A high performance liquid chromatography method for simultaneous determination of multiple human milk oligosaccharides

Abstract

The present invention discloses a high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, and relates to the technical field of analytical chemistry. Multiple human milk oligosaccharides include at least three of lactose, lactose-N-triose II, lactose-N-tetraose, and lactose-N-neotetraose, including (1) configuring standard solutions of different concentrations; (2) subjecting the standard solution and the test sample to quantitative measurement through high performance liquid chromatography, drawing a standard curve according to the test results of the standard solution, and combining the standard curve and the standard solution to quantitative analysis of the test sample. The present application adopts high performance liquid chromatography-differential detection method to achieve simultaneous separation and determination of multiple human milk oligosaccharides, which is low in cost, easy to operate, has low requirements on instruments, can complete separation and determination in a short time, greatly shortens the detection time, has stable peak time and good peak shape, is easy to carry out content determination, and has good sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component after lactose and lipids, with a content of approximately 5-15 g / L. HMOs consist of five monomers: D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid (sialic acid). These five monomers, when combined in varying proportions, can form thousands of HMOs, of which approximately 200 have been identified. Common HMOs include 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), lacto-N-neotetraose (LNnT), and lacto-N-tetraose (LNT). The reducing end of HMOs is a lactose structure, on which the sugar chain is extended to form HMOs with diverse structures. Based on the connected monomers, they can be mainly divided into three categories: (1) fucosylated neutral HMOs; (2) sialylated acidic HMOs; and (3) non-fucosylated neutral HMOs. Lacto-N-neotetraose (LNnT) belongs to the non-fucosylated neutral HMOs, with a content of approximately 0.5 g / L. LNnT is a linear tetrasaccharide (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) composed of D-galactose, N-acetylglucosamine, D-galactose and D-glucose connected by β1-4, β1-3 and β1-4 glycosidic bonds. It has a molecular weight of 707.63 and a molecular formula of C 26 H 45 NO 21 Lactose-N-tetraose (LNT) is a non-fucosylated neutral HMO, accounting for 6% of the total HMOs. LNT (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) is a linear tetrasaccharide composed of D-galactose, N-acetylglucosamine, D-galactose and D-glucose linked in sequence. Its content in breast milk of full-term and premature infants is approximately 0.79 g / L and 1.04 g / L, respectively. Its molecular formula is C 26 H 45 NO 21 , molecular weight is 707.63.

[0003] As a mixture, HMOs possess an unparalleled variety of biological functions. For example, they can act as prebiotics by promoting the growth of bifidobacteria and lactic acid bacteria, inhibiting the growth of pathogens, maintaining intestinal flora balance, and enhancing the body's resistance to infection. They can also act as receptor analogs, directly binding to pathogens, preventing the binding of pathogens to intestinal epithelial cells and the onset of infection. They are also potential antiviral drugs, with HMOs having been shown to be effective against viruses such as rotavirus, norovirus, and human immunodeficiency virus. Therefore, the development of highly sensitive and efficient separation methods for the simultaneous detection of lactose, lactose-N-triose II (LNT II), lactose-N-neotetraose (LNnT), or lactose-N-tetraose (LNT) is urgently needed. Summary of the Invention

[0004] The present invention provides a high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, thereby providing a detection method for simultaneously detecting the contents of lactose, lactose-N-triose II (LNT II) and lactose-N-neotetraose (LNnT) or lactose-N-tetraose (LNT), with high sensitivity and good separation effect.

[0005] In order to solve the above technical problems, one of the objectives of the present invention is to provide a high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein the multiple human milk oligosaccharides include at least three of lactose, lactose-N-triose II, lactose-N-tetraose, and lactose-N-neotetraose, comprising the following steps:

[0006] (1) Prepare standard solutions of lactose, lactose-N-triose II, lactose-N-tetraose or lactose-N-neotetraose at different concentrations;

[0007] (2) The standard solution and the test sample are quantitatively measured by high performance liquid chromatography, a standard curve is drawn based on the test results of the standard solution, and the test sample is quantitatively analyzed by combining the standard curve and the spectrum of the standard solution;

[0008] In step (2), the HPLC conditions are: guard column: NH2, 5μm, 4.6x10mm, analytical column uses NH2, 5μm, 4.6x 300mm, the mobile phase is 70% acetonitrile water isocratic elution, the mobile phase speed is 1mL / min, the column oven temperature is 40℃, the injection volume is 10μL, and the detector is a differential detector.

[0009] As a preferred solution, in step (2), the detection concentration range of the detection sample is 0.2-2 g / L.

[0010] As a preferred embodiment, the standard curve of lactose is Y=77114.4X-4116.57, with concentration as the abscissa and corresponding peak area as the ordinate.

[0011] As a preferred embodiment, the standard curve of the lactose-N-triose II is Y=48274X-2232.2, with concentration as the abscissa and the corresponding peak area as the ordinate.

[0012] As a preferred embodiment, the standard curve of lactose-N-tetraose is Y=48446X+906.53, with concentration as the abscissa and corresponding peak area as the ordinate.

[0013] As a preferred embodiment, the standard curve of lactose-N-neotetraose is Y=45857X-176.99, with concentration as the abscissa and corresponding peak area as the ordinate.

[0014] As a preferred embodiment, the concentration of the standard solution is 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L and 2 g / L.

[0015] As a preferred embodiment, in step (2), the test sample is a fermentation broth or a standard solution.

[0016] In order to solve the above technical problems, a second object of the present invention is to provide a high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides for application in the food field.

[0017] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0018] The present application adopts high performance liquid chromatography-differential detection method to achieve simultaneous separation and determination of lactose, lactose-N-triose II (LNT II) and lactose-N-neotetraose (LNnT) or lactose-N-tetraose (LNT), and accurate quantification. Compared with other high performance liquid chromatography detection methods, it is low in cost, simple to operate, and has low instrument requirements. The selected NH2 chromatographic column can achieve good separation effect for lactose, lactose-N-triose II (LNT II) and lactose-N-neotetraose (LNnT) or lactose-N-tetraose (LNT). The selected acetonitrile mobile phase has low polarity and fast elution speed for sugar compounds. Combined with the mobile phase and column oven temperature, the elution conditions are optimized to improve the separation efficiency. The established isocratic elution method can effectively separate various human milk oligosaccharides. The separation and determination of lactose, lactose-N-triose II (LNT II) and lactose-N-neotetraose (LNnT) or lactose-N-tetraose (LNT) can be completed within 20 minutes, greatly shortening the detection time. The peak elution time is stable and the peak shape is good, which is easy to determine the content. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : is the HPLC atlas of the lactose (Lac) standard solution of the present invention;

[0020] Figure 2 : is the HPLC spectrum of the lactose-N-triose II (LNTII) standard solution of the present invention;

[0021] Figure 3 : is the HPLC spectrum of the lactose-N-tetraose (LNT) standard solution of the present invention;

[0022] Figure 4 : is the HPLC spectrum of the lactose-N-neotetraose (LNnT) standard product of the present invention;

[0023] Figure 5 : This is the HPLC spectrum obtained by detecting sample A in Example 1 of the present invention;

[0024] Figure 6 : This is the HPLC spectrum obtained by detecting sample B in Example 2 of the present invention;

[0025] Figure 7 : This is the HPLC spectrum obtained by detecting sample C in Example 3 of the present invention;

[0026] Figure 8 : This is the HPLC spectrum obtained by detecting sample D in Example 4 of the present invention;

[0027] Figure 9 : This is the HPLC spectrum obtained by detecting the fermentation broth A in Example 5 of the present invention;

[0028] Figure 10 : This is the HPLC spectrum obtained by detecting the fermentation broth B in Example 6 of the present invention;

[0029] Figure 11 : This is the HPLC spectrum obtained by detecting sample E in Comparative Example 1 of the present invention;

[0030] Figure 12 : This is the HPLC spectrum obtained by detecting sample F in Comparative Example 2 of the present invention;

[0031] Figure 13 : HPLC spectrum obtained by detecting sample G in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Preparation Example

[0034] Preparation method of fermentation broth A: β-1,3-galactosyltransferase is introduced when constructing the plasmid, and the plasmid is transferred into the Escherichia coli BL21 (DE3) strain, which produces lactose-N-tetraose. Lactose and lactose-N-triose II are precursors for synthesizing tetrasaccharides. The lactose-N-tetraose (LNT)-producing strain is inoculated into 5 mL of LB liquid culture medium, cultured for 15 hours, and transferred (1% inoculation amount) to 50 mL of liquid LB culture medium. After culture for 6 hours, the strain is transferred to the fermentation medium. After culture for 6 hours, IPTG and lactose are added, and fermentation is carried out for 60 hours to produce fermentation broth A.

[0035] Preparation method of fermentation broth B: β-1,4-galactosyltransferase is introduced during plasmid construction, and the plasmid is transferred into the Escherichia coli BL21 (DE3) strain, which produces lactose-N-neotetraose. Lactose and lactose-N-triose II are precursors for synthesizing neotetraose. The lactose-N-neotetraose (LNnT)-producing strain is inoculated into 5 mL of LB liquid culture medium, cultured for 15 hours, and then transferred (1% inoculation amount) to 50 mL of liquid LB culture medium. After culture for 6 hours, the strain is transferred to the fermentation medium. After culture for 6 hours, IPTG and lactose are added, and fermentation is carried out for 60 hours to produce fermentation broth B.

[0036] The LB medium includes 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast extract, pH 7.2; the fermentation medium includes 2 g / L yeast extract, 30 g / L glycerol, 15 g / L tryptone, 17.9 g / L Na2HPO4·12H2O, 15 mg / L CaCl2·2H2O, 3 g / L KH2PO4, 0.1 vt% Triton-X 100, 2 g / L NH4Cl, 10 mg / L VB1, 1 g / L (NH4)2HPO4, 1 g / L MgSO4·7H2O, 2.2 g / L trisodium citrate·2H2O, and 10 ml / L trace element I; the trace element formula I includes 13.74 g / L sodium nitrilotriacetic acid, 5.6 g / L ammonium ferric citrate, and ZnSO4·7H2O. 0.9g / L, CoCl2·6H2O 0.2g / L, CuCl2·2H2O 0.1g / L, MnCl2·4H2O 1g / L, Na2MoO4·2H2O 0.2g / L, boric acid 0.1g / L.

[0037] Example 1

[0038] A high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides, including lactose (Lac), lactose-N-triose II (LNT II), and lactose-N-tetraose (LNT), comprises the following steps:

[0039] (1) Prepare standard solutions of lactose (Lac), lactose-N-triose II (LNT II), and lactose-N-tetraose (LNT);

[0040] Take 10 mg of lactose-N-triose II (Carbosynth ≥ 99%), lactose-N-tetraose (Carbosynth-90%), and lactose (McLean-98%), respectively, and put them into a 5 mL volumetric flask. Add appropriate amount of pure water to dissolve them, and dilute to 5 mL to prepare 2 g / L standard solutions. Dilute the standard solutions step by step to prepare standard solutions with concentrations of 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, and 2 g / L, respectively.

[0041] (2) Sample A and the standard solution were subjected to high performance liquid chromatography. 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, and 2 g / L of the standard solution were taken, and the concentrations were sequentially subjected to high performance liquid chromatography for quantitative measurement from low to high. The HPLC spectrum results of the standard solution are shown in FIG. Figure 1-3As shown; the test sample A is a standard mixed solution containing 8.51g / L lactose, 4.64g / L lactose-N-triose II, and 2.13g / L lactose-N-tetraose. The HPLC spectrum results of the test sample A are as shown Figure 5 As shown; HPLC conditions are: Guard column: NH2, 5μm, 4.6x10mm, analytical column uses NH2, 5μm, 4.6x 300mm, mobile phase is 70% acetonitrile water isocratic elution, mobile phase speed is 1mL / min, column oven temperature is 40℃, injection volume is 10μL;

[0042] (3) The standard curve was drawn with the concentration (c) as the horizontal axis and the corresponding peak area (A) as the vertical axis. The standard curves of lactose, lactose-N-triose II (LNT II) and lactose-N-tetraose (LNT) standards were prepared. As shown in Table 1 below, the detection concentration range of lactose, lactose-N-triose II and lactose-N-tetraose was 0.2-2 g / L, and the concentration and peak area showed a good linear relationship;

[0043] Table 1 - Linear regression equation of standard solution and detection limit, quantification limit and linear range of the method

[0044] Compound Detection concentration range (g / L) <![CDATA[R 2 ]]> Linear regression equation lactose 0.2-2 0.9998 Y=77114.4X-4116.57 Lactose-N-triose II 0.2-2 0.9996 Y=48274X-2232.2 Lactose-N-tetraose 0.2-2 0.9997 Y=48446X+906.53

[0045] (4) Based on the standard curve, the lactose, lactose-N-triose II (LNT II) and lactose-N-tetraose (LNT) in the test sample A were qualitatively and quantitatively analyzed, as shown in FIG. Figure 5 As shown, the calculated lactose content in the test sample A was 8.48 g / L, the lactose-N-triose II content was 4.62 g / L, and the lactose-N-tetraose content was 2.11 g / L.

[0046] Example 2

[0047] A high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides, including lactose (Lac), lactose-N-triose II (LNT II), and lactose-N-neotetraose (LNnT), comprises the following steps:

[0048] (1) Prepare standard solutions of lactose, lactose-N-triose II (LNT II), and lactose-N-neotetraose (LNnT);

[0049] Take 10 mg of lactose-N-triose II (Carbosynth ≥ 99%), lactose-N-neotetraose (Carbosynth ≥ 99%), and lactose (McLean-98%), respectively, and put them into a 5 mL volumetric flask. Add appropriate amount of pure water to dissolve them, and dilute to 5 mL to prepare 2 g / L standard solutions. Dilute the standard solutions step by step to prepare standard solutions with concentrations of 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, and 2 g / L, respectively.

[0050] (2) Sample B and the standard solution were subjected to high performance liquid chromatography. 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, and 2 g / L of the standard solution were taken, and the concentrations were sequentially subjected to high performance liquid chromatography for quantitative measurement from low to high. The HPLC spectrum results of the standard solution are shown in FIG. Figure 1-2 , 4; test sample B is a standard mixed solution containing 1.4 1g / L lactose, 2.42g / L lactose -N- triose II, 1.13g / L lactose -N- neotetraose, the HPLC spectrum of the test sample B is as shown Figure 6 As shown; HPLC conditions are: Guard column: NH2, 5μm, 4.6x 10mm, analytical column using NH2, 5μm, 4.6x 300mm, mobile phase is 70% acetonitrile water isocratic elution, mobile phase speed is 1mL / min, column oven temperature is 40℃, injection volume is 10μL;

[0051] (4) The standard curve was drawn with the concentration (c) as the abscissa and the corresponding peak area (A) as the ordinate. The standard curves of lactose, lactose-N-triose II (LNT II) and lactose-N-neotetraose (LNnT) standards were prepared. As shown in Table 2 below, the detection concentration range of lactose, lactose-N-triose II and lactose-N-neotetraose was 0.2-2 g / L, and the concentration and peak area showed a good linear relationship;

[0052] Table 2 - Linear regression equation of standard solution and detection limit, quantification limit and linear range of the method

[0053] Compound Detection concentration range (g / L) <![CDATA[R 2 ]]> Linear regression equation lactose 0.2-2 0.9998 Y=77114.4X-4116.57 Lactose-N-triose II 0.2-2 0.9996 Y=48274X-2232.2 Lactose-N-neotetraose 0.2-2 0.9996 Y=45857X-176.99

[0054] (5) Based on the standard curve, the lactose, lactose-N-triose II (LNT II) and lactose-N-neotetraose (LNnT) in the test sample B were qualitatively and quantitatively analyzed, as shown in Figure 5. Figure 6As shown, the calculated lactose content in the test sample B was 1.38 g / L, the lactose-N-triose II content was 2.38 g / L, and the lactose-N-neotetraose content was 1.11 g / L.

[0055] Example 3

[0056] A high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 1, except that in step (4), test sample C is used instead of test sample A. Test sample C is a standard mixed solution containing 1.72 g / L lactose-N-triose II and 0.91 g / L lactose-N-tetraose. The HPLC spectrum of test sample C is as follows: Figure 7 As shown, the calculated content of lactose-N-triose II in the test sample C is 1.69 g / L, and the content of lactose-N-tetraose is 0.93 g / L.

[0057] Example 4

[0058] A high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 2, except that in step (4), the test sample D is used instead of the test sample A. The test sample D is a standard mixed solution containing 1.89 g / L lactose-N-triose II and 1.32 g / L lactose-N-neotetraose. The HPLC spectrum result of the test sample D is as follows: Figure 8 As shown, the content of lactose-N-triose II in the test sample D was calculated to be 1.87 g / L, and the content of lactose-N-neotetraose was 1.30 g / L.

[0059] Example 5

[0060] A high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 1, except that in step (4), fermentation broth A is used instead of test sample A. Fermentation broth A sample pretreatment: 2 mL of fermentation broth A is boiled in 100°C boiling water for 10 minutes to remove protein, and centrifuged at 12000 rpm / min and 4°C for 10 minutes to obtain a supernatant, which is filtered through a 0.22 μm microporous filter membrane into a high performance liquid chromatograph for analysis; the HPLC spectrum of fermentation broth A is shown as follows: Figure 9 shown.

[0061] Combined with the standard curve in Table 1 and Figure 9 , the calculated lactose content in the fermentation broth A sample was 2.15 g / L, the lactose-N-triose II content was 2.31 g / L, and the lactose-N-tetraose content was 2.72 g / L.

[0062] Example 6

[0063] A high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 2, except that in step (4), fermentation broth B is used instead of test sample A, and fermentation broth B is diluted 10 times before detection. Fermentation broth B sample pretreatment: 2 mL of fermentation broth B is taken, boiled in 100°C boiling water for 10 minutes to remove protein, centrifuged at 12000 rpm / min and 4°C for 10 minutes to obtain a supernatant, filtered through a 0.22 μm microporous filter membrane into a high performance liquid chromatograph for analysis; the HPLC spectrum results of fermentation broth B are as follows: Figure 10 shown.

[0064] Combined with the standard curve in Table 2 and Figure 10 , the calculated lactose content in the fermentation broth B sample was 21.35 g / L, the lactose-N-triose II content was 14.41 g / L, and the lactose-N-neotetraose content was 19.24 g / L.

[0065] Comparative Example 1

[0066] A high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 1, except that in step (2), the column oven temperature in the high performance liquid chromatography conditions is 60°C, and in step (4), the test sample A is replaced by the test sample E, which is a standard mixed solution containing 0.5 g / L lactose-N-triose II (LNT II) and 0.5 g / L lactose-N-tetraose (LNT).

[0067] like Figure 11 As shown in the figure, the column oven temperature is too high, resulting in no peak graph of the test sample within 5-20 minutes, affecting the detection accuracy and efficiency.

[0068] Comparative Example 2

[0069] A high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 2, except that in step (2), the column oven temperature in the high performance liquid chromatography conditions is 60°C, and in step (4), the test sample A is replaced by the test sample F, which is a standard mixed solution containing 0.5 g / L lactose-N-triose II (LNT II) and 0.5 g / L lactose-N-neotetraose (LNnT).

[0070] like Figure 12 As shown in the figure, the column oven temperature is too high, resulting in no peak graph of the test sample within 5-25 minutes, affecting the detection accuracy and efficiency.

[0071] Comparative Example 3

[0072] A high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides, wherein each step and the reagents and process parameters used in each step are the same as those in Example 2, except that, in step (2), the mobile phase in the high performance liquid chromatography conditions is 70% acetonitrile water, and ammonia water is added to adjust the pH to about 7.3; in step (4), the test sample A is replaced by the test sample G, and the test sample G is replaced by a 0.8 g / L lactose-N-neotetraose (LNnT) standard solution.

[0073] like Figure 13 As shown in the figure, under the acetonitrile-ammonia mobile phase, the peak shape of the lactose-N-neotetraose (LNnT) standard is asymmetric, and the peak time is 23.303 min. The detection concentration is 0.72 g / L. The detection time is long and the baseline is unstable, which is not conducive to detection and will affect the test results.

[0074] The specific embodiments described above further illustrate the objectives, technical solutions, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. In particular, it should be noted that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention for those skilled in the art.

Claims

1. A high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides, characterized in that: The preparation of a variety of human milk oligosaccharides including lactose, lactose-N-triose II and lactose-N-neotetraose involves the following steps: (1) Prepare standard solutions of lactose, lactose-N-triose II, or lactose-N-neotetraose at different concentrations; (2) The standard solution and the test sample are quantitatively measured by high performance liquid chromatography, a standard curve is drawn based on the test results of the standard solution, and the test sample is quantitatively analyzed by combining the standard curve and the spectrum of the standard solution; In step (2), the HPLC conditions are: Guard column: Xtimate ® NH2, 5μm, 4.6 x 10mm, analytical column using Xtimate ® NH2, 5μm, 4.6 x 300mm, mobile phase is 70% acetonitrile water isocratic elution, mobile phase speed is 1mL / min, column oven temperature is 40°C, injection volume is 10μL, detector is differential refractive index detector; In step (2), the detection concentration range of the detection sample is 0.2-2 g / L, and the detection sample is fermentation broth.

2. The high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides according to claim 1, wherein: The standard curve of lactose is Y=77114.4X-4116.57, with concentration as the abscissa and the corresponding peak area as the ordinate.

3. The high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides according to claim 1, wherein: The standard curve of the lactose-N-triose II is Y=48274X-2232.2, with concentration as the horizontal axis and the corresponding peak area as the vertical axis.

4. The high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides according to claim 1, wherein: The standard curve of the lactose-N-neotetraose is Y=45857X-176.99, with concentration as the abscissa and the corresponding peak area as the ordinate.

5. The high performance liquid chromatography method for simultaneously determining multiple human milk oligosaccharides according to claim 1, wherein: The concentrations of the standard solutions are 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L and 2 g / L.

6. Application of a high performance liquid chromatography detection method for simultaneously determining multiple human milk oligosaccharides according to any one of claims 1 to 5 in the food field.

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