| Objective:To establish a method for determination of 2-propyl-4-pentenoic（4-ene-VPA）,Valproate’s toxic Metabolite, in plasma by RP-HPLC. And to explore the relationship between the plasma concentration of 4-ene-VPA and the adverse reaction, which will provide the reference to the rational clinical application of the Valproate（VPA）. Methods:（1） The plasma concentration of 4-ene-VPA in children with epilepsy was determined by RP-HPLC. An UV detector（256nm） and ZORBAXSB-C18 column（150mm ×4.6mm,5μm） were used. The mobile phase consisted of methanol-water（75:25）. The flow rate was 1.0 m L·min-1, and column temperature was 35℃. The plasma ample was extracted by ethyl acetate, and2,4’-Dibromoacetophenone was selected as derivative reagent. Octanoic acid and triethylamine were used as internal standard and catalysts, respectively. The system was put in thermostatic water bath at 70℃ for 30 minutes,and nitrogen gas was used to dry the samples. The residues were dissolved in 100 ml methanol and was filtered by 0.45μm microporous membrane. Then the sample was injected into the RP-HPLC for analysis.（2） The outpatients with epilepsy who need monitor the plasma concentration of VPA in our hospital were consulted. Then the patients under 18 years old who were taking VPA over seven days and reaching steady-state plasma concentration were included. In totally, 96 patients’ plasma samples were collected,including 53 males and 43 females. The mean age was（7.8 ± 4.8） years, and the mean body weight was（26.9 ± 15.9） ㎏, and the average height was（116.5 ± 32.3） cm.Then the patients’ biochemical values were recorded. The 96 patients were divided into five groups: ① The 0 to 2 years old group（n = 14）,② The 3 to 6 years old group（n = 30）, ③ The 7 to 10 years old group（n = 22）, ④The 11 to 14 years old group（n = 21）,⑤The 15 to 18 years old group（n = 9）. SPSS19.0 software was used for statistical analysis, and the measurement data were represented by sx)( ±.Results:The maximum absorption wavelength of 4-ene-VPA was determined at256 nm by Agilent 1260 DAD diode array detector. And the retention time of derivatives and the internal standard’s were 8.92 min and 13.15 min, respectively.Without endogenous substances interfering, the method was of good specificity. The linear range of 4-ene-VPA was 120μg·mL-1, which showed a good linear relationship. The standard curve was rxy=±=)9928.04119.02680.0(,and LLOQ was 1μg·mL-1（S/N>5）. At low,medium and high concentration(1μg·mL-1,4μg·mL-1and 16μg·mL-1, respectively), the recoveries were 114.5%, 109.3% and 97.1%,respectively. The inter-day relative standard deviation（RSD） was 4.62%, while the intra-day RSD was 5.85%. The low, medium and high concentration plasma samples were placed at-80℃ for three months, and the relative error was 8.6%, 0.8% and14.0%, respectively.①For 02years old group（n=14）, the concentration of VPA and4-ene-VPA were（66.6 ± 8.8）μg·mL-1 and（2.4 ± 0.3） μg·mL-1, respectively, and glutamic-pyruvic transaminase（ALT）was 20.92 ± 9.10. ②For 36 years old group（n= 30）, the concentration of VPA and 4-ene-VPA were（61.2 ± 4.7） μg·mL-1 and（2.2 ±0.1） μg·mL-1, respectively, and ALT was 15.27 ± 6.59. ③For 710 years old group（n= 22）, the concentration of VPA and 4-ene-VPA were（74.3 ± 7.0） μg·mL-1and（2.6 ±0.2） μg·mL-1, respectively, and ALT was 12.50 ± 5.46. ④For 1114 years old group（n = 21）, the concentration of VPA and 4-ene-VPA were（59.7 ± 5.0） μg·mL-11and（2.5± 0.2） μg·mL-1, respectively, and ALT was 14.57 ± 4.80. ⑤For 1517 years old group（n = 9）,the concentration of VPA and 4-ene-VPA were（63.8 ± 5.0） μg·mL-1and（2.8 ± 0.3） μg·mL-1, respectively, and ALT was 13.56 ± 4.80. In 02-year old group,the concentrations of 4-ene-VPA and ALT were of significant correlation（P = 0.018, r= 0.621）, While the VPA concentrations and ALT had no correlation. All other age groups, both the concentrations of 4-ene-VPA and VPA had no correlation with ALT.Conclusion: The method is accurate、sensitive and suitable for clinical routine monitoring of 4-ene-VPA. The RP-HLPC can determination epileptic’s plasma concentration of 4-ene-VPA who take a conventional-dose and reach a steady state concentration,the result is 2.46±0.84 μg·mL-1which can apply to Clinical. |
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