Study On The Key Problems Of Sample Pre-treatment Of Plasma Drug Concentration In Therapeutic Drug Monitoring

Therapeutic drug monitoring is the main method of individualized treatment.With the development of individualized treatment,clinical pharmacy workers need to monitor the blood concentration of a large number of clinical samples and more and more drugs.Therefore,the durability and reliability of analytical methods are problems that analysts must face.The commonly used sample pretreatment methods for blood drug concentration monitoring include protein precipitation method(PPT),liquid-liquid extraction method(LLE)and solid phase extraction method(SPE),but these methods often require multi-step operation,and complex operation steps will increase the error of measurement results.For the monitoring of some cytotoxic drugs,complicated operation steps increase the chance of contact between analysts and clinical samples,which poses certain safety risks.Therefore,simplifying the operation steps of sample pretreatment is of great significance to reduce the chance of sample exposure and improve the accuracy of analysis results.The existing sample pretreatment methods(PPT,LLE,SPE)have complicated operation steps,however,hollow fiber centrifugal ultrafiltration(HFCF-UF)operation is simple.It only needs one step of centrifugation to complete the sample pretreatment,and can be prepared into a semi-closed Device to reduce sample exposure.We use HFCF-UF technology to provide a simple and fast sample pretreatment method for clinical blood drug concentration monitoring.Membrane separation methods(equilibrium dialysis,ultrafiltration)are commonly used in the analysis of free drug concentration,but the non-specific binding(NSB)of the drug on the membrane is an important factor that limits its application.When drugs and membranes produce severe NSB,it will affect the accuracy and reliability of the determination results,which limits the use of these methods.The NSB problem of the membrane is a key problem in the sample pretreatment process.Therefore,we studied methods to reduce the adsorption of the membrane to the drug.By using Tween-80 to modify the hollow fiber ultrafiltration membrane,the membrane’s mycophenolic acid(MPA)NSB problem.Established a hollow fiber centrifugal ultrafiltration(HFCF-UF)HPLC-MS/MS method for monitoring the free drug concentration of MPA Part Ⅰ: Determination of Free Mycophenolic Acid Concentration in Plasma by Modified Hollow Fiber Centrifugal Ultrafiltration MethodObjective: To study the modification method to reduce the NSB of the drug and the hollow fiber ultrafiltration membrane,and to establish a pretreatment method for the determination of the free MPA concentration in plasma.Method: The polysulfone hollow fiber ultrafiltration membrane was modified with three types of surfactants(anionic cationic non-ionic),and the concentration and soaking time of the surfactants were optimized.The absolute recovery rate was used as the index to investigate the improvement of non-specific adsorption of various types of surfactantsResults: By immersing the polysulfone hollow fiber ultrafiltration membrane in the non-ionic surfactant Tween-80 at a concentration of 0.05g/m L for 3 hours,the absolute recovery of MPA is close to 100%.On this basis,HPLC-MS/ MS method is used to monitor the blood concentration of free mycophenolic acid.The linear range was 10.12-506.0 ng/m L,and the limit of quantification and detection were 10.12 and 3.04 ng/m L,respectively.The RSD value of intra-day and inter-day precision are less than 8.9%,and the absolute recovery rate is 99.2-107.6%.Conclusion: In this study,a polysulfone hollow fiber ultrafiltration membrane modified by twain-80 was used to treat blood samples,and the problem of NSB between polysulfone ultrafiltration membrane and MPA was solved.A simple and rapid blood concentration analysis method for free mycopholanolic acid was established by combining HPLM-MS/MS.Part Ⅱ: A simple and rapid method for determination of methotrexate concentration in plasmaObjective: Establish a simple and rapid method for the analysis of methotrexate plasma concentration.The established method is compared with the traditional method to determine the reliability of the method.Method: Take 500 μL of plasma sample into the hollow fiber centrifugal ultrafiltration device,add 20 μL of 5% trichloroacetic acid and vortex to mix,centrifuge at 3500 r/min for 15 min,and 10 μL of ultrafiltrate for chromatographic analysis.The established method is used for the determination of clinical samples,and the measurement results are compared with traditional methods.Results: After adding protein precipitation agent(20 μL 5%trichloroacetic acid),one-step centrifugal operation completes sample pretreatment.The absolute recovery rate is close to 100%.The calibration curve established by the control solution and the calibration curve obtained by spiked plasma samples were directly used for t test,and the results were not statistically different(P>0.05).On this basis,the HFCF-UF method was established for the determination of MTX plasma concentration.There was no statistical difference between the MTX blood concentration of 24 patients measured by this method and the traditional method.Conclusion: The MTX bound to plasma needs to be added with a release agent and then centrifuged in one step to complete the sample pre-processing.The entire sample pre-processing process is simple and fast,which is especially suitable for the determination of batch clinical blood samples.The established method can directly use the control solution instead of spiked plasma to establish the standard curve,avoiding the error caused by the difference between the plasma conditions of clinical patients and healthy people.Therefore,this method is particularly suitable for the determination of clinical blood drug concentration.