CN114740096A - Detection method of breast milk oligosaccharide - Google Patents
Detection method of breast milk oligosaccharide Download PDFInfo
- Publication number
- CN114740096A CN114740096A CN202210038222.7A CN202210038222A CN114740096A CN 114740096 A CN114740096 A CN 114740096A CN 202210038222 A CN202210038222 A CN 202210038222A CN 114740096 A CN114740096 A CN 114740096A
- Authority
- CN
- China
- Prior art keywords
- lactoyl
- injection concentration
- breast milk
- sample
- sodium borohydride
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention provides a detection method of breast milk oligosaccharide, which comprises the following steps: pretreating an emulsion sample, and reducing by using sodium borohydride to obtain a solution to be detected; reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution; measuring the standard substance solution by adopting high performance liquid chromatography-mass spectrometry, and establishing a standard curve; performing qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography, and performing quantitative analysis by adopting a mass spectrum combined with a standard curve; the chromatographic conditions are as follows: a chromatographic column: hypercarb porous graphite carbon chromatography; the mobile phase A is: an aqueous solution containing 0.05 to 0.15% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15% formic acid; gradient elution. The method can effectively separate isomers, can quantify oligosaccharides with extremely low concentration in breast milk, and has low quantification limit.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a detection method of breast milk oligosaccharide.
Background
Breast milk oligosaccharides (HMOs) are the 3 rd largest component of human breast milk, which is second to lactose and fat, and the content of the HMOs in the breast milk is 22-25 g/L, and the content of the HMOs in mature milk is 12-13 g/L. HMOs have important biological functions, and not only have the function of resisting intestinal pathogenic microorganism infection, but also have the function of maintaining intestinal microecological balance. The breast milk is the gold standard for preparing the infant milk powder, so the establishment of the detection method of the breast milk oligosaccharide is a necessary prerequisite for analyzing the components of the breast milk and further developing the more nutritional and healthy infant milk powder.
The pretreatment method of the sample in HMOs detection is to remove fat and protein in the breast milk. The fat removal method usually adopts a low-temperature centrifugation method, and the protein removal method mainly adopts an organic precipitation method and an ultrafiltration method.
The organic precipitation method is a method commonly used for removing protein in pretreatment of breast milk oligosaccharide, has simple operation and low cost, but takes longer time, needs precipitation at low temperature, and in the organic precipitation process, organic solvents, particularly ethanol and acetonitrile, can cause recrystallization of lactose and oligosaccharide, thereby reducing the recovery rate of HMOs.
The ultrafiltration method can effectively remove the protein in the breast milk, is simple and time-saving to operate and can reduce the loss of HMOs.
The current quantitative determination methods of HMOs comprise: high performance anion exchange chromatography, capillary electrophoresis chromatography, high performance liquid chromatography, and liquid chromatography-mass spectrometry. The various methods are as follows:
the high-efficiency anion exchange chromatography is widely applied to qualitative and quantitative analysis of breast milk oligosaccharides, particularly to separation of oligosaccharide isomers, but the pretreatment process is complicated, and neutral oligosaccharides and acidic oligosaccharides in the breast milk can be analyzed and detected only after being pre-separated by a silica gel column.
Capillary electrophoresis chromatography, if used with a UV detector, does not require derivatization, but can only detect a portion of the oligosaccharides, such as sialic acid-related oligosaccharides; if a fluorescence detector is used, various oligosaccharides including neutral and acidic oligosaccharides can be detected at the same time, and the isomers of various breast milk oligosaccharides can be separated at the same time, but a sample needs to be extracted and derivatized by using a graphite carbon stationary phase, the pretreatment process is complicated and time-consuming, and the sample loss is easily caused in the purification process.
Only a portion of breast milk oligosaccharides can be detected using ion chromatography, liquid chromatography, and mass spectrometry alone.
The liquid chromatography-mass spectrometry is widely used for qualitative and quantitative analysis and detection of breast milk oligosaccharides at present, but if an amino chromatographic column is used, the separation of oligosaccharide isomers is limited, and the quantitative limit is higher.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a method for detecting breast milk oligosaccharides, which can detect 21 breast milk oligosaccharides simultaneously, and has accurate and reliable results.
The invention provides a detection method of breast milk oligosaccharide, which comprises the following steps:
A) pretreating an emulsion sample, and reducing by using sodium borohydride to obtain a solution to be detected;
reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution;
B) measuring the standard substance solution by adopting high performance liquid chromatography-mass spectrometry, and establishing a standard curve; performing qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography, and performing quantitative analysis by adopting a mass spectrum combined with a standard curve;
the chromatographic conditions are as follows: a chromatographic column: hypercarb porous graphite carbon chromatography; the mobile phase A is: an aqueous solution containing 0.05 to 0.15% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15% formic acid; and (4) gradient elution.
Preferably, the milk sample is liquid milk or a milk powder solution obtained by dissolving milk powder in water.
Preferably, the chromatographic conditions are:
the chromatographic column is a Hypercarb porous graphite carbon chromatographic column with the diameter of 2.1 multiplied by 100mm and the diameter of 3 mu m; the temperature of the chromatographic column is 25-35 ℃; the flow rate is 0.2 mL/min; the amount of sample was 10. mu.L.
The mobile phase A is: an aqueous solution containing 0.1 to 0.12% by volume of formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.1-0.12 v% formic acid.
Preferably, the gradient elution is specifically:
0～2min，0％～7％B；2～20min，7％～12％B；20～30min，12％～30％B；30～35min，30～40％B；35～38min，40％～100％B；38～40min，100～0％B；40～45min，0％B。
preferably, the mass spectrometry conditions are:
an electrospray ion source is adopted; scanning negative ions; the electric spray is 25-30V, and the ion source temperature is 130-180 ℃.
Drying gas temperature: and (3) drying gas flow rate at 250-350 ℃: 10-15L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 250 to 350 ℃; flow rate of heat preservation gas: 10 to 15L/min.
Preferably, the breast milk oligosaccharide is any one or more of the following:
lactoyl-N-trisaccharide, lactoyl-N-tetrasaccharide, lactoyl-N-neotetraose, lacto-N-neohexaose, p-lacto-N-neohexaose, lactoyl-N-neooctaose, 2' -fucosyllactose, 3-fucosyllactose, difucosyllactose, lactoyl-N-fucopentaose I, lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose V, lactoyl-N-difuconeooctaose, lactoyl-N-difucohexaose I, lactoyl-N-difucohexaose II, lacto-N-difucosyl-hexaose, 3' -sialylated lactose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c.
Preferably, the injection concentration of the lactoyl-N-trisaccharide is 0.156 to 20 mu g/mL, the injection concentration of the lactoyl-N-tetraose is 0.39 to 50 mu g/mL, the injection concentration of the lactoyl-N-neotetraose is 0.156 to 20 mu g/mL, the injection concentration of the lactose-N-neohexaose and the para-lactose-N-neohexaose is 0.156 to 20 mu g/mL, the injection concentration of the lactoyl-N-neooctaose is 0.39 to 50 mu g/mL, the injection concentration of the 2' -fucose lactose is 0.39 to 50 mu g/mL, the injection concentration of the 3-fucose lactose is 0.39 to 50 mu g/mL, the injection concentration of the difucose lactose is 0.39 to 50 mu g/mL, the injection concentration of the lactoyl-N-fucose I is 0.39 to 50 mu g/mL, the injection concentration of lactoyl-N-fucopentaose II is 0.156-20 mug/mL, the injection concentration of lactoyl-N-fucopentaose V is 0.156-20 mug/mL, the injection concentration of lactoyl-N-difucooctaose is 0.39-50 mug/mL, the injection concentration of lactoyl-N-difucohexaose I is 0.39-50 mug/mL, the injection concentration of lactoyl-N-difucohexaose II is 0.156-20 mug/mL, the injection concentration of lactose-N-difucosyl neohexaose is 0.156-20 mug/mL, the injection concentration of 3 '-sialylated lactose is 0.156-20 mug/mL, the injection concentration of 6' -sialylated lactose is 0.39-50 mug/mL, the injection concentration of sialylated-lactose-N-tetraose a is 0.39-50 mug/mL, the injection concentration of the sialyl-lactose-N-tetraose b is 0.39-50 mu g/mL, and the injection concentration of the sialyl-lactose-N-tetraose c is 0.39-50 mu g/mL.
Preferably, the pretreatment of step a) is the removal of fat and protein;
the fat removal method specifically comprises the following steps: centrifuging for 10-15 min at the temperature of 2-5 ℃ at the speed of 12000-14000 rpm/min;
the protein removal method specifically comprises the following steps: and (3) passing the solution after the fat is removed through a 10kDa molecular weight filter, and centrifuging for 20-40 min at 12000-14000 rpm/min.
Preferably, the reduction of the milk sample in the step a) by using sodium borohydride specifically comprises:
adding sodium borohydride into the pretreated milk sample for reaction, adding acetic acid to stop the reaction, and filtering to obtain the product; the volume ratio of the pretreated milk sample to the sodium borohydride is (100-200): (100-200); the reaction temperature is 50-60 ℃; the reaction time is 30-40 min; the molar ratio of the sodium borohydride to the acetic acid is (0.5-1): (0.5 to 1).
Preferably, the reduction of the breast milk oligosaccharide standard substance in the step A) by using sodium borohydride specifically comprises the following steps:
adding sodium borohydride into a breast milk oligosaccharide standard substance for reaction, and adding acetic acid to stop the reaction to obtain the breast milk oligosaccharide standard substance; the reaction temperature is 50-60 ℃; the reaction time is 30-40 min; the molar ratio of the sodium borohydride to the acetic acid is (0.25-0.5): (0.25 to 0.5); the adding amount ratio of the breast milk oligosaccharide standard substance to the sodium borohydride is 1: 1.
Compared with the prior art, the invention provides a detection method of breast milk oligosaccharide, which comprises the following steps: A) pretreating an emulsion sample, and reducing by using sodium borohydride to obtain a solution to be detected; reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution; B) measuring the standard solution by adopting high performance liquid chromatography, and establishing a standard curve; performing qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography, and performing quantitative analysis by adopting a mass spectrum combined with a standard curve; the chromatographic conditions are as follows: a chromatographic column: hypercarb porous graphite carbon chromatography; the mobile phase A is: an aqueous solution containing 0.05 to 0.15% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15% formic acid; gradient elution. The method can effectively separate isomers, can quantify oligosaccharides with extremely low concentration in breast milk, and has low quantification limit. The method has the advantages of simple pretreatment and less HMO loss of the sample.
Drawings
FIG. 1 is a diagram of qualitative and quantitative analysis of breast milk oligosaccharides by an amino chromatography column-liquid chromatography-mass spectrometry;
FIG. 2 is a diagram showing qualitative and quantitative analysis of breast milk oligosaccharides by a porous graphite carbon column-liquid chromatography-mass spectrometry method;
FIG. 3a is a total ion flow graph of 21 breast milk oligosaccharide standards under initial elution conditions; b is a total ion flow diagram of 21 breast milk oligosaccharides mixed targets under the optimal gradient condition; the graph cd shows the oligosaccharide isomers under the optimal gradient condition.
Detailed Description
The invention provides a detection method of breast milk oligosaccharide, and a person skilled in the art can use the content of the text for reference and appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a method for detecting breast milk oligosaccharide, which comprises the following steps:
A) pretreating an emulsion sample, and reducing by using sodium borohydride to obtain a solution to be detected;
reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution;
B) measuring the standard solution by adopting high performance liquid chromatography, and establishing a standard curve; performing qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography, and performing quantitative analysis by adopting a mass spectrum combined with a standard curve;
the chromatographic conditions are as follows: a chromatographic column: hypercarb porous graphitic carbon chromatography; the mobile phase A is as follows: an aqueous solution containing 0.05 to 0.15% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15% formic acid; gradient elution.
The detection method of the breast milk oligosaccharide provided by the invention is particularly suitable for detecting the breast milk oligosaccharide in the milk powder, and the milk sample comprises but is not limited to liquid milk or a milk powder solution obtained by dissolving the milk powder in water. Wherein the liquid milk sample can be breast milk, cow milk, goat milk, etc.
The breast milk oligosaccharide of the invention is any one or more of the following:
lactoyl-N-trisaccharide, lactoyl-N-tetrasaccharide, lactoyl-N-neotetraose, lacto-N-neohexaose, p-lacto-N-neohexaose, lactoyl-N-neooctaose, 2' -fucosyllactose, 3-fucosyllactose, difucosyllactose, lactoyl-N-fucopentaose I, lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose V, lactoyl-N-difuconeooctaose, lactoyl-N-difucohexaose I, lactoyl-N-difucohexaose II, lacto-N-difucosyl-hexaose, 3' -sialylated lactose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c.
The milk sample of the invention is first pretreated.
According to the invention, the pretreatment is preferably a removal of fat and protein;
the fat removal method specifically comprises the following steps: centrifuging for 10-15 min at the temperature of 2-5 ℃ at the speed of 12000-14000 rpm/min;
the protein removal method specifically comprises the following steps: and (3) passing the solution after the fat is removed through a 10kDa molecular weight filter, and centrifuging for 20-40 min at 12000-14000 rpm/min.
And after pretreatment, reducing by using sodium borohydride to obtain a solution to be detected.
The reduction of the milk sample by using sodium borohydride specifically comprises the following steps:
and adding sodium borohydride into the pretreated milk sample for reaction, adding acetic acid to stop the reaction, and filtering to obtain the product.
The volume ratio of the pretreated milk sample to the sodium borohydride is preferably (100-200): (100-200); the reaction temperature is preferably 50-60 ℃; more preferably a water bath reaction at 60 ℃; the reaction time is 30-40 min; the molar ratio of the sodium borohydride to the acetic acid is (0.5-1): (0.5 to 1). The filtration is performed by adopting a 0.22 mu m filter membrane.
And (3) reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution.
The breast milk oligosaccharide standard substance is preferably reduced by sodium borohydride, and specifically comprises the following steps:
adding sodium borohydride into the breast milk oligosaccharide standard substance for reaction, and adding acetic acid to terminate the reaction to obtain the breast milk oligosaccharide standard substance; the reaction temperature is 50-60 ℃; the reaction time is 30-40 min; the molar ratio of the sodium borohydride to the acetic acid is (0.25-0.5): (0.25 to 0.5); the adding amount ratio of the breast milk oligosaccharide standard substance to the sodium borohydride is 1: 1.
After the breast milk sample is degreased and deproteinized, oligosaccharide is reduced, and sample loading detection can be performed through a filter membrane without solid-phase extraction and desalination.
The standard substance is the 21 oligosaccharide standard substances, 1-3mg of the standard substance is dissolved in 2-6 mL of ultrapure water, and the solution is uniformly mixed to obtain a primary standard substance solution which is stored at-20 to-80 ℃.
The establishing process of the standard solution with different concentrations is preferably as follows:
taking primary standard solutions of the group 1 standard sample, wherein the primary standard solutions comprise 60-120 mu L of each of lactyl-N-tetraose, lactyl-N-neooctaose, 2 '-fucosyllactose, 3-fucosyllactose, difucosyllactose, lactyl-N-fucopentaose I, lactyl-N-difucohexaose I, lacto-N-difucosyl-octaose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialylated-lacto-N-tetraose b and sialylated-lacto-N-tetraose c;
taking the group 2 standard products to comprise 24-48 mu L of each of lactyl-N-trisaccharide, lactyl-N-neotetraose, lacto-N-neohexaose, para-lacto-N-neohexaose, lacto-N-fucopentaose II, lacto-N-fucopentaose V, lacto-N-difuconeohexaose, lacto-N-difucohexaose II and 3' -sialylated lactose.
Mixing all the samples, concentrating in vacuum to dry, adding 200-400 mu L of ultrapure water, mixing uniformly, adding 200-400 mu L of sodium borohydride (0.25-0.5M) to react in a water bath at 60 ℃ for 30-40 min, adding 200-400 mu L of acetic acid (0.25-0.5M) to terminate the reaction, obtaining the samples of the group 1 with the concentration of 50 mu g/mL and the samples of the group 2 with the concentration of 20 mu g/mL, and taking 21 mixed samples to dilute into different concentrations step by step respectively.
And (4) measuring the standard substance solution by adopting high performance liquid chromatography, and establishing a standard curve. And carrying out qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography.
The chromatographic conditions of the invention are as follows:
the type and specification of the chromatograph are not limited in the present invention, and those skilled in the art are familiar with the present invention. Preferably, the column: hypercarb porous graphite carbon chromatography; 2.1X 100mm, 3 μm; the preferable temperature of the chromatographic column is 25-35 ℃; more preferably 28 to 32 ℃.
The high performance liquid chromatography adopts a porous graphite carbon chromatographic column with the diameter of 2.1 multiplied by 100mm and the diameter of 1.7 mu m, which is more beneficial to the separation of isomers in the breast milk oligosaccharide, and the high performance liquid chromatography has higher sensitivity and separation degree, so that the detection of the breast milk oligosaccharide is more accurate and effective.
The flow rate is 0.2 mL/min; the amount of sample was 10. mu.L.
The mobile phase A is: an aqueous solution containing 0.05 to 0.15% by volume of formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15 v% formic acid; preferably, the mobile phase a is: an aqueous solution containing 0.1 to 0.12% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.1-0.12% formic acid.
The gradient elution of the invention is preferably specifically:
0～2min，0％～7％B；2～20min，7％～12％B；20～30min，12％～30％B；30～35min，30～40％B；35～38min，40％～100％B；38～40min，100～0％B；40～45min，0％B。
and performing quantitative analysis by combining mass spectrum with a standard curve; and (5) quantifying by combining an external standard method with the peak area percentage.
According to the invention, the mass spectrometry conditions are preferably:
an electrospray ion source is adopted; scanning negative ions; the electrospray ionization is 25-30V, and the ion source temperature is 130-180 ℃.
Drying gas temperature: and (3) drying gas flow rate at 250-350 ℃: 10-15L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 250 to 350 ℃; flow rate of heat preservation gas: 10 to 15L/min.
More preferably:
an electrospray ion source; scanning negative ions; the mass spectrum parameters are as follows: drying gas temperature: 300 ℃, dry gas flow rate: 12L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 300 ℃; flow rate of heat preservation gas: 10-15L/min.
The sample injection concentration of the lactoyl-N-trisaccharide is 0.156-20 mu g/mL, the sample injection concentration of the lactoyl-N-tetraose is 0.39-50 mu g/mL, the sample injection concentration of the lactoyl-N-neotetraose is 0.156-20 mu g/mL, the sample injection concentration of the lactose-N-neohexaose and the para-lactose-N-neohexaose is 0.156-20 mu g/mL, the sample injection concentration of the lactoyl-N-neooctaose is 0.39-50 mu g/mL, the sample injection concentration of the 2' -fucose lactose is 0.39-50 mu g/mL, the sample injection concentration of the 3-fucose lactose is 0.39-50 mu g/mL, the sample injection concentration of the difucosyl lactose is 0.39-50 mu g/mL, the sample injection concentration of the lactoyl-N-pentaose I is 0.39-50 mu g/mL, the injection concentration of lactoyl-N-fucopentaose II is 0.156-20 mug/mL, the injection concentration of lactoyl-N-fucopentaose V is 0.156-20 mug/mL, the injection concentration of lactoyl-N-difucooctaose is 0.39-50 mug/mL, the injection concentration of lactoyl-N-difucohexaose I is 0.39-50 mug/mL, the injection concentration of lactoyl-N-difucohexaose II is 0.156-20 mug/mL, the injection concentration of lactose-N-difucosyl neohexaose is 0.156-20 mug/mL, the injection concentration of 3 '-sialylated lactose is 0.156-20 mug/mL, the injection concentration of 6' -sialylated lactose is 0.39-50 mug/mL, the injection concentration of sialylated-lactose-N-tetraose a is 0.39-50 mug/mL, the injection concentration of the sialyl-lactose-N-tetraose b is 0.39-50 mu g/mL, and the injection concentration of the sialyl-lactose-N-tetraose c is 0.39-50 mu g/mL.
According to the invention, through sample pretreatment, oligosaccharide is reduced into a sugar alcohol form, so that spectrogram complication caused by separation of alpha and beta isomers by a porous graphite carbon column can be avoided, and meanwhile, the response intensity of oligosaccharide detection is greatly improved. And establishing a standard curve on the basis of a standard substance in a high performance liquid chromatography and mass spectrometry mode, detecting the breast milk oligosaccharide in the sample loading sample in the same mode, and obtaining the content of the breast milk oligosaccharide by combining the standard curve.
The invention provides a method for detecting breast milk oligosaccharide, which comprises the following steps: A) pretreating an emulsion sample, and reducing by using sodium borohydride to obtain a solution to be detected; reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution; B) measuring the standard solution by adopting high performance liquid chromatography, and establishing a standard curve; performing qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography, and performing quantitative analysis by adopting a mass spectrum combined with a standard curve; the chromatographic conditions are as follows: a chromatographic column: hypercarb porous graphite carbon chromatography; the mobile phase A is as follows: an aqueous solution containing 0.05 to 0.15% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15% formic acid; gradient elution. The method can effectively separate isomers, can quantify oligosaccharides with extremely low concentration in breast milk, and has low quantification limit. The method has the advantages of simple pretreatment and less HMO loss of the sample. The invention omits the solid phase removing extraction process, thereby greatly reducing the loss of oligosaccharide. In the solid-phase extraction process, ultrapure water is generally used for desalting, and acetonitrile solutions with different concentrations are used for eluting breast milk oligosaccharide, so that on one hand, desalting is difficult to complete, and on the other hand, breast milk oligosaccharide is difficult to completely elute from an extraction column.
In order to further illustrate the present invention, the following examples are provided to describe the method for detecting breast milk oligosaccharides.
Example 1
and 2, taking 1mg of the 21 biological oligosaccharide standard products, dissolving the 21 biological oligosaccharide standard products in 2mL of ultrapure water, uniformly mixing to obtain a primary standard product solution, and storing at-80 ℃.
Step 3, taking the primary standard substance solutions of group 1 standard substances including lactyl-N-tetraose, lactyl-N-neooctaose, 2 '-fucosyllactose, 3-fucosyllactose, difucosyllactose, lactyl-N-fucopentaose I, lactyl-N-difucohexaose I, lacto-N-difucosyl-octaose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialylated-lacto-N-tetraose b and sialylated-lacto-N-tetraose c, 60 muL respectively, and taking group 2 standard substances including lactyl-N-trisaccharide, lacto-N-neotetraose, lacto-N-neohexaose and p-lacto-N-neohexaose, 24 mu L of each of lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose V, lactoyl-N-difucohexaose II and 3' -sialylated lactose is mixed, vacuum concentration is carried out until drying is carried out, 200 mu L of ultrapure water is added for even mixing, 200 mu L of sodium borohydride (0.25M) is added for reaction for 30min under a water bath at 60 ℃, 200 mu L of acetic acid (0.25M) is added for stopping reaction, the concentration of the group 1 standard sample is 50 mu g/mL, the concentration of the group 2 standard sample is 20 mu g/mL, and the 21 mixed standards are taken and respectively diluted step by step into different concentrations.
Step 4, establishing a standard curve for standard substance solutions with different concentrations by adopting a high performance liquid chromatography and mass spectrometry mode;
the high performance liquid chromatography adopts Hypercarb porous graphite carbon chromatography (2.1 × 100mm, 3 μm,) and takes 0.1% formic acid water solution (A) and 0.1% formic acid acetonitrile solution (B) as mobile phases; the gradient elution procedure was: 0-2min, 0% -7% of B; 2-20min, 7% -12% of B; 20-30min, 12% -30% B; 30-35min, 30-40% B; 35-38min, 40% -100% B; 38-40min, 100-0% B; 40-45min, 0% B; the flow rate was 0.2mL/min, and the column temperature was 30 ℃.
The mass spectrum adopts an electrospray ion source; scanning negative ions; the mass spectrum parameters are as follows: drying gas temperature: 300 ℃, drying gas flow rate: 12L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 300 ℃; flow rate of heat preservation gas: 10-15L/min.
And 5, carrying out high performance liquid chromatography and mass spectrometry on the sample by using the same detection method, and combining a standard curve to obtain the contents of the 21 breast milk oligosaccharides.
Example 2
The difference from example 1 is only the sample pretreatment, specifically:
and 2, taking 1mg of the 21 biological oligosaccharide standard products, dissolving the 21 biological oligosaccharide standard products in 2mL of ultrapure water, uniformly mixing to obtain a primary standard product solution, and storing at-80 ℃. .
Step 3, taking the primary standard substance solutions of group 1 standard substances including lactyl-N-tetraose, lactyl-N-neooctaose, 2 '-fucosyllactose, 3-fucosyllactose, difucosyllactose, lactyl-N-fucopentaose I, lactyl-N-difucohexaose I, lacto-N-difucosyl-octaose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialylated-lacto-N-tetraose b and sialylated-lacto-N-tetraose c, 60 muL respectively, and taking group 2 standard substances including lactyl-N-trisaccharide, lacto-N-neotetraose, lacto-N-neohexaose and p-lacto-N-neohexaose, 24 mu L of each of lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose V, lactoyl-N-difucohexaose II and 3' -sialylated lactose is mixed, vacuum concentration is carried out until the mixture is dried, 200 mu L of ultrapure water is added for even mixing, 200 mu L of sodium borohydride (0.25M) is added for reaction for 30min in a water bath at 60 ℃, 200 mu L of acetic acid (0.25M) is added for stopping the reaction, the concentration of the group 1 standard sample is 50 mu g/mL, the concentration of the group 2 standard sample is 20 mu g/mL, and the 21 mixed standard samples are respectively diluted step by step into different concentrations.
Step 4, establishing a standard curve for standard solutions with different concentrations by adopting a high performance liquid chromatography and mass spectrometry mode;
the high performance liquid chromatography adopts Hypercarb porous graphite carbon chromatography (2.1 × 100mm, 3 μm,) and takes 0.1% formic acid water solution (A) and 0.1% formic acid acetonitrile solution (B) as mobile phases; the gradient elution procedure was: 0-2min, 0% -7% of B; 2-20min, 7% -12% of B; 20-30min, 12% -30% B; 30-35min, 30-40% B; 35-38min, 40% -100% B; 38-40min, 100-0% B; 40-45min, 0% B; the flow rate was 0.2mL/min, and the column temperature was 30 ℃.
The mass spectrum adopts an electrospray ion source; scanning negative ions; the mass spectrum parameters are as follows: drying gas temperature: 300 ℃, dry gas flow rate: 12L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 300 ℃; flow rate of heat preservation gas: 10-15L/min.
And 5, carrying out high performance liquid chromatography and mass spectrometry on the sample by using the same detection method, and combining a standard curve to obtain the contents of the 21 breast milk oligosaccharides.
Example 3
The difference from example 1 is only the sample pretreatment, specifically:
And 2, taking 1mg of the 21 biological oligosaccharide standard products, dissolving the 21 biological oligosaccharide standard products in 2mL of ultrapure water, uniformly mixing to obtain a primary standard product solution, and storing at-80 ℃. .
Step 3, taking the primary standard substance solutions of group 1 standard substances including lactyl-N-tetraose, lactyl-N-neooctaose, 2 '-fucosyllactose, 3-fucosyllactose, difucosyllactose, lactyl-N-fucopentaose I, lactyl-N-difucohexaose I, lacto-N-difucosyl-octaose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialylated-lacto-N-tetraose b and sialylated-lacto-N-tetraose c, 60 muL respectively, and taking group 2 standard substances including lactyl-N-trisaccharide, lacto-N-neotetraose, lacto-N-neohexaose and p-lacto-N-neohexaose, 24 mu L of each of lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose V, lactoyl-N-difucohexaose II and 3' -sialylated lactose is mixed, vacuum concentration is carried out until the mixture is dried, 200 mu L of ultrapure water is added for even mixing, 200 mu L of sodium borohydride (0.25M) is added for reaction for 30min in a water bath at 60 ℃, 200 mu L of acetic acid (0.25M) is added for stopping the reaction, the concentration of the group 1 standard sample is 50 mu g/mL, the concentration of the group 2 standard sample is 20 mu g/mL, and the 21 mixed standard samples are respectively diluted step by step into different concentrations.
And 4, establishing a standard curve for the standard solutions with different concentrations by adopting a high performance liquid chromatography and mass spectrometry mode.
The high performance liquid chromatography adopts Hypercarb porous graphite carbon chromatography (2.1 × 100mm, 3 μm,) and takes 0.1% formic acid water solution (A) and 0.1% formic acid acetonitrile solution (B) as mobile phases; the gradient elution procedure was: 0-2min, 0% -7% B; 2-20min, 7% -12% of B; 20-30min, 12% -30% B; 30-35min, 30-40% B; 35-38min, 40% -100% B; 38-40min, 100-0% B; 40-45min, 0% B; the flow rate was 0.2mL/min, and the column temperature was 30 ℃.
The mass spectrum adopts an electrospray ion source; scanning negative ions; the mass spectrum parameters are as follows: drying gas temperature: 300 ℃, dry gas flow rate: 12L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 300 ℃; flow rate of heat preservation gas: 10-15L/min.
And 5, carrying out high performance liquid chromatography and mass spectrometry on the sample by using the same detection method, and combining a standard curve to obtain the contents of the 21 breast milk oligosaccharides.
Table 121 breast milk oligosaccharide standard MRM optimization results
LNT II, LNT, LNnT, LNnH + p-LNnH, LNnO, 2'FL, 3FL, LDFT, LNFP I, LNFP II, LNFP V, LNnDFH, LNDFH I, LNDFH II, DFLNH, 3' -SL, 6'-SL, LST a, LST b, LST c correspond to lacto-N-trisaccharide, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-neohexaose, p-lacto-N-neohexaose, lacto-N-neooctaose, 2' -fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-pentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose I, lacto-N-tetraose, and L-N-neohexaose in Table 1, respectively, lactoyl-N-difucohexaose II, lacto-N-difucosyl-octaose, 3 '-sialyllactose, 6' -sialyllactose, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c.
TABLE 221 concentrations of Standard solutions (. mu.g/mL)
Regression equation of 321 breast milk oligosaccharide standard products in table
As can be seen from Table 3, the 21 oligosaccharide standards showed good linearity (R2 ≧ 0.995) within the set concentration range. The precision of HPLC analysis detection method is good, and RSD (relative standard deviation) value of 21 saccharide components in repeated sample injection analysis is controlled within 10%.
Methodology validation
Standard recovery rate experiment
Preparing an unlabeled sample: taking a breast milk sample 200ul, 13000r/min, centrifuging for 15min at 4 ℃, removing upper-layer fat, taking out the lower clear liquid, passing the lower clear liquid through a 10kDa molecular weight filter, centrifuging for 30min at 13000rpm, taking 100 mu L of filtrate, adding 100 mu L of sodium borohydride (0.5M), reacting for 30min in a water bath at 60 ℃, adding 100 mu L of acetic acid (0.5M) to terminate the reaction, filtering through a 0.22 mu M filter membrane, and injecting samples. Make 3 repetitions
Preparation of a labeled sample: taking 200 mu L breast milk sample, respectively adding 21 kinds of oligosaccharides to correspondingly enhance the concentration, and preparing the rest pretreatment steps together with standard samples: after the labeling is added, 13000r/min is carried out, centrifugation is carried out for 15min at 4 ℃, then the fat on the upper layer is removed, the lower clear liquid is taken out, the lower clear liquid passes through a 10kDa molecular weight filter and is centrifuged for 30min at 13000rpm, 100 mu L of filtrate is taken, 100 mu L of sodium borohydride (0.5M) is added to react for 30min in water bath at 60 ℃,100 mu L of acetic acid (0.5M) is added to stop the reaction, and the mixture is filtered by a filter membrane of 0.22 mu M and injected. Repeat for 3 times.
TABLE 4 results of recovery test with standard addition
Method accuracy and precision experiment
The method is used for detecting the accuracy and the precision of the method of the 21 breast milk oligosaccharide standard substance mixed standard solutions under two concentrations of high (6.25 mug/mL, 2.5 mug/mL) and low (0.78 mug/mL and 0.313 mug/mL) for 3 times of repetition.
TABLE 5 results of the accuracy and precision tests
As can be seen from tables 4 and 5, the addition recovery rate and accuracy of 21 breast milk oligosaccharides were all between 80% and 120%, the accuracy of the sample pretreatment method was represented by the RSD value, the RSD values of the 21 oligosaccharides obtained by the experiment were all less than 10%, the accuracy of the method was represented by the coefficient of variation CV value, and the CV values of the 21 oligosaccharides obtained by the experiment were all less than 15%. Therefore, the method established in the experiment can be used for detecting and analyzing 21 oligosaccharides in breast milk, and breast milk samples (n ═ 6) of different lactation periods provided by 2 volunteers are selected, and the content of oligosaccharide in the breast milk is determined by the method for accurately determining breast milk oligomerization, and the determination results are shown in table 6.
TABLE 6 measurement of oligosaccharides in human colostrum
Note: "nd" indicates no detection.
Comparative example 1:
FIG. 1 shows qualitative and quantitative analysis of breast milk oligosaccharides by amino chromatography column-liquid chromatography-mass spectrometry. The method is used for detecting 12 breast milk oligosaccharides in a co-separation mode, wherein 4 isomers are included, and the linear range detected by the method is 0.78-100 mu g/mL.
FIG. 2 shows qualitative and quantitative analysis of breast milk oligosaccharides by a porous graphite carbon column-liquid chromatography-mass spectrometry method, wherein 21 breast milk oligosaccharides including 6 isomers are qualitatively and quantitatively analyzed together, and the linear detection range is 0.156-50 μ g/mL. Wherein the upper graph of FIG. 2 is a chromatogram for qualitatively and quantitatively analyzing total ion current of 21 breast milk oligosaccharides, and the lower graph is a test result graph of 6 isomers; therefore, the method is superior to the method in the background art no matter the capacity of separating the breast milk oligosaccharide isomer or the detection limit of the method.
Comparative example 2
Fig. 3a is a total ion flow diagram of 21 breast milk oligosaccharide standards under initial elution conditions, and the initial set mobile phase conditions are as follows: a was 0.1% formic acid water, B was 0.1% formic acid + acetonitrile, and the flow rate was 0.2 mL/min. Gradient elution is adopted for 0-21 min, 0-21% of B,21-32min, 12-40% of B,32-37min, 40-100% of B,37-42min, 100% of B,42-43min, 100-0% of B,43-55min and 0% of B. By varying the gradient and elution time, optimal gradient conditions were obtained: 0-2min, 0-7% of B,2-20min, 7-12% of B,20-30min, 12-30% of B,30-35min, 30-40% of B,35-38min, 40-100% of B,38-40min, 100-0% of B, 40-45min and 0% of B. The flow chart of the 21 breast milk oligosaccharide mixed target total ion flow under the optimal gradient condition is shown in figure 3 b; furthermore, after optimization, the initial elution time was shortened by 10 minutes and the oligosaccharide isomers were better separated, such as lsta, LSTb, LSTc (see fig. 3 cd).
Comparative example 3
In order to verify the optimal deproteinization method, the recovery rate of each oligosaccharide in the breast milk was determined after the organic precipitation method and the ultrafiltration method were applied to the same breast milk sample, respectively.
As shown in Table 7, the average recovery rate of the breast milk samples treated by the ultrafiltration method is higher than that of the samples treated by the ethanol precipitation method, which indicates that the oligosaccharide in the breast milk samples can be more greatly retained and the loss generated in the pretreatment can be reduced by treating the breast milk samples by the ultrafiltration method.
TABLE 7 comparison of Experimental results for pretreatment methods
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A method for detecting an oligosaccharide in breast milk, comprising:
A) pretreating the milk sample, and reducing by using sodium borohydride to obtain a solution to be detected;
reducing the breast milk oligosaccharide standard substance by using sodium borohydride to obtain a standard substance solution;
B) measuring the standard substance solution by adopting high performance liquid chromatography-mass spectrometry, and establishing a standard curve; performing qualitative analysis on the liquid to be detected by adopting a high performance liquid chromatography, and performing quantitative analysis by adopting a mass spectrum combined with a standard curve;
the chromatographic conditions are as follows: a chromatographic column: hypercarb porous graphitic carbon chromatography; the mobile phase A is: an aqueous solution containing 0.05 to 0.15% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.05-0.15% formic acid; gradient elution.
2. The detection method according to claim 1, wherein the milk sample is liquid milk or a milk powder solution obtained by dissolving milk powder in water.
3. The detection method according to claim 1, wherein the chromatographic conditions are:
the chromatographic column is a Hypercarb porous graphite carbon chromatographic column with the diameter of 2.1 multiplied by 100mm and the diameter of 3 mu m; the temperature of the chromatographic column is 25-35 ℃; the flow rate is 0.2 mL/min;
the mobile phase A is: an aqueous solution containing 0.1 to 0.12% formic acid; the mobile phase B is as follows: acetonitrile solution containing 0.1-0.12% formic acid.
4. The detection method according to claim 1, wherein the gradient elution is in particular:
0～2min，0％～7％B；2～20min，7％～12％B；20～30min，12％～30％B；30～35min，30～40％B；35～38min，40％～100％B；38～40min，100～0％B；40～45min，0％B。
5. the detection method according to claim 1, wherein the mass spectrometry conditions are:
an electrospray ion source is adopted; scanning negative ions; the electric spraying voltage is 25-30V, and the ion source temperature is 130-180 ℃;
drying gas temperature: at 250-350 ℃, drying gas flow rate: 10-15L/min; atomizer pressure: 15-25 psi; capillary voltage: 2-5 kV; temperature of insulating gas: 250 to 350 ℃; flow rate of heat preservation gas: 10 to 15L/min.
6. The method according to claim 1, wherein the breast milk oligosaccharide is any one or more of:
lactoyl-N-trisaccharide, lactoyl-N-tetrasaccharide, lactoyl-N-neotetraose, lacto-N-neohexaose, p-lacto-N-neohexaose, lactoyl-N-neooctaose, 2' -fucosyllactose, 3-fucosyllactose, difucosyllactose, lactoyl-N-fucopentaose I, lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose V, lactoyl-N-difuconeooctaose, lactoyl-N-difucohexaose I, lactoyl-N-difucohexaose II, lacto-N-difucosyl-hexaose, 3' -sialylated lactose, 6' -sialylated lactose, sialylated-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c.
7. The detection method according to claim 6, wherein the sample injection concentration of lactoyl-N-trisaccharide is 0.156 to 20 μ g/mL, the sample injection concentration of lactoyl-N-tetraose is 0.39 to 50 μ g/mL, the sample injection concentration of lactoyl-N-neotetraose is 0.156 to 20 μ g/mL, the sample injection concentration of lactose-N-neohexaose and para-lactose-N-neohexaose is 0.156 to 20 μ g/mL, the sample injection concentration of lactoyl-N-neooctaose is 0.39 to 50 μ g/mL, the sample injection concentration of 2' -fucosyllactose is 0.39 to 50 μ g/mL, the sample injection concentration of 3-fucosyllactose is 0.39 to 50 μ g/mL, the sample injection concentration of difucosyllactose is 0.39 to 50 μ g/mL, and the sample injection concentration of lactoyl-N-fucosylI is 0.39 to 50 μ g/mL, the injection concentration of lactoyl-N-fucopentaose II is 0.156-20 mug/mL, the injection concentration of lactoyl-N-fucopentaose V is 0.156-20 mug/mL, the injection concentration of lactoyl-N-difucooctaose is 0.39-50 mug/mL, the injection concentration of lactoyl-N-difucohexaose I is 0.39-50 mug/mL, the injection concentration of lactoyl-N-difucohexaose II is 0.156-20 mug/mL, the injection concentration of lactose-N-difucosyl neohexaose is 0.156-20 mug/mL, the injection concentration of 3 '-sialylated lactose is 0.156-20 mug/mL, the injection concentration of 6' -sialylated lactose is 0.39-50 mug/mL, the injection concentration of sialylated-lactose-N-tetraose a is 0.39-50 mug/mL, the injection concentration of the sialyl-lactose-N-tetraose b is 0.39-50 mu g/mL, and the injection concentration of the sialyl-lactose-N-tetraose c is 0.39-50 mu g/mL.
8. The detection method according to claim 1, wherein the pretreatment of step a) is removal of fat and protein;
the fat removal method specifically comprises the following steps: centrifuging for 10-15 min at the temperature of 2-5 ℃ at the speed of 12000-14000 rpm/min;
the protein removal method specifically comprises the following steps: and (3) passing the solution after the fat is removed through a 10kDa molecular weight filter, and centrifuging for 20-40 min at 12000-14000 rpm/min.
9. The detection method according to claim 1, wherein the reduction of the milk sample in step a) with sodium borohydride specifically comprises:
adding sodium borohydride into the pretreated milk sample for reaction, adding acetic acid to stop the reaction, and filtering to obtain the product; the volume ratio of the pretreated milk sample to the sodium borohydride is (100-200): (100-200); the reaction temperature is 50-60 ℃; the reaction time is 30-40 min; the molar ratio of the sodium borohydride to the acetic acid is (0.5-1): (0.5 to 1).
10. The detection method according to claim 1, wherein the breast milk oligosaccharide standard substance of step a) is reduced by sodium borohydride, specifically:
adding sodium borohydride into a breast milk oligosaccharide standard substance for reaction, and adding acetic acid to stop the reaction to obtain the breast milk oligosaccharide standard substance; the reaction temperature is 50-60 ℃; the reaction time is 30-40 min; the molar ratio of the sodium borohydride to the acetic acid is (0.25-0.5): (0.25 to 0.5); the ratio of the breast milk oligosaccharide standard substance to the sodium borohydride is 1: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210038222.7A CN114740096A (en) | 2022-01-13 | 2022-01-13 | Detection method of breast milk oligosaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210038222.7A CN114740096A (en) | 2022-01-13 | 2022-01-13 | Detection method of breast milk oligosaccharide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114740096A true CN114740096A (en) | 2022-07-12 |
Family
ID=82275964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210038222.7A Pending CN114740096A (en) | 2022-01-13 | 2022-01-13 | Detection method of breast milk oligosaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114740096A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107192771A (en) * | 2017-05-04 | 2017-09-22 | 中国农业科学院农产品加工研究所 | The quantitative method of breast milk oligosaccharide fast qualitative |
| CN107621399A (en) * | 2016-07-14 | 2018-01-23 | 北京三元食品股份有限公司 | A kind of method of oligosaccharide in detection breast milk |
| CN110161147A (en) * | 2019-06-19 | 2019-08-23 | 北京三元食品股份有限公司 | The high-throughput quantification measuring method of free oligosaccharides in cream |
| CN112526021A (en) * | 2020-03-13 | 2021-03-19 | 内蒙古伊利实业集团股份有限公司 | Method for detecting 2' -fucosyllactose in milk |
| CN113686992A (en) * | 2021-08-31 | 2021-11-23 | 黑龙江飞鹤乳业有限公司 | Method for detecting target breast milk oligosaccharide in formula food |
-
2022
- 2022-01-13 CN CN202210038222.7A patent/CN114740096A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107621399A (en) * | 2016-07-14 | 2018-01-23 | 北京三元食品股份有限公司 | A kind of method of oligosaccharide in detection breast milk |
| CN107192771A (en) * | 2017-05-04 | 2017-09-22 | 中国农业科学院农产品加工研究所 | The quantitative method of breast milk oligosaccharide fast qualitative |
| CN110161147A (en) * | 2019-06-19 | 2019-08-23 | 北京三元食品股份有限公司 | The high-throughput quantification measuring method of free oligosaccharides in cream |
| CN112526021A (en) * | 2020-03-13 | 2021-03-19 | 内蒙古伊利实业集团股份有限公司 | Method for detecting 2' -fucosyllactose in milk |
| CN113686992A (en) * | 2021-08-31 | 2021-11-23 | 黑龙江飞鹤乳业有限公司 | Method for detecting target breast milk oligosaccharide in formula food |
Non-Patent Citations (1)
| Title |
|---|
| SARAH M等: "Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies", ANAL BIOANAL CHEM, no. 406, pages 7925 - 7935 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107621399B (en) | Method for detecting oligosaccharide in breast milk | |
| AU2020102328A4 (en) | Method for determination of heterocyclic aromatic amine in oil | |
| CN110542735A (en) | method for high-throughput determination of multiple fat-soluble vitamins by ultra-high performance liquid mass spectrometry | |
| CN101290306B (en) | Milk and milk product tetracycline antibiotic residual quantity checking method | |
| CN110988193B (en) | Method for detecting advanced glycosylation end products in aquatic products | |
| CN112730723B (en) | Method for detecting 22 free amino acids in plasma by ultra-high performance liquid chromatography-tandem mass spectrometry | |
| Piestansky et al. | Ultrasensitive determination of serotonin in human urine by a two dimensional capillary isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry | |
| NL2029775B1 (en) | Method for detecting multiple advanced glycation end products in dairy products | |
| CN111239267B (en) | Method for detecting short-chain fatty acids in serum and lymph tissue based on GC-MS | |
| CN114965756A (en) | Method for detecting neurotransmitter substances | |
| CN108845063B (en) | Detection reagent combination and detection method of aquatic product additive | |
| CN114740096A (en) | Detection method of breast milk oligosaccharide | |
| CN112526021A (en) | Method for detecting 2' -fucosyllactose in milk | |
| CN111721861A (en) | Method for simultaneously detecting multiple heterocyclic amines in cured meat | |
| CN110068618B (en) | Detection method of intestinal flora metabolites related to nephropathy | |
| CN111239313A (en) | Rapid contour analysis method of human milk oligosaccharide | |
| CN116381087A (en) | Method for simultaneously detecting 17 kinds of hot processing hazardous substances in class 4 and application thereof | |
| CN113009002A (en) | Sample pretreatment method containing furfural substances and method for detecting content of furfural substances | |
| CN112924563B (en) | Derivatization-based breast milk oligosaccharide characterization and quantitative analysis method | |
| CN114137097A (en) | Method for detecting melatonin in milk by liquid chromatography-tandem mass spectrometry and performance evaluation thereof | |
| CN115144494B (en) | Method for detecting oligosaccharide in mammal milk | |
| CN112649516A (en) | Derivatization-based milk powder containing 4 human milk oligosaccharides and qualitative and quantitative method thereof | |
| CN110988210A (en) | Method for reducing nonspecific adsorption of fat-soluble vitamin 96-well plate | |
| CN114624338A (en) | Method for quantitatively analyzing free amino acids in biological sample by using liquid chromatography-tandem mass spectrometry | |
| CN110596279A (en) | Method for detecting biogenic amine in cheese |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |