液相色谱-质谱联用测定鼠血浆中的23-羟基白桦酸

 目的建立测定小鼠血浆中23-羟基白桦酸的液相色谱-质谱联用法(LC-MS)。方法取血浆,加入内标物枸橼苦素,经二氯甲烷-水(6∶1)提取后,40℃水浴中氮气吹干,残渣用甲醇溶解,进行LC-MS测定。色谱柱为Intersil C₈(2.1mm×250mm,3.5μm);体积分数为70%乙腈为流动相(其中三乙胺为体积分数0.05%);流速0.2mL·min^-1;柱温30℃;电喷雾离子源(ESI),负离子检测(23-HBA,m/z 471;柠檬苦素,m/z 469)。结果23-羟基白桦酸的线性范围为10～1000μg·L^-1(r=0.9998),最低定量限为10μg·L^-1,方法平均回收率>88%,日内、日间精密度(RSD)均小于8%。小鼠血浆样品-20℃放置至少3周内稳定。结论本方法专属性强,灵敏度高,线性范围宽,操作简便,适用于药动学研究。

Determination of 23-Hydroxybetulinic Acid in Mouse Plasma by Liquid Chromatogram-Mass Spectrometry

OBJECTIVE To develop a sensitive and specific LC-MS method for the determination of 23-hydroxybetulinic acid in mouse plasma.METHODS 23-Hydroxybetulinic acid and the internal standard limonin were extracted by liquid-liquid extraction. The organic layer was blowed by N₂ to dryness at 40 ℃.The residue was reconstituted in methanol. The LC-MS method was performed on an Intersil C₈ column(2.1 mm × 250 mm, 3.5 μm). The mobile phase consisted of 70% acetonitrile and 0.05% triethylamine. The flow rate was at 0.2 mL ·min^-1.The temperature of column was 30 ℃. Negative ion electrospray ionization(ESI)was used to form deprotonated molecules at m/z 471of 23-hydroxybetulinic acid and 469 of the internal standard limonin. RESULTS The linear calibration curve was observed in the concentration range of 10～1 000 μg·L^-1(r=0.999 8). The limit of quantitation of 23-hydroxybetulinic acid was 10 μg·L^-1.The relative recoveries were more than 88%.The inter and intra-day precisions(RSD)were below 8.0%. 23-Hydroxybetulinic acid was stable in plasma samples at -20 ℃ for at least 3 weeks.CONCLUSION The method is proved to be convenient, sensitive, rapid and suitable for pharmacokinetic study.