Simultaneous determination of four immunosuppressants in whole blood – A fast method on a Shimadzu tandem LCMS-8030 mass analyser

Significance of the Topic

A precise and rapid monitoring of immunosuppressant drugs in whole blood is critical for transplant recipients to maintain therapeutic efficacy and minimize toxicity. Conventional immunoassays often suffer from cross-reactivity with metabolites, whereas LC-MS/MS offers high specificity and sensitivity. Establishing a fast, reliable method supports clinical decision-making and improves patient outcomes.

Objectives and Overview of the Study

This work describes the development and validation of a kit-based LC-MS/MS method for simultaneous quantification of four key immunosuppressants—cyclosporine A, tacrolimus, sirolimus, and everolimus—in whole blood. The method, implemented on a Shimadzu LCMS-8030 triple quadrupole system, aims to deliver rapid analysis suitable for routine laboratory use.

Methodology and Instrumentation

Sample Preparation:

• Protein precipitation of whole blood using a commercial Chromsystems kit.
• Injection of supernatant into the LC-MS/MS system.

Chromatography and Detection:

• Shimadzu LCMS-8030 with online trapping to reduce matrix effects and ion suppression.
• Ultra-fast polarity switching (15 ms) and scan speed (15,000 u/sec).
• UFsweeper™ technology to minimize cross-talk.

Validation Parameters:

• Linearity: Calibration ranges of 25–900 µg/L for cyclosporine A, 2–40 µg/L for tacrolimus and everolimus, 2–50 µg/L for sirolimus. Correlation coefficients exceeded 0.999.
• Accuracy: Bias within ±10% and imprecision CV < 10% across four quality-control levels.
• Precision: Inter- and intra-day CVs ranged from 1.4% to 9.4%.
• Stability: QC samples remained stable over 14 days, demonstrating robust method performance.
• Method Comparison: Passing-Bablok regression against existing HPLC-MS/MS and immunoassays showed slopes near unity (0.97–1.03) and high correlation.

Main Results and Discussion

The method achieved excellent linearity, accuracy, and precision within therapeutic ranges for all four immunosuppressants. Online trapping effectively removed interfering matrix components, and rapid polarity switching enabled a total run time of under 5 minutes. Method comparison confirmed equivalence to established LC-MS/MS protocols and superiority over immunoassays in specificity.

Benefits and Practical Applications of the Method

This validated protocol offers:

• High throughput suitable for routine clinical laboratories.
• Reduced sample preparation time and minimal manual handling.
• Broad dynamic range covering sub-therapeutic to toxic concentrations.
• Reliable quantification to guide immunosuppressive therapy adjustments.

Future Trends and Opportunities

Advancements may include full automation of sample prep, integration with laboratory information systems, and extension to additional immunosuppressants or metabolites. High-resolution MS and microflow chromatography could further enhance sensitivity and reduce solvent consumption.

Conclusion

The described LC-MS/MS method on the Shimadzu LCMS-8030 platform provides a rapid, robust, and highly specific approach for simultaneous quantification of four major immunosuppressants in whole blood. Its performance characteristics support its adoption as a routine clinical assay.

Instrumentation Used

• Shimadzu LCMS-8030 triple quadrupole mass spectrometer with UFsweeper™ and rapid polarity switching.
• Chromsystems MassTox® immunosuppressant kit for sample preparation.

References

1. Vogeser M, Spöhrer U. Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry. Clin Chem Lab Med. 2006;44(9):1126-1130.
2. Chromsystems application manual, MassTox® Immunosuppressants. 2010.
3. Passing H, Bablok. A new biometrical procedure for testing the equality of measurements from two different analytical methods. J Clin Chem Clin Biochem. 1983;21(11):709-720.
4. Vogeser M, Seger C. A decade of HPLC-MS/MS in the routine clinical laboratory-goals for further developments. Clin Biochem. 2008;41(9):649-662.