Analysis of major components of residual bacitracin and colistin in food samples by liquid chromatography tandem mass spectrometry

A selective and sensitive liquid chromatography tandem mass spectrometric (LC-MS/MS) method has been developed for simultaneous determination of residual bacitracin and colistin in bovine milk samples. Milk samples were deproteinized and extracted with a mixture of trichloroacetic/formic acid. After centrifugation, bacitracin and colistin in the extracts were separated on a reversed phase Alltima BDS C18 column (250 mm × 2.1 mm, 5 μm) using a gradient elution programme of ammonium formate buffer (saturated ammonium formate:formic acid:acetonitrile:water, 1:5:50:950, v/v/v/v) and 0.1% formic acid in acetonitrile at 0.2 ml min−1. Using electrospray LC-MS/MS with time-scheduled multiple reaction monitoring (MRM), identification and quantification of the major components of the two polypeptides were performed based upon the intensities of mass fragments from the respective doubly charged precursor ions: bacitracin A at 712 → 199 amu and 712 → 227 amu, and colistin A at 586 → 101 amu, 586 → 202 amu and 586 → 241 amu, respectively, at the defined retention time windows and the matching of the specific tolerance of the relative abundance of the monitored ions. Polymyxin B, a polypeptide of colistin family, was employed as the internal standard. With an additional solid phase extraction procedure, the analytical method was applicable to the analysis of the two polypeptide antibiotics in animal's liver and tissue samples. The practical quantification limits of bacitracin A and colistin A were 100 and 50 μg kg−1; and mean intra-day (n = 7) and inter-day (n = 4) recoveries were in the range of 89.2-110% (R.S.D. < 10%), and 90.4-112% (R.S.D. < 13%) respectively in all matrices.