UPLC-MS/MS同时测定大鼠血浆中他莫西芬与其主要活性代谢物及药动学研究  

Simultaneous determination of tamoxifen and its main active metabolites in rat plasma by UPLC-MS/MS and pharmacokinetics study

作　　者：赵佳佳[1] 熊巍[1,2] 周晓丹[1] 王凌[1] 蒋学华[1] 

机构地区：[1]四川大学华西药学院,成都610041 [2]四川省烟草质量监督检测站,成都610041

出　　处：《药物分析杂志》2017年第7期1172-1180,共9页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立超高效液相色谱-串联质谱法测定大鼠血浆中他莫西芬及其2种主要活性代谢物的浓度,并应用于大鼠口服他莫西芬后的药动学研究。方法:血浆样品中加入适量氘代内标,以甲醇沉淀蛋白处理后,采用UPLC BEH Shield RP18(50 mm×2.1 mm,1.7μm)色谱柱分离,以水(含3.5 mmol·L^(-1)甲酸铵,甲酸调节pH至3.5)为流动相A,甲醇(含3.5 mmol·L^(-1)甲酸铵,甲酸调节pH至3.5)为流动相B;以0.5 mL·min^(-1)的流速进行梯度洗脱,柱温35℃,进样量2μL,样品分析时间为3 min。质谱采用多反应离子监测(MRM)的扫描模式,以电喷雾离子源(ESI)在正离子电离模式下进行测定。选择性监测质荷比为m/z 372.5→72.0(他莫西芬),m/z 388.5→72.0(4-羟基-他莫西芬),m/z 374.5→58.0(4-羟基-N-去甲基-他莫西芬),m/z 377.5→72.0(他莫西芬-d5),m/z 393.5→72.0(4-羟基-他莫西芬-d5),m/z 379.5→58.0(4-羟基-N-去甲基-他莫西芬-d5)。结果:他莫西芬、4-羟基-他莫西芬和4-羟基-N-去甲基-他莫西芬分别在0.956 8~382.7 ng·mL^(-1)、0.500 8~200.2 ng·mL^(-1)和0.500 4~200.2 ng·mL^(-1)浓度内呈良好的线性关系。方法学各项指标均符合要求。药动学参数分别为T_(1/2):(28.00±2.53)、(8.56±0.51)、(13.24±2.20)h,C_(max):(39.97±3.24)、(2.72±0.25)、(2.55±0.13)ng·mL^(-1),T_(max):(3.17±0.98)、(5.50±1.23)、(12.00±0.00)h,AUC_(0-t):(302.62±17.22)、(26.92±1.68)、(82.14±3.75)h·ng·mL^(-1),AUC_(0-∞):(344.19±16.72)、(32.22±1.50)、(88.00±4.91)h·ng·mL^(-1)。结论:本法经方法学验证,适用于大鼠血浆中他莫西芬及其主要活性代谢物的测定。Objectives： To develop a UPLC-MS/MS method for the simultaneous determination of tamoxifen and its 2 kinds of main active metabolites in rat plasma and to apply to the pharmacokinetic study of tamoxifen after oral administration in rats. Methods： After addition of deuterium internal standard, plasma samples were prepared by protein precipitation using methanol, and then were chromatographed on a UPLC BEH Shield RP18 column （ 50 mm× 2. 1 mm, 1.7 μm ）. Mobile phase A ： water （ with 3.5 mml· L^-1 ammonium formate and pH was adjusted to 3.5 by formic acid ） ; Mobile phase B ： methanol （ with 3.5 mmol· L^-1 ammonium formate and pH was adjusted to 3.5 by formic acid ）; the gradient elution was used with a flow rate of 0.5 mL· min^-1. Each analytical run was 3 minutes with injection volume 2μL. The column temperature was 35℃. The detection was performed by the multiple reactions monitoring （ MRM ） mode via the electrospray ionization source （ ESI ） operating in the negative ionization mode. Selected ion mass m/z 372.5 →72.0 （ tamoxifen, TAM ） , m/z 388.5 → 72.0 （ 4-hydroxy- tamoxifen, 4-OH-TAM ） , m/z 374. 5→ 58.0 （ 4-hydroxy-N-demethyl-tamoxifen, EDF ） , m/z 377.5 → 72.0 （ tamoxifen-d5, TAM-d5 ） , m/z 393.5 →72.0 （ 4-hydroxy-tamoxifen-ds, 4-OH-TAM-d5 ） , m/z 379.5 →58.0 （ 4-hydroxy-N-demethyl-tamoxifen-ds, EDF-d5 ） were monitored to quantify. Results： The analytes displayed a good linearity over the concentration of 0. 956 8-382.7 ng· mL^-1 for TAM, 0. 500 8-200.2 ng· mL^-1 for 4-OH-TAM and 0. 500 4-200. 2 ng· mL^-1 for EDF, respectively. The lower-limit-of-quantifications were 0. 956 8 ng· mL^-1, 0. 500 8 ng· mL^-1 and O. 500 4 ng· mL^-1 for TAM, 4-OH-TAM and EDF respectively. Validation of this method is in line with requirements. Pharmacokinetic parameters of TAM, 4-OH-TAM and EDF were as follow： T1/2 ：（ 28.00 ± 2.53 ）, （ 8.56 ± 0.51 ）, （ 13.24 ± 2.20 ） h, Cmax：（ 39.97 ± 3.24 ）, （ 2.72 ± 0.25 ）, （ 2.55 ±

关 键 词：他莫西芬 代谢产物 羟基他莫西芬 羟基去甲基他莫西芬 血药浓度 药动学 超高效液相色谱-串联质谱 

分 类 号：R917[医药卫生—药物分析学]