Quantification of immunosuppressants in human blood by LC-HRAM mass spectrometry for clinical research

Importance of the Topic

The accurate measurement of immunosuppressant drugs in blood is vital for therapeutic drug monitoring in organ transplant patients, ensuring optimal dosing to prevent graft rejection or drug toxicity. High-resolution mass spectrometry improves specificity and sensitivity compared to traditional immunoassays and low-resolution methods.

Objectives and Study Overview

This work aimed to implement and validate an LC-HRAM MS method for clinical research quantification of four key immunosuppressants—tacrolimus, sirolimus, everolimus, and cyclosporine A—in human whole blood. The method employs offline protein precipitation with isotopically labelled internal standards and an online solid-phase extraction (SPE) coupled to a dual-channel UHPLC system and a Q Exactive Plus Orbitrap mass spectrometer.

Used Instrumentation

• Thermo Scientific Vanquish Flex Duo UHPLC system with dual-channel configuration for direct LC or online SPE and analytical separation
• Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer with heated electrospray ionization (HESI-II)
• TraceFinder software version 5.1 for data acquisition and processing
• ClinMass® LC-MS/MS Complete Kit for Immunosuppressants in Blood (RECIPE Chemicals + Instruments)

Methodology

Sample preparation involved adding isotopically labelled standards to 100 µL blood, precipitating proteins with 220 µL precipitant, vortexing and centrifugation. The supernatant was transferred to vials for injection. A 50 µL injection was processed on the online SPE channel using SPE cartridges and analytical column supplied in the kit. The total chromatographic run time was 2.0 minutes. Mass spectrometry acquisition was performed in Full MS (70 000 resolution, m/z 800–1250) or Parallel Reaction Monitoring (PRM, 17 500 resolution) modes, using stepped collision energies to generate product ions for each analyte.

Main Results and Discussion

• Linearity: All analytes showed a linear response (R2 > 0.99) across calibration ranges up to ~1 250 ng/mL, with weighted 1/x regression.
• Lower Limits of Quantification (LLOQ): Achieved LLOQs of 0.625–2.27 ng/mL in Full MS and 0.262–2.27 ng/mL in PRM modes.
• Accuracy and Trueness: Bias between nominal and measured concentrations in kit controls was within ±5% for most analytes; external quality control samples met acceptance criteria.
• Precision: Intra-day CVs were below 5.4%; inter-day CVs did not exceed 6.5% across control levels.
• Carryover: No significant carryover observed in blank injections following high-level calibrators.

Benefits and Practical Applications

The workflow combines fast sample preparation, robust online SPE, and high-resolution Orbitrap detection to deliver reliable quantification for clinical research. The method supports high throughput, minimal carryover, and confident identification of analytes, making it suitable for research laboratories conducting therapeutic drug monitoring studies.

Future Trends and Applications

Emerging directions include further multiplexing of immunosuppressants and metabolites, integration of automated sample preparation modules, application of ion mobility separation for enhanced selectivity, and adoption of real-time data processing with machine learning algorithms to streamline therapeutic monitoring in precision medicine.

Conclusion

The described LC-HRAM mass spectrometry method demonstrates excellent performance for quantifying four immunosuppressant drugs in human blood, combining speed, accuracy, and sensitivity. The use of a comprehensive sample preparation kit and advanced Orbitrap technology ensures reliable data for clinical research applications.