CN107621399A - A kind of method of oligosaccharide in detection breast milk - Google Patents

A kind of method of oligosaccharide in detection breast milk Download PDF

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CN107621399A
CN107621399A CN201610556457.XA CN201610556457A CN107621399A CN 107621399 A CN107621399 A CN 107621399A CN 201610556457 A CN201610556457 A CN 201610556457A CN 107621399 A CN107621399 A CN 107621399A
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oligosaccharide
lactose
breast milk
liquid
saliva
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CN107621399B (en
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陈历俊
赵军英
魏京华
姜铁民
董学艳
李菊芳
张毅
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SANYUAN FOOD CO Ltd BEIJING
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SANYUAN FOOD CO Ltd BEIJING
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Abstract

The present invention relates to it is a kind of detect breast milk in oligosaccharide method, including to human milk samples carry out pre-treatment the step of, the pre-treatment is specially:Human milk samples are taken, are centrifuged off upper-layer fat, layer liquid is removed and adds alcohols solvent, after 40 DEG C~﹣ of ﹣, 100 DEG C of 5 15min of freezing, centrifugation, supernatant is taken, produces;Present invention also offers carry out method that is qualitative and quantitatively detecting to the oligosaccharide in the breast milk after pre-treatment using liquid matter method;The present invention establishes a kind of non-reduced breast milk LC-MS while the method for detecting a variety of oligosaccharide in breast milk, with pre-treatment is simple, fast and effectively advantage, mass spectrum uses Q Exactive detectors, the parameter of liquid phase gradient elution and the parameter of Mass Spectrometer Method is determined, give non-reduced breast milk oligosaccharide in the positive-ion mode Mass Spectrometer Method when ion information, the qualitative and quantitative detection of breast milk oligosaccharide can be performed well in.

Description

A kind of method of oligosaccharide in detection breast milk
Technical field
The invention belongs to technical field of analysis and detection, more particularly to a kind of method for detecting oligosaccharide in breast milk, more specifically Ground, it is related to a kind of method that oligosaccharide in breast milk is detected using liquid matter method.
Background technology
Breast milk oligosaccharide (human milk oligosaccharides, HMO) is that lactose and fat are only second in human breast milk 3rd big component of fat, content is up to 22~23g/L in colostrum, is 12~13g/L in ripe breast.HMO has important biology Function, not only there is the function of resisting enteropahtogenic microganism infection, also maintain the effect of intestinal microecology balance.Breast milk into Point be golden standard prepared by baby milk powder, therefore establish the detection method of breast milk oligosaccharide, be analysis breast milk composition and then The prerequisite of the exploitation more baby milk powder of nutrient health.
At present, the detection method of breast milk oligosaccharide mainly has four kinds:Chromatography of ions, high performance liquid chromatography, LC-MS, hair Cons electrophoresis chromatogram.Various methods are specific as follows:
1) if LC-MS can equipped with time-of-flight detector and corresponding chromatographic column (porous graphitic carbon chromatographic column is more) To detect up to 300 kinds of breast milk oligosaccharide;But LC-MS detection breast milk need to carry out complex pretreatment (Totten et al.,Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies,2014,Journal,406(Issue),7925-7935):Water is added to centrifuge NaBH is used in degreasing → plus ethanol takes off albumen →4Reduction →【Graphitic carbon stationary phase extracts:Deionized water washes desalination → elution (20% acetonitrile solution, 40% acetonitrile and 0.05% trifluoroacetic acid solution)】→ freeze-drying → deionized water dissolving → liquid matter Combination detection.
If 2) Capillary Electrophoresis chromatogram is furnished with fluorescence detector, the isomerism of a variety of breast milk oligosaccharide can be also separated simultaneously Body, if using UV detectors, can only detection part oligosaccharide, such as the oligosaccharide that sialic acid is related.Capillary Electrophoresis chromatogram is female Following (Albrecht et al., the CE-LIF-MS n profiling of oligosaccharides in human of breast processing milk and feces of breast-fed babies,2010,Journal,31(Issue),1264–1273):Add water from Heart degreasing → plus ethanol take off albumen →【Graphitic carbon stationary phase extracts:Deionized water washes desalination → elution (20% aqueous acetonitrile Liquid, 40% acetonitrile and 0.05% trifluoroacetic acid solution)】→ 9- amino pyrene -1,4,6- trisulfonic acids trisodium salt derives (overnight) → hair Thin electrochromatophoresis detection.
3) chromatography of ions, liquid phase and mass spectrum is used alone can only detection part breast milk oligosaccharide.
In terms of testing result, compared to Capillary Electrophoresis chromatogram, chromatography of ions, and exclusive use liquid phase or mass spectrum, liquid Matter combination method can detect more HMO species, but LC-MS detection HMO has problems with:
1) sample pre-treatments step is complex, is easily caused accidental error increase.Pre-treatment sampling amount is only 50 μ L, sample To pass through the steps such as reduction, SPE, freeze-drying, redissolution after degreasing, de- albumen.In solid phase extraction procedure, need by Different elutions, it is also easy to produce and elutes not thorough and cause sample loss;Opened in freezing dry process or after drying close During tube sealing, sample loss is also easily caused due to vacuum be present.Because sampling amount is less, and HMO contents therein are only 1% left The right side, therefore it is easily caused larger accidental error.
2) detector used in mass spectrum is mostly flight time (Time of Flight, TOF) detector, although TOF is applied Scope it is wider, but do not represent state-of-the-art mass detector.
Q Exactive detectors can substitute TOF at many aspects, and Q Exactive detections have been purchased in many laboratories Device, but so far not on the relevant report using Q Exactive detection breast milk oligosaccharide.
The content of the invention
The purpose of the present invention is for defect and deficiency present in prior art, there is provided oligosaccharide in one kind detection breast milk Method, this method include to breast milk carry out pre-treatment the step of, and in breast milk after pre-treatment oligosaccharide carry out it is qualitative And the step of quantitative analysis.
Specifically, the purpose of the present invention is achieved through the following technical solutions:
A kind of method of oligosaccharide in detection breast milk, including to human milk samples carry out pre-treatment the step of, the pre-treatment Specially:Human milk samples are taken, are centrifuged off upper-layer fat, layer liquid is removed and adds alcohols solvent, it is cold in 100 DEG C of 40 DEG C~﹣ of ﹣ After freezing 5-15min, centrifugation, supernatant is taken, is produced.
In foregoing pre-treatment, the first step is centrifuged for removing the fat in breast milk, and the parameter of noncentricity used is with can be effective The fat removed in breast milk is advisable.Currently preferred parameter of noncentricity is:Breast milk is centrifuged under the conditions of 2-8 DEG C, 2000-6000g Sample 5-30min, such a condition can ideally degreasing, avoid fat presence measurement result is disturbed, it is ensured that measure As a result precision.In concrete operations, different centrifugal conditions may be selected for different sample sizes, such as sample size is During 100 μ L or so, 20min can be centrifuged under the conditions of 2000g at 2 DEG C;During sample size 500-800 μ L or so, can with 4 DEG C, 13min is centrifuged under the conditions of 5000g, or at 8 DEG C, 20min etc. is centrifuged under the conditions of 6000g.
The alcohols solvent added in lower floor's liquid is preferably ethanol, and more preferably volume fraction is 60-70% The ethanol water of (most preferably 66.7%).The albumen in breast milk can be precipitated using ethanol as solvent, avoids albumen to oligomeric Sugar determination interferes., can be to greatest extent as precipitation solvent using ethanol compared with the acetonitrile used in document report Reduce the loss of oligosaccharide.
After ethanol precipitation albumen, deproteinized is removed using centrifugal method, preferable centrifugal condition is 10000-15000rpm Centrifuge 10-50min.Similarly, for the sample that sample size is larger, big centrifugation rate can be selected, longer centrifugation time, For example, when processing sample is 800 μ L or so, centrifugal condition can be to centrifuge 50min under 15000rpm.
Preferably pre-treating method of the invention is:The human milk samples 5- is centrifuged under the conditions of 2-8 DEG C, 2000-6000g 30min, upper strata material is removed, removes layer liquid and add ethanol water of 1.5-2.5 times equivalent to lower floor's liquid volume, After 70 DEG C~﹣ of ﹣, 90 DEG C of freezing 5-15min, 10-50min is centrifuged in 10000-15000rpm, supernatant is taken, produces.
Aforementioned sample pre-treatment has following advantage compared with the method that background section refers to:
(1) it is simple, quick, effective
Water soluble materials containing oligosaccharide are directly passed through filter membrane by the human milk samples of the present invention after degreasing takes off albumen (such as Angela Technologies;Nylon;0.22μm;It can be detected after 13mm) filtering.And background technology document In sample pre-treatments in, after breast milk degreasing takes off albumen, first pass around reduction, because reduction adds salt, therefore to use and consolidate Mutually extraction special purpose device desalination, desalination processes are due to using eluent to cause the concentration of oligosaccharide to reduce, so will be again by true Sky dries concentration, and after being redissolved with ultra-pure water, could pass through membrane filtration liquid quality detection, therefore compared with background document, Sample-pretreating method is simple in the established method of invention, and operating procedure is reduced, and at least saves 24h, sample can be completed in 2h The pre-treatment of product, realizes quick detection.
(2) HMO losses are few in sample
The present invention only has the filtering link before degreasing, de- albumen and the loading of human milk samples, and this three operating process are to mother The loss of newborn oligosaccharide is smaller.And due to SPE desalination, vacuum distillation concentration caused by sample reduction in background document Process, easily cause the loss of breast milk oligosaccharide.In solid phase extraction procedure, typically take first with ultrapure water desalination, then with difference The acetonitrile solution of concentration elutes breast milk oligosaccharide, and one side desalination is difficult to thoroughly, and another aspect breast milk oligosaccharide is difficult to thoroughly Eluted completely from extraction column.And in freeze-drying, when drying opening sample after terminating, easily cause the loss of sample.
(3) cost is low
The cost of inventive samples pre-treatment mainly includes ethanol reagent, centrifuge tube and filter membrane used, and in background document Solid-phase extraction device, disposable solid-phase extraction column (about 50 yuan /), acetonitrile eluent, Yi Jixi are also needed in addition to above-mentioned consumptive material Consumptive material used in de- and drying process, significantly increase the cost of sample pre-treatments.
Further, detection method of the present invention also includes using liquid matter method to oligomeric in the breast milk after pre-treatment Sugar carries out the step of qualitative and quantitative analysis, wherein, liquid phase uses Hypercarb porous graphitic carbon chromatographic columns, with (0.05%- 0.15%) formic acid-(2%-4%) acetonitrile-water is mobile phase A phase;(0.05-0.15%) formic acid-(85%-95%) acetonitrile-water For Mobile phase B phase, by 0min 100%A, 10-30min 84%A, 20-40min 100%B, 30-50min 100A% bar Part carries out gradient elution.
Preferably, the mobile phase A is mutually the acetonitrile-water of 0.1% formic acid -3%;Mobile phase B is mutually 0.1% formic acid -90% Acetonitrile-water.
" % " refers to the percentage by volume of formic acid or acetonitrile in mobile phase.
It is further preferred that the flow velocity of mobile phase is 100-300 μ L/min.
The mass spectrum of the present invention preferably uses Q Exactive detectors.
It is further preferred that mass spectrum includes following parameter:Using that can heat electric spray ion source, cation sweeps pattern entirely.
Still further preferably, mass spectrum also includes following parameter:
S type lens radio frequencies 50-70V;And/or 300-350 DEG C of capillary temperature;And/or spray voltage 3-4kV;With/ Or, 300-400 DEG C of heating-up temperature;And/or sheath throughput 30-40;And/or scanning throughput 0;And/or secondary air amount 5- 20;And/or scanning range 150-2000m/z.
Most preferably liquid quality detection condition of the invention is as follows:
Using Hypercarb porous graphitic carbon chromatographic columns, using the acetonitrile-water of 0.1% formic acid -3% as mobile phase A phase; The acetonitrile-water of 0.01% formic acid -90% is Mobile phase B phase, by 0min 100%A, 10-30min 84%A, 20-40min 100% B, 30-50min 100A% condition carry out gradient elution;Detected using Q Exactive detectors.
Using method of the present invention, it is possible to achieve to the qualitative and quantitative analysis of oligosaccharide in breast milk.
Specifically, during qualitative analysis, the molecular weight (being accurate to 0.0001) according to various oligosaccharide is carried out.Inventor's root According to the monomer composition of breast milk oligosaccharide in document, the molecular formula of each oligosaccharide is inferred to, and by mass spectrum software, be calculated The molecular weight of the oligosaccharide of document report, it is as a result as shown in table 1 below:
The accurate molecular weight result of calculation of the oligosaccharide of table 1
In table 1,2 ' FL are 2 ' rock algae lactose;6 ' SL are 6 ' sialyl lactoses;LDFT is lactodifucotetraose;6’SLN For sialyl-N-acetyllactosamine;LNT/LNnT is lacto-N-tetraose/lacto-N-neotetraose;LNFPII is lactose-N- rocks Algae pentose II;LST-c is saliva lacto-N-neotetraose;LNDFH I are the rock algae hexose I of lactose-N- two;LNH be lactose-N- oneself Sugar;F-LSTc is rock algae-saliva lacto-N-neotetraose;MFpLHN IV are fucosido-p- breast-N- hexoses IV;S-LNH is Saliva lactose-N- hexoses;DFpLNH II are two rock algae bases-p- breast-N- hexoses;4121a is low newly to detect undetermined breast milk Glycan;4320a is newly to detect undetermined breast milk oligosaccharide;FLNO is the sugar of rock algae lactose-N- eight;DFS-LNH is two rock algaes-saliva The new hexoses of liquid lactose base-N-;5031a is newly to detect undetermined breast milk oligosaccharide;FS-LNO is rock algae-saliva lactose base-N- Eight sugar;TFiLNO is the sugar of three fucoses-isolactose base-N- eight.
The method established of the present invention can carry out qualitative analyses to 18 kinds of breast milk oligosaccharide simultaneously, including accounting for breast milk ratio The larger rock algae lactose and saliva lactose of example, the present invention can qualitatively breast milk oligosaccharide specifically such as table 2, shown in table 3, " name in table Title is write a Chinese character in simplified form " ibid:
Single electric charge oligosaccharide in the breast milk of table 2
Double charge oligosaccharide in the breast milk of table 3
During quantitative analysis, using standard items, it can be achieved to determine above-mentioned 18 kinds of oligosaccharide using internal standard method or external standard method Amount analysis, present invention preferably employs external standard method to carry out quantitative analysis.
Specifically, the quantitative analysis comprises the following steps:
(1) testing sample solution is prepared:Human milk samples are taken, are centrifuged off upper-layer fat, layer liquid is removed and adds alcohols Solvent, after 40 DEG C~﹣ of ﹣, 100 DEG C of freezing 5-15min, centrifuging and taking, supernatant, produce;
(2) standard solution is prepared:Oligomeric saccharide is soluble in water, prepare the standard of 3-8 various concentrations gradient Product solution;
(3) standard working curve is drawn:The standard solution of step (2) is entered using any one foregoing liquid matter method condition Row detection, using concentration as abscissa, peak area is ordinate, draws standard working curve;
(4) quantitative analysis:Testing sample solution is detected using with step (3) identical liquid matter condition, according to mark Quasi- working curve, calculate the content of oligosaccharide.
Preferably, the invention provides to the larger 2 '-rock algae lactose of content in above-mentioned 18 kinds of oligosaccharide, 6 '-saliva breast Sugar, the method for the quantitative analysis of 3 '-rock algae lactose.Specifically, when the range of linearity of 2 '-rock algae lactose standard working curve is 0.5-20mg/mL, the range of linearity of 6 '-saliva lactose standard working curve are 0.5-50mg/mL, 3 '-rock algae lactose standard work When the range of linearity for making curve is 0.5-20mg/mL, the R of the standard curve of saliva lactose plus salts algae lactose2>=0.999, i.e., it is female The concentration of oligosaccharide in breast can carry out relative quantitative assay using this standard curve in this scope to it.It for details, reference can be made to Fig. 1-3, it can be seen that (left side accompanying drawing in Fig. 1-3), the R of standard curve when the range of linearity exceeds this scope2< 0.999, it is poor for quantitative analysis effect.
In the above-mentioned range of linearity, it is established that the regression equations of 3 kinds of breast milk oligosaccharide be specially:
2 '-rock algae lactose:Y=1 × 106X-39286, R2=0.999;
6 '-saliva lactose:Y=2 × 107x+6×106, R2=0.999;
3 '-rock algae lactose:Y=4 × 106X-11189, R2=1.
When the present invention establishes oligosaccharide in a kind of detection breast milk, the pre-treating method of breast milk, this method has preceding locate Reason is simple, fast and effectively advantage;The present invention has also set up using Liquid Chromatography/Mass Spectrometry while has detected a variety of oligosaccharide in breast milk Method, it is determined that the parameter of liquid phase gradient elution and Mass Spectrometer Method, give non-reduced breast milk oligosaccharide in positive ion mode Ion information during lower Mass Spectrometer Method, the qualitative and quantitative detection of breast milk oligosaccharide can be performed well in.
It on the basis of common sense in the field is met, above-mentioned each optimum condition, can be mutually combined, it is each preferably to produce the present invention Embodiment.
Brief description of the drawings
Fig. 1 is the standard curve of 2 '-salt algae lactose, and left side is curve of the range of linearity in 0.5-100mg/mL;Right side is Curve of the range of linearity in 0.5-20mg/mL;
Fig. 2 is the standard curve of 6 '-saliva lactose, and left side is curve of the range of linearity in 0.5-100mg/mL;Right side is Curve of the range of linearity in 0.5-50mg/mL;
Fig. 3 is the standard curve of 3 '-salt algae lactose, and left side is curve of the range of linearity in 0.5-100mg/mL;Right side is Curve of the range of linearity in 0.5-20mg/mL;
Fig. 4 is repeatability (peak area) result figure of breast milk oligosaccharide and standard items;
Fig. 5 be comparative example 1 testing result figure, top 2 '-rock algae lactose mass spectrogram, bottom 6 '-saliva lactose matter Spectrogram;
Fig. 6 be comparative example 2 testing result figure, top 2 '-rock algae lactose mass spectrogram, bottom 6 '-saliva lactose matter Spectrogram.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
The method of oligosaccharide, comprises the following steps in a kind of detection breast milk:
1) sample pre-treatments:100 μ L human milk samples are centrifuged off upper-layer fat through 2000g, 20min, 2 DEG C, remove a layer sample Liquid adds the ethanol of two volumes 66.7%, 80 DEG C of freezing 10min of ﹣, is centrifuged after taking-up, centrifugal condition 10000r/min, 10min, supernatant liquor is taken after centrifugation.Through 0.22 μm of membrane filtration before loading.
2) liquid-phase condition:
Chromatographic column:Hypercarb porous graphitic carbon chromatographic columns.
Mobile phase A phase:The acetonitrile-water of 0.1% formic acid -3%;
The acetonitrile-water of 0.1% formic acid of Mobile phase B phase -90%.
Gradient elution, condition are as shown in table 4:
The condition of gradient elution of table 4
3) Mass Spectrometry Conditions
Using positive ion mode;Scanning range:150-2000m/z;Electric spray ion source can be heated;Full scan data acquisition Pattern;Sheath throughput 30, secondary air amount 5, scanner flow 0, spray voltage 3kV, 300 DEG C of heating-up temperature, S type lens radio frequencies 50V, 300 DEG C of capillary temperature.
4) mass spectral results are analyzed
Qualitative analysis:Monokaryon oligosaccharide and double-core oligosaccharide are as shown in table 5 below and table 6 in breast milk:
Single electric charge oligosaccharide in the breast milk of table 5
Double charge oligosaccharide in the breast milk of table 6
Quantitative analysis:
(1) standard solution is prepared:By 2 '-rock algae lactose, 6 '-saliva lactose, soluble in water respectively, being configured to concentration is 1mg/mL mother liquor;3 kinds of mother liquors respectively take 100 μ L, are diluted with water to 1mL, obtain a dilution (concentration 100ppm);Respectively take dilution The μ L of liquid 500 are diluted with water to 1mL, obtain secondary dilution liquid (concentration 50ppm);Secondary dilution liquid is diluted respectively to obtain concentration difference For 20ppm, 10ppm, 5ppm, 1ppm, 0.5ppm 5 standard solutions;
(2) standard working curve is drawn:Three groups of standard solutions are analyzed respectively using above-mentioned liquid-phase condition, with dense It is that ordinate draws standard working curve to spend for abscissa, peak area, and as a result such as Fig. 1-3 (right part of flg) is shown.
(3) quantitative analysis:Testing sample is detected using above-mentioned detection method, according to corresponding molecular weight in mass spectrogram Calculated by peak area obtain the content of respective substance, as a result as shown in table 7:
Table 7:The content (mg/mL) of three kinds of materials in breast milk
Title Content
2 '-rock algae lactose 4.02
6 '-saliva lactose 0.37
3 '-rock algae lactose 0.39
Embodiment 2
The method of oligosaccharide, with embodiment 1, is differed only in, sample pre-treatments, gradient elution in a kind of detection breast milk, Mass Spectrometry Conditions are different, are specially:
1) sample pre-treatments:500 μ L human milk samples are centrifuged off upper-layer fat through 5000g, 13min, 4 DEG C, remove a layer sample Liquid adds two volumes, 66.7% ethanol, 80 DEG C of freezing 25min of ﹣, is centrifuged after taking-up, centrifugal condition 12000r/min, 25min, takes supernatant liquor after centrifugation, through 0.22 μm of membrane filtration before loading.
2) liquid phase gradient elution, as shown in table 8.
The condition of gradient elution of table 8
3) Mass Spectrometer Method
Using positive ion mode;Scanning range:150-2000m/z;Electric spray ion source can be heated;Full scan data acquisition Pattern;Sheath throughput 36, secondary air amount 10, scanner flow 0, spray voltage 3.4kV, 360 DEG C of heating-up temperature, S type lens Radio frequency 62V, 330 DEG C of capillary temperature.
4) mass spectral results are analyzed, such as table 9, shown in 10.
Single electric charge oligosaccharide in the breast milk of table 9
Double charge oligosaccharide in the breast milk of table 10
Embodiment 3
The method of oligosaccharide, with embodiment 1, is differed only in, sample pre-treatments, gradient elution in a kind of detection breast milk, Mass Spectrometry Conditions are different, are specially:
1) sample pre-treatments:800 μ L human milk samples are centrifuged off upper-layer fat through 6000g, 20min, 8 DEG C, remove a layer sample Liquid adds two volumes, 66.7% ethanol, 80 DEG C of freezing 50min of ﹣, is centrifuged after taking-up, centrifugal condition 15000r/min, 50min, takes supernatant liquor after centrifugation, through 0.22 μm of membrane filtration before loading.
2) liquid phase gradient elution, as shown in table 11.
The condition of gradient elution of table 11
3) Mass Spectrometer Method
Using positive ion mode;Scanning range:150-2000m/z;Electric spray ion source can be heated;Full scan data acquisition Pattern;Sheath throughput 40, secondary air amount 20, scanner flow 0, spray voltage 4kV, 400 DEG C of heating-up temperature, S type lens are penetrated Frequency 70V, 350 DEG C of capillary temperature.
4) mass spectral results are analyzed, as shown in table 12 and table 13.
Single electric charge oligosaccharide in the breast milk of table 12
Double charge oligosaccharide in the breast milk of table 13
Method validation
Recovery of standard addition is tested
It is not added with marking sample preparation:500 μ L human milk samples are centrifuged off upper-layer fat through 4000g, 15min, 4 DEG C, remove layer The μ L of sample liquid 400 add the ethanol of two volumes 66.7%, 80 DEG C of freezing 30min of ﹣, are centrifuged after taking-up, centrifugal condition 13000r/ Min, 30min, supernatant liquor is taken after centrifugation, film, sample introduction are crossed after being diluted with water.Do 3 repetitions.
Mark-on sample preparation:250 μ L human milk samples add the-FL of 150 μ L 2 ' (1mg/mL), 25 μ L3-FL (1mg/mL), 150 μ - the SL of L 6 ' (1mg/mL), upper-layer fat is centrifuged off through 4000g, 15min, 4 DEG C, removes the μ L of layer sample liquid 600 and add two volumes 66.7% ethanol, 80 DEG C of freezing 30min of ﹣, centrifuges after taking-up, centrifugal condition 13000r/min, 30min, is taken after centrifugation Layer clear liquid, crosses film, sample introduction after being diluted with water.Each mark product add different amounts, each 3 repetitions of addition.
Testing result is shown:The 2 '-FL rate of recovery is 70-90%, and the 3-FL rate of recovery is 72-93%, 6 '-SL recovery Rate is 100-120%.
Repeated experiment
Using same human milk samples and standard items, 12h is spaced, same sample is detected, compares the average value of response And deviation, determine the repeatability of method, totally 5 times, as a result as shown in Fig. 4 and table 14.As a result show, the HMO detection sides established The repeatability that method detects for breast milk is preferable.
Table 14:The average value and standard deviation of standard items and human milk samples
Comparative example 1
The detection method of the comparative example is with embodiment 1, and it differs only in sample-pretreating method difference, the comparative example Sample-pretreating method is:After breast milk degreasing takes off albumen, eluent is then removed simultaneously by SPE desalination, vacuum drying again It is used to detect after redissolution, it is specific as follows:Breast milk is centrifuged off upper-layer fat through 4000g, 15min, 4 DEG C, takes subnatant to add Two volumes, 66.7% ethanol, 80 DEG C of freezing 30min of ﹣, centrifuge, centrifugal condition 13000r/min, 30min, centrifuge after taking-up After take the μ L of supernatant liquor 200 →【Graphitic carbon stationary phase extracts:1. graphitic carbon activates:The trifluoroacetic acid of 80% acetonitriles of 9mL+0.1% Extraction column is rinsed, then with 9mL ultrapure waters;2. loading;3. desalination:36mL ultra-pure waters wash desalination;4. elute:9mL 20% acetonitrile solution elutes, and then the trifluoroacetic acid solution of 40% acetonitriles of 9mL+0.05% elutes】→ vacuum drying (24h) → Ultra-pure water is dissolved to 1mL → LC-MS detection, and testing result is as shown in Figure 5.
Comparative example 2
The detection method of the comparative example is with embodiment 1, and it differs only in sample-pretreating method difference, the comparative example Sample-pretreating method is:After breast milk degreasing takes off albumen, oligosaccharide therein is reduced, then taken off again by SPE Salt, vacuum drying removes eluent and is used to detect after redissolving, specific as follows:Breast milk is centrifuged off through 4000xg, 15min, 4 DEG C Upper-layer fat, take subnatant to add two volumes, 66.7% ethanol, 80 DEG C of freezing 30min of ﹣, centrifuged after taking-up, centrifugal condition For 13000r/min, 30min, take the μ L of supernatant liquor 100 to add 1900 μ L water after centrifugation, with 2mL NaBH4 reduction (1h) →【Stone Black carbon fixation mutually extracts:1. graphitic carbon activates:The trifluoroacetic acid of 80% acetonitriles of 9mL+0.1% rinses extraction column, is then surpassed with 9mL Pure water rinsing;2. loading;3. desalination:36mL ultra-pure waters wash desalination;4. elute:The acetonitrile solutions of 9mL 20% elute, then The trifluoroacetic acid solution of 40% acetonitriles of 9mL+0.05% elutes】→ vacuum drying (24h) → ultra-pure water is dissolved to 1mL → liquid matter connection With detection;Testing result is as shown in Figure 6.
It can be seen that from the result of comparative example 1 and comparative example 2:After the method processing of comparative example 1 and 2, due to processing During oligosaccharide lose, cause can't detect, or the signal detected weaker the problem of being difficult to quantitative analysis, enter one Step illustrates that pre-treating method of the present invention has and does not destroy oligosaccharide, contributes to the advantage of detection.
Although above the present invention is made to retouch in detail with general explanation, embodiment and experiment State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art 's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed Scope.

Claims (10)

  1. A kind of 1. method for detecting oligosaccharide in breast milk, it is characterised in that:The step of including carrying out pre-treatment to human milk samples, institute Stating pre-treatment is specially:Human milk samples are taken, are centrifuged off upper-layer fat, layer liquid is removed and adds alcohols solvent, in 40 DEG C of ﹣ After 100 DEG C of freezing 5-15min of~﹣, centrifugation, supernatant is taken, is produced.
  2. 2. according to the method for claim 1, it is characterised in that:The alcohols solvent is ethanol, and preferred volume fraction is 60- 70% ethanol water.
  3. 3. method according to claim 1 or 2, it is characterised in that the pre-treatment is specially:At 2-8 DEG C, 2000- The human milk samples 5-30min is centrifuged under the conditions of 6000g, removes upper strata material, layer liquid is removed and adds equivalent to subnatant The ethanol water of 1.5-2.5 times of body volume, after 70 DEG C~﹣ of ﹣, 90 DEG C of freezing 5-15min, centrifuged in 10000-15000rpm 10-50min, supernatant is taken, produced.
  4. 4. according to the method described in claim any one of 1-3, it is characterised in that methods described also includes using liquid matter method to preceding The step of oligosaccharide in breast milk after processing carries out qualitative and quantitative analysis;
    Wherein, liquid phase uses Hypercarb porous graphitic carbon chromatographic columns, with (0.05%-0.15%) formic acid-(2%-4%) second Nitrile-water is mobile phase A phase;(0.05-0.15%) formic acid-(85%-95%) acetonitrile-water is Mobile phase B phase, by 0min 100% A, 10-30min 84%A, 20-40min100%B, 30-50min 100A% condition carry out gradient elution;
    Preferably, the mobile phase A is mutually the acetonitrile-water of 0.1% formic acid -3%;Mobile phase B mutually for the acetonitrile of 0.1% formic acid -90% - Water.
  5. 5. according to the method for claim 4, it is characterised in that:Mass spectrum uses Q Exactive detectors.
  6. 6. the method according to claim 4 or 5, it is characterised in that mass spectrum includes following parameter:Using electron spray can be heated Ion gun, cation sweep pattern entirely;
    Preferably, in addition to:
    S type lens radio frequencies 50-70V;And/or 300-350 DEG C of capillary temperature;And/or spray voltage 3-4kV;And/or add Hot 300-400 DEG C of temperature;And/or sheath throughput 30-40;And/or scanning throughput 0;And/or secondary air amount 5-20; And/or scanning range 150-2000m/z.
  7. 7. according to the method described in claim any one of 4-6, it is characterised in that:The oligosaccharide of the qualitative analysis include 2 '- Rock algae lactose, 6 '-saliva lactose, 3 '-rock algae lactose, lactodifucotetraose, sialyl-N-acetyllactosamine, lactose-N- Tetrose/lacto-N-neotetraose, lacto-iV-fucopentaose II, saliva lacto-N-neotetraose, the rock algae hexose I of lactose-N- two, breast Sugar-N- hexoses, rock algae-saliva lacto-N-neotetraose, fucosido-p- breast-N- hexose IV, saliva lactose-N- hexoses, two rocks Algae base-p- breast-N- hexoses, the sugar of rock algae lactose-N- eight, the two rock algaes-new hexoses of saliva lactose base-N-, rock algae-saliva lactose base- N- eight is sugared, the one or more in three fucoses-sugar of isolactose base-N- eight;Preferably include above-mentioned 18 kinds.
  8. 8. according to the method for claim 4, it is characterised in that the quantitative analysis comprises the following steps:
    (1) testing sample solution is prepared:Human milk samples are taken, are centrifuged off upper-layer fat, layer liquid is removed and adds alcohols solvent, After 40 DEG C~﹣ of ﹣, 100 DEG C of freezing 5-15min, centrifugation, supernatant is taken, is produced;
    (2) standard solution is prepared:Oligomeric saccharide is soluble in water, and the standard items for preparing 3-8 various concentrations gradient are molten Liquid;
    (3) standard working curve is drawn:Standard solution using the liquid matter method described in claim 4 or 5 or 6 to step (2) Detected, using concentration as abscissa, peak area is ordinate, draws standard working curve;
    (4) quantitative analysis:Testing sample solution is detected using with step (3) identical liquid matter condition, according to standard work Make curve, calculate the content of oligosaccharide.
  9. 9. according to the method for claim 8, it is characterised in that:The oligosaccharide is selected from 2 '-rock algae lactose, 6 '-saliva breast Sugar, the one or more in 3 '-rock algae lactose.
  10. 10. according to the method for claim 9, it is characterised in that:The range of linearity of 2 '-rock algae lactose standard working curve is 0.5-20mg/mL, the range of linearity of 6 '-saliva lactose standard working curve are 0.5-50mg/mL, 3 '-rock algae lactose standard work The range of linearity for making curve is 0.5-20mg/mL.
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109270176A (en) * 2018-08-22 2019-01-25 中国农业科学院农产品加工研究所 Sheep cream oligosaccharides measuring method
CN110672756A (en) * 2019-11-07 2020-01-10 江南大学 Method for detecting content of 2' -fucosyllactose in milk powder
CN110672757A (en) * 2019-11-07 2020-01-10 江南大学 Pretreatment method for detecting content of 2' -fucosyllactose in milk powder
CN111239311A (en) * 2018-11-28 2020-06-05 中国科学院大连化学物理研究所 Analysis method of acid sugar and acid sugar derivative in infant excrement
CN111239313A (en) * 2018-11-29 2020-06-05 中国科学院大连化学物理研究所 Rapid contour analysis method of human milk oligosaccharide
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
CN112924563A (en) * 2019-12-05 2021-06-08 中国科学院大连化学物理研究所 Derivatization-based breast milk oligosaccharide characterization and quantitative analysis method
CN112986367A (en) * 2019-12-13 2021-06-18 内蒙古蒙牛乳业(集团)股份有限公司 Method for detecting acid lactooligosaccharide in mammal milk by capillary electrophoresis separation
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
CN114594169A (en) * 2020-12-03 2022-06-07 中国科学院大连化学物理研究所 Quantitative detection method for oligosaccharide content in breast milk
CN114740096A (en) * 2022-01-13 2022-07-12 澳优乳业(中国)有限公司 Detection method of breast milk oligosaccharide
CN114965750A (en) * 2021-07-19 2022-08-30 山东恒鲁生物科技有限公司 Qualitative and quantitative detection method for human lactooligosaccharide in dairy product
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CN115112799A (en) * 2022-06-30 2022-09-27 北京三元食品股份有限公司 Method for qualitatively detecting acid oligosaccharide in breast milk
CN115144494A (en) * 2022-06-28 2022-10-04 贵州大学 Method for detecting oligosaccharide in mammal milk
CN115480023A (en) * 2022-11-03 2022-12-16 中轻检验认证有限公司 Method for detecting content of monosaccharide in milk and milk product

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100003699A1 (en) * 2008-01-18 2010-01-07 Glykos Finland Ltd. Tissue carbohydrate compositions and analysis thereof
CN103149312A (en) * 2013-02-05 2013-06-12 东北农业大学 Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry
CN103877748A (en) * 2012-12-21 2014-06-25 戴安公司 Separation Of Glycans By Mixed-mode Liquid Chromatography
CN105246911A (en) * 2013-04-12 2016-01-13 丹麦科技大学 A mutant sialidase having trans-sialidase activity for use in production of sialylated glycans
CN105372414A (en) * 2015-10-09 2016-03-02 成都大熊猫繁育研究基地 Method for analyzing key factors in giant panda milk
WO2016044167A1 (en) * 2014-09-15 2016-03-24 Abbott Laboratories Methods for increasing muscle strength and mobility in subjects experiencing significant physical inactivity using gamma linolenic acid
CN105548433A (en) * 2015-12-17 2016-05-04 浙江出入境检验检疫局检验检疫技术中心 Method for simultaneous detection of a plurality of content of non-protein nitrogen-containing compounds in milk by liquid chromatography-tandem mass spectrometry

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100003699A1 (en) * 2008-01-18 2010-01-07 Glykos Finland Ltd. Tissue carbohydrate compositions and analysis thereof
CN103877748A (en) * 2012-12-21 2014-06-25 戴安公司 Separation Of Glycans By Mixed-mode Liquid Chromatography
CN103149312A (en) * 2013-02-05 2013-06-12 东北农业大学 Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry
CN105246911A (en) * 2013-04-12 2016-01-13 丹麦科技大学 A mutant sialidase having trans-sialidase activity for use in production of sialylated glycans
WO2016044167A1 (en) * 2014-09-15 2016-03-24 Abbott Laboratories Methods for increasing muscle strength and mobility in subjects experiencing significant physical inactivity using gamma linolenic acid
CN105372414A (en) * 2015-10-09 2016-03-02 成都大熊猫繁育研究基地 Method for analyzing key factors in giant panda milk
CN105548433A (en) * 2015-12-17 2016-05-04 浙江出入境检验检疫局检验检疫技术中心 Method for simultaneous detection of a plurality of content of non-protein nitrogen-containing compounds in milk by liquid chromatography-tandem mass spectrometry

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CAROLINE THUM ET AL.: "Composition and enrichment of caprine milk oligosaccharides from New Zealand Saanen goat cheese whey", 《JOURNAL OF FOOD COMPOSITION AND ANALYSIS》 *
CHARLES C. NWOSU ET AL.: "Comparison of the Human and Bovine Milk N-Glycome via High-Performance Microfluidic Chip Liquid Chromatography and Tandem Mass Spectrometry", 《JOURNAL OF PROTEOME RESEARCH》 *
SHUAI WU ET AL.: "Annotation and Structural Analysis of Sialylated Human Milk Oligosaccharides", 《JOURNAL OF PROTEOME RESEARCH》 *
YUANWU BAO ET AL.: "Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry", 《ANALYTICAL BIOCHEMISTRY》 *
魏京华等: "液相色谱-质谱法快速检测4 种乳源低聚糖", 《食品科学》 *

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