QuEChERS as alternative extraction procedure in doping analyses.

QuEChERS is a dispersive solid phase extraction commonly applied in food analysis for residues, such as pesticides or mycotoxins for more than 20 years. Due to the quick and easy sample preparation procedure, a QuEChERS method based on ammonium acetate combined with formic acid in acetonitrile was tested for the preparation of urine samples for doping control purposes. Testing urine samples with different pH and specific gravity, using the combination of 10 M ammonium acetate with 3% formic acid in acetonitrile, 312 out of 342 tested compounds could be extracted at their respective minimum required performance levels according to the World Anti-Doping Agency (WADA) technical documents. For nine selected analytes representing six categories of WADA's Prohibited List, we validated the QuEChERS extraction method fulfilling WADA's requirements for a confirmation procedure of the nonthreshold substances investigated. Especially for the intact stanozolol-glucuronides analyzed by high-resolution mass spectrometry, the described extraction method might be an alternative for confirmation procedures as it is time- and cost-saving compared with the commonly applied solid phase extraction.

Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

The use of RNA-based 5'-aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses.

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'-aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti-doping analyses.

Horseradish-peroxidase-conjugated anti-erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept.

Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non-specific cross-reactivity with secondary antibodies. An in-house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in-house anti-EPO-HRP conjugate was performed as well as the current two-step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in-house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control.

Physiological strain associated with high-intensity hypoxic intervals in highly trained young runners.

To examine the physiological strain associated with hypoxic high intensity interval training (HHIT), 8 highly trained young runners (age, 18.6 ± 5.3 years) randomly performed, 5 × 3-minute intervals in either normoxic (N, 90% of the velocity associated with VO(2max), vVO(2max)) or hypoxic (H, simulated 2,400-m altitude, 84% of νVO(2max)) conditions. Cardiorespiratory (ventilation [V(E)], oxygen consumption [V(O2)], heart rate [HR], oxygen saturation [SpO(2)]), rating of central perceived exertion (RPE(C)) responses, changes in neutrophils, erythropoietin (EPO), blood lactate ([La]) and, bicarbonate ([HCO(-)(3)]), vagal-related indices of HR variability (natural logarithm of the square root of the mean of the sum of the squares of differences [Ln rMSSD]) and maximal sprint and jump performances were compared after each session. Compared with N, H was associated with similar V(E) (Cohen's d ± 90% confidence limits, 0.0 ± 0.4, with % chances of higher/similar/lower values of 15/61/24) but at least lower VO(2) (-0.8 ± 0.4, 0/0/100), HR (-0.4 ± 0.4, 1/21/78), and SpO(2) (-1.8 ± 0.4, 0/0/100). Rating of perceived exertion was very likely higher (+0.5 ± 0.4, 92/8/0). Changes in [HCO(3)] (-0.6 ± 0.8, 5/13/83), [La] (+0.2 ± 0.4, 52/42/5), and EPO (+0.2 ± 0.4, 55/40/5) were at least possibly greater after H compared with those after N, whereas changes in neutrophils were likely lower (-0.5 ± 0.7, 4/15/81). Changes in 20-m sprint time (+0.20 ± 0.23, 49/50/1) were possibly lower after H. There was no clear difference in the changes in Ln rMSSD (+0.2 ± 1.7, 48/18/34) and jump (+0.3 ± 0.9, 60/25/15). In conclusion, although perceived as harder, HHIT is not associated with an exaggerated physiological stress in highly trained young athletes. The present results also confirm that HHIT may not be optimal for training both the cardiorespiratory and neuromuscular determinants of running performance in this population.