A black box illuminated: specific detection and quantification of vitamin D3 in small volume human milk samples using nanoLC‐ESI/MS/MS (623.8)

Abstract

Breastfeeding infants are at particular risk for vitamin D deficiency with human milk as their primary source of vitamin D. Serum 25‐OH‐D is typically used to characterize maternal and infant vitamin D deficiency. However, direct measurement of vitamin D in human milk is critical to illuminate the black box representing maternal vitamin D contribution to the infant during feeding. Here we have developed a sensitive and specific method to quantitate low femtomolar levels of vitamin D3, a common form in human milk, from an individual donor using 1 mL of human milk. The method combines initial saponification of human milk with liquid‐liquid extraction, followed by UPLC clean‐up, derivatization, and nanoLC‐MS/MS analysis. Deuterated D3 was used as internal standard. Full product ion spectra with accurate masses (±20 ppm) were obtained where MRM transitions of m/z 560.385 ‐> 298.119 and 563.404 ‐> 301.137 were used for quantification. Six milk samples from 3 mothers at two lactational stages, 2 weeks and 2 months, were analysed to assess method performance and give initial insight into vitamin D3 over lactation. Vitamin D3 concentrations increased from 0.09 ‐ 2.1 pmole/mL in 2‐week milk to 1.3 ‐ 2.7 pmole/mL in 2‐month milk. Milk from two of the mothers showed an approximate 10‐fold increase over the 6 weeks between sampling, while the third mother had higher initial values of D3 with a modest 30% increase between 2 weeks and 2 months.