Simultaneous analysis of immunosuppressive drugs in whole blood samples using LC-MS/MS

Importance of the topic

The accurate measurement of immunosuppressive drugs in whole blood is critical for transplant patients. Under- or over-dosing can lead to graft rejection or adverse effects such as infections. A streamlined, reliable assay that measures multiple drugs and metabolites in a single run enhances clinical decision making and laboratory efficiency.

Aims and Study Overview

This work aimed to develop and validate a single LC-MS/MS method for simultaneous quantification of six immunosuppressive compounds (tacrolimus, everolimus, sirolimus, cyclosporin A, mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG)) in whole blood. The study combined reagents and columns from a commercial kit (DOSIMMUNE) with optimized gradient conditions to ensure appropriate separation and sensitivity across all analytes.

Methodology and Instrumentation

A simplified sample preparation involved protein precipitation, centrifugation and direct injection of the supernatant. Calibration standards covered relevant therapeutic ranges for each analyte. QC samples were prepared by spiking whole blood with known concentrations.

• Instrument: Shimadzu Nexera X3 UHPLC coupled to LCMS-8050 triple quadrupole MS.
• Columns: DOSIMMUNE trap and analytical columns.
• Mobile phases: DOSIMMUNE Mobile Phase A and B with modified gradient for MPA/MPAG retention.
• Ionization: ESI negative mode for MPA/MPAG, ESI positive for other drugs.
• MRM transitions optimized for each analyte and internal standard.

Results and Discussion

Calibration curves for each compound exhibited excellent linearity (R² ≥ 0.99). Inter- and intra-day precision and accuracy for QC samples fell within acceptance criteria. All six analytes were quantified in patient whole blood samples with concentrations matching expected reference ranges. Chromatographic separation prevented co-elution, and the method achieved run times under 4 minutes per sample.

Benefits and Practical Applications

This unified assay reduces turnaround time and reagent consumption compared to separate methods. It supports therapeutic drug monitoring in clinical laboratories, enabling rapid dosage adjustments and better patient management. The method’s high throughput and robustness make it suitable for routine QA/QC in transplant centers.

Future Trends and Opportunities

Advances may include integration with automated sample preparation platforms and expansion to additional metabolites or biomarkers of immune status. Coupling with micro-flow LC or high-resolution MS could further improve sensitivity. Application of machine learning for peak integration and quality control may enhance data consistency.

Conclusion

A single LC-MS/MS assay was successfully developed for six key immunosuppressants and their metabolites in whole blood. The method demonstrated excellent performance, meeting clinical laboratory requirements for accuracy, precision and throughput.

Reference

• Goda T., Nakanishi T., Masuda J., Kimura N., Aizawa K. Simultaneous analysis of immunosuppressive drugs in whole blood samples using LC-MS/MS. Shimadzu Application Note TP-143.