Determination of Fat-Soluble Vitamins in Foods Using Agilent Chem Elut S Extraction with LC/DAD and LC/MS/MS Triple-Quadrupole

Significance of the topic

Fat-soluble vitamins A, D2/D3, and E play critical roles in nutrition, immune function, and clinical diagnostics. Reliable quantification in complex food samples is essential for quality control, regulatory compliance, and nutritional labeling.

Objectives and study overview

This study aimed to develop a fast, robust, and sensitive method for simultaneous determination of retinol (vitamin A), cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), and α-tocopherol (vitamin E) in diverse food matrices. Applications included infant formula, egg, canned tuna, and mushrooms.

Methodology

• Sample pretreatment by alkaline saponification at 75–80 °C for 45 min to release free vitamins and remove lipids.
• Supported liquid extraction (SLE) using Agilent Chem Elut S 12 mL cartridges with hexane containing antioxidant (BHT) to simplify cleanup and reduce coextractives.
• Evaporation and reconstitution in methanol prior to analysis.
• Calibration by isotopically labeled internal standards for D vitamins and external calibration for A and E.
• Validation of specificity, linearity (R2 > 0.99), accuracy (recoveries 70–130%), precision (RSD ≤ 20%), and instrument limits of quantification.

Instrumentation

• Agilent 1290 Infinity II UHPLC system with multisampler, high-speed pump, column oven, and diode array detector (DAD).
• Agilent Poroshell 120 SB-AQ column (2.1 × 150 mm, 2.7 µm), 45 °C, flow 0.25 mL/min, formic acid in water/methanol gradient.
• Agilent 6470 triple quadrupole LC/MS with Jet Stream electrospray ionization in positive mode, dynamic MRM acquisition.
• Data processing with Agilent MassHunter workstation.

Main results and discussion

The SLE approach reduced sample preparation time by 30–50%, lowered matrix residue compared to liquid–liquid extraction, and provided high recoveries and reproducibility. LOQs were established at 0.5 ng/mL for D2/D3, 50 ng/mL for A, and 0.5 µg/mL for E, with signal-to-noise ratios > 10. Good agreement between native and spiked recoveries was achieved across all tested matrices.

Benefits and practical applications

• Streamlined workflow with fewer transfer steps and reduced solvent use.
• High-throughput capability for routine food testing and nutritional analysis.
• Enhanced matrix cleanup and consistent cartridge performance.
• Simultaneous quantification of multiple fat-soluble vitamins from a single extract.

Future trends and opportunities

Further automation of SLE and sample handling can increase throughput. Integration with high-resolution mass spectrometry and improved data analytics will extend method applicability to emerging vitamins and bioactive lipids. Miniaturized and portable LC/MS platforms may enable on-site monitoring in food production and field studies.

Conclusion

The presented UHPLC-DAD-MS/MS method with Agilent Chem Elut S extraction offers a rapid, sensitive, and reproducible solution for comprehensive analysis of fat-soluble vitamins in complex food matrices, supporting regulatory and nutritional testing needs.

References

• Reddy P., Jialal I. Fat-Soluble Vitamins. StatPearls. 2022.
• Devries J.W., Silvera K.R. Determination of Vitamins A (Retinol) and E (Alpha-Tocopherol) in Foods by Liquid Chromatography: Collaborative Study. J. AOAC Int. 2002;85:424–434.
• Gill B.D., Indyk H.E. Analysis of Vitamin D2 and Vitamin D3 in Infant and Adult Nutritional Formulas by LC-MS/MS: A Multilaboratory Testing Study. J. AOAC Int. 2018;101:256–263.