Analysis of Free Inositol Stereoisomers in Dietary Supplements by Hydrophilic Liquid Chromatography using the Arc™ Premier System and ACQUITY™ QDa™ II Mass Detector

Significance of the topic

Inositol stereoisomers play vital roles in metabolic regulation and hormonal signaling. Myo-inositol and D-chiro-inositol are key components in nutraceuticals used to manage polycystic ovary syndrome, insulin resistance, and gestational diabetes. Accurate quantification of these isomers in dietary supplements is essential for ensuring product quality, efficacy, and regulatory compliance.

Objectives and Study Overview

This study aimed to develop a robust hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method for the simultaneous separation and quantification of seven free inositol stereoisomers in dietary supplements, utilizing the Waters Arc™ Premier System coupled with an ACQUITY™ QDa™ II Mass Detector.

Methodology and Instrumentation used

Standard and Sample Preparation:

• Prepared individual and mixed standard solutions of allo-, muco-, epi-, D-chiro-, neo-, myo-, and scyllo-inositol with deuterated myo-inositol-d6 as internal standard.
• Extracted supplement powders by aqueous dissolution, dilution, filtration, and conducted spiking for recovery assessment.

Chromatographic Conditions:

• Column: ACQUITY UPLC BEH Amide (1.7 µm, 2.1×150 mm) at 25 °C.
• Mobile Phase A: 90:10 ACN/water with 0.01% NH₄OH; B: 50:50 ACN/water with 0.01% NH₄OH and 20 mM NH₄HCO₃.
• Gradient elution, flow rate 0.35 mL/min, 23 min runtime, 2 µL injection volume.

Mass Spectrometry:

• ACQUITY QDa II Mass Detector in ESI- mode, 0.5 kV capillary voltage, 600 °C probe temperature, 7 V cone voltage.
• Selected ion recording at m/z 179 for analytes and 185 for internal standard.

Main Results and Discussion

The optimized method resolved all seven inositol stereoisomers in 23 minutes with a minimum resolution of 1.6 for the critical allo-/muco-pair. Calibration curves exhibited linearity (R² ≥ 0.998) over 0.05–15 µg/mL, with LOQs of 0.09–0.9 ppm. Precision (RSD < 1.1%) and accuracy (recoveries 98–106%) were demonstrated for myo- and D-chiro-inositol. Potential monosaccharide interferences were effectively eliminated by SIR detection. Method robustness was enhanced by optimizing mobile phase additives and implementing routine column maintenance.

Benefits and Practical Applications of the Method

• Simultaneous quantification of seven inositol stereoisomers across diverse supplement matrices.
• High throughput with a 23-minute run time and straightforward sample preparation.
• Enhanced selectivity and sensitivity via SIR detection minimizing interferences.
• Robust performance with stable backpressure and reproducible separations.

Future Trends and Opportunities

The method can be extended to biological fluids for pharmacokinetic and clinical studies, and adapted for bound inositol forms through enzymatic hydrolysis. Integration with high-resolution MS and automated sample handling could further increase throughput and broaden applications in research and quality control laboratories.

Conclusion

A HILIC-MS method using the Arc Premier System and ACQUITY QDa II Mass Detector provides a rapid, accurate, and reliable solution for simultaneous analysis of free inositol stereoisomers in dietary supplements, supporting rigorous QC and regulatory requirements.

References

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