A New Method For Liquid Chromatography Coupled With Tandem Mass Spectrometry Method By Detection Of Four Immunosuppressants In Whole Blood And Dried Blood Spots

Immunosuppressants are commonly used drugs that inhibit the abnormal immune response in the body and are often used in the postoperative treatment of organ transplantation.These drugs usually have a narrow therapeutic window and the drug concentration is closely related to hepatorenal toxicity,therefore,in order to maximise the curative effect and avoid the side effects,it is necessary to monitor the concentration of immunosuppressants clinically.Hitherto,LC-MS/MS has become the gold method for drug monitoring owing to remarkable precision,accuracy,and sensitivity.In the clinical application,most whole blood methods are used to detect one or two immunosuppresssants only,while the simultaneous detection of four drugs is relatively rare,the emerging dried blood spots sampling method using for high-throughput detection of immunosuppressants is even less reported.Based on the current status of clinical detection of immunosuppressants,this paper elaborated the steps of each condition from the establishment of liquid chromatography conditions,mass spectrometry conditions and sample pretreatment methods,thus,a high-throught LC-MS/MS was developed for simultaneous detection of cyclosporin A(Cs A),sirolimus(SIR),tacrolimus(TAC)and everolimus(EVE)in whole blood,which provided a more convenient and efficient immunosuppressants detection method for clinical application.What’s more,we have made a validation of the linearity,low limit of detection,selectivity,precision and matrix effects according to the guidelines defined by the Clinical and Laboratory Standards Institute(CLSI).All the results were good,the linearity of Cs A was 5.0 to 1250 ng/m L,and the linearity of SIR,TAC and EVE was 0.5 to 125 ng/m L.The precision and accuracy of the method were high,which fully met the clinical detection requirements of immunosuppressants.In addition,through the improvement and innovation of the whole blood method which developed previously in our laboratory,we have initially established a highthrought LC-MS/MS method,which has good precision,high sensitivity and specificity,for simultaneous detection of Cs A,SIR,TAC and EVE in DBS which has the advantages of small sample volume,convenient storage and transportation.This method requires only one drop of whole blood(20 μL)to accurately detect the concentration of four drugs.All the results of methodological validation were contented with the FDA guidelines.The linearity of Cs A was 20 to 1000 ng/m L,and that of SIR,TAC and EVE was 2 to 100 ng/m L.The extraction recovery was high(71 to 116%).DBS samples can be stored at room temperature for at least 5 days,indicating good stability of this method.The Passing-Bablok correlation test and Bland-Altman agreement analysis of dried blood spots method and whole blood method showed good results,proving the good repeatability of whole blood method and the applicability and feasibility of DBS method as an alternative for the whole blood method in the therapeutic drug monitoring of immunosuppressant in future clinical applications.