Establishment Of Monitoring Method For Determination Of Voriconazole In Human Serum And Clinical Application Of CYP2C19 Detection For Gene Polymorphism

ObjectiveTo establish a method for the determination of voriconazole in serum by automatic two-dimensional chromatography（2D-HPLC）.The method was used to monitor the concentration of voriconazole in patients,and a microsequencing kit was used to detect CYP2C19 genotype polymorphism.The relationship between voriconazole blood concentration,CYP2C19 gene polymorphism and treatment effect and adverse reactions were analyzed,and to provide reference for clinical rational use of voriconazole.Method（1）First-dimensional liquid chromatography was ASTON SC2（3.5 mm×25 mm,5μm）column,and the mobile phase was 20 mmol/L ammonium phosphate–acetonitrile-methanol（3∶1∶1,V∶V∶V,phosphoric acid adjusted pH=5.4）,and the flow rate was1.2 mL/min.The second-dimension column was SHIMADZU C18（4.6 mm×150 mm,5μm）column,and the mobile phase was 10mmol/L ammonium acetate（triethylamine adjusted to pH=7.0）-10mmol/L ammonium acetate（trifluoroacetic acid adjusted pH=3.0）-Methanol-Acetonitrile（30∶10∶10∶50,V∶V∶V∶V）,and the flow rate was1.0 mL/min.The middle column was ASTON SH middle column（3 mm×10 mm,5μm）,The auxiliary mobile phase was purified water.Sample was extracted by first-dimensional column,then captured by middle column and transferred to second-dimensional column for analysis.The detection wavelength was 256 nm,the temperature was 40℃and the injection volume was 200μL.（2）The voriconazole blood concentration test method was used to measure the voriconazole blood concentration in the hospital from March 2018 to February 2019,and a microsequencing kit was used to detect CYP2C19 genotype polymorphism.The patients included in the study were screened according to the inclusion criteria and exclusion criteria.The proposed safety and efficacy evaluation criteria were used for the study.Hardy-Weinberg genetic balance test was performed on CYP2C19 genotype using ECXEL 2016,and the results were analyzed by SPSS 21.0 software.Results（1）The linear relationship of vcriconazole was good in the range of 0.28²2.56μg/mL（r=0.9999）and the minimum quantitative limit was 0.28μg/mL.The recovery rate of method was from 99.01%to 104.62%,the extraction recoveriey rate was from85.81%to 89.82%.The RSD of inter-day and intra-day assays was less than 9%.The RSD of the quality control samples at room temperature for 24 h,repeated freeze-thaw and long-term cryopreservation stability was less than 9%,This method meets the requirements for serum sample determination.（2）A total of 57 patients were included in the study,including 35 males and 22females with an average age of 67.7±14.77 years old and body weight of 56.65±9.56 kg.There was no statistically significant effect of voriconazole plasma concentration on clinical efficacy（P>0.05）.but there was statistically significant significance effect of voriconazole plasma concentration on safety（P<0.05）.Among the 57 patients,28cases were EM patients,27 cases were IM patients,and 2 cases were PM patients.The genotype frequencies were consistent with Hardy-Weinberg equilibrium.The results showed that the C_minin of extensive metabolism,intermediate metabolism and poor metabolism was 2.78±1.56,3.09±1.39 and 7.09±0.16μg/mL.There was no significant difference of C_minin between extensive metabolism and intermediate metabolism（P>0.05）.There was significant differences of C_minin between poor metabolism and extensive metabolism or intermediate metabolism（P<0.05）.The treatment efficiency of extensive metabolism,intermediate metabolism and poor metabolism was 82.14%,77.78%and 100.00%,and the incidence of adverse reactions was 3.57%,7.41%and100.00%.The statistical analysis of the efficiency and safety by Fisher exact test showed that the effect of CYP2C19 gene polymorphism on the safety of voriconazole was statistically significant（P<0.05）,but the effect of CYP2C19 gene polymorphism on the treatment effenciency of voriconazole was no statistically significant（P>0.05）.Optimal scale regression showed that the metabolic difference caused by CYP2C19polymorphism was an important factor affecting the blood concentration of voriconazole.（3）A case of interaction between rifapentine and voriconazole was found in patients who were monitored for blood concentration.After combining the two drugs,the plasma concentration of voriconazole was as low as 0.415μg/mL.Conclusion（1）The method was simple,accurate and sensitive,it is suitable for determination of voriconazole concentration in serum.（2）The blood concentration of voriconazole was significantly related to safety.The CYP2C19 gene polymorphism is significantly associated with the safety of voriconazole,and the CYP2C19 genotype was an important factor affecting the blood concentration of voriconazole.（3）The interaction between rifapentine and voriconazole should be avoided.When there is a contradiction in treatment,other antifungal drugs such as amphotericin B and echinomycin should be selected according to the actual situation.When voriconazole must be used,the patient’s anti-tuberculosis regimen should be free of rifamycin.