Simultaneous determination of ß2-agonists in human urine by fast-gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 2-agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid/liquid extraction, followed by derivatization of the extract and detection of 2-agonists trimethylsilyl-derivatives by fast-gas chromatography/electron impact-mass spectrometry (fast-GC/EI-MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra- and inter-assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level (LCL). Fast-GC/MS sequences, based on the use of short columns, high carrier-gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/hour. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring (SIM) modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments.