A Demonstration of HRMS with CE Spread Function for Identification of Cytotoxic Contaminant Present in Marine Bacteria Extract for Natural Product Discovery

Significance of the Topic

Natural products derived from marine bacteria offer promising lead compounds for drug discovery, yet co-extracted contaminants can yield false positives or pose health risks. High-resolution accurate mass spectrometry (HRAMS) with enhanced fragmentation capability is therefore crucial for reliable identification and structural elucidation of trace compounds in complex microbial extracts.

Objectives and Study Overview

This study demonstrates an integrated workflow combining the Shimadzu LCMS-9030 quadrupole time-of-flight (Q-TOF) system with a novel collision energy (CE) spread function and the LabSolutions Insight Explore software. The goal is to efficiently detect, identify, and confirm a cytotoxic contaminant within an HPLC-purified extract of marine bacterial origin.

Methodology and Instrumentation

LC Conditions

• Column: Shim-pack GIST C18 (2.1×100 mm, 2 µm)
• Mobile phase: A – water/0.1% formic acid, B – methanol/0.1% formic acid (80% B isocratic)
• Flow rate: 0.4 mL/min
• Oven temperature: 40 °C
• Injection volume: 1 µL

MS Conditions (LCMS-9030 Q-TOF)

• Ionization: HESI at 4.5 kV
• Interface temperature: 300 °C; DL: 250 °C; Heat block: 400 °C
• Nebulizing gas: 2 L/min; Drying and heating gas: 10 L/min
• MS scan: positive mode m/z 100–600
• MS/MS: 6 targeted precursors m/z 50–500, CE 25 ± 17 V (ramping 8–42 V), dwell time 0.05 s, loop time 0.35 s

Main Results and Discussion

Four components (P1–P4) were resolved by LC-HRAMS, with P3 representing the major peak (>90%). The CE spread feature produced rich MS/MS spectra with numerous fragment ions across a broad collision energy range, enhancing structural insight compared to fixed-CE methods. Accurate mass measurements (<1 ppm MS, <2 ppm MS/MS) allowed empirical formula generation and database matching. LabSolutions Insight Explore automated formula prediction, in-silico fragmentation, and database searches (ChemSpider, PubChem), leading to confident identification of P3 as tributyl acetyl citrate. Minor peaks P2 and P4 were annotated as structural analogues, demonstrating the workflow’s capacity to characterize low-abundance species.

Benefits and Practical Application of the Method

• Enhanced fragment generation via CE spread accelerates unambiguous structural elucidation
• Automated software tools streamline formula prediction, database searching, and fragment annotation
• Applicable to quality control in natural product research and contaminant screening in complex matrices

Future Trends and Potential Applications

Ongoing developments may include integration of data-independent acquisition strategies, artificial intelligence-driven spectral interpretation, expansion of spectral databases for broader compound coverage, and coupling with high-throughput workflows for industrial quality control and environmental monitoring.

Conclusion

The combined use of an ultra-fast CE spread function on the LCMS-9030 Q-TOF and the LabSolutions Insight Explore software delivers a robust, reliable workflow for high-confidence identification of contaminants in marine bacterial extracts. This approach enhances fragment diversity, automates structural elucidation, and is well suited for complex natural product analyses.

References

1. Chua C.K., Jumhawan U., Shao H.C., Toinon D., Simplifying Natural Product Analysis with Data-Independent Acquisition Workflow, Appl News, 2021
2. Dalisay D.S., Saludes J.P., Gonzalvo J.A., Unpublished data, University of San Agustin, 2021
3. Shimadzu Corporation, LCMS-9030 Webinar Slides, 2021