Clean-up, detection and determination of salbutamol in human urine and serum

Abstract

Salbutamol {2-(tert-butylamino)-1-[4-hydroxy-3-(hydroxymethyl)phenyl]et hanol}, also known as albuterol, is clinically the most widely used β₂-adrenoceptor agonist in the treatment of bronchial asthma. During this study, we evaluated liquid–liquid extraction (LLE) and solid-phase extraction (SPE) in order to develop a reliable extraction method followed by analysis using liquid chromatography and gas chromatography. An assay is described which involves SPE as the clean-up method followed by gas chromatography-mass spectrometry to determine salbutamol levels in human serum after oral administration. The SPE method requires the use of a hyper-cross-linked styrene–divinylbenzene bonded phase (ENV+) without involving any sample pre-treatment to obtain 60–65% recoveries for salbutamol and terbutaline as the internal standard. Distilled water and 1% trifluoroacetic acid in methanol were found to be the most suitable washing solvent and eluting solvent, respectively. A detection limit of 2 ng mL⁻¹ was achieved by derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide to form trimethylsilyl (TMS)-salbutamol (m/z 369) and TMS-terbutaline (m/z 356). The relationship between the ratio of the peak area of salbutamol to that of the internal standard and concentration was linear for the range tested (2–200 ng mL⁻¹) and the correlation of coefficient was 0.9999 with a y-intercept not significantly different from zero. The inter-day relative standard deviation (RSD) was <10% for all three concentrations. The intra-day RSD was 14% for 2 ng mL⁻¹. This assay was then successfully applied to human serum samples obtained from clinical trials after oral administration of salbutamol.