Method for detecting oligosaccharide in breast milk through ion exchange chromatography tandem mass spectrometry

Abstract

The invention relates to the technical field of detection of breast milk oligosaccharides, and provides a method for detecting breast milk oligosaccharides by ion exchange chromatography tandem mass spectrometry, which comprises the following steps: detecting a breast milk sample by ion exchange chromatography tandem mass spectrometry to obtain at least 42 breast milk oligosaccharides. According to the technical scheme, the problem that high-throughput detection of breast milk oligosaccharide is difficult to realize in related technologies is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of human milk oligosaccharide detection, and in particular to a method for detecting human milk oligosaccharides using ion exchange chromatography tandem mass spectrometry. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third most abundant nutrient in breast milk, after lactose and fat. They are a mixture of 3 to 10 monosaccharides. They promote the proliferation of beneficial intestinal flora in newborns, protect against viruses, and enhance cognitive function in infants and young children, playing a vital 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 glycosylation composition and glycosidic linkage, approximately 200 different HMO structures have been discovered to date.

[0003] Because human milk oligosaccharides lack chromophores, have large molecular weights, and exhibit low sensitivity, traditional detectors such as ultraviolet (UV) and fluorescence are no longer suitable. In recent years, researchers have developed a variety of detection techniques for measuring human milk oligosaccharides in various samples, including capillary electrophoresis, high-performance liquid chromatography (HPLC), ion chromatography, and liquid chromatography-mass spectrometry. However, these methods still have shortcomings and deficiencies. For example, when using HPLC, HPLC requires derivatization due to the poor UV absorption of human milk oligosaccharides, resulting in complex procedures and high detection limits. Capillary electrophoresis also exhibits poor ability to distinguish isomers. Based on the electrochemical activity of carbohydrate molecules and their anionic state in strong alkaline solutions, ion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is gaining increasing application for the detection of human milk oligosaccharides. However, this method is currently limited to the detection of 6–8 specific human milk oligosaccharides of high interest and has yet to achieve high-throughput detection of multiple human milk oligosaccharides. In terms of high-throughput detection, the rapid development of mass spectrometry technology has been favored by the academic community. However, the currently reported mass spectrometry detection technologies for human milk oligosaccharides are mostly liquid chromatography-mass spectrometry. However, liquid chromatography has limited ability to separate human milk oligosaccharide isomers, making it difficult to achieve high-throughput detection. Summary of the Invention

[0004] The present invention proposes a method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry, which solves the problem of difficulty in achieving high-throughput detection of human milk oligosaccharides in related technologies.

[0005] The technical solutions of the present invention are as follows: The present invention provides a method for detecting human milk oligosaccharides by ion exchange chromatography-tandem mass spectrometry. The method uses ion exchange chromatography-tandem mass spectrometry to detect human milk samples, and at least 42 human milk oligosaccharides are detected.

[0006] In the present invention, the human milk oligosaccharides include the following types:

[0007] As a further technical solution, when performing ion exchange chromatography-tandem mass spectrometry detection, the mobile phase of the ion exchange chromatography is mobile phase A: water; mobile phase B: sodium hydroxide aqueous solution; mobile phase C: sodium acetate aqueous solution.

[0008] In the present invention, water, sodium hydroxide aqueous solution and sodium acetate aqueous solution are selected as the mobile phase. This mobile phase system has good stability, can effectively reduce interference, improve the detection resolution and sensitivity, and improve the efficiency, accuracy and reliability of human milk oligosaccharide detection.

[0009] As a further technical solution, the concentration of the sodium hydroxide aqueous solution is 150-200 mmol / L, and the concentration of the sodium acetate aqueous solution is 150-200 mmol / L.

[0010] In the present invention, the concentration of the sodium hydroxide aqueous solution in mobile phase B can be any value within 150-200 mmol / L. For example, it can be 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, 200 mmol / L, etc. Preferably, it is 200 mmol / L; the concentration of the sodium acetate aqueous solution in mobile phase C can be any value within 150-200 mmol / L. For example, it can be 150 mmol / L, 155 mmol / L, 165 mmol / L, 185 mmol / L, 195 mmol / L, 200 mmol / L, etc. Preferably, it is 200 mmol / L.

[0011] As a further technical solution, gradient elution is performed using the mobile phase. In terms of volume percentage, the gradient elution is specifically as follows:

[0012] In the present invention, by optimizing the elution gradient, peak shape overlap can be effectively avoided, the peak separation effect can be enhanced, the detection resolution can be further improved, and high-throughput detection of human milk oligosaccharides can be achieved.

[0013] As a further technical solution, when performing ion exchange chromatography-tandem mass spectrometry detection, the ion exchange chromatography uses an anion exchange column and a guard column matching the anion exchange column.

[0014] In the present invention, the anion exchange chromatography column and the matching guard column can be commercially available products. For example, the anion exchange chromatography column can be a Carbo Pac PA200 analytical column (inner diameter 3 mm, column length 250 mm), and the matching guard column can be a Carbo Pac PA200 guard column (inner diameter 3 mm, column length 50 mm).

[0015] As a further technical solution, when performing ion exchange chromatography tandem mass spectrometry detection, the column temperature of the ion exchange chromatography is 25-30 °C, the flow rate is 0.2-0.4 mL / min, and the injection volume is 20-25 μL.

[0016] In the present invention, the column temperature of the ion exchange chromatography can be any value between 25-30 °C. For example, it can be 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, etc., and preferably 30 °C; the flow rate can be any value between 0.2-0.4 mL / min. For example, it can be 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, etc., and preferably 0.4 mL / min; the injection volume can be any value between 20-25 μL. For example, it can be 20 μL, 22 μL, 25 μL, etc., and preferably 25 μL.

[0017] As a further technical solution, when performing ion exchange chromatography tandem mass spectrometry detection, the mass spectrometry conditions are as follows: electrospray ionization source, simultaneous scanning in positive and negative ion modes, spray voltage 3-3.5 kV, atomization temperature 320-325 °C, sheath gas flow rate 8.1-9 L / min, purge gas flow rate 3-3.9 L / min, scanning mode multi-reaction monitoring, resolution 2000, maximum ion injection time 100 ms, normalized collision energy 10-30 eV.

[0018] In the present invention, when performing mass spectrometry detection, an electrospray ionization source is used, and simultaneous scanning in positive and negative ion modes can take into account the ionization detection of human milk oligosaccharides with different properties in the sample, expanding the range of detectable human milk oligosaccharide species. The spray voltage is set at 3 - 3.5 kV. Within this voltage range, a stable charged droplet flow can be effectively formed from the sample solution, ensuring the smooth progress of the ionization process. If the voltage is too low, the sample cannot be fully ionized; if the voltage is too high, the ion source becomes unstable, generating excessive noise interference. The atomization temperature is controlled at 320 - 325 °C, and this temperature range helps to improve the ionization efficiency and ion transmission rate. The scanning mode is selected as multiple reaction monitoring, which can significantly improve the selectivity and sensitivity of the detection. The resolution is set at 2000, which can effectively distinguish ions with similar mass-to-charge ratios, making the detection results more accurate and reliable. The maximum ion injection time is set at 100 ms, which can ensure the detection sensitivity while avoiding the space charge effect caused by excessive ion injection and affecting the detection accuracy. The normalized collision energy is set at 10 - 30 eV, and this energy range can meet the fragmentation requirements of various human milk oligosaccharides, providing rich information for the structural identification of human milk oligosaccharides.

[0019] As a further technical solution, when performing ion exchange chromatography tandem mass spectrometry detection, after ion exchange chromatography separation, the ions in the mobile phase are removed, and then mass spectrometry detection is carried out.

[0020] In the present invention, by removing the ions in the mobile phase after ion exchange chromatography separation and then performing mass spectrometry detection, the compatibility problem between ion exchange chromatography and mass spectrometry is solved, and the contamination of the mass spectrometer by high-concentration ionic compounds is avoided. When removing the ions in the mobile phase, a membrane transfer method can be adopted, such as using a membrane suppressor as the ion removal device.

[0021] As a further technical solution, the human milk sample is a processed human milk sample, and the processing method includes the following steps: after the human milk sample is centrifuged for the first time, absolute ethanol is added to the supernatant, followed by a second centrifugation and filtration to obtain the processed human milk sample.

[0022] In the present invention, by processing the human milk sample, the preliminary enrichment and purification of the human milk components are achieved, and the relatively pure processed human milk sample obtained is more conducive to the accurate analysis of human milk oligosaccharides during detection.

[0023] As a further technical solution, the volume ratio of the human milk sample to absolute ethanol is 1:1 - 2.

[0024] In the present invention, the volume ratio of the human milk sample to absolute ethanol can be any value between 1:1 - 2. For example, it can be 1:1, 1:2, etc., and preferably 1:1. The absolute ethanol is preferably pre-cooled absolute ethanol, and the temperature of the pre-cooled absolute ethanol is 0 - 4 °C.

[0025] The working principle and beneficial effects of the present invention are: Unlike conventional methods such as ion exchange chromatography coupled with pulsed amperometric detection and liquid chromatography-mass spectrometry, the present invention utilizes ion exchange chromatography coupled with mass spectrometry, enabling efficient detection of numerous human milk oligosaccharides. This significantly increases detection throughput, enabling the detection of at least 42 human milk oligosaccharides. Compared to existing technologies, the present invention not only improves detection efficiency but also significantly expands the number of detectable human milk oligosaccharides, providing a more comprehensive and accurate data foundation for in-depth research on human milk oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Figure 1 This is the total ion current of 42 human milk oligosaccharides in Example 1; Figure 2 This is the extracted ion chromatogram of human milk oligosaccharides such as 3-FL in Example 1; Figure 3 This is the extracted ion chromatogram of human milk oligosaccharides such as 6-SG in Example 1; Figure 4 This is the extracted ion chromatogram of human milk oligosaccharides such as 3-SLN in Example 1; Figure 5 This is the extracted ion chromatogram of human milk oligosaccharides such as DFLNnH in Example 1; Figure 6 This is the extracted ion chromatogram of human milk oligosaccharides such as Le A trisaccharide in Example 1; Figure 7 This is the extracted ion chromatogram of human milk oligosaccharides such as LNDFH II in Example 1; Figure 8 This is the extracted ion chromatogram of human milk oligosaccharides such as LNFP II in Example 1; Figure 9 This is the extracted ion chromatogram of human milk oligosaccharides such as LNTri in Example 1; Figure 10 This is the chromatogram of control group 1 in comparative experiment 1; Figure 11 This is the chromatogram of control group 2 in comparative experiment 1; Figure 12 This is the chromatogram of the experimental group in comparative experiment 1; Figure 13 This is the total ion current of human milk oligosaccharides in the control group of comparative experiment 2; Figure 14 This is the total ion current diagram of human milk oligosaccharides in the experimental group in comparative experiment 2. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] In the following examples, 42 human milk oligosaccharide standards were purchased from Shanghai Saiers Biochemical Technology Co., Ltd., and detailed information is shown in Table 1 below; 50% sodium hydroxide aqueous solution was purchased from Yufeishier Technology Co., Ltd.; anhydrous sodium acetate was purchased from Sigma, Germany; and ultrapure water was chromatographic grade.

[0030] Table 1 Detailed information of each human milk oligosaccharide standard

[0031] Example 1 A method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry 1. Detection 1.1 Processing of breast milk samples Take 100 μL of breast milk sample in a 1.5 mL centrifuge tube, centrifuge at 4°C and 4000 rpm for 15 minutes, remove the upper fat layer, add 100 μL of pre-cooled anhydrous ethanol (temperature is 0°C) to the lower clear liquid, vortex for 30 seconds, centrifuge at 4°C and 11000 rpm for 15 minutes, and filter through a 0.2 μm filter membrane to obtain the processed breast milk sample.

[0032] 1.2 Solution preparation Preparation of single-label stock solutions: Accurately weigh 1.0 mg of each of the 42 human milk oligosaccharide standards and use ultrapure water as the solvent to prepare single-label stock solutions with a mass concentration of 1 mg / g. Dilute each single-label stock solution to a 500 ppm single-label stock solution and store at -20°C.

[0033] Preparation of mixed standard solution 1: Use a pipette to draw 100 μL each of the single standard stock solutions of 2-FL, LNDFH I, DFLNH b, LNH, LSTa, LNFP I, 3-SLN, Le y tetrasaccharide, and 3-GL, and add ultrapure water to prepare a 10 ppm mixed standard solution 1.

[0034] Preparation of mixed standard solution 2: Use a pipette to draw 100 μL each of the single-standard stock solutions of 3-FL, LNDFH II, DFpLNH, LNnH, LSTb, LNFP II, 6-SLN, blood group A trisaccharide, FLNH II, and 4-GL, and add ultrapure water to prepare a 10 ppm mixed standard solution 2.

[0035] Preparation of mixed standard solution 3: Use a pipette to aspirate 100 μL of each single standard stock solution of 3-SL, LNnDFH I, DFLNnH, pLNH, LSTc, LNFPIII, 3-SG, Le A trisaccharide, FpLNH III, and 6-GL, and add ultrapure water to prepare a 10 ppm mixed standard solution 3.

[0036] Preparation of mixed standard solution 4: Use a pipette to aspirate 100 μL of each single standard stock solution of 6-SL, LNnDFH II, DFpLNnH, pLNnH, LSTd, LNFPV, 6-SG, Le X trisaccharide, FLNH II, and α1-3,β1-4 galactotrisaccharide, and add ultrapure water to prepare a 10 ppm mixed standard solution 4.

[0037] Preparation of mixed standard solution 5: Use a pipette to aspirate 100 μL of each single standard stock solution of 2-FL, 3-FL, 3-SL, 6-SL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNDFH I, LNDFH II, LSTa, LSTd, LNTri, and DFL, and add ultrapure water to prepare a 10 ppm mixed standard solution 5.

[0038] 1.3 Chromatographic conditions Chromatograph: Thermo Dionex ICS-5000 ion chromatograph; Chromatographic column: CarboPac PA200 analytical column (inner diameter 3 mm, column length 250 mm), CarboPac PA200 guard column (inner diameter 3 mm, column length 50 mm); Column temperature: 30 °C; Flow rate: 0.4 mL / min; Injection volume: 25 μL; Mobile phase: Mobile phase A is water, mobile phase B is 200 mmol / L sodium hydroxide aqueous solution (prepared from 50% sodium hydroxide aqueous solution and ultrapure water), and mobile phase C is 200 mmol / L sodium acetate aqueous solution (prepared from anhydrous sodium acetate and ultrapure water); In terms of volume percentage, the elution gradient:

[0039] The chromatograph is connected in series with a conductivity detector, and an ERD 500 (micropore 2 mm) suppressor is added. After adjusting the current value to reduce the conductivity to below 5 μs, it is then introduced into the mass spectrometer for detection.

[0040] 1.4 Mass spectrometry conditions Mass spectrometer: Thermo Scientific™ TSQ Altis™ triple quadrupole mass spectrometer; Electrospray ionization source, simultaneous scanning in positive and negative ion modes; Spray voltage: 3.5 kV; Atomization temperature: 325 °C; Sheath gas flow rate: 8.1 L / min; Auxiliary gas flow rate: 4.2 L / min; Purge gas flow rate: 3 L / min; Ion transfer tube temperature: 325 °C; Scanning mode: Multiple reaction monitoring; Resolution: 2000; Maximum ion injection time: 100 ms; Isolation window: 4.00 m / z; Normalized collision energy: 10 eV, 20 eV, 30 eV.

[0041] 1.5 Detection results 42 human milk oligosaccharides in the processed human milk samples and mixed standard solutions 1 - 5 were detected by ion exchange chromatography - tandem mass spectrometry. The total ion current chromatogram of the 42 human milk oligosaccharides is as Figure 1 shown. The extracted ion chromatograms of each isomer among the 42 human milk oligosaccharides are as Figures 2 - 9 shown. It can be seen from Figures 1 - 9 that the 42 human milk oligosaccharides can all be well separated, which indicates that the method of the present invention using ion exchange chromatography - tandem mass spectrometry realizes the efficient detection of numerous human milk oligosaccharides, significantly improves the detection throughput, and can detect at least 42 human milk oligosaccharides.

[0042] The detection limits of the 42 human milk oligosaccharides are shown in Table 2 below.

[0043] Table 2 Detection limits of 42 human milk oligosaccharides

[0044] According to the data in Table 2, the detection limits of the 42 human milk oligosaccharides are relatively low, which indicates that the detection method of the present invention has high sensitivity.

[0045] Comparative experiment 1 Influence of elution gradient on the detection of human milk oligosaccharides Experimental group: The same as Example 1; Control group 1: By volume percentage, the elution gradient is as shown below, and the rest is the same as Example 1;

[0046] Control group 2: By volume percentage, the elution gradient is as shown below, and the rest is the same as Example 1;

[0047] The chromatogram of Control Group 1 is as shown in Figure 10 . It can be seen from Figure 10 that there are more peaks from 26 to 30 minutes, and the separation effect is not good. The chromatogram of Control Group 2 is as shown in Figure 11 . It can be seen from Figure 11 that the number of peaks decreases and becomes more crowded, and the separation effect is even worse. The chromatogram of the experimental group is as shown in Figure 2 . It can be seen from Figure 12 that not only the number of peaks within 26 - 30 minutes increases, but also the resolution increases, enabling high - throughput detection of human milk oligosaccharides.

[0048] Effect of the treatment method of breast milk samples on the detection of human milk oligosaccharides in Comparative Experiment 2 Experimental group: the same as Example 1; Control group: Replace pre - cooled absolute ethanol with an equal amount of acetonitrile, and the rest is the same as Example 1.

[0049] The total ion chromatogram of human milk oligosaccharides in the control group is as shown in Figure 13 . The total ion chromatogram of human milk oligosaccharides in the experimental group is as shown in Figure 14 . The peak areas of common human milk oligosaccharides are shown in Table 3 below. By analyzing the peak areas, it is found that there are 15 in total with relatively small peak areas in the control group, and they correspond to human milk oligosaccharides with relatively low contents in breast milk, and high - throughput detection of human milk oligosaccharides cannot be achieved.

[0050] Table 3 Effect of different treatment methods on the detection of human milk oligosaccharides

[0051] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting human milk oligosaccharides by ion exchange chromatography-tandem mass spectrometry, characterized in that, At least 42 human milk oligosaccharides were detected by ion exchange chromatography tandem mass spectrometry for breast milk samples.

2. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 1, wherein When performing the ion exchange chromatography tandem mass spectrometry detection, the mobile phase of the ion exchange chromatography is mobile phase A: water; mobile phase B: sodium hydroxide aqueous solution; mobile phase C: sodium acetate aqueous solution.

3. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 2, characterized in that, The concentration of the sodium hydroxide aqueous solution is 150 - 200 mmol / L, and the concentration of the sodium acetate aqueous solution is 150 - 200 mmol / L.

4. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 2, wherein Gradient elution is performed using the said mobile phase. In terms of volume percentage, the specific gradient elution is as follows: 。 5. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 2, wherein, When performing the ion exchange chromatography tandem mass spectrometry detection, the ion exchange chromatography uses an anion exchange column and a guard column matching the anion exchange column.

6. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 2, wherein When performing the ion exchange chromatography tandem mass spectrometry detection, the column temperature of the ion exchange chromatography is 25 - 30 °C, the flow rate is 0.2 - 0.4 mL / min, and the injection volume is 20 - 25 μL.

7. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 2, wherein, When performing the ion exchange chromatography tandem mass spectrometry detection, the mass spectrometry conditions are as follows: electrospray ionization source, simultaneous scanning in positive and negative ion modes, spray voltage 3 - 3.5 kV, atomization temperature 320 - 325 °C, sheath gas flow rate 8.1 - 9 L / min, purge gas flow rate 3 - 3.9 L / min, scanning mode is multiple reaction monitoring, resolution is 2000, maximum ion injection time 100 ms, and normalized collision energy is 10 - 30 eV.

8. A method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 2, wherein When performing the ion exchange chromatography tandem mass spectrometry detection, after the ion exchange chromatography detection is completed, the ions in the mobile phase are removed and then mass spectrometry detection is carried out.

9. A method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to any one of claims 1 to 8, characterized in that, The breast milk sample is a processed breast milk sample, and the processing method includes the following steps: after the breast milk sample is centrifuged for the first time, absolute ethanol is added to the supernatant, then centrifuged for the second time and filtered to obtain the processed breast milk sample.

10. The method for detecting human milk oligosaccharides by ion exchange chromatography tandem mass spectrometry according to claim 9, characterized in that, The volume ratio of the breast milk sample to absolute ethanol is 1:1 - 2.

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