Application Of Microdialysis And Stable Labelled Isotope In Study Of Pharmacokinetics Of Nicotine

1. ObjectiveWe 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. MethodsThe study contains four parts as follows2.1 Developing the detection method of nicotine and DL-nicotineTo 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 probeAssessing 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 studyThe 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 injectionThe 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 cataplasmThe 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 Result3.1 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 probe3.2.1 In vitro studyIn 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 studyThe 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 injectionThe 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 cataplasmThe 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 Conclusion4.1 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.