Extracellular and intracellular zilpaterol and clenbuterol quantification in Hep G2 liver cells by UPLC-PDA and UPLC-MS/MS

Abstract

Zilpaterol and Clenbuterol are β-adrenergic agonists that have been widely used to feed cattle. Although the use of Zilpaterol has been approved, Clenbuterol is still used illegally at unknown doses. However, the research of both substances has been based mainly on the evaluation of residues. To our knowledge, this is the first time that a cellular model using Hep G2 cells treated with Zilpaterol and Clenbuterol is presented as an alternative approach to quantify both drugs at the cellular level. Thus, a complete analytical methodology has been developed for the accurate quantitation of these β-adrenergic agonists in both cellular compartments. We propose the use of ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) for extracellular determinations while UPLC coupled to a tandem mass spectrometer (UPLC-MS/MS) for intracellular analysis. The methods were fully validated in terms of selectivity, linearity, accuracy, and precision, limits of detection and quantitation (LOD and LOQ, respectively), stability, carryover, and matrix effect. The method for intracellular content was linear ranging from 0.25 to 8 ng/mL while for extracellular content, the concentration of Zilpaterol and Clenbuterol ranged from 0.125 to 4 μg/mL, with correlation coefficients of R > 0.98 and >0.99, respectively. The combination of the two methodologies in the cellular model showed intracellular concentrations of 0.344 ± 0.06 μg/mL and 2.483 ± 0.36 μg/mL for Zilpaterol and Clenbuterol, respectively. Extracellular concentration was 0.728 ± 0.14 μg/mL and 0.822 ± 0.11 μg/mL for Zilpaterol and Clenbuterol, respectively. This work shows the potential applications of cellular modelling in the study of toxicity for the mentioned drugs.

Keywords: Clenbuterol; Extracellular compartment; Intracellular compartment; UPLC-MS/MS; UPLC-PDA; Zilpaterol.