Use of volumetric absorptive microsampling (VAMS) for the quantification of novel blood markers of EAAS doping

Code: 20D02FP

Volumetric Absorptive Microsampling (VAMS) is a recent microsampling technique used to obtain dried specimens of biological fluids that could represent in the near future a valid alternative to urine, whole blood and serum sampling for anti-doping purposes. Although similar to Dried Blood Spots (DBS), VAMS promise to bring a number of significant advantages over them: i) improving quantitative performance thanks to higher sample volume acccuracy; ii) obtaining the extraction yield and reproducibility without and impact of hematocrit (HCT) value; iii) enhancing the overall analytical performance, resulting in better correlatio with plasma values; iv) simplifying collection procedures by homogeneous samples with a lower reject rate; v) availability of 96-well formats, opening the access to automation. In this study, the suitability of VAMS, in 30µL format, as an innovative matrix for the measurement of novel highlighted blood markers of Endogenous Anabolic Androgenic Steroids (EAAS) doping will be evaluated. A UHPLC-MS/MS method for the quantification of a significant panel of circulating steroid hormones and their phase II metabolites will be developed. Mass spectrometric parameters and transitions will be fine-tuned to achieve the maximal sensitivity needed to detect ad quantify as many markers as possible. Chromatographic conditions will also be thoroughly optimized in terms of separation of isomers with the aim of finding the best compromise between resolution and analysis time. Validation procedure will be carried out following the WADA requirements. Various parameters, such as limits of detection and quantification, precision and accuracy, matrix effects, selectivity, repeatability and robustness will be investigated for each measured analyte. The developed analytical method will be transferred to a WADA-accredited laboratory and an inter-laboratory comparison will be performed. Finally, results will be discussed putting particular emphasis on the systematic comparison with more conventional hematological matrices, such as serum and plasma.

Main findings

A novel LC-MS/MS method for the measurement of 18 steroids panel, including main endogenous steroid hormones and androgen phase II metabolites, was developed and validated in compliance with WADA requirements for quantitative methods. A simple sample preparation procedure was optimized and resulted in extraction recoveries ranging from 69.8% to 92.5%, while showing ion suppression-related matrix effect around 30% for most of target analytes. The validation protocol allowed demonstrating the satisfactory performance of developed method in terms of selectivity (separation of isomers and exclusion of interferences), trueness, repeatability, precision, combined uncertainty, linearity range, LLOQ, carry-over and overall robustness. Finally, the method was successfully transferred to the Lausanne WADA-accredited laboratory. A stability study involving 20 healthy subjects (10 males and 10 females) demonstrated that steroid concentrations measured in VAMS samples stored at room temperature, 4°C, -20°C and -80°C, do not deviate from the values measured in baseline samples, with calculated mean percentage differences being always lower than Reference Change Value threshold which estimated method’s analytical variability. Moreover, the influence of up to three consecutive freeze and thaw cycles was evaluated as not significant. VAMS proved to be a valid collection strategy for measuring steroid hormones in blood that could be employed in doping control analysis with the goal of increasing sampling frequency dedicated to the newly implemented Blood Steroid Profile (BSP). The stability of steroid hormones in blood microsampling collected on VAMS support opens the way of interesting advantages for blood samples’ transportation and storage for anti-doping purposes. Nevertheless, prior to the introduction of VAMS sampling for BSP analysis, further studies aiming at evaluating the correlation between steroid concentration levels measured in VAMS (capillary whole blood) and in currently employed serum samples should be performed in the future to gather an exhaustive overview of VAMS potential in anti-doping context.

Publications/Presentations related to the project

Publications:

- F. Ponzetto, M. Parasiliti Caprino, L. Leoni, L. Marinelli, A. Nonnato, R. Nicoli, T. Kuuranne, E. Ghigo, G. Mengozzi, F. Settanni, LC-MS/MS measurement of endogenous steroid hormones and phase II metabolites in blood volumetric absorptive microsampling (VAMS) for doping control purposes, Clin. Chim. Acta (2024), under review.