A method for analyzing monosaccharide composition in polysaccharides

Abstract

The present invention discloses a method for analyzing the composition of monosaccharides in polysaccharides. Trifluoroacetic acid is added to the polysaccharide for hydrolysis to generate monosaccharides; the monosaccharides are dissolved in an organic solvent and then sodium hydroxide is added for alkalization to obtain alkalized monosaccharides; iodomethane is added to the alkalized monosaccharides for methylation to obtain methylated sugars, which are then analyzed by GC-MS. The entire polysaccharide processing process has mild conditions, simple operation, and is environmentally friendly. The analytical method proposed in the present invention can analyze the composition of ten monosaccharides in polysaccharides. It can be used not only for the analysis of aldoses, but also for the analysis of ketoses (fructose), with a clear chemical reaction equation; the analysis has high sensitivity and can complete qualitative and quantitative analysis of monosaccharides below 10μg.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide analysis, and particularly relates to a method for analyzing the monosaccharide composition in polysaccharides. Background Art

[0002] Sugars are important organic compounds found widely in nature and have garnered significant attention for their biological activities, including anti-tumor, antioxidant, and immune-enhancing properties. However, their structural characteristics, high polarity, and strong water solubility make them challenging to analyze qualitatively and quantitatively. Monosaccharide composition is a crucial component of primary polysaccharide structural research. Studying monosaccharide composition is crucial for understanding the structural characteristics, physicochemical properties, and structure-activity relationships of polysaccharides.

[0003] Traditional methods for analyzing the monosaccharide composition of Polygonatum sibiricum polysaccharides primarily utilize the reaction of a pre-column derivatization reagent (PMP) with the monosaccharides after polysaccharide hydrolysis, followed by separation and determination using HPLC (high-performance liquid chromatography). This method is simple, rapid, efficient, and sensitive, with superior sensitivity to the commonly used differential index detector (RID) and evaporative light scattering (ELSD) detectors in liquid chromatography. This method has the advantages of simplicity, practicality, good reproducibility, high sensitivity, and high throughput. Its principle is as follows:

[0004]

[0005] However, this method has the following shortcomings: 1. It is only applicable to the analysis of aldoses, not ketoses; 2. The sensitivity of the analysis is not high, and it can only achieve qualitative and quantitative analysis of about 100 μg of monosaccharides that must contain aldehyde groups.

[0006] Another approach is to use ion chromatography-pulsed amperometric detection to determine the sugar content in the sample. This method can determine the sugar content in the sample without derivatization. This method has high sensitivity, simple operation, and good separation. However, there are limitations on fluidity during ion chromatography determination. The fluidity needs to be an alkaline solution, electrochemically inert, and with a small background current under the working voltage. However, fructose and glucose are diastereomers, and keto-enol interconversion will occur under alkaline conditions. As a result, fructose will be converted into glucose during pre-column derivatization of monosaccharides, thereby interfering with the quantitative and qualitative determination of these two sugars.

[0007] There is also a gas chromatography (GC) method for determination. This method uses the fact that GC is very sensitive to the acetylation reaction of sugar nitrile derivatives. Acetic anhydride is used to generate sugar nitrile derivatives for polysaccharide analysis. The reaction principle of sugar nitrile derivatives is as follows:

[0008]

[0009] Using this method, researchers have established a gas chromatography method for determining the soluble and insoluble monosaccharides and total non-starch polysaccharides in foods with a fat content of less than 5%. They also used flame ionization on a wide-bore capillary column to detect acetonitrile sugar alcohol derivatives. This method is accurate but only suitable for the analysis of aldoses, not ketoses like fructose.

[0010] Based on the above problems, it is urgent to propose a widely used analysis method for monosaccharide components in polysaccharides. Summary of the Invention

[0011] In order to solve the above problems, the present invention discloses a method for analyzing the monosaccharide composition in polysaccharides, which comprises the following steps:

[0012] Trifluoroacetic acid is added to the polysaccharide to hydrolyze it into monosaccharides;

[0013] Dissolving the monosaccharide in an organic solvent and then adding sodium hydroxide for alkalization to obtain an alkalized monosaccharide;

[0014] adding methyl iodide to the alkalized monosaccharide to carry out a methylation reaction to obtain methylated sugar;

[0015] The methylated sugars were analyzed using GC-MS.

[0016] Furthermore, analyzing the methylated sugars using GC-MS comprises the following steps:

[0017] GC-MS was used to analyze various monosaccharide standards, and a standard curve of the chromatographic peak area versus concentration of each monosaccharide was drawn;

[0018] Determine the monosaccharide composition and configuration in the polysaccharide based on the GC-MS results of the monosaccharide standards and the GC-MS results of the methylated sugars;

[0019] The peak area in the polysaccharide sample was calculated according to the standard curve of each monosaccharide to determine the content of each monosaccharide component in the polysaccharide.

[0020] Furthermore, the feeding ratio of the polysaccharide and trifluoroacetic acid is 0.1-5 mL of 2 mol / L trifluoroacetic acid for every 20 mg of polysaccharide.

[0021] Furthermore, the hydrolysis conditions are: reaction for 6 hours under sealed conditions and a temperature of 100°C.

[0022] Furthermore, the organic solvent is acetone, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, etc. or dimethyl sulfoxide.

[0023] Furthermore, the mass ratio of the sodium hydroxide to the polysaccharide is 1:1-6.

[0024] Furthermore, the alkalization condition is: alkalization at 35° C. for 30 minutes.

[0025] Furthermore, the amount of iodomethane added is 1 mL of CH3I per 20 mg of polysaccharide.

[0026] Furthermore, the methylation reaction conditions are: sealing, avoiding light, and reacting for 12 hours at a temperature of 35°C.

[0027] Furthermore, the test conditions of the GC-MS are:

[0028] BR-17 flexible capillary column (0.25 μm × 250 μm × 30 m); carrier gas: high-purity helium (purity ≥ 99.999%), high-purity nitrogen (purity ≥ 99.999%); column flow rate: 1.0 mL / min; splitless injection was used, and the inlet temperature was 250°C; temperature program conditions: initial temperature of 50°C, increased to 100°C at a rate of 25°C / min, held for 3 min, and then increased to 184°C at a rate of 4°C / min;

[0029] EI ion source, electron energy 70 eV; ion source temperature 220°C; transfer line temperature 280°C; quadrupole temperature 40°C; full scan mode, scan range m / z 40-600; mass spectrum retrieval standard library: NIST 11.L.

[0030] Beneficial effects of the present invention:

[0031] The present invention hydrolyzes polysaccharides into monosaccharides, dissolves them in dimethyl sulfoxide, alkalizes them, and then completely methylates them and analyzes them by GC-MS, providing a new method for determining the monosaccharide composition in polysaccharide components. The entire polysaccharide treatment process has mild conditions, simple operation, and is environmentally friendly.

[0032] The analytical method proposed in the present invention can analyze the composition of ten monosaccharides in polysaccharides. This method can be used not only for the analysis of aldose, but also for the analysis of ketose (fructose), and has a clear chemical reaction equation; the analysis has high sensitivity and can complete qualitative and quantitative analysis of monosaccharides below 10μg.

[0033] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 A flow chart of a method for analyzing the monosaccharide composition in a polysaccharide according to an embodiment of the present invention is shown;

[0036] Figure 2 Shown are the GC-MS spectra of various monosaccharide standards in the examples of the present invention;

[0037] Figure 3 The structural formula of each monosaccharide standard after methylation in the embodiments of the present invention is shown;

[0038] Figure 4 The GC-MS spectrum of the polysaccharide and monosaccharide composition in the Polygonatum sibiricum extract according to the embodiment of the present invention is shown. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0040] The present invention hydrolyzes the polysaccharide, dissolves it with dimethyl sulfoxide, then alkalizes it, and then completely methylates it with CH3I. The monosaccharide in the polysaccharide can be determined by GC-MS analysis.

[0041] The reaction principle is as follows:

[0042]

[0043] That is, dimethyl sulfoxide forms dimethyl sulfoxide sulfonyl anion in an alkaline environment, and the dimethyl sulfoxide sulfonyl anion reacts with sugar to form sugar hydroxyl-sodium alcohol, and the sugar hydroxyl-sodium alcohol is then completely methylated by iodomethane to form methyl ether methylated sugar.

[0044] like Figure 1 As shown, the analysis method proposed by the present invention is carried out according to the following steps:

[0045] Trifluoroacetic acid is added to the polysaccharide to hydrolyze it to produce monosaccharides; the feeding ratio of polysaccharide to trifluoroacetic acid is 0.1-5 mL of 2 mol / L trifluoroacetic acid per 20 mg of polygonatum polysaccharide; the hydrolysis conditions are: reaction for 6 hours under sealed conditions and temperature of 100°C;

[0046] The monosaccharide is dissolved in an organic solvent and then sodium hydroxide is added for alkalization to obtain an alkalized monosaccharide; the organic solvent is acetone, N,N-dimethylformamide, N-methylpyrrolidone or dimethyl sulfoxide, preferably dimethyl sulfoxide, wherein the mass ratio of sodium hydroxide to polysaccharide is 1:1-6, and the alkalization conditions are: 35°C for 30 minutes;

[0047] Adding iodomethane to the alkalized monosaccharide for methylation reaction to obtain methylated sugar; the amount of iodomethane added is 1 mL of CH3I per 20 mg of Polygonatum sibiricum polysaccharide; the methylation reaction conditions are: in a sealed state, in the dark, for 12 hours, and at a temperature of 35°C;

[0048] The methylated sugars are analyzed using GC-MS. Specifically, a plurality of monosaccharide standards are first analyzed using GC-MS, and a standard curve of the chromatographic peak area versus concentration of each monosaccharide is plotted;

[0049] Determine the monosaccharide composition and configuration in the polysaccharide based on the GC-MS results of the monosaccharide standards and the GC-MS results of the methylated sugars;

[0050] The peak area in the polysaccharide sample was calculated according to the standard curve of each monosaccharide to determine the content of each monosaccharide component in the polysaccharide.

[0051] The above analysis method is exemplified below with reference to specific examples, and the present invention is described using Polygonatum sibiricum polysaccharide as an example.

[0052] Example 1 GC-MS measurement of various monosaccharide standards.

[0053] Ten different monosaccharide standards, including glucose (Glc), mannose (Man), fructose (Fru), galactose (Gal), rhamnose (Rha), ribose (Rib), xylose (Xyl), arabinose (Ara), gluconic acid (GlcA), and galacturonic acid (GalA), were subjected to GC-MS determination. The specific process is as follows:

[0054] 1.1 Weigh 5 mg of each monosaccharide standard and place it in a 10 mL Schlenk tube. Add 1 mL of dimethyl sulfoxide and sonicate for 10 minutes. Place it in a 10 mL Schlenk tube and add 30 mg of NaOH to alkalize for 30 minutes. Add 0.5 mL of CHCl while stirring. Seal the tube and protect from light. Incubate at 35°C for 12 hours. After the reaction is complete, add 2 mL of pure water to stop the reaction. Extract with the same volume of dichloromethane three times, concentrate and dry, dissolve in 2 mL of dichloromethane, and analyze by GC-MS.

[0055] 1.2GC-MS chromatographic conditions:

[0056] BR-17 elastic capillary column (0.25 μm × 250 μm × 30 m);

[0057] Carrier gas: high-purity helium (purity ≥99.999%), high-purity nitrogen (purity ≥99.999%); column flow rate: 1.0 mL / min; splitless injection, inlet temperature: 250°C; programmed temperature: initial temperature: 50°C, heated to 100°C at a rate of 25°C / min, held for 3 min, and then heated to 184°C at a rate of 4°C / min.

[0058] Mass spectrometry conditions: EI ion source, electron energy 70 eV; ion source temperature 220°C; transfer line temperature 280°C; quadrupole temperature 40°C. Full scan mode, scan range m / z 40–600; mass spectrum search standard library: NIST 11.L.

[0059] The GC-MS spectra of each monosaccharide standard after methylation are as follows Figure 2 As shown, Figure 2 The spectra corresponding to 1-12 are the spectra of the mixed standard of 10 monosaccharides, benzophenone, glucose, mannose, ribose, galactose, rhamnose, arabinose, fructose, glucuronic acid, galacturonic acid and xylose, respectively; Table 1 shows the peak time, chemical formula, molecular weight and ion fragmentation information of each monosaccharide standard after methylation.

[0060] Table 1 Methylation mass spectrometry data of 10 monosaccharides

[0061]

[0062]

[0063] The structural formula of each monosaccharide standard after methylation is as follows Figure 3As shown, glucose (Glc), mannose (Man), galactose (Gal), ribose (Rib), xylose (Xyl), arabinose (Ara), and gluconic acid (GlcA) all have α-D and β-D configurations, rhamnose (Rha) has α-L and β-L configurations, gluconic acid (GlcA) has one configuration, α-D-GlcA, and fructose (Fru) has a single configuration.

[0064] Example 2 takes Polygonatum sibiricum polysaccharide as an example to illustrate the analysis method proposed in the present invention.

[0065] 2.1 The polysaccharide of Polygonatum sibiricum was first treated: 2 mL of 2 mol / L trifluoroacetic acid was added, the tube was sealed and reacted at 100 ° C for 6 hours. After the reaction, the tube was centrifuged at 5000 rpm for 10 minutes, and the residual trifluoroacetic acid was removed by spin-drying with methanol three times. The acid hydrolyzate was added with dimethyl sulfoxide and ultrasonically dissolved for 10 minutes. The tube was placed in a 10 mL Schlenk tube and alkalized by adding 60 mg NaOH for 30 minutes. 1 mL of CHCl was added under stirring, the tube was sealed, protected from light, and reacted at 35 ° C for 12 hours. After the reaction, 2 mL of pure water was added to stop the reaction, and the tube was extracted with the same volume of dichloromethane three times. The tube was concentrated and spin-dried, dissolved in 2 mL of dichloromethane, and analyzed by GC-MS.

[0066] 2.2 GC-MS Chromatographic Conditions: BR-17 flexible capillary column (0.25 μm × 250 μm × 30 m); Carrier gas: high-purity helium (purity ≥99.999%), high-purity nitrogen (purity ≥99.999%); Column flow rate: 1.0 mL / min; Splitless injection, inlet temperature: 250°C; Temperature program: Initial temperature: 50°C, ramp to 100°C at 25°C / min, hold for 3 min, then ramp to 184°C at 4°C / min. Mass spectrometry conditions: EI ion source, electron energy: 70 eV; ion source temperature: 220°C; transfer line temperature: 280°C; quadrupole temperature: 40°C. Full scan mode, scan range: m / z 40–600; Mass spectral search standard library: NIST 11.L.

[0067] 2.3 Methodological investigation of the above analytical methods

[0068] 2.3.1 Linear Relationship Investigation: 5.00 mg of 10 monosaccharide standards were weighed and 10 standard solutions were prepared according to the method described in 1.1 of Example 1. The samples were loaded onto a GC-MS detector for analysis. The chromatographic peak areas of eight monosaccharides at varying concentrations were measured, and a standard curve was plotted plotting the chromatographic peak areas (Y) versus mass concentration (X) for the ten monosaccharides. Using mass concentrations S / N = 3 and S / N = 10 as the limits of detection and quantification, the ten monosaccharides were analyzed within the corresponding ranges. The linear ranges, regression equations, correlation coefficients, limits of detection, and limits of quantification for the ten monosaccharides were obtained and are shown in Table 2.

[0069] Table 2 Linear relationship of standard curves of 10 monosaccharides

[0070]

[0071]

[0072] 2.3.2 Precision determination: The methylated monosaccharide mixed standard was injected 8 times continuously for testing, and the relative standard deviation (RSD) of the 10 monosaccharide mixtures was calculated based on this.

[0073] 2.3.3 Stability determination: The methylated monosaccharide mixed standard was injected once at 0, 2, 12 and 24 hours, and the RSD of the 10 monosaccharides was calculated.

[0074] 2.3.4 Repeatability Determination: Accurately weigh 20.00 mg of Polygonatum sibiricum polysaccharide and prepare solutions according to the method in 2.1. Prepare 6 test solutions and inject them into the solution. Calculate the RSDs of the 10 monosaccharides determined in the pre-experimental study.

[0075] 2.3.5 Sample Recovery: Accurate determination and testing of polysaccharides in six samples were performed. Three different concentrations of 10 monosaccharides were mixed with the standard solution within the linear range for sample recovery testing. Each sample was injected three times, and the RSDs of the 10 monosaccharides determined in the preliminary experiment were calculated.

[0076] The determination results of precision, stability, repeatability and the calculation results of sample recovery are shown in Table 3.

[0077] Table 3 Precision, repeatability and stability of 10 monosaccharides

[0078]

[0079] 2.4 GC-MS was used to determine the content of polysaccharides in the extract of Polygonatum sibiricum. Figure 4 As shown, Figure 4Where 1 is glucose; 2 is mannose; 3 is galactose; 4 is glucuronic acid; 5 is galacturonic acid; and 6 is benzophenone. The peak areas of each monosaccharide in Polygonatum sibiricum polysaccharide were calculated using the standard curves for the 10 monosaccharides in Table 3 to determine the exact content of the polysaccharide components. The content and mole percentage of each polysaccharide component are shown in Table 4. The main component of Polygonatum sibiricum polysaccharide is glucose, with a content of 12.19 μg / mg and a mole percentage of 58.54 mol%. The second main component is galactose, with a content of 3.82 μg / mg and a mole percentage of 17.37 mol%.

[0080] Table 4 Determination of monosaccharide composition of Polygonatum sibiricum polysaccharide

[0081] monosaccharides Content (μg / mg) Mole percent (mol%) glucose 12.19 58.54 Mannose 2.06 9.91 Galactose 3.82 17.37 Glucuronic acid 2.12 9.45 Galacturonic acid 1.06 4.73

[0082] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing the monosaccharide composition in polysaccharides, characterized in that: The analytical method comprises the following steps: Trifluoroacetic acid is added to the polysaccharide for hydrolysis to generate monosaccharides; the feeding ratio of the polysaccharide to trifluoroacetic acid is 0.1-5 mL of 2 mol / L trifluoroacetic acid per 20 mg of polysaccharide; the hydrolysis conditions are: reaction for 6 hours under sealed conditions and temperature of 100°C; The monosaccharide is dissolved in an organic solvent and then sodium hydroxide is added for alkalization to obtain an alkalized monosaccharide; the mass ratio of sodium hydroxide to polysaccharide is 1:1-6; adding methyl iodide to the alkalized monosaccharide to carry out a methylation reaction to obtain methylated sugar; The methylated sugars were analyzed by GC-MS. The test conditions of GC-MS were programmed temperature conditions: the initial temperature was 50°C, the temperature was increased to 100°C at a rate of 25°C / min, maintained for 3 minutes, and then increased to 184°C at a rate of 4°C / min.

2. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1, characterized in that: The methylated sugars are analyzed by GC-MS, comprising the following steps: GC-MS was used to analyze various monosaccharide standards, and a standard curve of the chromatographic peak area versus concentration of each monosaccharide was drawn; Determine the monosaccharide composition and configuration in the polysaccharide based on the GC-MS results of the monosaccharide standards and the GC-MS results of the methylated sugars; The peak area in the polysaccharide sample was calculated according to the standard curve of each monosaccharide to determine the content of each monosaccharide component in the polysaccharide.

3. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1, characterized in that: The organic solvent is acetone, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide or dimethyl sulfoxide.

4. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1, characterized in that: The mass ratio of the sodium hydroxide to the polysaccharide is 1:1-6.

5. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1 or 4, characterized in that: The alkalization condition is: alkalization at 35° C. for 30 minutes.

6. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1, characterized in that: The amount of iodomethane added is 1 mL of CH3I per 20 mg of polysaccharide.

7. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1, characterized in that: The methylation reaction conditions are as follows: the reaction is carried out in a sealed container in the dark for 12 hours at a temperature of 35°C.

8. The method for analyzing the monosaccharide composition in polysaccharides according to claim 1, characterized in that: The test conditions of the GC-MS are: BR-17 elastic capillary column (0.25 μm × 250 μm × 30 m); Carrier gas: high-purity helium (purity ≥ 99.999%), high-purity nitrogen (purity ≥ 99.999%); Column flow rate: 1.0 mL / min; splitless injection was used, and the injection port temperature was 250 °C; EI ion source, electron energy 70 eV; ion source temperature 220 °C; transfer line temperature 280 °C; quadrupole temperature 40 °C; full scan mode, scan range m / z 40-600; mass spectrum retrieval standard library: NIST 11.L.

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