Simultaneous Analysis of Fat-Soluble Vitamins in Beverages Using Triple Quadrupole LC-MS/MS

Importance of the Topic

Fat-soluble vitamins A, D, E, and K are critical micronutrients for human health, playing key roles in vision, immune function, bone health, and antioxidant protection. Because they cannot be synthesized endogenously, accurate and sensitive measurement of these vitamins in dietary sources and beverages is essential for nutritional assessment, quality control, and research in food science and clinical nutrition.

Objectives and Study Overview

The study presents a rapid, simultaneous analytical method for quantifying thirteen fat-soluble vitamins—retinol, α- and β-carotene, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), α-, β/γ-, and δ-tocopherols, phylloquinone, menaquinone-4, and menaquinone-7—using triple quadrupole LC-MS/MS. Special focus was placed on resolving α- and β-carotene isomers and validating the method in commercial vegetable juice matrices.

Methodology and Instrumentation

Sample Preparation:

• Mix 1 g of vegetable juice with 5 g anhydrous sodium sulfate and ethanol.
• Shake and centrifuge at 4,000×g for 5 min; repeat extraction twice.
• Combine supernatants, dilute to 40 mL with ethanol, then dilute sample 1:100 and centrifuge at 15,000 rpm.

Chromatographic and Mass Spectrometric Conditions:

• UHPLC: Nexera X3 with Kinetex XB-C18 column (100 × 3.0 mm, 1.7 µm), 0.5 mL/min flow, 40 °C, gradient from 15% to 100% ethanol in methanol.
• MS/MS: Shimadzu LCMS-8050, APCI source, MRM transitions optimized for each vitamin, interface at 300 °C, heat block 200 °C.

Main Results and Discussion

The optimized method achieved baseline separation of α- and β-carotene, with calibration ranges from 0.1 to 1,000 ng/mL and repeatability RSDs below 10% at 1 ppb. Recovery experiments in two commercial vegetable juices yielded 96–103% for α-carotene and 97–112% for β-carotene, indicating minimal ion suppression and robust quantification across matrices. Concentrations measured aligned well with declared nutritional information.

Benefits and Practical Applications

This approach offers:

• Simultaneous quantification of 13 fat-soluble vitamins in a single run.
• High sensitivity and specificity via triple quadrupole detection.
• Elimination of time-consuming saponification, simplifying sample preparation.
• Wide dynamic range suitable for nutritional and QA/QC laboratories.

Future Trends and Opportunities

Further developments may include coupling with high-resolution mass spectrometry for untargeted vitamin derivatives, automating sample preparation and data processing, expanding to other food and biological matrices, and adopting greener solvents or microextraction techniques to enhance throughput and sustainability.

Conclusion

The described LC-MS/MS method on the LCMS-8050 and Nexera X3 platform provides a fast, reliable, and highly sensitive solution for comprehensive analysis of fat-soluble vitamins, including challenging isomer separations, enabling improved nutritional evaluation and quality control in the food and beverage industry.

Reference

Ministry of Health, Labour and Welfare. Dietary Reference Intakes for Japanese (2020).