Performance Comparison of Agilent 1290 Infinity Binary and Quaternary Systems as an MS Front-End

Significance of the Topic

Flavonoids are widespread dietary polyphenols with diverse biological properties, including antioxidant, anti-inflammatory and anticancer effects. Enzymatic methylation by flavonoid O-methyltransferases (OMTs) enhances metabolic stability, oral bioavailability and bioactivity of these compounds. Differentiation of structural isomers of methylated flavonoids is critical for understanding enzyme specificity and biological function, but isomeric complexity and scarcity of standards pose major analytical challenges.

Objectives and Study Overview

This study aimed to establish a robust UHPLC method coupled with high-resolution Q-TOF mass spectrometry for baseline separation and unambiguous identification of enzymatically generated methylated flavonol isomers. Using kaempferol and quercetin as model substrates, characteristic MS/MS fragmentation rules and retention behavior were defined to assign methylation positions. Ten methylated derivatives were identified, including four not previously reported in plant sources.

Methodology

Recombinant flavonol OMTs from Camellia sinensis (CsFOMT-1, CsFOMT-2, CsCOMT) were expressed in E. coli and purified. Kaempferol and quercetin (750 μmol each) were incubated with S-adenosyl-L-methionine in Tris-HCl buffer (pH 7.5) at 35 °C for 1 h. Reactions were quenched, extracted with ethyl acetate, dried and reconstituted in methanol. Samples were filtered and separated by reversed-phase UHPLC on a C18 column using a binary gradient of 0.5% formic acid/2 mM ammonium formate in water (solvent A) and acetonitrile (solvent B). High-resolution Q-TOF MS in positive ESI mode recorded full scan and collision-induced dissociation spectra for structural assignment.

Instrumentation

• Agilent 1290 Infinity LC System: binary pump with degasser, autosampler with thermostat, thermostatted column compartment, diode array detector
• Agilent ZORBAX Eclipse Plus C18 column (2.1×100 mm, 1.8 µm) at 30 °C
• Mobile phase A: 0.5% formic acid/2 mM ammonium formate; B: acetonitrile; flow rate 0.2 mL/min; gradient: 12→25% B (0–5 min), 25→75% B (5–20 min), 75→12% B (20–25 min)
• Injection volume 5 µL; post time 3 min
• Agilent 6530 Accurate-Mass Q-TOF MS with dual JetStream ESI: drying and sheath gas 8 L/min at 300 °C, nebulizer 35 psi, capillary 3500 V, fragmentor 130 V; reference ions m/z 121.0509 and 922.0098

Main Results and Discussion

Characteristic retro Diels–Alder (rDA) fragments and neutral loss of methyl radical or methane enabled positional assignment of methoxy groups. Methoxy at 4′ and 7 positions produced prominent [M+H–CH3]+ ions, whereas methylation at C3 or C5 yielded [M+H–CH4]+ fragments. UHPLC achieved baseline separation of monomethyl and dimethyl isomers, with retention times correlating to calculated logP values. Kaempferol reactions generated 5-MeK, 3-MeK, 7-MeK and 3,7-DMK. Quercetin reactions yielded 3-MeQ, 4′-MeQ, 3′-MeQ, 7-MeQ and two dimethyl isomers (3,3′-DMQ, 7,3′-DMQ). Four compounds have not been previously reported in plants.

Benefits and Practical Applications

• Allows confident assignment of methylation sites without authentic standards
• Supports high-throughput profiling of flavonoid methylation in plant metabolomics
• Facilitates functional characterization of OMT enzymes and discovery of bioactive methylated flavonoids

Future Trends and Potential Applications

Extension of this approach to other flavonoid subclasses and integration with metabolomic databases will enrich mapping of plant methylation networks. Incorporation of ion mobility or orthogonal separations may further resolve complex isomeric mixtures. The workflow can guide metabolic engineering and accelerate discovery of novel bioactive compounds.

Conclusion

The combined UHPLC-Q-TOF MS strategy provides robust separation and accurate identification of methylated flavonol isomers based on diagnostic fragmentation patterns and retention behavior. This method enables rapid profiling of OMT products and can be broadly applied to plant metabolite characterization and functional enzyme studies.

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