AU2023203422B1 - Method for qualitatively detecting acidic oligosaccharides in human milk - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 29
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- 150000002500 ions Chemical class 0.000 claims description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
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Abstract
The present application relates to a method for qualitatively detecting acidic oligosaccharides in
human milk, which is carried out successively according to the following steps: (1) centrifuging
human milk to remove upper-layer fats and proteins, and taking a middle layer of the centrifuged
product; (2) adding ethanol to the middle-layer product, and conducting standing reaction; (3)
centrifuging the reaction product and taking a supernatant; (4) adsorbing the supernatant via a
chromatography column, washing the column with ultrapure water, subsequently eluting the
column with a sodium chloride solution, and then collecting an eluent; and (5) analyzing the
eluent via liquid chromatography-mass spectrometry. The method for qualitatively detecting acidic
oligosaccharides in human milk provided by the present application can be used for correct
qualitative determination of 9 acidic oligosaccharides from a human milk sample through
improvement of the extraction process and liquid chromatography and mass spectrometry
parameters. Meanwhile, this method also has good repeatability, and is applicable to large-scale
analysis work of human milk samples.
Description
Description
Cross-Reference The present application claims priority to Chinese Patent Application No. 202210757416.2 filed with the China Patent Office on June 30, 2022, and entitled "METHOD FOR QUALITATIVELY DETECTING ACIDIC OLIGOSACCHARIDES IN HUMAN MILK", the disclosure of which is incorporated herein by reference.
Technical Field The present application relates to the field of detection, particularly to a method for qualitatively detecting acidic oligosaccharides in human milk.
Background Art In human milk, human milk oligosaccharides (HMOs) are short-chain polymers formed by connecting 3-10 monosaccharides via covalent bonds. HMOs are formed by polymerizing five basic monosaccharides, i.e., galactose (Gal), glucose (Glc), N-acetylgucosamine (GlcNAc), fucose (Fuc) and sialic acid (Sia). Among them, sialic acid is present in a form of N-acetylneuraminic acid (Neu5Ac) in human milk. According to their chemical charges, HMOs can be divided into two categories: neutral oligosaccharides consisting of monosaccharides without charges (Glu, Gal, GlcNAc and Fuc) and acidic oligosaccharides with Neu5Ac negative charge residues. The acidic oligosaccharides account for 12-14% of the total amount of HMOs. The human milk oligosaccharides have the functions of promoting brain development, regulating intestinal flora, resisting viruses, preventing necrotizing enterocolitis, regulating epithelial cells and immune cells and the like. The human milk may contain thousands of oligosaccharides, however, the types and contents of HMOs will change due to geographical differences, lactation stages, delivery modes, genetic factors and other factors. At present, qualitative and quantitative detections can be performed on HMOs through capillary electrophoresis, liquid chromatography, liquid chromatography-mass spectrometry, ion chromatography and other methods. Due to various types of HMOs and complicated change in their contents and types, simulating human milk with milk powder is particularly complex and difficult. Methods for qualitative detection of HMOs in human milk are established to study the change in contents of different types of oligosaccharides in human milk to summarize the change rule of the types and contents of HMOs, which will provide a basis for the addition of HMOs to infant formula in the future, so as to facilitate the preparation of infant formula similar to Chinese human milk. In view of this, the present application is provided hereby.
Summary of the Invention The objective of the present application is to provide a method for qualitatively detecting acidic oligosaccharides in human milk. The method can achieve the separation and extraction and qualitative detection of acidic oligosaccharides in human milk. In order to achieve the above objective, the present application provides a method for qualitatively detecting acidic oligosaccharides in human milk, which is successively carried out according to the following steps: (1) Centrifuging human milk to remove upper-layer fats and proteins, and taking a middle layer of the centrifuged product; (2) Adding ethanol to the middle-layer product, and conducting standing reaction; (3) Centrifuging the reaction product and taking a supernatant; (4) Adsorbing the supernatant via a chromatography column, washing the column with ultrapure water, subsequently eluting the column with a sodium chloride solution, and then collecting an eluent; and (5) Analyzing the eluent via liquid chromatography-mass spectrometry. Preferably or optionally, in step (1), the specific centrifuging conditions are as follows: centrifuging for 25-35 min at 8000-10000 rpm at 3-5 °C, preferably, the centrifuging conditions are as follows: centrifuging for 30 min at 10000 rpm at 4 °C. Preferably or optionally, in step (2), the volume ratio of the added ethanol to the middle-layer product obtained in step (1) is 2-3:1, preferably 2:1. Preferably or optionally, in step (2), the standing reaction is conducted at an ambient temperature of -17 °C to -23 °C for 25-35 min, preferably at an ambient temperature of -20 °C for 30 min. Preferably or optionally, in step (3), the specific centrifuging conditions are as follows: centrifuging for 25-35 min at 3000-5000 g at 3-5 °C, preferably, the centrifuging conditions are as follows: centrifuging for 30 min at 4000 g at 4 °C. Preferably or optionally, in step (4), the used chromatography column is Cleanert IC-A, and is activated by ultrapure water prior to adsorption. Preferably or optionally, in step (4), the concentration of the used sodium chloride solution is -60 mmol/L. Preferably or optionally, in step (5), the parameters of liquid chromatography in liquid chromatography-mass spectrometry are as follows: analytical column DionexIonPcTMAS19 (2 x
250 mm), protection column DionexlonPackTMAG19 (2 x 50 mm), an electrochemical detector, column temperature: 35 °C; mobile phase A: ultrapure water, mobile phase D: 50 mmol/L NaOH solution; suppressor current: 495 mA, flow rate: 2 mL/min; eluting conditions: 95 % A and 5 % D at0-2min; 80 %Aand20 %Dat25min; 70 %Aand30 %D at45 min; 95 %Aand5 %D at 45.1 min. Preferably or optionally, in step (5), the parameters of mass spectrometry in liquid chromatography-mass spectrometry are as follows: a Q Exactive detector is used for detection, an electric spray ion source is used, a spray voltage is 3.5 kV, a sheath gas pressure is 35 arb, an auxiliary gas pressure is 10 arb, a heating temperature is 350 °C, a capillary temperature is 320 °C, and a collision energy is 30 V, and Full MS/dd MS2 scanning is performed on a 550-2000 m/z substance in a negative ion mode. The method for qualitatively detecting acidic oligosaccharides in human milk provided by the present application can correctly perform qualitative detection on 9 acidic oligosaccharides from a human milk sample through improvement of the extraction process and liquid chromatography and mass spectrometry parameters. Meanwhile, this method also has good repeatability, and is applicable to large-scale analysis work of human milk samples.
Brief Description of the Drawings FIG.1 is a total ion chromatogram of acidic oligosaccharides in a negative ion mode according to a technical solution in Comparative Example 1; FIG. 2 is a total ion chromatogram of acidic oligosaccharides in a negative ion mode according to a technical solution in Example 1; FIG. 3 is a total ion chromatogram of acidic oligosaccharides 3'-SL and 6'-SL in a negative ion mode according to a technical solution in Comparative Example 1; FIG. 4 is a total ion chromatogram of acidic oligosaccharides 3'-SL and 6'-SL in a negative ion mode according to a technical solution in Example 1; FIG. 5 is a secondary mass spectrogram of 6'-SLNFP VI under a technical solution in Example 1; FIG. 6 is a secondary mass spectrogram of 6'-SLNFP VI under a technical solution in Comparative Example 2; FIG. 7 is a secondary mass spectrogram of 6'-SLNFP VI under a technical solution in Comparative Example 3; FIG. 8 is a secondary mass spectrogram of 6'-SLNFP VI under a technical solution in Comparative Example 4; FIG. 9 is a secondary mass spectrogram of 6'-SLNFP VI under a technical solution in Comparative Example 5;
FIG 10 is a secondary mass spectrogram of 3'-SL under a technical solution in Example 1; FIG 11 is a secondary mass spectrogram of 6'-SL under a technical solution in Example 1; FIG 12 is a secondary mass spectrogram of 6'-SLN under a technical solution in Example 1; FIG 13 is a secondary mass spectrogram of LSTc under a technical solution in Example 1; FIG 14 is a secondary mass spectrogram of DSLNT under a technical solution in Example 1; FIG 15 is a secondary mass spectrogram of MSLNnH under a technical solution in Example 1; FIG 16 is a secondary mass spectrogram of MSMFLNnH under a technical solution in Example 1; and FIG 17 is a secondary mass spectrogram of DSMFLNnH under a technical solution in Example 1.
Detailed Description of the Invention Next, specific embodiments of the present application will be described in detail. It should be understood that specific embodiments described herein are only for illustrating and explaining the present application, but not intended to limit the present application. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials, reagents and the like used in the following examples are commercially available. Example 1 This example provides a method for qualitatively detecting acidic oligosaccharides in human milk. 1 mL of human milk sample was taken and centrifuged at 10000 rpm at 4 °C to remove upper-layer fats and a small amount of proteins, a middle-layer product was taken and transferred to a centrifuge tube, absolute ethanol with a volume twice that of the middle-layer product was added into the centrifuge tube, and then the above materials were subjected to standing reaction for 30 min at -20 °C. After the reaction was completed, the reaction product was centrifuged for 30 min at 4000 g at 4 °C, and a supernatant was taken. The supernatant was added in a Cleanert IC-A column that was activated with 10 mL of ultrapure water and then subjected to standing for 10 min, neutral oligosaccharides and lactose were eluted with 5 mL of ultrapure water, then acidic oligosaccharides were eluted with 5 mL of 50 mmol/L sodium chloride solution, followed by collecting an eluent of this part. The collected eluent was analyzed via liquid chromatography-mass spectrometry. Wherein, the parameters of liquid chromatography were as follows: analytical column DionexIonPacTMAS19 (2x250 mm), protection column DionexIonPacTMAG19 (2x50 mm), an electrochemical detector, and column temperature: 35 °C. Mobile phase A: ultrapure water, mobile phase D: 50 mmol/L NaOH solution. Suppressor current: 495 mA, and flow rate: 2 mL/min.
Eluting conditions: 95 % A and 5 % D at 0-2 min; 80 % A and 20 % D at 25 min; 70 % A and % D at 45 min; 95 %A and 5 % D at 45.1 min. The parameters of mass spectrometry were as follows: a Q Exactive detector was used for detection. An electric spray ion source was used, wherein a spray voltage was 3.5 kV, a sheath gas pressure was 35 arb, an auxiliary gas pressure was 10 arb, a heating temperature was 350 °C, a capillary temperature was 320 °C, a collision energy was 30 V, and Full MS/dd MS2 mode scanning was performed on a 550-2000 m/z substance in a negative ion mode. Comparative Example 1 The rest steps in this Comparative Example were the same as those in Example 1 except that the mobile phase of liquid chromatography and elution conditions were different. In this Comparative Example, the mobile phase D selected a 200 mmol/L NaOH solution, and eluting conditions were as follows: 0-25 min, 5 %-20 % D; 25-50 min, 20 %-30 % D; 51-55 min, % D; 55.1-60 min, 5 % D. Effect Example 1 The total ion chromatogram of acidic oligosaccharides in Example 1 and Comparative Example 1 in a negative ion mode was determined. The results are as shown in FIG. 1 and FIG. 2. It can be seen from FIG. 1 that in the technical solution of Comparative Example 1, the acidic oligosaccharides were all eluted within 4-11 min, and therefore different types of acidic oligosaccharides were not separated well. Referring to FIG. 2, in the technical solution of Example 1, different types of acidic oligosaccharides were separated well by specific selection of eluting conditions and mobile phases. To further compare the effect of Example 1 with the effect of Comparative Example 1, acidic oligosaccharides 3'-SL and 6'-SL that are isomers with each other were selected as verification objects. As shown in FIGs. 3-4, under the liquid chromatography conditions of Comparative Example 1, 3'-SL and 6'-SL were not separated, however, 3'-SL and 6'-SL were completely separated after liquid chromatography conditions were optimized (i.e., the technical solution in Example 1), with good effect. Comparative Example 2 The rest steps in this Comparative Example were the same as those in Example 1 except that the collision energy of mass spectrometry was 20 V. Comparative Example 3 The rest steps in this Comparative Example were the same as those in Example 1 except that the collision energy of mass spectrometry was 25 V.
Comparative Example 4 The rest steps in this Comparative Example were the same as those in Example 1 except that the collision energy of mass spectrometry was 35 V. Comparative Example 5 The rest steps in this Comparative Example were the same as those in Example 1 except that the collision energy of mass spectrometry was 40 V. Effect Example 2 The secondary mass spectrograms of acidic oligosaccharides 6'-SLNFP VI in Example 1 and Comparative Examples 2-5 were plotted. The results are as shown in FIGs. 5-9. It can be seen from FIG. 5 that at the collision energy of 30 V (Example 1), the characteristic fragment ions 0,4A2-CO2 (m/z 306) were generated, and other B type, C type and cross-ring fracture fragment ions were generated at the same time, so as to achieve qualitative analysis of acidic oligosaccharide 6'-SLNFP VI, however, this effect cannot be achieved under other collision energies. Further, the optimal collision energies of other 8 acidic oligosaccharides were tested according to whether qualitative analysis can be carried out. The results are shown in Table 1. Table 1 Suitable collision energy of 8 oligosaccharides Number Name Ionization Precursor ion Collision energy mode
Si 3'-SL -H 632.20 20,25,30 S2 6'-SL -H 632.20 20,25,30
S3 6'-SLN -H 673.23 25,30 S4 LSTc -H 997.33 25,30
S6 DSLNT -H 1288.43 30,35 S7 MSLNnH -H 1362.47 30 S8 MSMFLNnH -H 1508.52 30,35
S9 DSMFLNnH -H 1654.58 30,35 It can be seen from Table 1 that qualitative analysis of all 8 acidic oligosaccharides can be achieved at the collision energy of 30 V by combining the optimal collision energies of each acidic oligosaccharide. The secondary mass spectrograms of each acidic oligosaccharide at the collision energy of 30 V are shown in FIGs.10-17. It can be seen from FIGs. 10-17 that at the collision of 30 V, the above 8 oligosaccharides can generate the characteristic fragment ions of this kind of oligosaccharide, and other B type, C type and cross-ring fracture fragment ions are generated at the same time, so as to achieve qualitative analysis of this kind of oligosaccharide.
Effect Example 3 A human milk sample was determined by using the method in Example 1. Qualitative determination results are shown in Table 2. Table 2 Determination results of acidic oligosaccharides in samples
Retention Precursor Characteristic Molecular Ionization Number Name time ion ion formula mode (min) m/z m/z C23H39N Si 3'-SL 11.96 -H 632.20 408,468 019 C23H39N S2 6'-SL 12.98 -H 632.20 306,410 019 C29H42N2 S3 6'-SLN 14.66 -H 673.23 306 019 C37H62N2 S4 LSTc 10.99 -H 997.33 306,572 029 6'-SLNFP C43H72N2 S5 10.08 -H 1143.39 306,572 VI 033 C48H79N3 S6 DSLNT 18.52 -H 1288.43 306,493 037 C51H85N3 S7 MSLNnH 9.92 -H 1362.47 281,306,572 039 MSMFLNn C57H95N3 S8 8.88 -H 1508.52 306,364,572 H 043 DSMFLNn C63H105N S9 8.43 -H 1654.58 306,656,572 H 3047
It can be seen from Table 2 that the method in Example 1 can be used for directionally performing qualitative determination on 9 acidic oligosaccharides in human milk. Further, this sample was repeatedly detected three times by using the method in Example 1, and the average response values and relative standard deviations were compared. The results are shown in Table 3. Table 3 Results of repeated tests
RSD Number Name Molecular formula Abundance (0%)
S1 3'-SL C23H39NO19 52659391 2.86 S2 6'-SL C23H39NO19 33601112 3.53
S3 6'-SLN C29H42N2019 716701 8.82 S4 LSTc C37H62N2029 16428183 5.01 S5 6'-SLNFP VI C43H72N2033 1668656 7.61 S6 DSLNT C48H79N3037 1229645 3.75 S7 MSLNnH C51H85N3039 1117577 5.52 S8 MSMFLNnH C57H95N3043 4281129 7.23 S9 DSMFLNnH C63H105N3047 324133 3.95
It can be seen from Table 3 that the relative standard deviations of acidic oligosaccharides in a human milk sample determined by using the method in Example 1 of the present application was lower. Therefore, this method has good repeatability. The foregoing describes preferred embodiments of the present application in detail. However, the present application is not limited to the specific details of the above embodiments, multiple simple deformations can be made to the technical solution of the present application within the scope of the technical concept of the present application, and these simple deformations are all included within the scope of protection of the present application. In addition, various different embodiments of the present application can be randomly combined, and these combinations should also be considered as the contents disclosed in the present application without prejudice to the idea of the present application.
Industrial practicability The present application provides a method for qualitatively detecting acidic oligosaccharides in human milk. Through improvement on the extraction process and liquid chromatography and mass spectrometry parameters, this method can correctly perform qualitative determination on 9 acidic oligosaccharides in a human milk sample. Meanwhile, this method has good repeatability, and is applicable for large-scale analysis work of human milk samples.
8A
Throughout this specification and the claims which follow, unless the context requires otherwise,
the word "comprise", and variations such as "comprises" and "comprising", will be understood to
imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of
any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to
any matter which is known, is not, and should not be taken as an acknowledgment or admission or
any form of suggestion that that prior publication (or information derived from it) or known matter
forms part of the common general knowledge in the field of endeavour to which this specification
relates.
Claims (6)
1. A method for qualitatively detecting acidic oligosaccharides in human milk, which is successively carried out according to the following steps: (1) Centrifuging human milk to remove upper-layer fats and proteins, and taking a middle layer of the centrifuged product; (2) Adding ethanol to the middle-layer product, and conducting standing reaction; (3) Centrifuging the reaction product and taking a supernatant; (4) Adsorbing the supernatant via a chromatography column, washing the column with ultrapure water, subsequently eluting the column with a sodium chloride solution, and then collecting an eluate; wherein, the used chromatography column is activated by ultrapure water prior to adsorption; the chromatography column is filled with hydrogen carbonate-type strong base anion exchange resin; and (5) Analyzing the eluate via liquid chromatography-mass spectrometry; wherein, the parameters of liquid chromatography in liquid chromatography-mass spectrometry are as follows: analytical column with a size of 2 x 250 mm, protection column with a size of 2 x 50 mm, an electrochemical detector, column temperature: 35 °C; mobile phase A: ultrapure water, mobile phase D: 50 mmol/L NaOH solution; suppressor current: 495 mA, flow rate: 2 mL/min; eluting conditions: 95 % A and 5 %D at 0-2 min; 80 %A and 20 %D at 25 min; 70 % A and 30 %D at min; 95 % A and 5 %D at 45.1 min; and the parameters of mass spectrometry in liquid chromatography-mass spectrometry are as follows: a detector is used for detection, an electric spray ion source is used, a spray voltage is 3.5 kV, a sheath gas pressure is 35 arb, an auxiliary gas pressure is 10 arb, a heating temperature is 350 °C, a capillary temperature is 320 °C, and a collision energy is 30 V, and Full MS/dd MS2 scanning is performed on a 550-2000 m/z substance in a negative ion mode; wherein, the analytical column is an anion exchange column with supermacroporous resin, the ion-exchange group of which is alkanol quaternary ammonium ion; the protection column is an anion exchange column with microporous resin, the ion-exchange group of which is alkanol quaternary ammonium ion; the detector is a detector that combines the parent ion selectivity of a quadrupole with high-resolution accurate mass number orbitrap detection.
2. The method for qualitatively detecting acidic oligosaccharides in human milk according to claim 1, wherein in step (1), the specific centrifuging conditions are as follows: centrifuging for -35 min at 8000-10000 rpm at 3-5 °C, preferably, the centrifuging conditions are as follows: centrifuging for 30 min at 10000 rpm at 4 °C.
3. The method for qualitatively detecting acidic oligosaccharides in human milk according to
claim 1, wherein in step (2), the volume ratio of the added ethanol to the middle-layer product
obtained in step (1) is 2-3:1, preferably 2:1.
4. The method for qualitatively detecting acidic oligosaccharides in human milk according to
claim 1, wherein in step (2), the standing reaction is conducted at an ambient temperature of
-17 °C to -23 °C for 25-35 min, preferably at an ambient temperature of -20 °C for 30 min.
5. The method for qualitatively detecting acidic oligosaccharides in human milk according to
claim 1, wherein in step (3), the specific centrifuging conditions are as follows: centrifuging for
-35 min at 3000-5000 g at 3-5 °C, preferably, the centrifuging conditions are as follows:
centrifuging for 30 min at 4000 g at 4 °C.
6. The method for qualitatively detecting acidic oligosaccharides in human milk according to
claim 1, wherein in step (4), the concentration of the used sodium chloride solution is 40-60
mmol/L.
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| PCT/CN2023/078186 WO2024001248A1 (en) | 2022-06-30 | 2023-02-24 | Method for qualitatively testing acidic oligosaccharides in breast milk |
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Non-Patent Citations (2)
| Title |
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| Gao, X., et al., 'Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis of Human Milk Neutral and Sialylated Free Oligosaccharides Using Girard’s Reagent P On-Target Derivatization'. * |
| Gnoth, M., et al., 'Human Milk Oligosaccharides Are Minimally Digested In Vitro', The Journal of Nutrition, 2000, Vol. 130(12), pages 30144-3020 * |
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