高效液相色谱-串联质谱法测定犬血浆中雌三醇的含量

HPLC-MS/MS Determination of Estriol in Canine Plasma

取犬血浆500μL,加入20.0μg·L-1氯霉素内标甲醇溶液50μL,用甲基叔丁基醚(MTBE)先后萃取2次,使样品中雌三醇(E3)溶入MTBE中,MTBE的加入量均为1.00mL,充分摇匀2.0min后,离心10min。收集并合并2次萃取的上清液,氮吹至干。加入甲醇100μL溶解残渣后再次离心10min,取上清液(10.0μL)进样进行色谱分离。用Agilent XDB-C18色谱柱为固定相,和以不同比例的乙腈(A)和水(B)的混合液作流动相进行梯度洗脱。串联质谱分析中采用电喷雾离子源负离子扫描和多反应监测模式。测得E3的线性范围在0.2~40.0μg·L-1之间,测定下限(10S/N)为0.2μg·L-1。在空白犬血浆中加入E3标准溶液进行回收率和精密度试验,测得日间回收率在93.3%~110%之间,测定值的日内和日间相对标准偏差(n=5)分别在1.6%~3.1%和4.2%~5.9%之间。

Estriol(E3)in canine plasma sample(500μL)with 50μL of 20.0μg·L-1 chloramphenicol-C3HOH solution added as internal standard,was extracted twice with 1.00 mL of methyl tert-butyl ether(MTBE)in each extraction.After mixing well for 2 min,the mixture was centrifuged for 10 min.The supernatants from each extraction were collected,combined and evaporated to dryness by N2-blowing.100μL methanol was added to dissolve the residue,and the solution obtained was centrifuged for 10 min.An aliquot of 10.0μL of the supernatant was taken and introduced into the instrument for HPLC separation on Agilent XDB-C18 column,with mixtures of acetonitrile(A)and water(B)in various ratios as mobile phase for gradient elution.In MS/MS analysis,ESI source with negative ion scanning mode and MRM were adopted.Linearity range for E3 was found between 0.2 to 40.0μg·L-1,with value of lower limit of determination(10 S/N)of 0.2μg·L-1.Tests for recovery and precision were made by adding E3 standard solutions to blank canine plasma sample and analyzed by the proposed method.Values of recovery(inter-day)found were in the range from 93.3%to 110%,and values of RSDs(n=5)found were in the ranges of 1.6%to 3.1%(intra-day)and 4.2%to 5.9%(inter-day).