Correlation Between The Concentration Of Methotrexate And Its Metabolites And Adverse Reactions

Objective:To establish a high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）method for the simultaneous determination of methotrexate（MTX）and its main metabolite 7-hydroxymethoxazole（7-OHMTX）;Which was compared with high performance liquid chromatography（HPLC）and chemiluminescent microparticle immunoassay（CMIA）,respectively;The patients with osteosarcoma（OS）,acute lymphoid leukemia（ALL）,and non-Hodgkin’s lymphoma（NHL）after High-dose methotrexate（HD-MTX）administration,to study the correlation between liver injury,myelosuppression and MTX and 7-OHMTX plasma concentrations,and to explore the clinical significance of simultaneous monitoring of MTX and 7-OHMTX plasma concentrations for predicting the side effects of MTX.Method:Aripiprazole was used as an internal standard（IS）.Protein precipitation was used for sample pretreatment with aripiprazole set as the internal standard;Separation was carried out on Waters Xbridge（C18 4.6*100mm,3.5μm）with flow rate of 0.3m L·min^-1 and mobile phase of0.05%formic acid aqueous solution-Methanol gradient;the detection was a Waters 2790tandem mass spectrometer with an ESI ion source and performing positive ion detection in multiple reaction monitoring mode（MRM）,the m/z of MTX,7-OHMTX,and internal standard was:456→308,472→324 and 448→285 respectively.130 plasma samples from patients receiving HD-MTX chemotherapy were used as the research object,and the results of HPLC-MS/MS method were compared with HPLC and CMIA methods for paired T-test,Passing-Bablock regression analysis and Bland-Altman analysis,respectively.Retrospective study on a total of 28 OS,ALLand NHL patients with HD-MTX treatment,MTX and 7-OHMTX plasma concentrations were determined simultaneously in 24 h,48 h and 72 h after HD-MTX administration by HPLC-MS/MS.Liver injury and myelosuppression index were collected within 1 week after HD-MTX adminstration,and then analyzed the correlation between the blood concentration of MTX and 7-OHMTX,the ratio of 7-OHMTX/MTX,the sum of MTX and 7-OHMTX plasma concentrations with liver damage and myelosuppression;Result:The result of methodological verification:the linear range of MTX and 7-OHMTX is:0.01～12.85μmol·L^-1,0.086～13.78μmol·L^-1,the intraday and interday of precision RSD（%）were less than15%,the extraction recovery rate was more than 76%.The result of consistent comparison:For the 0 h MTX concentration,the result of T-test indicate that there was a significant difference（P<0.0001）between HPLC-MS/MS and CMIA assays and non-significant difference（p>0.05）between HPLC-MS/MS and HPLC;for the 24h,48h and 72h MTX concentration,the result of T-test indicate that there was non-significant difference（P>0.05）between HPLC-MS/MS assay versus（vs.）HPLC and CMIA assays.The results of retrospective analysis:For OS patients,the severity of liver injury（Alanine aminotransferase,ALT）and the degree of HGB decline were significantly correlated with the 48 h 7-OHMTX/MTX ratio（P<0.01）;The degree of WBC decline was correlated with the 24h 7-OHMTX/MTX ratio but not significant（P=0.059）;there was no correlation between the degree of NE decline and the 7-OHMTX/MTX ratio.For ALL and NHL patients,the sum of MTX and 7-OHMTX plasma concentrations with liver damage and myelosuppression.There were a significant negative correlation between MTX concentration and liver injury at 48h（p=0.01）and correlation between 24h 7-OHMTX concentration and liver injury,but the difference was not statistically significant（P=0.227）.There was no correlation between HGB,WBC,NE and platelet decreased MTX and 7-OHMTX concentrations.Conclusion:The method had high specificity,high sensitivity,simple pretreatment,accurate and rapid,and can be used for clinical simultaneous determination of MTX and 7-OHMTX blood drug concentration.For 0h MTX concentration,the detected result by CMIA was low;for the 24h,48h and 72h MTX concentration,these three assays can replace each other.For OS patients,the 48h 7-OHMTX/MTX ratio is more clinically useful for predicting the occurrence of acute drug-induced liver injury induced by MTX.For ALL and NHL patients,the 48 h MTX concentration only had a reference value for ALT elevation.