HPLC-MS/MS法同时测定人血浆中地尔硫?和代谢物浓度及其在药动学研究中的应用

目的：建立在人血浆中同时测定地尔硫?（DTZ）及其活性代谢物去乙酰地尔硫?（M1）和N-去甲基地尔硫?（MA）浓度的HPLC-MS/MS方法，并应用于健康志愿者口服盐酸地尔硫?片的药动学研究。方法：以DTZ-d4、MA-d4和M1-d6为内标，经乙腈沉淀蛋白后，采用CAPCELL PAK ADME（50 mm×2.1mm，3μm）色谱柱，流动相为含0.1%甲酸水和乙腈，进行梯度洗脱分离。采用AB SCIEX API 4000质谱仪，电喷雾离子源（ESI），正离子扫描，多反应监测模式（MRM）进行检测。24名健康受试者单剂量空腹口服DTZ片30 mg后采集血浆样品，以HPLC-MS/MS法测定血浆中DTZ及其代谢物的浓度，计算药动学参数并进行统计分析。结果：DTZ、M1和MA质量浓度分别在0.1978~40.16、0.2511~5.017和0.2149~20.02ng·mL^-1范围内线性关系良好（r>0.9986）。方法学各项指标均符合要求。健康中国受试者单剂量空腹口服DTZ片30 mg后，DTZ、MA和M1的主要药动学参数依次为C_max（22.2±6.34）ng·mL^-1、（8.38±2.96）ng·mL^-1、（1.52±0.70）ng·mL^-1，T_max（3.19±1.73）h、（4.25±1.93）h、（7.06±1.97）h，AUC_0-∞（197.99±56.38）ng·h·mL^-1、（115.78±29.07）ng·h·mL^-1、（27.22±15.49）ng·h·mL^-1。结论：所建方法简便、快捷、灵敏，适用于地尔硫?及其代谢物的药动学研究。DTZ和MA的血药浓度-时间曲线显示双峰，提示二者在体内可能存在肝肠循环过程。  

Objective: To establish an HPLC-MS/MS method for simultaneous determination the concentration of diltiazem (DTZ)and its active metabolites, such as desacetyl-diltiazem (M1)and N-desmethyl-diltiazem (MA)in human plasma, and carry out pharmacokinetic study in Chinese healthy volunteers orally administrated DTZ. Methods: DTZ-d4, MA-d4 and M1-d6 were used as internal standard. After precipitation by acetonitrile, the sample was separated on the CAPCELL PAK ADME (including 50 mm×2.1 mm, 3 μm)chromatographic column through gradient elution by 0.1% formic acid and acetonitrile. Then the analytes were detected by the AB SCIEX API 4000 mass spectrometer with the electrospray ionization (ESI)sourcein multiple reaction monitoring (MRM)mode. Plasma samples were collected from 24 healthy subjects after single oral administration 30 mg DTZ tablets. The concentrations of DTZ and its metabolites in plasma were determined by HPLC-MS/MS. The pharmacokinetic parameters were calculated and statistically analyzed. Results: DTZ, M1 and MA were all in good linear relationships in the range of 0.200 0-40.00 ng·mL^-1, 0.250 0-5.000 ng·mL^-1 and 0.200 0-20.00 ng·mL^-1, respectively (r>0.998 6). All methodological indicators were in line with requirements. The main pharmacokinetic parameters of DTZ, MA and M1 in healthy Chinese volunteers after single oral administration of 30 mg DTZ tablets in fasting condition were as follows:C_max (22.2±6.34)ng·mL^-1, (8.38±2.96)ng·mL^-1, (1.52±0.70)ng·mL^-1; T_max (3.19±1.73)h, (4.25±1.93)h, (7.06±1.97)h; AUC_0-∞ (197.99±56.38) ng·h·mL^-1, (115.78±29.07)ng·h·mL^-1, (27.22±15.49)ng·h·mL^-1. Conclusion: This method is simple, rapid, sensitive and suitable for clinical pharmacokinetic study of DTZ and its metabolites. The concentration-time curve of DTZ and MA shows double peaks, indicating that they all pass through enterohepatic circulation. 

[1] ECHIZEN H, EICHELBAUM M. Clinical pharmacokinetics of verapamil, nifedipine and diltiazem[J]. Clin Pharmacokinet, 1986, 11(6):425
[2] CHAFFMAN M, BROGDEN RN. Diltiazem, a review of its pharmacological properties and therapeutic efficacy[J]. Drugs, 1985, 29(5):387
[3] SASEEN JJ, CARTER BL, BROWN TE, et al. Comparison of nifedipine alone and with diltiazem or verapamil in hypertension[J]. Hypertension,1996, 28(1):109
[4] GARCIA-PEREZ B, AYALA I, CASTELLS MT, et al. Effects of nifedipine, verapamil and diltiazem on serum biochemical parameters and aortic composition of atherosclerotic chickens[J]. Biomed Pharmacother, 2005, 59(1-2):1
[5] BUCKLEY MM, GRANT SM, GOA KL, et al. Diltiazem. A reappraisal of its pharmacological properties and therapeutic use[J]. Drugs,1990, 39(5):757
[6] MURATA K, YAMAHARA H, NODA K. Pharmacokinetics of diltiazem and its metabolites in dogs after oral administration of a multiparticulate sustained-release preparation[J]. Pharm Res, 1993.10(8):1165
[7] PIEPHO RW, BLOEDOW DC, LACZ JP, et al. Pharmacokinetics of diltiazem in selected animal species and human beings[J]. Am J Cardiol,1982, 49(3):525
[8] HERMANN P,RODGER SD,REMONES G,et al. Pharmacokinetics of diltiazem after intravenous and oral administration[J]. Eur J Clin Pharmacol,1983, 24(3):349
[9] NAKAMURA S,USUKI S,ITO Y,et al. Metabolic fate of diltiazem. Distribution, excretion and protein binding in rat and dog[J]. Arzneimittelforschung,1987, 37(11):1244
[10] YABANA H, NAGAO T, SATO M. Cardiovascular effects of the metabolites of diltiazem in dogs[J]. J Cardiovasc Pharmacol,1985, 7(1):152
[11] GUAN XF, LI DY, YIN WJ, et al. Popμlation pharmacokinetic modeling of diltiazem in Chinese renal transplant recipients[J]. Eur J Drug Metab Pharmacokinet,2018,43(1):55
[12] PATEL CG, LI L, GIRGIS S, et al. Two-way pharmacokinetic interaction studies between saxagliptin and cytochrome P450 substrates or inhibitors:simvastatin, diltiazem extendedrelease, and ketoconazole[J]. Clin Pharmacol, 2011, 2(3):13
[13] MOLDEN E,JOHANSEN PW,BOE GH,et al. Pharmacokinetics of diltiazem and its metabolites in relation to CYP2D6 genotype[J]. Clin Pharmacol Ther, 2002, 72(3):333
[14] MAYHEW BS, JONES DR, HALL SD. An in vitro model for predicting in vivo inhibition of cytochrome P4503A4 by metabolic intermediate complex formation[J]. Drug Metab Dispos, 2000, 28(9):1031
[15] 施孝金,钟明康,王宏图,等. HPLC法同时测定人血浆中地尔硫及其代谢产物[J].药物分析杂志, 1995, 15(6):20 SHI XJ, ZHONG MK, WANG HT, et al. Simultaneous assay fordiltiazem and deacetyldiltiazem in human plasma by HPLC[J]. Chin J Pharm Anal,1995, 15(6):20
[16] MOLDE E, HELEN B G, CHRISTENSEN H, et al. Highperformance liquid chromatography-mass spectrometry analysis of diltiazem and 11 of its phase I metabolites in human plasma[J]. J Pharm Biomed Anal,2003, 33(2):275
[17] QUAGLIA MG,DONATI E,FANALI S,et al. Analysis of diltiazem and its related substances by HPLC and HPLC/MS[J]. J Pharm Biomed Anal,2005, 37(4):695
[18] SMITH MS, VERGHESE CP, SHAND DG, et al. Pharmacokinetic and pharmacodynamic effects of diltiazem[J]. Am J Cardiol,1983, 51(8):1369