| Servier is a French company that had first developmented ivabradine an anti-angina drugs. A great number of researches have confirmed it's effective and security. It is pomissed by European Drug Enforcement Administration in 27 countries in November 2005 to treat people who had aβ-receptor blockers contraindicated or intolerant chronic stable anginaSymptoms. This drug is the first special defibrillators current (If) inhibitors, which can slow antrum room knot dot .But traditional medicines and its mechanism does not affect blood pressure, blood flow dynamics, force of cardiac muscle systole, conducts or ventricular bipolar.Aim: Our study was to explore and establish LC-MS/MS method (Liquid chromatography– tandem mass spectrometry) to detect ivabradine in human plasma for the first time in this field. Firstly clarify hydrochloric acid ivabradine healthy subjects in China, our researh discusses the dynamic characteristics of traditional Chinese medicine and drug in the body of absorption, distribution, metabolism and excretion of dynamic change characteristics, expounds its disposal pattern of hydrochloric acid ivabradine slice of stage II clinical trials research design and give the scheme determination of doses provide reference basis for its clinical application, provide theoretical basis. Methods: as the following three partsThe first part was to establish specific useful, high sensitivity, repeatability strong, accuracy, LC-MS/MS detecting methods. Detecting steps: in 10 mL centrifugal tube precision add blank plasma samples 1 mL, internal standard solution (2·g·mL-1) 50·L, spiral 30s blending. Add 4 mL ethyl acetate, whirlpool 3 min, in 4,000 r·min-1 centrifugal 10 min. Absorb organic layer to another clean centrifugal tube, 30℃in just to blow to nearly dry nitrogen flow. Scraps with 200·L mobile phase after soluble in 16000 r·min-1 for·8 min, absorb centrifugal supernatant fluid metastasize to the automatic sampler sample tube, the incoming sample amount 5·L, undertake LC - MS/MS analysis.The second part was to study the invivo pharmacokinetics characteristics of ivabradine hydrochloride tablets following a single dose administration in healthy volunteers. With 30 healthy cases randomly divided into three groups, each parallel of the healthy subjects 10 (half by half), low, medium and high three doses were oral 2.5 mg, 5.0 mg, 7.5 mg ivabradine hydrochloride tablet. 4mL blood smaples were collected according to the time schedule, which included a blank durg smaple just prior to dosing and then at 8, 15, 30 and 45 min and 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36, 48h after durg administration. Put them in heparin venous anticoagulant tubes, centrifugal separation out the plasma ,then put it in ordinary dry tube in - 20℃for preserving, to be measured. With LC-MS/MS to determine the concentration of ivabradine in plasma, use DAS2.0 to calculate pharmacokinetic parameters.The third part was to study the invivo pharmacokinetics characteristics of ivabradine hydrochloride tablets following multiple dosing administration in healthy volunteers. After single dose administration, the volunteers of high, medium and low three doses groups continue to be given medicine, every time give dosage for 2.5 mg, 5.0 mg, 7.5 mg, 2 times a day, continuous to be given medicine for 7 days. 4mL blood smaples were collected according to the time schedule, which included smaples just prior to dosing in the morning of the 4th to 7th days,and then at 8, 15, 30 and 45 min and 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36, 48h after durg administration in the 7th day. Put them in heparin venous anticoagulant tubes, centrifugal separation out the plasma ,then put it in ordinary dry tube in - 20℃for preserving, to be measured. With LC-MS/MS to determine the concentration of ivabradine in plasma, use DAS2.0 to calculate pharmacokinetic parameters.Results:1 The LC-MS/MS detection method of ivabradine in human plasma had highly accuracy,sensitive, specificitive, selectivity and good reappearance. the linear range of ivabradine were from 0.101 to 101 ng·mL-1; The lowest limit of quantification was 0.101 ng·mL-1; the intra-precision and intra-precision RSD of three concentrations were all less than 15.0 %, the draw recovery rates were above 70 %; medium effect; stability et al.that meet the criterion of determination of biological samples.2 The 30 cases had been given different single dose ivabradine. the pharmacokinetics parameter of ivabradine were Tmax:0.85±0.43 h,1.15±0.24h,1.00±0.46 h;Cmax:14.92±4.05 ng·mL-1,35.98±8.68 ng·mL-1,48.62±9.62 ng·mL-1;t1/2:3.87±1.30 h,5.03±1.19 h,5.00±1.84 h; AUC0-t:64.23±13.87 ng·mL-1,169.60±33.09 ng·mL-1,215.92±57.21 ng·mL-1;AUC0-∞:64.88±13.97 ng·mL-1,171.19±33.45 ng·mL-1,217.95±56.42 ng·mL-1;CLZ/F:0.04±0.01,0.03±0.01 L·h-1,0.04±0.01 L·h-1;VZ/F:0.23±0.09 L,0.21±0.03 L,0.26±0.10 L. The pharmacokinetics parameter of N-des-ivabradine were: Tmax:1.08±0.75 h,1.30±0.26 h,1.13±0.48 h;Cmax:1.81±0.73 ng·mL-1,3.11±1.61 ng·mL-1, 6.26±1.02 ng·mL-1;t1/2:8.15±3.09 h,9.64±1.32 h,10.78±2.67 h; AUC0-t:13.28±3.26 ng·mL-1,23.33±9.17 ng·mL-1,43.94±9.22 ng·mL-1;AUC0-∞:15.04±3.09 ng·mL-1,25.39±9.49 ng·mL-1,46.87±10.05 ng·mL-1;CLZ/F:0.17±0.04 L·h-1,0.24±0.13 L·h-1,0.17±0.03 L·h-1;Vz/F:2.11±1.31 L,3.22±1.54 L,2.50±0.38 L.3 The 30 cases had been given different multiple dose ivabradine. the pharmacokinetics parameter of ivabradine were:Tmax:0.85±0.44 h,1.08±0.53 h,1.05±1.05 h;Cmax:18.32±3.27 ng·mL-1,37.88±10.56 ng·mL-1,50.01±17.46 ng·mL-1;t1/2:4.55±1.62 h,6.68±2.05 h,7.15±1.90 h; AUC(0-48):93.40±22.86 ng·mL-1,209.89±71.26 ng·mL-1,326.49±135.05 ng·mL-1;AUCss:79.78±16.87 ng·mL-1,165.61±50.30 ng·mL-1,243.61±87.08 ng·mL-1;CL/F:0.03±0.01 L·h-1,0.03±0.01 L·h-1,0.03±0.01 L·h-1;Vd/F:0.18±0.06 L,0.25±0.09 L,0.27±0.15 L. The pharmacokinetics parameter of N-des-ivabradine were Tmax:0.83±0.47 h,1.23±0.80 h,1.30±1.43 h;Cmax:2.60±0.83 ng·mL-1,4.94±1.76 ng·mL-1,7.59±1.79 ng·mL-1;t1/2:11.47±2.83 h,14.25±2.44 h,13.79±3.23 h; AUC(0-48):30.15±9.77 ng·mL-1,58.05±19.92 ng·mL-1,102.19±20.72 ng·mL-1;AUCss:17.51±5.11 ng·mL-1,30.54±10.21 ng·mL-1,52.83±12.94 ng·mL-1;CL/F:0.09±0.05 L·h-1,0.09±0.05 L·h-1,0.07±0.01 L·h-1;Vd/F:1.33±0.30 L,1.83±0.76 L,1.37±0.34 L. The AF of three groups were:1.44±0.22,1.21±0.37,1.51±0.59.Conclusion:1 The LC-MS/MS detection method of ivabradine and N-des-ivabradine in human plasma had been established.The metod had highly accuracy,sensitive, specificitive, selectivity and good reappearance. It could be used for pharmacokinetics study of ivabradine and N-des-ivabradine.2 The Cmax and AUC0-t had linear correlation characteristic of single administration between the three groups of 2.5 mg group, 5.0 mg group,7.5 mg group.3 The Tmax and t1/2 of multiple administration were accordance with single administration, there were no accumulation of ivabradine and N-des-ivabradine in body.Under the three dose ivabradine maybe had a favourable safety. |
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