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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。
OBJECTIVE: To establish a method for the determination of tamoxifen and its two major active metabolites in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry and to study the pharmacokinetics of tamoxifen in rats. Methods: The deuterated internal standard was added to the plasma samples and treated with methanol precipitation. The samples were separated on a UPLC BEH Shield RP18 (50 mm × 2.1 mm, 1.7 μm) column with water (containing 3.5 mmol·L -1 ) Ammonium formate and formic acid to adjust the pH to 3.5) as mobile phase A, methanol (containing 3.5 mmol·L -1 ammonium formate and formic acid to adjust the pH to 3.5) as mobile phase B and 0.5 mL · min -1 ) Gradient elution, the column temperature 35 ℃, injection volume 2μL, sample analysis time was 3 min. Mass spectrometry was performed using multiple reactive ion monitoring (MRM) scanning mode with electrospray ionization (ESI) in positive ionization mode. The m / z 372.5 → 72.0 (tamoxifen), m / z 388.5 → 72.0 (4-hydroxy-tamoxifen), m / z 374.5 → 58.0 (4-hydroxy- M-z 377.5-72.0 (tamoxifen-d5), m / z 393.5-72.0 (4-hydroxy-tamoxifen-d5), m / z 379.5-> 58.0 (4-hydroxy-N-desmethyl-tamoxifen-d5). Results: The values of tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen were respectively 0.956 8 ~ 382.7 ng · mL -1 and 0.500 8 ~ 200.2 ng · ML ~ (-1) and 0.500 4 ~ 200.2 ng · mL ~ (-1) concentration showed a good linear relationship. Methodology indicators are in line with the requirements. The pharmacokinetic parameters were 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) and (88.00 ± 4.91) h · ng · mL ~ (-1). Conclusion: This method is validated by methodology and is suitable for the determination of tamoxifen and its major active metabolite in rat plasma.