CN109254101B - Method for purifying and analyzing breast milk acid oligosaccharide - Google Patents
Method for purifying and analyzing breast milk acid oligosaccharide Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to a method for purifying and analyzing breast milk acid oligosaccharide. The method is characterized in that: a sample is subjected to low-temperature centrifugal degreasing, an organic solvent is added for precipitation and protein removal, then the interference of lactose and neutral oligosaccharide is removed through an online purification column, the obtained acidic sugar is separated through a hydrophilic interaction chromatographic column, and the structure, the composition and the content information of the breast milk acidic sugar are obtained through evaporative light, electrospray, ultraviolet or mass spectrometry detection. The method has the characteristics of good selectivity, good stability, simple and controllable operation, high flux and the like, and is suitable for rapid and high-flux analysis of mammal milk and human milk samples.
Description
Technical Field
The invention relates to a method for purifying and analyzing breast milk acid oligosaccharide. The method is characterized in that: a sample is subjected to low-temperature centrifugal degreasing, an organic solvent is added for precipitation and deproteinization, acidic oligosaccharide is purified by column chromatography in an electrostatic repulsion mode, interference of lactose and neutral oligosaccharide is removed, and structural composition analysis is obtained by on-line HILIC column separation analysis, evaporative light, electrospray, ultraviolet or mass spectrum detection. The method has the characteristics of good selectivity, good stability, simple and controllable operation, high flux and the like, and is suitable for rapid analysis of a large number of breast milk samples.
Technical Field
Oligosaccharides are important components in breast milk and have important physiological functions. Plays a vital role in the normal growth and development of the infant, such as anti-inflammation, anti-infection, immune regulation and maintenance of intestinal flora balance. The oligosaccharide comprises acid oligosaccharide and neutral oligosaccharide, wherein the acid oligosaccharide is oligosaccharide which is often sialylated, has lower content than neutral oligosaccharide, and plays an irreplaceable role in vivo, such as preventing cancer, promoting normal development of infant brain, and the like. The types and contents of the acid oligosaccharide in different human and animal milks have certain differences.
The precondition for deeply knowing the biological and pharmacological activities of the acid oligosaccharide in breast milk is to analyze the composition, structure and content of the acid oligosaccharide in human and animal milk, so that the development of a simple, rapid and high-flux analysis method of the acid oligosaccharide is of great significance. However, due to the low content of acid oligosaccharides in breast milk oligosaccharides, there is a serious interference between lactose and neutral oligosaccharides during analysis, and the structure is very diverse, thereby increasing the difficulty of analysis. In recent years, research on analysis of breast milk acid oligosaccharides has been receiving attention.
At present, the main purification methods for breast milk acid oligosaccharide are 2 methods: (1) an off-line method is adopted, a large amount of lactose is removed by gel permeation, a large amount of neutral oligosaccharide is removed by ion exchange chromatography, and the purified acid oligosaccharide is further desalted and then analyzed. (2) An off-line PGC SPE column was used to remove a large amount of lactose and neutral oligosaccharides to obtain an acidic oligosaccharide fraction. However, the 2 methods described above have low selectivity and are tedious and time-consuming. Through an online SPE column chromatographic purification and analysis method, a large amount of lactose and neutral oligosaccharide interference is removed, the acid oligosaccharide fraction is directly subjected to HILIC column chromatographic analysis, and the method has the characteristics of high selectivity, good repeatability and high flux, and has certain advantages when being used for researching an acid oligosaccharide profiling method. Therefore, the invention designs a new breast milk acid oligosaccharide purification and analysis method, which is used for the rapid analysis of large-scale breast milk acid oligosaccharides.
Disclosure of Invention
The invention relates to a method for purifying and analyzing breast milk acid oligosaccharide. The invention is characterized in that: a sample is subjected to low-temperature centrifugal degreasing, organic solvent precipitation protein removal and column chromatography lactose and neutral oligosaccharide removal, the purified acid oligosaccharide is directly separated by an online HILIC column, and subjected to evaporative light, electrospray and ultraviolet or mass spectrometry detection to obtain structure, composition and content analysis.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for purifying and analyzing acidic oligosaccharide of breast milk comprises the steps of carrying out low-temperature centrifugal degreasing on a sample, adding an organic solvent for precipitation and protein removal, purifying the acidic oligosaccharide through column chromatography, collecting an acidic oligosaccharide fraction window, and continuously analyzing the acidic oligosaccharide by adopting an online HILIC column. The mobile phase is organic solvent and water or organic solvent and buffer saline solution, and isocratic or gradient elution conditions are used by optimizing chromatographic parameters.
1. The centrifugal degreasing step comprises the following steps: collecting breast milk sample, centrifuging at 0-10 deg.C for 1-500 min, preferably for 5-30 min, 500-50000g, preferably 2000-5000g, and removing upper layer lipid. Centrifuging for 1-5 times, and collecting the lower layer to obtain defatted breast milk sample.
2. The precipitation protein-removing step comprises the following steps: taking a defatted breast milk sample, adding 1-5 times of organic solvent by volume, mixing uniformly, standing for 2-48 hours at 0-10 ℃, centrifuging for 1-500 minutes, preferably 5-30 minutes at 500-10 ℃ for 50000g, preferably 2000-5000g, collecting supernatant, centrifuging for 1-5 times, and combining the supernatant to obtain a defatted deproteinized sample solution or a solid obtained by freeze-drying. The organic solvent is one or more of methanol, acetonitrile, ethanol and acetone.
3. The stationary phase of the column chromatography purification acid oligosaccharide and the stationary phase of the HILIC column are polar chromatography fillers, comprise silica gel or polar bonded silica gel fillers, and have the following structural formula:
wherein SiO is2Is silica gel, R is polar group, the purifying column is filledThe material is silica gel or filler with polar groups bonded on the surface of the silica gel, and the bonded polar groups comprise one or more than two of amphoteric amino acid, sulfonyl, carboxyl, zwitter ions and the like; the HILIC analytical column filler is a material with silica gel surface bonded with polar groups, and the bonded polar groups comprise one or more than two of amino acid, amino group, amido group, zwitterion and quaternary ammonium group;
4. the organic solvent in the eluent is one or more of methanol, acetonitrile, ethanol and acetone.
The buffer salt type and the concentration and pH value in the mobile phase are as follows:
a) ammonium formate buffer salt, concentration 1-200mM, pH 2-10;
b) ammonium acetate buffer salt at a concentration of 1-200mM, pH 2-10;
c) ammonium bicarbonate buffer salt with concentration of 1-200mM and pH of 2-10;
the mobile phase gradient was optimized as follows: and the mobile phase water or the buffer saline solution and the organic solvent are used as eluent, and the mixing ratio is 5/95-95/5.
7. The collection window of the acid oligosaccharide is 0-10BV, and the effluent liquid is collected.
8. The chromatographic operating parameters were optimized as follows: the inner diameter of the chromatographic column is 2.1-10 mm; the flow rate is 0.1-2 BV/min; the column temperature is 15-60 ℃; the detector is an ultraviolet detector, an electrospray detector, an evaporation light detector or a mass spectrometry detector.
The invention has the following advantages:
1. the selectivity is high. Aiming at the problems in the current oligosaccharide preparation, the invention provides a method for purifying acid oligosaccharide by using column chromatography to obtain better separation, and effectively solves the problems of insufficient selectivity and low purification efficiency of ion exchange chromatography or gel filtration chromatography.
2. The flux is high. The purification and analysis process is simple and rapid.
3. The repeatability is good. The chromatographic column stationary phase used in the invention has good stability and is easy to realize automation.
4. The method has wide application range and can be used for analyzing milk and human milk of different mammals.
Drawings
FIG. 1 is the online purification and analysis spectrum of breast milk acidic oligosaccharide in example 1;
FIG. 2 is an EIC overlay map of human milk acidic oligosaccharides obtained in example 1;
FIG. 3 is a comparison graph of the analysis of example 2 with and without the on-line SPE clean process, a-without SPE, b-with on-line SPE-HILIC.
Detailed Description
Example 1:
degreasing and deproteinizing process of human milk oligosaccharide
A certain amount of human milk is taken, centrifuged at 4000g at 4 ℃ for 60min, and upper lipid is removed. And (3) taking the lower water layer, adding 2 times of volume of absolute ethyl alcohol, uniformly mixing, standing at 4 ℃ for 12 hours, centrifuging at 4 ℃ for 4000g for 60min, centrifuging for 1 time, and collecting the upper liquid to obtain the defatted protein-removed oligosaccharide sample.
Human milk oligosaccharide purification and analysis process
And directly taking a liquid sample of the human milk oligosaccharide subjected to degreasing and protein removal for analysis. The sample size was 20. mu.L, and the SPE purification column used a Click TE-GSH column, the column specification was 2.1 × 50mm, the flow rate was 0.2mL/min, and the eluent was an acidic oligosaccharide-containing solution eluted at 80% acetonitrile in water for 5 min. And (3) enabling the SPE purification column and the HILIC analysis column to be in a serial state through a switching valve, and enabling the eluent to directly flow into the HILIC analysis column for collection. And after the acid oligosaccharide is captured into the HILIC analysis column, switching a switching valve to enable the SPE purification column and the HILIC analysis column to be in a parallel state, and analyzing and detecting the acid oligosaccharide by the HILIC analysis column through a buffer salt system. The specific conditions are as follows: the HILIC analysis column used XAmide column, the specification of the chromatographic column was 4.6 x 150mm, and the flow rate was 1.0 mL/min; the column temperature was 30 ℃ and mass spectrometric detection was carried out. Mobile phase a was acetonitrile, B was water, C was 100mM ammonium formate, pH 3.2. The captured acid oligosaccharide was further analyzed under the conditions of 0-30min, 80-50% A, and 10% C.
Through mass spectrum detection, lactose and neutral oligosaccharide are not detected, which indicates that the acid oligosaccharide is well purified through an on-line SPE-HILIC system, and 30 acid oligosaccharides are detected in the human milk oligosaccharide.
Example 2:
degreasing and deproteinizing process of human milk oligosaccharide
The procedure was as in example 1.
Human milk oligosaccharide purification and analysis process
And directly taking a liquid sample of the human milk oligosaccharide subjected to degreasing and protein removal for analysis. The sample volume was 20. mu.L, and the analysis was carried out directly on a HILIC column without purification using SPE column. The other conditions were the same as in example 1.
Through mass spectrum detection, besides acid oligosaccharide, lactose and neutral oligosaccharide are also detected, and the neutral oligosaccharide and the acid oligosaccharide appear in a chromatogram in a crossed manner, and the neutral oligosaccharide seriously interferes the analysis of the acid oligosaccharide. Therefore, it is important to purify the acid oligosaccharides to remove lactose and neutral oligosaccharides before analysis.
Example 3:
degreasing and deproteinizing process of goat milk oligosaccharide
Centrifuging a certain amount of goat milk at 4 deg.C 2000g for 5min, and removing upper lipid layer. And (3) taking the lower water layer, adding 4 times of acetone by volume, uniformly mixing, standing at 4 ℃ for 18 hours, centrifuging at 4 ℃ for 2000g for 10min, centrifuging for 2 times, collecting the upper liquid to obtain a liquid sample of the defatted and deproteinized oligosaccharide, and concentrating to be dry for later use.
Purification and analysis process of goat milk oligosaccharide
Weighing a sample of degreased and deproteinized goat milk oligosaccharide, preparing a solution with the concentration of 100mg/mL by using 50% acetonitrile water, wherein the sample volume is 100 mu L, the SPE purification column uses a chromatographic column with a silica gel surface bonded with aspartic acid groups, the inner diameter of the chromatographic column is 2.1 x 100mm, the flow rate is 0.2mL/min, and the 85% acetonitrile water is eluted for 10min isocratically. The eluent is a solution containing acid oligosaccharide. And (3) enabling the SPE purification column and the HILIC analysis column to be in a serial state through a switching valve, and enabling the eluent to directly flow into the HILIC analysis column for collection. And after the acid oligosaccharide is captured into the HILIC analysis column, switching a switching valve to enable the SPE purification column and the HILIC analysis column to be in a parallel state, and analyzing and detecting the acid oligosaccharide by the HILIC analysis column through a buffer salt system. The specific conditions are as follows: the HILIC analysis column uses a chromatographic column with a histidine acid group bonded on the surface of silica gel, the specification of the chromatographic column is 3.0 x 150mm, and the flow rate is 0.4 mL/min; the column temperature was 40 ℃ and mass spectrometric detection was carried out. Mobile phase a was acetonitrile, B was water, C was 50mM ammonium bicarbonate buffer, pH 8.0. The captured acid oligosaccharide was further analyzed under conditions of 0-35min, 85-40% A, and 20% C.
Through mass spectrum detection, lactose and neutral oligosaccharide are not detected, which indicates that the acid oligosaccharide is well purified through an on-line SPE-HILIC system, and 12 acid oligosaccharides are detected in the goat milk oligosaccharide. Sialylated oligosaccharides containing NeuGc were also detected.
Example 4:
degreasing and deproteinizing process of porcine lactooligosaccharide
Centrifuging a certain amount of pig milk at 4 deg.C for 60min at 5000g, and removing upper lipid layer. And (3) taking the lower water layer, adding acetonitrile with 2 times of volume amount, uniformly mixing, standing at 4 ℃ for 24 hours, centrifuging at 4 ℃ for 5000g for 60min, centrifuging for 4 times, and collecting the upper layer liquid to obtain the defatted deproteinized porcine lactooligosaccharide sample.
Purification and analysis process of porcine lactooligosaccharide
And directly taking a liquid sample of the defatted and deproteinized porcine lactooligosaccharide for analysis. The sample volume was 100. mu.L, the SPE purification column used silica gel column, the inner diameter of the column was 3.0X 150mM, the flow rate was 0.3mL/min, the mobile phase was A-methanol, B-water, C-200mM ammonium acetate buffer (pH 8.0), 0-4min, 90-50% A, 5% C remained unchanged. The eluent is a solution containing acid oligosaccharide. And (3) enabling the SPE purification column and the HILIC analysis column to be in a serial state through a switching valve, and enabling the eluent to directly flow into the HILIC analysis column for collection. And after the acid oligosaccharide is captured into the HILIC analysis column, switching a switching valve to enable the SPE purification column and the HILIC analysis column to be in a parallel state, and analyzing and detecting the acid oligosaccharide by the HILIC analysis column through a buffer salt system. The specific conditions are as follows: the HILIC analysis column uses arginine column, the specification of the chromatographic column is 4.6 x 250mm, and the flow rate is 1.0 mL/min; the column temperature was 35 ℃ and mass spectrometric detection was carried out. Mobile phase a was acetonitrile, B was water, C was 100mM ammonium bicarbonate buffer, pH 9.0. The captured acid oligosaccharide was further analyzed under the conditions of 0-35min, 85-40% A, and 10% C.
Through mass spectrum detection, lactose and neutral oligosaccharide are not detected, which indicates that the acid oligosaccharide is well purified through an on-line SPE-HILIC system, and 14 acid oligosaccharides are detected in the pig milk oligosaccharide.
Example 5:
degreasing and deproteinizing process of cow milk oligosaccharide
Centrifuging a certain amount of cow milk at 4500g at 4 deg.C for 10min to remove upper lipid layer. Taking the lower water layer, adding 2 times volume of ethanol, mixing, standing at 4 deg.C for 24 hr, centrifuging at 4 deg.C 4500g for 10min, collecting the upper layer solution to obtain defatted protein-removed milk oligosaccharide liquid sample, and concentrating to dry.
Purification and analysis process of cow milk oligosaccharide
Weighing a sample of the defatted deproteinized bovine milk oligosaccharide, preparing a solution with the concentration of 80mg/mL by using 60% ethanol water, wherein the sample volume is 100 mu L, the SPE purification column uses a silica gel column, the inner diameter of the chromatographic column is 2.1 x 50mm, the flow rate is 0.2mL/min, and the isocratic elution is carried out for 4min by using 75% acetonitrile water. The eluent is a solution containing acid oligosaccharide. And (3) enabling the SPE purification column and the HILIC analysis column to be in a serial state through a switching valve, and enabling the eluent to directly flow into the HILIC analysis column for collection. And after the acid oligosaccharide is captured into the HILIC analysis column, switching a switching valve to enable the SPE purification column and the HILIC analysis column to be in a parallel state, and analyzing and detecting the acid oligosaccharide by the HILIC analysis column through a buffer salt system. The specific conditions are as follows: an HILIC analytical column XAmini column with a chromatographic column specification of 3.0 x 150mm and a flow rate of 0.4 mL/min; the column temperature was 40 ℃ and mass spectrometric detection was carried out. The mobile phase A was acetonitrile, B was water, C was 100mM ammonium acetate buffer, pH 5.0. The captured acid oligosaccharide was further analyzed under the conditions of 0-25min, 85-40% A, and 10% C.
Through mass spectrum detection, lactose and neutral oligosaccharide are not detected, which indicates that the acid oligosaccharide is well purified through an on-line SPE-HILIC system, and 7 acid oligosaccharides are detected in cow milk oligosaccharide.
Example 6:
defatting and deproteinizing process of camel milk oligosaccharide
Centrifuging a certain amount of camel milk at 4 deg.C 3000g for 10min, and removing upper layer lipid. Taking the lower water layer, adding 2 times volume of ethanol, mixing, standing at 4 deg.C for 24 hr, centrifuging at 4 deg.C 3000g for 5min, collecting the upper layer liquid to obtain defatted camel milk oligosaccharide liquid sample, and concentrating to dry.
Purification and analysis process of camel milk oligosaccharide
Weighing a sample of the defatted and deproteinized camel lactooligosaccharide, preparing a solution with the concentration of 120mg/mL by using 70% acetonitrile water, wherein the sample volume is 50 mu L, the SPE purification column uses a silica gel column, the inner diameter of the chromatographic column is 2.1 x 50mm, the flow rate is 0.2mL/min, and the linear gradient elution is carried out for 6min by using 85-70% acetonitrile water. The eluent is a solution containing acid oligosaccharide. And (3) enabling the SPE purification column and the HILIC analysis column to be in a serial state through a switching valve, and enabling the eluent to directly flow into the HILIC analysis column for collection. And after the acid oligosaccharide is captured into the HILIC analysis column, switching a switching valve to enable the SPE purification column and the HILIC analysis column to be in a parallel state, and analyzing and detecting the acid oligosaccharide by the HILIC analysis column through a buffer salt system. The specific conditions are as follows: an HILIC analytical column XAmini column with a chromatographic column specification of 3.0 x 150mm and a flow rate of 0.4 mL/min; the column temperature was 40 ℃ and mass spectrometric detection was carried out. The mobile phase A was acetonitrile, B was water, C was 100mM ammonium acetate buffer, pH 5.0. The captured acid oligosaccharide was further analyzed under the conditions of 0-25min, 85-40% A, and 10% C.
Through mass spectrum detection, lactose and neutral oligosaccharide are not detected, which indicates that the acid oligosaccharide is well purified through an on-line SPE-HILIC system, and 6 acid oligosaccharides are detected in camel milk oligosaccharide.
Example 7:
degreasing and deproteinizing process of yak milk oligosaccharide
Centrifuging a certain amount of yak milk at 4 deg.C for 10min at 5000g, and removing upper lipid. And (3) taking the lower water layer, adding 2 times of ethanol in volume, mixing uniformly, standing at 4 ℃ for 10 hours, centrifuging at 4 ℃ for 15min at 5000g, collecting the upper liquid to obtain a liquid sample of the defatted deproteinized yak milk oligosaccharide, and concentrating to dryness for later use.
Purification and analysis process of yak milk oligosaccharide
Weighing a sample of the defatted deproteinized yak milk oligosaccharide, preparing a solution with the concentration of 120mg/mL by using 50% methanol water, wherein the sample volume is 50 mu L, the SPE purification column uses a Click Xion column, the inner diameter of the chromatographic column is 2.1 x 100mM, the flow rate is 0.2mL/min, 85% acetonitrile contains 10mM ammonium bicarbonate (pH 8.5) buffer salt, and the elution is carried out for 6min in an isocratic manner. The eluent is a solution containing acid oligosaccharide. And (3) enabling the SPE purification column and the HILIC analysis column to be in a serial state through a switching valve, and enabling the eluent to directly flow into the HILIC analysis column for collection. And after the acid oligosaccharide is captured into the HILIC analysis column, switching a switching valve to enable the SPE purification column and the HILIC analysis column to be in a parallel state, and analyzing and detecting the acid oligosaccharide by the HILIC analysis column through a buffer salt system. The specific conditions are as follows: a HILIC analytical column XAmide column with a chromatographic column specification of 3.0 x 150mm and a flow rate of 0.4 mL/min; the column temperature was 40 ℃ and mass spectrometric detection was carried out. The mobile phase A was acetonitrile, B was water, C was 100mM ammonium acetate buffer, pH 5.0. The captured acid oligosaccharide was further analyzed under the conditions of 0-25min, 80-40% A, and 10% C.
Through mass spectrum detection, lactose and neutral oligosaccharide are not detected, which indicates that the acid oligosaccharide is well purified through an on-line SPE-HILIC system, and 6 kinds of acid oligosaccharides are detected in yak milk oligosaccharide.
Table 1 example 1 acidic oligosaccharides detected in human milk
Table 2 acid oligosaccharides detected in sheep milk of example 3
| Peak number | Retention time/min | Composition of1 | Molecular weight | Name (R) |
| 1 | 20.50 | H2A1 | 633.2 | 3’- |
| 2 | 20.94/21.33 | H1N1A1 | 674.2 | 6'- |
| 3 | 21.60 | H2A1 | 633.2 | 6’- |
| 4 | 22.93 | H3A1 | 795.3 | |
| 5 | 23.23 | H3A1 | 795.3 | |
| 6 | 21.20 | H2G1 | 649.2 | 3’-S(Gc)L |
| 7 | 21.52/22.10 | H1N1G1 | 680.2 | 3’-S(Gc) |
| 8 | 22.16 | H2G1 | 649.2 | 6’-S(Gc) |
| 9 | 23.44 | H3G1 | 811.2 | |
| 10 | 25.35 | H2A2 | 924.3 | DSL |
| 11 | 26.31 | H2A1G1 | 940.3 | |
| 12 | 26.87 | H2G2 | 956.3 |
Table 3 shows the acid oligosaccharides detected in the pig milk of example 4
| Peak number | Retention time/min | Composition of1 | Molecular weight | Name (R) |
| 1 | 20.50 | H2A1 | 633.2 | 3’- |
| 2 | 20.94/21.33 | H1N1A1 | 674.2 | 6'- |
| 3 | 21.60 | H2A1 | 633.2 | 6’- |
| 4 | 22.93 | H3A1 | 795.3 | |
| 5 | 23.23 | H3A1 | 795.3 | |
| 6 | 23.27 | H2N1A1 | 836.3 | |
| 7 | 24.72 | H3N1A1 | 998.3 | LST a |
| 8 | 25.61 | H3N1A1 | 998.3 | |
| 9 | 19.77/21.17 | H1N1A1 | 674.2 | - |
| 10 | 22.24 | H2N1A1 | 836.3 | |
| 11 | 26.15 | H4N1A1 | 1160.4 | |
| 12 | 31.40 | H3A2 | 1086.3 | |
| 13 | 33.49 | H4N1A2 | 1451.5 | / |
| 14 | 34.29 | H4N1A2 | 1451.5 | / |
Table 4 shows the acid oligosaccharides detected in cow's milk of example 5
Table 5 example 6 acid oligosaccharides detected in camel milk
| Peak number | Retention time/min | Composition of1 | Molecular weight | Name (R) |
| 1 | 20.50 | H2A1 | 633.2 | 3’- |
| 2 | 20.94/21.33 | H1N1A1 | 674.2 | 6′- |
| 3 | 21.60 | H2A1 | 633.2 | 6’- |
| 4 | 22.93 | H3A1 | 795.3 | |
| 5 | 26.15 | H4N1A1 | 1160.4 | |
| 6 | 31.40 | H3A2 | 1086.3 |
Table 6 acid oligosaccharides detected in yak lactic acid of example 7
| Peak number | Retention time/min | Composition of1 | Molecular weight | Name (R) |
| 1 | 20.50 | H2A1 | 633.2 | 3’- |
| 2 | 20.94/21.33 | H1N1A1 | 674.2 | 6′- |
| 3 | 21.60 | H2A1 | 633.2 | 6’- |
| 4 | 22.93 | H3A1 | 795.3 | |
| 5 | 25.35 | H2A2 | 924.3 | |
| 6 | 31.40 | H3A2 | 1086.3 |
1：H：Hex；N：HexNAc；F：dHex；A：NeuNAc；G：NeuNGc.
Claims (6)
1. A method for purifying and analyzing breast milk acid oligosaccharide is characterized by comprising the following steps: carrying out centrifugal degreasing and protein precipitation on a breast milk sample, removing lactose and neutral oligosaccharide by using an SPE purification column to obtain an acidic oligosaccharide fraction, directly carrying out on-line analysis by using a hydrophilic interaction chromatography HILIC column, and carrying out mass spectrum detection to obtain structural component analysis;
the SPE purification column filler is silica gel or a filler of a silica gel surface bonded polar group, and the bonded polar group comprises one or more than two of amphoteric amino acid, sulfonyl, carboxyl and zwitter ion;
the HILIC analytical column filler is a material with a silica gel surface bonded with polar groups, and the bonded polar groups comprise one or more than two of amino acid, amino group, amide group, zwitterion and quaternary ammonium group;
A. when the filler of the SPE purification column is one or two materials of glutathione and aspartic acid groups bonded on the surface of silica gel, the mobile phase adopted in the step of purifying the acid oligosaccharide by the SPE purification column is an organic solvent and water, the filler of the HILIC column is one or two materials of amide and amino groups bonded on the surface of silica gel, and the mobile phase adopted in the step of analyzing the effluent liquid of the acid oligosaccharide by hydrophilic interaction chromatography is a buffered saline solution with pH of 2-6 and an organic solvent;
or B, when the filler of the SPE purification column is one or two materials of glutathione and aspartic acid groups bonded on the surface of silica gel, the mobile phase adopted in the step of purifying the acid oligosaccharide by the SPE purification column is organic solvent and water, the filler of the HILIC column is one or more group materials of lysine, arginine and histidine bonded on the surface of silica gel, and the mobile phase adopted in the step of analyzing the effluent of the acid oligosaccharide by hydrophilic interaction chromatography is buffered saline solution pH 2-10 and organic solvent;
or C, when the filler of the SPE purification column is a silica gel material, the mobile phase adopted in the step of purifying the acid oligosaccharide by the SPE purification column is an organic solvent and water, the filler of the HILIC column is one or two group materials of amino and amide bonded on the surface of the silica gel, and the mobile phase adopted in the step of analyzing the effluent of the acid oligosaccharide by hydrophilic interaction chromatography is a buffered saline solution with pH of 2-6 and an organic solvent;
or D, when the filler of the SPE purification column is a silica gel material, the mobile phase adopted in the step of purifying the acid oligosaccharide by the SPE purification column is an organic solvent and water, the filler of the HILIC column is one or more group materials of lysine, arginine or histidine bonded on the surface of the silica gel, and the mobile phase adopted in the step of analyzing the effluent of the acid oligosaccharide by hydrophilic interaction chromatography is a buffered saline solution with the pH value of 2-10 and an organic solvent;
or E, when the filler of the SPE purification column is a material bonded with cysteine groups on the surface of silica gel, the mobile phase adopted in the step of purifying the acid oligosaccharide by the SPE purification column is organic solvent and pH6-10 of a buffer salt solution, the filler of the HILIC column is one or more than two group materials bonded with quaternary ammonium groups, lysine, arginine or histidine on the surface of the silica gel, and the mobile phase adopted in the step of analyzing the effluent of the acid oligosaccharide by hydrophilic interaction chromatography is the buffer salt solution with pH6-10 and the organic solvent;
or F, when the filler of the SPE purification column is a material formed by bonding one or more groups of carboxyl, sulfonyl and phosphate on the surface of silica gel, the mobile phase adopted in the step of purifying the acid oligosaccharide by the SPE purification column is an organic solvent and water, the filler of the HILIC column is a material formed by bonding quaternary ammonium groups on the surface of silica gel, and the mobile phase adopted in the step of analyzing the effluent of the acid oligosaccharide by hydrophilic interaction chromatography is a buffered saline solution with the pH of 6-10 and an organic solvent;
the steps of purifying the acid oligosaccharide by the SPE purification column are as follows: taking a degreased protein-removed breast milk solid sample, dissolving the sample by using a sample dissolving solution, or directly taking a degreased protein-removed breast milk liquid sample, feeding the sample into an SPE purification column through an automatic sample injector, taking a hydrophilic material with an electrostatic repulsion effect as a chromatographic column stationary phase, taking a mobile phase of water and an organic solvent or a buffer saline solution and an organic solvent as a mobile phase, eluting by using an isocratic or gradient method, and directly feeding acidic oligosaccharide fraction into a series-connected analysis column on line for analysis;
the step of analyzing the effluent of the acid oligosaccharide by hydrophilic interaction chromatography comprises the following steps: directly introducing the acidic oligosaccharide effluent into a HILIC column on line, collecting the acidic oligosaccharide effluent into the HILIC column, separating and analyzing, eluting with water and organic solvent or buffered saline solution and organic solvent by isocratic or gradient method, and detecting;
the volume ratio of the sample dissolving solution to a mixed solution of one or more of organic solvents of methanol, acetonitrile, ethanol and acetone and water is 50-70%; the organic solvents in the eluent are: one or more than two of methanol, acetonitrile, ethanol, isopropanol and acetone;
the selected buffer salt types in the eluent and the concentrations thereof in the mobile phase are as follows:
a) ammonium formate buffer salt, concentration 1-200 mM;
b) ammonium acetate buffer salt with concentration of 1-200 mM;
c) ammonium bicarbonate buffer salt with concentration of 1-200 mM;
in the steps of purifying the acid oligosaccharide by the SPE purification column and analyzing the acid oligosaccharide by the hydrophilic chromatographic column, the volume ratio of mobile phase water and an organic solvent or a buffer saline solution and an organic solvent as an eluent is 5/95-95/5;
in the step of purifying the acid oligosaccharide by the SPE purification column, the inner diameter of the chromatographic column is 2.1-10mm, and the grain diameter of the filler is 1-40 mu m; the flow rate of the analysis method is 0.1-2 BV/min; collecting the acidic oligosaccharide fraction with a collection window of 0-10BV, and collecting the effluent;
optimizing chromatographic operating parameters including flow rate, column temperature and detector, and valve switching time, the specific operations are as follows:
a) the flow rate is 0.1-2 BV/min;
b) the column temperature is 15-60 ℃;
c) the detector is a mass spectrum;
d) the valve switching time is 0-10 BV.
2. The purification and analysis method of claim 1, wherein: the centrifugal degreasing step comprises: collecting breast milk sample, centrifuging at 0-10 deg.C for 1-500 min at 500-50000g, and removing upper lipid layer; centrifuging for 1-5 times, and collecting the lower layer to obtain defatted breast milk sample.
3. The purification and analysis method according to claim 2, wherein: the centrifugal degreasing step comprises: taking a breast milk sample, centrifuging for 5-30 minutes at the temperature of 0-10 ℃ and at the temperature of 2000-5000g, and removing upper-layer lipid; centrifuging for 1-5 times, and collecting the lower layer to obtain defatted breast milk sample.
4. The purification and analysis method of claim 1, wherein: the precipitation protein-removing step comprises: taking a defatted breast milk sample, adding 1-5 times of organic solvent by volume, mixing uniformly, standing for 2-48 hours at 0-10 ℃, centrifuging for 1-500 minutes at 500-50000g at 0-10 ℃, and collecting supernatant; centrifuging for 1-5 times, and mixing the upper layer solution to obtain defatted deproteinized sample solution or lyophilized solid; the organic solvent is one or more of ethanol, acetone, methanol and acetonitrile.
5. The purification and analysis method of claim 4, wherein: the precipitation protein-removing step comprises: taking a defatted breast milk sample, adding 1-5 times of organic solvent by volume, mixing uniformly, standing for 2-48 hours at 0-10 ℃, centrifuging for 5-30 minutes at 2000-5000g at 0-10 ℃, and collecting supernatant; centrifuging for 1-5 times, and mixing the upper layer solution to obtain defatted deproteinized sample solution or lyophilized solid; the organic solvent is one or more of ethanol, acetone, methanol and acetonitrile.
6. The purification and analysis method of claim 1, wherein: the breast milk sample is human milk and/or mammalian milk, i.e. one of ruminant, primate, carnivorous, haplopore.
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| CN112649516A (en) * | 2019-10-12 | 2021-04-13 | 中国科学院大连化学物理研究所 | Derivatization-based milk powder containing 4 human milk oligosaccharides and qualitative and quantitative method thereof |
| CN112730635A (en) * | 2019-10-28 | 2021-04-30 | 中国科学院大连化学物理研究所 | Liquid chromatography analysis method of oligosaccharide |
| CN112924563B (en) * | 2019-12-05 | 2022-11-15 | 中国科学院大连化学物理研究所 | Derivatization-based breast milk oligosaccharide characterization and quantitative analysis method |
| CN112526022A (en) * | 2020-11-27 | 2021-03-19 | 内蒙古伊利实业集团股份有限公司 | Method for detecting breast milk oligosaccharide in milk |
| CN114594169A (en) * | 2020-12-03 | 2022-06-07 | 中国科学院大连化学物理研究所 | Quantitative detection method for oligosaccharide content in breast milk |
| CN113533572A (en) * | 2021-07-19 | 2021-10-22 | 山东恒鲁生物科技有限公司 | Qualitative and quantitative detection method for human milk oligosaccharide in dairy products |
| CN115112799B (en) * | 2022-06-30 | 2023-02-28 | 北京三元食品股份有限公司 | Method for qualitatively detecting acid oligosaccharide in breast milk |
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