CN107192771A - The quantitative method of breast milk oligosaccharide fast qualitative - Google Patents
The quantitative method of breast milk oligosaccharide fast qualitative Download PDFInfo
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Abstract
The invention discloses a kind of quantitative method of breast milk oligosaccharide fast qualitative, mainly include the following steps that：Step 1, sample pre-treatments：150 250 μ l breast milks are taken, fat and protein is removed, final supernatant, plus ultra-pure water dilution is obtained, obtains loading sample；Step 2, to standard items use ultra performance liquid chromatography, mass spectrum, set up standard curve；Step 3, using ultra performance liquid chromatography by the loading sample breast milk oligosaccharide carry out different component separation, and utilize mass spectrum combined standard curve, carry out quantitative analysis, produce breast milk oligosaccharide content.The ultra performance liquid chromatography uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, with 8 10mmol/L ammonium formate solution (A) and acetonitrile (B) for mobile phase；Gradient elution program is：0 10min, 95% 75%B；10 15min, 75%B；15 20min, 75% 65%B；20 21min, 65% 10%B；21 24min, 10%B；24 25min, 10 95%B；25 35min, 95%B；Flow velocity is 0.3mL/min, 40 60 DEG C of column temperature.The present invention can 12 kinds of breast milk oligosaccharide of quick detection, and can be quantitative to 12 kinds of breast milk oligosaccharide.
Description
Technical field
The present invention relates to the detection field of breast milk oligosaccharide, more particularly to a kind of quantitative side of breast milk oligosaccharide fast qualitative
Method.
Background technology
Breast milk oligosaccharide has different physiological roles, can not only reduce the generation of infection, and can also effectively facilitate enteron aisle has
The propagation of bacteria group, suppresses pernicious bacteria growth indirectly, to maintain intestinal microecology to balance, so as to protect baby intestinal from cause
Germ attacks, but also with the effect for promoting baby intestinal locally and systemically immune system maturation, fraction breast milk oligosaccharide
Systemic immune response and preventing chronic inflammation can also be mitigated.In addition, also there is research to point out that oligosaccharide is passed with nerve synapse and nerve
Lead it is in close relations, can promote baby cognitive development, enhancing learning and memory ability；But breast milk oligosaccharide is shared in breast milk
Ratio only has 12/5000 to thousand/1000ths, and breast milk oligosaccharide constitutes more than kind more than 200, complicated, is unfavorable for mother
The research of newborn oligosaccharide, the detection for breast milk oligosaccharide is also provided with very big difficulty, and the country is for breast milk oligosaccharide now
Research relatively lag behind, detect that the method for breast milk oligosaccharide not only takes longer, and breast milk oligosaccharide can not be quantified,
A kind of quick detection breast milk oligosaccharide is provided, and can the method quantitative to oligosaccharide be necessary.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantage that at least will be described later.
It is a still further object of the present invention to provide a kind of quantitative method of breast milk oligosaccharide fast qualitative, it can quick detection
12 kinds of breast milk oligosaccharide, and can be quantitative to 12 kinds of breast milk oligosaccharide.
It is quantitative there is provided a kind of breast milk oligosaccharide fast qualitative according to object of the present invention and further advantage in order to realize
Method, mainly include the following steps that：
Step 1, sample pre-treatments：150-250 μ l breast milks are taken, fat and protein is removed, takes final supernatant, plus it is ultrapure
Water dilutes, and obtains loading sample.
Step 2, to standard items use ultra performance liquid chromatography, mass spectrum, set up standard curve.
Step 3, using ultra performance liquid chromatography by the loading sample breast milk oligosaccharide carry out different component separation,
And using mass spectrum with reference to the standard curve, carry out quantitative analysis, produce the breast milk oligosaccharide content.The ultra high efficiency liquid phase
Chromatogram uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, is flowing with 8-10mmol/L ammonium formate solution (A) and acetonitrile (B)
Phase；Gradient elution program is：0-10min, 95%-75%B；10-15min, 75%B；15-20min, 75%-65%B；20-
21min, 65%-10%B；21-24min, 10%B；24-25min, 10-95%B；25-35min, 95%B；Flow velocity is 0.3mL/
Min, 40-60 DEG C of column temperature.
Preferably, removal fatty described in step 1 is by the way of centrifugation, with 5000-7000r/min speed in 2-
At 5 DEG C, the breast milk is centrifuged after 8-12min, upper-layer fat is removed；The removal of the protein is molten after fat is removed
In liquid, 400-700 μ L acetonitriles are added, be vortexed ultrasound 8-12min after mixing, with 8000-12000r/min speed at 2-8 DEG C
8-12min is centrifuged, supernatant is taken.
Preferably, for the supernatant is further purified, the supernatant is centrifuged again, with 8000-11000r/
Min speed centrifuges 6-10min at 2-6 DEG C, takes final supernatant.
Preferably, mass spectrum described in step 3 uses electric spray ion source；Cation is scanned；The mass spectrometry parameters are：Dry
Gas temperature：100-200 DEG C, dry gas flow velocity：14-18L/min；Nebulizer pressure：30-40psi；Capillary voltage：2-
5kV；It is incubated gas temperature：250-350℃；It is incubated gas flow rate：10-15L/min.
Preferably, the breast milk oligosaccharide of the measure is 12 kinds of oligosaccharide, and the oligosaccharide is respectively：2- fucoses breast
Sugar, 3 fucose lactose, 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, lacto-N-fucopentaose III, breast
The sugar II of two rock algaes of acyl-N- six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a, lactose base tetrose b, lactose base
Tetrose c.
Preferably, standard items described in step 3 are 12 kinds of oligomeric saccharides, and the standard items take 1-3mg, will
It is dissolved in 1-3mL ultra-pure water, and primary criteria product solution is obtained after mixing, -50--80 DEG C of storages are placed in.
Preferably, the primary criteria product solution is taken to set up the standard solution of various concentrations.
Preferably, the process of setting up of the standard solution of the various concentrations is：Take the 2- fucoses lactose, 3 rocks
Each 8-12 μ L of primary criteria product solution of algae sugar lactose, and 60-100 μ L ultra-pure waters are respectively added, two mixed samples are obtained, concentration is equal
For 100mg/L, described two mixed samples are taken to be diluted to different concentration step by step respectively；Take the 3- sialyl lactoses, 6 salivas
Yogurt sugar, lacto-N-fucopentaose I, lacto-N-fucopentaose III, the sugar II of two rock algaes of lactoyl-N- six, lactoyl-N- hexoses, two
Saliva LNT, lactose base tetrose a, lactose base tetrose b, each 2-8 μ L of lactose base tetrose c, and respectively add the super of 30-80 μ L
Pure water is mixed, and is obtained ten mixed samples, concentration is 50mg/L, is taken described ten mixed samples to be diluted to step by step respectively different
Concentration.
Preferably, according to the mixed sample of the various concentrations, the standard curve is set up.
The present invention at least includes following beneficial effect：
By by sample pre-treatments, and the mode centrifuged twice, make the purity of breast milk oligosaccharide solution higher, to a certain degree
The degree of accuracy of testing result is ensure that, and by ultra performance liquid chromatography, mass spectrographic mode, mark is set up on the basis of standard items
Directrix curve, detects the breast milk oligosaccharide in the loading sample, and obtain the mother with reference to the standard curve in the same way
The content of newborn oligosaccharide, the ultra performance liquid chromatography uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, is more beneficial for breast milk
Oligosaccharide is separated, and the ultra performance liquid chromatography speed, sensitivity and separating degree are higher, substantially reduce analysis time so that
The detection of breast milk oligosaccharide is more quick, more precisely, recycles the standard curve, with reference to testing result, can be achieved to institute
State 12 kinds of breast milk oligosaccharide to quantify, more conducively breast milk oligosaccharide area research gos deep into, and breast milk oligosaccharide of the present invention is quick
The method of qualitative, quantitative can accurately and effectively detect 12 kinds of breast milk oligosaccharide, and can be accurately obtained 12 kinds of breast milk oligosaccharide
Content.
The further advantage of the present invention, mesh, mark and feature embody part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is the process chart of the quantitative method of breast milk oligosaccharide fast qualitative of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or many
The presence or addition of individual other elements or its combination.
As shown in figure 1, the present invention provides a kind of breast milk oligosaccharide fast qualitative quantitative method, mainly including following step
Suddenly：
Step 1, sample pre-treatments：150-250 μ l breast milks are taken, fat and protein is removed, takes final supernatant, plus it is ultrapure
Water dilutes, and obtains loading sample.
Step 2, to standard items use ultra performance liquid chromatography, mass spectrum, set up standard curve.
Step 3, using ultra performance liquid chromatography by the loading sample breast milk oligosaccharide carry out different component separation,
And using mass spectrum with reference to the standard curve, carry out quantitative analysis, produce the breast milk oligosaccharide content.The ultra high efficiency liquid phase
Chromatogram uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, is flowing with 8-10mmol/L ammonium formate solution (A) and acetonitrile (B)
Phase；Gradient elution program is：0-10min, 95%-75%B；10-15min, 75%B；15-20min, 75%-65%B；20-
21min, 65%-10%B；21-24min, 10%B；24-25min, 10-95%B；25-35min, 95%B；Flow velocity is 0.3mL/
Min, 40-60 DEG C of column temperature.
In such scheme, step 1 is by by sample pre-treatments, and the mode centrifuged twice, makes breast milk oligosaccharide solution
Purity it is higher, ensure that the degree of accuracy of testing result to a certain degree, and by ultra performance liquid chromatography, mass spectrographic mode,
Standard curve is set up on the basis of standard items, the breast milk oligosaccharide in the loading sample is detected in the same way, and combine
The standard curve obtains the content of the breast milk oligosaccharide, and the ultra performance liquid chromatography uses 2.1 × 100mm, 1.7 μm of amino
Chromatographic column, is more beneficial for the separation of breast milk oligosaccharide, and the ultra performance liquid chromatography speed, sensitivity and separating degree are higher, significantly
Shorten analysis time.
In one preferred scheme, fatty removal is by the way of centrifugation described in step 1, with 5000-7000r/min speed
Degree centrifuges the breast milk after 8-12min at 2-5 DEG C, removes upper-layer fat；The removal of the protein is to remove fat
In solution afterwards, 400-700 μ L acetonitriles are added, be vortexed ultrasound 8-12min after mixing, and exists with 8000-12000r/min speed
8-12min is centrifuged at 2-8 DEG C, supernatant is taken.
In such scheme, because the breast milk oligosaccharide has hydrophily, by the way of centrifugation, water oil reservoir is separated,
Fat is removed, then pure acetonitrile is added in the solution for removing fat, makes protein precipitation, recycles the mode of centrifugation further will
Precipitating proteins are separated, with the breast milk oligosaccharide solution purified.
In one preferred scheme, for the supernatant is further purified, the supernatant is centrifuged again, with 8000-
11000r/min speed centrifuges 6-10min at 2-6 DEG C, takes final supernatant.
In such scheme, the supernatant is centrifuged again, breast milk oligosaccharide solution is further purified, and improve pure
Change efficiency.
In one preferred scheme, mass spectrum uses electric spray ion source described in step 3；Cation is scanned；The mass spectrometry parameters
For：Dry gas temperature：100-200 DEG C, dry gas flow velocity：14-18L/min；Nebulizer pressure：30-40psi；Capillary
Voltage：2-5kV；It is incubated gas temperature：250-350℃；It is incubated gas flow rate：10-15L/min.
In such scheme, the mass spectrum way choice, the more conducively accuracy of testing result, first to the standard items
Mass spectrum sets up the standard curve, then to the loading sample mass spectrum, is easy to quantifying for the follow-up breast milk oligosaccharide, standard items
Mass spectrum multiple-reaction monitoring optimum results are as shown in table 1.
In one preferred scheme, the breast milk oligosaccharide of the measure is 12 kinds of oligosaccharide, and the oligosaccharide is respectively：2- rocks
Algae sugar lactose, 3 fucose lactose, 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, lactoyl-N- rocks algae five
Sugar III, the sugar II of two rock algaes of lactoyl-N- six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a, lactose base tetrose b,
Lactose base tetrose c.
In one preferred scheme, standard items described in step 3 are 12 kinds of oligomeric saccharides, and the standard items take
1-3mg, is dissolved in 1-3mL ultra-pure water, and primary criteria product solution is obtained after mixing, -50--80 DEG C of storages are placed in
Hide.
In one preferred scheme, the primary criteria product solution is taken to set up the standard solution of various concentrations.
In one preferred scheme, the process of setting up of the standard solution of the various concentrations is：Take the 2- fucoses breast
Sugar, each 8-12 μ L of primary criteria product solution of 3 fucose lactose, and 60-100 μ L ultra-pure waters are respectively added, two mixed samples are obtained,
Concentration is 100mg/L, takes described two mixed samples to be diluted to different concentration step by step respectively；Take the 3- salivas yogurt
Sugar, 6 sialyl lactoses, lacto-N-fucopentaose I, lacto-N-fucopentaose III, the sugar II of two rock algaes of lactoyl-N- six, lactoyl-N-
Hexose, two saliva LNTs, lactose base tetrose a, lactose base tetrose b, each 2-8 μ L of lactose base tetrose c, and respectively add 30-80
μ L ultra-pure water is mixed, and is obtained ten mixed samples, concentration is 50mg/L, is taken described ten mixed samples to be diluted to step by step respectively
Different concentration.
In one preferred scheme, according to the mixed sample of the various concentrations, the standard curve is set up.
In such scheme, the method for the quick measure breast milk oligosaccharide is directed to 12 kinds of breast milk oligosaccharide,
12 kinds of breast milk oligosaccharide content difference in human milk samples is larger, therefore is divided into two parts and does mixed sample, builds difference
The standard solution of concentration, as shown in table 2, according to the standard solution of various concentrations in table 2 set up standard curve and described in
The equation of standard curve, as shown in table 3；The mass spectral results are combined with the standard curve, you can to 12 kinds of breast milks
Oligosaccharide is quantified.
The quantitative method of the breast milk oligosaccharide fast qualitative is as shown in Figure 1.
Embodiment 1
Step 1, take 150 μ L breast milks to add after isometric ultra-pure water, 5000r/min, 2 DEG C of centrifugation 8min, remove upper strata
Fat, then adds 400 μ L pure acetonitrile, is vortexed after mixing and takes supernatant after 8min, 8000r/min, 2 DEG C of centrifugation 12min of ultrasound,
Supernatant is centrifuged again, with 8000r/min speed at 2 DEG C, the supernatant is centrifuged after 12min, final supernatant is taken,
Loading is can be used to after adding ultra-pure water dilution.
Step 2,12 kinds of standard items will be purchased take 0.5mg, be dissolved in 0.5mL ultra-pure water, after mixing it is primary
Standard solution, places and is stored at -50 DEG C.
Step 3, each 8 μ L of primary criteria product solution for taking 2- fucoses lactose, 3 fucose lactose, and it is super respectively to add 60 μ L
Pure water, obtains two mixed samples, and concentration is 100mg/L, takes described two mixed samples to be diluted to step by step respectively different dense
Degree；Take the 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lactoyl-N-
The sugar II of two rock algae six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a, lactose base tetrose b, lactose base tetrose c
Each 2 μ L, and 30 μ L ultra-pure water mixing is respectively added, ten mixed samples are obtained, concentration is 50mg/L, take described ten mixing marks
Sample is diluted to different concentration step by step respectively.
Step 4, the standard solution to various concentrations, using ultra performance liquid chromatography, mass spectrographic mode, set up standard bent
Line.
The ultra performance liquid chromatography uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, molten with 8mmol/L ammonium formate
Liquid (A) and acetonitrile (B) are mobile phase；Gradient elution program is：0-10min, 95%-75%B；10-15min, 75%B；15-
20min, 75%-65%B；20-21min, 65%-10%B；21-24min, 10%B；24-25min, 10-95%B；25-
35min, 95%B；Flow velocity is 0.3mL/min, 40 DEG C of column temperature.
The mass spectrum uses electric spray ion source；Cation is scanned；The mass spectrometry parameters are：Dry gas temperature：100
DEG C, dry gas flow velocity：14L/min；Nebulizer pressure：30psi；Capillary voltage：2kV；It is incubated gas temperature：250℃；Protect
Wet rate of flow of fluid：10L/min.
Step 5, again with same detection method to loading sample ultra performance liquid chromatography, mass spectrum, combined standard curve, i.e.,
Obtain 12 kinds of breast milk oligosaccharide contents.
Embodiment 2
Step 1, take 200 μ L breast milks to add after isometric ultra-pure water, 6000r/min, 4 DEG C of centrifugation 10min, remove upper strata
Fat, then adds 600 μ L pure acetonitrile, is vortexed after mixing and is taken after 10min, 10000r/min, 4 DEG C of centrifugation 10min of ultrasound
Clearly, then by supernatant centrifuge, with 10000r/min speed at 4 DEG C, the supernatant is centrifuged after 10min, taken on final
Clear liquid, loading is can be used to after adding ultra-pure water dilution.
Step 2,12 kinds of standard items will be purchased take 1mg, be dissolved in 1mL ultra-pure water, after mixing primary criteria
Product solution, places and is stored at -80 DEG C.
Step 3, each 10 μ L of primary criteria product solution for taking 2- fucoses lactose, 3 fucose lactose, and it is super respectively to add 80 μ L
Pure water, obtains two mixed samples, and concentration is 100mg/L, takes described two mixed samples to be diluted to step by step respectively different dense
Degree；Take the 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lactoyl-N-
The sugar II of two rock algae six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a, lactose base tetrose b, lactose base tetrose c
Each 5 μ L, and 50 μ L ultra-pure water mixing is respectively added, ten mixed samples are obtained, concentration is 50mg/L, take described ten mixing marks
Sample is diluted to different concentration step by step respectively.
Step 4, the standard solution to various concentrations, using ultra performance liquid chromatography, mass spectrographic mode, set up standard bent
Line.
The ultra performance liquid chromatography uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, molten with 9mmol/L ammonium formate
Liquid (A) and acetonitrile (B) are mobile phase；Gradient elution program is：0-10min, 95%-75%B；10-15min, 75%B；15-
20min, 75%-65%B；20-21min, 65%-10%B；21-24min, 10%B；24-25min, 10-95%B；25-
35min, 95%B；Flow velocity is 0.3mL/min, 50 DEG C of column temperature.
The mass spectrum uses electric spray ion source；Cation is scanned；The mass spectrometry parameters are：Dry gas temperature：150
DEG C, dry gas flow velocity：16L/min；Nebulizer pressure：35psi；Capillary voltage：3kV；It is incubated gas temperature：300℃；Protect
Wet rate of flow of fluid：12L/min.
Step 5, again with same detection method to loading sample ultra performance liquid chromatography, mass spectrum, combined standard curve, i.e.,
Obtain 12 kinds of breast milk oligosaccharide contents.
Embodiment 3
Step 1, take 250 μ L breast milks to add after isometric ultra-pure water, 7000r/min, 2 DEG C of centrifugation 8min, remove upper strata
Fat, then adds 700 μ L pure acetonitrile, is vortexed after mixing and is taken after 12min, 12000r/min, 8 DEG C of centrifugation 8min of ultrasound
Clearly, then by supernatant centrifuge, with 12000r/min speed at 8 DEG C, the supernatant is centrifuged after 8min, final supernatant is taken
Liquid, loading is can be used to after adding ultra-pure water dilution.
Step 2,12 kinds of standard items will be purchased take 3mg, be dissolved in 3mL ultra-pure water, after mixing primary criteria
Product solution, places and is stored at -80 DEG C.
Step 3, each 12 μ L of primary criteria product solution for taking 2- fucoses lactose, 3 fucose lactose, and respectively add 100 μ L
Ultra-pure water, obtains two mixed samples, and concentration is 100mg/L, takes described two mixed samples to be diluted to step by step respectively different
Concentration；Take the 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lactoyl-
The sugar II of bis- rock algaes of N- six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a, lactose base tetrose b, lactose base tetrose
Each 8 μ L of c, and 80 μ L ultra-pure water mixing is respectively added, ten mixed samples are obtained, concentration is 50mg/L, take described ten mixing
Standard specimen is diluted to different concentration step by step respectively.
Step 4, the standard solution to various concentrations, using ultra performance liquid chromatography, mass spectrographic mode, set up standard bent
Line.
The ultra performance liquid chromatography uses 2.1 × 100mm, 1.7 μm of amino chromatographic columns, molten with 10mmol/L ammonium formate
Liquid (A) and acetonitrile (B) are mobile phase；Gradient elution program is：0-10min, 95%-75%B；10-15min, 75%B；15-
20min, 75%-65%B；20-21min, 65%-10%B；21-24min, 10%B；24-25min, 10-95%B；25-
35min, 95%B；Flow velocity is 0.3mL/min, 60 DEG C of column temperature.
The mass spectrum uses electric spray ion source；Cation is scanned；The mass spectrometry parameters are：Dry gas temperature：200
DEG C, dry gas flow velocity：18L/min；Nebulizer pressure：40psi；Capillary voltage：5kV；It is incubated gas temperature：350℃；Protect
Wet rate of flow of fluid：15L/min.
Step 5, again with same detection method to loading sample ultra performance liquid chromatography, mass spectrum, combined standard curve, i.e.,
Obtain 12 kinds of breast milk oligosaccharide contents.
The oligomeric saccharide MRM optimum results of 1 12 kinds of breast milks of table
2FL, 3FL, 3SL, 6SL, LNFP I, LNFP III, LNDFH II, LNnH, DSLNT, LSTa, LSTb, LSTc divide in table 1
Dui Ying not 2- fucoses lactose, 3 fucose lactose, 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, breast
Acyl-N- rock algaes pentasaccharides III, the sugar II of two rock algaes of lactoyl-N- six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a,
Lactose base tetrose b, lactose base tetrose c.
The concentration (mg/L) of 2 12 kinds of standard solutions of table
3 12 kinds of breast milk oligosaccharide mark product regression equations of table
From table 3,12 kinds of oligomeric saccharides are in good linear relationship (R in setting concentration range2≥0.99)。
UPLC (ultra high efficiency liquid chromatography) analyzing detecting method precision is good, and 12 kinds of carbohydrate contents repeat RSD (the relative marks of sample introduction analysis
Quasi- deviation) value control within 10%.
To verify feasibility and the degree of accuracy of the assay method, do mark-on reclaims experiment to be estimated, take breast milk 200
μ L are handled according to pre-treating method, and 12 kinds of oligosaccharide compositions therein are quantified according to the mass spectrum liquid-phase condition optimized
Analysis, acquisition is not added with standard specimen product analysis result, and aforesaid operations is repeated 3 times, and discharges error, according to each oligosaccharide measured into
Divide the horizontal mark-on of actual content, carry out sample detection, testing result is as shown in table 4.
4 12 kinds of breast milk oligosaccharide rate of recovery testing results of table
As shown in Table 4, the TIANZHU XINGNAO Capsul of 12 kinds of breast milk oligosaccharide is between 80%~120%, sample pre-treatments side
The accuracy of method is represented with RSD values, and the RSD values of experiment 12 kinds of oligosaccharide of gained are respectively less than 10%.Therefore, this experiment is set up
Method can be used for the detection and analysis of 12 kinds of oligosaccharide in breast milk, and from the breast milk that 5 volunteers provide, with described quick
The content that the oligomeric method of breast milk determines oligosaccharide in breast milk is determined, measurement result is as shown in table 5.
Oligosaccharide measurement result in the people's colostrum of table 5
Note："-" represents not report.
Result of the test is shown in Table 5, shows the measurement result of 12 kinds of breast milk oligosaccharide in the measurement range obtained by consulting literatures
Within, therefore the assay method can be used for the measure of breast milk oligosaccharide.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (9)
1. a kind of quantitative method of breast milk oligosaccharide fast qualitative, wherein, mainly include the following steps that：
Step 1, sample pre-treatments：Take 150-250 μ l breast milks, remove fat and protein, take final supernatant, plus ultra-pure water is dilute
Release, obtain loading sample；
Step 2, to standard items use ultra performance liquid chromatography, mass spectrum, set up standard curve；
Step 3, using ultra performance liquid chromatography by the loading sample breast milk oligosaccharide carry out different component separation, and profit
With mass spectrum with reference to the standard curve, quantitative analysis is carried out, the breast milk oligosaccharide content is produced.The ultra performance liquid chromatography
Using 2.1 × 100mm, 1.7 μm of amino chromatographic columns, with 8-10mmol/L ammonium formate solution (A) and acetonitrile (B) for mobile phase；
Gradient elution program is：0-10min, 95%-75%B；10-15min, 75%B；15-20min, 75%-65%B；20-
21min, 65%-10%B；21-24min, 10%B；24-25min, 10-95%B；25-35min, 95%B；Flow velocity is 0.3mL/
Min, 40-60 DEG C of column temperature.
2. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 1, wherein, fatty removal described in step 1
By the way of centrifugation, with 5000-7000r/min speed at 2-5 DEG C, the breast milk is centrifuged after 8-12min, in removal
Layer fat；The removal of the protein is in the solution after removing fat, to add 400-700 μ L acetonitriles, is vortexed after mixing and surpasses
Sound 8-12min, centrifuges 8-12min at 2-8 DEG C with 8000-12000r/min speed, takes supernatant.
3. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 2, wherein, for the supernatant is further purified
Liquid, the supernatant is centrifuged again, and 6-10min is centrifuged at 2-6 DEG C with 8000-11000r/min speed, is taken on final
Clear liquid.
4. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 1, wherein, mass spectrum described in step 3 is using electricity
Esi ion source；Cation is scanned；The mass spectrometry parameters are：Dry gas temperature：100-200 DEG C, dry gas flow velocity：14-
18L/min；Nebulizer pressure：30-40psi；Capillary voltage：2-5kV；It is incubated gas temperature：250-350℃；It is incubated gas
Flow velocity：10-15L/min.
5. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 1, wherein, the breast milk oligosaccharide of the measure
For 12 kinds of oligosaccharide, the oligosaccharide is respectively：2- fucoses lactose, 3 fucose lactose, 3- sialyl lactoses, 6 saliva yogurts
Sugar, lacto-N-fucopentaose I, lacto-N-fucopentaose III, the sugar II of two rock algaes of lactoyl-N- six, lactoyl-N- hexoses, two salivas
LNT, lactose base tetrose a, lactose base tetrose b, lactose base tetrose c.
6. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 5, wherein, standard items described in step 3 are institute
12 kinds of oligomeric saccharides are stated, the standard items take 1-3mg, are dissolved in 1-3mL ultra-pure water, primary mark is obtained after mixing
Quasi- product solution, is placed in -50--80 DEG C of storages.
7. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 6, wherein, take the primary criteria product solution
Set up the standard solution of various concentrations.
8. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 7, wherein, the standard items of the various concentrations
The process of setting up of solution is：Each 8-12 μ L of primary criteria product solution of the 2- fucoses lactose, 3 fucose lactose are taken, and respectively
60-100 μ L ultra-pure waters are added, two mixed samples are obtained, concentration is 100mg/L, described two mixed samples difference are taken step by step
It is diluted to different concentration；Take the 3- sialyl lactoses, 6 sialyl lactoses, lacto-N-fucopentaose I, lactoyl-N- rock algaes
Pentasaccharides III, the sugar II of two rock algaes of lactoyl-N- six, lactoyl-N- hexoses, two saliva LNTs, lactose base tetrose a, lactose base tetrose
B, each 2-8 μ L of lactose base tetrose c, and 30-80 μ L ultra-pure water mixing is respectively added, ten mixed samples are obtained, concentration is 50mg/
L, takes described ten mixed samples to be diluted to different concentration step by step respectively.
9. the quantitative method of breast milk oligosaccharide fast qualitative as claimed in claim 8, wherein, according to the mixed of the various concentrations
Standard specimen is closed, the standard curve is set up.
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