Simultaneous Analysis of Immunosuppressive and Antifungal Drugs in Human Blood Plasma Using LC/MS/MS

Importance of the Topic

Liquid chromatography–mass spectrometry (LC–MS/MS) enables precise simultaneous quantitation of immunosuppressive and antifungal agents in plasma, critical for therapeutic drug monitoring in transplant patients who require both immunosuppression and prophylaxis against opportunistic fungal infections.

Objectives and Study Overview

• Establish a single LC–MS/MS assay for mycophenolic acid (MPA), voriconazole (VRC), itraconazole (ITC), and hydroxyitraconazole (OH-ITC).
• Validate method performance according to regulatory guidelines for accuracy, precision, linearity, and matrix effects.
• Demonstrate applicability in patient plasma samples.

Methodology and Instrumentation

Control plasma aliquots were spiked with target analytes and deproteinized by centrifugation. The supernatant was analyzed using a Shimadzu Nexera LC system coupled to an LCMS-8050 triple quadrupole mass spectrometer operating in positive ESI mode with MRM transitions optimized for each compound and corresponding deuterated internal standards.

• Column: Shim-pack Scepter C18, 50 mm × 2.1 mm, 3 μm.
• Mobile phase: 10 mmol/L formic acid/ammonium formate in water (A) and methanol (B).
• Gradient: 55% B to 100% B over 0.9 min, return to 55% by 2.11 min, total run 3 min.
• Flow: 0.45 mL/min; column temp: 40 °C; injection: 5 μL.
• MS conditions: interface 300 °C, desolvation line 250 °C, heat block 400 °C, gas flows optimized.

Main Results and Discussion

Calibration showed excellent linearity (R²>0.998) across wide concentration ranges (MPA 100–20 000 ng/mL, VRC 50–10 000 ng/mL, ITC and OH-ITC 5–1 000 ng/mL). Accuracy at the lower limit of quantitation and QC levels was within ±15%, and intra-run precision (%RSD) remained below 15%. Matrix effect evaluation across six plasma sources demonstrated minimal variability. Patient sample analysis confirmed selective detection without significant interferences, yielding clinically relevant concentration data for therapeutic monitoring.

Benefits and Practical Applications

• Streamlines simultaneous monitoring of immunosuppressants and antifungals in transplant recipients.
• Reduces sample volume and total analysis time compared to separate assays.
• Enhances laboratory efficiency and supports timely dose adjustments for optimized patient care.

Future Trends and Opportunities

Advances in automated sample preparation and high-throughput LC–MS/MS could further increase assay throughput. Expanding the panel to include additional immunosuppressive drugs or metabolites will support comprehensive therapeutic profiling. Integration with real-time data analytics may enable predictive dosing models and personalized therapy adjustments.

Conclusion

The validated LC–MS/MS method offers robust, precise, and accurate simultaneous quantitation of key immunosuppressive and antifungal agents in plasma. Its implementation in clinical laboratories can significantly improve therapeutic drug monitoring in transplant patient management.

Reference

• US FDA, Bioanalytical Method Validation: Guidance for Industry (2018).
• JP MHLW, Guideline on Bioanalytical Method Validation in Pharmaceutical Development (2013).