LC-MS法测定人血浆中的三氟柳及其活性代谢物_2-羟基-4-三氟甲基苯甲酸

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;0.15~6 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)，流速0.4 mL·min^-1;三氟柳监测离子为 ^-(m/z 247.0)，内标对乙酰氨基酚监测离子为 ^-(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/z 205.0)，内标水杨酸监测离子为 ^-(m/z137.0)。结果：血浆中三氟柳浓度测定方法的线性范围为0.01~20.37 μg·mL^-1，血浆中HTB浓度测定方法的线性范围为0.7~159.9 μg·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 develop a reliable liquid chromatography-mass spectrometry(LC-MS)method for the determination of triflusal and its active metabolite 2-hydroxy-4-(trifluoromethyl)benzoic acid(HTB)in human plasma. Methods: The stability of triflusal in human plasma was evaluated when formic acid of different concentrations was used as the esterase inhibitor.The plasma concentration of triflusal and HTB was determined by LC-MS,respectively.Separation of the analytes was achieved on a Hedera ODS-2 column and MS determination was carried out in electrospray ionization mode.The stability of triflusal in human plasma was kept by adding 20 μL of 4% formic acid to an aliquot of 400 μL of plasma,and then triflusal was extracted with acetyl acetate.Triflusal was eluted with a mobile phase of acetonitrile(A)-5 mmol·L^-1 ammonium acetate solution containing 0.1 % acetic acid(B)using 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 deprotonated ions were at m/z 247.0 and 150.0 for monitoring for triflusal and the internal standard(IS)paracetamol,respectively.The protein precipitation method was used for HTB sample treatment.HTB in the treated samples was separated with a mobile phase of methanol-5 mmol·L^-1 ammonium acetate solution containing 3% formic acid(75∶ 25)at a flow rate of 0.25 mL·min^-1.The deprotonated ions were at m/z205.0 and 137.0 for monitoring for HTB and salicylic acid(IS)respectively. Results: The calibration ranges were 0.01- 20.37 μg·mL^-1 and 0.7-159.9 μg·mL^-1 for triflusal and HTB,respectively.As the lower limit of quantitation for triflusal(0.01 μg·mL^-1)was lower than that by the previous reported Methods(0.03 μg·mL^-1),the elimination half-life of triflusal in human could be determined more accurately.The current method was successfully applied to determine the concentration levels of triflusal and HTB in human plasma. Conclusion: The Methodscan be used for determination of triflusal and HTB in human plasma in pharmacokinetics and bioequivalence studies.