levels (MRPL) are fulfilled, and the method was validated for its utilisation as initial testing procedure in doping controls. Finally, the approach was applied to authentic post‐administration study urine samples (for insulins and gonadorelin) in order to provide proof of principle. [ABSTRACT FROM AUTHOR]

This study aimed to reveal correlation, calculate significance, and discover the regression equation of rice production to Green House Gas (GHG) emission in North Sulawesi Province. The data on GHG emissions from rice cultivation (Gg CO2eq) was obtained from the Ministry of Environment and Forestry of Indonesia. Data on rice production from wetland and dryland (Gg) was from the BP Statistical Review annual period of 2000-2021, both for North Sulawesi Province. Data analysis of correlation coefficient, F-test for Regression, and Simple Regression Analysis will be processed with the help application of MS Excel. The results show that the correlation between rice production and emission of rice cultivation in North Sulawesi Province is 0.53 and classified as a moderate correlation. The coefficient of determination stated that the emission of rice cultivation could be explained by about 28.6% from rice production. Therefore, rice production is statistically significant to the emission of rice cultivation with a 5% confidence level for North Sulawesi Province. Furthermore, this study found a regression equation, emission of rice cultivation is 112.67 + 0.516 times rice production. [ABSTRACT FROM AUTHOR]

The article explores the question whether the way in which Juri Lotman uses the categories of semiotic explosion and unpredictability enables and necessitates the need to give space not only to different descriptions, but also to various self-descriptions (auto-communications) of religion in culture. In other words, the question is posed whether his concept of the semiosphere aids in making sense of the synchronic and diachronic contradictions and controversies in religion - both within what is perceived as a religion and between what are understood to be religions. The focus lies especially on whether Lotman's semiotic theory of culture has a potential of advancing mutual recognition and supportive respect, that is - solidaristic tolerance between different religious and non-religious ways of being human. The claim is made that this is indeed the case as he models the human situation so that the 'outside of the system' is not understood only as a continuation of reality, but visualizes and includes within the system a space for a radical intrusion of possibility - for the truly unpredictable - on the level of culture, humanity, as well as the individual. Therefore, Lotman's theory of culture could be developed further, in the direction of a translation device between different cultures (of religion), opening up new perspectives for dealing with the challenging semiotic situation in which we all find ourselves living in today. [ABSTRACT FROM AUTHOR]

An introduction is presented on the role of religion in the semiosphere, exploring the dynamic nature of religious texts and their importance in the context of Juri Lotman's semiotic theory.

Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes' urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033—substances listed under anabolic substances (S1) on the World Anti-Doping Agency's Prohibited List—in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02–0.40 ng/mL) and in sf (0.01–0.25 ng/mL) as well as of LGD-4033 in bp (0.21–2.00 ng/mL) and in sf (0.03–0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine. [ABSTRACT FROM AUTHOR]

Rationale: Exhaled breath (EB) was found to be a promising matrix in the field of sports drug testing due to the non-invasive and non-intrusive sampling procedure, but significant inter-individual variations regarding detected drug concentrations have been observed in previous studies. To investigate whether the detectability of doping agents in EB is affected by sex or tobacco smoking, two administration studies were conducted with male and female smokers and nonsmokers concerning the elimination of the beta blocker propranolol and the stimulant pseudoephedrine into EB. Methods: Following the administration of 40 mg propranolol or 30 mg pseudoephedrine, a total of 19 participants, including female and male nonsmokers as well as female and male smokers, collected EB and dried blood spot (DBS) samples over a period of 24 h. Respective analyte concentrations were determined using liquid chromatography and high-resolution tandem mass spectrometry, and semiquantitative assays were characterized with regard to selectivity, limit of detection and identification, precision, linearity, and carryover. Results: Both propranolol and pseudoephedrine were identified in postadministration EB samples from female and male nonsmokers as well as female and male smokers, and the maximum detected drug levels ranged from 9 to 2847 pg/ cartridge for propranolol and from 26 to 4805 pg/cartridge for pseudoephedrine. The corresponding DBS levels were in a range of 4-30 ng/mL for propranolol and 55-186 ng/mL for pseudoephedrine. Conclusions: Neither the consumption of cigarettes nor the sex appears to represent a decisive criterion as to the detectability of propranolol or pseudoephedrine in EB, but inter-individual variations regarding the detected drug levels were observed among all studied population groups. [ABSTRACT FROM AUTHOR]

Aims: Current evidence of the impact of acute exercise on insulin levels in individuals with type 1 diabetes remains controversial. Therefore, we conducted a systematic review and meta‐analysis to explore exercise‐induced changes in insulin levels. Materials and Methods: We conducted a systematic review (until 05 November 2023) and meta‐analysis exploring the effect of exercise on insulin concentration in individuals with type 1 diabetes. We included randomised cross‐over studies for rapid‐acting insulin and pre‐ and post‐studies for long‐acting insulin in individuals with type 1 diabetes performing any type of acute exercise and had a control condition. The exercise‐induced change in insulin levels was the outcome of interest. When possible, the mean differences (MDs) in insulin levels were pooled using the DerSimonian and Laird random effect method. Risk of bias was assessed for each included study. Results: Seventeen trials, encompassing 186 participants with type 1 diabetes, were included in the systematic review. Twelve out of 17 studies included participants on rapid‐acting insulin regimens and used a cross‐over design, whereas five out of 17 single‐arm studies included participants on (ultra)long‐acting insulin. Seven out of 12 studies on rapid‐acting insulins and all the single‐arm studies were at high risk of bias. Results suggest a statistically significant, small‐to‐moderate increase of rapid‐acting insulin after 30 min of exercise (MD of 18.44 [95% CI 0.02; 36.86; I2 0%] pmol/L); meanwhile, findings on (ultra)long‐acting insulin were inconclusive. Conclusions: A small‐to‐moderate increase of insulin levels in studies including rapid‐acting insulin was found after a bout of physical exercise in individuals with type 1 diabetes. However, current gaps in high‐quality evidence challenge our understanding of insulin kinetics around exercise. [ABSTRACT FROM AUTHOR]

Rationale: Exhaled breath (EB) has been demonstrated to be a promising alternative matrix in sports drug testing due to its non-invasive and non-intrusive nature compared with urine and blood collection protocols. In this study, a pilot-test system was employed to create drug-containing aerosols simulating EB in support of the analytical characterization of EB sampling procedures, and the used analytical method was extended to include a broad spectrum of prohibited substances. Methods: Artificial and authentic EB samples were collected using sampling devices containing an electret filter, and doping agents were detected by means of liquid chromatography and tandem mass spectrometry with unispray ionization. The analytical approach was characterized with regard to specificity, limits of detection, carry-over, recovery and matrix effects, and the potential applicability to routine doping controls was shown using authentic EB samples collected after single oral dose applications of glucocorticoids and stimulants. Results: The analytical method was found to be specific for a total of 49 model substances relevant in sports drug testing, with detection limits ranging from 1 to 500 pg per cartridge. Both ion suppression (-62%) and ion enhancement (+301%) effects were observed, and all model compounds applied to EB sampling devices were still detected after 28 days of storage at room temperature. Authentic EB samples collected after the oral administration of 10 mg of prednisolone resulted in prednisolone findings in specimens obtained from 3 out of 6 participants up to 2 h. In octodrine, dimethylamylamine (DMAA) and isopropylnorsynephrine post-administration EB samples, the drugs were detected over a period of 50, 48, and 8 h, respectively. Conclusions: With the analytical approach developed within this study, the identification of a broad spectrum of prohibited doping agents in EB samples was accomplished. Application studies and stability tests provided information to characterize EB as a potential matrix in sports drug testing. [ABSTRACT FROM AUTHOR]

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone–releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized "hematocrit effect." [ABSTRACT FROM AUTHOR]

Background: Peptide-derived drugs represent an emerging class of prohibited substances in professional sports and, thus, in modern doping controls. After parental administration (e.g. subcutaneous, intravenous), these drugs undergo various metabolic processes, which degrade them to biologically active or inactive peptides. Knowledge about these metabolic processes and the hereby produced metabolites plays a key role in successful doping controls due to the effective design of analytical assays under consideration of optimal analytical targets. Unfortunately, the complexity of biological matrix (e.g. blood or urine) complicates the immediate identification of relevant metabolites due to the enormous excess of naturally occurring peptides and their degradation products. Methods: In this study, a strategy employing in-vitro metabolism of stable isotope-labeled peptides producing characteristic reporter ions derived from labeled immonium ions is shown. The in-vitro experiments were performed with human skin tissue microsomes (S9), and model drugs representing prohibited peptide hormones were synacthen, insulin, and corticorelin (respectively, their stable isotope-labeled analogs). After generic sample preparation, the metabolites were identified by means of liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS) in an untargeted approach. Results and conclusions: For all three model peptides, several metabolic products were readily identified. While insulin and corticorelin were found to be comparably stable, synacthen was fully degraded, yielding a plethora of metabolic products. A proof of concept concerning the transferability of the obtained data was accomplished by analyzing plasma samples collected post-administration of recombinant human insulin, corroborating the presence of a skin protease-indicative insulin metabolite in vivo. [ABSTRACT FROM AUTHOR]

Rationale: The mitochondrial open reading frame of 12S rRNA type‐c (MOTS‐c) peptide was recently discovered and described to control metabolic homeostasis through AMPK activation along with AICAR accumulation. Consequently, it appears advisable to monitor the potential use of synthetic MOTS‐c in sports, and a detection method suitable for sports drug testing purposes is necessary. Methods: For the detection of MOTS‐c in doping control plasma samples, a test method employing liquid chromatography and mass spectrometry (LC/MS) was developed. Following optimization, the assay was comprehensively validated and additional parameters such as the (long‐term) stability and in vitro metabolism of the peptide were evaluated. In order to determine endogenous MOTS‐c reference limits, the results generated by LC/MS‐based detection were compared with those obtained with a commercially available enzyme‐linked immunosorbent assay (ELISA). Results: The LC/MS‐based test method was fully validated for quantitative results interpretation according to the World Anti‐Doping Agency's International Standard for Laboratories (WADA's ISL). It was found to be specific and sensitive, enabling a lower limit of detection (LLOD) for hMOTS‐c in plasma at 100 pg/mL. Following optimization, animal MOTS‐c analogues and four metabolites as well as two oxidation products were implemented. However, endogenous levels of a reference population of 20 healthy subjects studied by ELISA experiments (45.9–218.5 ng/mL) could not be confirmed by LC/MS. Conclusions: A mass spectrometric detection assay for MOTS‐c in human plasma samples was developed and successfully validated according to WADA's ISL, providing an additional tool for future doping control purposes. Besides MOTS‐c, the assay also includes four in vitro derived metabolites and two oxidation products, which might further improve the traceability of the drug. The analytical approach was compared with a commercially available ELISA, and considerable differences in measured MOTS‐c levels were observed. [ABSTRACT FROM AUTHOR]

In 2017 the conference "From Conflict to Communion" was jointly organised by the Roman Catholic Church in Estonia and the Estonian Evangelical Lutheran Church. The conference was inspired by a study report of the international Lutheran and Catholic joint commission. The document "From Conflict to Communion" (= CC; 2013), today available in 15 languages, contributed significantly to the celebration of the 500th anniversary of Reformation in ecumenical spirit. The article is divided into three parts. It starts with a descriptive part, providing a brief overview of the document. The second part is analytical, offering a critical reflection on the assumptions, method and purpose of CC and commenting on some of the emphases of the document. The third part includes a constructive reflection on the significance of CC for the Catholic Lutheran relations in particular, as well as on its significance in a wider ecumenical context. CC proposes for study and critical discussion a jointly told story about the events of the 16th century Reformation, a joint view on the theology of Martin Luther, a jointly given summary and evaluation of the dialogue between Lutherans and Catholics, of its results, current state and perspectives. The article claims that it is also important to read the document as a response to the questions such as how to understand an ecumenical dialogue and the ecumenical movement and why and how to take part of it. CC contains disenchanting and inspiring ideas for the communication between all churches and Christians. CC is thought-provoking vis-a-vis partners who have not explicitly condemned each other on doctrinal grounds but whose conflict and mutual non-recognition have been or are manifest in more or less concealed ignoring of each other. CC contributes to understanding the crucial importance of ecumenical conversations and theological research. [ABSTRACT FROM AUTHOR]

In several lignite-fired power plants of the ‘Vattenfall Generation AG’ in Germany components of P91/P92 material are used in long-time operation. About this experience in operation of selected components will be reported. In this context own experimental results of a research project in the damage evolution will be presented. The project ‘Damage development III’ was edited together with the MPA Stuttgart and was supported by Vattenfall and AVIF. The aim of the project was to improve the knowledge about the process of creep damage by experimental tests and additional numerical calculations. An instruction was given for planning, implementation and analysis of recurrent investigations on components consisting of 9% Cr steels which are subjected to high operation loading. Finally, the damage phenomena are presented by two case studies, a damage in a pipe bend due to faulty heat treatment and the creep-crack assessment of a lack of side-wall fusion in a reheater weld by fracture mechanics. [ABSTRACT FROM PUBLISHER]

In DWI, multi-exponential signal analysis can be used to determine signal underlying diffusion components. However, the approach is very complex due to the inherent low SNR, the limited number of signal decay data points, and the absence of appropriate acquisition parameters and standardized analysis methods. Within the scope of this work, different methods for multi-exponential analysis of the diffusion signal in the kidney were compared. To assess the impact of fitting parameters, a simulation was conducted comparing the free non-negative (NNLS) and rigid non-linear least square (NLLS) fitting methods. The simulation demonstrated improved accuracy for NNLS in combination with area-under-curve estimation. Furthermore, the accuracy and stability of the results were further enhanced utilizing optimized parameters, namely 350 logarithmically spaced diffusion coefficients within [0.7, 300] × 10−3 mm2/s and a minimal SNR of 100. The NNLS approach shows an improvement over the rigid NLLS method. This becomes apparent not only in terms of accuracy and omitting prior knowledge, but also in better representation of renal tissue physiology. By employing the determined fitting parameters, it is expected that more stable and reliable results for diffusion imaging in the kidney can be achieved. This might enable more accurate DWI results for clinical utilization. [ABSTRACT FROM AUTHOR]

The numbers of vitamin D inadequacies has reportedly increased in the general population, especially in the Northern hemisphere. However, routine measurement of 25(OH) vitamin D is usually associated with a substantial effort due to the requirement of a venous blood sample taken by medical professionals. Thus, the objective of this work is to develop and validate an easy and minimal-invasive method, using a microsampling technique for autonomous blood collections by medically untrained individuals. The assay enables a simplified monitoring of the vitamin D -status in both, risk group and normal population throughout the year. For this purpose, a simple methanol extraction without derivatization combined with a UHPLC-HRMS method was developed to quantify 25(OH)D2 and 25(OH)D3 in capillary blood. For sample collection, a 20 μl Mitra® device with VAMS® technology is used. By employing the six-fold deuterium-labelled 25(OH)D3 as internal standard, the validated assay provides accurate (<10%) and precise (<11%) results. With a LOQ of 5 ng/ml, the approach also proved sensitive enough to adequately identify potential vitamin D deficiencies (< 12 ng/ml), and proof-of-concept analyses of authentic VAMS® samples (n = 20) yielded test results in the expected blood concentration range. Implementing VAMS® sampling for vitamin D -status monitoring enables a higher frequency due to a simplified, straightforward, and time-effective sample collection. VAMS® assures accurate sample volumes because of its absorptive capacities and, thus, area bias and homogeneity issues associated with conventional DBS are avoided. Regular monitoring of 25(OH)D status throughout the year supports people in high-risk groups for vitamin D -deficiency by early identifying inadequacies and, thus, preventing adverse health consequences. • Bioanalytical method for determination of 25OHD 2 and 25OHD 3 using volumetric blood microsampling. • Sensitive quantification of 25OHD without derivatization. • Precise and accurate quantitative results superior to non-volumetric dried blood spots (DBS). • Established approach facilitates regular vitamin D status-monitoring e.g. in risk groups. [ABSTRACT FROM AUTHOR]

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR’s function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptors (PPARs), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) are regulators of transcriptional processes and effects of exercise and pseudo-exercise situations. Compounds occasionally referred to as endurance exercise mimetics such as AdipoRon and 112254, both adiponectin receptor agonists, can be used to simulate the physiology of endurance exercise via pathways including these transcriptional regulators. Adiponectin supports fatty acid utilization and triglyceride-content reduction in cells and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells. In routine doping control analysis, knowledge about phase-I and -II metabolic products of target analytes is essential. Hence, in vitro- and in vivo -metabolism experiments are frequently employed tools in preventive doping research to determine potential urinary metabolites for sports drug testing purposes, especially concerning new, (yet) unapproved compounds. In the present study, in vitro assays were conducted using human liver microsomal and S9 fractions, and rat in vivo experiments were performed using both AdipoRon and 112254. For AdipoRon, obtained samples were analyzed using liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry with both electrospray ionization or atmospheric-pressure chemical ionization techniques. Overall, more than five phase I-metabolites were found in vitro and in vivo , including particularly monohydroxylated and hydrogenated species. No phase II-metabolites were found in vitro ; conversely, signals suggesting the presence of glucuronic acid or other conjugates in samples collected from in vivo experiment were observed, the structures of which were however not conclusively identified. Also for 112254, several phase-I metabolites were found in vitro , e.g . monohydroxylated and demethylated species. Here, no phase II-metabolites were observed neither using in vitro nor in vivo samples. Based on the generated data, the implementation of metabolites and unmodified drug candidates into routine doping control protocols is deemed warranted for comprehensive sports drug testing programs until human elimination study data are available. [ABSTRACT FROM AUTHOR]

Dried blood spots (DBS) represent a sample matrix collected under minimal-invasive, straightforward and robust conditions. DBS specimens have been shown to provide appropriate test material for different analytical disciplines, e.g. , preclinical drug development, therapeutic drug monitoring, forensic toxicology and diagnostic analysis of metabolic disorders in newborns. However, the sample preparation has occasionally been reported as laborious and time consuming. In order to minimize the manual workload and to substantiate the suitability of DBS for high sample-throughput, the automation of sample preparation processes is of paramount interest. In the current study, the development and validation of a fully automated DBS extraction method coupled to online solid-phase extraction using the example of nicotine, its major metabolites nornicotine, cotinine and trans -3′-hydroxycotinine and the tobacco alkaloids anabasine and anatabine is presented, based on the rationale that the use of nicotine-containing products for performance-enhancing purposes has been monitored by the World Anti-Doping Agency (WADA) for several years. Automation-derived DBS sample extracts were directed online to liquid chromatography high resolution/high mass accuracy tandem mass spectrometry, and target analytes were determined with support of four deuterated internal standards. Validation of the method yielded precise (CV <7.5% for intraday and <12.3% for interday measurements) and linear (r 2 > 0.998) results. The limit of detection was established at 5 ng mL −1 for all studied compounds, the extraction recovery ranged from 25 to 44%, and no matrix effects were observed. To exemplify the applicability of the DBS online-SPE LC–MS/MS approach for sports drug testing purposes, the method was applied to authentic DBS samples obtained from smokers, snus users, and e-cigarette users. Statistical evaluation of the obtained results indicated differences in metabolic behavior depending on the route of administration (inhalative versus buccal absorption) in terms of the ratio of nicotine and nornicotine. [ABSTRACT FROM AUTHOR]

The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). The aim of the present study was to develop a method for the simultaneous detection of four different GHRHs and respective metabolites from human plasma by means of immunoaffinity purification and subsequent nano-ultrahigh performance liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. The target analytes included Geref (Sermorelin), CJC-1293, CJC-1295, and Egrifta (Tesamorelin) as well as two metabolites of Geref and CJC-1293, which were captured from plasma samples using a polyclonal GHRH antibody in concert with protein A/G monolithic MSIA™ D.A.R.T.'S® (Disposable Automation Research Tips) prior to separation and detection. The method was fully validated and found to be fit for purpose considering the parameters specificity, linearity, recovery (19-37 %), lower limit of detection (<50 pg/mL), imprecision (<20 %), and ion suppression/enhancement effects. The analytes' stability and metabolism were elucidated using in vitro and in vivo approaches. EDTA blood samples were collected from rats 2, 4, and 8 h after intravenous administration of GHRH (one compound per test animal). All intact substances were detected for at least 4 h but no anticipated metabolite was confirmed in laboratory rodents' samples; conversely, a Geref metabolite (GHRH) was found in a human plasma sample collected after subcutaneous injection of the drug to a healthy male volunteer. The obtained results demonstrate that GHRHs are successfully detected in plasma using an immunoaffinity-mass spectrometry-based method, which can be applied to sports drug testing samples. Further studies are however required and warranted to account for potential species-related differences in metabolism and elimination of the target analytes [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The analysis of low-molecular-mass peptides in doping controls has become a mandatory aspect in sports drug testing and, thus, the number of samples that has to be tested for these analytes has been steadily increasing. Several peptides <2 kDa with performance-enhancing properties are covered by the list of prohibited substances of the World Anti-Doping Agency including Desmopressin, LH-RH, Buserelin, Triptorelin, Leuprolide, GHRP-1, GHRP-2, GHRP-3, GHRP-4, GHRP-5,GHRP-6, Alexamorelin, Ipamorelin, Hexarelin, ARA-290, AOD-9604, TB-500 and Anamorelin. With the presented method employing direct urine injection into a liquid chromatograph followed by ion-mobility time-of-flight mass spectrometry, a facile, specific and sensitive assay for the aforementioned peptidic compounds is provided. The accomplished sensitivity allows for limits of detection between 50 and 500 pg/mL and thus covers the minimum required performance level of 2 ng/mL accordingly. The method is precise (imprecision <20%) and linear in the estimated working range between 0 and 10 ng/mL. The stability of the peptides in urine was tested, and -20°C was found to be the appropriate storage temperature for sports drug testing. Finally, proof-of-concept was shown by analysing elimination study urine samples collected from individuals having administered GHRP-6, GHRP-2, or LHRH. [ABSTRACT FROM AUTHOR]

The total oxidation of methane was studied over a Pd-Pt/Al 2 O 3 catalyst with selected variations of typical gaseous emission components of natural gas engines being oxygen, methane, water, carbon dioxide, higher hydrocarbons, CO, NO x , and SO 2 . The light-off, durability and reactivation of deactivated samples were studied. A continuous deactivation of the catalyst was observed in methane/air. In situ XAS revealed a Pd oxidation state +2 under these conditions. No pronounced changes in BET surface area, noble metal dispersion, and oxidation state were observed for the deactivated samples. However, the deactivation is accompanied by segregation of Pt and Pd in core–shell bimetallic particles. This deactivation did not occur in the presence of NO x . The catalyst could furthermore be reactivated in the presence of NO x as well as by the reduction in hydrogen. Even a small addition of SO 2 was observed to have a pronounced negative impact on the catalyst activity and durability. This deactivation is attributed to the blocking of active noble metal sites by sulfur compounds, because the number of active sites is drastically reduced as observed by CO-chemisorption measurements. [ABSTRACT FROM AUTHOR]

The misuse of insulin for performance enhancement in sport or as toxic agent has frequently been reported in the past. In contrast to synthetic insulin analogues, the administration of recombinant human insulin is hardly recognized by mass spectrometry. The present study was designed to uncover the misuse of recombinant human insulin for doping control purposes as well as for forensic applications. It is hypothesized that an altered metabolite profile of circulating insulin prevails after subcutaneous administration due to exposure of insulin to epidermal proteases. In vitro experiments with skin tissue lysates (S9 fraction and microsomes), different biological fluids (urine, serum, plasma) and recombinant human insulin were performed and the deriving metabolites were characterized by liquid chromatography coupled to high resolution mass spectrometry (HRMS). Afterwards, authentic blood samples of patients suffering from diabetes mellitus and a control group of healthy humans were analysed. Therefore, a method using protein precipitation, ultrafiltration and antibody-coated magnetic beads for purification with subsequent separation by nano-scale liquid chromatography coupled a Q Exactive mass spectrometer was applied. Several metabolites of insulin with C-terminally truncated sequences of the B-chain (and A-chain in minor extent) were identified within this study. Here, the DesB30 human insulin represents the major metabolite in all experiments. This metabolite is frequently found in urine samples due to degradation processes and, thus, disqualifies this matrix for the intended purposes. In contrast, blood samples do commonly not contain DesB30 insulin, which was corroborated by data obtained from the control group. In post-administration blood samples, minute but distinct amounts (approx. 50 pg mL −1 ) of DesB30 insulin were found and suggest the use of this analyte as potential marker for subcutaneous human insulin administration, supporting the attempts to uncover illicit recombinant human insulin administrations. [ABSTRACT FROM AUTHOR]

The development of a new class of erythropoietin mimetic agents (EMA) for treating anemic conditions has been initiated with the discovery of oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus stimulating erythropoiesis. The most promising amino acid sequences have been mounted on various different polymeric structures or carrier molecules to obtain highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic profiles. Concomitant with creating new therapeutic options, erythropoietin mimetic peptide (EMP)-based drug candidates represent means to artificially enhance endurance performance and necessitate coverage by sports drug testing methods. Therefore, the aim of the present study was to develop a strategy for the comprehensive detection of EMPs in doping controls, which can be used complementary to existing protocols. Three model EMPs were used to provide proof-of-concept data. Following EPO receptor-facilitated purification of target analytes from human urine, the common presence of the cysteine-flanked core structure of EMPs was exploited to generate diagnostic peptides with the aid of a nonenzymatic cleavage procedure. Sensitive detection was accomplished by targeted-SIM/data-dependent MS analysis. Method characterization was conducted for the EMP-based drug peginesatide concerning specificity, linearity, precision, recovery, stability, ion suppression/enhancement, and limit of detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating the multi-analyte testing capability of the presented approach. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Dried blood spot (DBS) sampling, a technique used for taking whole blood samples dried on a filter paper, was initially reported in 1963 by Robert Guthrie. While the diagnostic analysis of metabolic disorders in newborns was the focus of investigations at that time, the number of established applications for preclinical drug development, toxicological studies, and therapeutic drug monitoring increased enormously in the last decades. As a consequence of speed, simplicity, and minimal invasiveness, DBS recommends itself as the preferential technique in sports drug testing. The present approach highlights for the first time the development of a screening assay for the analysis of the synthetic human adrenocorticotropic hormone tetracosactide hexaacetate (Synacthen) in DBS using liquid chromatography tandem mass spectrometry. Highly purified sample extracts were obtained by an advanced sample preparation procedure including the addition of an internal standard (d-tetracosactide) and immunoaffinity purification. The method's overall recovery was 27.6 %, and the assay's imprecision was calculated between 8.1 and 17.9 % for intraday and 12.9 to 20.5 % for interday measurements. Stability of the synthetic peptide in DBS was shown for at least 10 days at room temperature and presents a major benefit, since a rapid degradation in conventionally applied matrices such as urine or plasma is well known. With a limit of detection of 50 pg/mL, a detection window of several hours is expected considering reported steady-state plasma levels of 300 pg/mL after intramuscular application of Synacthen Depot (1 mg). The analysis of authentic DBS samples within the scope of an administration study with 250 μg Synacthen (short stimulation test) demonstrated the great potential of the developed assay to simplify the analysis of Synacthen for doping control purposes. [ABSTRACT FROM AUTHOR]

The surface activation of high-density polyethylene powder using a remote plasma process at atmospheric pressure is investigated. The developed circulating fluidized bed plasma reactor enables to carry fine-grained powder particles repetitively through the afterglow of 64 dielectric barrier discharges of argon with O2 and CO2 admixtures. The evolution of particle size distribution during conveying in the reactor is monitored by laser diffraction and reveals that only a small particle fraction is lost during the process. The contact angle of activated powder samples steadily decreases with increasing treatment time. The surface activation is most effective for O2 admixtures of 0.25 vol% and CO2 admixtures of above approximately 1 vol%, respectively. [ABSTRACT FROM AUTHOR]

Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time. [ABSTRACT FROM AUTHOR]

Hadron properties are modified when the hadron is embedded in a nuclear medium. Here we discuss the Gerasimov-Drell-Hearn, GDH, sum rule for polarised photoproduction from a polarised nucleon within a polarised nuclear target. Strong enhancement is expected with the suppression of the proton and nucleon resonance masses and enhancement of the proton’s anomalous magnetic moment in medium. This could be tested in polarised photoproduction experiments with interesting targets being polarised deuterons, 3 He, 6 Li and 7 Li. The largest contribution to the GDH sum rule comes from the Δ resonance excitation. In existing data with polarised deuterons and 3 He the Δ excitation is shifted to slightly lower energy when compared to model predictions where the Δ is treated with its free mass. [ABSTRACT FROM AUTHOR]

Purpose: The desire to increase the athletic performance, to 'optimize' an individual's appearance, and to complement but also to arguably substitute exercise by means of drugs and drug candidates has generated a considerable (illicit) market for compounds such as anabolic-androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely developed for therapeutic use, their abuse/misuse generates enormous health risks, which has necessitated comprehensive controls of compound trafficking by customs and anti-doping authorities. Methods: From 2012 to 2013, the Bureau of Customs Investigation confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010 and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new challenges in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Results: Among the 337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of which being categorized as doping agents by the World Anti-Doping Agency (WADA). A total of 83.7 % accounted for steroidal substances (predominantly testosterone, trenbolone, and nandrolone and corresponding esters), 12.8 % accounted for peptide hormones and growth factors (predominantly hGH and growth hormone releasing peptides (GHRPs)), 3.2 % of the products contained hormones and metabolic modulators, and 0.3 % accounted for diuretic agents. Outstanding findings were the detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin β4, and a fusion protein of unknown biological activity. Conclusions: Trafficking of considerable amounts of arguably performance and/or body-enhancing compounds has been observed during the past 4 years, the majority of which is categorized as relevant to sports drug testing. Several substances are of fake/non-approved nature and represent enormous health risks to the 'customer'. [ABSTRACT FROM AUTHOR]

Highlights: [•] Whole blood dried on filter paper – a minimal invasive sampling technique. [•] Screening of anabolic steroid esters in dried blood spots for doping control analysis. [•] Direct detection of exogenous testosterone via the intact esters. [•] Enhancement of the assay sensitivity by preparation of the methyloxime derivatives. [•] Liquid chromatography coupled to high sensitivity mass spectrometry. [ABSTRACT FROM AUTHOR]

Aim The aim was to evaluate the influence of post surface design and luting system on bond strength of quartz-fiber-reinforced composite posts ( QFRCPs) luted to root canal dentin. Materials and methods Single-rooted bovine teeth ( n = 650) were randomly assigned (13 groups, n = 50), sectioned, endodontically treated, filled, and post space (length 8 mm) prepared. Custom-made plain-surfaced fiber posts ( PSXRO) and (both RTD) macroretentive Macro-Lock Post Illusion X- RO ( MLXRO) were inserted into the post spaces using six luting systems: Ketac Cem (KC), Fuji Plus (FP), RelyX Unicem, Multilink Primer_Multilink, Sealbond Ultima_CoreCem, and LuxaBond_LuxaCore Z. As control, a titanium post was cemented with KC. After water storage (24 h, 37°C), pull-out test was performed, followed by failure mode assessment. Bond strength was calculated in MPa and analyzed using anova, Dunnett-T3-test, and Student's t-test with Bonferroni correction. Results Post design and luting system significantly influenced the bond strength [ MPa] ( P < 0.05). Compared with the control 4.3 (1.5), all test groups exhibited higher bond strengths ( P < 0.05), except for group PSXRO/KC 4.2 (1.0). The remaining bond strengths were PSXRO: FP 8.6 (1.5), RelyX Unicem 10.4 (3.4), Multilink Primer_Multilink 12.7 (3.0), SealBond Ultima_CoreCem 12.7 (3.0), LuxaBond_LuxaCore Z 15.7 (2.5), and MLXRO: KC 7.2 (2.2), FP 13.4 (2.5), RelyX Unicem 9.2 (2.9), Multilink Primer_Multilink 12.5 (4.5), SealBond Ultima_CoreCem 13.7 (4.6), LuxaBond_LuxaCore Z 20.6 (2.2). The bond strengths of MLXRO were higher than those of PSXRO when luted with KC, FP, and LuxaBond_LuxaCore Z ( P < 0.05). Conclusion The post surface design and luting system selection influenced the bond strength of conventionally and adhesively luted QFRCPs to bovine root canal dentin. [ABSTRACT FROM AUTHOR]

RATIONALE AICAR (5-aminoimidazole-4-carboxamide 1β-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse is suspected by measuring its urinary concentration and comparing the observed level with naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for the determination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated. METHODS Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study. RESULTS The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h. CONCLUSIONS Determination of thee CIRs is the method of choice to distinguish between an endogenous and an exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

• Several prohibited substances in sports are enantiomers. • especially for threshold violations the enantiomeric signature is relevant. • enantiomeric determination provides also information about the source. Analytics employed in modern doping controls are designed to cover an extensive range of rather diverse classes of substances, all of which are banned in sport according to the list of prohibited substances and methods of doping, resulting from their potential to be performance-enhancing and/or harmful to health. Many of these bioactive substances or their metabolites are chiral, which are comprehensively characterized and, if appropriate analytical approaches are applied, can be clearly identified. In sports drug testing, the enantiomeric composition of relevant compounds is not considered in all instances, although differences of isomers concerning their biological activity have been established. To date, the separation of stereoisomers in doping controls is only applied for selected target compounds, but with the development of efficient chiral chromatographic stationary phases, the added value of information on e.g. racemic shifts during the metabolic biotransformation reactions of drugs has been recognized. The immense variability of the substance classes represents however a major challenge, especially because both 'classic' doping agents belonging to the category of lower molecular mass molecules (e.g. stimulants, β 2 -agonists, betablockers, corticoids, etc.) as well as larger molecules from the category of peptides and proteins necessitate consideration. In the present (mini)review, the current status of analytical techniques in the field of doping control analysis of stereoisomers is highlighted and critically reviewed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Urine samples have been the predominant matrix for doping controls for several decades. However, owing to the complementary information provided by blood (as well as serum or plasma and dried blood spots (DBS)), the benefits of its analysis have resulted in continuously increasing appreciation by anti-doping authorities. On the one hand, blood samples allow for the detection of various different methods of blood doping and the abuse of erythropoiesis-stimulating agents (ESAs) via the Athlete Biological Passport; on the other hand, targeted and non-targeted drug detection by means of chromatographic–mass spectrometric methods represents an important tool to increase doping control frequencies out-of-competition and to determine drug concentrations particularly in in-competition scenarios. Moreover, blood analysis seldom requires in-depth knowledge of drug metabolism, and the intact substance rather than potentially unknown or assumed metabolic products can be targeted. In this review, the recent developments in human sports drug testing concerning mass spectrometry-based techniques for qualitative and quantitative analyses of therapeutics and emerging drug candidates are summarized and reviewed. The analytical methods include both low and high molecular mass compounds (e.g., anabolic agents, stimulants, metabolic modulators, peptide hormones, and small interfering RNA (siRNA)) determined from serum, plasma, and DBS using state-of-the-art instrumentation such as liquid chromatography (LC)–high resolution/high accuracy (tandem) mass spectrometry (LC-HRMS), LC–low resolution tandem mass spectrometry (LC-MS/MS), and gas chromatography–mass spectrometry (GC-MS). [ABSTRACT FROM AUTHOR]

Abstract: Doping control analytical laboratories for human sports predominantly employ nowadays chromatographic–mass spectrometric test methods for routine, high throughput screening and confirmation assays concerning low and high molecular mass analytes. Liquid chromatography–(tandem) mass spectrometry [(LC–MS(/MS)] and particularly ultrahigh pressure liquid chromatography (UHPLC)–MS/MS instruments have become devices of choice due to their indispensable capabilities that compensate for limitations inherent to other commonly used strategies such as immunological and gas chromatography–(tandem) mass spectrometry [(GC–MS(/MS)]-based detection methods. UHPLC–MS/MS-based assays at low mass spectrometric resolution have been established allowing for fast and sensitive targeted analyses focusing on pre-selected target analytes with diagnostic precursor–product ion pairs. Combining UHPLC to high resolution/high accuracy MS(/MS) further expanded the targeted approach (i.e., plotting extracted ion chromatograms of protonated or deprotonated molecules as well as product ions measured with accurate masses) toward non-targeted analyses enabling also retrospective data mining. In this review, recent applications of UHPLC–MS/MS in sports drug testing procedures published between 2008 and 2012 are presented and advantages as well as limitations in a short- and long-term perspective are discussed. [Copyright &y& Elsevier]

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 β2-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. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 μU/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog®, Novolog®, Apidra®, Lantus®, and Levemir® in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics. [ABSTRACT FROM AUTHOR]

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity. [ABSTRACT FROM AUTHOR]

As recently reported, dried blood spot (DBS) analysis is an advantageous technique for doping control purposes due to the minimal invasive sample collection, the simple and economic manner, as well as the low susceptibility to manipulation. Its general applicability to the sports drug testing arena has been shown for analytes of various substance classes, all of which comprise exclusively low molecular mass compounds. The aim of the present study was to investigate whether the technique of DBS analysis is applicable also to (pegylated) peptides with relevance for doping controls. As target analyte, peginesatide (Omontys, Hematide), a recently approved pegylated erythropoietin-mimetic peptide of approximately 45 kDa, tested for the treatment of anaemia in patients with renal failure, was chosen, which has been prohibited in elite sports due to its assumed endurance enhancing effects. Therefore, a detection method for peginesatide employing DBS was developed based on extraction, proteolytic digestion and cation-exchange purification followed by liquid chromatography-tandem mass spectrometry analysis. Eventually, the assay was validated for qualitative purposes and proved to be specific, sensitive (limit of detection, 10 ng/mL) and precise (relative standard deviations below 18 %), demonstrating the general suitability of DBS analysis in sports drug testing also for (pegylated) peptides. [ABSTRACT FROM AUTHOR]

Abstract: Erythropoiesis-stimulating agents (ESAs) have frequently been confessed to be illicitly used in elite sports due to their endurance enhancing effects. Recently, peginesatide, the first representative of a new generation of ESAs, referred to as Erythropoietin (EPO)-mimetic peptides, obtained approval in the USA under the trade name Omontys® for the treatment of anaemic patients. Lacking sequence homology with EPO, it consists of a pegylated homodimeric peptide of approximately 45kDa, and thus, specific approaches for the determination of peginesatide in blood were developed as conventional detection assays for EPO do not allow for the analysis of the EPO-mimetic peptides. However, as urine specimens are the most frequently provided doping control samples and pharmacokinetic studies conducted in rats and monkeys revealed the excretion of the pegylated peptide into urine, a detection method for peginesatide in urine would be desirable. A mass spectrometric assay in human urine was developed consisting of protein precipitation with acetonitrile followed by proteolytic digestion after the removal of the acetonitrile fraction under reduced pressure. Purification and concentration of the resulting proteotypic target peptide was accomplished by means of solid-phase extraction on strong cation-exchange resin prior to liquid chromatographic–tandem mass spectrometric analysis. Method validation was performed for qualitative purposes and demonstrated specificity, precision, linearity as well as sufficient sensitivity (limit of detection: 0.5ng/ml) while proof-of-concept for the applicability of the assay for the determination of peginesatide in authentic urine samples was obtained by analyzing animal in vivo specimens collected after a single i.v. administration of peginesatide over a period of 4 days. [Copyright &y& Elsevier]

Abstract: In the present study, a screening assay was developed comprising 11 prohibited peptides (<1.5kDa) that are sufficiently purified from urine using weak cation exchange with subsequent determination of all substances by means of nanoUHPLC separation coupled to high resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH), Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin, Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable to further target analytes or metabolites. The method was validated with a main focus on qualitative result interpretation considering the parameters specificity, linearity (0–500pg/mL), recovery (45–95%), precision (<20% at 100pg/mL), limits of detection (2–10pg/mL), robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion study urine samples for LHRH, Desmopressin and GHRP-2. [Copyright &y& Elsevier]

In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0-20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80-120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing. [ABSTRACT FROM AUTHOR]

Abstract: For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30–32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1–29)) and CJC-1295 (GH-RH analogue), LongR3-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (∼25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled. [Copyright &y& Elsevier]

Abstract: With the constantly increasing sensitivity and robustness of liquid chromatography–mass spectrometry-based instruments combined with enhanced reproducibility as well as mass accuracy and resolution, LC–MS(/MS) has become an integral part of sports drug testing programs particularly concerning the detection of peptide hormones. Although several of the relevant peptidic drugs such as insulins (Humalog LisPro, Novolog Aspart, etc.), growth hormone releasing peptides (GHRPs, e.g., GHRP-2, GHRP-6, Hexarelin, etc.), and insulin-like growth factors (e.g., IGF-1, IGF-2, long-R3-IGF-1) are currently analyzed using dedicated top-down analytical procedures, i.e. employing specifically tailored sample preparation procedures followed by targeted LC–MS(/MS) measurements focusing on intact analytes, first approaches towards multi-analyte methods have been established. These allow the determination of the prohibited substances in blood and urine doping control specimens following therapeutic applications. In addition, the use of new complementary devices such as ion mobility analyzers, e.g., in hybrid mass spectrometers yielded promising data for the differentiation of isobaric insulins, which outlines the potential to further accelerate and multiplex doping control analytical assays to meet the continuously increasing demands of rapid and unambiguous test methods. Moreover, the potential of LC–MS/MS to target recombinant peptide hormones such as human growth hormone using bottom-up approaches has been demonstrated by targeting proteotypic peptides that unambiguously differentiate the recombinant molecule from the naturally occurring and endogenously produced analog. [Copyright &y& Elsevier]