Abstract
A multi‐residue gradient liquid chromatographic (LC) separation was developed for five β‐agonists (fenoterol, metaproterenol, terbutaline, salbutamol and clenbuterol) in human plasma and detection was made using LC/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) in the selected‐ion monitoring (SIM) mode. Detection limits for the protonated molecules of the β‐agonists were <2.5 ppb in plasma or 100 pg on‐column and responses were highly linear over the range 2.5–50 ppb. A solid‐phase extraction (SPE) procedure, yielding recoveries of 67–104% from plasma spiked at 5 ppb with the five analytes, was also developed. In‐source collision‐induced dissociation was used to produce mass spectra containing diagnostic fragment ions and a common mechanism of fragmentation for all the β‐agonists was identified. The LC/APCI‐MS method was unable to determine salbutamol at levels <10 ppb in spiked plasma. To determine whether this resulted from low SPE efficiency or matrix interference, LC/APCI‐MS/MS was performed in the multiple reaction monitoring (MRM) mode to enhance the selectivity of the analysis. The MRM results showed that high recoveries were obtained for salbutamol, confirming the detrimental effect of co‐eluting interferences in the LC/MS analysis. This study demonstrates the feasibility of using SPE, gradient LC separation and highly sensitive/selective detection using LC/APCI‐MS and MS/MS for the multi‐residue analysis of β‐agonists at low ppb levels in a biological matrix.