Simultaneous Quantitative Analysis of Five Immunosuppressants in Whole Blood Using LC-MiniMS

Therapeutic drug monitoring of immunosuppressants is essential for organ transplant recipients, yet routine testing still relies heavily on centralized, costly mass spectrometry platforms. To improve the accessibility of decentralized clinical testing, a rapid multicomponent assay for five commonly used immunosuppressants in human whole blood was developed and validated using a self-built miniature liquid chromatography-ion trap mass spectrometry (LC-MiniMS) system. Protein precipitation was adopted as the sample preparation strategy, and response surface methodology was used to systematically optimize key pretreatment and mass spectrometric parameters. Under optimized conditions, the method required only 9 min from injection to completion of analysis. The assay was validated in terms of selectivity, linearity, sensitivity, accuracy, precision, matrix effect, and stability. Good linearity was obtained across the required concentration ranges for tacrolimus, sirolimus, mycophenolic acid, everolimus, and cyclosporine A, with all correlation coefficients greater than 0.99. The limits of quantification were adequate for clinical monitoring, and the accuracy and precision at low, medium, and high quality control levels met the acceptance criteria for therapeutic drug monitoring. The normalized matrix factors remained close to unity, indicating that the method effectively minimized endogenous matrix interference in whole blood. Stability assessment showed that the analytes exhibited acceptable stablity under short-term storage, repeated freeze-thaw cycles, and long-term frozen storage conditions. To further verify clinical applicability, 30 real whole-blood samples were analyzed in parallel on LC-MiniMS and a QTRAP 6500+ platform. The two platforms exhibited good agreement, confirming that the miniature platform could provide reliable quantitative results comparable to those of a conventional laboratory mass spectrometer. Overall, this work demonstrated that LC-MiniMS enables rapid, accurate, and practical multianalyte therapeutic drug monitoring in whole blood. The approach not only reduced dependence on centralized instrumentation but also provided a feasible pathway toward point-of-care and distributed mass spectrometric testing. Although the present study focused on five immunosuppressants in whole blood, the workflow and optimization strategy could be extended to other clinically relevant drug panels and sample types. This work also highlighted several practical advantages. The method uses a simple protein precipitation workflow rather than a labor-intensive extraction procedure, which shortens turnaround time and lowers the barrier to routine use. The response-surface optimization strategy improved analytical sensitivity while maintaining run stability, indicating that the same framework could be adapted for other multianalyte assays on miniature instruments.