Chiral liquid chromatography–mass spectrometry (LC–MS/MS) method development for the detection of salbutamol in urine samples

• Chiral separation of salbutamol enantiomers.
• Clean and efficient sequential reversed phase-PBA SPE sample clean-up.
• Application to porcine urine samples.

A sequential solid-phase extraction (SPE) method was developed and validated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) for the detection and quantification of salbutamol enantiomers in porcine urine. Porcine urine samples were hydrolysed with β-glucuronidase/arylsulfatase from Helix pomatia and then subjected to a double solid-phase extraction (SPE) first using the Abs-Elut Nexus SPE and then followed by the Bond Elut Phenylboronic Acid (PBA) SPE. The salbutamol enantiomers were separated using the Astec CHIROBIOTIC™ T HPLC column (3.0 mm × 100 mm; 5 μm) maintained at 15 °C with a 15 min isocratic run at a flow rate of 0.4 mL/min. The mobile phase constituted of 5 mM ammonium formate in methanol. Salbutamol and salbutamol-tert-butyl-d9 (internal standard, IS) was monitored and quantified with the multiple reaction monitoring (MRM) mode. The method showed good linearity for the range of 0.1–10 ng/mL with limit of quantification at 0.3 ng/mL. Analysis of the QC samples showed intra- and inter-assay precisions to be less than 5.04%, and recovery ranging from 83.82 to 102.33%.