Assessing limits of detection in qualitative methods: A simple implementation of logistic regression in a web‐based R Shiny application environment and its potential in toxicology and doping control

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Higenamine is prohibited in sports as a β2‐agonist by the World Anti‐Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single‐dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo‐conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo‐conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7‐sulfate. In addition, the option to include complementary biomarkers of diet‐related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

Section: Methodsmentioning

confidence: 99%

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing

Rubio

Thomas

Euler

et al. 2023

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“…Limit of identification (LOI), the lowest concentration in which the TD2023IDCR criteria is met at 95% identification rate, was estimated by a sigmoid fit function approach. 23 The stability of the reconstituted samples maintained on autosampler at 20 C was assessed by the reinjection, after 24 and 48 h, of all samples prepared on the first day of validation. For evaluation of carryover, one urine sample with spiked cobalt at a concentration of 100 ng/mL was injected before two solvent blanks.…”

Section: Evaluation Of Sample Digestionmentioning

confidence: 99%

Coordination chemistry applied to anti‐doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry

dos Santos,

Carneiro,

Braz

et al. 2023

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Cobalt was included on the World Anti‐Doping Agency Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia‐inducible factor. Although it has proven benefits for performance enhancement, the unavailability of inductively coupled plasma–mass spectrometry on routine of the accredited laboratories is a factor that reduces its applicability in anti‐doping analysis. Therefore, an analytical method for quantification of urinary cobalt as its diethyldithiocarbamate complex by liquid chromatography coupled with high‐resolution tandem mass spectrometry was developed and validated. Palladium was proposed as internal standard and rhodium as a complexation control. A microwave‐assisted acid digestion of the urine samples was essential, not only to eliminate the matrix effect but mainly to avoid the non‐specific bond of cobalt to endogenous molecules. A linear method was obtained over the studied range from a negative urine control to a spiked concentration of 25 ng/mL, with an estimated limit of quantification of 2.5 ng/mL, and an adequate combined standard uncertainty of 11.4%. Considering that all reagents are commercially available, the proposed strategy is feasible to be included on routine sample preparation. Monitoring urinary cobalt concentrations globally opens the perspective to support the anti‐doping system to define a suitable threshold value and to understand its potential misuse by athletes seeking for performance improvement.

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

Miyamoto,

Ota,

Sato

et al. 2024

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.