CN107621399A - A kind of method of oligosaccharide in detection breast milk - Google Patents
A kind of method of oligosaccharide in detection breast milk Download PDFInfo
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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
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)
- 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. 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. 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. 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. according to the method for claim 4, it is characterised in that：Mass spectrum uses Q Exactive detectors.
- 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. 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. 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. 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. 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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