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.

先前报道的LC-MS方法定量定量组织中的8-α-羟基-mutilin（头孢菌素的标记残留物）均使用了伪MRM过渡（从质子化分子离子到质子化分子离子，m / z 337→337）。在寻找产物离子时，导致检测的选择性和灵敏度欠佳。通过在碱性介质中使用电喷雾负电离，我们首次发现了对8-α-羟基-mutilin，m / z的高度选择性和灵敏的真正MRM过渡335→179.通过这种新发现的MRM过渡和使用截短侧耳素作为内标，已经开发出了一种非常灵敏，选择性强且功能强大的LC-MS / MS方法，并经过验证可用于定量家兔中的8-α-羟基-mutilin组织（肌肉，肝脏，肾脏和脂肪）。与以前发表的方法相比，选择性和灵敏度得到了显着提高。对于已验证的浓度范围（0.2–10 ppm或0.2–10μg/ g），批内和批间准确度（偏差百分比）分别为-5.0至3.1和-4.9至3.0。批内和批间精度的％CV分别为2.2到6.6和4.7到8.3。经验证的方法已成功用于支持兔子的两项GLP组织残留耗竭研究。