Simultaneous detection of testosterone, nandrolone, and boldenone esters in dried blood spots for doping control in sports by liquid chromatography-tandem mass spectrometry.

Testosterone, nandrolone, and boldenone, which are listed as doping substances on the World Anti-Doping Agency Prohibited List, are mostly available commercially in esterified forms. Isotope ratio mass spectrometry (IRMS) represents a key tool for identifying these substances, as they are hydrolyzed and discharged in the urine as pseudo-endogenous substances. However, IRMS, which comprises a complicated process, cannot achieve the direct detection of steroid esters in blood samples. These substances can be detected using dried blood spots (DBSs), reducing the impact of esterase hydrolysis. Here, a simultaneous liquid chromatography-tandem mass spectrometry method for detecting 28 steroid (13 testosterone, nine nandrolone, and six boldenone) esters was developed using three DBS types of samples, including a cellulose paper and polymer. The substances were first derivatized with methyloxime to increase their sensitivities (the limits of detection were <0.1-0.4, <0.1-0.9, and <0.1-0.9 ng/mL for the testosterone, nandrolone, and boldenone esters, respectively). Further, the DBS absorbents were verified since the effect of interferences depended on it. Next, a study involving seven participants was conducted to detect intramuscularly administered testosterone enanthate (100 mg). Polymer and cellulose papers were used to collect blood from their upper arms and fingertips, respectively, and testosterone enanthate was identified and detectable at both blood-collection sites for up to 144 and 216 h, respectively. Furthermore, testosterone enanthate was detectable in the DBS samples stored under refrigeration after 6 months, indicating the stable nature of DBS.

Doping control analysis of trimetazidine in dried blood spot.

Dried blood spot (DBS) analysis has been an inherent part of sports drug testing through the technological advancements of the past decade. Trimetazidine, a non-threshold banned substance, is excreted into urine after a dose of the permitted drug lomerizine. Therefore, a lomerizine-specific metabolite (M6) is analyzed to confirm the origin of trimetazidine in traditional urine analysis. Application studies were conducted to develop an analytical method for trimetazidine applicable to DBS. These studies comprise (1) the effect of different sampling sites on the detection of trimetazidine, (2) the determination of the appropriate trimetazidine level required for DBS analysis, and (3) differentiating between trimetazidine and lomerizine use. A high-resolution mass spectrometric method for detecting trimetazidine in DBS was validated. After oral administration of trimetazidine (n = 7), venous and capillary blood (fingertip and upper arm) were spotted on cellulose paper. Trimetazidine could be identified in DBS in all subjects up to 60 h after administration. The limit of detection was 0.05 ng/ml, and the limit of identification was 0.06 ng/ml, suggesting the minimum required performance level of 0.2 ng/ml. In the fingertip capillary blood, biases of 9.7% (vs. upper arm) and 13.0% (vs. vein) were observed in the trimetazidine intensity; however, there were no concerns in the qualitative analysis. After administering lomerizine (n = 10), the intact lomerizine has a strong peak intensity in blood compared to trimetazidine. Contrary to urine analysis, the M6 was less detectable in blood. Laboratories should confirm intact lomerizine whenever trimetazidine is identified in DBS.

Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games.

The doping control analyses at the XXXII Olympic Games (July 23 to August 8, 2021) and the XVI Paralympic Games (August 24 to September 5, 2021) held in Tokyo, Japan, after a year of delay due to the COVID-19 pandemic are summarized in this paper. A new satellite facility at the existing World Anti-Doping Agency (WADA)-accredited Tokyo laboratory was established and fully operated by 278 staff, including 33 Tokyo laboratory staff, 49 international experts, and 196 Japanese temporary staff. The numbers of urine samples were 5081 (Olympics) and 1519 (Paralympics), and the numbers of blood samples were 1103 (Olympics) and 500 (Paralympics). The laboratory could prepare for analysis in advance using a paperless chain-of-custody system, allowing for faster turnaround time reporting. For the first time, a new polymerase chain reaction method for detecting erythropoietin (EPO) gene doping was used. The laboratory also analyzed blood samples for detecting steroid esters following the spotting of collected venous EDTA blood onto dried blood spot cards. Moreover, full-scan data acquisition using high-resolution mass spectrometers was performed for all urine samples, allowing for detecting traces of doping substances, which are not currently analyzed in the subsequent data processing. The presence of some prohibited substances was confirmed, resulting in 8 atypical findings (ATFs) and 11 adverse analytical findings (AAFs), including homologous blood transfusion (2 cases) and recombinant EPO in the blood (1 case), at the Olympics, whereas 2 ATFs and 10 AAFs were reported at the Paralympics.

Analysis of tretoquinol and its metabolites in human urine by liquid chromatography-tandem mass spectrometry.

Tretoquinol (trimetoquinol), a ß2-agonist, has been explicitly listed on the World Anti-Doping Agency Prohibited List 2019 since January 2019; however, it has been distributed as an antiasthmatic on the medical market. This study aimed to develop a liquid chromatography-tandem mass spectrometric method for the quantification of tretoquinol (free form plus glucuronide) in human urine for doping control purposes. An excretion study (n = 6) of tretoquinol hydrochloride hydrate (6 mg) was performed, and urine samples were collected prior to oral administration and during the first 48 h, along with spot urine samples at 7 and 14 days after administration. All the urine samples were analysed using the developed method. The limit of detection for the developed method was 0.03 ng/mL. The inter-day precision for the target analyte was excellent (2.7% to 9.2%), and the inter-day accuracy of target analyte was -0.6% to -3.6%. In all subjects, tretoquinol (free form plus glucuronide conjugate) was identified up to 48 h after administration. The maximum concentrations were in the range of 12.4-78.8 ng/mL and the mean concentration was 55.3 ng/mL. The metabolites O-methylated tretoquinol, tretoquinol sulphate and O-methylated tretoquinol sulphate could be also identified in human urine after administration. The longest-lasting urinary metabolite of tretoquinol currently known, O-methylated tretoquinol, is also likely to be a useful marker in doping controls.