The validation of an LC-MS/MS method for the quantification of vitamin D metabolites in human milk and their biological variability in Gambian women

Vitamin D is required for healthy growth and development, but data on human milk vitamin D content is limited. We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of vitamin D metabolites in human milk, and its application in samples collected on two consecutive days from women in rural Gambia. Vitamin D compounds were extracted from 1 mL of milk by liquid-liquid extraction and derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis by LC-MS/MS. The limit of quantification was 0.05 nmol/L for vitamin D₂, 0.025 nmol/L for vitamin D₃ and 0.1 nmol/L for 25(OH)D₂ and 25(OH)D₃. Within- and between-day imprecision was <12 % for all analytes except vitamin D₂ (14 %).

From all data combined, geometric mean (-/+ 1 SD) vitamin D₃ concentration was 0.94 (0.43, 1.80) nmol/L and for 25(OH)D₃ 0.32 (0.23, 0.42) nmol/L. The within-person (intra-individual) coefficient of variation (%CV) was 32 % and 12 % for vitamin D₃ and 25(OH)D₃, respectively. Between-person (inter-individual) %CVs were 89 % and 34 % for vitamin D₃ and 25(OH)D₃, respectively. There was no significant association between vitamin D metabolite concentrations and milk fat (creamatocrit). Mean vitamin D content of human milk as ARA averaged 42 IU/L with 25(OH)D₃ responsible for around two-thirds of the biological activity. In conclusion, this work describes a reliable LC-MS/MS method for quantification of vitamin D and 25(OH)D in low volumes of human milk providing a platform for future work. This study contributes to current understanding of variability of milk vitamin D content.