Measuring Choline and its Metabolites in Human Plasma Using an LC/MS/MS System with Fully Automated Sample Preparation Module

Significance of the Topic

Choline metabolism produces trimethylamine (TMA) by intestinal bacteria and trimethylamine N-oxide (TMAO) by hepatic enzymes; TMAO is emerging as a biomarker linked to cardiovascular disease risk and prognosis, underscoring the need for accurate quantification in plasma samples.

Objectives and Study Overview

This study presents and validates a fully automated LC/MS/MS workflow combining the CLAM-2030 sample prep module with an LCMS-8060 system to simultaneously quantify choline, TMA, and TMAO in human plasma, aiming to reduce manual steps and variability while maintaining analytical performance.

Instrumentation Used

• Sample preparation: CLAM-2030 module for automated deproteination and internal standard addition
• Liquid chromatography: Shimadzu Nexera X2 with Shim-pack Velox HILIC column (50 × 2.1 mm, 2.7 μm) at 40 °C, flow rate 0.4 mL/min, 3 μL injection volume, gradient from 70 to 5% acetonitrile
• Mass spectrometry: Shimadzu LCMS-8060 with positive-mode ESI, MRM transitions optimized for choline, TMA, TMAO and their isotopic standards

Methodology

Plasma samples were directly loaded into the CLAM-2030 for automated precipitation and addition of choline-d13, TMA-d9, and TMAO-d9 internal standards. Calibration curves were prepared by serial dilution in water, covering 1–1000 nmol/L for choline, 10–1000 nmol/L for TMA, and 10–10000 nmol/L for TMAO. Data acquisition employed MRM, with analysis cycles of approximately 5 minutes per sample, enabling parallel sample prep and LC/MS/MS analysis for high throughput.

Main Results and Discussion

Calibration curves showed outstanding linearity (R2 > 0.997) and accuracy (86–96%) at the lowest concentration levels. Precision was within 14% RSD for standards. In human plasma (n = 3), measured concentrations were 938 nmol/L choline (2.8% RSD), 685 nmol/L TMA (1.1% RSD), and 655 nmol/L TMAO (4.4% RSD). Automated preparation reduced variability and facilitated reproducible quantification.

Benefits and Practical Applications

• Reduces labor and manual handling time through full automation
• Minimizes operator-induced variability for consistent quantitative results
• Enables high-throughput analysis with ~5 minutes per sample turnaround
• Applicable to clinical diagnostics, biomedical research, and large-scale biomarker studies

Future Trends and Applications

The integration of automated sample preparation with mass spectrometry is expected to expand into multiplexed biomarker panels, coupling with microfluidic devices, and implementation in routine clinical laboratories and epidemiological research to support personalized medicine.

Conclusion

The CLAM-2030 and LCMS-8060 combination offers a robust, reliable, and fully automated platform for the simultaneous quantification of choline, TMA, and TMAO in human plasma, delivering high throughput and reproducible performance suitable for research and clinical applications.

References

• Wang Z, Hazen SL, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011;472:57–63.
• Yazaki Y, Aizawa K, et al. Ethnic difference in association of outcomes with trimethylamine N-oxide in acute heart failure patients. ESC Heart Fail. 2020;7(5):2373–2378.