Original Paper

Analytical and Bioanalytical Chemistry

, Volume 403, Issue 5, pp 1433-1440

First online:

A rapid analytical method for cholecalciferol (vitamin D3) in fortified infant formula, milk and milk powder using Diels–Alder derivatisation and liquid chromatography–tandem mass spectrometric detection

  • Grant A. AbernethyAffiliated withFonterra Research Centre, Fonterra Co-operative Group Limited Email author 

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A method for analysing vitamin D3 (VD3, cholecalciferol) has been established and validated. This method is rapid and cost effective and is intended for use in quality control in the manufacture of fortified infant formulae and milk powders. Milk or reconstituted milk powder was solubilised in methanol and extracted in one step into isooctane, which was separated by centrifugation. A portion of the isooctane layer was then transferred, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione was added to derivatise VD3. The analyte was then re-extracted into a small volume of acetonitrile and analysed by reverse-phase chromatography. Detection was by triple quadrupole mass spectrometer using a selective transition, m/z 560 → 298. An internal standard, deuterium-labelled VD3, was used to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies. The method has been subjected to a single-laboratory validation and has been found to be linear, highly selective and accurate with respect to National Institute of Standards and Technology Standard Reference Material 1849, analyte spiking experiments and comparison with an LC–UV-based method. The repeatability standard deviation was 4.23 %. Significantly for routine laboratories, the method returns results within 2 h, generates minimal waste and minimises health and safety concerns to the analyst.


Vitamin D3 Cholecalciferol Diels–Alder derivatisation 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) Infant formula Milk