Quantification of immunosuppressants in human whole blood by liquid chromatography-tandem mass spectrometry for clinical research

Significance of the Topic

The precise measurement of immunosuppressant levels in human whole blood is critical for patient management after organ transplantation and for research into therapeutic drug monitoring. Combining multiple analytes in a single rapid assay supports clinical decision making and improves laboratory throughput while preserving accuracy and sensitivity.

Objectives and Study Overview

This work describes the development and validation of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to quantify four key immunosuppressants—cyclosporin A, tacrolimus, sirolimus, and everolimus—in human whole blood. The approach integrates minimal sample preparation with online solid phase extraction for streamlined analysis within a two-minute total runtime.

Methodology and Instrumentation

Sample preparation begins with protein precipitation and addition of isotopically labeled internal standards, followed by online SPE on a Transcend II TLX-1 system. Chromatographic separation employs RECIPE SPE cartridges and analytical columns. Detection is achieved on a Thermo Scientific TSQ Endura triple quadrupole mass spectrometer with heated electrospray ionization operating in positive mode. Two selected reaction monitoring transitions per analyte allow simultaneous quantification and confirmation. Data processing is performed using Thermo Scientific TraceFinder 3.3 software.

Main Results and Discussion

Linearity was demonstrated over broad concentration ranges for each analyte, with correlation coefficients (R2) exceeding 0.99. Lower limits of quantification were determined as 14.0 ng/mL for cyclosporin A, 0.85 ng/mL for everolimus, 1.87 ng/mL for sirolimus, and 0.31 ng/mL for tacrolimus. Accuracy assessments using external proficiency test samples yielded biases between −21.5% and +15.1%. Intra-assay precision (%CV) remained below 9.9% at all concentration levels, and inter-assay precision did not exceed 12.9%, meeting stringent criteria for research use.

Benefits and Practical Applications

• Simultaneous quantification of four immunosuppressants in a single, rapid run.
• Minimal offline sample handling reduces potential for error and increases throughput.
• High sensitivity and broad dynamic range suitable for clinical research and therapeutic drug monitoring studies.

Future Trends and Applications

Advances in online sample preparation and mass spectrometric detection will continue to shorten analysis times while enhancing multiplexing capabilities. Integration with laboratory information management systems and the use of high-resolution MS may further improve selectivity, enabling broader panels of analytes and metabolite profiling in transplant pharmacokinetics.

Conclusion

The described LC-MS/MS method provides a robust, accurate, and precise tool for quantifying cyclosporin A, everolimus, sirolimus, and tacrolimus in human whole blood. Its streamlined workflow and rapid two-minute analysis support high-throughput clinical research applications.

Reference

• De Nardi C., Kern K., Peters S. Quantification of immunosuppressants in human whole blood by liquid chromatography-tandem mass spectrometry for clinical research. Technical Note 64916, Thermo Fisher Scientific, 2017.