A Novel Methodology for the Quantification of B-Vitamers in Breast Milk

Abstract

With this report we present the development, validation and application of an analytical methodology for the quantification of 18 water soluble vitamers and secreted or biological forms in breast milk. On a relatively low amount of breast milk (200 µL), we applied isotope dilution-based sample preparation based on a combination of enzymatic treatment and protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Compounds separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. To perform the quantification of 18 water soluble vitamers, procured pooled breast milk was used to build matrix-matched calibration curves, as labelled internal standards were not available for each vitamer. The analytical approach has been validated according to the EMA guidelines. The overall performance of the method was considered adequate, with 0.3-28.3% and 0.9-32.6% intra and inter-day precision respectively and averaged accuracy reaching 92.2-107.5%. In addition, performed freeze/thaw stability studies showed the potential degradation of some vitamers. We therefore recommend particular attention in sample collection with rather having dedicated aliquots with small volumes. The feasibility of this analytical approach has been evaluated by quantifying various breast milk samples that were procured from an external supplier. The main forms found in breast milk were thiamine monophosphate for B 1 , flavin adenine dinucleotide for B 2 , nicotinamide for B 3 , pyridoxal for B 6 and 5-methyl tetrahydrofolic acid for B 9 . In addition, we newly reported nudifloramide as B 3 form present in breast milk. With this analytical approach, it will give more confidence to provide a comprehensive assessment of the presence of water soluble vitamins in breast milk. This will enable the accurate evaluation of the nutritional requirements of infants.

A Rapid Chemiluminescence Assay for Measurement of Folate in Small Volumes of Breast Milk

“…First, we only used 5-MeTHF as an internal standard, whereas one cannot exclude that other bioactive forms exist in breast milk, such as folic acid found in supplemented food. Indeed, results with LC-MS/MS suggest that this form could be present in significant amounts in breast milk [10]. Moreover, we were unable to identify the reason for and nature of an unexpected small increase in folate signal when undiluted samples were heated to denature FBP.…”

“…A previous report suggested that microbiological assays might lead to measurement of overestimated folate levels in breast milk [11]. We thus chose instead to compare the presented method with an LC-MS/MS method recently developed in house [10]. It is important to notice that this methodology detects and quantifies two major folate forms with vitamin B 9 activity, namely 5-MeTHF and folic acid.…”

“…The LC-MS/MS method quantifies 5-MeTHF with an accuracy of 99% with an inter-day precision of 4.9 percent. For folic acid, accuracy is 105.8% [10]. A series of 28 randomly selected breast milk samples was analyzed with our FOLA method and with the LC-MS/MS methodology in parallel.…”

“…Some studies have used high performance liquid chromatography (HPLC) to quantify folates in breast milk [10,11,12,13]. With this technique, typically 5-MeTHF and folic acid are measured, while bacteriological assays likely quantify a larger range of folate forms (including mono- and polyglutamates) but without distinguishing individual forms.…”

“…For validation purposes, we compared values with those obtained in parallel using a recently validated LC-MS based methodology developed in-house. [10]…”