Establishment Of The Methods For Measurement Mycophenolic Acid And Pirarubicin In Biological Samples And Clinical Application

Objective:1.To establish an HPLC-UV method for simultaneous determination of the concentrations of mycophenolic acid and its main metabolites MPAG and Ac MPAG in human plasma,which used to the monitoring of therapeutic drugs in transplant patients.2.To establish an HPLC-MS method to conduct collision studies on MPA,MPAG and Ac MPAG,and to predict the pyrolysis rule of mass spectrometry.3.To establish an HPLC-UV method for simultaneous determination of pirorubicin,honokiol and pirorubicin concentrations in human plasma,which used for the monitoring of concentration,curative effect and therapeutic drugs after the combined use of Traditional Chinese medicine.4.To establish an HPLC-UV method for determination of pirorubicin concentrations in human urine,which used to study the changes of concentration,absorption of drugs and monitoring of therapeutic drugs before and after chemotherapy for bladder cancer patients.Methods:1.MPA and its metabolites in plasma are simultaneously determined,which liquid chromatographic condition of the mobile phase was methanol-prurified water（10 mmol/L Na H₂PO₄,p H 3.00）with gradient elution.The flow rate was 1.0 m L/min.The detection wavelength was 215 nm and the column temperature was 40℃.Estazolam was used as internal standard and metaphosphate-acetonitrile was used as protein precipitator.2.MPA,MPAG and Ac MPAG were analyzed by mass spectrometry with electrospray ion source is used to adjust parameters to optimize the parent ion and fragment ion,and multireaction monitoring mode is used for mass spectrometry analysis.The results were analyzed using Mass Hunter software.3.Plasma pirobilin and honokiol were determined with the mobile phase of（A）aqueous solution（5 mmol/L Na H₂PO₄,0.3%三乙胺,p H 2.50）-（B）methanol:acetonitrile（1:4）,with isocratic elution.The flow rate is 1.0m L/min,and detection wavelength is 254 nm.Epirubicin was used as the internal standard,and the samples were pre-treated by ethyl acetate secondary liquid-liquid extraction.4.Urine was detected with the mobile phase was（A）aqueous solution（5 mmol/L KH₂PO₄,0.3%三乙胺,p H 2.70）-（B）methanol with isoelution.And the flow rate is 1.0 m L/min.The detection wavelength is 254 nm.The method of adding methanol to centrifuge to remove protein and ion was selected for the pretreatment of biological urine samples of pirobilin.5.To collect blood samples of MPA valley concentration from outpatients who received MMF treatment after kidney transplantation;To collect blank urine samples,chemotherapeutic solution and 0.5 h urine samples frome bladder cancer patients.Results:1.The linear range of MPA,MPAG and Ac MPAG in plasma was 0.1953～25.00μg/m L,0.3906～50.00μg/m L,0.1953～25.00μg/m L respectively.The precision RSD of the quality control samples was less than 15%,and the relative recovery of accuracy was between 85%and 110%.MPA,MPAG,Ac MPAG and internal standard extraction recovery can meet the requirements.The samples remain stable under the required environment.The method is simple,rapid and can meet the requirements of clinical drug monitoring and application.2.The structure of MPAG and Ac MPAG were similar in mass spectrolysis,but the ion rearrangement of debris was different.MPA,MPAG and Ac MPAG all crack the same group with the increase of collision energy,and have similar cracking rules.3.The linear range of pirobilin and honokiol in plasma was 0.31～40.00μg/m L,0.20～25.75μg/m L respectively.The precision RSD of the quality control samples was less than 15%,and the relative recovery of accuracy was between 85%and 110%.The extraction recovery rate is well.The samples remain stable under the required environment.The method is simple,reliable,and can meet the requirements of clinical drug monitoring and application.4.The linear range of pirobilin and honokiol in urine was 2.00～200.00μg/m L respectively.The precision RSD of the quality control samples was less than 15%,and the relative recovery of accuracy was between 85%and110%.The analysis method is rapid and simple,which is suitable for monitoring drug concentration in clinical patients.5.The blood concentration of MPA ranged from 0.35 to 6.65μg/m L,which was different from the recommended therapeutic grain concentration,with significant differences among individuals.But no adverse reactions occurred.The urine concentration of pirarubicin in patients with bladder cancer ranged from 21.81 to 290.57μg/m L half an hour after perfusion.Due to the differences among individuals,there were also differences in absorption rates.No obvious adverse reaction occurred after the treatment,and the therapeutic effect was better.Conclusions:The established HPLC-UV method has good precision,accuracy,high sensitivity,simple operation,and can quickly determine the concentration of the substance to be detected.It is suitable for clinical drug monitoring.MPA,MPAG and Ac MPAG showed similar cracking rules,which provided the basis for further metabolism of MPA in vivo,structure of metabolites,optimization of mass spectrometry method,and spectral cleavage of similar compound substances.In renal transplant patients,there were individual differences in the blood drug concentration of MPA,which was different from the clinically recommended therapeutic grain concentration.Therefore,it was necessary to conduct therapeutic drug monitoring.There were differences in the absorption rate of pirorubicin among patients with bladder cancer.The detection and analysis of concentration changes before and after perfusion was beneficial to the adjustment of chemotherapy regimens and the monitoring of chemotherapy drugs,which was of certain significance to the adjustment of chemotherapy regimens and relevant clinical studies.