BBSS: A ROBUST AND REPRODUCIBLE LIPID DIA UPLC/MS USING WATERS CONNECT WITH THE XEVO Q-TOF G3

Significance of the Topic

This study addresses the need for reliable, high-throughput lipidomic analyses in research and clinical settings. Robust and reproducible detection of lipid species supports biomarker discovery, quality control, and process optimization in pharmaceutical and biomedical laboratories.

Goals and Overview of the Study

The main objective was to evaluate a data-independent acquisition (DIA) UPLC/MS workflow using the waters_connect platform coupled to a Xevo G3 Q-Tof mass spectrometer. The study aimed to demonstrate streamlined sample processing, accurate lipid identification, quantification, and reporting within a unified informatics environment.

Methodology

Sample Preparation:

• 100-fold diluted AVANTI EquiSPLASH internal standard in isopropanol (IPA).
• Protein precipitation of plasma extracts with 100× EquiSPLASH in IPA.
• Calibration curves generated with AVANTI Odd-Chain LIPIDOMIX at 10×, 20×, and 50× concentrations spiked into pooled human plasma.

Chromatography and Acquisition:

• 12-minute reversed-phase UPLC gradient using ACQUITY Premier UPLC CSH C18 column.
• Mobile phases: A (60:39:1 acetonitrile:water:ammonium formate, 0.1% formic acid), B (90:9:1 IPA:acetonitrile:ammonium formate, 0.1% formic acid).
• Positive and negative electrospray ionization DIA on Xevo G3 Q-Tof.

Data Processing:

• Automated mass calibration and detector setup via waters_connect.
• Identification using the Waters Lipidomics CCS Library and high-energy fragmentation spectra.
• Reporting templates and optional export to third-party tools via API (e.g., Lipostar).

Used Instrumentation

• ACQUITY Premier UPLC I-Class system.
• ACQUITY Premier UPLC CSH C18 (2.1 × 100 mm, 1.7 µm) column.
• Xevo G3 Q-Tof mass spectrometer.
• waters_connect software platform.

Results and Discussion

• Retention time reproducibility showed CVs <0.5% across 125 injections.
• Signal response CVs <5% for multiple endogenous lipid classes, including outlier data.
• Mass accuracy stability averaged <0.57 ppm (positive mode) and <1.93 ppm (negative mode) over 75–125 injections.
• Calibration curves for odd-chain phospholipids exhibited high linearity (R² >0.97) within biological concentration ranges.

Benefits and Practical Applications

• High throughput analysis with a 12-minute run time supports large sample batches.
• Robust and reproducible workflow minimizes operator intervention.
• Comprehensive lipid identification including head group and fatty acyl fragment confirmation.
• Seamless integration with third-party data analysis tools enhances flexibility.

Future Trends and Opportunities

Advancements may include expanded lipid libraries, deeper integration of machine learning for automated annotation, multiplexed high-resolution platforms for even greater throughput, and translation of lipidomics workflows into routine clinical diagnostics.

Conclusion

The evaluated DIA UPLC/MS workflow with waters_connect and Xevo G3 Q-Tof delivers a simple, flexible, and highly reproducible lipidomics solution. It provides accurate identification, quantitation, and reporting, addressing key needs in research and quality control settings.

References

1. Munjoma N. et al. High Throughput LC-MS Platform for Large Scale Screening of Bioactive Polar Lipids in Human Plasma and Serum. J Proteome Res. 2022;21(11):2596-2608.
2. Goracci L. et al. LipoStar, a Comprehensive Platform-Neutral Cheminformatics Tool for Lipidomics. Anal Chem. 2017;89(11):6257-6264.