Accurate LC-MS Bioanalytical Quantification of Itaconic Acid Using the Automated Andrew+ Pipetting Robot for Ostro Sample Preparation

Importance of the Topic

Accurate quantification of low-abundance metabolites such as itaconic acid in complex biological matrices is essential for understanding inflammatory processes and metabolic pathways. Robust sample cleanup and reproducible workflows reduce variability and improve data quality, supporting both research and regulatory bioanalysis requirements.

Study Objectives and Overview

This application note demonstrates the fully automated preparation, extraction, and LC-MS/MS quantification of itaconic acid from human plasma. The study evaluates performance metrics of the Andrew+ Pipetting Robot combined with the Waters Ostro Protein Precipitation and Phospholipid Removal 96-well Plate, comparing automation against manual procedures.

Methodology and Instrumentation

The automated workflow uses OneLab Software to coordinate liquid handling and vacuum steps on the Andrew+ deck. Key steps include:

• Preparation of calibration standards (0.5–100 ng/mL) and quality controls (0.75, 7.5, 75 ng/mL)
• Protein precipitation with organic solvent and phospholipid removal on Ostro plates
• Vacuum-assisted sample passage, followed by evaporation under nitrogen and reconstitution
• LC-MS/MS analysis with an ACQUITY PREMIER System and Xevo TQ-S micro mass spectrometer to mitigate metal chelation and maximize recovery

Main Results and Discussion

The automated method achieved an excellent calibration linearity with R² = 0.997 across a 200-fold concentration range. Recovery experiments demonstrated:

• Low and high QC recoveries of 100% and 99%, surpassing manual workflow results
• Intra-day accuracy between 104.9–112.8% and precision (RSD) of 1.3–3.6% (N = 4)

These metrics meet or exceed accepted bioanalytical validation criteria, confirming the reliability of the automated approach.

Practical Benefits and Applications

Automation delivers multiple advantages:

• Consistent pipetting accuracy and reduced operator-to-operator variability
• Enhanced throughput by freeing analysts for parallel tasks
• Software-guided user actions with visual and video prompts to reduce procedural errors
• Modular consumable placement using magnetic "domino" carriers for deck stability

This solution is well suited for high-volume bioanalytical labs performing routine LC-MS quantification of small molecules.

Future Trends and Applications

Advancements in laboratory automation will focus on integrating real-time feedback, machine learning for protocol optimization, and expanding compatibility across diverse sample types. Coupling automated liquid handling with data-driven decision support tools can further streamline method development and QC monitoring for emerging biomarkers.

Conclusion

The Andrew+ Pipetting Robot paired with Ostro 96-well plates provides a reliable, high-throughput workflow for itaconic acid quantification. Automation improved recovery, precision, and overall assay robustness compared to manual techniques, offering a scalable solution for bioanalytical laboratories.

Reference

1. Wells DA. Automation Tools and Strategies for Bioanalysis. In: Progress in Pharmaceutical and Biomedical Analysis. Elsevier; 2003. p. 135–197.
2. Brennan K, et al. Bioanalytical LC-MS Quantification of Itaconic Acid: A Potential Metabolic Biomarker of Inflammation. Waters Application Note 720006683; 2019.
3. Bansal S, DeStefano A. Key Elements of Bioanalytical Method Validation for Small Molecules. AAPS Journal. 2007;9(1):E109–E114.