HPLC-MS／MS法测定C6胶质瘤荷瘤大鼠脑组织中替尼泊苷的浓度

Determination of Teniposide in C6 Glioma Tumor-Bearing Rat Brain Tissue by Sensitive Liquid Chromatography/Tandem Mass Spectrometry

目的建立测定C6胶质瘤荷瘤大鼠脑组织中替尼泊苷含量的高效液相色谱-串联质谱联用法。方法脑组织匀浆后用乙酸乙酯-二氯甲烷(4∶1)液-液提取,流动相重组离心后进样分析。色谱柱为LichrospherC18(2.1mm×100mm,5μm)柱,依托泊苷为内标,以甲醇-0.1%甲酸的5mmol·L-1乙酸铵(70∶30)为流动相,流速0.2mL.min-1,柱温35℃,以多反应离子监测方式检测:替尼泊苷[M+NH4]+,m/z674/383;内标:依托泊苷[M+NH4]+,606/229。结果替尼泊苷和内标依托泊苷的保留时间分别在2.7和2.2min,替尼泊苷的线性范围为3～300μg·L-1。提取回收率大于75%,方法回收率大于90%,日内、日间RSD<10%(n=5)。结论本试验方法简便快速,适用于替尼泊苷体液的浓度测定,利于新药临床研究。

OBJECTIVE To establish a rapid HPLC-MS/MS method for the determination of teniposide concentration in C6 glioma tumor-bearing rat brain tissue. METHODS The samples were extracted from homogenated brain tissue with ethyl acetate-dichlormethane（4 ： 1） by twice liquid-liquid extraction, and the sample was injected after reconstitution with mobile phase . Chromatograph was carried out on a Lichrospher C18 （2.1 minx 100 mm, 5 pm） column, using etoposide as internal standard, and the concentrations of teniposide were determined. The mobile phase was methyl alcohol-0.5 mmol·L^-1 ammonium acetate （contain 0.1% formic acid） （70 ： 30）. The flow rate was set at 0.2 mL·min^-1. Column temperature was 35 ℃ and the samples were analyzed by mass spectrometry in the multitude reaction ion monitoring mode using the target [M＋NH4]^＋ ions m/z 674/383 for teniposide and 606/229 for internal standard etoposide. RESULTS The retention times of teniposide and etopside were 2.7 and 2.2 min, respectively. A good linearity was shown in the range of 3-300 μg·L^-1. The extraction recovery was over 75% and the method recovery was over 90%. The interday or intraday precisions were less than 10% in replicate analysis （n=5）. CONCLUSION This HPLC-MS/MS method is simple, quick. It is suitable for the determination of teniposide humoral concentration, and clinical investigation.