Improved Simple Sample Pretreatment Method for Quantitation of Major Human Milk Oligosaccharides Using Ultrahigh Pressure Liquid Chromatography with Fluorescence Detection

Abstract

Human milk oligosaccharides (HMOs) maintain and promote infant health. Most of the current methods for HMOs quantitation require labor-intensive and time-consuming steps for sample preparation. This study presents two very simple and easy-to-operate pretreatment methods, requiring either ultrafiltration or centrifugation to separate free oligosaccharides from whole fat human milk and other milk matrix before oligosaccharides labeling for quantifying HMOs using ultrahigh pressure liquid chromatography with fluorescence detection. A single chromatography run quantified 15 sialylated and neutral HMOs with high sensitivity (with an LOD less than 8 pg for all HMOs tested: about 1 pg for 2′-fucosyllactose, 3-fucosyllactose, 4′-galactosyllactose, 3′-galactosyllactose, and 6′-galactosyllactose) and good linearity with coefficient of correlation above 0.999. Accuracy and precision were satisfactory for both pretreatment methods. Overall, the centrifugation pretreatment was efficient and reliable for samples with high levels of oligosaccharides, and the ultrafiltration pretreatment was especially suitable for samples with low oligosaccharide abundance.

Changes in HMO Concentrations throughout Lactation: Influencing Factors, Health Effects and Opportunities

“…The data from these studies conducted in the Chinese population varied considerably. The results reported by Elwakiel et al, 2018 [ 1 ] were over twice as high as the results reported by Huang et al, 2019 [ 24 ] and Ma et al, 2018 [ 25 ] at each stage of lactation ( Table 3 ). This may be explained, at least in part, by the different analytical methodologies and the times of breast milk collection used to analyze HMOs.…”

“…Given this study did not include the North region of China (e.g., Inner Mogolia/Hohhot), it is still unknown whether there may be any differences in the concentrations and composition of HMOs between different ethnic Chinese groups or populations with different dietary patterns (which have not been reported in the studies). It is important to note that the study by Austin et al, 2016 [ 32 ] only reported ten individual HMOs, therefore the total HMOs based on the sum of the ten individual HMOs were lower than any of the results reported by Elwakiel et al [ 1 ], Huang et al [ 24 ] and Ma et al [ 25 ] and cannot be directly compared ( Table 3 ).…”

“…This may be explained, at least in part, by the different analytical methodologies and the times of breast milk collection used to analyze HMOs. The study conducted by Elwakiel et al, 2018 [ 1 ], used capillary electrophoresis-laser-induced fluorescence (CE-LIF), while the study reported by Ma et al, 2018 [ 25 ] used HPLC-MRM-MS, and the study by Huang et al, 2019 [ 24 ] used the UHPLC-FL method.…”

“…The different geographic regions and potentially ethnic diversities in China where the milk samples were collected could also explain the variation in the concentrations of HMOs, e.g., the breast milk samples in the study by Elwakiel et al, 2018 [ 1 ] were collected in Hohhot (North of China), in Beijing (Northeast) by Huang et al, 2019 [ 24 ] and in Guangzhou (South) by Ma et al, 2018 [ 25 ]. However, when breast milk samples were collected from 3 different cities in China (from Northeast to South: Beijing, Suzhou, Guangzhou) and analyzed within the same study by Austin et al, 2016 [ 32 ], no difference was observed in the HMO compositions.…”

Infant Formula Supplemented with Biotics: Current Knowledge and Future Perspectives

“…HMO 3′-GL is naturally present in human milk and was already isolated from human milk in 1988 [ 40 ]. Recently, a single chromatography run using an improved sample pre-treatment method and ultrahigh pressure liquid chromatography with fluorescence detection quantified 15 sialylated and neutral HMOs with high sensitivity, identifying 3′-GL and 6’-GL in colostrum, transitional, and mature human milk [ 108 ]. Human milk from mothers with preterm delivery revealed 3′-GL concentrations of 4–28.82 µg/mL (median 10.44) at 1 w postpartum, whereas concentrations ranged from 4–32.97 µg/mL (median 12.34) in colostrum and 4–20.73 µg/mL (median 4) at 8 w postpartum from mothers with term delivery [ 46 ].…”