LC/MS法测定犬血浆中的水飞蓟宾浓度

目的建立犬血浆中水飞蓟宾浓度的LC-MS测定方法。方法取犬血浆400μl,加200μl磷酸氢二钠(0.1mol.L-1)和50μl内标萘酚(10mg·mL-1),3ml乙醚萃取后取上清液挥干,100μl甲醇复溶,离心并取10μl进行LC-MS测定。色谱条件:色谱柱为ODS-C18柱(2.1mm×150mm,5μm,Phenomenex,USA),流动相为甲醇:2%乙酸水=55∶45,流速为0.2ml.min-1;质谱条件:电喷雾离子化(ESI)方式,以选择性负离子方式检测,水飞蓟宾和内标萘酚的选择检测离子质荷比分别为m/z481.00(M-H]-)和m/z142.92(M-H]-)。结果水飞蓟宾的线性范围为25～8000μg.L-1,定量下限(LOQ)为25μg.L-1。准确度、精密度及回收率均符合有关要求。结论本方法专属性强,检测限低,灵敏度高,线性关系良好,方法简便快捷、经济,适用于水飞蓟宾药代及毒代动力学研究。

Determination of silybin in beagle dog plasma by LC/MS

Aim To develop an LC-MS method for determination of silybin in beagle dog plasma.Methods The plasma samples were analyzed by an LC-MS method after liquid-liquid extraction procedure.In brief,400 μl each plasma was placed in a digestion tube with 200 μl of Na2HPO4(0.1 mol·L-1)and 50 μl of naphthol(10 mg·L-1)which were used as the internal standard.Subsequently,3 ml of aether was added to the tube and rigorously vortexed for 1 min.After centrifugation at 5 000 r·min-1 for 10 min to precipitate the proteins 2 ml of supernatant was transferred to fresh test tubes and evaporated under nitrogen gas flow of 40℃.Residues were dissolved in 100 μl of mobile phase and centrifuged at 12 000 r·min-1 for 5 min.Thereafter,10 μl aliquots were injected into the LC/MS for analysis.Separation was performed on a reversed phase C18 column(2.1 mm×150 mm,5 μm,Phenomenex,USA).The mobile phase was composed of a mixture of 2% acetic acid and methanol(45 ∶55%,V/V).The flow rate was 0.2 ml·min-1 and the column temperature maintained at 35℃.The instrument was tuned and optimized for the transmission of the nominal negative ion of silybin and naphtha at m/z 481.00 and 142.92.Mass spectrometer was performed by the negative ion mode and the selected ion monitoring(SIM).Results In these analytical conditions,the retention time for silybin and naphthol were about 6.793 min and 9.835 min,respectively.The assay had a calibration range from 25 to 8 000 μg·L-1 and the lowest limit of quantification(LOQ)of 25 μg·L-1 for silybin.The accuracy,precision,sensitivity,stability and recovery of the method fulfilled the guideline of the analytical validation criteria.Conclusion The present results indicate that this method appeared to be a convenient,sensitive and suitable approach for pharmacokinetic study of silybin in beagle dogs.