Simultaneous Determination of Vitamin D2 and D3 in Milk

Význam tématu

The quantification of vitamins D2 and D3 in milk products is crucial for nutritional labeling and public health. Vitamin D supports calcium and phosphorus metabolism and exists as ergocalciferol in plants and cholecalciferol in animal-derived foods. Accurate measurement at low fortification levels helps prevent deficiency and ensures compliance with food regulations.

Cíle a přehled studie / článku

This study presents a sensitive and reproducible LC/MS/MS method for simultaneous determination of vitamin D2 and D3 in fortified milk. Using isotopically labeled internal standards and derivatization, the aim was to achieve detection limits below 1 ng/g and high repeatability across diverse milk matrices common in commercial testing laboratories.

Použitá instrumentace

• Agilent 1290 Infinity II High Speed Pump
• Agilent 1290 Infinity II MCT and vial sampler
• Agilent 6470 Triple Quadrupole LC/MS with AJS electrospray ion source

Použitá metodika

Sample preparation involves:

• Weighing 2.0 g of milk powder and spiking with deuterated internal standards and pyrogallol antioxidant.
• Saponification with 50% ethanolic KOH at 70 °C for one hour to liberate vitamin D from fats.
• Liquid-liquid extraction using isooctane, followed by aqueous washing.
• Derivatization with PTAD in acetonitrile under dark conditions to enhance MS sensitivity.
• Final dilution with water and acetonitrile before syringe filtration.

Chromatographic separation is performed on a Poroshell 120 EC-C18 column at 40 °C with a gradient from 50:50 water/formic acid to 100% methanol at 0.3 mL/min. Detection uses positive ion MRM transitions specific for D2, D3 and their labeled analogues.

Hlavní výsledky a diskuse

The method demonstrates:

• Limit of quantitation of 0.4 ng/g for both vitamins, with clear baseline separation of D2 and D3 peaks.
• Excellent linearity from 0.4 to 40 ppb (R2 > 0.999 for both analytes).
• Repeatability below 2.5% RSD at a 10 ppb spike level across five milk samples.
• Recoveries averaging 98% for vitamin D2 and 85% for vitamin D3 under fortification conditions.

Matrix blanks showed no interfering signals at the target retention times, confirming method specificity in complex dairy matrices.

Přínosy a praktické využití metody

This LC/MS/MS approach provides high sensitivity and robustness for routine analysis in quality control and research laboratories. The use of isotopic standards and derivatization ensures accurate quantitation even in variable fat-content samples, supporting regulatory compliance and nutritional assessment of fortified foods.

Budoucí trendy a možnosti využití

Future developments may include automated sample handling to increase throughput, application to other fat-soluble vitamins, and integration into multi-vitamin screening workflows. Advances in high-resolution mass spectrometry and data processing could further improve detection limits and reduce analysis time.

Závěr

A validated Agilent 1290 Infinity II–6470 LC/TQ method enables reliable simultaneous determination of vitamins D2 and D3 in milk at low concentration levels. Its high sensitivity, reproducibility, and specificity make it suitable for routine fortification monitoring and regulatory testing.

Reference

• Gill P, Patel A, Steiner G et al. Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single Laboratory Validation, First Action 2016.05. Journal of AOAC International. 2017;100(1).
• Agilent Technologies. Estimation of Fat-soluble Vitamins, Ergocalciferol, and Cholecalciferol in Edible Oil. Application note 5991-8328EN. 2017.