Rapid Analysis of Cyclosporine A, Everolimus, Sirolimus, and Tacrolimus Drugs in Whole Blood Using an Agilent Triple Quadrupole LC/MS/MS System with Automated Online Sample Cleanup

Importance of the Topic

Monitoring immunosuppressive agents such as cyclosporine A, everolimus, sirolimus and tacrolimus is essential in clinical practice to ensure therapeutic efficacy and avoid toxicity. Rapid, highly sensitive and specific assays support timely dosing decisions and improve patient outcomes, particularly in transplant medicine where narrow therapeutic windows demand accurate drug quantification.

Objectives and Study Overview

The study aimed to develop and validate a high-throughput LC-MS/MS method capable of simultaneously quantifying four structurally related immunosuppressants in whole blood within a two-minute run time. Key goals included achieving low detection limits, broad linear ranges, robust performance over thousands of injections and minimal sample preparation complexity.

Methodology and Instrumentation

A simple protein precipitation protocol was used, mixing 100 µL whole blood with a zinc sulfate:methanol solution (ratio 1:4) containing deuterated or analog internal standards. Samples were processed on an Agilent 1260 Infinity LC system configured for online cleanup with a trapping column (ZORBAX Eclipse Plus C18) and an analytical Poroshell 120 EC-C18 column, both maintained at 60 °C. The LC was coupled to an Agilent 6460 or 6470 Triple Quadrupole Mass Spectrometer with JetStream technology. Two binary pumps facilitated loading/washing and analytical elution with ammonium acetate/formic acid buffers in water and methanol. MRM transitions were optimized for each analyte and its internal standard to ensure specificity and sensitivity.

Main Results and Discussion

• Linearity: Calibration curves were linear from 1.95 to 2 000 ng/mL for cyclosporine A and 0.10 to 100 ng/mL for everolimus, sirolimus and tacrolimus, with correlation coefficients above 0.995.
• Sensitivity: Lower limits of quantitation (LLOQs) of 1.95 ng/mL for cyclosporine A and 0.10 ng/mL for the other drugs were achieved.
• Accuracy and Precision: BioRad QC samples over 14 days and four operators yielded accuracies between 94 % and 107 % and CVs below 14 % across all levels.
• Robustness: Alternating calibration and stress-test sets (over 11 000 injections on 6460 and 14 000 on 6470) showed consistent peak areas with less than 10 % variation before column or autosampler maintenance.

Benefits and Practical Applications

The presented approach offers a two-minute assay combining rapid sample throughput and streamlined preparation. Automated online cleanup reduces matrix effects and instrument contamination, making the workflow suited for clinical and high-volume analysis in therapeutic drug monitoring and pharmacokinetic studies.

Future Trends and Applications

• Further miniaturization of sample preparation and integration with microfluidic devices.
• Expansion to high-resolution mass spectrometry for metabolite profiling.
• Implementation of data-driven quality control and real-time decision support.
• Multiplexed assays coupling additional immunosuppressants or biomarkers.

Conclusion

The developed Agilent LC-MS/MS method achieves sensitive, precise and robust quantitation of four critical immunosuppressive drugs in whole blood within a rapid two-minute run time, demonstrating suitability for routine clinical and research laboratories.

Reference

No formal literature references were provided in the original document.