Simultaneous Determination of Fat-Soluble Vitamins in Beverages Using a Triple Quadrupole Mass Spectrometer

Importance of the Topic

Assessing fat-soluble vitamins in food and beverages is critical due to their essential roles in vision, immunity, bone health, and antioxidant protection. These compounds cannot be synthesized by the human body and require accurate measurement for nutritional assessment and quality control.

Objectives and Study Overview

This study aimed to develop a rapid and sensitive method for simultaneous quantification of 13 fat-soluble vitamins—including provitamin A isomers—in beverage matrices. Key objectives included:

• Simultaneous detection of vitamins A (retinol, α- and β-carotene), D (ergocalciferol, cholecalciferol), E (α-, β/γ-, δ-tocopherols), and K (phylloquinone, menaquinones 4 and 7).
• Individual separation and quantification of structurally similar α- and β-carotene.
• Validation through calibration range, repeatability, and recovery experiments in commercial vegetable juices.

Methodology

Sample preparation involved adding anhydrous sodium sulfate to vegetable juice, extracting twice with ethanol, diluting 1:100, and a final centrifugation step. Alkaline hydrolysis was omitted given the lack of esterified carotenoids.

Used Instrumentation

• Shimadzu Nexera X3 ultra-high-speed liquid chromatograph.
• Shimadzu LCMS-8050 triple quadrupole mass spectrometer with APCI source.
• Kinetex XB-C18 column (100 mm × 3.0 mm, 1.7 µm).
• Mobile phases: methanol and ethanol gradient; flow rate 0.5 mL/min; column temperature 40 °C.
• MRM transitions optimized for each vitamin compound.

Results and Discussion

• Calibration was linear over 0.1–1000 ng/mL for most vitamins, with repeatability below 10% (n = 5).
• Distinct chromatographic separation and MRM transitions enabled reliable quantification of α- and β-carotene isomers.
• Recovery rates in two commercial vegetable juices ranged from 97% to 113% for α-carotene and 68% to 112% for β-carotene, indicating minimal ion suppression.
• Total analysis time was under 14 minutes per run, achieving high sensitivity and broad dynamic range.

Benefits and Practical Applications

• Simultaneous multi-vitamin analysis reduces analytical time and resource consumption.
• Simplified sample preparation without hydrolysis streamlines laboratory workflows.
• High sensitivity and precision support nutritional labeling, QA/QC, and research applications in food and beverage industries.

Future Trends and Potential Applications

Future developments may include expanding this approach to diverse food and supplement matrices, integrating automation for high-throughput screening, adopting greener extraction solvents, and combining quantitative vitamin profiling with metabolomic analyses for comprehensive nutritional insights.

Conclusion

A robust LC-MS/MS protocol was established for the rapid, precise, and simultaneous quantification of 13 fat-soluble vitamins, including individual analysis of α- and β-carotene. The method offers excellent sensitivity, reproducibility, and recovery in beverage samples without complex hydrolysis steps.

References

1. Ministry of Health, Labour and Welfare – Dietary Reference Intakes for Japanese 2020.