Fully automated analysis of immunosuppressant drugs in whole blood using stable isotope labeled internal standards

Significance of the topic

The monitoring of immunosuppressant drugs is essential in transplant medicine to maintain drug levels within a narrow therapeutic window. Overexposure can lead to toxicity and long term complications while underexposure risks graft rejection. Therapeutic drug monitoring (TDM) supports personalized dosing, enhances patient safety, and improves clinical outcomes. Liquid chromatography tandem mass spectrometry (LC MS MS) offers superior specificity, sensitivity, and the ability to quantify multiple analytes simultaneously, addressing limitations of conventional immunoassays.

Objectives and study overview

This work presents a fully automated LC MS MS method for quantifying four key immunosuppressants – Cyclosporin A, Tacrolimus, Sirolimus, and Everolimus – in whole blood. The primary goals were to integrate an automated sample preparation module with high performance LC MS MS detection and to validate the workflow for routine clinical TDM.

Methodology and instrumentation

Sample preparation was performed using the Clinical Laboratory Automated sample preparation Module (CLAM 2000) combined with a Shimadzu LCMS 8050 system. A DOSIMMUNE reagent kit provided stable isotope labeled internal standards. Key steps included:

• Automated loading of hemolyzed whole blood and calibrators
• Addition of extraction buffer and isotope labeled internal standards
• Mixing, centrifugation, and filtration in the CLAM 2000 platform
• Online trapping on a DOSIMMUNE C8 column followed by separation on a DOSIMMUNE C18 analytical column at 65 °C using isocratic elution
• Detection by electrospray ionization in positive mode with multiple reaction monitoring transitions for each analyte and its labeled standard

Main results and discussion

Validation demonstrated linear calibration curves over clinical ranges with correlation coefficients above 0.998. Accuracy fell between 90 and 110 percent and intra run precision (CV) remained below 16.5 percent across all quality control levels. A comparison of patient samples analyzed by LC MS MS and a commercial immunoassay for Tacrolimus showed strong agreement (slope 0.9437, R² = 0.9567). The automated protocol reduced hands on time, improved reproducibility, and minimized risk of manual error.

Benefits and practical applications

This automated LC MS MS approach offers multiple advantages:

• Simultaneous quantification of four immunosuppressants in a single run
• Enhanced specificity and reduced cross Reactivity compared to immunoassays
• Stable isotope labeled standards boost data accuracy and precision
• High throughput capability with lower reagent consumption and cost savings
• Reduced operator workload and improved laboratory safety

Future trends and opportunities

Advances in LC MS MS automation and kit based solutions will further streamline TDM workflows. Potential developments include expanding panels to additional drugs, integrating data processing with laboratory information systems, and applying machine learning for dose adjustment recommendations. Portable mass spectrometry and point of care solutions may also emerge for rapid bedside monitoring.

Conclusion

The fully automated DOSIMMUNE CLAM 2000 LC MS MS method provides reliable, high throughput monitoring of key immunosuppressants in whole blood. Strong validation performance and excellent agreement with immunoassays support its implementation in clinical laboratories, offering improved analytical quality, cost efficiency, and better patient management.