Comprehensive Analysis of Primary and Secondary Metabolites in Citrus Fruits Using an Automated Method Changeover UHPLC System and LC/MS/MS System [LCMS-8050]

Importance of the Topic

The comprehensive profiling of both primary and secondary metabolites in foods underpins advances in nutritional science, quality control and plant breeding. In citrus fruits, metabolites such as organic acids, amino acids, sugars, carotenoids and flavonoids determine sensory attributes and functional properties. Automated coupling of multiple chromatographic methods expands analytical throughput and enables simultaneous detection of diverse compound classes.

Objectives and Overview of the Study

This study aimed to establish and demonstrate an automated LC/MS/MS workflow for simultaneous quantification of major primary metabolites (organic acids, amino acids, sugars) and secondary metabolites (carotenoids, flavonoids) in seven citrus cultivars. Emphasis was placed on comparing flesh versus peel composition and differentiating cultivars based on metabolic profiles.

Methodology and Instrumentation

Sample Preparation:

• Seven citrus types (mikan, ponkan, shiranui, amakusa, hassaku, buntan, hyuganatsu); flesh and peel separated, homogenized.
• Sequential extraction: water/ethanol for polar metabolites; hexane for non­polar carotenoids.
• Centrifugation, filtration, dilution tailored for organic acids, sugars, carotenoids.

Chromatography and Mass Spectrometry:

• UHPLC Nexera system with automated method changeover among three chromatographic setups.
• Columns: pentafluorophenyl (Discovery HS F5-3), C18 (Inertsil ODS-4), amino (Asahipak NH2P-50 2D).
• Mobile phases include aqueous formic acid, acetonitrile, acetonitrile/isopropanol.
• Triple quadrupole LCMS-8050 with electrospray ionization (ESI +/–) operated in MRM mode.
• Calibration curves defined across relevant concentration ranges for each analyte class.

Main Results and Discussion

Analytical Performance:

• MRM chromatograms confirmed robust separation and detection of >50 compounds in a single run sequence.
• Calibration showed linearity over 2–4 orders of magnitude for all analytes.

Flesh vs. Peel Comparison:

• PCA score plots clearly separated flesh and peel samples (PC1 30 %, PC2 17 %).
• Peel contained ~5× higher flavonoid levels; flesh contained ≥30× more carotenoids.
• Sugar composition differed despite similar total sugar content: flesh richer in fructose and glucose relative to sucrose.

Cultivar Differentiation:

• PCA of flesh metabolites distinguished mikan and hybrids, driven by elevated carotenoid levels.
• Quantification confirmed mikan hybrids had highest β-cryptoxanthin and β-carotene.

Benefits and Practical Applications

The automated LC/MS/MS workflow streamlines high­throughput metabolome profiling for food quality assessment, breeding selection and nutritional research. Simultaneous analysis reduces sample handling and instrument downtime, supporting routine QA/QC in food industry laboratories.

Future Trends and Possibilities

Emerging directions include:

• Expansion to broader metabolite coverage via high­resolution MS and data‐dependent acquisition.
• Integration with chemometric and machine learning tools for predictive quality assessment.
• Application to additional food matrices and process monitoring.
• Miniaturization and online extraction to further enhance throughput.

Conclusion

This work demonstrates a flexible, automated UHPLC-MS/MS platform capable of comprehensive profiling of primary and secondary metabolites in citrus fruits. The approach offers high sensitivity, reproducibility and throughput, facilitating advanced studies in food science and agriculture.

Instrument Used

• UHPLC Nexera system (Shimadzu) with automated method scouting.
• LCMS-8050 triple quadrupole mass spectrometer (Shimadzu) with ESI and MRM capability.
• Columns: Discovery HS F5-3; Inertsil ODS-4; Asahipak NH2P-50 2D.