The Building Of Ultra-high Performance Liquid Chromatographic Method To Determine The Concentration Of Methotrexate, Folinic Acid,5-methylenetetrahydrofolate And The Preliminary Study Of Its Clinical Application

Objective:1. A method of Ultraviolet and Fluorescence Ultrahigh pressure liquid chromatography wasset up to detect Methotraxate, Calcium Folinate and5-methyltetrahydrofolate blood druglevel in plasma.2.To study the relation MTX、CF and5-MTHF blood drug level changing character toplasma creatinine and approach the clinical significance in children patient of acutelymphoblastic leukemia after large dose.Method:1.The MTX dissociated by means of chromatographic column, mobile phase used MethylCyanides-40mol. L-1potassium dihydrogen phosphate balanced solution(93：7，balancedsolution used by phosphoric acid, pH=4.5), ultraviolet detecting(wave length was304nm),standard curve quantitation; CFand5-MTHF dissociated by means of chromatographiccolumn, mobile phase Methyl Cyanides-40mol. L-1potassium dihydrogen phosphatebalanced solution(93：7，balanced solution used by phosphoric acid, pH=4.5), fluorescencedetector(excitation wave length was297nm emanation wave length was303nm,), standardcurve quantitation.2.70ALL patients,venous blood was collected by test tube including EDTA decoagulant,centrifuged3minutes to separate plasma.Used the UPLC to detect MTX,CF and5-MTHFconcentration, and analyzed functional parameter Cr through auto-biochemistry machine; besides, analyzed the relationship of MTX、CF和5-MTHF concentration and Cr.Result:1. The retention time of MTX、 CF、5MTHF was2.465,2.878,3.448minutes, thedetectability was0.5,0.05,0.05mg/L, there were linear correlation among of them, the r2was0.9996、0.9998、0.9990(n=6); various coefficient of recovery of concentration,within-day precision and day to day precision accorded to methodology.2.①In the blood serum of Children patients,there were the relation of MTX、CF、5-MTHF blood drug level and Cr.②There were direct correlation of Cr and MTX, while there were inverse correlation ofCr and5-MTHF, CF. there were statistical significance in the correlations.Conclusion:1.The method detected MTX、CF、5-MTHF blood drug level was stable,highreproducibility, and it can be measured in clinic.2.After used MTX,the highly the blood drug level of MTX,the highly of Cr;while thehighlythe blood drug level of CF、5-MTHF, the lower the Cr.It suggested that CF can save theinjuried to CF obviously.It was conformity with literature report.