High Definition Data Directed Analysis (HD-DDA)

Importance of the topic

In complex biological, environmental, and industrial analyses, acquiring high‐quality MS/MS spectra from trace‐level analytes is essential for confident identification and quantification. High Definition Data Directed Analysis (HD-DDA) addresses these challenges by enhancing sensitivity and speed in routine LC/MS/MS workflows.

Objectives and overview of the study

The study aimed to:

• Boost routine discovery workflows through increased MS/MS sensitivity.
• Implement real‐time decision making to maximize the number of high‐quality MS/MS events per injection.
• Lower detection limits across a wide m/z range for complex samples.

HD-DDA was demonstrated on the Waters SYNAPT G2-Si HDMS platform, comparing performance against standard Data Directed Analysis.

Methodology and instrumentation

The experimental setup comprised:

• Waters SYNAPT G2-Si HDMS mass spectrometer with T-Wave™ ion mobility separation.
• Wideband Enhancement transmission mode to synchronize TOF pusher timing with fragment release.
• UPLC system using a 2.1 × 100 mm column for peptide or small‐molecule separations.

During HD-DDA runs, up to 15 precursors per MS survey scan are selected based on intensity and mobility‐derived charge separation. Each MS/MS event terminates upon reaching a predefined TIC target or time threshold, enabling rapid return to survey mode.

Results and discussion

Major outcomes include:

• Up to tenfold increase in duty cycle for fragment ions, particularly in the 200–800 m/z range, leading to greatly enhanced sensitivity.
• Faster decision cycles, allowing more MS/MS acquisitions per chromatographic run without extending analysis time.
• Improved selectivity via ion mobility separation, which discriminates analyte ions from background signals before fragmentation.
• Rich fragmentation spectra for low‐abundance peptides, demonstrated by comparing Wideband‐enhanced MS/MS with conventional acquisition.

Benefits and practical applications

HD-DDA enables:

• Comprehensive profiling in proteomics and metabolomics by capturing more MS/MS spectra in a single injection.
• Lower detection limits in trace contaminant screening for environmental and food safety labs.
• Improved workflow efficiency in QA/QC and pharmaceutical research through robust, high‐sensitivity acquisition.

Future trends and opportunities

Emerging directions include integrating machine learning for adaptive precursor selection, extending HD-DDA to ultra‐high resolution platforms, and combining with advanced separation techniques to deepen structural characterization.

Conclusion

HD-DDA on the SYNAPT G2-Si delivers a potent combination of intelligent acquisition, ion mobility separation, and Wideband Enhancement to achieve unmatched MS/MS sensitivity and throughput in complex sample analysis.

Reference

Tomczyk N, Wallace A, Richardson K, Grzyb A, Wildgoose J. High Definition Data Directed Analysis (HD-DDA). Waters Corporation; 2013.