Quantifying the human milk oligosaccharides 2’‐fucosyllactose and 3‐fucosyllactose in different food applications by high‐performance liquid chromatography with refractive index detection

Abstract

In recent years, production of biosynthesized human milk oligosaccharides (HMOs) has become scalable to industrial standards. As a result, infant formula fortified with 2'-fucosyllactose (2'-FL), the most abundant HMO in human breast milk, is now commercially available. 2'-FL and 3-fucosyllactose (3-FL), another abundant HMO, are thought to be beneficial for infant health and development. Products containing HMOs are projected to expand in the future, showing the need for robust, easily applicable analytical methods for the quantitative assessment of HMOs in different food applications. We present here a validated high-performance liquid chromatography method for the quantification of 2'-FL and 3-FL in whole milk, infant formula, and cereal bars. The sample preparation was simple dispersion and extraction of the sample. The samples were analyzed by hydrophilic interaction liquid chromatography with refractive index detection and a runtime of 19 min. The method had a high degree of linearity (R 2 > 0.9995) in the range 0.2 to 12 mg/mL. The recovery for 2'-FL was 88% to 105% and for 3-FL 94% to 112%. The limit of detection (LOD) for whole milk was 0.1 mg/mL for 2'-FL and 0.2 mg/mL for 3-FL. In infant formula and cereal bars, the LOD was 0.6 mg/g for both 2'-FL and 3-FL. To show the practical application of this method, it was successfully utilized in stability studies of 2'-FL and 3-FL in whole milk, UHT milk, and yoghurt. The method provides a means of simultaneous and robust quantification of 2'-FL and 3-FL in various food matrices with high accuracy and high reproducibility.Practical Application: 2'-Fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are two of the most abundant human milk oligosaccharides (HMOs) present in human breast milk. We present a fast HPLC method for the robust quantification of these two compounds in infant formula, whole milk, UHT milk, cereal bars, and yoghurt. This method can easily be set up by food producers and researchers to analyze the dosage of 2'-FL and 3-FL in their product or perform shelf life studies in different food applications.

Xây dựng và thẩm định phương pháp phân tích hàm lượng một số oligosaccharide từ sữa mẹ (HMOs) trong thực phẩm bổ sung bằng kỹ thuật sắc ký lỏng khối phổ (LC-MS/MS)

“…Kỹ thuật sắc ký lỏng với nhiều loại detector khác nhau đã và đang được sử dụng rộng rãi để xác định các thành phần trong thành phẩm và bán thành phẩm. Các hình thức phát hiện và định lượng cũng đa dạng từ phân tích trực tiếp bằng RI [5], ELSD [6], HPAEC [7][8][9] MS/MS [10] đến phân tích gián tiếp bằng PDA và FLD [7].…”

“…Dựa trên một số tài liệu tham khảo [5][6][7][8] và tính chất phân cực của nhóm HMOs [1], 02 loại cột HILIC và cột Amide đã được lựa chọn để khảo sát điều kiện tách trên sắc ký lỏng của các chất phân tích. Ngoài ra, do nền mẫu là TPBS dạng lỏng và dạng bột thường chứa hàm lượng lớn protein, lipid, vitamin, khoáng chất,... bước chiết và làm sạch mẫu trước khi phân tích trên LC-MS/MS là cần thiết.…”

The Impact of Dietary Fucosylated Oligosaccharides and Glycoproteins of Human Milk on Infant Well-Being

“…Moreover, recent reports [16,157] show that interest in the addition of fucosylated oligosaccharides, mainly 2 -FL, is growing not only for infant formula, but also in highly specialized food preparations that can be used in the clinic and which are extremely important for the health of the general public. Additionally, to meet the needs, new techniques have been developed that allow the determination of fucosylated milk oligosaccharides such as 2 -FL and 3-FL not only in breast milk, but also to control levels in commercially available infant formulas [164]. Such a modern analytical approach also has potential application to carry out stability tests in various food applications.…”

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control

“…Furthermore, conventional oligosaccharide sample preparation techniques-such as labeling-required overnight steps that were undesirable during process control applications in biotechnology settings. One of the most commonly used fluorescent labels, APTS, provided both excellent fluorescent characteristics and the necessary charge for the electromigration with as short as 1-h derivatization time, i.e., readily suitable for routine process analytical applications [35][36][37][38][39][40].…”