Method for detecting glucosidic bond in polysaccharide structure and application thereof

Abstract

The invention relates to a method for detecting glucosidic bonds in a polysaccharide structure and application thereof, and the method comprises the following steps: carrying out methylation derivation on polysaccharide and a standard substance, then detecting through GC-MS, and calibrating the type of the detected glucosidic bonds by using relative retention time; the calculation formula of the relative retention time is as follows: the relative retention time = the chromatographic peak appearance time of the polysaccharide glycosidic bond / the chromatographic peak appearance time of the standard substance. According to the method for detecting the glycosidic bonds in the polysaccharide structure and the application of the method, the detection result is calibrated through the relative retention time, the'drift 'of a chromatographic peak caused by factors such as an instrument or a chromatographic state is greatly avoided, and the accuracy and reproducibility of polysaccharide structure identification are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide structure analysis, and in particular to a method for detecting glycosidic bonds in a polysaccharide structure and an application thereof. Background Art

[0002] Polysaccharides are a type of long-chain polymer formed by multiple monosaccharide molecules connected by glycosidic bonds. They are a very important type of macromolecule in cells. Carbohydrates are important components of receptor molecules on cell membranes, and are participants in functions such as cell recognition and information transmission. They are a type of non-specific broad-spectrum immunomodulator and an important biological material, and are widely involved in various life activities.

[0003] The biological activity of polysaccharides is closely related to their specific chemical structure. The chemical structure of polysaccharides is the basis of biological activity, and changes in structure directly affect the strength of their biological activity. The identification of polysaccharide structure is the premise for revealing its structure-activity relationship and the mechanism of biological activity, and it is also the basis for further modification and derivatization of polysaccharides to improve their activity and commercial value. However, the complexity of polysaccharide structure brings great difficulties to the structural analysis process, especially the qualitative and quantitative analysis of glycosidic bonds. First, there are many types of monosaccharides, the basic units that make up polysaccharides, including triose, tetrose, pentose, hexose, heptose, etc. Secondly, there are many isomers of sugars in nature, such as glucose, galactose, mannose and fructose. Finally, each monosaccharide can form a variety of glycosidic bonds. Taking glucose as an example, common glycosidic bond types include 1,3-glucose bond, 1,4-glucose bond, 1,6-glucose bond, 1,3,6-glucose bond and 1,4,6-glucose bond. In order to analyze the sites of glycosidic bonds in polysaccharide molecules, chemical methods were commonly used in the early days: periodic acid oxidation and Smith degradation, and the possible glycosidic bond connection mode was indirectly inferred based on the reaction products. Due to the long reaction time, poor reproducibility, and the large impact of experimental operation standardization on the accuracy of the results, chemical analysis has become an alternative or auxiliary verification method.

[0004] In recent years, with the popularization of gas chromatography-mass spectrometry (GC-MS), instrumental analysis of polysaccharide structure has gradually become the mainstream, and the analysis method combining chemical method with instrumental method has made up for the shortcomings of single means to detect polysaccharide structure. After chemical derivatization, the sample mass spectrum can be analyzed by GC-MS to identify the structure such as glycosidic bond type, sugar chain sequence and sugar ring form. With the increase of complexity of natural polysaccharides, the mass spectra of some monosaccharide isomers (such as glucose and galactose) are very similar, resulting in deviations in the results of qualitative and quantitative analysis of glycosidic bonds by GC-MS and reduced accuracy. Therefore, it is considered to improve the accuracy of GC-MS identification of glycosidic bonds by comparing mass spectra combined with gas chromatography elution time (RT). However, changes in instrument status and chromatographic conditions will affect RT. Even for the same instrument and the same chromatographic parameters, the RT of the same glycosidic bond will also slightly "drift" as the instrument (especially the chromatographic column) ages. This has brought great interference to the further analysis of polysaccharide structure (such as the connection order of glycosidic bonds, etc.).

[0005] Therefore, how to accurately identify the polysaccharide structure has become an important limiting factor in the development and utilization of polysaccharide resources. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for detecting glycosidic bonds in polysaccharide structures and its application. The detection results are calibrated by relative retention time, which greatly avoids the "drift" of chromatographic peaks caused by factors such as instruments or chromatographic states, and greatly improves the accuracy and reproducibility of polysaccharide structure identification.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for detecting glycosidic bonds in a polysaccharide structure, the method comprising the following steps: methylating and derivatizing a polysaccharide and a standard, and then detecting the polysaccharide and a standard by GC-MS, and calibrating the type of glycosidic bonds detected by relative retention time;

[0009] The calculation formula of the relative retention time is: the chromatographic peak elution time of the polysaccharide glycosidic bond / the chromatographic peak elution time of the standard.

[0010] The present invention creatively uses relative retention time to calibrate the detected glycosidic bond types, which can minimize the interference of external factors such as instrument status and chromatographic conditions on the results, reduce the impact of chromatographic peak "drift", and can effectively separate glycosidic bonds of different isomers, greatly improving the accuracy and reproducibility of glycosidic bond detection in polysaccharides.

[0011] Preferably, the glycosidic bond type is determined based on the characteristics of the mass spectrum.

[0012] Preferably, the calibration step includes: comparing the relative retention time of the test product with the relative retention time of the sample with known structure; judging whether they have the same glycosidic bond according to the difference between the relative retention time of the test product and the relative retention time of the sample with known structure.

[0013] Preferably, the calibration standard for determining the same glycosidic bond is that the relative retention time of the test product differs from the relative retention time of the sample with a known structure by 0-0.1 min, for example, 0 min, 0.02 min, 0.04 min, 0.06 min, 0.08 min or 0.1 min.

[0014] Preferably, the molar ratio of the glycosidic bonds of the polysaccharide is calculated by the relative peak area of ​​the chromatographic peak.

[0015] Preferably, the calculation formula of the molar ratio of the glycosidic bonds is:

[0016] Glycosidic bond molar ratio (%) = (relative molar amount / total relative molar amounts of each component) × 100.

[0017] Preferably, the calculation formula of the relative molar amount is: relative molar amount=chromatographic peak area / derivative molecular weight.

[0018] Preferably, the polysaccharides are divided into neutral polysaccharides and acidic polysaccharides according to the total amount of uronic acid in the polysaccharides.

[0019] Preferably, the neutral polysaccharide comprises a polymer having a total amount of uronic acid less than 10%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%.

[0020] Preferably, the acidic polysaccharide comprises a polymer having a total amount of uronic acid of not less than 10%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0021] Preferably, the methylation includes partial methylation and complete methylation.

[0022] Preferably, the source of the polysaccharide includes any one of plants, animals or microorganisms, or a combination of at least two of them.

[0023] Preferably, the methylation derivatization step comprises:

[0024] The neutral polysaccharide or standard is dissolved, a methylation reagent is added for methylation, and a glycosidic bond hydrolysis reagent is added for reduction and acetylation.

[0025] Preferably, the concentration of the neutral polysaccharide after dissolution is 0.2-4 mg / mL, for example, it can be 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL or 4 mg / mL.

[0026] Preferably, the dissolving solvent comprises a combination of sodium hydroxide and dimethyl sulfoxide.

[0027] Preferably, the concentration of the solvent is 0.1-1 g / mL, for example, it can be 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 0.6 g / mL, 0.7 g / mL, 0.8 g / mL, 0.9 g / mL or 1 g / mL, etc.

[0028] Preferably, the methylating agent comprises methyl iodide.

[0029] Preferably, the final concentration of the methylation reagent is 10-40%, for example, 10%, 15%, 20%, 25%, 30%, 35% or 40%, etc.

[0030] Preferably, the glycosidic bond hydrolyzing agent comprises any one of trifluoroacetic acid or formic acid.

[0031] Preferably, the final concentration of the glycosidic bond hydrolyzing agent is 1-4 M, for example, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M or 4 M.

[0032] Preferably, the reducing reagent includes any one of or ammonia water.

[0033] Preferably, the final concentration of the reducing agent is 0.2-2M, for example, 0.2M, 0.5M, 1M, 1.5M or 2M.

[0034] Preferably, the reagent for the acetylation reaction comprises acetic anhydride.

[0035] Preferably, the final concentration of the reagent for the acetylation reaction is 90-99.9%, for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, etc.

[0036] Preferably, the temperature of the acetylation reaction is 80-120°C, for example, it can be 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C or 120°C.

[0037] Preferably, the acetylation reaction time is 1-3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h or 3 h.

[0038] Preferably, the acidic polysaccharide is pretreated before methylation derivatization, and the pretreatment step includes: dissolving the acidic polysaccharide, dividing the sample into two equal parts, adding a first reducing agent and a second reducing agent respectively, dialyzing and drying to obtain two pretreated products.

[0039] The present invention creatively adopts different methylation derivatizations according to the different properties of polysaccharides. In the present invention, the acidic polysaccharide is pretreated and reduced by two reducing agents, which can significantly improve the accuracy of detection.

[0040] Preferably, the final concentration of the acidic polysaccharide is 0.5-8 mg / mL, for example, it can be 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL or 8 mg / mL.

[0041] Preferably, the dissolving solvent is deionized water.

[0042] Preferably, the first reducing agent comprises sodium borohydride.

[0043] Preferably, the final concentration of the first reducing agent is 0.5-6M, for example, it can be 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, 5.5M or 6M, etc.

[0044] Preferably, the second reducing agent comprises sodium borodeuteride.

[0045] Preferably, the final concentration of the second reducing agent is 0.5-6M, for example, it can be 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, 5.5M or 6M, etc.

[0046] Preferably, the dialysis time is 2-48 h, for example, it can be 2 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h or 48 h.

[0047] Preferably, the standard comprises any one of an internal standard or an external standard.

[0048] Preferably, the internal standard comprises any one of inositol, n-octane, n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane, n-tetracosane, n-hexacosane, n-octacosane, n-triacontane, n-dotriacontane, n-tetratriacontane, n-hexatriacontane, n-octatriacontane or n-tetracontane, or a combination of at least two thereof.

[0049] Preferably, the external standard comprises any one or a combination of at least two of inositol, n-octane, n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane, n-tetracosane, n-hexacosane, n-octacosane, n-triacontane, n-dotriacontane, n-tetratriacontane, n-hexatriacontane, n-octacosane or n-tetracontane.

[0050] The present invention creatively adopts inositol as a standard substance. Inositol is a cyclohexanehexol, the structure of which is similar to that of polysaccharides and is easy to identify. It can be used as an internal standard or an external standard for detection.

[0051] Preferably, the purity of the standard is 98-100%, for example, it can be 98%, 98.5%, 99%, 99.5% or 100%.

[0052] Preferably, the final concentration of the standard is 0.1-2 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL or 2 mg / mL.

[0053] Preferably, the internal standard is mixed with the polysaccharide and then subjected to GC-MS.

[0054] Preferably, the external standard and the polysaccharide are subjected to GC-MS independently.

[0055] Preferably, the polysaccharide is purified before detection.

[0056] Preferably, the purification operation includes any one or two operations of protein removal, pigment removal or fat removal.

[0057] The object involved in the present invention is a purified polysaccharide with high sugar content and low impurity content, which has little interference with polysaccharide structural analysis and is suitable for structural analysis of polysaccharides from most sources (such as plant polysaccharides, animal polysaccharides or microbial polysaccharides).

[0058] Preferably, the polysaccharide purity after purification is not less than 40%, for example, it can be 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0059] Preferably, the injection volume of the GC-MS is 0.5-5 μL, for example, it can be 0.5 μL, 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL or 5 μL.

[0060] Preferably, the detection temperature of the GC-MS is 100-300°C, for example, it can be 100°C, 150°C, 200°C, 250°C or 300°C.

[0061] Preferably, the carrier gas of the GC-MS comprises helium.

[0062] The present invention creatively adopts helium as the carrier gas of GC-MS. As a protective gas, helium has a relatively stable property, is easy to separate from other molecular components, has an enrichment effect on the sample, and has simple ion fragmentation and does not interfere with the detection results.

[0063] Preferably, the mass scanning range of the GC-MS is 30-350 m / z, for example, 30-300 m / z, 50-350 m / z, 100-350 m / z, etc.

[0064] In a second aspect, the present invention provides an application of the method for detecting glycosidic bonds in a polysaccharide structure described in the first aspect to identifying glycosidic bonds in polysaccharides.

[0065] Compared with the prior art, the present invention has at least the following beneficial effects:

[0066] 1. The object of the present invention is purified polysaccharides, which have high sugar content and low impurity content, have little interference with polysaccharide structural analysis, and are suitable for structural analysis of polysaccharides from most sources (such as plant polysaccharides, animal polysaccharides or microbial polysaccharides).

[0067] 2. The present invention corrects the elution time of the chromatographic peak by an internal standard or an external standard, thereby obtaining the relative retention time of the elution peaks of different glycosidic bonds, which greatly avoids the "drift" of the chromatographic peak caused by factors such as the instrument or chromatographic state, and greatly improves the accuracy and reproducibility of polysaccharide structure identification.

[0068] 3. The present invention adopts a combination of chemical method and instrumental method to make up for the defects of detecting polysaccharide structure by a single method, and can reduce the difficulty of polysaccharide structure analysis while improving the sensitivity of glycosidic bond detection.

[0069] 4. The polysaccharide spectra corresponding to glycosidic bonds of different types or connection modes are different, and the relative retention times are also different, that is, the characteristic spectra of polysaccharides correspond to their structures one by one. Therefore, by collating the spectra information of glycosidic bonds of different types or connection modes into a database, and using software to screen and match the database, the automated and rapid identification of batches of unknown polysaccharide structures can be achieved, which greatly facilitates the analysis of polysaccharide structures and has broad commercial value and guiding significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the chromatogram result of neutral plant polysaccharide A in Example 1.

[0071] Figure 2 This is the chromatogram result of neutral microbial polysaccharide B in Example 2.

[0072] Figure 3This is the chromatogram result of the external standard inositol in Example 3.

[0073] Figure 4 This is the chromatogram result of neutral polysaccharide C in Example 3.

[0074] Figure 5 This is the chromatogram result of acidic plant polysaccharide D of Example 4 (hydrogen substitution).

[0075] Figure 6 This is the chromatogram result of acidic plant polysaccharide D in Example 4 (deuterated).

[0076] Figure 7 This is the chromatogram result of the external standard normal alkane in Example 5.

[0077] Figure 8 This is the chromatogram result of neutral plant polysaccharide A and internal standard normal alkane in Example 6.

[0078] Fig. 9 This is the chromatogram result of neutral plant polysaccharide A in Example 7.

[0079] Fig.10 This is the chromatogram result of neutral plant polysaccharide A in Example 8.

[0080] Fig.11 This is the chromatogram result of neutral microbial polysaccharide B in comparative example 1. DETAILED DESCRIPTION

[0081] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0082] Sources of reagents used in the following examples:

[0083] Normal alkanes: from Anpu Laboratory, item number: CDAA-M-690077-BA-1ml

[0084] Neutral plant polysaccharide A: derived from Coptis chinensis.

[0085] Neutral microbial polysaccharide B: derived from Flammulina velutipes polysaccharide.

[0086] Neutral animal polysaccharide C: derived from Chinese round field snail.

[0087] Acidic plant polysaccharide D: derived from passion fruit peel.

[0088] The extraction and preparation methods of the above-mentioned polysaccharides A, B, C and D are the same, all of which adopt water extraction and alcohol precipitation to obtain crude polysaccharides, followed by impurity removal (pigment removal, protein removal and defatting) to improve the purity of the polysaccharides, and then DEAE ion purification and gel molecular sieve purification are used respectively to obtain the corresponding neutral plant polysaccharide A, neutral microbial polysaccharide B, neutral animal polysaccharide C and acidic plant polysaccharide D. The purity of the polysaccharides after purification is not less than 40%.

[0089] Example 1

[0090] This embodiment provides a method for detecting glycosidic bonds in polysaccharide structures

[0091] Use 0.1g / mL NaOH / DMSO reagent to dissolve neutral plant polysaccharide A to a concentration of 1.5mg / mL, dissolve inositol as an internal standard to a concentration of 1.5mg / mL, add iodomethane to a concentration of 20% to completely methylate the free hydroxyl groups, add trifluoroacetic acid to the methylated product to a concentration of 2.5M to hydrolyze the glycosidic bonds, then add NaBD4 to a concentration of 0.8M for reduction reaction, and finally add acetic anhydride to a concentration of 95% and react at 100°C for 3h for acetylation reaction to obtain partially methylated sugar alcohol acetyl derivatives.

[0092] Then GC-MS detection was performed, and the detection conditions were: injection volume 0.5 μL, detection temperature 100°C to 300°C, carrier gas was high-purity helium, and mass scanning range (m / z): 30 to 350.

[0093] The chromatogram of neutral plant polysaccharide A is shown in Figure 1 The relative retention time △RT and molar percentage of each glycosidic bond were calculated according to the sample chromatogram, and the type of glycosidic bond was analyzed according to the corresponding mass spectrum. The results are shown in Table 1.

[0094] Table 1

[0095]

[0096]

[0097] Figure 1 The chromatographic peak at the peak time of 24.529 min is the chromatographic signal after the internal standard inositol is derivatized. As shown in Table 1, the glycosidic bond of neutral plant polysaccharide A is mainly 4-Glc(p), with a molar percentage of more than 70%, and also contains part of 6-Gal(p), t-Glc(p), 4,6-Glc(p) and a small amount of 2,6-Gal(p), t-Fuc(p) and 6-Glc(p).

[0098] Example 2

[0099] This embodiment provides a method for detecting glycosidic bonds in polysaccharide structures

[0100] Use 1g / mL NaOH / DMSO reagent to dissolve neutral microbial polysaccharide B to a concentration of 0.5mg / mL, dissolve inositol as an external standard to a concentration of 2mg / mL, add iodomethane to a concentration of 25% to completely methylate the free hydroxyl groups, add trifluoroacetic acid to a concentration of 1M to the methylated product to hydrolyze the glycosidic bonds, then add NaBD4 to a concentration of 1.5M for reduction reaction, and finally add acetic anhydride to a concentration of 90%, and finally add acetic anhydride to react at 120°C for 2.5h for acetylation reaction to obtain partially methylated sugar alcohol acetyl derivatives.

[0101] Then, GC-MS detection was performed under the following conditions: injection volume 1 μL, detection temperature 100°C to 300°C, carrier gas was high-purity helium, and mass scanning range (m / z): 30 to 350.

[0102] Table 2

[0103] Glycosidic bond type Peak time (min) Relative retention time ∆RT Peak area Mole percentage (%) t-Rha(p) 6.518 0.266 3432614 8.53 t-Man(p) 8.298 0.339 1054373 2.62 6-Man(p) 12.967 0.530 6863803 17.06 6-Gal(p) 14.793 0.604 25487647 63.35 2,6-Gal(p) 18.899 0.772 3397091 8.44 Inositol 24.476 1.000 / /

[0104] Figure 2 This is the chromatogram result of neutral microbial polysaccharide B. Figure 3 The chromatographic peak at the peak time of 24.476min is the chromatographic signal after the external standard inositol is derivatized. As can be seen from Table 2, the glycosidic bond of neutral microbial polysaccharide B is mainly 6-Gal(p), with a molar percentage of more than 60%, and also contains part of 6-Man(p), t-Rha(p), 2,6-Gal(p) and t-Man(p). Combined with Table 1 in Example 1, it can be seen that although the peak time deviation of 6-Gal(p) under different conditions is large (the difference is 0.262min), the relative retention time difference after calibration is small (the difference is 0.01). It shows that the method for identifying the glycosidic bond of polysaccharides has high accuracy and good reproducibility.

[0105] Example 3

[0106] This embodiment provides a method for detecting glycosidic bonds in polysaccharide structures

[0107] Use 0.5g / mL NaOH / DMSO reagent to dissolve neutral animal polysaccharide C to a concentration of 3.5mg / mL, dissolve inositol as an external standard to a concentration of 2mg / mL, add iodomethane to a concentration of 40% to completely methylate the free hydroxyl groups, add formic acid to the methylated product to a concentration of 4M to hydrolyze the glycosidic bonds, then add NaBD4 to a concentration of 2M for reduction reaction, and finally add acetic anhydride to a concentration of 98% and react at 80°C for 2h for acetylation reaction to obtain partially methylated sugar alcohol acetyl derivatives.

[0108] Then GC-MS detection was performed, and the detection conditions were: injection volume 2 μL, detection temperature 100°C to 300°C, carrier gas was high-purity helium, and mass scanning range (m / z): 30 to 350.

[0109] Table 3

[0110] Glycosidic bond type Peak time (min) Relative retention time ∆RT Peak area Mole percentage (%) t-Glc(p) 8.56 0.350 3826850 14.33 4-Glc(p) 13.667 0.559 20926034 72.12 3,4-Glc(p) 15.787 0.646 551798 1.76 4,6-Glc(p) 17.855 0.731 3475143 11.09 3,4,6-Glc(p) 20.255 0.829 234570 0.70 Inositol 24.432 1.000 / /

[0111] Figure 3 The chromatographic peak at the peak time of 24.432 min is the chromatographic signal after the external standard inositol is derivatized. Figure 4 It is the chromatogram of neutral animal polysaccharide C. As can be seen from Table 3, the glycosidic bond of neutral animal polysaccharide C is mainly 4-Glc(p), with a molar percentage of more than 70%, and it also contains some t-Glc(p), 4,6-Glc(p) and a small amount of 3,4-Glc(p), 3,4,6-Glc(p). Combined with Table 1 in Implementation Case 1, it can be seen that although the peak time deviation of 4,6-Glc(p) under different conditions is large (the difference is 0.039min), the relative retention time difference after calibration is small (the difference is 0.01). It shows that this method has high accuracy and good reproducibility in identifying the glycosidic bonds of polysaccharides.

[0112] Example 4

[0113] This embodiment provides a method for detecting glycosidic bonds in polysaccharide structures

[0114] Acidic plant polysaccharide D was dissolved in deionized water to a concentration of 8 mg / mL, and the sample was divided into two equal parts. NaBH4 (also known as hydrogen substitution) was added to a concentration of 4 M and NaBD4 (also known as deuterium substitution) was added to a concentration of 2.5 M to reduce uronic acid, followed by dialyzation and freeze-drying.

[0115] Use 0.1g / mL NaOH / DMSO reagent to dissolve the above hydrogenated and deuterated lyophilized products to a concentration of 6mg / mL, dissolve inositol to a concentration of 1.5mg / mL, add iodomethane to a concentration of 30% to completely methylate the free hydroxyl groups, add trifluoroacetic acid to a concentration of 2.5M to the methylated product to hydrolyze the glycosidic bonds, then add NaBD4 to a concentration of 0.5M for reduction reaction, and finally add acetic anhydride to a concentration of 99% and react at 100°C for 3h for acetylation reaction to obtain partially methylated sugar alcohol acetyl derivatives.

[0116] Then, GC-MS detection was performed under the following conditions: injection volume 5 μL, detection temperature 100°C to 300°C, carrier gas was high-purity helium, and mass scanning range (m / z): 30 to 350.

[0117] Table 4

[0118]

[0119] The chromatographic peak at 24.515 min is the chromatographic signal of the external standard inositol after derivatization. As shown in Table 4, the glycosidic bond of acidic plant polysaccharide D is mainly 4-Gal(p)-UA, with a molar percentage of more than 80%, and also contains some t-Gal(p)-UA, a small amount of 2,4-Rha(p), t-Ara(f) and 3,6-Gal(p). Figure 5 Hydrogen peak chromatogram and Figure 6 In the deuterated elution chromatogram, although there are deviations in the elution time of some glycosidic bonds (for example, the elution time of 4-Gal(p)-UA in the hydrogen elution chromatogram and the deuterated elution chromatogram differs by 0.011), the relative retention time after calibration is almost the same (the difference is less than 0.001). This shows that the method has high accuracy and good reproducibility in identifying the glycosidic bonds of polysaccharides.

[0120] Example 5

[0121] This example provides a method for detecting glycosidic bonds in polysaccharide structures. The only difference between this method and Example 2 is that normal alkanes are used as standard substances. The rest are the same as Example 2. GCMS detection is performed. The chromatogram of the standard normal alkane detection is as follows: Figure 7 As shown in the figure, under the same detection conditions, the peak shape of normal alkanes is abnormal, with severe tailing, making it difficult to calculate and analyze. The results show that it is difficult to use standard normal alkanes as external standards for accurate data calibration.

[0122] Example 6

[0123] This example provides a method for detecting glycosidic bonds in polysaccharide structures. The difference between this example and Example 1 is that normal alkanes are used. The normal alkanes contain 17 even-numbered normal alkanes mixed standard (C8-C40) as standard products. The rest is the same as Example 1. GCMS detection is performed, and the chromatogram is as follows: Figure 8 As shown in the figure, the use of n-alkane standard as internal standard will interfere with the normal peaking of sugar structure in the sample, resulting in abnormal peak shape of chromatographic peak, severe tailing, and difficulty in calculation and analysis. The results show that it is difficult to use standard n-alkane as internal standard for accurate data calibration.

[0124] Example 7

[0125] This embodiment provides a method for detecting glycosidic bonds in polysaccharide structures. The difference between this embodiment and embodiment 1 is that the reaction temperature for adding acetic anhydride is 50°C, and the rest is the same as embodiment 1. GCMS detection chromatogram is as follows Fig. 9 As shown, the response values ​​of the sample and internal standard are weakened, the baseline noise fluctuation is enhanced, the sample spectral signal and quality are deteriorated, and it is difficult to calculate and analyze.

[0126] The results show that too low a temperature will cause abnormal sample chromatograms, making them difficult to detect and analyze.

[0127] Example 8

[0128] This embodiment provides a method for detecting glycosidic bonds in polysaccharide structures. The difference between this embodiment and embodiment 1 is that the reaction temperature for adding acetic anhydride is 180°C, and the rest is the same as embodiment 1. GCMS detection chromatogram is as follows Fig.10 As shown, too high a temperature causes sample degradation, so the sample response value becomes weak and difficult to calculate and analyze.

[0129] The results show that too high a temperature will cause sample degradation and make analysis impossible.

[0130] Comparative Example 1

[0131] This comparative example provides a method for detecting glycosidic bonds in polysaccharide structures. The difference between this method and Example 2 is that the relative retention time is not calculated, and only the gas chromatography peak time is used for judgment. The rest is the same as Example 2. The chromatogram of the external standard inositol is as follows: Fig.11 As shown, the peak time of inositol is 23.18 min, which is 1.296 min different from the peak time of inositol in Example 2. Judging by the peak time of gas chromatography, the peak time difference is large, and the glycosidic bond of the sample is difficult to determine.

[0132] In summary, the present invention provides a method for detecting glycosidic bonds in polysaccharide structures and its application, which calibrates the detection results by relative retention time, greatly avoiding the "drift" of chromatographic peaks caused by factors such as instruments or chromatographic states, and greatly improving the accuracy and reproducibility of polysaccharide structure identification.

[0133] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting glycosidic bonds in a polysaccharide structure, characterized in that: The method comprises the following steps: methylating and derivatizing the polysaccharide and the standard, and then detecting them by GC-MS, and calibrating the detected glycosidic bond type by relative retention time; The calculation formula of the relative retention time is: relative retention time = chromatographic peak elution time of polysaccharide glycosidic bond / chromatographic peak elution time of standard product.

2. The method according to claim 1, characterized in that The glycosidic bond type is determined based on the characteristics of the mass spectrum; Preferably, the calibration step includes: comparing the relative retention time of the test product with the relative retention time of the sample with known structure; judging whether they have the same glycosidic bond according to the difference between the relative retention time of the test product and the relative retention time of the sample with known structure; Preferably, the calibration has the same glycosidic bond judgment standard as follows: the relative retention time of the test product differs from the relative retention time of the sample with a known structure by 0-0.1 min.

3. The method according to claim 1 or 2, characterized in that: The molar ratio of the glycosidic bonds of the polysaccharide is calculated by the relative peak area of ​​the chromatographic peak; Preferably, the calculation formula of the molar ratio of the glycosidic bonds is: Glycosidic bond molar ratio (%) = (relative molar amount / total relative molar amount of each component) × 100; Preferably, the calculation formula of the relative molar amount is: relative molar amount=chromatographic peak area / derivative molecular weight.

4. The method according to any one of claims 1 to 3, characterized in that The polysaccharide includes neutral polysaccharide and acidic polysaccharide; Preferably, the neutral polysaccharide comprises a polymer having a total uronic acid content of less than 10%; Preferably, the acidic polysaccharide comprises a polymer having a total amount of uronic acid not less than 10%; Preferably, the methylation includes partial methylation and complete methylation; Preferably, the source of the polysaccharide includes any one of plants, animals or microorganisms, or a combination of at least two of them.

5. The method according to any one of claims 1 to 4, characterized in that The methylation derivatization step comprises: Dissolve the neutral polysaccharide or standard, add a methylation reagent for methylation, add a glycosidic bond hydrolysis reagent for reduction and acetylation; Preferably, the concentration of the neutral polysaccharide after dissolution is 0.2-4 mg / mL; Preferably, the solvent is an aqueous solution containing sodium hydroxide and / or dimethyl sulfoxide; Preferably, the concentration of the solvent is 0.1-1 g / mL; Preferably, the methylation reagent comprises methyl iodide; Preferably, the final concentration of the methylation reagent is 10-40%; Preferably, the glycosidic bond hydrolysis reagent includes any one of trifluoroacetic acid or formic acid; Preferably, the final concentration of the glycosidic bond hydrolysis reagent is 1-4M; Preferably, the reducing reagent includes any one of sodium borodeuteride or ammonia water; Preferably, the final concentration of the reducing agent is 0.2-2M; Preferably, the reagent for the acetylation reaction comprises acetic anhydride; Preferably, the final concentration of the reagent for the acetylation reaction is 90-99.9%; Preferably, the temperature of the acetylation reaction is 80-120°C; Preferably, the acetylation reaction time is 1-3 hours.

6. The method according to any one of claims 1 to 5, characterized in that The acidic polysaccharide is pretreated before methylation derivatization, and the pretreatment steps include: dissolving the acidic polysaccharide, treating two portions of the acidic polysaccharide solution with a first reducing agent and a second reducing agent respectively, and dialyzing and drying to obtain two portions of pretreated products; Preferably, the final concentration of the acidic polysaccharide is 0.5-8 mg / mL; Preferably, the dissolving solvent is deionized water; Preferably, the first reducing agent comprises sodium borohydride; Preferably, the final concentration of the first reducing agent is 0.5-6M; Preferably, the second reducing agent comprises sodium borodeuteride; Preferably, the final concentration of the second reducing agent is 0.5-6M; Preferably, the dialysis time is 2-48 hours.

7. The method according to any one of claims 1 to 6, characterized in that The standard substance includes any one of an internal standard or an external standard; Preferably, the internal standard comprises any one or a combination of at least two of inositol, n-octane, n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane, n-tetracosane, n-hexacosane, n-octacosane, n-triacontane, n-dotriacontane, n-tetratriacontane, n-hexatriacontane, n-octacosane or n-tetracontane; Preferably, the external standard comprises any one or a combination of at least two of inositol, n-octane, n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane, n-tetracosane, n-hexacosane, n-octacosane, n-triacontane, n-dotriacontane, n-tetratriacontane, n-hexatriacontane, n-octacosane or n-tetracontane; Preferably, the purity of the standard is 80-100%; Preferably, the final concentration of the standard is 0.1-2 mg / mL; Preferably, the internal standard is mixed with the polysaccharide and then subjected to GC-MS detection; Preferably, the external standard and the polysaccharide are detected independently by GC-MS.

8. The method according to claims 1-7, characterized in that: The polysaccharide is purified before detection; Preferably, the purification operation includes any one or two operations of protein removal, pigment removal or fat removal; Preferably, the polysaccharide has a purity of not less than 40% after purification.

9. The method according to claims 1-8, characterized in that The injection volume of the GC-MS is 0.5-5 μL; Preferably, the detection temperature of the GC-MS is 100-300°C; Preferably, the carrier gas of the GC-MS comprises helium; Preferably, the mass scanning range of the GC-MS is 30-350 m / z.

10. Use of the method for detecting glycosidic bonds in a polysaccharide structure according to any one of claims 1 to 9 in identifying glycosidic bonds in polysaccharides.

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