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

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


Celkové hodnotenie

Vedecká práca
Prevedenie (dizajn)
Diskusná interakcia
PoužívateľVedecká prácaDizajnDiskusná interakcia
PharmDr. Andrea Horniaková100%100%100%
PharmDr. Ondrej Štefánik100%100%100%
RNDr. Monika Švecová100%100%100%
Ing. Zuzana Brnoliaková PhD.100%100%100%
Mgr. Veronika Kucháriková100%100%100%
PharmDr. Orsolya Hrubá100%100%100%
Mgr. Nikolett Nemcová100%100%100%
Mgr. Bohuš Kubala100%100%100%
RNDr. Zora Krivošíková PhD.100%100%100%
Mgr. Ihar Karatkevich100%100%100%
RNDr. Michaela Abrahamovská100%100%100%
Mgr. Kristián Slíž100%100%100%
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 Mikuš1,2
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

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.


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


What are the main benefits of the selected online SPE approach in your work?

Thank you for your question! The developed fully integrated SPE–UHPLC–MS/MS approach represents an attractive solution for the monitoring of trace clenbuterol levels in human urine matrices. The sample preparation is reduced to a simple dilution by diluting the sample with water and adding an internal standard solution. The main benefits include (i) minimum sample handling and preparation (due to the online-SPE loading and washing step), (ii) short analysis time and high sample throughput (due to the UHPLC separation), (iii) high sensitivity (due to the triple quadrupole detection) and (iv) high selectivity (due to the SPE–UHPLC–MS/MS arrangement with online sample preparation).

Thank you for an interesting poster presentation.

Clenbuterol is known to be an antiastmatic drug with a known mechanism of action (beta 2 agonist). Why is clenbuterol considered an anabolic in your work? 


Thank you for your question!  For skeletal muscle, beta-2 receptor activation results in nonsteroidal cytoplasmic PI3K/Akt/mTOR/p70SK6 anabolic pathway activation and FOXO catabolic pathway inhibition. These actions shift the protein turnover balance of the cells toward greater synthesis and less degradation.

Greetings, I do have question about clenbuterol: is it possible to quantifiy how often is it in use for professionals within different sport´s types? What are the clenbuterol acts and impacts on organism (in the meaning of practical physiological outcomes)? Thank you in advance for your reply, ZB

Thank you for your interesting questions!

Ad. 1) Yes. WADA annually publishes statistical reports called Anti-Doping Testing Figures Report as reported by the WADA-accredited laboratories. Link:

Ad. 2) Clenbuterol is an orally bioavailable, directly acting sympathomimetic agent with a mainly β-adrenergic activity that binds to β2 receptors with high affinity and selectivity. When clenbuterol binds to β2-adrenoceptors, it activates adenylyl cyclase, leading to an increase in the intracellular concentration of the second messenger cyclic adenosine monophosphate (cAMP) and the activation of protein kinase A (PKA). In the tracheobronchial tree, cAMP and PKA inhibit smooth muscle contraction by opening K+ channels and by down-regulating myosin light-chain kinase activity. For this reason, the most important therapeutic action of clenbuterol is relaxation of the smooth muscle of the airways in the management of reversible obstruction of the airways, such as bronchial asthma and chronic obstructive pulmonary disease. Furthermore, continued use of high oral doses of clenbuterol can elicit an off-target anabolic response in skeletal muscle and a lipolytic response in adipose tissue.

Greetings, thank you for your thorough reply. All the best for your further research. Cordially, ZB

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