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

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.