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

Significance of Topic

The combined use of immunosuppressive agents and antifungal drugs is critical to prevent organ rejection and opportunistic infections after transplantation. Reliable simultaneous quantification of these compounds in plasma supports therapeutic drug monitoring, optimizes dosing, and minimizes adverse interactions.

Objectives and Study Overview

This work describes the development and validation of a rapid LC-MS/MS assay for concurrent measurement of mycophenolic acid (MPA), voriconazole (VRC), itraconazole (ITC) and its active metabolite hydroxyitraconazole (OH-ITC) in human blood plasma.

Methodology

Sample Preparation:

• Aliquot 40 µL plasma and add 160 µL internal standard solution containing deuterated analogues (MPA-d3, VRC-d3, ITC-d4, OH-ITC-d4) in acetonitrile/methanol (90/10, v/v).
• Vortex for 60 s and centrifuge at 10 000 × g for 10 min.
• Collect 150 µL supernatant for injection.

Chromatography and Mass Spectrometry:

• Column: Shim-pack Scepter C18 Metal Free (50 × 2.1 mm, 3 µm) at 40 °C.
• Mobile Phases: A: 10 mmol/L formic acid + 10 mmol/L ammonium formate in water; B: same buffer in methanol.
• Gradient: 55% B (0–0.55 min) to 100% B (0.9–2.1 min), return to 55% B by 2.11 min; flow 0.45 mL/min; run time 3 min.
• Injection volume: 5 µL; ESI-positive mode; optimized MRM transitions and collision energies for each analyte and internal standard.

Used Instrumentation

• Nexera LC system coupled to LCMS-8050 triple quadrupole mass spectrometer (Shimadzu).
• Shim-pack Scepter C18 Metal Free column.
• Standard centrifuge and vortex mixer for sample pretreatment.

Main Results and Discussion

The assay demonstrated excellent linearity (R² > 0.998) over calibration ranges: MPA 100–20 000 ng/mL, VRC 50–10 000 ng/mL, ITC and OH-ITC 5–1 000 ng/mL. Accuracy was 92.8–103.9% and precision (%RSD) below 8.9% across QC levels. Matrix effects were minimal; no significant interference was observed in patient plasma samples. Chromatograms showed clear separation and reliable quantitation of all targets.

Benefits and Practical Applications

• Simultaneous analysis reduces turnaround time and sample volume.
• High sensitivity and specificity support robust therapeutic drug monitoring in transplant recipients.
• Validated according to US FDA and JP MHLW guidelines, ensuring regulatory compliance.

Future Trends and Potential Applications

Advances may include automated sample preparation, multiplexed panels covering additional immunosuppressants and antifungals, high-resolution mass spectrometry for broader metabolite profiling, and integration into point-of-care monitoring platforms for personalized dosing.

Conclusion

This validated LC-MS/MS method provides a fast, accurate, and reliable tool for simultaneous quantification of MPA, VRC, ITC, and OH-ITC in plasma, enhancing therapeutic drug monitoring and patient care in post-transplant therapy.

References

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