Measurement of immunosuppressants, Tacrolimus, Sirolimus, Everollmus and Cyclosporine A from whole blood using on-line SPE and LCMS-8030

Significance of Immunosuppressant Monitoring

Immunosuppressants such as tacrolimus, sirolimus, everolimus and cyclosporine A play a pivotal role in preventing organ rejection in transplant patients and managing immune-mediated disorders. Their narrow therapeutic windows demand precise, rapid and reliable quantification directly from whole blood, ensuring optimal dosing and patient safety.

Objectives and Study Overview

This work aimed to develop and validate a streamlined on-line solid-phase extraction (SPE) coupled with ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for measuring four key immunosuppressants in whole blood. The focus was on achieving fast separation, high sensitivity and robust quantitation suitable for routine therapeutic drug monitoring (TDM).

Methodology and Workflow

The developed protocol integrates a Nexera UHPLC system with a 6-port switching valve for on-line SPE, directly processing whole blood samples. Separation of analytes utilized pre-selected SPE and analytical columns from the ClinMass® Complete Kit (RECIPE Chemicals). Detection employed multiple-reaction-monitoring (MRM) in positive electrospray ionization mode, measuring the [M+NH4]+ precursor ions with two transitions per compound to ensure specificity.

Used Instrumentation

• Shimadzu LCMS-8030 triple quadrupole mass spectrometer
• Nexera UHPLC system with 6-port 2-way switching valve
• ClinMass® Complete Kit for Immunosuppressants in Whole Blood (RECIPE Chemicals)

Results and Discussion

The method achieved baseline separation of all four immunosuppressants in under two minutes. Calibration curves exhibited excellent linearity (R2 ≥ 0.992) over clinically relevant concentration ranges: tacrolimus (1.34–23.5 µg/L), sirolimus (1.33–23.9 µg/L), everolimus (1.33–23.9 µg/L) and cyclosporine A (24.9–1264 µg/L). Limits of detection and quantitation met TDM requirements. On-line SPE reduced sample preparation steps, enhancing throughput and reproducibility.

Benefits and Practical Applications

• Rapid turnaround: total run time under 2 minutes.
• High sensitivity and specificity via dual MRM transitions.
• Minimal manual sample handling through automated on-line SPE.
• Compatibility with commercial TDM kits simplifies method deployment in clinical laboratories.

Future Trends and Potential Uses

Emerging directions include expanding the panel to additional immunosuppressants or metabolites, integrating high-throughput robotics for large-scale clinical studies, and coupling with data analytics platforms for real-time dosing adjustments. Advances in microfluidic SPE cartridges and miniaturized MS sources may further reduce analysis time and solvent consumption.

Conclusion

The combined on-line SPE UHPLC-MS/MS method on the Nexera/LCMS-8030 platform delivers a fast, sensitive and reliable approach for quantifying major immunosuppressants directly from whole blood. Its robustness and simplicity make it well suited for routine therapeutic drug monitoring in transplant and immunology laboratories.

References

1. Shimadzu Corporation. Measurement of immunosuppressants from whole blood using on-line SPE and LCMS-8030. First Edition, September 2013.
2. RECIPE Chemicals + Instruments GmbH. ClinMass® Complete Kit for Immunosuppressants in Whole Blood, MS1000.