摘要目的：建立测定人血浆中尼莫地平的毛细管气相色谱电子捕获检测法，并用本法研究尼莫地平片剂在健康人体内的药代动力学及相对生物利用度。方法：色谱柱为25 m×0.2 mm ID OV-101熔融石英毛细管柱，检测器为⁶³Ni电子捕获检测器。内标为尼群地平，血浆样品在碱性条件下用正己烷—乙酸乙酯(1∶1)提取。结果：浓度在2.0～150.0 ng^.ml^-1与峰面积比呈良好线性关系，γ=0.99989。人血浆中尼莫地平的最低检出浓度为0.1 ng^.ml^-1，方法重现性好，提取回收率大于80%。10名志愿者随机交叉口服单剂量100 mg二种国产尼莫地平片剂后，以本法测定其体内过程符合一室模型。二种片剂的AUC_0→∞。C_max，T_max均无显著差异。结论：尼莫地平血药浓度测定结果表明两种尼莫地平片剂生物等效，A片剂对B片剂的相对生物利用度为102.0%。

DETERMINATION OF NIMODIPINE AND ITS PHARMACOKINETICS

IN HUMAN PLASMA BY CAPILLARY GAS

CHROMATOGRAPHY USING ELECTRON-CAPTURE DETECTION

Ding Li(Ding L), Gao Ling(Gao L)¹, Wang Guangji(Wang GJ)¹,

Zhang Zhengxing(Zhang ZX) and An Dengkui (An DK)

(Department of Pharmaceutical Analysis, ¹Research Center of Pharmacokinetics,

China Pharmaceutical University, Nanjing 210009)

ABSTRACTAIM: To develope a capillary GC-ECD method for the study of phamacokinetics and relative bioavailability of nimodipine tablet in human body.METHODS: Chromatography was performed on a 25 m×0.2 mm ID 0.2 μm film thickness OV-101 fused-silica capillary column coupled with a ⁶³Ni electron-capture detector. The carrier gas was highly pure nitrogen. The flow rate of carrier gas in the capillary column was 1.8 ml·min^-1. The split ratio was 1∶33. The flow rate of make-up gas was 17 ml·min^-1. The temperatures of the column, injector and detector were 260℃, 270℃ and 300℃, respectively. The internal standard was nitrendipine. After making alkaline with 1 mol·L^-1 NaOH solution, the plasma was extracted with n-hexane — ethyl acetate (1∶1).RESULTS: A good linearity was obtained from 2.0 ng·ml^-1 to 150.0 ng·ml^-1 of nimodipine in human plasma with a correlation coefficient of 0.99989. The detection limit of nimodipine in human plasma was 0.1 ng·ml^-1. The extraction recovery was more than 80%. The pharmacokinetics of nimodipine was determined by this GC-ECD method following a single oral dose of 100 mg of two kinds of domestic nimodipine tablets given to each of 10 volunteers in an open randomized two-way crossover design. The results showed that the plasma concentration-time courses of the two kinds of tablets conformed to one compartment model. There was no significant difference between the two formulations in the AUC_{0→∞→∞}, C_max and T_max.CONCLUSION: The established GC-ECD method was found to be a good method for determination of nimodipine in human plasma. The results of statistical analysis showed that the two formulations of nimodipine were biologically equivalent. The relative bioavailability of tablet A was 102.0% compared with that of tablet B.

KEY WORDSnimodipine; capillary GC-ECD; pharmacokinetics

尼莫地平为扩张脑血管、解除脑血管痉挛的一种1，4-二氢吡啶类钙离子通道拮抗剂，临床上主要用于治疗有明显症状的老年性脑功能损伤和由于蛛网膜下腔出血后脑血管痉挛引起的缺血性神经损伤、偏头痛及轻、中度高血压等疾病。目前血浆中尼莫地平的含量测定方法多采用HPLC法^［1～4］，但多数存在来自血浆的内源性物质的干扰，使方法的专属性受影响，双内标法虽能部分解决杂峰干扰问题^［3］，但较繁琐。此外，这些方法检测灵敏度不高，血浆中尼莫地平的最低检出浓度只能达到2～5 ng^.ml^-1，某些受试者服药6～8 h后已无法检出尼莫地平^［3,4］。Krol等^［1］虽最早报道了血浆中尼莫地平的GC测定法，但该法需将尼莫地平氧化成吡啶类似物后再测定，方法繁琐且不能区分原形药物及其代谢产物，故不适于体内样品测定方法。此后，Jakobsen等^［5］报道了血浆中尼莫地平的填充柱气相色谱电子捕获检测法，该法使尼莫地平的检测灵敏度提高了一个数量级，血浆中尼莫地平的最低检出浓度可达0.5 ng^.ml^-1，但该法中内标尼群地平并未与杂质峰达到基线分离。本文设计了血浆中尼莫地平的高分辨毛细管气相色谱电子捕获检测法，此法不但改善了色谱分离，而且使检测灵敏度得到进一步提高，血浆中尼莫地平的最低检出浓度可达0.1 ng^.ml^-1。用此法对10名健康志愿者口服国内两家药厂生产的尼莫地平片剂后的体内过程进行了研究，统计学结果表明两种片剂生物等效。

实验部分

1仪器与试药

Shimadzu GC-14A气相色谱仪；Shimadzu C-R6A数据处理机；Shimadzu ⁶³Ni电子捕获检测器；正己烷、乙酸乙酯、甲醇均为分析纯；尼莫地平和尼群地平(江苏省药品检验所提供)；尼莫地平片剂(常州聚荣制药有限公司，批号970813，20 mg/片，以下简称A片；上海信谊药厂，批号E970701，20 mg/片以下简称B片)。

2色谱条件

色谱柱为25 m×0.2 mm ID OV-101熔融石英毛细管柱，膜厚0.2 μm；载气为高纯度氮气，柱中载气流速为1.8 ml^.min^-1，分流比为1∶33，尾吹为17 ml^.min^-1，柱室、气化室及检测器的温度分别为260，270，300℃。电子捕获检测器参数为脉冲电流1 nA，量程1。

3血药浓度测定方法及最低检测浓度

于暗室中，取血浆1 ml置离心管中，精密加入1 μg^.ml^-1尼群地平甲醇溶液35 μl，1 mol^.L^-1 NaOH 0.5 ml，涡旋10 s，加乙酸乙酯—正己烷(1∶1) 6 ml，涡旋3 min，于4000 r^.min^-1离心5 min。取有机相5 ml于50℃水浴，氮气流吹干，用甲醇50 μl溶解残渣，精密吸取2 μl，按上述色谱条件进样分析。色谱图见图1。尼莫地平及内标尼群地平的出峰时间分别为12.6 min及7.6 min，血浆中杂质峰不干扰样品测定，以尼莫地平计理论塔板数为1.1×10⁵。在上述条件下，测得血浆中尼莫地平最低检出浓度为0.1 ng^.ml^-1(S/N=3)。

Fig 1Gas chromatograms of blank plasma (a), blank plasma with nitrendipine and nimodipine (b), volunteer plasma after oral administration of nimodipine tablets (c). 1. Nitrendipine; 2. Nimodipine.

4人体实验方案

4.1受试者选择10名健康男性受试者，体重(67.6±7.7) kg，年龄(22.1±0.7) a，经生化检验证实肝、肾功能正常，心电图检查正常，精神状态良好。受试者在被告知所有与药物有关的可能的不良反应后，签署受试者知情同意书，保持随时退出实验的权利。实验前一周及实验期间未服用其它任何药物及烟和酒，用药前12 h及用药后4 h内禁食，实验期间统一饮食。

4.2给药方案及血样采集采用双交叉试验设计方案，即10名男性健康受试者，按体重分组随机分成A，B两组，随机一半先服A片，后服B片；另一半先服B片，后服A片。剂量均为100 mg(5×20 mg)，两种制剂间隔一周。于早上7∶00空腹服药100 mg(5×20 mg)，用温开水200 ml送服。于服药后0.33，0.67，1.0，1.5，2.0，3.0，4.0，6.0，8.0和10.0 h于肘静脉取血3.0 ml，置肝素化试管中，4000 r^.min^-1离心5 min，取血浆1.0 ml供血样分析。

4.3数据分析将所得血药浓度-时间数据用PKBP-N1程序计算药物的药代动力学参数，再取均值±s。A片的相对生物利用度以B片为对照，非房室模型依赖性参数AUC和清除率(Cl/F)分别按下列各式计算：

AUC_0→10=Σ(C_i+C_i-1)×(T_i-T_i-1)/2

AUC_0→∞=Σ(C_i+C_i-1)×(T_i-T_i-1