Study On Fluctuation Of Microdialysis Recovery In Pharmacokinetics Of Transdermal Delivery System And Application Of Internal Standard Methord

1 ObjectiveMicordialysis is a kind of in vivo, realtime and online sampling method from extracellular fluid (ECF). We can calculate the concentration of the drug organization using the concentration of dialysate collected in the outflow side and the recovery. Microdialysis recovery will change during the test in vivo, so the method used before testing the recovery only once before or after the experiment (or the average of the two) can not correct the changes of recovery. The concentration of the drug surrounding the probe calculated by such changing recovery is suspected. So it is necessary to establish an on line method to determine the recovery at each sampling point.The objective of this study is to explore the factors inducing the fluctuation of recovery and to evaluate the feasibility of using internal standard method for the determination of nicotine recovery in microdialysis. Pharmacokinetics of two kind of nicotine patches was study using the research results.2 MethodsThis research includes four sections:2.1 Establishment of analytical method of samplesMethod of RP-HPLC with ultraviolet detector was applied to detect the content of nicotine and codeine phosphate. The chromatographic conditions were optimized and the methodology was investigated.2.2 Study on the fluctuation of recovery and factors inducing the fluctuationThe factors including the molecular weight and pKa of Alkaloid, pre-processing of the perfusate, perfusion flow rate, media concentration around the probe and temperature on the recovery of nicotine in vitro were investigated. The stability of recovery in vitro and in vivo was also investigated. The recovery of the four probes before and after the in vivo experiment was compared in vitro.2.3 Study on evaluation of the feasibility of using internal standard method for the determination of nicotine recovery in microdialysisThe appropriate internal standard was selected in the four alkaloids by examining the separation of chromatographic peaks of internal standard and nicotine and the stability of the proportion P of the recovery (or delivery) of nicotine to that of internal standard.Nicotine and codeine phosphate were dissolved in Ringer's solution. Nicotine, codeine phosphate and the mixture of them were perfused through the probe separately to calculate the proportion of the recovery (or delivery) of nicotine to that of codeine phosphate. And then codeine was perfused through the probe which was immersed in nicotine solution with different concentrations to calculate the proportion, too. In another condition nicotine was dissolved in rat plasma. The rat plasma protein binding rate was determined by using retrodialysis and internal standard method in vitro.Nicotine, codeine phosphate and the mixture of them were perfused through the probe separately to calculate the proportion of the recovery (or delivery) of nicotine to that of codeine phosphate in the subcutaneous tissue and plasma of rats. The delivery of codeine phosphate before and after administration of nicotine patches was compared with t test.2.4 Study on pharmacokinetic of two kinds of nicotine patches in vivoPermeation enhancer was selected by using uniform design. The optimized combination of propylene glycol (PG), Azone and oleic acid (OA) was studied.The probe was planted in the jugular vein and skin at the back of the rat. The flow rate of the perfusate was 1.0μl/min. These two kinds of nicotine patches were put on the surface of the back of the rat respectively and the dialysate sampling was collected during 16h. The concentration of nicotine in the tissue was calculated by the recovery of nicotine detected by internal standard method. The drug concentration-time curve was draw to analyse nicotine's pharmacokinetics behavior. The pharmacokinetics parameters were calculated using non-compartment model.3 Results3.1 Establishment of analytical method of samplesTwo chromatographic conditions were established according to the different drug concentrations in vivo and in vivro. The results showed that the two chromatographic peaks of nicotine and codeine phosphate separate from each other and have good symmetry. There was good linear relation between integral quantity of peak area and sample size. The limit of quantification and the limit of detection for nicotine and codeine phosphate met the requirement. 3.2 Study on the fluctuation of recovery and factors inducing the fluctuationThe molecular weight of the five alkaloids and their delivery in vitro microdialysis showed a good linear rerationship with the correlation coefficient r= 0.996. The relationship between delivery and PKa of alkaloids was not found yet. Perfusate on the ultrasound, filtration and other pre-treatment was able to improve recovery of nicotine in vitro (n=4). Recovery of nicotine determined by gain and by loss were almost the same at each flow rate studied in vitro. Recovery decreased as the flow rate increased (n=4). Nicotine recovery was independent of concentration over the concentration range investigated (n=4). Recovery increased as the temperature increased (n=5). The result of stability test indicated that the stability period of the probe is longer than 24h. The result of recovery was (60.5±1.5)%. The recovery of nicotine detected in vivo was (21±7)%. After the experiment in vivo, the recovery in vitro decreased with the maximum decrease of 46.8%.3.3 Study on evaluation of the feasibility of using internal standard method for the determination of nicotine recovery in microdialysisThe proportion of the recovery (or delivery) of nicotine to that of codeine phosphate was 1.358～1.414 when the drugs were dissolved in Ringer's solution, which changed in the range of 4%. The proportion was fairly stable and independent from the concentration of nicotine around the probe. Protein binding rate determined by internal standard method (39.15%) was almost the same as that determined by retrodialysis (39.80%). The P-values calculated in the same animal through the same probe were almost the same, but changed in different animals or different probes. The result of t test showed that there was no significant difference between the delivery of codeine phosphate determined before and after administration.3.4 Study on pharmacokinetic of two kinds of nicotine patches in vivoThe optimal combination was PG:Azone:OA=10%:4.5%:1%.Pharmacokinetic analysis showed that MRT of nicotine after the administration of the two kinds of nicotine patches determined in the blood was higher than that determined in the skin, and MRT of nicotine determined after the administration of the saled nicotine patches was higher than that determined after the administration of self-made nicotine cataplasm. It suggested that the retention time of nicotine in the blood is longer than that in the skin after the administration of the two kinds of nicotine patches. The retention time of nicotine determined after the administration of saled nicotine patches was longer than that determined after the administration of self-made nicotine cataplasm. It indicated that the two kinds of nicotine patches met the requirements of smoking cessation. The saled nicotine patches can long maintain a lower blood concentration in a little dose which met the requirements of smoking cessation.4 Conclusions 1. The molecular weight of the five alkaloids was in inverse proportion to their delivery in vitro microdialysis. The relationship between delivery and PKa of alkaloids was not found yet.2. Pre-treatment of perfusate, velocity of the perfusate and temperature would influence recovery. Nicotine recovery was independent of concentration over the concentration range investigated.3. Recovery of nicotine was fluctuated in rats, which was more stable in vitro. After the experiment in vivo, the recovery in vitro decreased.4. Codeine phosphate can be used as the internal standard in determining the recovery of nicotine.5. The proportion of the recovery (or delivery) of nicotine to that of codeine phosphate was fairly stable in vitro and in vivo and independent from the concentration of nicotine around the probe. The delivery of codeine phosphate was not affected by the administration. The internal standard method is an effective way in the determination of nicotine recovery.6. The retention time of nicotine in the blood is longer than that in the skin after the administration of the two kinds of nicotine patches. The retention time of nicotine determined after the administration of saled nicotine patches was longer than that determined after the administration of self-made nicotine cataplasm. It indicated that the two kinds of nicotine patches met the requirements of smoking cessation. The saled nicotine patches can long maintain a lower blood concentration in a little dose which met the requirements of smoking cessation.