LC-MS/MS法测定去甲斑蝥素在大鼠体内的组织分布

Distribution of norcantharidin determined by LC-MS/MS in rat tissues

目的建立高效液相色谱-串联质谱(LC-MS/MS)分析方法,从低、中、高3种剂量研究去甲斑蝥素(NCTD)在体内变化特征。方法液相色谱分离采用CAPCELL PAK MG II C_(18)色谱柱,乙腈-水(含0.1%甲酸)为流动相,梯度洗脱,流速0.2 mL/min,柱温为室温。质谱检测采用ESI离子源,离子对监测(MRM)模式下测定(离子对n/z 169.3→123.1,NCTD;m/z 267.1→135.1,内标利巴韦林)。大鼠尾静脉注射给药后于特定时间点处死动物,取各组织,匀浆,沉淀蛋白后,采用LC-MS/MS法测定NCTD在大鼠各组织中的浓度。结果 NCTD在心、肝、肺中的线性范围为(0.01~10.00)mg/L,在脾和肾中的线性范围为(0.10~10.00)mg/L。各组织样品的日内与日间精密度均小于14%,提取回收率在81.0%~106%之间。3种剂量NCTID静注给药后在大鼠体内呈现相似的动态变化过程(脾除外),给药后1 h达最大浓度,随后快速消除。NCTD在肝和肾中分布较高,且呈剂量依赖性。结论本文建立了一种简便、灵敏、准确的LC-MS/MS测定方法,可用于NCTD大鼠体内组织分布研究。NCTD高剂量给药时,其在肾中较高的浓度可能与肾毒性有关,为临床合理用药提供了参考依据。

Objective To establish a analysis method of LC-MS/MS to study the internal change features of noreantharidin （NCTD） in high dose, medium dose and low dose. Methods The HPLC procedure was performed on the chromatographic column of CAPCELL PAK MG Ⅱ C18 at room temperature. The mobile phase was acetonitrile-water （ containing 0.1% formic acid） in gradient elution, and flow velocity was 0.2 mL/min. A tandem mass spectrometer equipped with electrospray ionization （ESI） source was used for detecting in the mode of multiple reaction monitoring （ MRM, ion pair m/z 169.3→123.1 for NCTD and ion pair m/z 267.1→135.1 for internal standard ribavirin）. The concentration of NCTD was detected in different rat tissues by using LC-MS/MS. Results The linear range of NCTD was （0.01 - 10.00） p,g/mL in heart, liver and lung, and （0.10 -10.00） μg,/mL in spleen and kidney. Intra-day RSD and inter-day RSD were less than 14% in all groups and extraction recovery was between 81.0% and 106%. After intraw^nous injection of NCTD in 3 dose levels, the dynamic changes in all tissues （except of spleen） were similar. The concentration of NCTD reached to the peak after one hour and then rapidly decreased. The distribution level of NCTD was higher in liver and kidney showing a dose- dependent manner. Conclusion The LC-MS/MS method is simple, sensitive and accurate, which can be used for study the distribution of NCTD in rat tissues. When dose of NCTD is higher, its concentration is also higher in kidney, which may be related to its renal toxicity. The study will provide a reference for rational administration of NCTD in clinic.