Quantification of four immunosuppressants in human blood by LC-HRAM-MS for clinical research

Importance of the Topic

The accurate and rapid quantification of immunosuppressants in human blood is essential for therapeutic drug monitoring in organ transplant recipients. Proper dosing of cyclosporine A, everolimus, sirolimus, and tacrolimus prevents graft rejection and minimizes toxicity.

Study Objectives and Overview

This work describes the development and validation of a high-throughput LC-HRAM-MS method for simultaneous quantification of four key immunosuppressants in whole blood. The primary goals were to achieve a short run time, simple sample preparation, and reliable analytical performance suitable for clinical research.

Methodology and Instrumentation

Sample preparation relied on a two-step offline protein precipitation protocol:

• Mix 100 µL whole blood with 150 µL 0.1 M zinc sulfate solution and vortex.
• Add 200 µL methanol containing isotopically labeled internal standards; vortex and centrifuge at 10 000 × g for 10 min.
• Transfer 100 µL supernatant for LC-MS analysis.

Chromatography was performed on a Vanquish Flex Binary UHPLC with a Hypersil GOLD 30 × 2.1 mm, 1.9 µm column at 70 °C using a water–formic acid–ammonium formate and methanol–formic acid–ammonium formate gradient. Detection employed full-scan acquisition on an Orbitrap Exploris 120 mass spectrometer with HESI in positive mode.

Instrument Used

• Thermo Scientific Vanquish Flex Binary UHPLC system with Hypersil GOLD 30 × 2.1 mm, 1.9 µm column
• Thermo Scientific Orbitrap Exploris 120 high-resolution accurate-mass spectrometer with heated electrospray ionization
• TraceFinder 5.1 data processing software

Main Results and Discussion

The method delivered a 1.3-minute run time with retention times around 0.9–1.0 min. Seven-point calibration curves showed linearity across therapeutic ranges (R² ≥ 0.9916) using 1/x weighting. Intra-assay precision (%CV) was ≤11% and accuracy bias fell within ±14%. No significant carryover was observed.

Benefits and Practical Applications

• High throughput: 1.3-minute analysis per sample accelerates batch processing.
• Simple sample prep: two-step protein precipitation reduces labor and potential losses.
• High specificity and sensitivity: HRAM detection ensures accurate quantification at low concentrations.
• Robust performance: meets clinical research requirements for precision, accuracy, and carryover.

Future Trends and Potential Applications

Potential developments include automation of sample preparation to further improve throughput, expansion to additional immunosuppressive or co-medication panels, coupling with clinical decision software, and integration into point-of-care platforms. Advances in HRAM technology may enable simultaneous metabolite profiling.

Conclusion

This LC-HRAM-MS method provides a rapid, precise, and accurate tool for quantifying cyclosporine A, everolimus, sirolimus, and tacrolimus in human blood. Its simple workflow, short runtime, and robust validation make it well suited for high-throughput clinical research and therapeutic drug monitoring applications.