VAMS and StAGE as innovative tools for the enantioselective determination of clenbuterol in urine by LC-MS/MS.

• Dried urine is obtained by volumetric absorptive microsampling (VAMS). • The microsamples are treated by an original stop-and-go extraction (StAGE) procedure. • Five different polysaccharide-based chiral stationary phases (CSPs) are evaluated. • Enantioselective LC-MS/MS is carried out on a cellulose tris(4-methylbenzoate) column. • Successful application to real urine samples, future anti-doping and clinical study perspectives. Clenbuterol is a chiral, selective β 2 -adrenergic agonist. It is administered as a racemic mixture for therapeutic purposes (as a bronchodilator or prospective neuroprotective agent), but also for non-therapeutic uses (athletic performance enhancement, cattle growth promotion). Aim of the present study is to develop an original, enantioselective workflow for the analysis of clenbuterol enantiomers in urine microsamples. An innovative miniaturised sampling procedure by volumetric absorptive microsampling (VAMS) and a microsample pretreatment strategy based on stop-and-go extraction (StAGE) tips were developed and coupled to an original, chiral analytical method, exploiting liquid chromatography with triple quadrupole detection (LC-MS/MS). The method was validated, with satisfactory results: good linearity (r2 ≥ 0.9995) and LOQ values (0.3 ng/mL) were found over suitable concentration ranges. Extraction yield (>87 %), precision (RSD < 4.3 %) and matrix effect (85–90 %) were all within acceptable levels of confidence. After validation, the method was applied to the determination of clenbuterol in dried urine sampled by VAMS from patients taking the drug for therapeutic reasons. Analyte content ranged from 0.8 to 2.5 ng/mL per single enantiomer, with substantial retention of the original drug racemic composition. The VAMS-StAGE-LC-MS/MS workflow seems to be suitable for future application to anti-doping testing of clenbuterol in urine. [ABSTRACT FROM AUTHOR]