Extraction of a Range of Immunosuppressants from Whole Blood Using ISOLUTE® SLE+ Prior to LC-MS/MS Analysis

Significance of the Topic

Immunosuppressants such as sirolimus, tacrolimus, everolimus and cyclosporin A play a vital role in transplantation and autoimmune disease management. Reliable therapeutic drug monitoring is essential to avoid rejection or immunodeficiency. Supported liquid extraction (SLE+) combined with LC-MS/MS offers a robust alternative to traditional immunoassays and liquid-liquid extraction, reducing cross-reactivity and improving throughput.

Objectives and Study Overview

This application note evaluates the performance of the ISOLUTE SLE+ 400 µL supported liquid extraction plate for isolating four immunosuppressants from whole blood prior to quantification by LC-MS/MS.

Methodology and Instrumentation

• Sample Preparation: 50 µL whole blood diluted with 250 µL water, vortexed, and centrifuged at 12 000 RPM.
• Supported Liquid Extraction: 275 µL supernatant loaded onto ISOLUTE SLE+ plate; two sequential 600 µL ethyl acetate elutions under gravity and vacuum; evaporation at 30 °C; reconstitution in 25:75 water:acetonitrile (100 µL).
• LC Conditions: Waters Acquity UPLC BEH C18 column (50×2.1 mm, 1.7 µm), 10 °C sample tray, 60 °C column, gradient of 2 mM ammonium formate with 0.1% formic acid in water (A) and methanol (B), 10 µL injection.
• MS Conditions: Premier XE triple quadrupole with ESI+, desolvation at 450 °C, ion source at 150 °C, optimized MRM transitions for quantification and qualification.

Key Results and Discussion

Recoveries ranged from 60% to 97% with RSDs below 10% for all analytes, demonstrating excellent precision and reproducibility. The SLE+ workflow eliminated emulsion formation and delivered clean extracts. Extracted ion chromatograms showed well-resolved, interference-free peaks for each compound at 80 ng/mL.

Benefits and Practical Applications

• Improved analyte recovery and precision compared to immunoassays.
• No cross-reactivity, reducing risk of false positives.
• Streamlined sample preparation with reduced solvent use and processing time.
• Cost-effective solution for routine therapeutic drug monitoring in clinical and research laboratories.

Future Trends and Opportunities

Integration of SLE+ into automated, high-throughput platforms; expansion to broader panels of drugs and biomarkers; coupling with high-resolution mass spectrometry for multiplexed assays; development of greener, more sustainable extraction solvents.

Conclusion

The ISOLUTE SLE+ extraction method provides a reliable, efficient, and reproducible approach for quantifying key immunosuppressants by LC-MS/MS, enhancing therapeutic drug monitoring accuracy and laboratory productivity.

Reference

Biotage Application Note AN758 (2013)