Analysis of Cyclosporine, Everolimus, Sirolimus, and Tacrolimus in Whole Blood for Clinical Research

Significance of the Topic

Cyclosporine, everolimus, sirolimus and tacrolimus are key immunosuppressive drugs used in organ transplantation and autoimmune disease research. Accurate quantification in whole blood is critical for pharmacokinetic and pharmacodynamic studies given their narrow therapeutic windows and high variability.

Study Objectives and Overview

This application note describes the development of a rapid and sensitive UPLC-MS/MS method to quantify these four analytes in clinical research settings. The study aims to deliver high selectivity, low sample volume requirements, and fast turnaround to support large-scale pharmacological investigations.

Methodology and Instrumentation

• Sample Preparation: Protein precipitation of 50 µL whole blood with zinc sulfate, followed by addition of stable isotope internal standards and centrifugation to obtain the supernatant.
• Chromatography: Waters ACQUITY UPLC HSS C18 SB column (2.1×30 mm, 1.8 µm) with a water-methanol gradient containing ammonium acetate and formic acid. Total run time was 1.8 minutes per injection.
• Mass Spectrometry: Detection on a Xevo TQD tandem quadrupole in positive electrospray mode, using multiple reaction monitoring for each analyte and matching internal standard transitions.
• Data Processing: MassLynx software with TargetLynx Application Manager for calibration curve fitting, quantification and quality control review.

Key Results and Discussion

• Analytical Sensitivity: Quantification limits of 8 ng/mL for cyclosporine, 0.5 ng/mL for everolimus, 1 ng/mL for sirolimus and 0.5 ng/mL for tacrolimus, with bias below 15% and precision below 20% RSD.
• Precision and Repeatability: Total precision and repeatability across low to high quality control levels remained under 9.1% RSD over five days.
• Linearity: The method demonstrated linear response from approximately 19 to 1662 ng/mL for cyclosporine and 0.7 to 35 ng/mL for everolimus, sirolimus and tacrolimus.
• Matrix Effects and Dilution Integrity: Internal standards compensated for matrix enhancement (response ratios around 1.02–1.04). High-concentration samples diluted 1:1 with blank blood retained bias below 10%.
• Interference Assessment: No significant interference from endogenous compounds or co-administered immunosuppressants, with analyte recoveries within 85–115% criteria.
• External Quality Assurance: Analysis of 33 NEQAS proficiency samples yielded mean deviations within ±12% of consensus values for all four analytes.

Benefits and Practical Applications

This UPLC-MS/MS protocol offers high selectivity, minimal sample volume and rapid throughput, making it ideal for therapeutic drug monitoring, clinical pharmacology trials and routine research laboratory workflows.

Future Trends and Potential Applications

Further automation of sample preparation, expansion to high-resolution mass spectrometry platforms and inclusion in multiplex assay panels could extend this approach to additional immunosuppressant metabolites and biomarkers, supporting precision medicine initiatives.

Conclusion

A robust, high-throughput UPLC-MS/MS method has been validated for simultaneous quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. The workflow ensures speed, sensitivity and precision for diverse clinical research applications.

References

• Seger C et al. Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee. Ther Drug Monit. 2016;38:170–189.