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目的:建立测定人血浆中三氟柳及其代谢物2-羟基-4-三氟甲基苯甲酸(HTB)的LC-MS方法。方法:考察不同浓度甲酸水溶液作为酯酶抑制剂时三氟柳血浆样品的稳定性,最终以4%甲酸水溶液作为酯酶抑制剂。以LC-MS法分别测定三氟柳及HTB的血药浓度,使用Hedera ODS-2色谱柱,ESI方式。三氟柳用乙酸乙酯提取后测定,流动相为乙腈(A)-含0.1%醋酸的5 mmol·L-1醋酸铵水溶液(B)系统,进行梯度洗脱(0~0.1 min,20%A;0.1~0.15 min,20%A→30%A;0.15~6min,30%A;6~6.5 min,30%A→100%A;6.5~9.5 min,100%A;9.5~10 min,100%A→20%A;10~13.7 min,20%A),流速0.4mL·min-1;三氟柳监测离子为[M-H]-(m/z 247.0),内标对乙酰氨基酚监测离子为[M-H]-(m/z 150.0)。HTB用乙腈沉淀后测定,流动相为甲醇-含3%甲酸的5 mmol·L-1醋酸铵水溶液(75∶25),流速0.25 mL·min-1;HTB监测离子为[M-H]-(m/z 205.0),内标水杨酸监测离子为[M-H]-(m/z137.0)。结果:血浆中三氟柳浓度测定方法的线性范围为0.01~20.37μg·mL-1,血浆中HTB浓度测定方法的线性范围为0.7~159.9μg·mL-1。以本法测定的三氟柳定量下限(0.01μg·mL-1)低于文献报道定量下限(0.03μg·mL-1),可以更准确地估算药物的消除半衰期。本方法成功地运用于血药浓度的测定。结论:本方法均可用于药代动力学以及人体生物等效性试验人血浆中三氟柳及HTB浓度的测定。
Objective: To establish a LC-MS method for the determination of trifloral and its metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) in human plasma. Methods: To investigate the stability of Triflusal sodium plasma samples with different concentrations of formic acid aqueous solution as esterase inhibitor. Finally, 4% formic acid aqueous solution was used as esterase inhibitor. Plasma concentrations of trifolin and HTB were determined by LC-MS, using a Hedera ODS-2 column and ESI. Trifluralin was extracted with ethyl acetate and the mobile phase consisted of acetonitrile (A) - 5 mmol·L -1 ammonium acetate aqueous solution (B) containing 0.1% acetic acid and gradient elution (0 ~ 0.1 min, 20% A 0.1-0.15 min 20% A 30% A 0.15-6 min 30% A 6-6.5 min 30% A 100% A 6.5- 9.5 min 100% A 9.5-10 min, 100% A → 20% A; 10 ~ 13.7 min, 20% A) at a flow rate of 0.4 mL · min-1; the monitoring ion of triflusal was [MH] The ion was [MH] - (m / z 150.0). HTB was determined by acetonitrile precipitation. The mobile phase consisted of methanol - 5 mmol·L-1 ammonium acetate aqueous solution (75:25) containing 3% formic acid at a flow rate of 0.25 mL · min-1. The monitored ion of HTB was [MH] / z 205.0), the internal standard salicylic acid monitor ion is [MH] - (m / z 137.0). Results: The linear range of Trifluralin concentration in plasma was 0.01 ~ 20.37μg · mL-1, and the linear range of plasma HTB concentration was 0.7 ~ 159.9μg · mL-1. The lower limit of determination of triflurofloxacin (0.01μg · mL-1) by this method is lower than the lower limit of quantification (0.03μg · mL-1) reported in the literature, so that the elimination half-life of the drug can be estimated more accurately. The method was successfully applied to the determination of plasma concentration. Conclusion: This method can be used for the determination of triflusal and HTB in human pharmacokinetics and human bioequivalence test.