Enhanced Specificity Provides Increased Quantitative Accuracy When Using SONAR for the Analysis of Low Level Lipids in Plasma-Based Extracts

Importance of the Topic

Quantitative analysis of lipids in biological matrices is crucial for biomarker discovery, clinical diagnostics, and metabolic research. Traditional MRM-based LC-MS workflows can suffer from interference due to co-eluting species, leading to false positives and inaccurate quantitation. Data-independent acquisition (DIA) offers enhanced spectral clarity, and SONAR mode extends these benefits by coupling a sliding quadrupole window with high-resolution time-of-flight detection to improve specificity and quantitative accuracy at low analyte levels.

Study Objectives and Overview

This study aimed to evaluate the dynamic range, limit of detection (LOD), limit of quantification (LOQ), and quantitative linearity of low-abundance lipid species in pooled human plasma extracts using SONAR. Deuterated lipid standards spanning multiple classes were spiked into plasma at ratios from 1:1 to 1:100,000 to assess method performance across a wide concentration range.

Methodology and Instrumentation

Lipid extraction was performed by protein precipitation with isopropanol (1:2, v/v), incubation, and centrifugation. Spiking used SPLASH LipidoMix standards at 350 to 2 µg/mL. Chromatographic separation employed a 20 min reversed-phase gradient on an ACQUITY UPLC CSH C18 column (1.7 µm, 2.1 × 100 mm). Mass spectrometry was conducted on a Xevo G2-XS QTof in SONAR mode with a 10 Da quadrupole window scanning 350–950 m/z and TOF scanning 50–1200 m/z at 0.1 s per scan. Comparative DIA data using MSE were also acquired. Data processing and extraction were performed in Skyline.

Main Results and Discussion

SONAR provided cleaner fragmentation spectra by isolating co-eluting precursors, reducing interference and improving peak fidelity. The method achieved an average LOD of 0.001 µg/mL and LOQ of 0.1 µg/mL, comparable to targeted MRM assays. Representative deuterated diglyceride (DG(15:0/18:1) d7) transitions exhibited excellent linearity (R2 0.985–0.991) over a >20-fold dilution range. Enhanced spectral clarity was demonstrated for lysophosphatidylethanolamine (LPE(18:1)d7), with additional transitions detected due to the sliding window approach.

Benefits and Practical Applications

• High specificity reduces false positives in complex lipidomes
• Wide dynamic range suitable for low-abundance analytes
• Full-scan DIA data allow posthoc selection of transitions
• High duty cycle supports high-throughput studies

Future Trends and Opportunities

SONAR DIA is poised to expand into large-scale cohort studies and multi-omics workflows. Integration with advanced informatics and machine learning may further enhance lipid identification and quantitation. Real-time DIA acquisition and miniaturized LC-MS platforms represent potential developments to increase throughput and accessibility.

Conclusion

SONAR mode on a high-resolution QTof mass spectrometer delivers improved spectral clarity, sub-microgram detection capabilities, and robust quantitative performance for low-level lipids in complex plasma extracts. This approach combines the flexibility of DIA with targeted quantitation accuracy, offering a powerful tool for lipidomics research.

Reference

• A. King, L. Gethings, R. Plumb, I. Wilson, Increased Specificity, Improved Lipidomic Clarity, and Identification Confidence with SONAR. Waters Application Note. 720006036EN. 2017.
• Sarafian et al., Bile Acid Profiling and Quantification in Biofluids using Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry, Anal. Chem., 2015.