High-coverage analysis method for human milk oligosaccharides based on liquid chromatography-high resolution mass spectrometry

Abstract

This invention relates to the field of human milk oligosaccharide (HMO) detection technology, and proposes a high-coverage analytical method for HMOs based on high-resolution liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The method includes the following steps: detecting HMOs in the sample solution using UPLC-HRMS, comparing fragment ions, retention times, and the precise mass number and isotopic distribution of the primary precursor ion to achieve structural identification of HMOs in the breast milk sample; the chromatographic conditions in the UPLC-HRMS method include: using 50 mmol / L ammonium formate aqueous solution as mobile phase A, using pure acetonitrile as mobile phase B, and gradient elution; the content of mobile phase B is expressed as a volume percentage. This technical solution solves the problems of low coverage and low sensitivity in HMO detection and analysis in related technologies.

Description

Technical Field

[0001] This invention relates to the field of human milk oligosaccharide detection technology, specifically to a high-coverage analysis method for human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant nutrient in breast milk after lactose and fat, and are a mixture of 3 to 10 monosaccharides. HMOs can promote the proliferation of beneficial gut microbiota in newborns, resist viruses, and improve cognitive function in infants and young children, playing an important role in their healthy growth. HMOs are composed of five basic sugar units: glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), fucose (Fuc), and N-acetylneuraminic acid (sialic acid, Neu5Ac). Due to differences in glycosyl composition, glycosidic bond linkage, and other factors, approximately 200 different HMO structures have been discovered to date. However, most current analytical methods for HMOs are limited to seven HMOs: 2'-fucosylvose (2'-FL), 3-fucosylvose (3-FL), lactose difucosylate (LDFT), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL), resulting in low HMO coverage.

[0003] Therefore, it is necessary to propose a high-coverage analytical method for human milk oligosaccharides (HMOs) that can achieve high coverage in HMO detection, good sensitivity, and stability. This is of great significance for laying the foundation for high-coverage analysis and regularity research of human milk oligosaccharides. Summary of the Invention

[0004] This invention proposes a high-coverage analysis method for human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry, which solves the problems of low coverage and low sensitivity in the detection and analysis of human milk oligosaccharides in related technologies.

[0005] The technical solution of the present invention is as follows:

[0006] This invention proposes a high-coverage analysis method for human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry, which includes the following steps: using UPLC-HRMS to detect human milk oligosaccharides in the sample solution, comparing fragment ions, retention time, and the precise mass number and isotope distribution of primary precursor ions to achieve structural identification of human milk oligosaccharides in the human milk sample;

[0007] The chromatographic conditions in the UPLC-HRMS method include: using 50 mmol / L ammonium formate aqueous solution as mobile phase A, using pure acetonitrile as mobile phase B, and using gradient elution. The content of mobile phase B is expressed as a volume percentage. The gradient elution program is as follows:

[0008] 0~32.0min, 85%~55%B; 32.0~32.2min, 55%~15%B; 32.2~36.0min, 15%B; 36.0~36.2min, 15%~85%B; 36.2~40.5min, 85%B.

[0009] As a further technical solution, the chromatographic conditions in the UPLC-HRMS method also include: column temperature 58~60℃.

[0010] As a further technical solution, the chromatographic conditions in the UPLC-HRMS method also include a flow rate of 0.3~0.4 mL / min.

[0011] As a further technical solution, the chromatographic conditions in the UPLC-HRMS method also include: injection volume of 1.8~2μL.

[0012] As a further technical solution, the mass spectrometry conditions in the UPLC-HRMS include: using electrospray ionization positive ion or electrospray ionization negative ion mode for mass spectrometry detection, and the electrospray ionization source parameters are: sheath gas flow rate of 39~40L / min.

[0013] As a further technical solution, the parameters of the electrospray ionization source also include: the temperature of the ion transmission tube is 315~320℃.

[0014] As a further technical solution, the parameters of the electrospray ionization source also include: an atomization temperature of 445~450℃.

[0015] As a further technical solution, the mass spectrometry detection method is Full-MS or data-dependent secondary scan mode dd-MS2, the collision energy CEs adopt step-normalized energy levels of 20eV, 40eV, and 60eV, and the first-order mass-to-charge ratio scan range is 300~2000.

[0016] When using the electrospray ionization positive ion mode for detection, the spray voltage is 3700V;

[0017] When using the electrospray ionization negative ion mode for detection, the spray voltage is 3000V.

[0018] As a further technical solution, the sample solution to be tested includes a breast milk sample, a breast milk oligosaccharide mixed standard solution, and a quality control sample solution.

[0019] As a further technical solution, the human milk oligosaccharide mixed standard solution is divided into mixed standard solution 1, mixed standard solution 2, mixed standard solution 3, mixed standard solution 4, and mixed standard solution 5.

[0020] The standards in the mixed standard 1 solution include: LNB, Lewis X triose, 3'-SG, 2'-FL, Le Ytetraose, LNTri, α1-3,β1-4 Galactotriose, 3'-SL, LNnT, α1-3,β1-4,α1-3 Galactotriose, 3'-SL-3-FL, LHFP I, LNDFH II, DSLNT, and DF-pLNnH;

[0021] The standards in the mixed standard 2 solution include: Blood group A triose, 3-FL, 4'-GL, Le Btetraose, Blood Group A tetraose type V, S-Le X, LDFT, GNT, 6'-SL, LNFP II, LST d, DSL, pLNH, F-LNH II and DF-LNnH;

[0022] The standards in the mixed standard 3 solution include: Lewis A triose, Blood group B triose, 6'-SG, 6'-SLN, 3'-GL, LNT, LNFP III, LST a, pLNnH, LNDFH I, F-pLNH III, DF-LNH a, LNnO, and LacNAC;

[0023] The standards in the mixed standard 4 solution include: GNB, 6'-GL, S-Le A, LNFP VI, LST b, LNnDFH I, LNH, S-LNFP V, DF-pLNH II, and TF-LNH;

[0024] The standards in the mixed standard 5 solution include: Blood Group A pentaose, LNFP V, Blood Group B pentaose, LST c, LNnH, LNnDFH II, F-LSTa, F-LNH I, α-Heptasaccharide, and DF-LNH b.

[0025] In this invention, the human milk oligosaccharide mixed standard solution includes a total of 64 HMOs, covering 16 isomers. The Chinese names, English names, abbreviations, CAS numbers, molecular formulas, and monoisotope mass numbers of these 64 HMOs are as follows:

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] As a further technical solution, the sample solution to be tested includes a breast milk sample, and the breast milk sample undergoes a sample pretreatment process before testing, specifically:

[0033] A1. After thawing the original breast milk sample in a water bath, add the mixed standard solution as an internal standard, mix well, and centrifuge to obtain the intermediate aqueous phase;

[0034] A2. Add acetonitrile to the intermediate aqueous phase, mix well, let stand, centrifuge to obtain the supernatant;

[0035] A3. The supernatant is freeze-dried under vacuum and then reconstituted to obtain a breast milk sample.

[0036] As a further technical solution, the mixed standard solution includes sucrose pentose with an internal standard concentration of 2.5 mg / g and sucrose octaose with an internal standard concentration of 0.125 mg / g.

[0037] As a further technical solution, the method for preparing the quality control sample solution includes the following steps: mixing the transition milk sample and the mature milk sample evenly, vortexing for 10 minutes to obtain the quality control sample solution.

[0038] In this invention, the transitional milk sample and mature milk sample were obtained from the transitional milk and mature milk of 19 mothers from Harbin Shangde Hospital and Qiqihar Jianhua Hospital. All the transitional milk and mature milk of these 19 mothers were mixed and vortexed for 10 minutes, and then dispensed into 1.5 mL tubes on average.

[0039] As a further technical solution, the preparation method of the human milk oligosaccharide mixed standard solution includes the following steps:

[0040] B1. Weigh LNB, Lewis triose, 3-FL, 4'-GL, Le B tetraose, BloodGroup A tetraose type V, S-Le a, pLNnH, LNDFH I, F-pLNH III. Add 1.0 mg each of DF-LNH a, LNnO, and LacNAC standards to ultrapure water to prepare a single-standard stock solution with a mass concentration of 1 mg / g, and store at -20℃ for later use;

[0041] B2. Weigh 0.10 mg each of GNB, 6'-GL, S-Le A, LNFP VI, LST b, LNnDFH I, LNH, S-LNFP V, DF-pLNH II, TF-LNH, Blood group A pentaose, LNFP V, Blood Group B pentaose, LST c, LNnH, LNnDFH II, F-LSTa, F-LNH I, α-Heptasaccharide, and DF-LNH b standards, add them to ultrapure water, and prepare a single-standard stock solution with a mass concentration of 0.1 mg / g. Store at -20℃ for later use.

[0042] B3. Take 20 μL each of the single standard stock solutions of LNB, Lewis X triose, 3'-SG, 2'-FL, Le Y tetraose, LNTri, α1-3,β1-4Galactotriose, 3'-SL, LNnT, α1-3,β1-4,α1-3 Galactotetraose, 3'-SL-3-FL, LHFP I, LNDFH II, DSLNT, and DF-pLNnH, add ultrapure water, and prepare mixed standard solution 1 with a mass concentration of 67 ppm;

[0043] B4. Take 20 μL of each of the single standard stock solutions of Blood Group A triose, 3-FL, 4'-GL, Le B tetraose, Blood Group Atetraose type V, S-Le X, LDFT, GNT, 6'-SL, LNFP II, LST d, DSL, pLNH, F-LNH II and DF-LNnH, add ultrapure water, and prepare mixed standard 2 solution with a mass concentration of 67 ppm.

[0044] B5. Take 20 μL of each of the single standard stock solutions of Lewis A triose, Blood group B triose, 6'-SG, 6'-SLN, 3'-GL, LNT, LNFP III, LST a, pLNnH, LNDFH I, F-pLNH III, DF-LNH a, LNnO, and LacNAC, add ultrapure water, and prepare a mixed standard solution with a mass concentration of 67 ppm.

[0045] B6. Take 40 μL of each of the single standard stock solutions of GNB, 6'-GL, S-Le A, LNFP VI, LST b, LNnDFH I, LNH, S-LNFP V, DF-pLNHII, and TF-LNH, add ultrapure water, and prepare a mixed standard 4 solution with a mass concentration of 10 ppm.

[0046] B7. Take 40 μL of each of the single standard stock solutions of Blood Group A pentaose, LNFP V, Blood Group B pentaose, LST c, LNnH, LNnDFH II, F-LSTa, F-LNH I, α-Heptasaccharide, and DF-LNH b, add ultrapure water, and prepare a mixed standard solution with a mass concentration of 10 ppm.

[0047] As a further technical solution, the comparison of fragment ions includes comparison in positive ion mode and comparison in negative ion mode.

[0048] As a further technical solution, in the comparison under the positive ion mode, the addendum ions include [M+H]. + [M+NH4] + [M+Na] + [M+2NH4] 2+ [M+2Na] 2+ Addition ions;

[0049] In the comparison under the negative ion mode, the added ions include [MH]. - [M-2H] 2-and [M+HCO2] - Addition ions.

[0050] In this invention, the inventors identified 268 types of human milk oligosaccharides by their English names, abbreviations, molecular formulas, monoisotope mass numbers, and their corresponding [M+H] values. + [M+NH4] + [M+Na] + [M+2NH4] 2+ [M+2Na] 2+ [MH] - [M+HCOO] - The precise mass numbers are imported into TraceFinder to form a self-built database. After detecting human milk oligosaccharides in the sample solution using UPLC-HRMS, the structure of human milk oligosaccharides in breast milk samples can be effectively identified by comparing fragment ions, retention times, and the precise mass numbers and isotopic distribution of primary precursor ions. Since double-charged ions frequently appear when the molecular weight is large, [M+2NH4] is added for human milk oligosaccharides with a molecular weight >800. 2+ [M+2Na] 2+ [M-2H] 2- The addition method is used for HMOs whose glycosidic bond linkage is not clearly defined but whose monosaccharide composition is known. They are named using the notation "N / A5330", where "N" represents neutral oligosaccharide, "A" represents acidic oligosaccharide, 5 represents the number of hexose groups, 3 represents the number of fucose groups, 3 represents the number of N-acetylglucosamine groups, and 0 represents the number of sialic acid groups. Information on these 268 human milk oligosaccharides is as follows:

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] In this invention, the neutral oligosaccharide is in positive ion mode [M+Na] + In negative ion mode [MH] - The response is high. However, oligosaccharides with larger molecular weights [MH] exhibit this characteristic. - The response is much weaker than [M+Na]. + Because of the presence of ions, neutral oligosaccharides are typically chosen to be [M+Na]. + [MH] was selected as the optimal adduct ion for analysis. Conversely, acidic oligosaccharides such as 3'-SL, 6'-SL, and DSL showed high responses in both positive and negative ion modes, but the response in the positive ion mode decreased with increasing degree of polymerization and molecular weight. For example, DSLNT did not have a high-response adduct ion in the positive ion mode. Therefore, [MH] was chosen as the optimal acidic oligosaccharide. - It was analyzed as the optimal addit ion.

[0067] Oligosaccharides from human milk with a molecular weight close to 1000 or greater typically exhibit a higher frequency of double charge. Almost all human milk oligosaccharides with a molecular weight greater than 1500 [M+2Na] are oligosaccharides. 2+ The response has become the optimal additive ion, and it increases with increasing mass number [M + 2Na]. 2+ It has become the dominant addition ion, with a strength far exceeding that of single-charged ions. Combined with experimental findings, it has been discovered that ions with mass numbers close to 1000, such as LNH and DSL, also exhibit [M+2Na]. 2+ And its strength is similar to that of [M+Na]. + They are evenly matched.

[0068] The working principle and beneficial effects of this invention are as follows:

[0069] This invention establishes a high-coverage analytical method for HMOs using ultra-high performance liquid chromatography-tandem Orbitrap mass spectrometry (UPLC-HRMS). A specific chromatographic elution program is employed, combined with HRMS to acquire HMO information. By comparing fragment ions, retention times, and the precise mass number of the primary precursor ion, the structure of oligosaccharides in the sample is identified. This method can analyze 186 HMOs and exhibits good sensitivity, precision, and stability, laying a technical foundation for high-coverage analysis and regularity research of human milk oligosaccharides. Using 50 mmol / L ammonium formate aqueous solution as mobile phase A and pure acetonitrile as mobile phase B, along with a specific elution program, significantly improves the accuracy of HMO structure identification, avoids peak overlap, and prevents the difficulty in distinguishing HMO structures based solely on retention time. Attached Figure Description

[0070] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0071] Figure 1 This is a first-order mass spectrum in positive ion mode of solution 1 of the HMOs mixed standard solution in Example 1;

[0072] Figure 2 This is a first-order mass spectrum in positive ion mode for each component in the mixed standard solution 1 of the HMOs mixed standard solution in Example 1;

[0073] Figure 3 This is a first-order mass spectrum in positive ion mode of solution 2 of the HMOs mixed standard solution in Example 1;

[0074] Figure 4 This is a first-order mass spectrum in positive ion mode for each component in the mixed standard solution 2 of the HMOs mixed standard solution in Example 1;

[0075] Figure 5 This is a first-order mass spectrum in positive ion mode of solution 3 of the HMOs mixed standard solution in Example 1;

[0076] Figure 6 This is a first-order mass spectrum in positive ion mode for each component in the mixed standard solution 3 of the HMOs mixed standard solution in Example 1;

[0077] Figure 7 This is a first-order mass spectrum in positive ion mode of solution 4 of the HMOs mixed standard solution in Example 1;

[0078] Figure 8 This is a first-order mass spectrum in positive ion mode for each component in the mixed standard solution 4 of the HMOs mixed standard solution in Example 1;

[0079] Figure 9 This is a first-order mass spectrum in positive ion mode of the mixed standard solution 5 in the HMOs mixed standard solution in Example 1;

[0080] Figure 10 This is a first-order mass spectrum in positive ion mode for each component in the mixed standard solution 5 of the HMOs mixed standard solution in Example 1;

[0081] Figure 11 Mass spectra of isomers LNT and LNnT under the chromatographic conditions of Comparative Example 1;

[0082] Figure 12 Mass spectra of isomers LNT and LNnT under the chromatographic conditions of Example 1;

[0083] Figure 13 Mass spectra of isomers F-LNH Ⅰ, F-LNH Ⅱ and F-pLNH Ⅲ under the chromatographic conditions of Comparative Example 1;

[0084] Figure 14 Mass spectra of isomers F-LNH Ⅰ, F-LNH Ⅱ and F-pLNH Ⅲ under the chromatographic conditions of Example 1;

[0085] Figure 15 Mass spectra of isomers DF-LNH a, DF-LNnH, DF-LNH b, DF-pLNH Ⅱ and DF-pLNnH under the chromatographic conditions of Comparative Example 1;

[0086] Figure 16 Mass spectra of isomers DF-LNH a, DF-LNnH, DF-LNH b, DF-pLNH Ⅱ and DF-pLNnH under the chromatographic conditions of Example 1;

[0087] Figure 17 For positive ion mode QC samples, the signal intensity reference value NL is 1.30 × 10⁻⁶. 9 Total ion current of TIC Figure 1 ;

[0088] Figure 18 For positive ion mode QC samples, the signal intensity reference value NL is 1.30 × 10⁻⁶. 9 Total ion current of TIC Figure 2 ;

[0089] Figure 19 For positive ion mode QC samples, the signal intensity reference value NL is 1.50 × 10⁻⁶. 9 Total ion current of TIC Figure 3 ;

[0090] Figure 20For the negative ion mode QC sample, the signal intensity reference value NL is 3.07 × 10⁻⁶. 9 Total ion current of TIC Figure 1 ;

[0091] Figure 21 For the negative ion mode QC sample, the signal intensity reference value NL is 3.06 × 10⁻⁶. 9 Total ion current of TIC Figure 2 ;

[0092] Figure 22 For the negative ion mode QC sample, the signal intensity reference value NL is 3.14 × 10⁻⁶. 9 Total ion current of TIC Figure 3 ;

[0093] Figure 23 PCA analysis chromatograms of HM9-2423, HM9-2431 and quality control sample solutions are shown; PC1 on the horizontal axis represents principal component 1 and PC2 on the vertical axis represents principal component 2.

[0094] Figure 24 The total ion chromatogram of the quality control sample solution in positive ion mode;

[0095] Figure 25 This is the total ion chromatogram of the quality control sample solution in negative ion mode. Detailed Implementation

[0096] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0097] In the following examples, comparative examples, and experimental cases, the quality control sample solution is also referred to as the QC sample solution;

[0098] The original breast milk samples HM9-2423 and HM9-2431 were obtained from healthy lactating mother volunteers in the Beijing area.

[0099] Example 1

[0100] The high-coverage analysis method for human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry includes the following steps: the human milk oligosaccharides in the sample solution are detected by UPLC-HRMS, and the structure of human milk oligosaccharides in the human milk sample is identified by comparing fragment ions, retention time, and the precise mass number and isotope distribution of the primary precursor ion.

[0101] The chromatographic conditions for the UPLC-HRMS method are as follows:

[0102] Chromatographic conditions in UPLC-HRMS include:

[0103] Column temperature 60℃;

[0104] Flow rate: 0.4 mL / min;

[0105] Injection volume: 2 μL;

[0106] Using 50 mmol / L ammonium formate aqueous solution as mobile phase A and pure acetonitrile as mobile phase B, the contents of mobile phase A and mobile phase B are expressed as volume percentages. The elution method is gradient elution, and the gradient elution program is as follows:

[0107] 0~32.0min, 85%~55%B; 32.0~32.2min, 55%~15%B; 32.2~36.0min, 15%B; 36.0~36.2min, 15%~85%B; 36.2~40.5min, 85%B.

[0108] Throughout the analysis, the sample solution was placed in an autosampler at 15°C.

[0109] After separation by liquid chromatography, the samples were analyzed by mass spectrometry using a Q Exactive plus mass spectrometer. Electrospray ionization (ESI) positive ion and electrospray ionization (ESI) negative ion modes were employed for mass spectrometry detection. The ESI source parameters were as follows: for ESI positive ion mode, the spray voltage was 3700 V (ESI+); for ESI negative ion mode, the spray voltage was 3000 V (ESI-); the sheath gas flow rate was 40 L / min; the ion transfer tube temperature was 320 °C; and the nebulization temperature was 450 °C. A full scan / data-dependent two-stage scan (Full-MS / dd-MS2) mode was used, with collision energies (CEs) set to normalized levels of 20 eV, 40 eV, and 60 eV; the first-order mass-to-charge ratio scan range was 300–2000.

[0110] The preparation method of the quality control sample solution includes the following steps: the transition milk sample and the mature milk sample (taken from the transition milk and mature milk of 19 mothers from Harbin Shangde Hospital and Qiqihar Jianhua Hospital) are mixed evenly and vortexed for 10 min to obtain the quality control sample solution.

[0111] The preparation method of the human milk oligosaccharide mixed standard solution (HMOs mixed standard solution) includes the following steps:

[0112] B1. Weigh LNB, Lewis triose, 3-FL, 4'-GL, Le B tetraose, BloodGroup A tetraose type V, S-Le a, pLNnH, LNDFH I, F-pLNH III. Add 1.0 mg each of DF-LNH a, LNnO, and LacNAC standards to ultrapure water to prepare a single-standard stock solution with a mass concentration of 1 mg / g, and store at -20℃ for later use;

[0113] B2. Weigh 0.10 mg each of GNB, 6'-GL, S-Le A, LNFP VI, LST b, LNnDFH I, LNH, S-LNFP V, DF-pLNH II, TF-LNH, Blood group A pentaose, LNFP V, Blood Group B pentaose, LST c, LNnH, LNnDFH II, F-LSTa, F-LNH I, α-Heptasaccharide, and DF-LNH b standards, add them to ultrapure water, and prepare a single-standard stock solution with a mass concentration of 0.1 mg / g. Store at -20℃ for later use.

[0114] B3. Take 20 μL each of the single standard stock solutions of LNB, Lewis X triose, 3'-SG, 2'-FL, Le Y tetraose, LNTri, α1-3,β1-4Galactotriose, 3'-SL, LNnT, α1-3,β1-4,α1-3 Galactotetraose, 3'-SL-3-FL, LHFP I, LNDFH II, DSLNT, and DF-pLNnH, add ultrapure water, and prepare mixed standard solution 1 with a mass concentration of 67 ppm;

[0115] B4. Take 20 μL of each of the single standard stock solutions of Blood Group A triose, 3-FL, 4'-GL, Le B tetraose, Blood Group Atetraose type V, S-Le X, LDFT, GNT, 6'-SL, LNFP II, LST d, DSL, pLNH, F-LNH II and DF-LNnH, add ultrapure water, and prepare mixed standard 2 solution with a mass concentration of 67 ppm.

[0116] B5. Take 20 μL of each of the single standard stock solutions of Lewis A triose, Blood group B triose, 6'-SG, 6'-SLN, 3'-GL, LNT, LNFP III, LST a, pLNnH, LNDFH I, F-pLNH III, DF-LNH a, LNnO, and LacNAC, add ultrapure water, and prepare a mixed standard solution with a mass concentration of 67 ppm.

[0117] B6. Take 40 μL of each of the single standard stock solutions of GNB, 6'-GL, S-Le A, LNFP VI, LST b, LNnDFH I, LNH, S-LNFP V, DF-pLNHII, and TF-LNH, add ultrapure water, and prepare a mixed standard 4 solution with a mass concentration of 10 ppm.

[0118] B7. Take 40 μL of each of the single standard stock solutions of Blood Group A pentaose, LNFP V, Blood Group B pentaose, LST c, LNnH, LNnDFH II, F-LSTa, F-LNH I, α-Heptasaccharide, and DF-LNH b, add ultrapure water, and prepare a mixed standard solution with a mass concentration of 10 ppm.

[0119] The process of preparing breast milk samples for testing includes sample pretreatment, specifically:

[0120] A1. After thawing the original breast milk sample HM9-2423 in a water bath at 4℃, 500mL of the sample was transferred to an EP tube, and 10uL of a mixed standard solution was added as an internal standard. The mixture was thoroughly mixed and centrifuged at 4℃ and 10000r / min for 20min to obtain the intermediate aqueous phase. The mixed standard solution contained 2.5mg / g of fructooligosaccharide and 0.125mg / g of fructooligosaccharide as internal standards.

[0121] A2. Add 1 mL of acetonitrile to the intermediate aqueous phase, mix well, let stand in a -20℃ refrigerator for 1 h, and centrifuge at 4℃ and 10000 r / min for 10 min to obtain the supernatant.

[0122] A3. The supernatant was freeze-dried under vacuum and reconstituted with 200 μL of pure water to obtain breast milk sample HM9-2423.

[0123] The pretreatment process for breast milk sample HM9-2431 is the same as that for breast milk sample HM9-2423 before testing.

[0124] Test 1: Inject solutions 1, 2, 3, 4, and 5 of the HMOs mixed standard solution into the sample respectively, and record the first-order mass spectrum in positive ion mode. The first-order mass spectrum of solution 1 in positive ion mode is shown below. Figure 1 As shown, the first-order mass spectra of each component in the mixed standard 1 solution in positive ion mode are as follows: Figure 2 Shown (LNB, 3'-SG, 2'-FL, α1-3,β1-4 Galactotriose, Lewis Figure 2 (The corresponding mass spectrum is marked in the image); the first-order mass spectrum of the mixed standard 2 solution in positive ion mode is shown below. Figure 3 As shown, the first-order mass spectra of each component in the mixed standard 2 solution under positive ion mode are as follows: Figure 4 Shown (3-FL, 4'-GL, Blood group Atriose, 6'-SL, LDFT, Le B tetraose, Blood Group A tetraose type V, GNT, S-Le Figure 4 (The corresponding mass spectrum is marked in the image); the primary mass spectrum of the mixed standard 3 solution in positive ion mode is shown below. Figure 5 As shown, the first-order mass spectra of each component in the mixed standard 3 solution under positive ion mode are as follows: Figure 6 As shown (LacNAc, 6'-SG, Blood group B triose, 3'-GL, Lewis A triose, 6'-SLN, LNT, LNFP III, LST a, LNDFH I, pLNnH, F-pLNH III, DF-LNH a, LNnO have been... Figure 6 (The corresponding mass spectrum is marked in the image); the primary mass spectrum of the mixed standard 4 solution in positive ion mode is shown below. Figure 7 As shown, the first-order mass spectra of each component in the mixed standard 4 solution in positive ion mode are as follows: Figure 8 As shown (GNB, 6'-GL, S-Le A, LNFP VI, LST b, LNnDFH I, LNH, S-LNFP V, DF-pLNH II, TF-LNH have been...), Figure 8 (The corresponding mass spectrum is annotated in the image); the primary mass spectrum of the mixed standard 5 solution in positive ion mode is shown below. Figure 9 As shown, the first-order mass spectra of each component in the mixed standard 5 solution in positive ion mode are as follows: Figure 10 Shown (Blood group A pentaose, Blood Group B pentaose, LNFP V, LST c, LNnDFH II, LNnH, F-LSTa, a-Heptasaccharide, F-LNH I, DF-LNH b have been Figure 10 (The corresponding mass spectrum is annotated in the image).

[0125] The retention times of 64 HMOs in the mixed standard solution of HMOs in positive ion mode and negative ion mode are shown in Table 1, and the detection limits of 64 HMOs in positive ion mode are shown in Table 2.

[0126] Table 1. Retention times of 64 HMOs in positive and negative ion modes.

[0127]

[0128]

[0129] Table 2. Detection limits of 64 HMOs in positive ion mode

[0130]

[0131]

[0132] As shown in Tables 1 and 2, the detection method has high sensitivity, with the detection limit for most HMOs below 100 ppb. By observing the retention time of the isomers, it was found that many isomers, such as LNB, 3'-SG, 2'-FL, 3'-SL, Le Y tetraose, S-Le X, and S-LNFP V, can be well separated by chromatography. However, the separation of some isomers, such as LST a, LNDFHI, LNH, F-LNHI, and DF-LNH a, which have a high degree of polymerization, is relatively poor.

[0133] Databases for positive and negative ion modes were established in TraceFinder. The abbreviations and chemical formulas of HMOs, along with fragment ions and retention times obtained from high-resolution mass spectrometry data of 64 HMO standards, were imported into the databases. Comparisons of fragment ions under positive and negative ion modes were then performed. The positive ion mode covers [M+H]. + [M+NH4] + [M+Na] + [M+2NH4] 2+ [M+2Na] 2+ Five additive ions; negative mode coverage [MH] - [M-2H] 2- and [M+HCO2] - Three adduct ions; detection of HMO types in breast milk samples;

[0134] The information on HMOs identified under the chromatographic conditions and positive ion mode in Example 1 is shown in Table 3.

[0135] The information on HMOs identified under the chromatographic conditions and negative ion mode in Example 1 is shown in Table 4.

[0136] Example 2

[0137] The only difference between this embodiment and Example 1 is that, in this embodiment, the chromatographic conditions for the UPLC-HRMS method are: column temperature 58℃; flow rate 0.3 mL / min; and injection volume 1.8 μL.

[0138] In the UPLC-HRMS mass spectrometry conditions, the sheath gas flow rate was 39 L / min, the ion transfer tube temperature was 315 °C, and the nebulization temperature was 445 °C.

[0139] Comparative Example 1

[0140] The only difference between this comparative example and Example 1 is that the chromatographic conditions in the UPLC-HRMS method are different in this comparative example, specifically:

[0141] Chromatographic conditions in UPLC-HRMS include:

[0142] Column temperature 60℃;

[0143] Flow rate: 0.4 mL / min;

[0144] Injection volume: 4 μL;

[0145] A 10 mmol / L ammonium formate aqueous solution (containing 0.1 wt% formic acid) was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The contents of mobile phase A and mobile phase B were expressed as volume percentages. The elution method was gradient elution, and the gradient elution program was as follows:

[0146] 0~35.0min, 85%~55%B; 35.0~36.5min, 55%~10%B; 36.5~39.5min, 10%B; 39.5~43.1min, 10%~85%B; 43.1~55.0min, 85%B.

[0147] Throughout the analysis, the sample solution was placed in an autosampler at 4°C.

[0148] The information of HMOs identified under the chromatographic conditions and positive ion mode in Comparative Example 1 is shown in Table 3.

[0149] The HMOs identified under the chromatographic conditions and negative ion mode in Comparative Example 1 are shown in Table 4.

[0150] Table 3. Information on HMOs identified under two chromatographic conditions in Example 1 and Comparative Example 1 in positive ion mode.

[0151]

[0152]

[0153] Table 4. HMOs identified under two chromatographic conditions in Example 1 and Comparative Example 1 in negative ion mode.

[0154]

[0155]

[0156] The types of HMOs can be identified by their precise mass numbers. Then, based on the number of peaks eluted under the chromatographic conditions of Example 1 and Comparative Example 1, the number of HMO isomers can be obtained.

[0157] The results showed that, using the same screening process and with strict manual verification of the results, 104 HMOs were identified in positive ion mode and 97 HMOs were identified in negative ion mode in Comparative Example 1, with 67 HMOs identified repeatedly in both positive and negative modes, for a total of 134 HMOs identified; in Example 1, 126 HMOs were identified in positive ion mode and 130 HMOs were identified in negative ion mode, with 70 HMOs identified repeatedly in both positive and negative modes, for a total of 186 HMOs identified.

[0158] In addition, the separation effects of LNT and LNnT isomers under the chromatographic conditions of Comparative Example 1 and Example 1 in negative ion mode are as follows: Figure 11 (Mass-to-charge ratio m / z = 707.24841) Figure 12(Mass-to-charge ratio m / z = 707.24841) As shown, the separation effects of the F-LNH I, F-LNHII, and F-pLNH III isomers under the chromatographic conditions of Comparative Example 1 and Example 1 are as follows: Figure 13 (Mass-to-charge ratio m / z = 1218.43851) Figure 14 (Mass-to-charge ratio m / z = 1218.43851) As shown, the separation effects of the isomers of DF-LNH a, DF-LNH b, DF-pLNH II, DF-LNnH and DF-pLNnH under the chromatographic conditions of Comparative Example 1 and Example 1 are as follows: Figure 15 (Mass-to-charge ratio m / z = 1364.49642) and Figure 16 As shown in the figure (mass-to-charge ratio m / z=1364.49642), comparing the separation of the same isomers by the two chromatographic methods, the isomer separation effect under the chromatographic conditions of Example 1 is the best.

[0159] Experiment Example 1: Methodological Validation

[0160] The analytical method of Example 1 was verified by instrument stability and repeatability experiments.

[0161] 1. Instrument stability

[0162] The preparation method of the quality control sample solution is the same as in Example 1.

[0163] Testing: The quality control sample solution was tested using the UPLC-HRMS method, and the test conditions were the same as in Example 1.

[0164] The total ion chromatogram (TIC) of the quality control sample solution was repeatedly detected three times in positive ion mode, as shown below. Figures 17-19 As shown;

[0165] The total ion chromatogram (TIC) of the quality control sample solution was repeatedly measured three times under negative ion mode, as shown below. Figures 20-22 As shown;

[0166] Analysis: By comparing the TIC spectra of the quality control sample solution under different mass spectrometry modes, the response intensity and retention time of each chromatographic peak basically overlapped, indicating that the instrument response was stable throughout the experiment, the variation caused by instrument error was small, and the experimental data results were reliable.

[0167] 2. PCA chart

[0168] UPLC-HRMS was used to detect human milk oligosaccharides in breast milk samples HM9-2423, HM9-2431, and quality control sample solutions. The test conditions were the same as in Example 1. After detection, principal component analysis (PCA) was performed. Figure 23As shown in the figure (2423 represents breast milk sample HM9-2423, 2431 represents breast milk sample HM9-2431, and QC represents quality control sample solution), the experimental results show that the quality control sample solution group can be tightly aggregated in both positive and negative ion modes, indicating that the experiment has good repeatability.

[0169] 3. Method precision

[0170] The quality control sample solution was tested using UPLC-HRMS under the same conditions as in Example 1. The test was repeated three times, and the peak areas of the six HMOs were statistically analyzed. The results are shown in Table 5.

[0171] Table 5 Precision test results

[0172]

[0173] The total ion chromatogram of the quality control sample solution in positive ion mode is shown below. Figure 24 As shown, the total ion chromatogram of the quality control sample solution in negative ion mode is as follows. Figure 25 As shown in Table 6, the detailed peak areas of HMOs identified in the quality control sample solutions under positive and negative ion modes are presented in Table 6.

[0174] Table 6. Detailed peak areas of HMOs identified in quality control sample solutions in positive and negative ion modes.

[0175]

[0176]

[0177] Table 6 shows that most of the neutral oligosaccharides present in high amounts in breast milk have 1 to 5 substituents, but the most common are mono- and di-fucosylated neutral oligosaccharides. Conversely, acidic oligosaccharides, such as 3'-SL and LST a monosialic acid-substituted oligosaccharides, are more abundant in breast milk, while di- or tri-substituted oligosaccharides are extremely rare. Oligosaccharides with a degree of polymerization >10, such as TF-LNO and DF-LND, are also relatively common in breast milk, indicating that compounds with even higher degrees of polymerization may still exist, requiring further exploration of new structures.

[0178] Experimental Example 2: Detection of Actual Breast Milk Samples

[0179] 1. Statistical analysis of the proportion of oligosaccharide response intensity in the identification results

[0180] To more intuitively and concisely identify the HMOs present in high amounts in breast milk, a treemap was constructed based on the proportions of their group and hierarchical relationships for all identified oligosaccharides. The response intensity of each oligosaccharide compound was displayed by its area size.

[0181] Results analysis: The types of human milk oligosaccharides with high content in breast milk samples HM9-2423 and HM9-2431 were completely consistent. Therefore, it can be inferred that the HMOs with high content in breast milk should include: 2'-FL, 3-FL, 3'-GL, LNnT, LeB tetraos, LNFP III, iso-LNnFP, LDFT, LNDFH II, DF-pLNH II, F-pLNH III and TF-pLNH, a total of 12 types.

[0182] 2. Analysis of differences between groups

[0183] (1) Univariate statistical analysis

[0184] Univariate statistical analysis is one of the most commonly used statistical analysis methods. Based on univariate analysis, differential analysis is performed on all detected oligosaccharides (including unidentified oligosaccharides).

[0185] Based on differential oligosaccharides with FC>1.5 or FC<0.67 and p value<0.05, the results showed that compared with the breast milk sample HM9-2431 group, 34 HMOs were downregulated and 21 HMOs were upregulated at higher levels in the breast milk sample HM9-2423 group.

[0186] (2) Differential oligosaccharide bioinformatics analysis - cluster analysis

[0187] Subsequent bioinformatics analyses, including cluster analysis and correlation analysis, were performed on the selected oligosaccharides with significant differences (the oligosaccharides must simultaneously meet the criteria of OPLS-DA VIP>1 and p value<0.05, and have a qualitative name).

[0188] To show the relationship between the test samples and the differences in oligosaccharide content in different test samples, the content of all test samples and differential oligosaccharides was subtracted from the average value of their respective groups and then divided by the root mean square of the group number for standardization. Then, the distance matrix was calculated and hierarchical clustering was used for cluster analysis.

[0189] Cluster analysis results showed that there were significant differences in the composition of breast milk samples HM9-2431 and HM9-2423. The established method has the advantages of high efficiency and high coverage, laying the foundation for subsequent research on the variation law of breast milk oligosaccharides.

[0190] In addition, cluster analysis results showed that the content of different human milk oligosaccharides differed significantly between breast milk samples HM9-2431 and HM9-2423, indicating that these two groups of breast milk may belong to the secretory phase and non-secretive phase, respectively. The content of human milk oligosaccharides can be affected by many factors such as region, secretory phase, blood type and diet.

[0191] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-coverage analytical method for human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: The UPLC-HRMS method was used to detect human milk oligosaccharides in the sample solution. By comparing fragment ions, retention time, and the precise mass number and isotope distribution of the primary precursor ion, the structure of human milk oligosaccharides in the human milk sample was identified. The identified human milk oligosaccharides numbered 186. The chromatographic conditions in the UPLC-HRMS method include: using 50 mmol / L ammonium formate aqueous solution as mobile phase A, using pure acetonitrile as mobile phase B, and using gradient elution. The content of mobile phase B is expressed as a volume percentage. The gradient elution program is as follows: 0~32.0min, 85%~55%B; 32.0~32.2min, 55%~15%B; 32.2~36.0min, 15%B; 36.0~36.2min, 15%~85%B; 36.2~40.5min, 85%B.

2. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 1, characterized in that, The chromatographic conditions in the UPLC-HRMS method also include: column temperature 58~60℃.

3. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 1, characterized in that, The chromatographic conditions in the UPLC-HRMS method also include a flow rate of 0.3~0.4 mL / min.

4. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 1, characterized in that, The chromatographic conditions in the UPLC-HRMS method also include: injection volume of 1.8~2 μL.

5. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 1, characterized in that, The mass spectrometry conditions in the UPLC-HRMS method include: mass spectrometry detection using electrospray ionization positive ion or electrospray ionization negative ion mode, and the electrospray ionization source parameters include: sheath gas flow rate of 39~40 L / min.

6. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 5, characterized in that, The parameters of the electrospray ionization source also include: the temperature of the ion transport tube is 315~320℃.

7. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 5, characterized in that, The parameters of the electrospray ionization source also include: atomization temperature of 445~450℃.

8. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 1, characterized in that, The sample solution to be tested includes breast milk samples, and the breast milk samples undergo a sample pretreatment process before testing, specifically: A1. After thawing the original breast milk sample in a water bath, add the mixed standard solution as an internal standard, mix well, and centrifuge to obtain the intermediate aqueous phase; A2. Add acetonitrile to the intermediate aqueous phase, mix well, let stand, centrifuge to obtain the supernatant; A3. The supernatant is freeze-dried under vacuum and then reconstituted to obtain a breast milk sample.

9. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 1, characterized in that, The comparison of fragment ions includes comparison in positive ion mode and comparison in negative ion mode.

10. The method for high-coverage analysis of human milk oligosaccharides based on high-resolution liquid chromatography-mass spectrometry according to claim 9, characterized in that, In the contrast under the positive ion mode, the adduct ions include [M+H]. + [M+NH4] + [M+Na] + [M+2NH4] 2+ [M+2Na] 2+ Addition ions; In the comparison under the negative ion mode, the added ions include [MH]. - [M-2H] 2- and [M+HCO2] - Addition ions.

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