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

Importance of the Topic

Metabolomics plays a critical role in clinical research and drug development by profiling small molecule metabolites in biological samples. Efficient and reproducible sample preparation is essential to achieve reliable quantitative results and to handle high sample throughput without introducing operator dependent variability.

Objectives and Study Overview

This study demonstrates the use of a fully automated sample preparation module coupled to an LC/MS/MS system for simultaneous analysis of primary metabolites in human plasma. The aim is to evaluate analytical performance, reproducibility, and operational efficiency of the combined CLAM-2030 and LCMS-8060 platform under a routine clinical testing scenario.

Methodology and Instrumentation

The workflow uses a CLAM-2030 module to perform protein precipitation, centrifugation, and supernatant recovery automatically. Prepared samples are transferred to a Nexera X2 UHPLC system paired with the LCMS-8060 triple quadrupole mass spectrometer. Chromatographic separation employs a reversed phase column with gradient elution using 0.1 percent formic acid in water and acetonitrile at 0.25 milliliter per minute flow rate and 2 microliter injection volume. Electrospray ionization in positive and negative modes supports multiple reaction monitoring transitions from a primary metabolite method package. Key instrument settings include a drying gas flow of 10 liters per minute, heat block temperature of 400 degrees Celsius, and interface temperature of 300 degrees Celsius.

Key Results and Discussion

A total of 55 primary metabolites including amino acids, organic acids, nucleosides, and lipids were detected in QC plasma samples. Peak area relative standard deviations were below 10 percent for 60 percent of analytes and below 20 percent for 93 percent. Normalizing against a D3–creatine internal standard improved reproducibility, with 67 percent of compounds under 10 percent RSD and 95 percent under 20 percent. Continuous analysis of 220 clinical samples over 22 days yielded a 5.9 percent RSD for the internal standard without instrument tuning or column change, demonstrating system stability.

Benefits and Practical Applications

• Automated sample preparation reduces hands on time and operator errors.
• Enhanced reproducibility through internal standard normalization.
• High throughput capability supports large study cohorts in clinical metabolomics.

Future Trends and Applications

Advances may include integration with data analysis pipelines for real time biomarker discovery, expansion to other biofluids or tissue extracts, and adoption of novel chromatographic phases for broader metabolome coverage. Further development could focus on multiplexed sample handling and coupling with high resolution mass analyzers.

Conclusion

The combination of the CLAM-2030 automated module with the LCMS-8060 system enables robust, high throughput quantitation of primary metabolites in plasma. The platform delivers consistent performance over extended runs, reduces manual workload, and supports reliable clinical research applications.

References

1. Imoto E and Kawakami D Application News Shimadzu Corporation December 2021