HILIC-MS/MS Analysis of Free Inositol Stereoisomers in Foods

Importance of the Topic

Accurate measurement of free inositol stereoisomers in foods is vital because these cyclitols play key roles in cellular signaling, metabolic regulation, and infant nutrition. An imbalance of isomers may contribute to metabolic and neurological disorders, making robust analytical methods indispensable for food quality control, nutritional research, and clinical nutrition studies.

Objectives and Study Overview

The primary goal was to adapt and validate a hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method, originally developed for dietary supplements, for application to complex food matrices including dairy and plant-based milks, grains, and infant formulas. Method improvements targeted enhanced matrix cleanup and interference reduction.

Sample Preparation and Methodology

• Protein precipitation: Samples (milk, cornmeal, formula) were treated with Carrez I and II reagents to remove proteins.
• Filtration and centrifugation: Supernatants were filtered (0.45 µm PVDF) and centrifuged at 20 000 × g, 4 °C.
• Internal standard addition: Final extracts were spiked with an isotopically labeled inositol standard.

Instrumentation Used

• LC: Arc™ Premier System with Quaternary Solvent Manager, Sample Manager, and Column Manager
• Column: ACQUITY UPLC™ BEH Amide, 1.7 μm, 2.1 × 150 mm
• MS/MS: Xevo™ TQ-S cronos triple quadrupole with electrospray ionization (ESI−)
• Software: MassLynx™ 4.2

Main Results and Discussion

• Method improvements: Incorporation of protein precipitation and MRM detection delivered recoveries of 89–118% (myo-inositol) and 96–109% (D-chiro-inositol) in food samples.
• Linearity and sensitivity: Calibration curves (2nd-order polynomial, 1/x weighting) exhibited R² ≥ 0.999; LOQs were 0.75 mg/100 mL for myo-inositol and 0.50 mg/100 mL for D-chiro-inositol in whole milk.
• Precision and ruggedness: Intra- and interday RSDs were ≤ 7.5%; column lot comparison by ANOVA showed no significant differences (p > 0.05).
• Specificity: MRM transitions and ion ratio monitoring confirmed analyte identity and eliminated interference from monosaccharides.

Benefits and Practical Applications

This validated HILIC-MS/MS method offers high specificity, accuracy, and precision for routine quantitation of free inositol stereoisomers in diverse food matrices. It supports food manufacturers, clinical laboratories, and research institutions in nutrient profiling and quality assurance.

Future Trends and Opportunities

• Expansion to all nine inositol stereoisomers and bound forms via hydrolysis steps.
• Automation and high-throughput workflows for large sample cohorts.
• Application to biomarker discovery in clinical studies and personalized nutrition research.

Conclusion

The optimized HILIC-MS/MS approach combining protein precipitation, robust HILIC separation, and triple quadrupole detection meets stringent performance criteria for analyzing free inositol stereoisomers in complex foods, enabling reliable data for quality control and nutritional investigations.

References

1. Ogasa K. et al. J. Nutr. Sci. Vitaminol. 21, 129–135 (1975)
2. Sanz M.L. et al. Food Chem. 84, 133–135 (2004)
3. Monnard I. et al. Anal. Bioanal. Chem. 412, 7871–7880 (2020)
4. AOAC Int. Appendix F, Official Methods (2016)
5. EU SANTE/11312/2021 v2026 guidelines