衍生化LC-MS测定人血浆中消旋卡多曲活性代谢物浓度及生物等效性

Determination of Racecadotril’s Active Metabolite by a New Derivative LC-MS Method and Their Bioequivalence in Healthy Chinese Volunteers

关键词:  消旋卡多曲  LC/MS  血药浓度  药动学  生物等效性  衍生化

Key words:  racecadotril  LC/MS  plasma concentration  pharmacokinetics  bioequivalence  derivarization

摘要:

目的 建立一种衍生化LC-MS检测人血浆中消旋卡多曲活性代谢物thiorphan(TP)浓度，同时评价消旋卡多曲2种规格散剂(10 mg?袋^-1和30 mg?袋^-1)和国产颗粒剂在健康人体内的药动学及生物等效性。方法 采用3制剂3周期二重3×3拉丁方设计，18名健康男性志愿者交叉单剂量口服受试制剂2种规格消旋卡多曲散(10 mg?袋^-1和30 mg?袋^-1)和参比制剂消旋卡多曲颗粒剂各300 mg后，采用衍生化LC-MS测定不同时间血浆中活性代谢物TP浓度，用DAS药动学程序进行药动学参数的计算及生物等效性评价。血浆样品中加入p-BPB衍生化试剂，得到稳定的TP衍生化产物，经乙酸乙酯萃取后，选替米沙坦作内标。色谱柱为Zorbax SB-C₁₈(150 mm×2.1 mm，5 μm)；流动相为5 mmol?L^-1甲酸铵(甲酸调节pH至3.0)-乙腈(45︰55)；流速为0.2 mL?min^-1；采用ESI+SRM方式监测；TP衍生化产物m/z 452.10[M+H]⁺，内标替米沙坦m/z 515.30[M+H]⁺。结果 TP线性范围为6.28~1 256 ng?mL^-1，最低检测浓度为6.28 ng?mL^-1，提取回收率在77.5%~80.4%，日内、日间精密度RSD<15%。受试制剂消旋卡多曲散(10 mg?袋^-1)，消旋卡多曲散(30 mg?袋^-1)和参比制剂消旋卡多曲颗粒的主要药动学参数：T_max分别为(1.8±1.2)，(1.9±1.3)和(0.7±0.2)h，t_{1/2分别为(1.4±0.4)，(1.2±0.7)和(1.1±0.8)h，Cmax分别为(802.0±356.5)，(804.2±459.8)和(845.6±285.8)ng?mL^-1，AUC0-8分别为(1 617.0±532.6)，(1 628.9±672.1)和(1 621.0±532.2)ng?h?mL^-1，AUC0-∞分别为(1690.8±567.2)，(1673.8±681.7)和(1 649.4±561.4)ng?h?mL^-1。2种受试制剂的相对生物利用度分别为99.8%和100.5%。结论 本方法灵敏，有效，可准确检测人体血浆中TP的浓度。2种受试制剂和参比制剂具有生物等效性。}

Abstract:

OBJECTIVE To establish a LC/MS test method for determining the concentration of thiorphan(TP, the active metabolite of racecadotril) in the human plasma, the pharmacokinetics and bioequivalence of two racecadotril pulvis(10 mg, 30 mg) and racecadotril granule(10 mg) in healthy human body. METHODS A single oral dose 300 mg of three preparations was randomly given to 18 healthy volunteers in a three cycle duplex 3×3 latin square crossover design. The concentration of TP in plasma, which was an active metabolite of racecadotril, was determined by LC-MS at different time. The pharmacokinetics parameters were calculated and the bioequivalence of three formulations were evaluated by DAS program. The p-BPB was added to the plasma as a derivative agent to achieve a stable TP-derivative. The TP-derivative was then extracted from the plasma using ethyl acetate. The method used telmisartan as internal standard material. In the test method, Zorbax SB-C₁₈(150 mm×2.1 mm, 5 μm) was used as chromatogram column, 5 mmol?L^-1 ammonium formate(adjust pH to 3.0 with formic acid)-acetonitrile (45∶55) as mobile phase, the flow rate 0.2 mL?min^-1, ESI+SRM as detector. RESULTS The analytical method validation of TP was as follows: linear range 6.28-1 256 ng?mL^-1, detection limit 6.28 ng?mL^-1, extraction rate 77.5%-80.4%, precision of within and between day(RSD) both <15%. The main pharmacokinetic parameters of racecadotril pulvis(10 mg), racecadotril pulvis(30 mg) and reference granules were as follows: T_max (1.8±1.2), (1.9±1.3) and (0.7±0.2)h, t_1/2 (1.4±0.4), (1.2±0.7) and (1.1±0.8)h, C_max(802.0±356.5), (804.2±459.8) and (845.6±285.8)ng?mL^-1, AUC_0-8(1 617.0±532.6), (1 628.9±672.1) and (1 621.0± 532.2)ng?h?mL^-1, AUC_0-∞ (1 690.8±567.2), (1 673.8±681.7) and (1 649.4±561.4)ng?h?mL^-1, respectively. The relative bioavailability of 2 test pulvis versus reference granule were 99.8% and 100.5%, respectively. CONCLUSION The test method of determining the concentration of TP in human plasma is sensitive and precise. Study shows that the two test racecadotril pulvises are bioequivalent to reference granule.