Confidence in Your Calibrators: MassTrak™ Immunosuppressant Calibrator and Quality Control Sets for the LC-MS/MS Analysis of Cyclosporine, Everolimus, Sirolimus, and Tacrolimus

Importance of the Topic

Therapeutic monitoring of immunosuppressant drugs such as cyclosporine, everolimus, sirolimus and tacrolimus is essential to ensure efficacy and safety in transplant and autoimmune patients. Traditional immunoassays can suffer from cross-reactivity and limited accuracy at low drug levels. As clinical laboratories increasingly adopt liquid chromatography–tandem mass spectrometry (LC-MS/MS) for these measurements, reliable calibrators and quality control materials become critical for standardized, traceable results and inter-laboratory harmonization.

Objectives and Study Overview

This application note evaluates the analytical performance of Waters MassTrak™ Immunosuppressant Calibrator and Quality Control Sets (IVD) in a proof-of-concept LC-MS/MS workflow. The study aims to demonstrate linearity, sensitivity, precision, accuracy and external quality assessment alignment for all four immunosuppressant compounds when using a validated protein precipitation protocol and a UPLC-MS/MS platform.

Methodology

Sample Preparation:

• Start with 50 µL whole blood; add 200 µL 0.1 M ZnSO₄, mix 5 s.
• Add 500 µL internal standard solution, mix 20 s, then centrifuge at 4696 g for 2 min.

Calibrators and QCs:

• Lyophilized blood samples spiked with each drug across multi-point ranges (e.g., cyclosporine 25–1200 ng/mL; others 1–50 ng/mL).

Quantitation:

• Calibration standards reconstituted per IFU and processed alongside QCs.
• Analyte concentrations determined against weighted regression curves.

Used Instrumentation

• ACQUITY UPLC I-Class FL system with 2.1×30 mm HSS C18 SB column (1.8 µm) at 55 °C.
• Xevo TQ-S micro triple quadrupole mass spectrometer in positive ESI mode (0.8 kV).
• Mobile phases: 2 mM ammonium acetate with 0.1% formic acid in water (A) and methanol (B).

Key Results and Discussion

• Linearity: Coefficients of determination (r²) ≥ 0.9988 for all drugs across calibration ranges.
• Sensitivity: Signal-to-noise (S/N) ≥ 10 at lowest calibrator levels in multiple runs.
• Precision: Total CV ≤ 7.1% across low, mid, high QC levels over five days (n=25 each).
• Accuracy: QC recoveries ranged 94.5–103.6% relative to nominal concentrations.
• External QA: Bland-Altman analysis versus UK NEQAS samples showed method bias within ±7.4% for all analytes, underscoring metrological traceability.

Benefits and Practical Applications

• ISO 17511-compliant value assignment ensures traceability.
• Lyophilized format reduces preparation time and enhances stability.
• Supports method harmonization across clinical laboratories.
• Enables robust monitoring in transplant and immunosuppression management.

Future Trends and Potential Applications

Ongoing developments in LC-MS/MS aim to reduce sample volume requirements, increase multiplexing of additional immunosuppressants and metabolites, and integrate automated sample preparation workflows. Expanded standardization across global quality schemes and integration with laboratory information systems will further improve result consistency and patient care.

Conclusion

The Waters MassTrak™ Immunosuppressant Calibrator and QC Sets demonstrated excellent performance for LC-MS/MS measurement of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. The combination of linear response, high sensitivity, precision ≤ 7.1% CV and accurate alignment with external QA schemes validates these materials as reliable standards for clinical immunosuppressant monitoring.

References

No external literature references were provided in the original document.