CN114594169A - Quantitative detection method for oligosaccharide content in breast milk - Google Patents
Quantitative detection method for oligosaccharide content in breast milk Download PDFInfo
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- CN114594169A CN114594169A CN202011404481.4A CN202011404481A CN114594169A CN 114594169 A CN114594169 A CN 114594169A CN 202011404481 A CN202011404481 A CN 202011404481A CN 114594169 A CN114594169 A CN 114594169A
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- lacto
- breast milk
- oligosaccharide
- lnfp
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- 238000001514 detection method Methods 0.000 title claims abstract description 8
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- 208000005623 Carcinogenesis Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229910052681 coesite Inorganic materials 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052906 cristobalite Inorganic materials 0.000 description 1
- 230000015961 delipidation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 229910052682 stishovite Inorganic materials 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 229910052905 tridymite Inorganic materials 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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Abstract
The invention relates to a rapid quantitative inspection method of free oligosaccharide in breast milk. The method is characterized in that after a sample is subjected to low-temperature centrifugation and degreasing and is deproteinized by adding an organic solvent, a quantitative analysis method of chromatography-mass spectrometry combination established by 24 oligosaccharide standard substances is used for realizing comprehensive and rapid quantification of oligosaccharides in breast milk. The method has the characteristics of high flux, comprehensive quantification, high sensitivity, good accuracy and good stability. Is suitable for comprehensive and rapid quantitative detection of breast milk oligosaccharide of mammals.
Description
Technical Field
The invention relates to a method for measuring the absolute content of free oligosaccharide in breast milk, which is suitable for the rapid and high-throughput analysis of breast milk samples.
Technical Field
Oligosaccharides derived from breast milk play a vital role in the growth and development of infants, such as anti-inflammatory, anti-infective, immunomodulating and maintaining intestinal flora balance. Wherein the acid oligosaccharide plays an irreplaceable role in vivo, such as preventing carcinogenesis, promoting normal development of infant brain, and the like.
The method can accurately and quickly carry out quantitative analysis on the oligosaccharides in the breast milk, and is a necessary means for further research on the oligosaccharides in the breast milk. The high-flux quantitative detection can comprehensively analyze the oligosaccharide content in breast milk, complete the analysis of the oligosaccharide content in a large number of crowd samples and track the change of the oligosaccharide content along with the lactation period to obtain the queue sample information, which provides favorable information for further developing the biological function research of the oligosaccharide and better utilizing the human milk oligosaccharide. However, due to the complex composition, complex and diverse oligosaccharide types and structures and difficult isomer separation and identification of breast milk, high-throughput accurate quantification of various isomers has a great challenge.
Tandem mass spectrometry can provide more compound information, for the identification and the ration of isomer provide help, liquid chromatogram and mass spectrometry are established ties, can provide effectual former grade separation for the mass spectrometry, and reasonable reduction flows out altogether for mass spectrometry quantitative stability accuracy promotes. Therefore, the invention designs a novel method for rapidly and quantitatively detecting the content of the breast milk oligosaccharide, which realizes the high-flux absolute quantification of the oligosaccharide in 24 days by utilizing the liquid chromatogram and mass spectrum to be connected in series and adjusting and optimizing the liquid phase separation condition and mass spectrum parameter information.
Disclosure of Invention
The invention relates to a rapid quantitative inspection method of free oligosaccharide in breast milk. The method is characterized in that after a sample is subjected to low-temperature centrifugation and degreasing and is added with an organic solvent to remove protein, a quantitative analysis method of chromatography-mass spectrometry combined established by 24 oligosaccharide standard substances is used for realizing comprehensive and rapid quantification of oligosaccharide in breast milk.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for rapidly quantifying oligosaccharide content in breast milk comprises the following steps: and (3) selecting appropriate chromatographic separation filler by using 24 oligosaccharide standard substances, separating under appropriate liquid phase conditions, drawing a standard curve by using a mass spectrum multi-channel quantitative method established by the standard substances, and quantitatively monitoring the free oligosaccharides in the breast milk after pretreatment to obtain the absolute content information of the free oligosaccharides in the breast milk.
The 24 oligosaccharide standards are shown in figure 1.
Structural information of 24 oligosaccharide standard products
1. The chromatographic separation filler used includes, but is not limited to, silica gel or a filler with polar groups bonded on the surface of the silica gel, and the structural formula of the filler is as follows
Wherein SiO2 is silica gel, R is polar group, and has one or more of amino acid, amide, amino group, carboxyl group, glycosyl, zwitterion, etc
2. The organic solvent in the eluent is one or more of methanol, acetonitrile, ethanol and acetone.
3. The buffer salt type and its concentration and pH in the mobile phase are as follows:
a) ammonium formate buffer salt, concentration 0-200mM, pH 2.0-7.0;
b) ammonium acetate buffer salt, concentration 0-200mM, pH 2.0-7.0;
c) ammonium bicarbonate buffer salt with concentration of 0-200mM and pH of 6.0-9.0;
4. the mobile phase gradient was optimized as follows:
and using mobile phase water or a buffered saline solution and an organic solvent as eluent, wherein the mixing ratio is 5/95-95/5.
5. The breast milk homogenization step is as follows: thawing frozen breast milk sample at 2-20 deg.C; carrying out ultrasonic treatment for not less than 1 minute, carrying out vortex oscillation for not less than 5 seconds, repeating the steps for not less than 1 time, and taking out a sample middle layer to finish homogeneous sampling of breast milk;
6. the low-temperature lipid removal step of breast milk comprises the following steps: taking a homogeneous breast milk sample, centrifuging for 5-500 minutes at 50000g of 500-;
7. the method for removing protein by precipitation of the breast milk sample comprises the following steps: taking the breast milk sample after low-temperature degerming, adding organic solvent with volume not less than 1 times, mixing uniformly, standing at 0-10 ℃ for 2-48 hours, centrifuging at 0-10 ℃ for 500-50000g for not less than 5 minutes, taking out supernatant, and completing the precipitation and protein removal treatment of the breast milk sample
The invention has the following advantages
1. The quantitative oligosaccharide has multiple varieties and high accuracy. The method can quantitatively cover oligosaccharide molecules with the mass concentration of more than 95% in breast milk, and can comprehensively reflect the content of the oligosaccharide in the breast milk.
2. High flux, quick and simple pretreatment process, short analysis and quantification time and suitability for analyzing a large amount of samples.
3. The quantitative accuracy is high, the repeatability is good, and the method is proved by methods such as precision, recovery rate and the like, and has good stability and repeatability.
4. Has wide application range, and can be used for absolute quantitative analysis of milk and human milk of different mammals
Drawings
FIG. 1 is the structural information of 24 oligosaccharide standards;
FIG. 2 is a graph of TIC of 24 standards by liquid phase separation;
Detailed Description
Example 1:
homogenization process of breast milk:
taking a certain amount of breast milk to naturally thaw at 4 ℃, ultrasonically homogenizing for 10 minutes, performing vortex oscillation for 1 minute, and then taking out the middle layer to finish the homogeneous sampling of the breast milk;
low temperature delipidation process of breast milk:
taking a homogeneous breast milk sample, centrifuging at 4 ℃ for 10 minutes at 10000g, taking out a lower layer transparent or semitransparent solution after the centrifugation is finished, and repeating the centrifugation twice to finish the low-temperature fat removal process of the breast milk;
the process of removing protein by precipitation of the breast milk sample:
taking the breast milk sample after low-temperature quality removal, adding 3 times of methanol in volume, uniformly mixing, standing at 10 ℃ for 2 hours, centrifuging at 4 ℃ for 10 minutes at 10000g, and taking out supernatant to finish precipitation protein removal of the breast milk sample;
thus, the pretreatment of the breast milk, namely the impurity removal and extraction process of the breast milk oligosaccharide is completed.
Example 2:
liquid phase separation process of 24 oligosaccharide standard products
A Waters BEH XAmide chromatographic column (2.1 x 150mm, 1.7 μm) was used for the experiment
Acetonitrile/aqueous ammonium acetate as mobile phase, ammonium acetate concentration 50mM
The gradient conditions were as follows:
wherein A is ACN/H2O/50mM ammonium acetate in a volume ratio of 71/19/10, B: ACN/H2O/50mM ammonium acetate in a volume ratio of-20/70/10
The column temperature of the chromatographic column is 40 ℃, and the flow rate is 0.3mL/min
FIG. 2 is a graph of TIC of 24 standards by liquid phase separation;
example 3:
drawing quantitative curve of 24 oligosaccharide standard products
The standard product is configured into mixed standard according to requirements and divided into a high content group and a low content group, and the specific information is as follows
Preparing high-content and low-content mixed standards of 1mg/mL respectively, diluting the mixed standards into 6 concentrations by using acetonitrile water (volume concentration is 70 percent) (see a table), and performing liquid phase separation as shown in an example 2;
the quantitative curve and quantitative limit of the standard product and the detection limit thereof are shown in the following table
The quantitative and qualitative detection ion pairs are:
Claims (5)
1. a method for quantitatively detecting the content of oligosaccharide in breast milk comprises the steps of carrying out liquid chromatography separation and mass spectrometry detection by using 24 oligosaccharide standard substances through chromatographic-mass spectrometry, taking retention time as a horizontal coordinate, extracting a response value of an ion pair as a vertical coordinate, establishing a standard curve of the standard substances, carrying out chromatographic-mass spectrometry analysis on free oligosaccharide in breast milk after pretreatment, and substituting the standard curve to obtain absolute content information of the 24 free oligosaccharide in breast milk;
the 24 oligosaccharide standards include:
3’SL(3’-Sialyllactose)、6’SL(6’-Sialyllatose)、LST-b(Lacto-Sialyl-tetraose b)、LST-c((Lacto-sialyl-tetraose)、DSLNT(Disialyl-LNT)、3’SLNFP-II(3’-Sialyl-Lacto-N-fucopentaose II)、6’SLNFP-VI(6’-Sialyl-Lacto-N-fucopentaose II)、2’FL(2’-Fucosyllactose)、3-FL(3-Fucosyllactose)、LNT(Lacto-N-tetraose)、LNnT(Lacto-N-neotetraose)、DFL(Difucosyl-Lacto)、LNFP-I(Lacto-N-fucopentaose I)、LNFP-III(Lacto-N-fucopentaose III)、LNFP-II(Lacto-N-fucopentaose II)、LNnDFH-II(Lacto-N-neo-difucohexaose II)、LNDFH-II(Lacto-N-difucohexaose II)、LNDFH-I(Lacto-N-difucohexaose I)、LNnDFH-I(Lacto-N-neo-difucohexaose I)、MFLNnH(Monofucosyl-Lacto-N-neo-hexaose)、MFLNH-III(Monofucosyl-Lacto-N-hexaose III)、MFLNH-I(Monofucosyl-Lacto-N-hexaose I)、DFpLNnH(Difucosyl-para-Lacto-N-neo-hexaose)、DFLNH[a](Difucosyl-Lacto-N-hexaose)。
2. the method of claim 1, wherein the chromatographic separation filler includes, but is not limited to, silica gel or one or more fillers having polar groups bonded to the surface of the silica gel, and the bonded polar groups include one or more of amino acids, amides, amino groups, carboxyl groups, and glycosyl zwitterions.
3. The process of claim 1, wherein the liquid phase separation conditions used comprise:
a) mobile phases include, but are not limited to, water; and one or more of acetonitrile, methanol, ethanol, acetone and isopropanol in an organic solvent;
b) the additive comprises one or more of ammonium formate buffer salt, ammonium acetate buffer salt, formic acid, acetic acid and trifluoroacetic acid, and the concentration of the buffer salt in the mobile phase is 0-200 mM;
c) the inner diameter of the chromatographic column is 2.1-10 mm;
d) the mobile phase is water and/or a buffered salt solution and an organic solvent; the ratio of water and/or a buffer salt solution to an organic solvent as an eluent is 5/95-95/5;
e) the flow rate is 0.1-2 BV/min;
f) the column temperature is 15-60 ℃.
4. The method of claim 1, wherein the standard establishment procedure is used for quantitative and qualitative detection of ion pairs as follows: 2' FL:487.13>325.02,523.13>178.94
3-FL:487.1>322.9,523.13>178.94
3’SL:632.18>289.98
6’SL:632.1>289.95
DFL:632.9>325.1
LNT:706.3>202
LNnT:706.3>263
DSLNT:643.64>290.1
LNFP-I:852.3>325
LNFP-III:852.3>364.1
LNFP-II:852.3>348.1
LSTb:997.25>290
LSTc:997.25>289.9
3’SLNFPII:1143.25>290.1,1143.25>348.1
6’SLNFPVI:1143.2>290.1
LNnDFH-II:998.30>364.02
LNDFH-II:998.3>348
LNDFH-I:998.3>348.01,998.3>325.10
LNnDFH-I:998.3>325.1
MFLNnH:1217.4>364,1217.4>526.2
MFLNH-III:1217.4>672,1217.4>364.1
MFLNH-I:1217.4>526.1
DFpLNnH:1363.45>364.1,1363.45>875.2
DFLNH[a]:1363.5>202,1363.5>325。
5. The method according to claim 1, wherein the pretreatment process before breast milk treatment comprises homogenization, low-temperature lipid removal, precipitation and protein removal,
1) the breast milk homogenization step is as follows: thawing frozen breast milk sample at 2-20 deg.C; carrying out ultrasonic treatment for not less than 1 minute, carrying out vortex oscillation for not less than 5 seconds, repeating the ultrasonic vortex step for not less than 0 time, and taking out a sample middle layer to complete homogeneous sampling of breast milk;
2) the low-temperature lipid removal step of breast milk comprises the following steps: taking a homogeneous breast milk sample, centrifuging for 5-500 minutes at 50000g under minus 4-10 ℃, repeating for no less than 1 time, and taking out a lower layer transparent or semitransparent solution to finish low-temperature quality removal of the breast milk sample;
3) the method for removing protein by precipitation of the breast milk sample comprises the following steps: taking the breast milk sample after low-temperature mass removal, adding organic solvent with volume not less than 1 times, mixing uniformly, standing for 2-48 hours at 0-10 ℃, centrifuging for not less than 5 minutes at 0-10 ℃ and 50000g, and taking out supernatant to finish the precipitation protein removal treatment of the breast milk sample; the organic solvent is: one or more of acetonitrile, ethanol, methanol, acetone, isopropanol and isopropanol.
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