HPLC荧光法测定盐酸曲马多及其片剂人体生物等效性

Determination of Tramadol in Human Plasma by HPLC with Fluorescence Detection and Its Application to Bioequivalence Study

目的建立HPLC荧光检测法测定曲马多及活性代谢物O-去甲基曲马多在血浆中的浓度,研究曲马多片在健康人体中的药动学及生物等效性。方法采用随机交叉自身对照试验设计,20名健康男性受试者单次po 100 mg曲马多口腔崩解片和曲马多片后,按规定时间采集肘静脉血,血样经液.液萃取处理,以0.03 mol·L^(-1)四硼酸钠(含0.5%三乙胺,磷酸调pH至4.0)-甲醇(75:25)为流动相,色谱柱为Diamonsil C_(18)柱(4.6 mm×200mm,5μm),流速1.0 mL·min^(-1),柱温50℃,荧光检测的激发波长275 nm,发射波长304 nm,测定曲马多和O-去甲基曲马多的血药浓度,并计算两制剂的主要药动学参数及相对生物利用度。结果测定血浆中曲马多和O-去甲基曲马多的最低检测限均为1.0μg·L^(-1),曲马多和O-去甲基曲马多分别在1.0～600.0和1.0～300.0μg·L^(-1)内线性关系良好;血药浓度测定的日内、日间精密度RSD均小于5%;测得曲马多口腔崩解片和曲马多片的血样中曲马多的主要药动学参数为:t_(max)(2.2±1.0)和(1.9±0.9)h,ρ_(max) (350.4±66.0)和(339.0±73.2)μg·L^(-1),t_(1/2)(6.9±1.8)和(6.8±1.8)h,AUC_(0-t)(3 953±1 550)和(3 703±1 310)μg·h·L^(-1);血样中O-去甲基曲马多的主要药动学参数为:t_(max)(3.4±1.7)和(3.0±1.5)h,ρ_(max)(51.5±20.7)和(50.2±19.4)μg·L^(-1),t_(1/2)(7.8±2.0)和(7.5±1.8)h,AUC_(0-t)(744±200)和(691±141)μg·h·L^(-1);曲马多口腔崩解片的相对生物利用度以曲马多计算为(106.4±16.4)%,以O-去甲基曲马多计算为(107.5±17.2)%。结论建立的HPLC荧光法灵敏、准确;测定结果经统计学分析曲马多口腔崩解片和曲马多片为生物等效制剂。

OBJECTIVE To develop a HPLC-fluorescence method for determining tramadol（TMD）and the active metabolite O- demethyltramadol（ODT） in human plasma and to study pharmacokinetics and relative bioavailability of tramadol tablets in healthy Chinese volunteers. METHODS A single dose of 100 mg tramadol orally disintegrating tablet and reference tramadol tablet was given to 20 healthy male volunteers according to a randomized cross-over design. The concentration of TMD and ODT in plasma was determined by HPLC with fluorescence detection after liquid-liquid extraction. A reverse phase C18 column （Diamonsil, 4. 6 mm×200 mm, 5μm） was used. The mobile phase was borax buffer （0.03 mol·L^-1 , containing 0. 5 % triethylamine, pH 4.0） -methanol（75 ： 25） , and was delivered at 1.0 mL·min^-1. The column temperature was maintained at 50℃. Fluorescence detection wavelength was set at 275 nm（excitation） and 304 nm（emission）. The pharmacokinetics and bioavailability were studied. RESULTS The linearity of TMD and ODT were 1.0 - 600. 0 and 1.0 - 300. 0 μg·L^-1, respectively. The quantity limit of TMD and ODT both were 1.0 μg·L^-1. Intra- and inter-day precisions were both less than 5%. The TMD pharmacokinetic parameters of tested and reference tablet were tmax（2.2±1.0）and （1.9±0.9） h, ρmax（350.4±66.0）and （339.0±73.2）μg·L^-1, t1/2（6.9±1.8）and （6.8±1.8）h, AUC0-t （3953±1550 ） and （ 3703± 1310 ） μg ·h·L^-1 . The ODT pharmacokinetic parameters of tested and reference tablet were tmax（ 3.4±1.7） and （3.0±1.5） h, ρmax（51.5±20.7） and （50.2±19.4）μg·L^-1, t1/2（7.8±2.0）and（7.5±1.8） h, AUC0-t（ 744 ± 200） and （691 ± 141 ）μg·h·L^-1. The relative bioavailability of tested to reference tablets calculated by TMD and ODT was（ 106.4±16.4 ） % and （ 107. 5 ± 17. 2） % respectively. CONCLUSION A sensitive and accurate HPLC method was developed. The comparative bioequivalence study demonstrate that the two preparations are bioequivalent.