Quantitative urine spot microsamples for the chiral analysis of clenbuterol by capillary electrokinetic chromatography

The application of quantitative volumetric dried urine spot (qDUS) sampling and analysis to the enantioselective determination of the β2-adrenergic agonist clenbuterol is described herein for the first time. The chiral determination is obtained by capillary electrokinetic chromatography (CEKC) coupled to tandem mass spectrometry (MS/MS), using carboxymethyl-β-cyclodextrin as the chiral selector and sample stacking by electrokinetic injection. Innovative qDUS was carried out by means of Capitainer®B 50 devices, which were confirmed to be able to produce volumetrically accurate 50-µL urine spots and applying an accelerated drying protocol by dehumidified air. After solvent extraction, the samples were analysed by the CEKC-MS/MS method. The analytical workflow was validated according to current international guidelines, with good results in terms of linearity (r^2 = 0.9994 for both enantiomers), extraction yields (≥ 87 %), intra- and inter-day precision RSD (≤ 11.8 %) and matrix effect (≤ 11 %). Sample stability was high (≥ 97 %) even though qDUS were stored at room temperature for 90 days. Application to the analysis of real qDUS samples from patients undergoing therapy with racemic clenbuterol was successful, with negligible detected enantiomeric excess, as expected. The developed analytical platform based on qDUS coupled to CEKC-MS/MS is thus suitable for application to enantioselective determination of clenbuterol in different settings, including toxicological, forensic and therapeutic drug monitoring ones.