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Method for detecting oligosaccharides in breast milk

An oligosaccharide, breast milk technology, applied in the field of analysis and detection, can solve the problems of not representing mass spectrometry detectors, incomplete elution, large accidental errors, etc., and achieve the effect of simple method, reduced operation steps, and rapid detection.

Active Publication Date: 2018-01-23
BEIJING SANYUAN FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 3) Using ion chromatography, liquid chromatography and mass spectrometry alone can only detect part of the breast milk oligosaccharides
In the process of solid phase extraction, it needs to be eluted with different eluents, which may cause sample loss due to incomplete elution; during the freeze-drying process or when the sealed tube is opened after drying, the vacuum may also easily lead to sample loss
Due to the small amount of sampling and the HMO content in it is only about 1%, it is easy to cause large accidental errors
[0009] 2) Most of the detectors used in mass spectrometry are Time of Flight (TOF) detectors. Although TOF has a wide range of applications, it does not represent the most advanced mass spectrometer detectors.
[0010] Q Exactive detector can replace TOF in many aspects, and many laboratories have purchased Q Exactive detector, but so far there is no relevant report on using Q Exactive to detect breast milk oligosaccharides

Method used

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  • Method for detecting oligosaccharides in breast milk
  • Method for detecting oligosaccharides in breast milk
  • Method for detecting oligosaccharides in breast milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] A method for detecting oligosaccharides in breast milk includes the following steps:

[0069] 1) Sample pretreatment: 100μL of breast milk sample was centrifuged at 2000g, 20min, 2℃ to remove the upper fat, take the lower sample solution and add double volume of 66.7% ethanol, freeze at ﹣80℃ for 10min, centrifuge after taking it out, centrifuge at 10000r / min, Take the supernatant after centrifugation for 10 min. Filter through 0.22μm filter membrane before loading.

[0070] 2) Liquid phase conditions:

[0071] Column: Hypercarb porous graphite carbon column.

[0072] Mobile phase A: 0.1% formic acid-3% acetonitrile-water;

[0073] The mobile phase B is 0.1% formic acid-90% acetonitrile-water.

[0074] Gradient elution, the conditions are shown in Table 4:

[0075] Table 4 Gradient elution conditions

[0076]

[0077]

[0078] 3) Mass spectrometry conditions

[0079] Adopt positive ion mode; scanning range: 150-2000m / z; heating electrospray ion source; full scan data acquisition mod...

Embodiment 2

[0094] A method for detecting oligosaccharides in breast milk is the same as in Example 1, except that sample pretreatment, gradient elution, and mass spectrometry conditions are different, specifically:

[0095] 1) Sample pretreatment: 500μL of breast milk sample was centrifuged at 5000g, 13min, 4℃ to remove the upper fat, take the lower layer sample solution and add twice the volume, 66.7% ethanol, freeze at -80℃ for 25min, take it out and centrifuge, centrifuge at 12000r / min , 25min, take the supernatant after centrifugation, filter through 0.22μm filter membrane before loading.

[0096] 2) Liquid phase gradient elution, as shown in Table 8.

[0097] Table 8 Gradient elution conditions

[0098]

[0099] 3) Mass detection

[0100] Adopt positive ion mode; scanning range: 150-2000m / z; heated electrospray ion source; full scan data acquisition mode; sheath gas flow 36, auxiliary gas flow 10, scanner flow 0, spray voltage 3.4kV, heating temperature 360 ℃, S-type lens radio frequency 62...

Embodiment 3

[0108] A method for detecting oligosaccharides in breast milk is the same as in Example 1, except that sample pretreatment, gradient elution, and mass spectrometry conditions are different, specifically:

[0109] 1) Sample pretreatment: 800μL of breast milk sample was centrifuged at 6000g, 20min, 8℃ to remove the upper fat, take the lower layer sample solution and add double volume, 66.7% ethanol, freeze at ﹣80℃ for 50min, take it out and centrifuge, centrifuge at 15000r / min Take the supernatant after centrifugation for 50 min, and filter it through a 0.22μm filter before loading.

[0110] 2) Liquid phase gradient elution, as shown in Table 11.

[0111] Table 11 Gradient elution conditions

[0112]

[0113] 3) Mass detection

[0114] Adopt positive ion mode; scanning range: 150-2000m / z; heated electrospray ion source; full scan data acquisition mode; sheath gas flow rate 40, auxiliary gas flow rate 20, scanner flow rate 0, spray voltage 4kV, heating temperature 400℃ , S-type lens radi...

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Abstract

The invention relates to a method for detecting oligosaccharides in breast milk. The method includes a step of breast milk sample pretreatment, namely taking a breast milk sample, centrifugally removing upper fat, taking lower liquid, adding alcohol solvent, freezing for 5-15min at a temperature ranging from -40 DEG C to -100 DEG C, centrifuging and taking supernatant. The invention further provides a method for qualitative and quantitative detection of oligosaccharides in the pretreated breast milk according to a liquid chromatography-mass spectrometry method. The method for simultaneous detection of various oligosaccharides in non-reducing breast milk by liquid chromatography-mass spectrometry is established and has advantages of effectiveness, quickness and simplicity in pretreatment. AQ Exactive detector is adopted for mass spectrometry, liquid-phase gradient elution parameters and mass spectrometric detection parameters are determined, ion information of non-reducing breast milkoligosaccharides in mass spectrometric detection under a positive ion mode is given, and the method can be well applied to qualitative and quantitative detection of the oligosaccharides in the breastmilk.

Description

Technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a method for detecting oligosaccharides in breast milk, and more specifically, to a method for detecting oligosaccharides in breast milk by adopting a liquid mass method. Background technique [0002] Human milk oligosaccharides (HMO) is the third largest component in human breast milk after lactose and fat. The content in colostrum is as high as 22-23g / L, and in mature milk it is 12-13g / L. HMO has an important biological function, not only has the function of resisting intestinal pathogenic microorganism infection, but also has the function of maintaining the intestinal microecological balance. Breast milk composition is the gold standard for the preparation of infant milk powder, so the establishment of a detection method for breast milk oligosaccharides is a necessary prerequisite for the analysis of breast milk composition and the development of more nut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N1/40G01N30/14G01N30/02
Inventor 陈历俊赵军英魏京华姜铁民董学艳李菊芳张毅
Owner BEIJING SANYUAN FOOD
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