Establishment of micromethods for macronutrient contents analysis in breast milk

Abstract

Commercially available milk analysers were originally developed for use in the dairy industry, but they are now used to analyse macronutrient content of breast milk in clinical studies and routine care of the premature or very low birthweight (VLBW) infants. Due to the different composition of cow and breast milk, these devices need to be validated against reference methods before they can be used in daily routine. However, current reference methods require a sample volume of 30-100 mL to analyse fat, protein and lactose. It is not feasible to obtain this volume of milk for research purposes, especially from VLBW infants as lactation may be delayed or impaired and the limited volume of breast milk must be provided to the infant. To support validation of milk analysers in both clinical and research settings, the aim of this study is to establish and validate micromethods for precise macronutrient analysis in small volume of breast milk and conduct a feasibility study of the micromethods as a post-validation. Methods include a modified Mojonnier ether extraction (fat), elemental analysis (protein) and ultra-performance liquid chromatography-tandem mass spectrometry (lactose). We were able to downsize volumes required for analysis of fat, protein and lactose to 1 mL, 260 μL and 100 μL; corresponding coefficients of variation are 1.7, 1.8 and 2.3%, respectively. The presented methods allow for reliable and precise analyses of macronutrients in ≤1.5 mL of breast milk and will be used to validate milk analysers.

Microbiota at Multiple Body Sites during Pregnancy in a Rural Tanzanian Population and Effects of Moringa-Supplemented Probiotic Yogurt

“…Fat was extracted from the milk using Mojonnier ether extraction and then gravimetrically analyzed to get the total fat content (34). The protein content of the breast milk was determined as per the methods of Choi et al (34). The true protein content was determined by subtracting the nonprotein nitrogen from the total nitrogen and multiplying this by 6.25 (35).…”

“…The lactose content was measured as described by Fusch et al (33). Fat was extracted from the milk using Mojonnier ether extraction and then gravimetrically analyzed to get the total fat content (34). The protein content of the breast milk was determined as per the methods of Choi et al (34).…”

Target fortification of breast milk: levels of fat, protein or lactose are not related

“…The breast milk samples were collected on two different occasions to cover different time points of lactation and various gestational ages at the time of sample collection. The samples were obtained from two previous studies that aimed to investigate the feasibility to implement target fortification in clinical practice . The first set of samples (n = 120) was collected from 40 voluntary donors (7 term and 33 preterm pregnancies).…”

“…Analyses of milk samples were performed using validated micro‐methods (1.5 mL of total sample volume) . The methods were based on a modified ether Mojonnier extraction (1 mL) for fat, elemental analysis (260 μ L) for true protein and UPLC‐MS/MS (100 μ L) for lactose with corresponding CV's (coefficients of variation) of 1.7, 1.8 and 2.3%, respectively.…”

Promoting healthy growth and nutrition in preterm infants: A challenge for clinicians and researchers

“…2) showed that growth was closely related to milk intake in the target fortification group (r2 = 0.68) whereas it was not related in the standard fortification group (r2 = 0.02). During this project we have also established and validated micro-methods that enabled us to measure all macronutrient content of breast milk in 1.5 ml of sample volume [14]. We also established a new ultra performance liquid chromatography (UPLC)/ mass spectrometry (MS)/MS method for the rapid determination of lactose [15].…”