Quantitative analysis of vitamin D and its main metabolites in human milk by supercritical fluid chromatography coupled to tandem mass spectrometry

Abstract

A novel method to quantitate vitamin D and its main metabolites (vitamin D3, vitamin D2, and their 25-hydroxy metabolites) in breast milk by supercritical fluid chromatography has been developed and fully validated. A small volume of sample (1 mL) is subjected to ethanolic protein precipitation and liquid-liquid extraction. Final extracts are derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione and vitamin D derivatives analyzed by supercritical fluid chromatography hyphenated to tandem mass spectrometry with atmospheric pressure chemical ionization. Multiple reaction monitoring is used for quantitation. Separation conditions were optimized using a gradient of methanol-water-ammonium formate into carbon dioxide. Make-up solvent was methanol containing ammonium formate. The quantitation limit reached levels as low as 50 pmol/L, with intra- and inter-day relative standard deviations lower than 15% and 20% for all analytes. Accuracy was evaluated by spiking experiments and was well within acceptability ratios (± 15%). The method was then applied to a subset of commercially available human milk samples. The newly developed method provides opportunities to determine the nutritional status of mother-infant dyads from a non-invasive measure, or for interventional or observational studies building knowledge on the composition of human milk. Graphical abstract.

Keywords: Human milk; Supercritical fluid chromatography; Tandem mass spectrometry; Vitamin D.

Graphical abstract

Fig. 1

Chemical structure of vitamin D parent forms and main circulating metabolite 25-hydroxy vitamin D, together with C3 inactive epimer

Fig. 2

Chromatographic separation of 25-hydroxy vitamin D-PTAD derivatives and their C3 inactive epimer on a Waters CSH^TM Fluoro Phenyl column (1.7 μm, 3 × 100 mm). Gradient of methanol/water (98:2, v/v) containing 10 mM ammonium formate as organic modifier on carbon dioxyde. Column temperature 45 °C, Atmospheric Back Pressure Regulator 128 bar. Make-up solvent was 10 mM ammonium formate in methanol, flow rate 0.4 mL/min

Fig. 3

Effect of injection volume on peak shape of vitamin D2 and vitamin D3 on a Waters Acquity® UPC2TM CSHTM Fluoro-Phenyl, 3.0 × 100 mm, 1.7 μm column. Gradient elution with methanol containing ammonium formate as organic modifier

Fig. 4

MRM chromatograms of a calibrating solution (2 ng/mL) and a spiked human milk (20 ng/100 mL)