| ObjectiveSodium valproate is widely used in clinical practice and is a first-line broad-spectrum antiepileptic drug.Epilepsy patients need to take drugs for a long time,and the poor effect of drug treatment is likely to lead to disease recurrence or even aggravation.In order to ensure the therapeutic effect of patients and reduce adverse reactions,this paper will establish a methodology for monitoring the plasma concentration of Valproic Acid（VPA）,determine the steady-state plasma concentration of clinical patients,and analyze the related factors affecting the plasma concentration of Valproic Acid.Mitochondrial DNA Polymeraseγ（POLG）genotype polymorphism was detected in patients with abnormal liver function,suspected Mitochondrial diseases,and patients who had failed conventional treatment,and the mutation frequency of this gene was calculated by combining with literature data.Finally,combined with blood drug concentration monitoring and genetic testing,economic evaluation was conducted on the medication regimen for epilepsy patients,and a safe,effective and economic individualized medication regimen was formulated.Method（1）The chromatographic conditions were as follows:Agilent Proshell 120 EC C18（3.0 mm×100 mm,2.7μm）,ultra-pure water（containing 0.01%acetic acid）-methanol as mobile phase,flow rate 0.5 m L·min-1.Using the electrospray ion source（ESI）for 0-3.5min:to MS,3.5-6min:To Waste,negative ion mode detection,multiple reaction monitoring（MRM）scanning mass spectrometry conditions,valproic acid m/z 143.3→143.3,internal standard diclofenac m/z 294.3→250.1.A Liquid Chromatography Tandem Mass Spectrometry（LC-MS/MS）method was established for the determination of valproic acid concentration in blood.（2）The patients with poor Therapeutic effect after the routine dose of sodium valproate(20-30 mg·kg-1)were monitored for the steady-state blood Drug concentration,and the dose of Drug was adjusted,and the Therapeutic Drug Monitoring（TDM）was performed again when the steady-state blood Drug concentration was reached.Clinical data of patients including gender,age,daily dose of VPA,underlying diseases and combined with other antiepileptic drugs were collected,and factors affecting blood drug concentration of patients were obtained by one-way analysis of variance and multiple linear regression analysis.（3）For patients with abnormal liver function,suspected mitochondrial disease or unsatisfactory therapeutic effect after dose adjustment,digital fluorescence in situ hybridization（FISH）method was used to determine POLG gene polymorphism.The leucocyte precipitates were extracted with 1×NH4Cl pretreatment solution and stored in nucleic acid purification reagent.1.5μl treated white blood cell samples were added into the sequencing reaction universal kit and put into a fluorescence detector for detection.POLG gene polymorphism was analyzed,mutation frequency of POLG gene was calculated,and medication was adjusted according to the results.（4）Economic evaluation was conducted on 97 patients diagnosed with epilepsy and treated with different treatment regiments by retrospective analysis.The patients were divided into experiential treatment group,TDM group,genome group and TDM+genome group.The frequency of epileptic seizure within 1 year under different treatment regiments,the situation of re-hospitalization due to epileptic seizure,and the adverse reactions related to epileptic treatment during treatment were observed.The total Cost（C）and Effectiveness（E）of the patients during the treatment of epilepsy were counted,and cost-effectiveness analysis（C/E）was conducted.The cost of blood drug concentration test and gene test was reduced by 30%for sensitivity analysis.Results（1）Through methodological verification,the serum valproic acid concentration had a good linear relationship in the range of 5-200μg·m L-1,and the standard curve equation was Y=0.4291X-2.6914（R2=0.999）.The Relative standard deviation（RSD）of intra-batch and inter-batch precision was less than 5%,and the Relative Error（RE）was within the range of±4%.The extraction recoveries of valproic acid at low,medium and high concentrations were more than 90%,and the extraction recoveries of diclofenac,the internal standard,were（98.05±2.71）%.Substrate had little effect on the determination of valproic acid and internal standard diclofenac.RE was less than±15%under the conditions of 24 h storage at room temperature,repeated freeze-thaw and long-term freezing at-80℃.This method has the characteristics of high specificity,high reproducibility and good stability.（2）A total of 105 patients receiving sodium valproate for epilepsy were included in this study.The mean steady-state plasma concentration was（59.24±38.25）μg·m L-1,and the proportion of patients in the effective therapeutic concentration range was44.76%.Patients whose blood concentration was not in the effective therapeutic range may lead to poor therapeutic effect.After adjusting the dosage,the mean steady-state plasma drug concentration of 105 patients was（71.62±21.83）μg·m L-1,and the concentration compliance rate was 79.05%.The clinical data of patients with single factor analysis of variance and multivariate linear regression analysis,the basic information model is statistically significant,patients with age,VPA daily dose,diarrhea,vomiting,heart failure are the key factors influencing the patients blood drug concentration（P<0.005）,which had the greatest influence the daily dose of valproic acid sodium（t=5.145）.（3）POLG gene detection was performed in 38 patients,among whom 22 patients still had poor efficacy after dosage adjustment,and 16 patients had abnormal liver function or suspected mitochondrial disease.The results showed that there were 37patients with wild-type GG and CC gene loci in POLG A467T and W748S,and 1 patient had homozygous mutation AA and GG in POLG A467T and W748S genes.The mutation frequency of POLG gene was 0.88%.At the same time,one patient with POLG gene mutation was replaced with carbamazepine,and the other patients with poor efficacy were treated with VPA dose adjustment or combined with other antiepileptic drugs.（4）According to the frequency of epileptic seizures before and after treatment,the total effective rate of the experiential treatment group,TDM group,genome and TDM+genome were 80.00%,93.33%,93.30%and 95.45%,respectively.The cost-effectiveness ratio（C/E）is obtained from total cost（C）/total efficiency（E）.The total cost（C）and C/E values of the experiential treatment group were compared with those of the other three groups,and there were statistically significant differences in the total cost（C）and C/E values of the TDM group and TDM+genome between the experiential treatment group and the group（P<0.05）.Sensitivity analysis showed that the results after the price of the two detection technologies was reduced by 30%were not significantly different from those before the adjustment,indicating that the results were reliable.Conclusion（1）The method of plasma concentration of valproic acid established in this study conforms to the requirements of biological sample analysis,and can quickly,conveniently and accurately detect the steady-state plasma concentration of valproic acid in clinical samples,meet the requirements of clinical detection,and lay a research foundation for the guidance of individualized drug use.（2）The dosage of sodium valproate is different among patients of different ages,and the daily dose of sodium valproate has a great influence on the blood concentration of patients.The dosage of valproic acid should be adjusted according to the actual situation of patients.The blood concentration of most patients with poor therapeutic effect is not within the effective range,so the dosage can be adjusted according to the results of blood concentration monitoring.（3）In this study,digital fluorescence in situ hybridization was used to determine POLG gene polymorphism in epilepsy patients,which was simple and accurate.The mutation frequency of the POLG genotype was low in 38 patients.Patients with POLG gene mutation are recommended to change the treatment drugs,and patients without POLG gene mutation should adjust the dose or medication regimen according to the results of TDM and clinical manifestations.（4）Blood concentration monitoring can ensure that the blood concentration of patients is within the effective therapeutic concentration range,and can reduce the cost of epilepsy treatment in patients in the long-term treatment.At the same time,individualized medication combined with the results of genetic testing can improve the treatment efficiency,reduce adverse reactions and reduce medical costs. |
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