Determination Of Dihydromyricetin In RP - HPLC And Its Oral And Metabolic Kinetics In Beagle Dogs

AIM:To study the extraction, concentrating, detected methods, the limit of quantitity and limit of detection of DMY in plasma and linear relationship, precision, accuracy and stability of detection method to establish a reverse phase HPLC-UV analytical method for DMY in plasma.Sametimes, it is also to investigate the pharmacokinetics parameters of DMY orally administrated to Beagle dogs.METHODS:To determine the ultraviolet spectra of DMY.Two solvents of acetonitrile and ethyl acetate were used to extract DMY in plasma at room temperature or in ice water bath, and temperature and time of dryness by nitrogen flow was choosed to confirm treated condition of Plasma samples.Reverse phase HPLC-UV, isocratic elution or gradient elution by acetonitrile-phosphoric acid(0.1%) as mobile phase, C18 collumn as immobile phase and carbamazepine as internal standard, was used to examine systemic suitability, specificity, limit of detection, limit of quantity and linear relationship of detection for DMY. The precision, accuracy and stability of detected method were assayed in three concentrations of DMY in plasma and RSDs should be less than 15%.Three doses of DMY(37.5,75 and 150mg/kg) were orally administrated to Beagle dogs and blood was taked at ten time points before and after administration to measure concentration of DMY and to evaluate pharmacokinetics parameters of DMY in plasma by pharmacokinetics model of DAS software.RESULTS:both of the maximum absorption of DMY and a comparatively large absorption of carbamazepine was at 292nm in ultraviolet spectra,therefore 292nm was choosed as the detection wave length of DMY. Extratction rate of DMY from plasma was significantly higher by ethyl acetate than acetonitrileextraction,it was nearly to 80% by ethyl acetate for three times and it was higher at a low temperature. Recovery rate of DMY in plasma was higher at lower temperature and in shorter time while dryness of nitrogen gas flow. A gradient program of elution by acetonitrile-phosphoric acid(0.1%) was used as follows:the initial elution condition was 15:85(v/v), linearly changed to 38:62 at 13min,38:62 from 13min to 16 min, linearly changed to 15:85 at 20min. The flow rate was 1.0 mL/min.The injecting volume was 10μL. Good HPLC chromatograms with a baseline separation of compounds in plasma were obtained within 20min.The chromatographic peaks of DMY and carbamazepine as internal standard standards were found before 15min and they were not disturbed by endogenous substance.No other chromatographic peak was found after 20min. A good systemic suitability and specificity was shown. The calibration curves were linear in the range of 40-1000μg/L.The limits of quantity and the limits of detection were 40μg/L and 0.5ng(injecting 5μL in concentration of 20μg/L), respectively. The inter-day and intra-day precision and accury were well determined at concentrations of 80,200,800μg/L for DMY and the RSD was less than 10%.DMY plasma samples were stable at room temperature for 1 hour and in refrigerator for 2 weeks, but unstable at room temperature for two hours and freezing-thawing.The extraction percentage and relative recovery of 3 concentrations were over the range of 79.1%±5.5%-83.6%±4.9%,95.9±3.5%-109.1%±1.9%, respectively.The accury of quality control samples during detection was in the range of 85%-115%,and the RSD was less than 10%.Drug concentrations in plasma increased with dose increasing after oral administration of DMY(37.5mg/kg,75 mg/kg,150mg/kg). Blood drug level descended to 1/10-1/20 peak concentration level at 6-8h after oral administration. Plasma drug concentrations were greatly different from individuals.Analyzed by statistical moment parameters, AUC increased with dose increasing of DMY, MRTwas not significant related with dose, t1/2 of DMY was 1.0-1.75h,Tmax was about lh,Cmax parallelly increased with dose,Vz/F was maxlmum in middle dose and minimum in high dose,CLz/F decreased with dose increasing.CONCLUSIONS:Temperature could be a big factor affecting stability of DMY in plasma samples.So DMY samples in plasma should be treated at lower temperature and stored in refrigerator after collection. This study provide a sensitive and accurate method for DMY HPLC-UV detection with C18 Collumn as immobile phase and acetonitrile-orthophosphoric acid(0.1%) as mobile phase. Plasma drug concentrations and metabolic process differ greatly among individuals after oral administration of DMY to Beagle dogs.