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

Significance of the Topic

Monitoring immunosuppressive drugs such as cyclosporine, everolimus, sirolimus, and tacrolimus is critical due to their narrow therapeutic windows and high pharmacokinetic variability among patients. Accurate measurement in whole blood supports research into dosing strategies, therapeutic drug monitoring, and optimization of transplant outcomes.

Objectives and Overview of the Study

This study aimed to develop a rapid and robust analytical method for the simultaneous quantification of four immunosuppressants in small volumes of human whole blood. The focus was on minimizing sample preparation time, reducing blood volume requirements, and achieving high throughput for clinical research applications.

Methodology and Instrumentation

Sample preparation involved protein precipitation of 50 µL whole blood by adding ZnSO4 and acetonitrile containing stable-isotope internal standards. The supernatant was directly injected into the UPLC system.
Equipment used:

• Chromatography: Waters ACQUITY UPLC I-Class with FL system equipped with an ACQUITY UPLC HSS C18 SB column (2.1 × 30 mm, 1.8 µm) at 55 °C
• Mobile phases: aqueous 2 mM ammonium acetate with 0.1% formic acid and methanol with 2 mM ammonium acetate plus 0.1% formic acid
• Injection volume: 20 µL, run time: 1.5 minutes (1.9 minutes injection-to-injection)
• Mass spectrometry: Xevo TQ-S micro operated in ESI+ mode with MRM transitions for each analyte and internal standard

Main Results and Discussion

The method achieved baseline separation of all four analytes within 1.5 minutes, with total analysis time under two minutes from injection to injection and no observable carryover. Analytical performance included:

• Sensitivity: Lower limit of quantification demonstrated acceptable precision (≤20% CV) and accuracy (≤15% bias) for concentrations as low as single-digit ng/mL levels
• Precision and repeatability: Total precision and intra-day repeatability ≤8.4% RSD across low, medium, and high quality control levels
• Linearity: Verified over ranges of 19.3–1500 ng/mL for cyclosporine and 0.77–39.0 ng/mL for everolimus, sirolimus, and tacrolimus
• Dilution integrity: Biases ≤10.4% following 1:1 dilution with blank whole blood
• Recovery: Extraction efficiencies between 93.2–108.4%
• Matrix effects: Effectively compensated by isotope-labeled internal standards, with post-column infusion confirming no significant ion suppression or enhancement at analyte retention times
• Selectivity: No significant interference from common endogenous substances, and external quality assurance samples showed agreement with trimmed means (all-laboratory means)

Benefits and Practical Applications

The proposed method offers:

• High throughput capacity suitable for clinical and pharmacokinetic research due to short analysis time
• Low sample volume requirement, beneficial for pediatric and serial sampling studies
• Cost-effective and straightforward sample preparation without solid-phase extraction
• High analytical selectivity and sensitivity supporting accurate therapeutic drug monitoring research

Future Trends and Potential Applications

Anticipated developments include extending the assay to additional immunosuppressants or biomarkers, integrating automation for sample preparation, and applying the workflow to real-time patient monitoring in transplant centers. Advances in high-throughput mass spectrometry may further reduce analysis time and improve data quality.

Conclusion

This UPLC-MS/MS method enables fast, precise, and sensitive simultaneous measurement of four key immunosuppressants in whole blood using minimal sample volume. Its robust performance and streamlined protocol make it an attractive tool for clinical research and pharmacokinetic studies.

Reference

Crossart A., Isbel M., Schuder C., Campbell S.B., Staatz C.E. Pharmacokinetic and Pharmacodynamic Considerations in Relation to Calcineurin Usage in Elderly Kidney Transplant Recipients. Frontiers in Pharmacology. 2021;12:635165.