Method for detecting human milk oligosaccharides

Abstract

The application discloses a detection method of breast milk oligosaccharide. The application provides a detection method of breast milk oligosaccharide, which comprises the step of detecting a to-be-detected sample by using a capillary electrophoresis-laser-induced fluorescence-based detection method, wherein the running buffer used in the capillary electrophoresis-laser-induced fluorescence-based detection method comprises Tris-base, EDTA, urea, and more than 2% (m / V) and less than 4% (m / V) PVP and more than 5% (m / V) and less than 15% (m / V) boric acid. The detection method provided by the application can be applied to the separation and detection of acidic and neutral HMOs isomers in the to-be-detected sample (for example, breast milk and infant formula milk powder samples).

Description

Technical Field

[0001] This invention belongs to the field of human milk oligosaccharide detection, specifically relating to a method for detecting human milk oligosaccharides, and more specifically to a CE-LIF method for simultaneously separating and detecting the content of nine human milk oligosaccharides in dairy products. Background Technology

[0002] Human milk oligosaccharides (HMOs) are important bioactive compounds in breast milk, ranking as the third largest solid component after lactose and lipids, with a concentration typically ranging from 5 to 20 g / L. These complex carbohydrates play multiple roles in infant health, including promoting beneficial gut microbiota and regulating immune responses. Due to their unique functional properties, HMOs are increasingly being added to infant formula and dietary supplements to mimic the protective effects of breast milk. Currently, over 200 HMOs have been identified, with the structures of more than 140 elucidated. The structure of HMOs is mainly composed of five monosaccharide building blocks, with the core backbone being lactose (Lac) at the reducing end. Fucoidose (Fuc) or sialic acid (Neu5Ac) residues can be further introduced through different glycosidic bond linkages to form isomers with the same molecular weight but different structures. These isomers mainly include positional isomers, such as 2'-fucosylvose (2'-FL) and 3-fucosylvose (3-FL), and 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL); they also include linker isomers, such as lactose-N-tetrasaccharide (LNT) and lactose-N-neotetrasaccharide (LNnT). This high degree of structural complexity and diversity of isomers presents significant technical challenges to the analysis and detection of HMOs.

[0003] Currently, researchers have employed various analytical techniques to characterize HMOs, but their complex structures, lack of UV-absorbing groups, and the fact that some components are electrically neutral present unique challenges for analysis and detection. In recent years, liquid chromatography-mass spectrometry (LC-MS) has been increasingly widely used in HMO analysis, especially LC-MS methods using porous graphitized carbon as the stationary phase, which can achieve simultaneous quantitative analysis of neutral and acidic HMOs, significantly improving analytical throughput and structural resolution. However, this method still faces challenges in distinguishing structurally very similar linked isomers. Furthermore, traditional chromatographic methods generally use large amounts of organic mobile phase, generating waste liquid that not only increases operating costs but also reduces the environmental friendliness of the method. Overall, although existing analytical methods have made significant progress in HMO detection, how to achieve efficient separation of key isomers in complex matrices while simultaneously considering analytical throughput, sensitivity, and green chemistry requirements remains a pressing research challenge.

[0004] Given the wide concentration range of HMOs in breast milk, detection methods must possess both a broad linear dynamic range and high sensitivity. Capillary electrophoresis (CE), with its high separation efficiency, short analysis time, low sample consumption, and simple pretreatment, has become an effective supplementary platform to chromatographic methods. To address the challenge of optical detection of HMOs due to the lack of chromophores or fluorophores, laser-induced fluorescence (LIF) detection technology offers a solution. By derivatizing HMOs using fluorescent labels (such as trisodium 8-aminopyrene-1,3,6-trisulfonate, APTS), not only are the necessary charges provided for their migration in an electric field, but the detection sensitivity of CE is also significantly improved.

[0005] Specific buffer solutions are crucial for the separation efficiency of CE (ecological electrochemical chromatography). Reference 1 discloses that while conventional CE matrices (such as HR-NCHO) can separate LNT and LNnT, they cannot effectively distinguish between 2'-FL and 3-FL. Adding a background electrolyte containing borate provides higher separation through borate-diol complexation. The difference in the three-dimensional structure of 2'-FL and 3-FL leads to different complexing abilities with borate, resulting in a difference in apparent mobility and achieving effective separation. Furthermore, CE technology itself is characterized by miniaturization and low waste volume, making it more in line with the requirements of green analytical chemistry.

[0006] Although existing studies have used CE to detect HMOs, there are still limitations.

[0007] Poor separation results: Reference 2 developed a laser-induced fluorescence detection capillary electrophoresis (CE-LIF) method, but it was only used to analyze neutral sugars in breast milk. In this study, 300 mM borate buffer was used as the background electrolyte to separate five selected oligosaccharides and other sugars. Furthermore, the validation of this method was limited to linearity and sensitivity estimation. Reference 3 developed a capillary electrophoresis method with laser-induced fluorescence detection; however, this method only achieved the detection of three oligosaccharides (DSLNT, 3'-SL, and 6'-SL) in colostrum samples.

[0008] Existing instrument detection methods all involve complex reaction systems: at the separation level, there is currently no single CE condition that can simultaneously separate all key HMO isomers. Therefore, the same sample needs to be analyzed twice, using both conventional and borate-type CE matrices, to achieve comprehensive separation of HMO isomers. To date, there is no CE separation method with fluorescence detection that can be used to simultaneously separate and detect the major neutral HMOs and acidic HMOs composed of sialic acid.

[0009] References

[0010] Citation 1: Sarkozy D, Borza B, Domokos A, Varadi E, Szigeti M, Meszaros-Matwiejuk A, Molnar-Gabor D, Guttman A. Ultrafast High-Resolution Analysis of Human Milk Oligosaccharides by Multicapillary Gel Electrophoresis[J]. Food Chemistry, 2021, 341:128200。

[0011] Citation 2: Song J-F, Weng M-Q, Wu S-M, Xia Q-C. Analysis of neutralsaccharides in human milk derivatized with 2-aminoacridone by capillary electrophoresis with laser-induced fluorescence detection[J]. Analytical biochemistry, 2002, 304(1): 126-129。

[0012] Citation 3: Rusin M, , , Dobrowolska-Iwanek J, Huras H, , et al. Development of a capillary electrophoresis method with laser-induced fluorescence detection for the characterization of oligosaccharides in human milk[J]. Monatshefte für Chemie-Chemical Monthly, 2024, 155(8): 825-834。 Summary of the Invention

[0013] The problem the invention aims to solve

[0014] As mentioned earlier, existing CE-LIF technology has a key bottleneck: conventional CE matrices (such as commercially available HR-NCHO matrices) can separate LNT / LNnT, but cannot separate 2'-FL / 3-FL; although borate matrices can solve the 2'-FL / 3-FL separation problem, they can lead to LNT / LNnT co-migration, requiring two analyses of the same sample to cover the key isomers.

[0015] It can be seen that existing technologies are insufficient for the efficient simultaneous separation and accurate detection of nine HMOs in breast milk and infant formula, namely 6'-sialyl lactose (6'-SL), disialial lactose-N-tetrasaccharide (DSLNT), 3'-sialyl lactose (3'-SL), 3-fucosylvose (3-FL), 3,2'-difucosylvose (DFL), lactose-N-neotetrasaccharide (LNnT), 2'-fucosylvose (2'-FL), lactose-N-tetrasaccharide (LNT), and lactose-N-fucopentose I (LNFP I). There is an urgent need to develop new detection methods to overcome these limitations.

[0016] Solution for solving the problem

[0017] [1]. A method for detecting human milk oligosaccharides, wherein the method for detecting human milk oligosaccharides includes a step of detecting a sample to be tested using a detection method based on capillary electrophoresis-laser induced fluorescence, wherein the running buffer used in the detection method based on capillary electrophoresis-laser induced fluorescence contains Tris-base, EDTA, urea, and greater than 2% (m / V) and less than 4% (m / V) of PVP and greater than 5% (m / V) of boric acid.

[0018] [2]. According to the method for detecting human milk oligosaccharides described in [1], wherein the running buffer contains less than 15% (m / V) boric acid.

[0019] [3]. The method for detecting human milk oligosaccharides according to [1] or [2], wherein the running buffer contains 70-100 mmol / L Tris-base, 1-5 mmol / L EDTA and / or 2-10 mol / L urea.

[0020] [4]. The method for detecting human milk oligosaccharides according to any one of [1]-[3], wherein, before the step of detecting the sample to be tested using the detection method based on capillary electrophoresis-laser induced fluorescence, a step of pretreatment of the sample to be tested is further included; optionally, the pretreatment step includes a sample purification step and / or a sample derivatization step.

[0021] [5]. According to the method for detecting human milk oligosaccharides described in [4], in the sample purification step, the sample to be tested is purified by alcohol precipitation and solid phase extraction column; and / or, in the sample derivatization step, the sample to be tested is derivatized by reductive amination derivatization method.

[0022] [6]. The method for detecting human milk oligosaccharides according to any one of [1]-[5], wherein, in the step of detecting the sample to be tested using a detection method based on capillary electrophoresis-laser induced fluorescence, the capillary electrophoresis is performed at a voltage of not less than 15 kV, preferably at a voltage of not less than 20 kV.

[0023] [7]. The method for detecting human milk oligosaccharides according to any one of [1]-[6], wherein, in the step of detecting the sample to be tested using a detection method based on capillary electrophoresis-laser induced fluorescence, the capillary in the capillary electrophoresis is operated at a temperature greater than 25 °C, preferably at a temperature less than 35 °C, and / or, the sample pan is maintained at a temperature of 10-25 °C.

[0024] [8]. The method for detecting human milk oligosaccharides according to any one of [1]-[7], wherein the capillary is a chemically inert and hydrophobic capillary; preferably, the capillary is a capillary with a fluorocarbon polymer coating.

[0025] [9]. The method for detecting human milk oligosaccharides according to any one of [1]-[8], wherein the sample to be tested includes milk and / or dairy products.

[0026]

[10] . A method for detecting human milk oligosaccharides according to any one of [1]-[9], wherein the human milk oligosaccharides include neutral human milk oligosaccharides and acidic human milk oligosaccharides composed of sialic acid; optionally, the human milk oligosaccharides include at least one of lactose-N-fucopentose I (LNFP I), lactose-N-tetrasaccharide (LNT), 2'-fucosyllactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), 3,2'-difucosyllactose (DFL), disialyllactose-N-tetrasaccharide (DSLNT), 3-fucosyllactose (3-FL), 3'-sialic acid lactose (3'-SL) and 6'-sialic acid lactose (6'-SL).

[0027] The effects of the invention

[0028] The detection method provided by this invention can be applied to the separation and detection of acidic and neutral HMO isomers in samples to be tested (such as breast milk and infant formula samples).

[0029] Compared with the prior art, the detection method of the present invention has the following advantages.

[0030] In some embodiments, this invention provides a capillary electrophoresis method and its running buffer for the separation and quantification of nine human milk oligosaccharides in breast milk and infant formula, solving problems such as low separation efficiency, severe peak overlap, and the need for double analysis of the same sample in existing methods. The running buffer used in this detection method contains PVP reagent, which causes polymer chains to intertwine, forming transient sieve pores that effectively separate oligosaccharide molecules, thereby improving the separation degree of human milk oligosaccharides.

[0031] In some embodiments, the running buffer used in the detection method of the present invention affects the interaction between the analyte and the buffer by adjusting the amount of boric acid added, thereby reducing sample diffusion. Especially in scenarios with narrow separation bandwidth, it can significantly improve peak sharpness and resolution. On the other hand, the unique chemical properties of boric acid enable it to form complexes with analytes with cis-diol structures (such as sugars and some polysaccharide-modified biomacromolecules), changing the effective charge / size ratio of these analytes and thus adjusting their electrophoretic migration rate, achieving selective separation of components that are difficult to separate by traditional methods.

[0032] In some embodiments, the running buffer used in the detection method of the present invention achieves the separation and quantification of at least nine human milk oligosaccharides by combining specific amounts of PVP and boric acid.

[0033] In some preferred embodiments, the selection of μSIL-FC coated capillary tubes, the adjustment of key electrophoresis parameters, and / or the combination of fluorescence detection and sample derivatization in the detection method of the present invention can achieve efficient separation of at least nine human milk oligosaccharides in breast milk and infant formula. Compared with traditional HR-NCHO matrix and borate-containing matrix methods, the present invention shows significant advantages in separating human milk oligosaccharides with similar structures (such as LNT / LNnT, 2'-FL / 3-FL), and is suitable for scenarios such as breast milk nutrient analysis, infant formula quality control and label compliance verification, and component detection of oligosaccharide functional foods, possessing good versatility, stability, and promotional value.

[0034] In some preferred embodiments, the precipitation by supercooled ethanol and the purification by the amino column involved in the detection method of the present invention can effectively remove proteins, lactose, and other potentially interfering substances from the sample to be tested (e.g., breast milk or infant formula). Washing with an aqueous solution containing a specific proportion of acetonitrile maximizes lactose removal, retains HMOs, and prevents low-abundance HMO peaks from being masked by interference. Attached Figure Description

[0035] Figure 1Electrophoretic spectra of mixed standards under different PVP concentrations (2%, 3%, 4%) in separation buffer.

[0036] Figure 2 Electrophoretic spectra of mixed standards under different boric acid concentrations (1%, 5%, 10%) in separation buffer.

[0037] Figure 3 Electrophoretic spectra of mixed standards under different separation voltages (15, 20, 30 kV).

[0038] Figure 4 Electrophoretic spectra of mixed standards under different separation temperatures (20, 25, 30, 35 ℃).

[0039] Figure 5 Linear range of 9 human milk oligosaccharide mixed standards.

[0040] Figure 6 Electrophoretic spectra of breast milk samples.

[0041] Figure 7 Electrophoretic spectra of infant formula samples. Detailed Implementation

[0042] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0043] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0044] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0045] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0046] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0047] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0048] Methods for detecting human milk oligosaccharides

[0049] In some aspects of this invention, a method for detecting human milk oligosaccharides is provided, the method comprising the step of detecting a sample using a capillary electrophoresis-laser induced fluorescence detection method, wherein the running buffer used in the capillary electrophoresis-laser induced fluorescence detection method comprises Tris-base, EDTA, urea, and 2.5-3.5% (m / V) PVP and greater than 5% (m / V) boric acid.

[0050] The method for detecting human milk oligosaccharides provided by this invention can separate and quantify at least nine types of human milk oligosaccharides, solving the problems of low separation efficiency, severe peak overlap, and double analysis of the same sample in existing methods.

[0051] Sample source

[0052] In the detection method provided by the present invention, there are no special restrictions on the test sample. For example, it can be milk, dairy products or any other sample containing human milk oligosaccharides, such as food samples.

[0053] In this invention, "milk" refers to the liquid produced by the mammary glands of mammals, such as human (breast milk), cow (e.g., dairy cow), goat, sheep, or camel.

[0054] In this invention, "dairy products" refers to any food in which one of its main ingredients is based on milk.

[0055] In this invention, there are no particular restrictions on the source of "dairy products," which can refer to food products produced by animals such as cattle, goats, sheep, yaks, horses, camels, and other mammals.

[0056] Examples of dairy products include low-fat milk (e.g., 0.1%, 0.5%, or 1.5% fat), nonfat milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, skim milk products, high-fat products, condensed milk, fresh cream, cheese, ice cream and dessert products, probiotic beverages, or probiotic yogurt-type beverages. "Milk powder" refers to artificial dairy products made by evaporating milk to dryness. In some preferred embodiments of the invention, the samples in the detection method are derived from cow or sheep milk powder products, as well as breast milk.

[0057] Preprocessing

[0058] In the detection method of the present invention, before the sample to be tested is detected by the detection method based on capillary electrophoresis-laser induced fluorescence, a pretreatment step of the sample to be tested is included.

[0059] In some implementations, the pretreatment step includes a sample purification step and a sample derivatization step.

[0060] Sample purification steps

[0061] In some embodiments, the sample purification step of the detection method provided by the present invention reduces the presence of proteins, lactose, salts and other possible interferences in the sample by means of alcohol precipitation and solid phase extraction column (e.g., normal phase silica bonded amino solid phase extraction column, hereinafter referred to as: amino column).

[0062] In some specific implementation schemes, during the sample purification step, the sample to be tested is weighed, and supercooled ethanol is added to remove protein impurities. The supernatant is centrifuged, and acetonitrile solution is added to construct an organic phase dissolution system. The solution is purified using an amino column, and an appropriate amount of the eluent is taken, dried under nitrogen, and the dried sample is awaiting subsequent derivatization.

[0063] In some embodiments, the ratio of the test sample to supercooled ethanol is 50–300 mg : 100–600 μL. In some preferred embodiments, the mass-to-volume ratio of the test sample to supercooled ethanol is 1:2.

[0064] In other embodiments, the ratio of the test sample to supercooled ethanol is 0.5–3 mL : 1–6 mL. In some preferred embodiments, the volume ratio of the test sample to supercooled ethanol is 1:2.

[0065] In some exemplary embodiments, for milk powder samples, the milk powder can be mixed with water to form a substantially homogeneous system, followed by subsequent necessary processing. For example, 200-800 mg of milk powder can be mixed with 1-5 mL of water.

[0066] In some embodiments, for breast milk samples, 100±10 mg of breast milk sample is weighed into a centrifuge tube, and 200±20 μL of supercooled ethanol is added, preferably maintaining a sample solution to supercooled ethanol mass-to-volume ratio of 1:2, and the mixture is thoroughly mixed. The mixture is centrifuged at 8000±1000 rpm / min for 5±2 min. 210±10 μL of the supernatant is then taken into a centrifuge tube, and 1.89±0.09 mL of acetonitrile is added, preferably maintaining a supernatant to acetonitrile volume ratio of 1:9, and the mixture is thoroughly mixed and collected for purification.

[0067] In some exemplary embodiments, for a breast milk sample, 100 mg of breast milk sample is weighed into a 1.5 mL centrifuge tube, 200 μL of supercooled ethanol is added, and the mixture is thoroughly mixed. The mixture is centrifuged at 8000 rpm / min for 5 min. 210 μL of the supernatant is then transferred to a 15 mL centrifuge tube, 1.89 mL of acetonitrile is added, the mixture is thoroughly mixed, and the mixture is collected for purification.

[0068] In some embodiments, for the milk powder sample, weigh 500±100 mg of milk powder into a centrifuge tube, add 3±1 mL of water at 40±5 °C, vortex until the milk powder is completely dissolved, and then add water to a final volume of 5 mL. Take 1±0.2 mL of the sample solution and add 2±0.4 mL of supercooled ethanol, preferably maintaining a volume ratio of sample solution to supercooled ethanol of 1:2, and mix thoroughly. Centrifuge at 8000±1000 rpm / min for 5±2 min. Take 1.2±0.2 mL of the supernatant into a centrifuge tube, add 10.8±1.8 mL of acetonitrile, preferably maintaining a volume ratio of supernatant to acetonitrile of 1:9, mix thoroughly, and collect for purification.

[0069] In some exemplary embodiments, for a milk powder sample, 500 mg of milk powder is weighed into a 15 mL centrifuge tube, 3 mL of 40 °C water is added, and the mixture is vortexed until the milk powder is completely dissolved. Water is then added to bring the volume to 5 mL. 1 mL of the sample solution is taken and 2 mL of supercooled ethanol is added, and the mixture is thoroughly mixed. The mixture is centrifuged at 8000 rpm for 5 min. 1.2 mL of the supernatant is taken into a 15 mL centrifuge tube, 10.8 mL of acetonitrile is added, and the mixture is thoroughly mixed. The mixture is then collected and purified.

[0070] In some implementations, after the amino column is activated, the sample is loaded and the amino column is eluted with an aqueous solution of acetonitrile containing ammonium formate to remove lactose.

[0071] In some preferred embodiments, the ammonium formate content in the acetonitrile aqueous solution containing ammonium formate is 5-20 mM, preferably 10-20 mM, for example 10 mM, 15 mM, 20 mM.

[0072] In some preferred embodiments, the acetonitrile aqueous solution containing ammonium formate is an 80-90% (V / V) acetonitrile aqueous solution, preferably an 83-86% (V / V) acetonitrile aqueous solution, such as an 83% (V / V) acetonitrile aqueous solution, such as an 84% (V / V) acetonitrile aqueous solution, such as an 85% (V / V) acetonitrile aqueous solution, such as an 86% (V / V) acetonitrile aqueous solution.

[0073] In some exemplary embodiments, the amino column is activated with 3 mL of 1% (v / v) trifluoroacetic acid aqueous solution and 3 mL of 85% (v / v) acetonitrile aqueous solution, respectively. The sample is passed through the column and eluted twice with 3 mL of 85% (v / v) acetonitrile aqueous solution containing 15 mM ammonium formate (pH 4.4). The eluent is then collected and brought to a final volume of 3.0 mL with 1% (v / v) trifluoroacetic acid aqueous solution, and vortexed. 200 μL of the eluent is taken and dried under nitrogen at 50 °C. The dried sample is then left for subsequent derivatization.

[0074] In some embodiments, the detection scheme of the present invention effectively removes proteins, lactose, and other potentially interfering substances from the sample to be tested (e.g., breast milk or infant formula) through precipitation with supercooled ethanol and purification by an amino column. In some preferred embodiments, rinsing with an aqueous solution containing a specific proportion of acetonitrile maximizes lactose removal, retains HMOs, and avoids the masking of low-abundance HMO peaks by interference.

[0075] Sample derivatization steps

[0076] In some embodiments, in the sample derivatization step of the detection method provided by the present invention, a reductive amination derivatization method is used to derivatize the sample obtained in the sample purification step.

[0077] In some specific implementations, the derivatizing reagent APTS is added to the sample obtained in the sample purification step, acetic acid is added in addition, and then sodium cyanoborohydride, a reducing agent, is added, and the mixture is heated until the liquid evaporates to dryness.

[0078] In some specific implementations, during the sample purification step, 18±3 μL of 48±5 mM APTS (5±1 mg APTS dissolved in 200±50 μL of 20±5% (V / V) acetic acid) is added to the vortex-dried sample, along with an additional 5±2 μL of acetic acid. Then, 2±1 μL of 1 mol / L sodium cyanoborohydride is added, and the mixture is vortexed to redissolve. After redissolving, the mixture is heated at 60±5 °C with the lid off until the liquid evaporates to dryness.

[0079] In some specific implementations, during the sample purification step, 18 μL of 48 mMAPTS (5 mg APTS dissolved in 200 μL of 20% (V / V) acetic acid) is added to the evaporated sample, along with an additional 5 μL of acetic acid. Then, 2 μL of sodium cyanoborohydride is added, and the mixture is vortexed to redissolve the sample before heating at 60 °C with the lid off until the liquid evaporates to dryness.

[0080] In some implementations, 100 μL of deionized water is added for reconstitution after the sample derivatization step is completed (after labeling).

[0081] Detection method of capillary electrophoresis-laser induced fluorescence

[0082] Run buffer

[0083] In this specification, the capillary electrophoresis-laser induced fluorescence detection method is also referred to as the laser-induced fluorescence detection capillary electrophoresis method, the capillary electrophoresis method with laser-induced fluorescence detection, or CE-LIF. The capillary electrophoresis-laser induced fluorescence detection method is described, for example, in reference 1.

[0084] In some preferred embodiments, the running buffer (the matrix for capillary electrophoresis) contains 2.1-3.9% (m / V) PVP, preferably 2.2-3.8% (m / V) PVP, more preferably 2.3-3.7% (m / V) PVP, even more preferably 2.4-3.6% (m / V) PVP, and even more preferably 2.5-3.5% (m / V) PVP.

[0085] In some preferred embodiments, the run buffer (the matrix for capillary electrophoresis) contains 2.8-3.3% (m / V) PVP, such as 2.8% (m / V) PVP, 2.9% (m / V) PVP, 3% (m / V) PVP, 3.1% (m / V) PVP, 3.2% (m / V) PVP, and 3.3% (m / V) PVP.

[0086] In some implementations, polyvinylpyrrolidone (PVP) has the CAS No. 9003-39-8 and the molecular formula (C6H9NO). n The structural formula is as follows: .

[0087] In some implementations, the weight-average molecular weight (Mw) of PVP is 1,000,000-2,000,000, preferably 1,000,000-1,500,000, such as 1,100,000, 1,300,000, and 1,500,000.

[0088] Adding PVP reagent, especially a specific amount of PVP, to the running buffer used in the detection method of the present invention causes the polymer chains to intertwine, forming transient sieves that screen oligosaccharide molecules and effectively improve the separation degree of human milk oligosaccharides.

[0089] In some preferred embodiments, the running buffer contains less than 15% (m / V) boric acid.

[0090] In some preferred embodiments, the running buffer contains 5-15% (m / V) boric acid.

[0091] In some further preferred embodiments, the running buffer contains 8-12% (m / V) boric acid, such as 8% (m / V) boric acid, 9% (m / V) boric acid, 10% (m / V) boric acid, 11% (m / V) boric acid, or 12% (m / V) boric acid.

[0092] In some implementations, boric acid has the following CAS No.: 10043-35-3; molecular formula: H3BO3; and structural formula: .

[0093] Adding a specific amount of boric acid to the running buffer used in the method of this invention affects the interaction between the analyte and the buffer, reducing sample diffusion. This significantly improves peak sharpness and resolution, especially in scenarios with narrow separation bandwidths. Furthermore, the unique chemical properties of boric acid allow it to form complexes with analytes possessing cis-diol structures (such as sugars and some polysaccharide-modified biomolecules), altering the effective charge / size ratio of these analytes and thus adjusting their electrophoretic migration rate, achieving selective separation of components that are difficult to separate using conventional methods. Moreover, specific amounts of boric acid and PVP further enhance the separation effect.

[0094] In some embodiments, the running buffer contains 70-100 mmol / L Tris-base, preferably 75-95 mmol / L Tris-base, more preferably 80-95 mmol / L Tris-base, such as 80 mmol / L Tris-base, 85 mmol / L Tris-base, or 89 mmol / L Tris-base.

[0095] In some implementations, Tris-base has the following CAS No.: 77-86-1; molecular formula: NH2C(CH2OH)3; and structural formula: .

[0096] In some embodiments, the running buffer contains 1-5 mmol / L EDTA, preferably 1-4 mmol / L EDTA, more preferably 1-3 mmol / L EDTA, such as 1 mmol / L EDTA, 2 mmol / L EDTA, or 3 mmol / L EDTA.

[0097] In some implementations, EDTA has the following CAS No.: 60-00-4; molecular formula: (HO2CCH2)2NCH2CH2N(CH2CO2H)2; and structural formula: .

[0098] In some embodiments, the running buffer contains 2-10 mol / L urea, preferably 2-8 mol / L urea, more preferably 2-6 mol / L urea, such as 2 mol / L urea, 4 mol / L urea, or 6 mol / L urea.

[0099] In some implementation schemes, urea has the following CAS No.: 57-13-6; molecular formula: NH2CONH2; and structural formula: .

[0100] Capillary electrophoresis

[0101] In some embodiments, the capillary electrophoresis is performed at a voltage of not less than 15 kV, preferably not less than 20 kV.

[0102] In some preferred embodiments, the capillary electrophoresis is performed at a voltage of 20-30 kV, preferably 25-30 kV, such as 25 kV, 28 kV, or 30 kV.

[0103] In some exemplary embodiments, the sample to be tested is injected at 3±1 kV for 10±3 seconds. After injection, a voltage ramp is set for 2±1 minutes to rise to a voltage of 20-30 kV, preferably 30 kV.

[0104] In some embodiments, the capillary is operated at a temperature greater than 25 °C during capillary electrophoresis.

[0105] In some embodiments, the capillary is operated at a temperature of less than 35 °C during capillary electrophoresis.

[0106] In some preferred embodiments, the capillary electrophoresis is performed at a temperature of 27-33 °C, preferably 28-32 °C, more preferably 29-31 °C, for example 29 °C, 30 °C, 31 °C.

[0107] In some embodiments, the sample pan is maintained at a temperature of 10-25 °C during capillary electrophoresis, preferably 10-20 °C, more preferably 12-18 °C, for example 12 °C, 15 °C, 18 °C.

[0108] In some embodiments, the capillary may be a chemically inert and hydrophobic capillary.

[0109] In some embodiments, the capillary may be a capillary with a fluorocarbon (FC) polymer coating.

[0110] In some embodiments, the capillary is commercially available, such as the μSIL-FC capillary from Agilent Technologies. In some exemplary embodiments, the capillary may have an inner diameter of 50 μm, a total length of 50 cm, and an effective length of 40 cm.

[0111] In this invention, there are no particular limitations on the instruments used for the capillary electrophoresis-laser induced fluorescence detection method. For example, commercially available instruments with capillary electrophoresis separation modules and / or LIF detection modules can be used.

[0112] In some exemplary embodiments, the instrument used for the capillary electrophoresis-laser induced fluorescence detection method may be a Sciex 8000 plus analysis system (Sciex) equipped with a solid-state laser induced fluorescence detector (488 nm excitation wavelength and 520 nm emission filter).

[0113] In some exemplary embodiments, the excitation wavelength is set to 488 nm and the emission wavelength is set to 520 nm.

[0114] In some preferred embodiments, the detection method provided by the present invention, by selecting μSIL-FC coated capillaries, adjusting key electrophoresis parameters, and / or combining fluorescence detection and sample derivatization, can achieve efficient separation of nine human milk oligosaccharides in breast milk and infant formula.

[0115] Construction of standard curve

[0116] Establishing a standard curve determines the basis for quantitative analysis comparison, and also determines the detection limit and quantitation limit of the detection system or method.

[0117] The detection method of the present invention also includes the step of constructing a standard curve. Specifically, a series of standard substance working solutions of different concentrations are prepared using standard substances of different human milk oligosaccharides. After going through the above-mentioned sample derivation steps and the detection method of capillary electrophoresis-laser induced fluorescence, a linear relationship between the calibration peak area and the standard substance concentration is constructed, and then the content of each human milk oligosaccharide in the sample to be tested is calculated.

[0118] Human milk oligosaccharides

[0119] HMOs are typically composed of 3 to 14 monosaccharides. HMOs consist of five basic monosaccharide units: glucose, galactose, N-acetylglucosamine, fucose, and N-acetylneuraminic acid. They usually contain a lactose core with a reduced end, which is elongated along with the other three monosaccharide units by the action of several glycosyltransferases. The number of monosaccharides in an HMO is generally 3 to 14.

[0120] HMOs can be classified into neutral HMOs (terminally L-fucose) and acidic HMOs (terminally sialic acid) based on whether they are sialylated.

[0121] In some embodiments, the detection method provided by the present invention can simultaneously detect neutral human milk oligosaccharides and acidic human milk oligosaccharides composed of sialic acid.

[0122] In some embodiments, the detection method provided by the present invention can detect at least one of lactose-N-fucopentose I (LNFPⅠ), lactose-N-tetrasaccharide (LNT), 2'-fucosyllactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), 3,2'-difucosyllactose (DFL), disialyllactose-N-tetrasaccharide (DSLNT), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

[0123] In some embodiments, the detection method provided by the present invention can simultaneously detect lactose-N-fucopentose I (LNFP I), lactose-N-tetrasaccharide (LNT), 2'-fucosyllactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), 3,2'-difucosyllactose (DFL), disialyllactose-N-tetrasaccharide (DSLNT), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

[0124] Exemplary implementation

[0125] In some exemplary embodiments, the present invention provides a method for detecting human milk oligosaccharides based on capillary electrophoresis-laser induced fluorescence detection, which achieves efficient separation and accurate quantification of nine HMOs in breast milk and infant formula. The specific steps are as follows.

[0126] 1. Preparation of running buffer: The running buffer is an aqueous solution containing 89 mmol / L tris-hydroxymethylaminomethane (Tris-base), 2 mmol / L ethylenediaminetetraacetic acid (EDTA), greater than 2% (m / V) and less than 4% (m / V) polyvinylpyrrolidone (PVP), 4 mol / L urea and greater than 5% (m / V) boric acid.

[0127] 2. Selection and pretreatment of capillary columns: Coated capillary columns are selected; before use, they are pressure-washed with hydrochloric acid (HCl) solution; before each injection, they are pressure-washed with HCl solution, deionized water and running buffer respectively.

[0128] 3. Electrophoresis analysis conditions: (1) Sample injection method: electric sample injection; (2) Operating conditions: set the separation voltage, capillary temperature, sample plate temperature and electrophoresis time respectively; (3) Detector: fluorescence detector.

[0129] 4. Sample purification: Accurately weigh the breast milk sample or infant formula sample (infant formula samples need to be prepared as a premixed solution with added standard), add supercooled ethanol to remove protein impurities. Centrifuge and collect the supernatant, add acetonitrile solution to construct an organic phase dissolution system. Purify the solution using an amino column, take an appropriate amount of eluent, blow dry with nitrogen at 50 ℃, and allow the dried sample to undergo derivatization.

[0130] 5. Sample derivatization and labeling: After rotary evaporation, add the derivatization reagent APTS, then add acetic acid, followed by the reducing agent sodium cyanoborohydride. Heat with the lid off until the liquid evaporates to dryness. After labeling, reconstitute with deionized water. Dilute breast milk and infant formula samples 20 times before injection. Inject the mixed standard solution directly.

[0131] 6. Preparation of Standard Curves: Prepare a mixed standard solution 1 containing 9 HMOs: 6'-SL, DSLNT, 3'-SL, 3-FL, DFL, LNnT, 2'-FL, LNT, and LNFPⅠ. Dilute the mixed standard stock solution 1 to obtain mixed standard working solutions of 0.1, 0.5, 1, 2, and 5 μg / mL. Purify and derivatize the mixed standard working solutions according to steps 4 and 5.

[0132] 7. Quantitative analysis: The derivatized and labeled solution is separated and detected according to the electrophoretic analysis conditions described in step 3. A linear relationship is established between the calibration peak area and the concentration of the mixed standard working solution in step 5. The content of each HMO in the breast milk sample or infant formula sample is calculated.

[0133] Capillary electrophoresis primarily relies on hydrodynamic volume and charge ratio to separate samples, providing excellent resolution for most isomers. In some preferred embodiments, this invention proposes a capillary electrophoresis method with laser-induced fluorescence detection and a derivatization step. Detection conditions (injection method, instrument conditions, and buffer concentration) are optimized by analyzing a mixture of nine selected HMO standard solutions. Ultimately, the optimal separation of HMOs was determined using a background electrolyte (BGE) containing 3% (m / V) polyvinylpyrrolidone (PVP) and 4 M urea as functional additives and 10% (m / V) borate, at 30 kV and 30°C, using a 50 μm inner diameter fluorocarbon (FC) capillary with electrodynamic injection.

[0134] Example

[0135] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0136] The materials and methods used in subsequent embodiments are as follows.

[0137] 1. Sample materials

[0138] Urea (Catalog No. U5128), sodium cyanoborohydride (Catalog No. 296813; 1 mol / L solution), ethylenediaminetetraacetic acid (EDTA, Catalog No. E6758), tris-hydroxymethylaminomethane (Tris-base, Catalog No. T1503), boric acid (Catalog No. B0394), polyvinylpyrrolidone (PVP, Catalog No. 437190; Mw1300000), trisodium 8-aminopyrene-1,3,6-trisulfonic acid (APTS, Catalog No. 09341), acetic acid (HAc, Catalog No. 695092), trifluoroacetic acid (TFA, Catalog No. T6508), and ammonia (NH3•H2O, Catalog No. 30501) were purchased from Sigma-Aldrich, Inc., USA. Acetonitrile (ACN, Catalog No. A955-4) and formic acid (FA, Catalog No. A117) were purchased from Thermo Fisher Scientific, Inc., USA. Lactose-N-fucopentose I (LNFP I), lactose-N-tetrasaccharide (LNT), 2'-fucosyllactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), 3,2'-difucosyllactose (DFL), disialylate-N-tetrasaccharide (DSLNT), 3-fucosyllactose (3-FL), 3'-sialylatesose (3'-SL), 6'-sialylatesose (6'-SL), and lactose were purchased from Wuhan Sugar Intelligence Co., Ltd., China. Deionized water was produced by Merck Milli-Q water purification systems. μSIL-FC capillary tubes (catalog number 194-8111) with an inner diameter of 50 μm were purchased from Agilent Technologies, USA. ProElutNH2 amino column (catalog number 63305) was purchased from Dima Technologies, USA.

[0139] 2. Reagent preparation

[0140] The running buffer was an aqueous solution containing 89 mmol / L Tris-base, 2 mmol / L EDTA, 3% (m / V) PVP, 4 mol / L urea and 10% (m / V) boric acid.

[0141] 1% (V / V) trifluoroacetic acid aqueous solution: Take 500 μL of trifluoroacetic acid and add water to make up to 50 mL.

[0142] 90% (V / V) acetonitrile aqueous solution: Measure 45 mL of acetonitrile, add 5 mL of water, and mix well.

[0143] 85% (V / V) acetonitrile aqueous solution: Measure 42.5 mL of acetonitrile, add 7.5 mL of water, and mix well.

[0144] 100 mmol / L ammonium formate (pH 4.4): Measure 80 mL of water, add 378 μL of formic acid, adjust the pH to 4.4 with ammonia, and add water to bring the volume to 100 mL.

[0145] 85% acetonitrile aqueous solution containing 15 mmol / L ammonium formate (pH 4.4): Measure 42.5 mL of acetonitrile, add 7.5 mL of 100 mmol / L ammonium formate (pH 4.4), and mix well.

[0146] 3. Sample preparation

[0147] 3.1 Preparation of HMOs Standards

[0148] In subsequent examples, the following nine HMOs were selected as research subjects. Firstly, they are highly abundant in breast milk; secondly, based on their structure and chemical properties, they represent the three major classes of HMOs in breast milk: fucosylated, sialic acid-modified, and unmodified. 1 mg of each of 6'-SL, DSLNT, 3'-SL, 3-FL, DFL, LNnT, 2'-FL, LNT, and LNFP I was weighed and dissolved in 1 mL of water to prepare a 0.1 mg / mL HMOs mixed standard solution 1. Mixed standard stock solution 1 was diluted 10-fold with acetonitrile to prepare a 10 μg / mL mixed standard stock solution 2. Diluted with 90% (v / v) ACN, mixed standard working solutions of 0.1, 0.5, 1, 2, and 5 μg / mL were obtained.

[0149] 3.2 Preparation of breast milk samples

[0150] Weigh 100 mg of breast milk sample into a 1.5 mL centrifuge tube, add 200 μL of supercooled ethanol, and mix well. Centrifuge at 8000 rpm / min for 5 min. Take 210 μL of the supernatant into a 15 mL centrifuge tube, add 1.89 mL of acetonitrile, mix well, and collect for purification.

[0151] 3.3 Preparation of Infant Formula Samples

[0152] Accurately weigh 500 mg of infant formula into a 15 mL centrifuge tube, add 3 mL of 40 °C water, vortex until the infant formula is completely dissolved, and then add water to a final volume of 5 mL. Take 1 mL of the sample solution and add 2 mL of supercooled ethanol, mix well. Centrifuge at 8000 rpm / min for 5 min. Take 1.2 mL of the supernatant into a 15 mL centrifuge tube, add 10.8 mL of acetonitrile, mix well, and collect for purification.

[0153] 4. Sample purification conditions

[0154] Purification: The amino column was activated with 3 mL of 1% (v / v) trifluoroacetic acid aqueous solution and 3 mL of 85% (v / v) acetonitrile aqueous solution, respectively. 2 mL of the mixed standard (or 2 mL of breast milk sample and 10 mL of infant formula sample) was passed through the column and washed twice with 3 mL of 85% acetonitrile solution containing 15 mM ammonium formate (pH 4.4). The eluent was then collected and brought to a final volume of 3.0 mL with 1% (v / v) trifluoroacetic acid aqueous solution, and vortexed. 200 μL of the eluent was dried under nitrogen at 50 °C. The dried sample was then left to undergo derivatization. The mixed standard and sample were processed simultaneously.

[0155] 5. Sample derivatization conditions

[0156] After vortexing to dryness, add 18 μL of 48 mM APTS (5 mg APTS dissolved in 200 μL of 20% (v / v) acetic acid), add an additional 5 μL of acetic acid, then add 2 μL of 1 mol / L sodium cyanoborohydride. Vortex to reconstitute, then heat at 60 °C with the lid off until the liquid evaporates to dryness. After labeling, add 100 μL of deionized water to reconstitute. Breast milk and infant formula samples are diluted 20-fold for injection, while mixed standards are injected directly.

[0157] 6. Capillary electrophoresis

[0158] The Sciex 8000 plus analytical system (Sciex) is equipped with a solid-state laser-induced fluorescence detector (488 nm excitation wavelength and 520 nm emission filter) for all capillary electrophoresis separations. The separation buffer is a borate-based buffer (containing PVP and urea) for HMO separation. μSIL-FC capillaries (50 μm inner diameter, 50 cm total length, 40 cm effective length) are operated at 30 °C. The sample tray is maintained at 15 °C. Separation is performed at a constant normal operating voltage of 30 kV. The excitation wavelength is set to 488 nm. The emission wavelength is set to 520 nm. New capillaries are rinsed with 0.1 mol / L HCl for 10 min before use. The injection program is as follows: rinse the capillaries with 0.1 mol / L HCl for 3 min, then rinse with water for 2 min, and rinse with running buffer for 5 min. The sample is injected at 3 kV for 10 seconds. After injection, a 2-minute ramp is set up, increasing the voltage to 30 kV.

[0159] 7. Data Processing

[0160] The content of each HMO in the breast milk sample, X1 (mg / 100g), is calculated according to formula (1); the content of each HMO in the infant formula sample, X2 (mg / 100g), is calculated according to formula (2).

[0161] (1).

[0162] (2).

[0163] In the formula: X1 and X2 are the contents of each HMO in breast milk and infant formula, respectively, in milligrams per 100 grams (mg / 100 g); m1 and m2 are the masses of breast milk and infant formula, respectively, in grams (g); C1 and C2 are the concentrations of each HMO in breast milk and infant formula solutions obtained from the standard curve, in milligrams per milliliter (μg / mL); n1 and n2 are the dilution factors (30) of the breast milk sample and (60) of the infant formula sample; V is the loading volume of breast milk and infant formula samples purified using an amino column in a 90% (V / V) ACN system, in milliliters (mL).

[0164] Example 1: Effect of PVP concentration on the detection of HMOs in dairy products

[0165] The steps are as follows.

[0166] 1. Preparation of running buffer: The running buffer is an aqueous solution containing 89 mmol / L Tris-base, 2 mmol / L EDTA, PVP, 4 mol / L urea and 10% (m / V) boric acid, with PVP added at 2%, 3% and 4% (m / V), respectively.

[0167] 2. Selection and pretreatment of capillary columns: Use μSIL-FC coated capillaries with an inner diameter of 50 μm (total length 50 cm, effective length 40 cm); before first use, rinse the new capillaries with 0.1 mol / L HCl at 50 psi for 10 min; before each injection, rinse the capillaries with 0.1 mol / L HCl at 50 psi for 3 min, rinse with water at 50 psi for 2 min, and rinse with buffer at 50 psi for 5 min.

[0168] 3. Electrophoresis analysis conditions: The Sciex CESI 8000 plus analysis system equipped with a solid-state laser-induced fluorescence detector was used for detection. The excitation wavelength was set to 488 nm and the emission wavelength was set to 520 nm. Electric injection was used, and the sample was injected at 3 kV for 10 s. A 2-min voltage ramp was set to increase the voltage to 30 kV. Separation was carried out at a constant normal voltage of 30 kV for 50 min. The sample plate temperature was 15 ℃ and the capillary temperature was 30 ℃.

[0169] 4. Sample purification: Weigh 1 mg of 6'-SL, DSLNT, 3'-SL, 3-FL, DFL, LNnT, 2'-FL, LNT, and LNFP I, respectively, and dissolve them in 10 mL of water to prepare a 0.1 mg / mL HMOs mixed standard stock solution 1. Dilute mixed standard stock solution 1 10 times with acetonitrile to prepare a 10 μg / mL mixed standard for purification. Activate the amino column with 3 mL of 1% (v / v) trifluoroacetic acid aqueous solution and 3 mL of 85% (v / v) acetonitrile aqueous solution, respectively. Transfer 2 mL of the mixed standard to the column and wash twice with 3 mL of 85% (v / v) acetonitrile aqueous solution containing 15 mM ammonium formate (pH 4.4). Elute with 3 mL of 1% (v / v) trifluoroacetic acid aqueous solution and collect the eluent. Make up to 3.0 mL with 1% (v / v) trifluoroacetic acid aqueous solution and vortex to mix. Take 200 μL of eluent, blow dry with nitrogen at 50 °C, and wait for derivatization of the dried sample.

[0170] 5. Sample derivatization and labeling: After vortexing to dryness, add 18 μL of 48 mM APTS (5 mg APTS dissolved in 200 μL of 20% (v / v) acetic acid), add an additional 5 μL of acetic acid, then add 2 μL of 1 mol / L sodium cyanoborohydride. Vortex to reconstitute, then heat at 60 °C with the lid off until the liquid evaporates to dryness. After labeling, add 100 μL of deionized water to reconstitute, mix with the standard solution, and inject directly into the sample.

[0171] 6. Capillary electrophoresis: Separation and detection were performed using a Sciex 8000 plus analysis system (Sciex) equipped with a solid-state laser-induced fluorescence detector, following the electrophoresis conditions in step 3.

[0172] Experimental results: such as Figure 1 As shown, PVP concentration does not affect the peak order. Increasing PVP concentration has varying effects on different HMOs; higher concentrations improve the separation of 3-FL and Lac. At 2% PVP concentration, 3-FL and Lac peaks are not separated from 2'-FL and LNnT peaks, while at 4% PVP concentration, the separation of 3'-SL and 3-FL in the HMO spectrum deteriorates. At 3% PVP concentration, the migration times of the nine HMOs and lactose show good reproducibility, and each sugar is completely separated, with a shorter detection time compared to 4% PVP concentration. At 3% PVP concentration, acidic oligosaccharides with sialic acid groups (3'-SL, 6'-SL, and DSLNT) achieve good separation in the range of 24 to 33 min.

[0173] Example 2: Effect of boric acid concentration on the detection of HMOs in dairy products

[0174] The steps are as follows: basically the same as those in Example 1, except for the following steps.

[0175] 1. Preparation of running buffer: The running buffer is an aqueous solution containing 89 mmol / L Tris-base, 2 mmol / L EDTA, 3% (m / V) PVP, 4 mol / L urea and boric acid, with the boric acid added at 1%, 5% and 10% (m / V), respectively.

[0176] Experimental results: such as Figure 2 As shown, the concentration of boric acid significantly regulates the peak elution order and separation efficiency of HMOs in capillary electrophoresis. This may be because at low boric acid concentrations, the binding capacity has a greater impact, while at high boric acid concentrations, the stability of the complexes has a greater impact. At a 1% boric acid concentration, the LNFP I and LNT peaks overlap and cannot be effectively separated; at a 5% boric acid concentration, not only is the separation efficiency of LNFP I / LNT not improved, but the electrophoretic peaks of 3-FL / 2'-FL also overlap. However, at a 10% boric acid concentration, all target HMOs exhibit sharp, single, symmetrical peaks without broad peaks or double peaks, indicating a significant improvement in isomer separation efficiency. As typical polyhydroxy compounds, HMOs can undergo a reversible reaction with borate to form stable anionic complexes. The specificity and stability of this complexation reaction are directly affected by the substituent type, charge distribution, hydroxyl spatial position, and glycosidic bond configuration of the HMO molecule, thereby altering the electrophoretic mobility differences of different HMO molecules in an electric field. The boric acid concentration can further amplify or reduce these mobility differences by regulating the equilibrium state of the complexation reaction (binding capacity and complex stability), ultimately achieving the selective separation of HMOs with different structures.

[0177] Example 3: The effect of capillary voltage on the detection of HMOs in dairy products

[0178] The steps are as follows: basically the same as those in Example 1, except for the following steps.

[0179] 1. Preparation of running buffer: The running buffer is an aqueous solution containing 89 mmol / L Tris-base, 2 mmol / L EDTA, 3% (m / V) PVP, 4 mol / L urea and 10% (m / V) boric acid.

[0180] 3. Electrophoresis analysis conditions: The Sciex CESI 8000 plus analysis system equipped with a solid-state laser-induced fluorescence detector was used for detection. The excitation wavelength was set to 488 nm and the emission wavelength was set to 520 nm. Electric injection was used, and the sample was injected at 3 kV for 10 s. A 2-minute voltage ramp was set, and the voltage was increased to 15, 20, and 30 kV respectively. Separation was carried out at a constant normal voltage for 70 min. The sample plate temperature was 15 ℃ and the capillary temperature was 30 ℃.

[0181] Experimental results: The instrument voltage directly determines the migration speed of electrophoresis. Too low a voltage leads to excessively long migration times, while too high a voltage accelerates the migration speed, affecting the separation effect. For example... Figure 3 As shown, at a voltage of 15 kV, the peak shapes of HMOs are relatively broad. Although all nine HMOs can be completely separated, the migration time is long, resulting in low efficiency. Increasing the voltage to 20 kV significantly reduces the migration time of HMOs. At a voltage of 30 kV, the migration time of HMOs is the shortest, the separation speed is the fastest, and the resolution is good. Furthermore, 30 kV is the maximum adjustable voltage of the system, at which point the current is approximately -22 μA, the peak shape is sharpest, and the separation is good, making it suitable for high-throughput analysis.

[0182] Example 4: Effect of capillary temperature on the detection of HMOs in dairy products

[0183] The steps are basically the same as those in Example 1, except for the following steps.

[0184] 1. Preparation of running buffer: The running buffer is an aqueous solution containing 89 mmol / L Tris-base, 2 mmol / L EDTA, 3% (m / V) PVP, 4 mol / L urea and 10% (m / V) boric acid.

[0185] 3. Electrophoresis analysis conditions: The Sciex CESI 8000 plus analysis system equipped with a solid-state laser-induced fluorescence detector was used for detection. The excitation wavelength was set to 488 nm and the emission wavelength was set to 520 nm. Electric injection was used, and the sample was injected at 3 kV for 10 s. A 2-min voltage ramp was set to increase the voltage to 30 kV. Separation was carried out at a constant normal voltage of 30 kV for 50 min. The sample plate temperature was 15 ℃, and the capillary temperatures were set to 20, 25, 30, and 35 ℃, respectively.

[0186] Experimental results: such as Figure 4As shown, at 20 ℃ and 25 ℃, the peak migration times of HMOs are too long, resulting in low analytical efficiency, and the 3-FL and 3'-SL peaks overlap, leading to poor separation. At 30 ℃, the peak migration times of HMOs are shorter than at 20 ℃ and 25 ℃, ensuring sufficient time for efficient separation of each HMO while avoiding excessively long analysis cycles. However, at 35 ℃, the peak migration times of HMOs are too short, leading to peak compression and insufficient separation (e.g., poor separation between the LNnT peak and the 2'-FL peak). Therefore, at 30 ℃, the separation of each HMO can be guaranteed while shortening the analysis time and improving analytical efficiency.

[0187] Application Example 1: Detection of HMOs in breast milk

[0188] The steps are as follows: basically the same as those in Example 1, except for the following steps.

[0189] 1. Preparation of running buffer: The running buffer is an aqueous solution containing 89 mmol / L Tris-base, 2 mmol / L EDTA, 3% (m / V) PVP, 4 mol / L urea and 10% (m / V) boric acid.

[0190] 4. Purification of breast milk samples: Accurately weigh 100 mg of breast milk sample into a 1.5 mL centrifuge tube, add 200 μL of supercooled ethanol, and mix well. Centrifuge at 8000 rpm / min for 5 min. Take 210 μL of the supernatant into a 15 mL centrifuge tube, add 1.89 mL of acetonitrile, mix well, and collect for purification.

[0191] The amino column was activated with 3 mL of 1% (v / v) trifluoroacetic acid aqueous solution and 3 mL of 85% (v / v) acetonitrile aqueous solution, respectively. A 2 mL sample of breast milk was passed through the column and washed twice with 3 mL of 85% (v / v) acetonitrile aqueous solution containing 15 mM ammonium formate (pH 4.4). The eluent was then collected and brought to a final volume of 3.0 mL with 1% (v / v) trifluoroacetic acid aqueous solution, and vortexed. 200 μL of the eluent was dried under nitrogen at 50 °C, and the dried sample was left for derivatization.

[0192] 5. Derivatization and labeling of breast milk samples: After vortexing to dryness, add 18 μL of 48 mM APTS (5 mg APTS dissolved in 200 μL of 20% (v / v) acetic acid), add an additional 5 μL of acetic acid, then add 2 μL of 1 mol / L sodium cyanoborohydride. Vortex to reconstitute, then heat at 60 °C with the lid off until the liquid evaporates to dryness. After labeling, add 100 μL of deionized water to reconstitute, and dilute the breast milk sample 20 times before injection.

[0193] 6. Capillary electrophoresis: Separation and detection were performed using a Sciex 8000 plus analysis system (Sciex) equipped with a solid-state laser-induced fluorescence detector, following the electrophoresis conditions in step 3 (Example 1).

[0194] Construction of the standard curve: 1 mg of 6'-SL, DSLNT, 3'-SL, 3-FL, DFL, LNnT, 2'-FL, LNT, and LNFPⅠ were dissolved in 10 mL of water to prepare a 0.1 mg / mL HMOs mixed standard solution 1. Mixed standard stock solution 1 was diluted 10-fold with acetonitrile to prepare a 10 μg / mL mixed standard stock solution 2. Diluted with 90% (V / V) ACN, mixed standard working solutions of 0.1, 0.5, 1, 2, and 5 μg / mL were obtained. 2 mL of each mixed standard working solution was purified by passing through an NH2 column. 200 μL of the eluent was dried under nitrogen at 50 °C, labeled, and reconstituted with 100 μL of water. The mixed standard was then directly injected into the sample.

[0195] 7. The content of each HMO in the breast milk sample, X1 (mg / 100g), is calculated using the formula: .

[0196] In the formula: X1—content of each HMO in breast milk, in milligrams per 100 grams (mg / 100 g); m1—mass of breast milk, in grams (g); C1—concentration of each HMO in the breast milk sample obtained from the standard curve, in micrograms per milliliter (μg / mL); n1—dilution factor of the breast milk sample (30); V1—volume of breast milk sample purified using an amino column in a 90% (V / V) ACN system, in milliliters (mL).

[0197] Experimental Results: The detection method employed in this invention can separate HMOs within a 20-40 min range and can be applied to breast milk matrix. This invention reduces interference from proteins, lactose, salts, and other potential contaminants through pretreatment methods such as alcohol precipitation to remove proteins and amino column purification. Electrophoresis patterns of the nine HMOs and lactose standards are shown below. Figure 5 As shown in Table 1, calibration curves were constructed by relating the peak areas of HMO mixed standards to their corresponding concentrations. The calibration curves exhibited good linearity in the range of 0.1–5 μg / mL, with a correlation coefficient R0. 2 All are greater than 0.995, and the average relative residual standard deviation error is less than 5%.

[0198] Electrophoresis pattern of breast milk sample as shown Figure 6As shown in Table 2, to evaluate the accuracy and repeatability of the breast milk matrix detection method, breast milk samples containing a certain concentration of HMOs were tested 10 times to obtain the accurate HMO content. Three concentrations of HMOs (low, medium, and high) within the linear range were added to this sample, and each spiked concentration was tested 10 times. The results show that the recoveries of all HMO components were between 93.3% and 100.8%; the RSD of the 10-time repeatability precision at all three levels was less than 4.6%, meeting the relevant requirements of GB 5009.295-2023. The overall recovery rate and precision meet the quantitative requirements for the use of this method in breast milk.

[0199] When evaluating the sensitivity of the breast milk matrix detection method, ultrafiltration (molecular weight cutoff of 10 kDa) was used to remove substances below 10 kDa (including HMOs) from the matrix, serving as a sample matrix blank. By adding a known concentration of HMOs solution to the blank treatment solution, repeated trials were conducted to determine the lowest HMOs concentration that the instrument could recognize. This concentration was measured 10 times and used as the limit of detection (LOD) for HMOs. HMOS LOD HMOS The limit of quantitation (LOQ) was 3.3 times that of the target value. HMOS The results are shown in Table 2. The detection limit and quantitation limit of the method of this invention are at extremely low concentration levels, which can cover the range of trace to conventional concentrations of HMOs in actual samples, meeting the requirements of accuracy, sensitivity and stability in separation and detection, and can be used for high-throughput analysis of actual samples.

[0200] Table 1. Linear range of HMO detection methods

[0201]

[0202] Table 2. Validation of methods for detecting HMOs in breast milk (n=10)

[0203]

[0204] Application Example 2: Detection of HMOs in infant formula

[0205] The steps are as follows: They are basically the same as those in Application Example 1, except for the following steps.

[0206] 4. Purification of infant formula sample: Accurately weigh 500 mg of infant formula into a 15 mL centrifuge tube, add 3 mL of 40 ℃ water, vortex until the infant formula is completely dissolved, and then add water to a final volume of 5 mL. Take 1 mL of the sample solution and add 2 mL of supercooled ethanol, mix well. Centrifuge at 8000 rpm / min for 5 min. Take 1.2 mL of the supernatant into a 15 mL centrifuge tube, add 10.8 mL of acetonitrile, mix well, and collect for purification.

[0207] The amino column was activated with 3 mL of 1% (v / v) trifluoroacetic acid aqueous solution and 3 mL of 85% (v / v) acetonitrile aqueous solution, respectively. 10 mL of infant formula sample was passed through the column and washed twice with 3 mL of 85% (v / v) acetonitrile aqueous solution containing 15 mM ammonium formate (pH 4.4). The eluent was then collected and brought to a final volume of 3.0 mL with 1% (v / v) trifluoroacetic acid aqueous solution, and vortexed. 200 μL of the eluent was dried under nitrogen at 50 °C, and the dried sample was left for derivatization.

[0208] 7. The content of each HMO in the infant formula sample is calculated using the formula X2 (mg / 100g): .

[0209] In the formula: X2—content of each HMO in the infant formula, in milligrams per 100 grams (mg / 100 g); m2—mass of the infant formula, in grams (g); C2—concentration of each HMO in the infant formula solution obtained from the standard curve, in micrograms per milliliter (μg / mL); n2—dilution factor of the infant formula sample (60); V2—volume of the infant formula sample in the 90% (V / V) ACN system after purification using an amino column, in milliliters (mL).

[0210] Experimental Results: The detection method used in this invention can be applied to infant formula matrices, separating HMOs within a 20-40 minute range. The electrophoretic spectra of the infant formula samples are shown below. Figure 7 As shown in Table 3, the accuracy, repeatability, and sensitivity of the detection method applied to infant formula matrix were evaluated. The recoveries of all HMOs components were between 90.7% and 101.2%; the RSD of the 10-times repeatability precision at all three levels was less than 4.5%, meeting the relevant requirements of GB 5009.295-2023. This invention's method can achieve infant formula quality control and label compliance verification by comparing the HMO content in infant formula with the claimed fortification content.

[0211] Table 3. Validation of methods for detecting HMOs in infant formula (n=10)

[0212]

[0213] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0214] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for detecting human milk oligosaccharides, characterized in that, The method for detecting human milk oligosaccharides includes the step of detecting the sample using a capillary electrophoresis-laser-induced fluorescence-based detection method. The running buffer used in the capillary electrophoresis-laser induced fluorescence detection method contains Tris-base, EDTA, urea, and PVP of greater than 2% (m / V) and less than 4% (m / V) and boric acid of greater than 5% (m / V) and less than 15% (m / V). The concentration of Tris-base is 70-100 mmol / L, the concentration of EDTA is 1-5 mmol / L, and the concentration of urea is 2-10 mol / L; In the step of detecting the sample using the detection method based on capillary electrophoresis-laser induced fluorescence, the capillary electrophoresis is performed at a voltage of not less than 15 kV.

2. The method for detecting human milk oligosaccharides according to claim 1, characterized in that, Before the step of detecting the sample using the detection method based on capillary electrophoresis-laser induced fluorescence, a step of pre-treatment of the sample is also included. The pretreatment steps include sample purification and / or sample derivatization.

3. The method for detecting human milk oligosaccharides according to claim 2, characterized in that, In the sample purification step, the sample to be tested is purified using alcohol precipitation and solid-phase extraction columns; and / or, In the sample derivatization step, the sample to be tested is derivatized using a reductive amination derivatization method.

4. The method for detecting human milk oligosaccharides according to claim 1, characterized in that, In the step of detecting the sample using the capillary electrophoresis-laser induced fluorescence detection method, the capillary is operated at a temperature greater than 25 °C during capillary electrophoresis, and / or the sample pan is maintained at a temperature of 10-25 °C.

5. The method for detecting human milk oligosaccharides according to claim 1, characterized in that, The capillary is a chemically inert and hydrophobic capillary.

6. The method for detecting human milk oligosaccharides according to claim 1, characterized in that, The samples to be tested include milk and / or dairy products.

7. The method for detecting human milk oligosaccharides according to claim 1, characterized in that, The human milk oligosaccharides include neutral human milk oligosaccharides and acidic human milk oligosaccharides composed of sialic acid.

8. The method for detecting human milk oligosaccharides according to claim 7, characterized in that, The human milk oligosaccharides include at least one of lactose-N-fucopentose I, lactose-N-tetrasaccharide, 2'-fucosylated lactose, lactose-N-neotetrasaccharide, 3,2'-difucosylated lactose, disialactose-N-tetrasaccharide, 3-fucosylated lactose, 3'-sialactose, and 6'-sialactose.

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