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.
The Journal of steroid biochemistry and molecular biology 2024 ; 245: 106633.
Jones KS, Meadows SR, Billing G, Koulman A, Prentice A
DOI : 10.1016/j.jsbmb.2024.106633
PubMed ID : 39547286
PMCID :
URL : https://linkinghub.elsevier.com/retrieve/pii/S096007602400181X
Abstract
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 D, 0.025 nmol/L for vitamin D and 0.1 nmol/L for 25(OH)D and 25(OH)D. Within- and between-day imprecision was <12 % for all analytes except vitamin D (14 %). From all data combined, geometric mean (-/+ 1 SD) vitamin D concentration was 0.94 (0.43, 1.80) nmol/L and for 25(OH)D 0.32 (0.23, 0.42) nmol/L. The within-person (intra-individual) coefficient of variation (%CV) was 32 % and 12 % for vitamin D and 25(OH)D, respectively. Between-person (inter-individual) %CVs were 89 % and 34 % for vitamin D and 25(OH)D, 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)D 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.
Lay Summary
The World Health Organisation (WHO) recommends exclusive breast-feeding for the first six months of life, but there is a lack of information on the micronutrient content of human milk, including vitamin D which is required for healthy growth and development. The lack of data is partly due to the limited availability of reliable measurement methods. This paper describes a sensitive and accurate liquid chromatography tandem mass spectrometry method to measure human milk vitamin D. We used the method to measure the vitamin D content of human milk in samples collected from women in rural Gambia. Samples were collected on two consecutive days from 23 women to find how vitamin D concentration was different between days and between women. We showed that vitamin D3 (cholecalciferol) levels were higher and more variable in concentration than its metabolite 25-hydroxyvitamin D3 (calcidiol). The concentration of vitamin D in these samples was low and would only contribute approximately one tenth of the recommended daily intake for infants. This study contributes to the current understanding of human milk vitamin D content and the method provides a platform for future work in this area.