Measurement of urinary cobalt as its complex with 2‐(5‐chloro‐2‐pyridylazo)‐5‐diethylaminophenol by liquid chromatography–tandem mass spectrometry for the purpose of anti‐doping control

Abstract

Cobalt is well known for its ability to stimulate erythropoiesis via stabilization of hypoxia-inducible factors. In sports, this can provide a competitive benefit to athletes, so the World Anti-Doping Agency prohibits the use of cobalt in any form except its cobalamin vitamers. As of now, cobalt in biological fluids is detected by inductively coupled plasma mass spectrometry (ICP-MS), a technique which has very limited availability in anti-doping laboratories. Therefore, a quantitative method based on liquid chromatography-tandem mass spectrometry capable of measuring urinary cobalt in the form of its complex with 2-(5-chloro-2-pyridylazo)-5-diethylaminophenol (5-Cl-PADAP) has been developed and validated. A cobalt complex with deuterium-labeled 5-Cl-PADAP was used as internal standard. The method was found linear over the concentration range of 5-500 ng/ml with a combined standard uncertainty less than 10% at 15, 200, and 450 ng/ml. Stability of cobalt ions in urine was investigated over the course of 2 months; the concentration

Value of amniotic fluid homocysteine assay in prenatal diagnosis of combined methylmalonic acidemia and homocystinuria, cobalamin C type

“…In this context, we retrospectively reviewed 248 at-risk fetuses with prenatal diagnostic data collected over a 10-year period to analyze the value of Hcy in the prenatal diagnosis of cblC defect. We first assessed the imprecisions of Hcy assay, the CV W , CV B and CV Bias values were all less than 10% [23] and proved that Hcy assay in AF was stable. Moreover, to interpret the results of Hcy assay in AF, the relative errors of the selected two levels (2.0 and 16.0 μmol/L) of Hcy were − 2.5% and 2.8%, respectively.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2020/2021

“…Maximum urinary cobalt concentrations of 478 ng/ml were observed following cobalt chloride applications, while cyanocobalamin at 1 mg/day dosing did not result in significant alterations of the urinary cobalt concentrations. As (LC)‐ICP‐MS is rarely available in anti‐doping laboratories, and yet analyses for cobalt appear particularly relevant considering the facile and cheap access to cobalt chloride, Sobolevsky and Ahrens 133 suggested to employ routinely available LC‐MS/MS systems for the quantification of urinary cobalt after complexation with 2‐(5‐chloro‐2‐pyridylazo)‐5‐diethylaminophenol. Urine was prepared for analysis by acidic hydrolysis and, following neutralization, cobalt complexation, and weak cation exchange SPE purification.…”

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

“…12 It was also demonstrated that the short-term administration of 10 or 5 mg/day for 3 weeks of inorganic cobalt provides erythropoietic stimulation. 13,14 Although it has been shown its effectiveness as a performance enhancement agent, relatively low attention has been provided to the cobalt issue on last years, 6,[12][13][14][15][16][17][18] which turns its potential misuse unknown. On the largest reference population study with athletes' samples conducted so far, Reichel et al found elevated (i.e., above 10 ng/mL) urinary cobalt concentrations for 1.9% of the analyzed samples, with a highest value of 948.0 ng/mL obtained for a collected urine from Indonesia.…”

“…15 Moreover, methods employing chromatography coupled to mass spectrometry, a traditional and readily available technique in antidoping laboratories, are yet scarce on the literature. [18][19][20][21] The described strategies employ a complexation reaction followed by a sample extraction to make possible the detection and, in some cases, the quantification of inorganic cobalt in biological matrices. The most common ligands used so far are the dithiocarbamate derivatives, [19][20][21] but the difficulty in obtaining an adequate internal standard (ISTD) turns some of the developed methods only for qualitative purposes.…”

“…A recent quantitative method employing an elegant approach with an in-house synthetized ligand and an isotopically labeled analog as ISTD was proposed by Sobolevsky and Ahrens. 18 Therefore, the main objective of the study was to develop and validate, based on the WADA guidelines, an analytical method for cobalt quantification as its diethyldithiocarbamate (C 4 H 10 NCS 2 , DDC) complex by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS). Palladium was employed as an ISTD and rhodium as a complexation control.…”