尼古丁药动学中的微透析与稳定同位素标记联用技术研究
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摘要
1目的
本课题研究属于国家自然科学基金项目《中药药动学中的微透析与稳定同位素标记联用技术研究》(编号：30772791)的部分内容。
本研究的目的是：探索将稳定同位素内标法结合微透析技术应用于经皮给药与注射给药系统研究的可行性,并考察与方法学相关的体内外影响因素,建立起利用“稳定同位素内标物”与微透析采样技术进行中药局部与整体药动学研究的一般方法。通过利用稳定同位素标记物作为内标物,实现微透析探针回收率的动态监测,解决常规微透析研究中回收率的可变性问题。模型药物是尼古丁(Nicotine),内标物是氘代尼古丁(DL-Nicotine)。
2方法
本课题的实验研究分为四个部分：
2.1尼古丁与氘代尼古丁的分析方法建立
建立液质联用(LC-MS/MS)法测定尼古丁与氘代尼古丁浓度的方法。尼古丁进样量在0.0107-10.7000μg／mL范围内,氘代尼古丁进样量0.0101-10.1480μg／mL范围内考察了方法的线性关系与日内、日间精密度,以及尼古丁与氘代尼古丁的稳定性。
2.2微透析探针的回收率与释放率研究
由于探针透析膜的透过性是双向的,如果把物质透入探针内部的过程称作“回收”,那么物质透出探针的过程则可称为“释放”。本部分研究主要内容是,分别在体外与体内环境下,对待测药物回收率与内标物释放率进行研究。(注：微透析的体内外实验中,灌流液与透析媒体均以人工细胞外液——Krebs-Ringer溶液为介质进行配制。)
2.2.1体外研究
通过改变灌流液流速与透析媒体(尼古丁)的浓度,分别考察灌流液流速与透析媒体浓度对尼古丁回收率与氘代尼古丁释放率的影响,测定氘代尼古丁释放率与尼古丁回收率之间的比例关系(P)。另外还考察了尼古丁回收率的日内重现性和氘代尼古丁释放率的日内稳定性(重现性试验通过在8个不同浓度的透析媒体中进行,循环4次测定尼古丁回收率,同时在全过程中测定氘代尼古丁释放率)。
2.2.2体内研究
家兔为实验动物,腹部皮下植入探针,灌流液为含氘代尼古丁的Krebs-Ringer溶液,浓度为172ng／mL,流速为1μL／mL。通过减量法测定氘代尼古丁在10h内的释放率,考察其体内稳定性。另外,通过比较体内实验前后氘代尼古丁(内标物)的释放率变化,评价探针的可重复利用性。
2.3尼古丁的体内过程研究
家兔为实验动物,耳缘静脉与腹部皮下植入探针,灌流液为含氘代尼古丁的Krebs-Ringer溶液,浓度为172ng／mL,流速为1μL／mL。尼古丁的给药途径分别为肌肉注射与皮下注射,每种给药方式下同时进行血液微透析实验及皮肤微透析实验,取样过程保持家兔清醒状戊(非自由活动),取样时间为10h。通过测定氘代尼古丁释放率,同步校正计算尼古丁回收率,实现体内尼古丁浓度的动态监测。
根据结果绘制药时曲线并进行房室模型分析及药动学参数计算。
2.4两种尼古丁透皮制剂的药动学研究
结合稳定同位素内标法与微透析采样技术,对尼古丁的两种不同透皮制剂——市售尼古丁贴剂(力克雷)与自制尼古丁巴布剂的体内药动学过程进行了研究。
家兔为实验动物,耳缘静脉与腹部皮下植入探针,灌流液为含氘代尼古丁的Krebs-Ringer溶液,浓度为172ng／mL,灌流液流速为1μL／mL。将两种透皮制剂分别贴于家兔已脱毛的腹部皮肤(给药部位处于半透膜位置上方),同时进行血液微透析实验及皮肤微透析实验,取样过程保持家兔清醒状态(非自由活动),取样时间在20h以上。通过测定氘代尼古丁释放率,同步校正计算尼古丁回收率,实现体内尼古丁浓度的动态监测。
根据结果绘制药时曲线并进行房室模型分析及药动学参数计算。
3结果
3.1建立了尼古丁与氘代尼古丁的含量测定方法——液质联用法
液相条件：色谱柱：Hypersil Gold C18柱(150×2.1mm,3μm,Thermo,USA)；流动相：甲醇-2mmol／L乙酸铵(70：30)；流速：0.21mL／min；进样量：5μL。
质谱条件：离子源为大气压电喷雾离子源(ESI)；喷雾电压：3.0kV；鞘气压力：25psi；辅助气流量：5arbitrary units;金属离子传输毛细管温度：300℃；碰撞能量：25eV；正离子检测；扫描方式：选择离子反应监测(SRM),用于定量分析的离子对为m／z 163／130(尼古丁),m／z 166／130(氘代尼古丁)。
在该条件下,峰形良好,杂质无干扰,出峰时间约2.7min。尼古丁进样量在0.0107-10.7000μg／mL范围内线性关系良好,线性回归方程：y=5560.95x+37828.8,r=0.9997,r=0.9997,定量下限：0.0107μg／mL(S／N>10)。氘代尼古丁进样量在0.0101-10.1480μg／mL范围内线性关系良好,线性回归方程：y=5074.83x+50473.3,r=0.9999,定量下限：0.0101μg／mL(S／N>10)
精密度试验中,低、中、高三个浓度的质控样品的日内精密度RSD分别为：4.2%、4.5%、2.7%(尼古丁)；3.8%、7.3%、3.9%(氘代尼古丁)。日间紧密度RSD分别为：11%、1.2%、3.6%(尼古丁)；6.4%、2.3%、4.6%(氘代尼古丁)。在室温放置12h和-20℃反复冻融3次的低、中、高三个浓度的标准品溶液稳定性良好。
3.2微透析探针的回收率研究
3.2.1体外研究
尼古丁回收率、氘代尼古丁释放率均随灌流液的流速增加而降低。同一流速下测得的尼古丁回收率与氘代尼古丁释放率无显著性差异,两者近似相等。尼古丁回收率、氘代尼古丁释放率均与透析媒体浓度无关。同一浓度的透析媒体中测得的尼古丁回收率与氘代尼古丁释放率无显著性差异,两者近似相等。尼古丁回收率具有良好的日内重现性；氘代尼古丁释放率具有良好的日内稳定性。由此说明,在尼古丁微透析研究中可以通过氘代尼古丁(内标物)的释放率校正尼古丁(待测药物)的回收率。
3.2.2体内研究
在连续10h体内取样过程中,氘代尼古丁的释放率为31.7912%±5.3%(mean±SD,n=4),在一定的范围内摆动。
3.3尼古丁的体内过程研究
肌肉注射尼古丁(剂量：11.99mg／kg)后,通过血液微透析取样分析,测得尼古丁的在血液中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程。
皮下注射尼古丁(剂量：7.19mg／kg)后,通过血液微透析取样分析,测得尼古丁在血液中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程。
3.4两种尼古丁透皮制剂的药动学研究
尼古丁贴剂(剂量：11.31mg／kg)给药后,通过血液微透析取样分析,在血液中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程。
自制巴布剂(剂量：191.68mg／kg)给药后,通过血液微透析取样分析,在血液中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程。
4结论
4.1建立了“稳定同位素内标法”,实现了待测药物回收率的动态监测,有效提高药动学数据的真实性与准确性。
4.2建立了液质联用法(LC-MS／MS)同时测定微透析样品中尼古丁与氘代尼古丁浓度的方法,为“稳定同位素内标法”的应用奠定了分析基础。
4.3建立了在家兔体内多部位植入探针的方法,同时进行血液与皮下微透析实验,实现了尼古丁整体药代动力学与局部药代动力学的对比研究。
4.4建立了微透析技术与稳定同位素标记技术联用进行中药整体药动学及局部药动学研究的一般方法学。将“稳定同位素内标法”应用于尼古丁的微透析研究中,对尼古丁的经皮给药系统与注射给药系统进行了药动学研究,方法可行。
1. Objective
We introduce stable labelled isotope technic to prepare interior marker for the first time. It makes the mensuration of microdialysis probe's recovery momently by combining microdialysis and stable labelled isotope technic. The new method won't bring error which formerly microdialysis does.
The objective of the present study is to investigate the liberative character and variational rule of concentration in blood and skin after muscle injection and transdermal del ivery systerm. The main objective of our research is to establish a routine method for entire and local pharmacokinetics study by using microdialysis and stable labelled isotope technic. The omdeldrug is nicotine.
2. Methods
The study contains four parts as follows
2.1 Developing the detection method of nicotine and DL-nicotine
To develope a LC-MS/MS method for simultaneous determination of nicotine and DL-nicotine in microdialysis samples. Find the linear equation between peak area and microdialysis sample size during 0.140-53.085ng(nicotine) and 0.148-50.19ng (DL-nicotine). Investigating the intra and inter-day precision and stability.
2.2 Study of recovery and delivery of microdialysis probe
Assessing the recovery and delivery of microdialysis probe in vitro and in vivo. The perfusate is DL-nicotine with Krebs-Ringer solution.
2.2.1 In vitro study
Assessing perfusate rate(0.5,1,2,3,4μL/min) and medium drug (nicotine) concentration(142,570,1140,2280ng/mL) that may affect recovery and delivery of the probe. Determine intra-day reproductivity of nicotine of microdialysis probe and stability of DL-nicotine of microdialysis probe. (Carring through 8 kinds of different medium drug concentration and determining delivery of DL-nicotine through the entire process in order to investigate the stability of DL-nicotine delivery.)
2.2.2 in vivo study
The experiment animal is rabbit. The probe is planted in the skin at the abdomen. Assessing method of delivery of DL-nicotine is retrodialysis. Determine stability of delivery and affecting fators. Finally, feasibility of reusing probe is also investigated by comparing the delivery of DL-nicotine in vivo before and after.
2.3 Pharmacokinetics study of nicotine by muscle injection and subcutaneous injection
The experiment animal is rabbit. The microdialysis probe should be planted in vein at ear and skin at the abdomen. The perfusate is DL-nicotine with Krebs-Ringer solution. The flow rate is 1u L/mL. Open microdialysis investigation in blood and skin at the abdomen simultaneously after muscle injection and subcutaneous injection. Keep animal awake but immovable through 10h. Detect the delivery of DL-nicotine in order to calculate recovery.of nicotine.
To analyse the pharmacokinetic compartmental model and pharmacokinetics parameters by DAS 2.0.
2.4 Pharmacokinetics study of nicotine patch and nicotine cataplasm
The experiment animal is rabbit. To investigate nicotine patch (nicorette) and nicotine cataplasm by combining microdialysis and stable labelled isotope technic. The microdialysis probe should be planted in vein at ear and skin at the abdomen. The perfusate is DL-nicotine with Krebs-Ringer solution.The flow rate is 1μL/mL. Open microdialysis investigation in blood and skin at the abdomen simultaneously after nicotine patch (nicorette) and nicotine cataplasm were given. Keep animal awake but immovable over 20h. Detect the delivery of DL-nicotine in order to calculate recovery of nicotine.
To analyse the pharmacokinetic compartmental model and pharmacokinetics parameters by DAS 2.0.
3 Result
3.1 Developing a LC-MS/MS method for simultaneous determination of nicotine and DL-nicotine in microdialysis samples.
HPLC separation was carried out on a Hypersil Gold C18 column (150mmx2.1mm, 3μm) with the mobile phase consisting of methanol and 2mmol/L ammonium acetate 70:30. The flow rate was 0.21mL/min. Mass spectra were measured by a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source, in positive ion model. In selected reaction monitoring (SRM) mode, the transitions of m/z 163 to 130, m/z 166 to 130 was used to quantify nicotine and DL-nicotine, respectively. Resultes:The calibration curve was in good linearity over the range of 0.0107-10.7000μg/mL (Nicotine), and 0.0101-10.1480μg/mL(DL-Nicotine). The lower limit of quantification is 0.0107μg/mL (Nicotine, S/N>10), and 0.0101μg/mL (DL-Nicotine, S/N>10). Both the intra and inter-day precisions (RSD) were less than 15%.
3.2 Assessment of recovery of microdialysis probe
3.2.1 In vitro study
In vitro recovery of nicotine and in vitro delivery of DL-nicotine from microdialysis probes determined by gain and by loss were comparable at each flow rate studied. Recovery and delivery decrease exponentially as the flow rate increased(n=4).
nicotine recovery and DL-nicotine delivery are independent of concentration over the concentration range investigated(n=4). And the results of nicotine recovery and DL-nicotine delivery keep accordant basicly. The linearity regression equation between dialysate concentration and medium concentration is y=0.3303x+3.7797, correlation coefficient is 0.9976.
The result of stability test (n=4, flow rate:3μL/mL) indicates that the stability period of the probe is longer than 7h. The result of recovery is 32.5845±2.3114%(mean±SD).
The result of reproductivity test (n=4, flow rate:3μL/mL)shows that recovery of nicotine is stable.
3.2.2 In vivo study
The result of DL-nicotine delivery is 31.7912±5.2769 (mean±SD),swing in a certain range.
3.3 Pharmacokinetics study of nicotine by muscle injection and subcutaneous injection
The result showed that it was one compartment model in blood and two compartment model in skin at the abdomen at the dose of 11.99mg/kg after muscle injection. And two compartment model in blood and one compartment model in skin at the abdomen at the dose of 7.19mg/kg after muscle injection.
3.4 Pharmacokinetics study of nicotine patch and nicotine cataplasm
The result showed that it was two compartment model in blood and one compartment model in skin at the abdomen at the dose of 11.31mg/kg after nicotine patch was given. And it was two compartment model in blood and one compartment model in skin at the abdomen at the dose of 191.68mg/kg after nicotine cataplasm was given.
4 Conclusion
4.1 Combining microdialysis and stable labelled isotope technic for the first time successly. It is feasible to investigate pharmacokinetics of nicotine after muscle injection and subcutaneous injection by the new method.
4.2 Developing a LC-MS/MS method first for simultaneous determination of nicotine and DL-nicotine in microdialysis samples which lay a foundation for the application of stable labelled isotope interior marker.
4.3 Fulfill a comprehensive pharmacokinetics study of microdialysis and stable labelled isotope interior marker in vitro and in vivo including the connection between the recovery and delivery of the probe, and influence, reproduction and stability.
4.4 Carrying the microdialysis study in blood and in skin. And real izing the comparative study between complete and local pharmacokinetics in rabbits.
4.5 Establishing a routine method for complete and local pharmacokinetics study by using microdialysis and stable labelled isotope technic.
本课题研究属于国家自然科学基金项目《中药药动学中的微透析与稳定同位素标记联用技术研究》(编号：30772791)的部分内容。
本研究的目的是：探索将稳定同位素内标法结合微透析技术应用于经皮给药与注射给药系统研究的可行性,并考察与方法学相关的体内外影响因素,建立起利用“稳定同位素内标物”与微透析采样技术进行中药局部与整体药动学研究的一般方法。通过利用稳定同位素标记物作为内标物,实现微透析探针回收率的动态监测,解决常规微透析研究中回收率的可变性问题。模型药物是尼古丁(Nicotine),内标物是氘代尼古丁(DL-Nicotine)。
2方法
本课题的实验研究分为四个部分：
2.1尼古丁与氘代尼古丁的分析方法建立
建立液质联用(LC-MS/MS)法测定尼古丁与氘代尼古丁浓度的方法。尼古丁进样量在0.0107-10.7000μg／mL范围内,氘代尼古丁进样量0.0101-10.1480μg／mL范围内考察了方法的线性关系与日内、日间精密度,以及尼古丁与氘代尼古丁的稳定性。
2.2微透析探针的回收率与释放率研究
由于探针透析膜的透过性是双向的,如果把物质透入探针内部的过程称作“回收”,那么物质透出探针的过程则可称为“释放”。本部分研究主要内容是,分别在体外与体内环境下,对待测药物回收率与内标物释放率进行研究。(注：微透析的体内外实验中,灌流液与透析媒体均以人工细胞外液——Krebs-Ringer溶液为介质进行配制。)
2.2.1体外研究
通过改变灌流液流速与透析媒体(尼古丁)的浓度,分别考察灌流液流速与透析媒体浓度对尼古丁回收率与氘代尼古丁释放率的影响,测定氘代尼古丁释放率与尼古丁回收率之间的比例关系(P)。另外还考察了尼古丁回收率的日内重现性和氘代尼古丁释放率的日内稳定性(重现性试验通过在8个不同浓度的透析媒体中进行,循环4次测定尼古丁回收率,同时在全过程中测定氘代尼古丁释放率)。
2.2.2体内研究
家兔为实验动物,腹部皮下植入探针,灌流液为含氘代尼古丁的Krebs-Ringer溶液,浓度为172ng／mL,流速为1μL／mL。通过减量法测定氘代尼古丁在10h内的释放率,考察其体内稳定性。另外,通过比较体内实验前后氘代尼古丁(内标物)的释放率变化,评价探针的可重复利用性。
2.3尼古丁的体内过程研究
家兔为实验动物,耳缘静脉与腹部皮下植入探针,灌流液为含氘代尼古丁的Krebs-Ringer溶液,浓度为172ng／mL,流速为1μL／mL。尼古丁的给药途径分别为肌肉注射与皮下注射,每种给药方式下同时进行血液微透析实验及皮肤微透析实验,取样过程保持家兔清醒状戊(非自由活动),取样时间为10h。通过测定氘代尼古丁释放率,同步校正计算尼古丁回收率,实现体内尼古丁浓度的动态监测。
根据结果绘制药时曲线并进行房室模型分析及药动学参数计算。
2.4两种尼古丁透皮制剂的药动学研究
结合稳定同位素内标法与微透析采样技术,对尼古丁的两种不同透皮制剂——市售尼古丁贴剂(力克雷)与自制尼古丁巴布剂的体内药动学过程进行了研究。
家兔为实验动物,耳缘静脉与腹部皮下植入探针,灌流液为含氘代尼古丁的Krebs-Ringer溶液,浓度为172ng／mL,灌流液流速为1μL／mL。将两种透皮制剂分别贴于家兔已脱毛的腹部皮肤(给药部位处于半透膜位置上方),同时进行血液微透析实验及皮肤微透析实验,取样过程保持家兔清醒状态(非自由活动),取样时间在20h以上。通过测定氘代尼古丁释放率,同步校正计算尼古丁回收率,实现体内尼古丁浓度的动态监测。
根据结果绘制药时曲线并进行房室模型分析及药动学参数计算。
3结果
3.1建立了尼古丁与氘代尼古丁的含量测定方法——液质联用法
液相条件：色谱柱：Hypersil Gold C18柱(150×2.1mm,3μm,Thermo,USA)；流动相：甲醇-2mmol／L乙酸铵(70：30)；流速：0.21mL／min；进样量：5μL。
质谱条件：离子源为大气压电喷雾离子源(ESI)；喷雾电压：3.0kV；鞘气压力：25psi；辅助气流量：5arbitrary units;金属离子传输毛细管温度：300℃；碰撞能量：25eV；正离子检测；扫描方式：选择离子反应监测(SRM),用于定量分析的离子对为m／z 163／130(尼古丁),m／z 166／130(氘代尼古丁)。
在该条件下,峰形良好,杂质无干扰,出峰时间约2.7min。尼古丁进样量在0.0107-10.7000μg／mL范围内线性关系良好,线性回归方程：y=5560.95x+37828.8,r=0.9997,r=0.9997,定量下限：0.0107μg／mL(S／N>10)。氘代尼古丁进样量在0.0101-10.1480μg／mL范围内线性关系良好,线性回归方程：y=5074.83x+50473.3,r=0.9999,定量下限：0.0101μg／mL(S／N>10)
精密度试验中,低、中、高三个浓度的质控样品的日内精密度RSD分别为：4.2%、4.5%、2.7%(尼古丁)；3.8%、7.3%、3.9%(氘代尼古丁)。日间紧密度RSD分别为：11%、1.2%、3.6%(尼古丁)；6.4%、2.3%、4.6%(氘代尼古丁)。在室温放置12h和-20℃反复冻融3次的低、中、高三个浓度的标准品溶液稳定性良好。
3.2微透析探针的回收率研究
3.2.1体外研究
尼古丁回收率、氘代尼古丁释放率均随灌流液的流速增加而降低。同一流速下测得的尼古丁回收率与氘代尼古丁释放率无显著性差异,两者近似相等。尼古丁回收率、氘代尼古丁释放率均与透析媒体浓度无关。同一浓度的透析媒体中测得的尼古丁回收率与氘代尼古丁释放率无显著性差异,两者近似相等。尼古丁回收率具有良好的日内重现性；氘代尼古丁释放率具有良好的日内稳定性。由此说明,在尼古丁微透析研究中可以通过氘代尼古丁(内标物)的释放率校正尼古丁(待测药物)的回收率。
3.2.2体内研究
在连续10h体内取样过程中,氘代尼古丁的释放率为31.7912%±5.3%(mean±SD,n=4),在一定的范围内摆动。
3.3尼古丁的体内过程研究
肌肉注射尼古丁(剂量：11.99mg／kg)后,通过血液微透析取样分析,测得尼古丁的在血液中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程。
皮下注射尼古丁(剂量：7.19mg／kg)后,通过血液微透析取样分析,测得尼古丁在血液中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程。
3.4两种尼古丁透皮制剂的药动学研究
尼古丁贴剂(剂量：11.31mg／kg)给药后,通过血液微透析取样分析,在血液中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程。
自制巴布剂(剂量：191.68mg／kg)给药后,通过血液微透析取样分析,在血液中的动力学过程属二房室模型,尼古丁的分布和消除为一级动力学过程；通过皮下微透析取样分析,尼古丁在皮下组织中的动力学过程属一房室模型,尼古丁的分布和消除为一级动力学过程。
4结论
4.1建立了“稳定同位素内标法”,实现了待测药物回收率的动态监测,有效提高药动学数据的真实性与准确性。
4.2建立了液质联用法(LC-MS／MS)同时测定微透析样品中尼古丁与氘代尼古丁浓度的方法,为“稳定同位素内标法”的应用奠定了分析基础。
4.3建立了在家兔体内多部位植入探针的方法,同时进行血液与皮下微透析实验,实现了尼古丁整体药代动力学与局部药代动力学的对比研究。
4.4建立了微透析技术与稳定同位素标记技术联用进行中药整体药动学及局部药动学研究的一般方法学。将“稳定同位素内标法”应用于尼古丁的微透析研究中,对尼古丁的经皮给药系统与注射给药系统进行了药动学研究,方法可行。
1. Objective
We introduce stable labelled isotope technic to prepare interior marker for the first time. It makes the mensuration of microdialysis probe's recovery momently by combining microdialysis and stable labelled isotope technic. The new method won't bring error which formerly microdialysis does.
The objective of the present study is to investigate the liberative character and variational rule of concentration in blood and skin after muscle injection and transdermal del ivery systerm. The main objective of our research is to establish a routine method for entire and local pharmacokinetics study by using microdialysis and stable labelled isotope technic. The omdeldrug is nicotine.
2. Methods
The study contains four parts as follows
2.1 Developing the detection method of nicotine and DL-nicotine
To develope a LC-MS/MS method for simultaneous determination of nicotine and DL-nicotine in microdialysis samples. Find the linear equation between peak area and microdialysis sample size during 0.140-53.085ng(nicotine) and 0.148-50.19ng (DL-nicotine). Investigating the intra and inter-day precision and stability.
2.2 Study of recovery and delivery of microdialysis probe
Assessing the recovery and delivery of microdialysis probe in vitro and in vivo. The perfusate is DL-nicotine with Krebs-Ringer solution.
2.2.1 In vitro study
Assessing perfusate rate(0.5,1,2,3,4μL/min) and medium drug (nicotine) concentration(142,570,1140,2280ng/mL) that may affect recovery and delivery of the probe. Determine intra-day reproductivity of nicotine of microdialysis probe and stability of DL-nicotine of microdialysis probe. (Carring through 8 kinds of different medium drug concentration and determining delivery of DL-nicotine through the entire process in order to investigate the stability of DL-nicotine delivery.)
2.2.2 in vivo study
The experiment animal is rabbit. The probe is planted in the skin at the abdomen. Assessing method of delivery of DL-nicotine is retrodialysis. Determine stability of delivery and affecting fators. Finally, feasibility of reusing probe is also investigated by comparing the delivery of DL-nicotine in vivo before and after.
2.3 Pharmacokinetics study of nicotine by muscle injection and subcutaneous injection
The experiment animal is rabbit. The microdialysis probe should be planted in vein at ear and skin at the abdomen. The perfusate is DL-nicotine with Krebs-Ringer solution. The flow rate is 1u L/mL. Open microdialysis investigation in blood and skin at the abdomen simultaneously after muscle injection and subcutaneous injection. Keep animal awake but immovable through 10h. Detect the delivery of DL-nicotine in order to calculate recovery.of nicotine.
To analyse the pharmacokinetic compartmental model and pharmacokinetics parameters by DAS 2.0.
2.4 Pharmacokinetics study of nicotine patch and nicotine cataplasm
The experiment animal is rabbit. To investigate nicotine patch (nicorette) and nicotine cataplasm by combining microdialysis and stable labelled isotope technic. The microdialysis probe should be planted in vein at ear and skin at the abdomen. The perfusate is DL-nicotine with Krebs-Ringer solution.The flow rate is 1μL/mL. Open microdialysis investigation in blood and skin at the abdomen simultaneously after nicotine patch (nicorette) and nicotine cataplasm were given. Keep animal awake but immovable over 20h. Detect the delivery of DL-nicotine in order to calculate recovery of nicotine.
To analyse the pharmacokinetic compartmental model and pharmacokinetics parameters by DAS 2.0.
3 Result
3.1 Developing a LC-MS/MS method for simultaneous determination of nicotine and DL-nicotine in microdialysis samples.
HPLC separation was carried out on a Hypersil Gold C18 column (150mmx2.1mm, 3μm) with the mobile phase consisting of methanol and 2mmol/L ammonium acetate 70:30. The flow rate was 0.21mL/min. Mass spectra were measured by a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source, in positive ion model. In selected reaction monitoring (SRM) mode, the transitions of m/z 163 to 130, m/z 166 to 130 was used to quantify nicotine and DL-nicotine, respectively. Resultes:The calibration curve was in good linearity over the range of 0.0107-10.7000μg/mL (Nicotine), and 0.0101-10.1480μg/mL(DL-Nicotine). The lower limit of quantification is 0.0107μg/mL (Nicotine, S/N>10), and 0.0101μg/mL (DL-Nicotine, S/N>10). Both the intra and inter-day precisions (RSD) were less than 15%.
3.2 Assessment of recovery of microdialysis probe
3.2.1 In vitro study
In vitro recovery of nicotine and in vitro delivery of DL-nicotine from microdialysis probes determined by gain and by loss were comparable at each flow rate studied. Recovery and delivery decrease exponentially as the flow rate increased(n=4).
nicotine recovery and DL-nicotine delivery are independent of concentration over the concentration range investigated(n=4). And the results of nicotine recovery and DL-nicotine delivery keep accordant basicly. The linearity regression equation between dialysate concentration and medium concentration is y=0.3303x+3.7797, correlation coefficient is 0.9976.
The result of stability test (n=4, flow rate:3μL/mL) indicates that the stability period of the probe is longer than 7h. The result of recovery is 32.5845±2.3114%(mean±SD).
The result of reproductivity test (n=4, flow rate:3μL/mL)shows that recovery of nicotine is stable.
3.2.2 In vivo study
The result of DL-nicotine delivery is 31.7912±5.2769 (mean±SD),swing in a certain range.
3.3 Pharmacokinetics study of nicotine by muscle injection and subcutaneous injection
The result showed that it was one compartment model in blood and two compartment model in skin at the abdomen at the dose of 11.99mg/kg after muscle injection. And two compartment model in blood and one compartment model in skin at the abdomen at the dose of 7.19mg/kg after muscle injection.
3.4 Pharmacokinetics study of nicotine patch and nicotine cataplasm
The result showed that it was two compartment model in blood and one compartment model in skin at the abdomen at the dose of 11.31mg/kg after nicotine patch was given. And it was two compartment model in blood and one compartment model in skin at the abdomen at the dose of 191.68mg/kg after nicotine cataplasm was given.
4 Conclusion
4.1 Combining microdialysis and stable labelled isotope technic for the first time successly. It is feasible to investigate pharmacokinetics of nicotine after muscle injection and subcutaneous injection by the new method.
4.2 Developing a LC-MS/MS method first for simultaneous determination of nicotine and DL-nicotine in microdialysis samples which lay a foundation for the application of stable labelled isotope interior marker.
4.3 Fulfill a comprehensive pharmacokinetics study of microdialysis and stable labelled isotope interior marker in vitro and in vivo including the connection between the recovery and delivery of the probe, and influence, reproduction and stability.
4.4 Carrying the microdialysis study in blood and in skin. And real izing the comparative study between complete and local pharmacokinetics in rabbits.
4.5 Establishing a routine method for complete and local pharmacokinetics study by using microdialysis and stable labelled isotope technic.
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