Determination of Vitamin D and Previtamin D in Food Products

Importance of the Topic

Vitamin D is a critical fat soluble nutrient that supports calcium and phosphate balance and promotes healthy bone mineralization. Regulatory agencies worldwide now require accurate labeling of vitamin D content in foods and supplements. Traditional analytical approaches often overlook the reversible thermal isomer previtamin D, leading to potential errors when samples experience heat during processing or storage. A robust analytical method that quantifies both vitamin D and previtamin D is essential for reliable nutritional labeling and quality control.

Objectives and Study Overview

• Develop a streamlined LC MS workflow to measure vitamin D3, vitamin D2 and their thermally produced isomers previtamin D3 and previtamin D2 in food matrices
• Establish calibration and quantitation schemes that incorporate previtamin D to improve accuracy over standard methods
• Validate the method with reference materials and real food samples including dairy powders, infant formulas, oatmeal, chocolate and fish oil

Methodology and Instrumentation

The method combines saponification, liquid liquid extraction, derivatization with PTAD and UPLC separation followed by triple quadrupole MS detection in positive ESI mode. Previtamin D isomerization is controlled by heating or room temperature treatments to determine relative response factors.

• UPLC System ACQUITY UPLC H Class
• Column ACQUITY UPLC BEH C18 2.1 x 50 mm 1.7 µm maintained at 40 °C
• Mobile Phase A water 0.1 percent formic acid, B acetonitrile 0.1 percent formic acid, gradient run time 8.5 minutes
• Mass Spectrometer Xevo TQ S micro triple quadrupole, ESI positive, MRM transitions for PTAD derivatives
• Software MassLynx version 4.1

Main Results and Discussion

Retention times for vitamin D PTAD derivatives and previtamin D PTAD derivatives were well separated at 3.50 and 3.67 minutes. Calibration plots for D3 and D2 showed excellent linearity (R2 > 0.997) over 1 to 500 ppb. Limits of quantitation in oatmeal were 0.01 mg/kg for D3 and 0.02 mg/kg for D2. Using NIST 1849a reference material, the method achieved 102.6 percent accuracy and 2.4 percent RSD. Spiking experiments in infant formula and oatmeal yielded recoveries between 98 and 117 percent. Comparison of quantitation strategies demonstrated that including previtamin D reduced bias from 11–12 percent down to 1–2 percent under different heating histories.

Benefits and Practical Application

• Eliminates quantitation errors caused by thermal isomerization during sample handling or storage
• Delivers low detection limits suited for regulatory compliance and nutrition labeling
• Applicable across diverse food matrices including dairy, infant formulas, cereals, chocolate and oils
• Improves data reliability for quality control laboratories and research studies

Future Trends and Applications

Automation of saponification and derivatization steps may increase throughput. High resolution mass spectrometry and ambient ionization techniques could further simplify sample preparation. Expanding the approach to additional vitamin D metabolites and related compounds will deepen understanding of vitamin D bioavailability in complex foods. Standardization of response factor determination may support interlaboratory reproducibility.

Conclusion

This study presents an enhanced LC MS method that quantifies both vitamin D and previtamin D in food products. By incorporating the isomeric contribution, the approach delivers improved accuracy, reproducibility and regulatory compliance. Its robust performance across multiple matrices makes it a valuable tool for food testing laboratories and quality assurance programs.

Reference

1. Vitamin D Fact Sheet for Health Professionals National Institutes of Health
2. Holden JM and Lemar LE American Journal of Clinical Nutrition 2008 88 suppl 551S 553S
3. Food Labeling Revision of the Nutrition and Supplement Facts Labels FDA Federal Register Vol 81 No 103 May 27 2016
4. AOAC Official Method 2016.05 Official Methods of Analysis AOAC International 20th Edition 2016
5. Keverling Buisman JA Hanewald KH Mulder FJ Roborgh JR Keunig KJ Journal of Pharmaceutical Sciences 1968 57 1326 1329