Establishment Of RP-HPLC Methods For Determination Of Valproic Acid And Metabolites And A Preliminary Study On The Relationship Between Serum Concentration And CYP2C9~*3

objective: Simultaneous determination the plasma concentration of VPA and its metabolites 2-Propyl-2-pentenoic（2-ene-VPA）and 2-Propyl-4-pentenoic（4-ene-VPA）using the reversed phase high performance liquid chromatography（RPHPLC）.Analysis of the relationship between VPA dose and the plasma concentration of VPA,2-ene-VPA and 4-ene-VPA,the effects of gender on serum concentrations of VPA,2-ene-VPA and 4-ene-VPA,the relationship of cytochrome P4502C9（CYP 2C9）gene polymorphism with the plasma concentration of VPA and 4-ene-VPA,to provide reference for better individualized medication.Methods: Choosed the under 18 years epilepsy paediatric patients who taken VPA alone,without abnormal liver function or organic disease.Taken VPA everday,after steady-state concentration,phlebotomize in the second morning and then to test such as:（1）Simultaneous determination the plasma concentration of VPA,2-ene-VPA and 4-ene-VPA by RP-HPLC.Octanoic acid was selected as an internal standard,acidification by sulfuric acid,n-hexane extraction,nitrogen gas dry the samples,acetonitrile dissolution,2,4’-Dibromoacetophenone as derivatization reagent,triethylamine catalysis,at 80 degrees water bath derivatization 30min;ZORBAX SB-C18 column（4.6mm×150mm,5μm）was uesd,an UV detector was 258 nm,the mobile phase consisted of acetonitrilemethanol-water was 59:13:28,the flow rate was 1.0 mL·min^-1,column temperature was 35 ℃.（2）Detected the genotypes of CYP2C9 of 74 patients by blood direct PCR kit;（3）Analyzed the effects of VPA dosage,sex and age influenced on the serum concentrations of VPA,2-ene-VPA and 4-ene-VPA.Analysis the relationship between genotypes of CYP2C9 and VPA,4-ene-VPA plasma concentration.（4）The measurement data was expressed as mean±SD.Using SPSS 20.0 for windows analyzed the relationship between VPA dose and the plasma concentration of VPA,2-eneVPA and 4-ene-VPA,the relationship between plasma concentration of VPA and 2-ene-VPA,4-ene-VPA.Investigating the influence of age and gender on plasma concentration of VPA,2-ene-VPA and 4-ene-VPA,the relationship between CYP2C9 genotype and plasma concentration of VPA and 4-ene-VPA by independent samples T-test and one-way ANOVA respectively.Result:（1）RP-HPLC analysis method of VPA,2-ene-VPA and 4-ene-VPA: retention times of VPA,2-ene-VPA,4-ene-VPA and Octanoic acid（internal standard,IS）were 11.6 min,9.4 min,8.6 min and 12.4 min respectively.The area ratio of VPA,2-ene-VPA and 4-ene-VPA with IS peak had good linear relationship with concentration,linear range of VPA was 5.00²00.00 μg·mL-1,the standard curve was yVPA=0.2169 xVPA+0.6286（r=0.9995,n=5）;linear range of 2-ene-VPA was 0.50¹6.00 μg·mL-1,the standard curve was y2-ene-VPA=0.3206 x2-ene-VPA+0.1041（r=0.9991,n=5）;linear range of 4-ene-VPA was 0.50¹6.00 μg·mL-1,the standard curve was y4-ene-VPA=0.4357 x4-ene-VPA+0.3531（r=0.9996,n=5）and LLOQ was5.00 μg·mL-1,0.50 μg·mL-1,0.50 μg·mL-1 respectively（S/N>5）.The extrac-tion recovery rate at low,medium and high concentration of VPA,2-ene-VPA and 4-eneVPA was（74.60%,81.44%,81.97%）,（76.82%,83.26%,78.57%）and（79.46%,83.26%,79.36%）respectively.Inter-day,intra-day and stability relative standard deviation RSD was less than 5%.（2）CYP2C9 gene band size was 315 bp,the wild type allele CYP2C9*1 and the mutant allele CYP2C9*3 frequency was 95.25% and4.75% respectively,the wild type homozygote（CYP2C9*1/*1）and the mutant heterozygote（CYP2C9*1/*3）genotype frequency was 90.5% and 9.5% respectively;（3）A total cases of 74 epilepsy paediatric patients were selected,the mean values of the age was（6.5±4.1）years old and the range values of the VPA doses were（18.34±5.86）mg·kg-1;（4）The relationship between VPA doses administered and the concentration of VPA,2-ene-VPA and 4-ene-VPA: The VPA dosage were positively correlated with VPA plasma concentrations（R=0.330,P<0.05）and has no significant correlation with the plasma concentrations of 2-ene-VPA and 4-ene-VPA（P=0.175,R=0.160）,（P=0.131,R=0.186）;（5）The influence of gender on plasma concentration of VPA,2-ene-VPA and 4-ene-VPA:（1）There are 74 cases epilepsy paediatric patients,the males and females VPA doses was（18.01±5.58）mg·kg-1and（19.02±6.18）mg·kg-1 respectively,the VPA dosage has no statistical significance between two groups（P=0.505）;（2）The plasma concentrations of VPA,2-ene-VPA and 4-ene-VPA in males were（70.15±24.79）μg·mL-1,（3.56±1.51）μg·mL-1,（1.07±0.40）μg· mL-1,in females were（76.23±23.61）μg·mL-1,（3.39±1.70）μg·mL-1,（1.08±0.39）μg·mL-1respectively,the two groups plasma concentrations had no statistical significance（P=0.346）,（P=0.682）,（P=0.970）.（6）The effect of genotypes on plas-ma concentration of VPA and 4-ene-VPA:（1）The VPA dosage of group A（CYP2C9*1/*1）and group B（CYP2C9*1/*3）were（17.89±5.66）mg·kg-1 and（22.12± 5.15）mg·kg-1respectively,and the VPA dosage has no statistical significance bet-ween two groups（P=0.062）.（2）The plasma concentrations of VPA in the two groups（group A=70.94±25.28 μg·mL-1,group B=79.93±12.94 μg·mL-1）had no statistical significance（P=0.359）.（3）The plasma concentrations of 4-ene-VPA in the two groups（group A=1.11±0.39 μg·mL-1,group B=0.76±0.29 μg·mL-1）had significant statistical significance（P= 0.025）.Conclusion:（1）We has been simultaneous determinated the plasma concentration of VPA,2-ene-VPA and 4-ene-VPA by RP-HPLC.It can be used to determine the drug concentration in patients with epilepsy after taking VPA and to analyze the efficacy and side effects of drugs;（2）CYP2C9*1/*3 phenotype reduces the activity of CYP2C9 and affects the metabolism of valproic acid,which is an important factor affecting the efficacy and adverse reactions.