Vitamin B12 quantification in human milk – Beyond current limitations using liquid chromatography and inductively coupled plasma – Mass spectrometry

•New method for vitamin B12 quantification in human milk based on cobalt detection.•Method validation demonstrates its reliability at levels as low as 40 pmol/L.•No difference is seenbetween microbiological assay and the newly developed method.•Method can be used to determine vitamin B12 in population observational studies. Vitamin B12 plays a key role in human biological functions and is vital in the neurological development of infants. The assessment of the vitamin B12 intake in exclusively breastfed babies depends on the reliability of its determination in milk. In this report, we present a new accurate and robust method for quantification of vitamin B12 in human milk. A highly specific sample preparation is applied, associated with chromatographic separation and detection by ICP-MS. Excellent sensitivity and accuracy are reported, with recovery values well within acceptability limits (80–120%), within- and between-day variability are lower than 10% and 15% respectively. Strong correlation with a microbiological assay was observed (r2 = 0.9) within the validation range (40–1000 pmol/L, corresponding to 54 to 1355 ng/L). The method can be used to routinely monitor vitamin B12 in clinical or population observational studies, determine infant’s intake or assess efficacy of mother’s supplementation.