CN115480023B - Method for detecting content of monosaccharide in milk and milk product - Google Patents
Method for detecting content of monosaccharide in milk and milk product Download PDFInfo
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- CN115480023B CN115480023B CN202211365388.6A CN202211365388A CN115480023B CN 115480023 B CN115480023 B CN 115480023B CN 202211365388 A CN202211365388 A CN 202211365388A CN 115480023 B CN115480023 B CN 115480023B
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- 238000001514 detection method Methods 0.000 claims abstract description 25
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- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims description 8
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- 238000012360 testing method Methods 0.000 description 1
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- 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
- G01N30/96—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 using ion-exchange
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- 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
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- 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
- G01N30/96—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 using ion-exchange
- G01N2030/965—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 using ion-exchange suppressor columns
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a method for detecting the content of monosaccharide composition of breast milk oligosaccharide in milk and dairy products, and relates to the field of food detection. The method comprises the following steps: preparing a standard solution: weighing a monosaccharide standard substance, adding a solvent for dissolving, and preparing into a standard solution with a required concentration; preparing a test solution: taking milk prepared from milk, liquid dairy products or solid dairy products, adding acetic acid solution for hydrolysis, adding acetonitrile for precipitating protein, centrifuging, taking supernatant, adding trifluoroacetic acid solution for hydrolysis, fixing volume, filtering and purifying the fixed volume liquid to obtain a test solution; content determination: and measuring the peak areas of the monosaccharides in the standard solution and the test solution by an ion chromatography-tandem mass spectrometry method, and calculating the content of the monosaccharides according to a standard curve method. The detection method provided by the invention has good linearity, and the method has good sensitivity, stability and repeatability, and is suitable for accurately measuring five constituent monosaccharides of breast milk oligosaccharide in milk and dairy products.
Description
Technical Field
The invention relates to the field of food detection, in particular to a method for detecting the content of monosaccharide composition of breast milk oligosaccharide in milk and dairy products.
Background
Human Milk Oligosaccharides (HMOs) are the third solid component contained in Human Milk, and can promote the growth of beneficial bacteria in the intestinal tract, allow the intestinal tract to produce short-chain fatty acids which are vital to the health of infants, and further effectively enhance the immunity of infants. HMOs in human breast milk consist of 5 monomers: d-glucose (D-glucose, glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (sialic acid, sia), wherein N-acetylneuraminic acid (Neu 5 Ac) is the predominant form of sialic acid.
At present, methods for detecting oligosaccharide content in food are divided into two methods, one is that firstly, oligosaccharide is decomposed into micromolecular monosaccharide through hydrolysis or enzymolysis and the like, and then the oligosaccharide content is calculated and reduced; the other method is to directly carry out corresponding detection on standard substances of oligosaccharides one by one. However, over 200 breast milk oligosaccharides have been identified, and if each HMOs is directly measured, more standards are required, which is difficult and costly. In addition, sialic acid is a natural brain nutrient, can promote the intelligence development of infants, improve memory and play an important role in resisting intestinal infection; l-fucose can promote the development of the nervous system and the immune system of infants, and is also an important component of the fucose structure of HMOs. In view of the fact that specific monosaccharides regulate different types of developmental processes and sialic acid and L-fucose in cow's milk and goat's milk are greatly different from that of breast milk in terms of content, type and structure, infant formula powder based on cow's milk or goat's milk is attracting more and more attention as a substitute for breast milk in terms of nutrition and health, and therefore, detection of the content of specific types of monosaccharides is also important.
The conventional method is difficult to detect the constituent monosaccharides in the breast milk oligosaccharide, and because the monosaccharide compounds have similar properties, the monosaccharide compounds have the characteristics of strong polarity, high water solubility, low solubility in common organic reagents, low volatility and lack of color development and fluorescent groups. Currently, methods for detecting monosaccharides mainly include Thin Layer Chromatography (TLC), gas Chromatography (GC), high Performance Liquid Chromatography (HPLC), and Ion Chromatography (IC). Among them, thin Layer Chromatography (TLC) has low sensitivity and poor resolution although it is simple to operate. High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) require derivatization to reduce polarity, either by introducing uv absorption or by fluorescence emitting groups. Sometimes, for a complex substrate, incomplete reaction or other derivatives generated in the derivatization process affect the accuracy of the detection result. And the two methods have more and harsher derivatization conditions and complex operation, and are easily interfered by coexisting compounds and ions in a sample. Non-derivatized saccharide compounds are generally in reverse phase C 18 Cannot be retained on a stationary phase and cannot be effectively separated, so that co-outflow is easy to occur by using liquid chromatography or liquid chromatography-tandem mass spectrometry, so that the qualitative difficulty or sensitivity is causedAnd (5) degree difference. Relatively speaking, the Ion Chromatography (IC) for detecting the monosaccharide compound has the advantages of no need of derivatization treatment, good separation effect, wide linear range and high sensitivity. However, the milk and dairy product matrixes are complex, and the ion chromatography qualitative and quantitative means are single, so that the detection sensitivity is low. Accurate determinations can be made if retention time is the only qualitative criterion, requiring the test compound to achieve baseline separation.
The prior art discloses an ultra-high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously determining 12 saccharide compounds of rhamnose, xylose, arabinose, fructose, mannose, glucose, galactose, mannitol, ribose, fucose, glucuronic acid and galacturonic acid in spirulina polysaccharide hydrolysate (Zhaodan et al, "ultra-high performance liquid chromatography-tandem mass spectrometry determination of monosaccharide composition of spirulina polysaccharide" (35.4 in 2017): 8), however, because of the high saccharide polarity, the monosaccharide composition is usually difficult to be retained on a reversed phase liquid chromatography column and the peak shape is poor, so that effective separation cannot be achieved, in addition, milk and dairy product matrixes are complex, and the content of various composition monosaccharides of breast milk oligosaccharides cannot be accurately determined by referring to the method.
The technical problem to be solved by the technical personnel in the field is how to provide a method which has strong selectivity and high sensitivity and can accurately measure the contents of various monosaccharide compositions of milk oligosaccharide in milk and dairy products. At present, no scheme for detecting constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products by adopting ion chromatography-tandem mass spectrometry (IC-MS/MS) is reported.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect of high difficulty in detecting the constituent monosaccharides in the breast milk oligosaccharides in the prior art, so that a novel method for detecting the content of the constituent monosaccharides in the breast milk oligosaccharides in milk and dairy products is provided.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a method for detecting the content of constituent monosaccharides of breast milk oligosaccharides in milk and dairy products, wherein the constituent monosaccharides are at least one of L-fucose, galactose, glucose, N-acetylglucosamine and sialic acid, and the method comprises the following steps:
(1) Preparing a standard solution: weighing a monosaccharide standard substance, adding a solvent for dissolving, and preparing into a standard solution with a required concentration;
(2) Preparing a test solution: taking milk prepared from milk, liquid dairy products or solid dairy products, adding acetic acid solution for hydrolysis, adding acetonitrile for precipitating protein, centrifuging, taking supernatant, adding trifluoroacetic acid solution for hydrolysis, fixing volume, filtering and purifying the fixed volume liquid to obtain a test solution;
(3) And (3) content determination: measuring the peak areas of monosaccharide in the standard solution and the test solution by ion chromatography-tandem mass spectrometry, calculating the content of monosaccharide according to a standard curve method,
wherein the chromatographic conditions comprise: mobile phase A: water, mobile phase B:250mmol/L NaOH solution, mobile phase C:500mmol/L NaAc solution, according to the following gradient elution procedure: 0.00 to 12.00min, A:94%, B:6%, C:0 percent; 12.00 to 12.10min, A:94% → 30%, B:6% → 40%, C:0% → 30%;12.10 to 17.00min, A:30%, B:40%, C:30 percent; 17.00 to 17.10min, A:30% → 60%, B:40%, C:30% → 0%;17.10 to 22.00min, A:60%, B:40%, C:0 percent; 22.00 to 22.10min, A:60% → 94%, B:40% → 6%, C:0 percent; 22.10 to 27.00min, A:94%, B:6%, C:0 percent.
Further, in the step (3), an ion chromatograph and a tandem mass spectrometer are adopted for detection, the ion chromatograph is connected with the tandem mass spectrometer, a three-way pipe is arranged between the ion chromatograph and the tandem mass spectrometer, and one end of the three-way pipe, which is not communicated with the ion chromatograph and the tandem mass spectrometer, is used for collecting waste liquid.
Further, in the step (3), the chromatographic conditions include: dionex ICS3000 ion chromatograph; dionex IonPac PA10 type anion analysis column, 250 mm. Times.4 mm; dionex IonPac PA10 type guard column, 50 mm. Times.4 mm; dionex ADRS 600 anion suppressor, 4mm, external water mode, suppression current 297mA.
Further, in the step (3), the chromatographic conditions include: the column temperature is 25 to 30 ℃; the temperature of the detection pool is 30 to 35 ℃; the sample size is 20 to 25 mu L; the flow rate was 0.6 to 0.8mL/min.
Further, in the step (3), the chromatographic conditions include: the column temperature is 30 ℃; the temperature of the detection pool is 35 ℃; the sample volume is 25 mu L; the flow rate was 0.8mL/min.
Further, in step (3), the mass spectrometry conditions include: an ion source: an electrospray ion source; ionization mode: a negative ion mode; the scanning mode comprises the following steps: a multi-stage reaction monitoring mode; the flow rate into the tandem mass spectrometer is adjusted to be 0.3 to 0.4mL/min.
Further, in step (3), the mass spectrometry conditions include: the flow rate into the tandem mass spectrometer was adjusted to 0.4mL/min.
Further, in step (3), the mass spectrometry conditions include: a Waters TQ Detector triple quadrupole tandem mass spectrometer; capillary voltage 3.00kV; the flow rate of the gas in the taper hole is 50-150L/h; the flow rate of the collision gas is 0.15mL/min; the outlet potential of the collision pool is 5V; the desolventizing temperature is 500 ℃; the flow rate of the desolventizing gas is 700-1000L/h.
Further, in step (3), the mass spectrometry conditions include: the gas flow rate of the taper hole is 50L/h; the flow rate of the desolventizing gas is 700L/h.
Further, in step (3), the mass spectrometry conditions include:
l-fucose quantitation ion pair: m/z 163.0/89.10, collision energy 7V; l-fucose qualitative ion pair: m/z 163.0/59.10, collision energy 12V; the taper hole voltage is 17V;
galactose quantitative ion pair: m/z 179.0/89.10, collision energy 10V; galactose qualitative ion pair: m/z 179.0/59.10, collision energy 18V; the taper hole voltage is 15V;
glucose quantification ion pair: m/z 179.0/89.10, collision energy 9V; glucose qualitative ion pair: m/z 179.0/59.10, collision energy 12V; the taper hole voltage is 15V;
n-acetylglucosamine quantitative ion pair: m/z 220.2/119.20, collision energy 7V; n-acetylglucosamine qualitative ion pair: m/z 220.2/89.20, collision energy 12V; the taper hole voltage is 18V;
n-acetylneuraminic acid quantification of ion pairs: m/z 308.2/87.20, collision energy 18V; n-acetylneuraminic acid qualitative ion pair: m/z 308.2/290.20, collision energy 7V; cone voltage 19V.
Further, in the step (1), the solvent is a methanol aqueous solution containing 10mmol/L of ammonium acetate, and the volume ratio of water to methanol in the methanol aqueous solution is 4:1.
further, in the step (2), the volume ratio of the milk or liquid dairy product to the acetic acid solution to the acetonitrile to the trifluoroacetic acid solution is 200:200:400:1, the mass fraction of the acetic acid solution is 1%, and the concentration of the trifluoroacetic acid solution is 0.1mol/L.
Further, in the step (2), the centrifugation conditions are as follows: the rotating speed is 8000r/min, and the time is 10min; the conditions for hydrolysis by adding trifluoroacetic acid solution are as follows: the temperature is 80 ℃ and the time is 20 to 60min.
Further, in the step (2), the conditions for adding the trifluoroacetic acid solution for hydrolysis are as follows: the temperature is 80 deg.C, and the time is 40min.
Further, in the step (2), the volume is determined by using a methanol aqueous solution containing 10mmol/L of ammonium acetate, and the volume ratio of water to methanol in the methanol aqueous solution is 4:1, the volume ratio of the milk or liquid dairy product to the constant volume liquid is 1:50.
further, in the step (2), the filtering and purifying conditions are as follows: and (3) sequentially passing the constant volume solution through a 0.22 mu m filter membrane and an IC-RP solid phase extraction column, and collecting filtrate.
Further, in the step (2), when the sample to be detected is a solid dairy product, the method for preparing the milk from the sample to be detected comprises the following steps: and (3) putting the solid dairy product into ultrapure water, and heating to fully dissolve the solid dairy product, wherein the mass-volume ratio of the solid dairy product to the ultrapure water is 0.1g/mL.
The technical scheme of the invention has the following advantages:
1. the invention provides a method for detecting the content of monosaccharide composition of breast milk oligosaccharide in milk and dairy products by ion chromatography-tandem mass spectrometry (IC-MS/MS) for the first time, combines the advantages of high separation capability of ion chromatography to polar substances, high selectivity and high sensitivity monitoring capability of triple quadrupole mass spectrometry and capability of providing relative molecular mass and structural information, and not only can realize the separation of monosaccharide and matrix impurities, but also can confirm the structure of the monosaccharide and the matrix impurities by verifying the separation and detection of five monosaccharide compositions (L-fucose, galactose, glucose, N-acetylglucosamine and sialic acid) of breast milk oligosaccharide in a complex matrix of milk and dairy products, thereby improving the sensitivity and accuracy of detection.
2. The detection method provided by the invention has good linearity, and the method has good sensitivity, stability and repeatability, is suitable for accurately measuring five constituent monosaccharides of breast milk oligosaccharide in milk and dairy products, and provides powerful technical support for quality control of production enterprises and supervision of food quality safety departments.
3. The invention preferably adds and configures ADRS 600 type suppressors in the ion chromatograph because the ion chromatograph mobile phase is strong alkaline substances (NaOH, naAc), which can not volatilize when using ESI source to measure, the corrosive substance can block the sample inlet, the damage to the apparatus is larger, when measuring, the difficult-to-volatilize buffer salt needs to be avoided entering the mass spectrum detector, the electrolytic suppressor in the ion chromatograph can realize on-line desalting, and the influence of salt or alkali in the eluent on the ESI source and the damage to the mass spectrum component are eliminated. By adjusting the current of the suppressor, the suppressor electrolyzes external water to generate H + Na by cation exchange membrane + And H + Exchange and then discharge to waste liquid, and finally enter the mass spectrum, wherein the product is a product after being inhibited by the inhibitor, pure water and a small amount of acetic acid, thereby eliminating the influence of inorganic acid and alkali on the mass spectrum detector.
Drawings
FIG. 1 is a schematic diagram of the connections of an apparatus used in an embodiment of the present invention;
FIG. 2 is a total ion flow diagram of a standard mixture formulated in accordance with the present invention;
FIG. 3 is a statistical chart of peak areas corresponding to five monosaccharides in a test solution obtained by different hydrolysis times according to the present invention;
FIG. 4 is a graph of the results of the present invention using different columns, wherein a is a total ion flow graph using a Dionex CarbonPac PA1 column; b is a total ion flow diagram of a chromatographic column adopting a Dionex CarbonPac PA 20; c is a total ion flow diagram using a Dionex CarbonPac PA10 column.
Detailed Description
The technical features of the present invention will be further described in detail with reference to specific examples.
1. Standard article
L-Fucose (L-Fucose, purity 98%, beijing Solarbio technologies Co., ltd.), galactose (Galactose, purity 99.9%, tanzhain ink quality testing standard substance center), glucose (Glucose, purity 98.7%, tanzhain ink quality testing standard substance center), N-acetylglucosamine (N-Acetyl-D-glucopyranosine, purity 98%, beijing Solarbio technologies Co., ltd.), N-acetylneuraminic acid (N-Acetyl serine, purity 98%, beijing Solarbio technologies Co., ltd.).
2. Apparatus and device
Dionex ICS3000 ion chromatograph (Dionex corporation, usa), amperometric detector and Chromeleon 6.70 chromatography workstation; waters TQD triple quadrupole tandem mass spectrometer (MS/MS) (Waters, japan) equipped with an electrospray ionization source (ESI) and a Labsolutions Ver. 5.91 chromatography workstation; an ultrasonic cleaner (KQ-500 DE, kunshan ultrasonic instruments Co., ltd.); dionex IonPac TM PA10 type anion analysis column (250 mm. Times.4 mm, thermo Co., USA); dionex IonPac TM Protective columns of PA10 type (50 mm. Times.4 mm, thermo company, USA); dionex ADRS 600 anion suppressor (4 mm, thermo corporation, USA); vortex mixer (IKA, germany); milli-Q ultrapure water purification systems (Millipore, USA); 0.22 μm filter (Shanghai' an spectral science apparatus Co., ltd.); IC-RP solid phase extraction cartridge (BonaeIjel technologies).
3. Sample pretreatment
Weighing 1g of infant formula milk powder to be tested in 10mL of ultrapure water, heating to 50 ℃, and fully dissolving the milk powder; sucking 200 μ L of milk powder solution into a centrifuge tube, adding 200 μ L of acetic acid solution with mass fraction of 1%, adding 400 μ L of acetonitrile to precipitate protein, mixing, ice-bathing for 10min, centrifuging for 10min at 8000r/min, and transferring the supernatant into a new centrifuge tube; adding 1mL of 0.1mol/L trifluoroacetic acid solution into the supernatant, hydrolyzing at 80 ℃ for 40min, taking out and cooling to room temperature; diluting to 10mL with a methanol aqueous solution containing 10mmol/L ammonium acetate (volume ratio of water to methanol is 4; and (3) sequentially passing the constant volume solution through a 0.22-micron filter membrane and an IC-RP solid phase extraction column, and collecting filtrate to obtain a test solution.
4. Conditions of the apparatus
Chromatographic conditions are as follows: dionex ICS3000 ion chromatograph; dionex IonPac TM PA10 type anion analytical column, 250mm × 4mm; dionex IonPac TM PA10 type guard post, 50mm x 4mm; dionex ADRS 600 anion suppressor, 4mm, external water mode, suppression current 297mA; the column temperature is 30 ℃; the temperature of the detection pool is 35 ℃; the sample size is 25 mu L; a mobile phase A: water, mobile phase B:250mmol/L NaOH solution, mobile phase C: a500 mmol/L NaAc solution, flow rate 0.8mL/min, was eluted according to the following gradient elution procedure:
TABLE 1 gradient elution procedure
Mass spectrum conditions: a Waters TQ Detector triple quadrupole tandem mass spectrometer; an ion source: an electrospray ion source; the scanning mode is as follows: a multi-stage reaction monitoring mode; ionization mode: a negative ion mode; capillary voltage 3.00kV; cone gas flow is 50L/h; the flow rate of the collision gas is 0.15mL/min; the desolventizing temperature is 500 ℃; the flow rate of the desolventizing gas is 700L/h. System control and data processing were performed using analytical software (MassLynx, version 4.1). The flow rate into the tandem mass spectrometer was 0.4mL/min.
The mass spectral parameters of the five monosaccharides are shown in table 2.
TABLE 2 Mass Spectrometry parameters for five monosaccharides
The instrument connection is shown in figure 1. The detection system mainly comprises an ion chromatograph and a tandem mass spectrometer, eluent enters the ion chromatograph through a pump, standard solution and test solution enter the ion chromatograph through an injector, a substance to be detected is separated through a chromatographic column of PA10 model, and a Dionex ADRS 600 anion suppressor removes salt on line, so that the influence of salt or alkali in the eluent on an ESI source and the damage to mass spectrum components are eliminated. Because the optimal flow rate of the PA10 chromatographic column is 0.8mL/min, if the flow rate directly enters a mass spectrum detector, a solvent can be incompletely volatilized, unatomised liquid drops can possibly enter a mass spectrum to pollute the interior of the mass spectrum, and meanwhile, background noise is increased to influence detection, a three-way pipe is arranged before the liquid drops enter the mass spectrum detector to split the liquid, part of the liquid is led out as waste liquid, the flow rate entering the mass spectrum is kept at 0.4mL/min, and the liquid drops can enter the mass spectrum at the flow rate suitable for mass spectrum detection under the condition of ensuring good separation of a detected substance, so that a good detection result is obtained.
5. Separation effect of five monosaccharides
Five monosaccharide standards (fucose, galactose, glucose, N-acetylglucosamine and N-acetylneuraminic acid) were dissolved in a methanol aqueous solution (water: methanol = 4) containing 10mmol/L ammonium acetate to obtain a standard mixture, wherein the mass concentration of each monosaccharide was 5mg/L, and the total ion flow diagram of fucose, galactose, glucose, N-acetylglucosamine and N-acetylneuraminic acid is shown in fig. 2 (the peak positions of each monosaccharide are represented by the following compounds: 1-L-fucose, 2-galactose, 3-glucose, 4-N-acetylglucosamine and 5-N-acetylneuraminic acid). As can be seen from FIG. 2, the baseline separation of the five monosaccharides is realized by adopting the above instrument conditions, the peak pattern is good, and the method can be used for detecting the content of the polysaccharide formed by breast milk oligosaccharides in the infant formula milk powder.
6. Linear range, detection limit and quantitation limit
Weighing five monosaccharide standards 1mg respectively, dissolving in a 1mL volumetric flask, adding a solvent (10 mmol/L ammonium acetate-containing methanol aqueous solution, wherein the volume ratio of water to methanol in the methanol aqueous solution is 4; diluting with the same solvent to obtain intermediate stock solution, and storing at 4 deg.C in dark place; diluting the intermediate stock solution with the same solvent step by step to prepare a series of mixed standard solutions with the concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 10.0 mug/L respectively, and preparing the mixed standard solutions for use.
And detecting the peak area of each standard solution monosaccharide under the chromatographic and mass spectrum conditions, and drawing a standard working curve of the monosaccharide by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate. The detection Limit (LOD) of the method is determined with a signal-to-noise ratio of 3, and the quantification Limit (LOQ) of the method is determined with a signal-to-noise ratio of 10. The evaluation results are shown in table 3.
TABLE 3 Linear Range and detection Limit evaluation results for five monosaccharides
As shown in Table 3, the linear relationship of the five monosaccharides is good, and the correlation coefficient is greater than 0.995.
7. Precision degree
Weighing five monosaccharide standards, preparing a standard solution with the concentration of 5mg/L by using a methanol water solution containing 10mmol/L ammonium acetate (the volume ratio of water to methanol is 4): 5.8% of L-fucose, 3.1% of galactose, 5.5% of glucose, 5.2% of N-acetylglucosamine and 3.2% of N-acetylneuraminic acid, and the result shows that the precision of the method is good.
8. Recovery rate of added standard
Taking a blank milk powder sample, adding a standard sample according to three levels of 50%, 100% and 200% of the background content of the sample, preparing a sample solution according to the pretreatment method of the sample (diluting the sample solution by 5 times and injecting sample on the basis of the method), measuring the content of five monosaccharides in a standard sample by adopting a standard curve method under the conditions of the chromatogram and the mass spectrum, taking 6 parallel samples, averaging the measurement results, and calculating the recovery rate and the relative standard deviation, wherein the standard recovery rate = (the measurement value of the standard sample solution-the measurement value of the sample solution)/the standard addition amount x 100%, and the results are shown in Table 4.
TABLE 4 recovery rates and phases of five monosaccharides in milk powderFor standard deviation of (n=6）
9. Sample assay
Example 1
The samples to be tested in this example were two commercially available infant formulas (nos. 1 and 2) without breast milk oligosaccharide and two commercially available infant formulas (nos. 3 and 4) with breast milk oligosaccharide. The content of five monosaccharides in four samples was determined by a standard curve method under the aforementioned chromatographic and mass spectrometric conditions, and each sample was measured in parallel 3 times, with the results being the average of the 3 measurements, as shown in table 5.
TABLE 5 Breast milk oligosaccharide composition monosaccharide content in infant milk formula (n = 3)
The infant formula milk powder 1 is a formula milk powder with goat milk powder as a base, the L-fucose content is higher, which indicates that the goat milk powder contains more L-fucosylated neutral oligosaccharides, so the types and the content of the acidic breast milk oligosaccharides can be properly increased when artificial addition is carried out. The infant formula 2 is a formula based on milk powder, and has no detectable L-fucose content and a high N-acetylneuraminic acid content. The fact that the content of the acidic oligosaccharide containing sialic acid residues in the milk powder is high is proved, and nutrition can be balanced by adding the type and the content of the neutral oligosaccharide. The infant formula milk powder 3 and the infant formula milk powder 4 are milk powder samples which are from the same brand and are added with HMOs in different age groups, the content difference of five monosaccharides of the infant formula milk powder 3 and the milk powder samples is small, the content of the milk powder 3 in the higher age group is slightly lower than that of the milk powder 4 in the lower age group, the change trend of the content of the oligosaccharide in breast milk in human body milk is the same, and the contents of the acidic oligosaccharide and the neutral oligosaccharide are relatively balanced.
Example 2
The present example provides a method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in breast milk, which is as described in example 1, and is different from the method described in example 1 only in that the sample is pretreated by: sucking 200 μ L of breast milk into a centrifuge tube, adding 200 μ L of acetic acid solution with mass fraction of 1%, adding 400 μ L of acetonitrile to precipitate protein, mixing, ice-bathing for 10min, centrifuging for 10min at 8000r/min, and transferring the supernatant into a new centrifuge tube; adding 1mL of 0.1mol/L trifluoroacetic acid solution into the supernatant, hydrolyzing at 80 ℃ for 40min, taking out and cooling to room temperature; diluting to 10mL with a methanol aqueous solution containing 10mmol/L ammonium acetate (volume ratio of water to methanol is 4; and (4) filtering the constant volume solution through a 0.22-micron filter membrane, and collecting filtrate to obtain a test solution.
Example 3
In this example, the content of monosaccharide in breast milk oligosaccharide in the infant formula milk is determined according to example 1, except that the hydrolysis conditions of the trifluoroacetic acid solution added in the pretreatment method of the sample are as follows: hydrolyzing at 80 deg.C for 20min.
Example 4
In this example, the content of monosaccharide in breast milk oligosaccharide in the infant formula milk is determined according to example 1, except that the hydrolysis conditions of the trifluoroacetic acid solution added in the pretreatment method of the sample are as follows: hydrolyzing at 80 deg.C for 30min.
Example 5
In this example, the content of monosaccharide in breast milk oligosaccharide in the infant formula milk is determined according to example 1, except that the hydrolysis conditions of the trifluoroacetic acid solution added in the pretreatment method of the sample are as follows: hydrolyzing at 80 deg.C for 50min.
Example 6
In this example, the content of monosaccharide in breast milk oligosaccharide in the infant formula milk is determined according to example 1, except that the hydrolysis conditions of the trifluoroacetic acid solution added in the pretreatment method of the sample are as follows: hydrolyzing at 80 deg.C for 60min.
The test solutions prepared in examples 1 and 3 to 6 (all prepared using infant formula) were measured under the above-mentioned chromatographic and mass spectrometric conditions, and the peak areas were recorded, as shown in FIG. 3, it can be seen that the hydrolysis effect of monosaccharide was the best when the solution of trifluoroacetic acid was added for hydrolysis at 80 ℃ for 40min.
Example 7
In this example, the monosaccharide content of breast milk oligosaccharide in the infant formula was measured by referring to example 1, except that a Dionex CarbonPac PA1 chromatographic column was used in the chromatographic conditions.
Example 8
In this example, the monosaccharide content of breast milk oligosaccharide in the infant formula was measured by referring to example 1, except that a Dionex CarbonPac PA20 chromatographic column was used as the chromatographic condition.
The monosaccharide content of breast milk oligosaccharides in the infant formula milk is detected according to the methods of example 1 and examples 7 to 8, and the result is shown in fig. 4, wherein a is a total ion flow diagram of a Dionex CarbonPac PA1 chromatographic column; b is a total ion flow diagram of a chromatographic column adopting a Dionex CarbonPac PA 20; c is a total ion flow diagram using a Dionex CarbonPac PA10 column. As can be seen from the figure, dionex CarbonPac PA1 cannot completely separate galactose and glucose, and the accurate determination of the content of the substance to be detected is influenced; the detection time of the Dionex CarbonPac PA20 chromatographic column is long, and the peak shape is poor; the Dionex IonPac PA10 can realize the baseline separation of the peak shapes of the five monosaccharides and is suitable for detecting the content of the five monosaccharide components. The compounds represented by the peak positions in the figure are as follows: 1-L-fucose, 2-galactose, 3-glucose, 4-N-acetylglucosamine and 5-N-acetylneuraminic acid.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (13)
1. A method for detecting the content of constituent monosaccharides of breast milk oligosaccharides in milk and dairy products, wherein the constituent monosaccharides are at least one of L-fucose, galactose, glucose, N-acetylglucosamine and sialic acid, and the method comprises the following steps:
(1) Preparing a standard solution: weighing a monosaccharide standard substance, adding a solvent for dissolving, and preparing into a standard solution with a required concentration;
(2) Preparing a test solution: taking milk prepared from milk, liquid dairy products or solid dairy products, adding acetic acid solution for hydrolysis, adding acetonitrile for precipitating protein, centrifuging, taking supernatant, adding trifluoroacetic acid solution for hydrolysis, fixing volume, filtering and purifying the fixed volume liquid to obtain a test solution;
(3) Content determination: measuring the peak areas of monosaccharide in the standard solution and the test solution by an ion chromatography-tandem mass spectrometry, calculating the content of the monosaccharide according to a standard curve method,
wherein the chromatographic conditions comprise: mobile phase A: water, mobile phase B:250mmol/L NaOH solution, mobile phase C:500mmol/L NaAc solution, elution was carried out according to the following gradient elution procedure: 0.00 to 12.00min, A:94%, B:6%, C:0 percent; 12.00 to 12.10min, A:94% → 30%, B:6% → 40%, C:0% → 30%;12.10 to 17.00min, A:30%, B:40%, C:30 percent; 17.00 to 17.10min, A:30% → 60%, B:40%, C:30% → 0%;17.10 to 22.00min, A:60%, B:40%, C:0 percent; 22.00 to 22.10min, A:60% → 94%, B:40% → 6%, C:0%;22.10 to 27.00min, A:94%, B:6%, C:0 percent.
2. The method for detecting the content of monosaccharide composition in milk and milk oligosaccharide in milk product according to claim 1, wherein in step (3), an ion chromatograph and a tandem mass spectrometer are adopted for detection, the ion chromatograph and the tandem mass spectrometer are connected, a three-way pipe is arranged between the ion chromatograph and the tandem mass spectrometer, and the three-way pipe is not communicated with one end of the ion chromatograph and one end of the tandem mass spectrometer for collecting waste liquid.
3. The method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products according to claim 1, wherein in the step (3), the chromatographic conditions comprise: ion chromatograph Dionex ICS3000 type; dionex IonPac PA10 type anion analytical column, 250 mm. Times.4 mm; dionex IonPac PA10 type guard column, 50 mm. Times.4 mm; dionex ADRS 600 anion suppressor, 4mm, external water mode, suppression current 297mA;
the column temperature is 25 to 30 ℃; the temperature of the detection pool is 30 to 35 ℃;
the sample size is 20 to 25 mu L; the flow rate was 0.6 to 0.8mL/min.
4. The method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products according to claim 3, wherein in the step (3), the chromatographic conditions comprise: the column temperature is 30 ℃; the temperature of the detection pool is 35 ℃; the sample size is 25 mu L; the flow rate was 0.8mL/min.
5. The method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products according to claim 1, wherein in the step (3), the mass spectrometry conditions comprise: an ion source: an electrospray ion source; ionization mode: a negative ion mode; the scanning mode comprises the following steps: a multi-stage reaction monitoring mode; the flow rate into the tandem mass spectrometer is adjusted to be 0.3 to 0.4mL/min.
6. The method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products according to claim 5, wherein in the step (3), the mass spectrometry conditions comprise: the flow rate into the tandem mass spectrometer was adjusted to 0.4mL/min.
7. The method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products according to claim 1, wherein in the step (3), the mass spectrometry conditions comprise: a Waters TQ Detector triple quadrupole tandem mass spectrometer; capillary voltage 3.00kV; the gas flow of the taper hole is 50 to 150L/h; the flow rate of collision gas is 0.15mL/min; the outlet potential of the collision pool is 5V; the desolventizing temperature is 500 ℃; the flow rate of the desolventizing gas is 700 to 1000L/h.
8. The method for detecting the content of constitutional monosaccharides of breast milk oligosaccharides in milk and dairy products according to claim 7, wherein in the step (3), the mass spectrometry conditions comprise: the gas flow rate of the taper hole is 50L/h; the flow rate of the desolventizing gas is 700L/h.
9. The method for detecting the content of monosaccharide composition in milk and dairy products according to claim 1, wherein in the step (1), the solvent is an aqueous methanol solution containing 10mmol/L ammonium acetate, and the volume ratio of water to methanol in the aqueous methanol solution is 4:1.
10. the method for detecting the content of monosaccharides that constitute breast milk oligosaccharides in milk and dairy products according to claim 1, wherein in step (2), the volume ratio of milk or liquid dairy product to acetic acid solution to acetonitrile to trifluoroacetic acid solution is 200:200:400:1, the mass fraction of the acetic acid solution is 1%, and the concentration of the trifluoroacetic acid solution is 0.1mol/L; the centrifugation conditions were: the rotating speed is 8000r/min, and the time is 10min; the conditions for hydrolysis by adding trifluoroacetic acid solution are as follows: the temperature is 80 ℃, and the time is 20 to 60min; and (3) carrying out volume fixing by using a methanol aqueous solution containing 10mmol/L of ammonium acetate, wherein the volume ratio of water to methanol in the methanol aqueous solution is 4:1, the volume ratio of the milk or liquid dairy product to the constant volume liquid is 1:50.
11. the method for detecting the content of monosaccharide composition in milk and dairy products according to claim 10, wherein in the step (2), the hydrolysis conditions of the trifluoroacetic acid solution are as follows: the temperature is 80 deg.C, and the time is 40min.
12. The method for detecting the content of the constituent monosaccharides of human milk oligosaccharides in milk and dairy products according to claim 1, wherein in step (2), the filtering and purifying conditions are as follows: and (3) sequentially passing the constant volume solution through a 0.22 mu m filter membrane and an IC-RP solid phase extraction column, and collecting filtrate.
13. The method for detecting the content of monosaccharide composition in milk and milk oligosaccharide in milk product according to claim 1, wherein in the step (2), when the sample to be detected is a solid milk product, the method for preparing the sample to be detected into milk comprises the following steps: and (3) putting the solid dairy product into ultrapure water, and heating to fully dissolve the solid dairy product, wherein the mass-volume ratio of the solid dairy product to the ultrapure water is 0.1g/mL.
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| CN110672756B (en) * | 2019-11-07 | 2020-12-29 | 江南大学 | Method for detecting content of 2' -fucosyllactose in milk powder |
| 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 |
| WO2022089707A1 (en) * | 2020-10-30 | 2022-05-05 | Mille International Aps | A method of producing a human milk oligosaccharide (hmo) |
| 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 |
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| 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 |
| CN114994214A (en) * | 2022-06-30 | 2022-09-02 | 北京三元食品股份有限公司 | Method for qualitatively detecting neutral oligosaccharide in breast milk |
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