超快速液相色谱-串联质谱法测定辛伐他汀的血药浓度及其生物等效性研究

Determination of Simvastatin in Human Plasma by UFLC-MS /MS and Its Application to Bioequivalence Study

目的建立检测人血浆中辛伐他汀的超快速液相色谱一串联质谱法（UFLC—MS／MS），从而考察2种辛伐他汀片的相对生物利用度。方法血浆样品经甲基叔丁基醚液液萃取后，经Shim—packXR—ODS色谱柱分离，采用梯度洗脱模式，流动相为水（A）-乙腈（B），梯度条件为O．01～3min，B：80％～90％；3～3．5min，B：90％～95％，3．51～5min，B：80％。多反应离子监测（MRM）模式进行定量分析，用于监测的离子反应对分别为m／z441．2—325．1（辛伐他汀）和m／z427．2—a25．2（内标洛伐他汀）。20名男性健康受试者随机双周期双交叉方式分别口服单剂量辛伐他汀试验制剂及参比制剂40nlg后于不同的时间点采血，并按照所建立的方法进行含量测定，并通过PhoenixWinNonlin6．0软件评价2种制剂的药动学参数及生物利用度。结果血浆中辛伐他汀在0．1—20ng·mL。内线性关系良好（0．9954），最低检测限为0．1ng·mL，提取回收率63．9％～83．8％．日内精密度〈7．1％．日间精密度〈10．0％。受试制剂和参比制剂的主要药动学参数分别为：1／2（3．96±1．65）和（3．77±1．75）h；（8．364±4．990）和，（33．39±14．55）ng·h·mL-1。以AUCo计算，受试制剂辛伐他汀的相对生物利用度为（97．3±24．3）％。结论本方法简单，快速，灵敏，成功应用于辛伐他汀生物等效性研究，辛伐他汀受试制剂相对参比制剂生物等效。

OBJECTIVE To develop a UFLC-MS/MS method for the determination of simvastatin in human plasma, so as to in- vestigate the relative bioavailability of two kinds of simvastatin tablets. METHODS After liquid-liquid extraction by methyl ten-butyl ether, the analytes were seperated on a Shim-pack XR-ODS column with a gradient elution by the mobile phase of water（A） and aceto- nitrile（B）. Conditions of the gradient elution were as follows ： 0. 01 - 3 min, B ：80% - 90% ; 3 - 3.5 min, B ：90% - 95% ,3.51 - 5 min, B ：80%. Multiple reaction monitor（MRM） was used to determine the concentration of simvastatin（ m/z 441.2--＊325.1 ）and the internal standard, lovastatin（ m/z 427.2±325.2）. A single oral dose of 40 mg test or reference preparation was given to 20 healthy volunteers in a randomized crossover design. Blood samples were obtained and analyzed by the validated UFLC-MS/MS method, and the pharmacokinetics and bioavailability were evaluated by Phoenix WinNonlin 6. 0 software. RESULTS The calibration curve of simvas- tatin was linear over 0. 1 - 20 ng mL - J （ r = 0. 995 4） with the lower limit of quantity of 0. 1 ng mL - 1. The absolute recoveries were between 63.9% and 83.8% , the extraction recoveries were 63.9% - 83.8% , and inter- and intra-day RSDs were less than 7.1% and 10. 0%, respectively. The main pharmacokinetic parameters of simvastatin of the test and reference tablets were as follows ： t1/2 were （ 3.96 ± 1.65 ） and （ 3.77 ± 1.75 ） h ;Pm= were （ 8. 364 1 ± 4. 989 8 ） and （ 8.439 9 ± 4. 856 6 ） ng mL - 1; tox were （ 1.71 ± 1. 19 ） and （ 1.74 ± 1.10） h ; AUC0 were （ 29.74 ± 13.05 ） and （ 31.16 ± 13.92 ） ng h mL- 1 ; AUCo.~ were （ 32. 35 ± 14.56 ） and （ 33.39 ± 14. 55 ）ng h mL- 1 , respectively. The relative bioavailability of the test to reference tablets was （97. 3 ＋ 24. 3 ）%. CONCLUSION The two preparations are bioequivalent. The established method is successfully used for the bioequivalenee study of simvastatin prepa- rations.