Improving the selectivity and sensitivity for quantifying 8-α-hydroxy-mutilin in rabbit tissues by using basic mobile phases and negative ionization

Abstract Previously reported LC–MS methods for quantifying 8-α-hydroxy-mutilin (a marker residue of tiamulin) in tissues all used a pseudo MRM transition (from protonated molecular ion to protonated molecular ion, m / z 337 → 337) due to difficulties in finding a product ion, leading to suboptimal selectivity and sensitivity for detection. By using electrospray negative ionization in a basic medium, we, for the first time, found a highly selective and sensitive true MRM transition for 8-α-hydroxy-mutilin, m / z 335 → 179. With this newly found MRM transition and the use of pleuromutilin as the internal standard, a very sensitive, selective, and robust LC–MS/MS method has been developed and validated for quantifying 8-α-hydroxy-mutilin in rabbit tissues (muscle, liver, kidney, and fat). In comparison with the previously published methods, the selectivity and sensitivity were significantly improved. For the concentration range validated (0.2–10 ppm or 0.2–10 μg/g), the within-run and between-run accuracies (% bias) ranged from −5.0 to 3.1 and −4.9 to 3.0, respectively. The% CV ranged from 2.2 to 6.6 and 4.7 to 8.3 for within-run and between-run precisions, respectively. The validated method was successfully used to support two GLP tissue residue depletion studies in rabbits.