Integration of mycophenolic acid and its metabolite analysis in plasma using LC-MS/MS with full-automated sample preparation

Significance of the topic

Mycophenolic acid (MPA) and its glucuronide metabolite (MPA-G) are key immunosuppressive agents used in transplant medicine. Accurate, high-throughput quantification of these compounds in plasma is critical for therapeutic drug monitoring, quality control, and pharmacokinetic studies. Traditional sample preparation methods are labor-intensive and prone to variability, creating a demand for robust, automated workflows.

Objectives and Study Overview

This study aimed to integrate analysis of MPA and MPA-G into a single, fully automated LC-MS/MS workflow. Using the DOSIMYCO™ sample preparation kit and Shimadzu’s CLAM-2000 automation platform, the authors developed and validated a rapid method for plasma analysis. Performance metrics—linearity, precision, accuracy, and throughput—were assessed over clinically relevant concentration ranges.

Methodology and Instrumentation

• Automated Sample Preparation: CLAM-2000 performed protein precipitation with methanol, followed by filtration and direct transfer to the LC system without manual intervention.
• Chromatography: A two-column setup used a DOSIMYCO™ C8 trap column and a DOSIMYCO™ C18 analytical column (2.1×50 mm, 5 μm) heated to 65 °C, achieving separation in 1.5 minutes.
• Mass Spectrometry: Shimadzu LCMS-8045 triple quadrupole with ESI in positive and negative modes. Key MRM transitions were optimized for MPA (m/z 338.10 > 207.10) and MPA-G (m/z 514.10 > 207.10), with isotopically labeled internal standards.
• LC Conditions: Isocratic trap flow at 2 mL/min and analysis flow at 0.8 mL/min; mobile phases and cleaning solvents tailored for DOSIMYCO™ kit compatibility.

Main Results and Discussion

• Linearity: Calibration curves for MPA (0.1–50 mg/L) and MPA-G (1–250 mg/L) exhibited r² > 0.99, confirming excellent linear response.
• Precision and Accuracy: At low, medium, and high QC levels, intra-run CVs ranged from 4.1% to 9.5%, with accuracy within ±10% of nominal concentrations.
• Throughput: Automated preparation reduced hands-on time from over 1 hour to approximately 5 minutes per sample, enabling parallel processing of multiple vials and continuous LC-MS/MS analysis.
• Reproducibility: Triplicate analyses demonstrated consistent performance across the entire concentration range, underscoring method robustness.

Benefits and Practical Applications

• Enhanced Lab Efficiency: Dramatic reduction in manual labor and turnaround time for clinical and research laboratories.
• Minimized Human Error: Automated handling ensures reproducible sample processing and data quality.
• High Sensitivity: Adaptable for low-level detection needed in pharmacokinetic and toxicology studies.
• Scalability: Suitable for high-throughput therapeutic drug monitoring and routine QA/QC in pharmaceutical manufacturing.

Future Trends and Opportunities

• Multiplexed Analysis: Expansion to additional drug metabolites or biomarkers in a single run.
• Miniaturization: Integration with microfluidic sample prep for further throughput gains and reduced solvent consumption.
• AI-Driven Workflows: Automated data processing and predictive maintenance to optimize system performance.
• Regulatory Compliance: Validation for diagnostic use and adoption in regulated environments.

Conclusion

The automated DOSIMYCO™/CLAM-2000 LC-MS/MS method delivers rapid, precise, and accurate quantification of mycophenolic acid and its glucuronide in plasma. By eliminating manual extraction steps, this approach enhances laboratory productivity and data reliability, meeting the needs of high-volume clinical and pharmaceutical settings.

References

1. Minohata T, Moreau S, Dayot F, Hoeffler J-F. Integration of mycophenolic acid and its metabolite analysis in plasma using LC-MS/MS with fully automated sample preparation. ALSACHIM SAS & Shimadzu Corporation; Research Use Only.