Simple and Sensitive Analysis of Clenbuterol in Urine Matrices by UHPLC-MS/MS Method with Online-SPE Sample Preparation

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ISBN: 978-80-974608-0-8

Simple and Sensitive Analysis of Clenbuterol in Urine Matrices by UHPLC-MS/MS Method with Online-SPE Sample Preparation

Kristián Slíž1,2 , Dominika Olešová3 , Juraj Piešt’anský4 , Peter Mikuš ,
1 Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University Bratislava, Odbojárov 10, 832 32 Bratislava, Slovakia
2 Toxicologic and Antidoping Centre, Faculty of Pharmacy, Comenius University Bratislava, Odbojárov 10, 832 32 Bratislava, Slovakia
3 Slovak Academy of Sciences, Biomedical Research Center, Institute of Experimental Endocrinology, Dúbravská Cesta 9, 845 45 Bratislava, Slovakia
4 Department of Galenic Pharmacy, Faculty of Pharmacy, Comenius University Bratislava, Odbojárov 10, 832 32 Bratislava, Slovakia
sliz7@uniba.sk

Clenbuterol is one of the most misused anabolic agents in professional sports. Therefore, the monitoring of clenbuterol in body fluids such as human urine is related to the development of rapid, selective and sensitive analytical methods that produce reliable results. In this work, these requirements were met by a two-dimensional separation method based on online solid-phase extraction  coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (SPE–UHPLC–MS/MS). The developed method provides favorable performance parameters, and it is characterized by minimum manual steps (only dilution and the addition of an internal standard) in the sample preparation. A limit of quantification (LOQ) of 0.1 ng/mL, excellent linearity (0.9999), remarkable precision (1.26% to 8.99%) and high accuracy (93.1% to 98.7%) were achieved. From a practical point of view, the analytical performance of the validated SPE–UHPLC–MS/MS method was demonstrated on blinded spiked urine samples from ten healthy volunteers. The estimated concentrations of clenbuterol were in accordance with their corresponding nominal values, as supported by the precision and accuracy data (relative standard deviation ≤5.4%, relative error ≤11%).The fulfillment of the World Anti-Doping Agency’s screening and confirmation criteria indicates that the proposed method is suitable for implementation in routine use in toxicologic and antidoping laboratories. Due to its high orthogonality and separation efficiency, the SPE–UHPLC–MS/MS method should also be easily adapted to the separation of structurally related compounds (such as clenbuterol metabolites). Thus, future antidoping applications could also include monitoring of clenbuterol metabolites, providing a longer detection widow.

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This work was supported by the projects VEGA 1/0514/22, KEGA 027UK-4/2020 and FaF/5/2023.