A method for identifying polysaccharide iron

Abstract

The invention discloses a kind of polysaccharide iron identification method, comprise the steps:1) decomplexation, polysaccharide iron test sample is dissolved in water, adds phosphate decomplexation, precipitates, and takes supernatant;2) hydrolysis, takes step 1) in supernatant, with acid adjustment pH to acidity, heating is hydrolyzed, after the completion of hydrolysis, cooling, with alkali adjustment pH to 4 6, filters, and obtains polysaccharide acid solution;3) polysaccharide end group detection in polysaccharide iron, is detected using Agilent high performance liquid chromatograph, using the mixed solution of the polysaccharide sodium of glucose, corresponding polysaccharide iron as reference substance, detection step 2) whether the relative retention time of the polysaccharide acid sodium in hydrolysis solution is consistent with that in reference substance, to determine the polysaccharide acid category of polysaccharide iron;4) molecular weight determination of polysaccharide in polysaccharide iron. Using high performance liquid chromatography to determine the molecular weight and the R group of polysaccharide in polysaccharide iron, the purpose of differentiating the specific kind structure of polysaccharide is finally reached.

Description

Technical Field

[0001] The present invention relates to the technical field of compound analysis, in particular to a method for identifying polysaccharide iron. Background Art

[0002] Different polysaccharides can be synthesized using different polysaccharides. The differences in polysaccharides are divided into differences in molecular weight and differences in R groups. Regarding the identification of polysaccharide iron, most current methods only perform iron identification and sugar identification on the test sample. Sugar identification only uses chemical reactions of sugars to determine whether the test sample contains sugars, without identifying the type of sugar, so it is impossible to distinguish different polysaccharide irons. Summary of the Invention

[0003] The purpose of the present invention is to solve the above problems and design a polysaccharide iron identification method, which uses high performance liquid chromatography to determine the molecular weight and R group of polysaccharides in polysaccharide iron, and ultimately achieves the purpose of identifying the specific type and structure of polysaccharides.

[0004] The technical solution of the present invention to achieve the above-mentioned purpose is: a polysaccharide iron identification method comprising the following steps:

[0005] 1) Decomplexation: dissolve the polysaccharide iron sample in water, add phosphate to decomplex, precipitate, and collect the supernatant;

[0006] 2) hydrolysis: taking the supernatant in step 1), adjusting the pH to acidic with acid, heating for hydrolysis, cooling after the hydrolysis is completed, adjusting the pH to 4-6 with alkali, and filtering to obtain a polysaccharide acid solution;

[0007] 3) Detection of polysaccharide end groups in polysaccharide iron was performed using an Agilent high performance liquid chromatograph, with a mixed solution of glucose and sodium polysaccharide corresponding to polysaccharide iron as a reference substance, and the relative retention time of the polysaccharide acid in the hydrolysis solution in step 2) was detected to be consistent with that of the sodium polysaccharide acid in the reference substance to determine the polysaccharide acid category of the polysaccharide iron;

[0008] 4) Determination of the molecular weight of the polysaccharide in the polysaccharide iron: The molecular weight of the polysaccharide is determined by the molecular exclusion method; the polysaccharide iron is taken, phosphate is added to decomplex it until it turns off-white, the polysaccharide iron is taken out and cooled, and the polysaccharide is diluted to the scale with purified water, filtered, and the filtrate is sampled and tested.

[0009] In the above scheme: in step 1), the ratio of the amount of water added to the weight of the test sample during decomplexation is 8-15 ml / g.

[0010] In the above scheme: in step 1), the phosphate during decomplexation is potassium dihydrogen phosphate and its crystalline hydrate.

[0011] In order to distinguish different polysaccharide irons, the present invention adopts high performance liquid chromatography to detect the molecular weight of polysaccharides (polysaccharide general formula: Gn-R, where G represents glucose, n represents the number of glucose, and R represents the terminal connecting group of polysaccharide) in polysaccharide iron and the R structure of polysaccharide (R is -OCH2(CHOH)4CH2OH (corresponding hydrolysis product is glucose) or -OCH2(CHOH) n COOH (the corresponding hydrolysis products are polysaccharide acids and their salt compounds)), thereby achieving the purpose of distinguishing different polysaccharide irons.

[0012] When the polysaccharide iron decomplexing solution is used for further hydrolysis, potassium dihydrogen phosphate should be used as the decomplexing salt. In the experiment, it was surprisingly found that when the amount of polysaccharide iron was the same, the detection response value of the polysaccharide acid or polysaccharide salt obtained by further hydrolysis with potassium dihydrogen phosphate decomplexing solution was much higher than that when sodium dihydrogen phosphate was used.

[0013] In the above scheme, the mass ratio of the added amount of the phosphate to the test sample is 0.005-0.08 mol / g, preferably 0.014-0.018 mol / g.

[0014] In the above scheme: the decomplexation temperature is above 80°C.

[0015] In the above scheme: the phosphate for decomplexation in step 4) is sodium dihydrogen phosphate or potassium dihydrogen phosphate, and the mass ratio of the added amount of phosphate to the test sample is 0.005-0.08 mol / g.

[0016] In the above scheme: in the hydrolysis step, acid is used to adjust the pH to 1-2, the hydrolysis temperature is controlled at 140-180° C., and the hydrolysis time is 60-100 minutes.

[0017] In the above scheme, the HPLC conditions are as follows: a XAmi de column; a mobile phase of 10 mmol / L disodium hydrogen phosphate-acetonitrile (20:80), the pH of 10 mmol / L disodium hydrogen phosphate being adjusted to 2.8 with phosphoric acid; a flow rate of 1.0 ml / min; and a column temperature of 35°C; a UV detector with a detection wavelength of 200 nm.

[0018] In the above scheme: the molecular weight determination of polysaccharide in polysaccharide iron includes the following steps:

[0019] 1) Chromatographic conditions

[0020] A hydrophilic spherical polymer was used as a filler, a Shodex OHpak column was selected, a 0.71% sodium sulfate solution was used as the mobile phase, and the pH was adjusted to 4.5±0.3 with phosphoric acid; the column temperature was 30°C; the flow rate was 1.0 ml per minute; the differential refractive index detector temperature was 40°C; and the injection volume was 20 μl.

[0021] 2) Preparation of test solution

[0022] After the decomplexation is completed, the reaction solution is taken out, cooled, diluted to the scale with purified water, filtered, and the filtrate is sampled for detection;

[0023] 3) Molecular weight reference solution: Take 5-6 glucose molecular weight references and prepare a solution of about 5 mg / ml as the molecular weight reference solution.

[0024] 4) Establishment of standard curve

[0025] Inject the molecular weight reference solution, record the chromatogram, and use GPC software to draw a standard curve. The correlation coefficient R must not be less than 0.998.

[0026] 5) Determination of molecular weight of the test sample

[0027] Take the test sample solution for testing, record the chromatogram, and calculate the weight-average molecular weight of the test sample using the molecular weight standard curve.

[0028] Beneficial effect: The present invention adopts high performance liquid chromatography to detect the molecular weight of polysaccharide (polysaccharide general formula: Gn-R, wherein G represents glucose, n represents the number of glucose, and R represents the terminal connecting group of polysaccharide) in polysaccharide iron and the R structure of polysaccharide (R is -OCH2(CHOH)4CH2OH (corresponding hydrolysis product is glucose) or -OCH2(CHOH) n COOH (the corresponding hydrolysis products are polysaccharide acids and their salt compounds)), thereby achieving the purpose of distinguishing different polysaccharide irons, the operation is simple and applicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the detection diagram of the polysaccharide terminal groups in Example 1.

[0030] Figure 2 This is the molecular weight detection standard curve of Example 1.

[0031] Figure 3 This is the polysaccharide end group detection diagram of Example 2.

[0032] Figure 4 This is the molecular weight detection standard curve of Example 2.

[0033] Figure 5 This is a diagram of polysaccharide terminal group detection after decomplexation with sodium dihydrogen phosphate according to the method of Example 1. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings.

[0035] Example 1

[0036] Polysaccharide iron, CAS number: 57680-55-4

[0037] 1) Polysaccharide terminal group detection in polysaccharide iron:

[0038] Agilent high performance liquid chromatography was used for detection, and a mixed solution of glucose and the corresponding sodium glucoheptonic acid was used as a reference substance to detect whether the retention time of the polysaccharide acid in the polysaccharide acid solution was consistent with that of the sodium glucoheptonic acid in the reference substance, so as to determine the polysaccharide acid category of the polysaccharide iron;

[0039] Polysaccharide iron decomplexation and polysaccharide hydrolysis

[0040] Take 10 g of polysaccharide iron with CAS number: 57680-55-4, add 80 ml of water and heat at 80 ° C to dissolve, add about 24 g of potassium dihydrogen phosphate (0.176 mol), heat at 80 ° C to decomplex until gray-green, centrifuge and precipitate, take the supernatant, adjust the pH to about 1.5 with concentrated hydrochloric acid, seal, place at 160 ° C for hydrolysis reaction for 80 minutes, take out and cool, adjust the pH to about 4.5 with 5 mol / L sodium hydroxide, and filter to obtain the glucoheptonic acid solution.

[0041] Take 10 g of polysaccharide iron with CAS number: 57680-55-4, add 80 ml of water and heat at 80 ° C to dissolve, add about 24 g of sodium dihydrogen phosphate, heat at 80 ° C to decomplex until gray-green, centrifuge and precipitate, take the supernatant, adjust the pH to about 1.5 with concentrated hydrochloric acid, seal, place at 160 ° C for hydrolysis reaction for 80 minutes, take out and cool, adjust the pH to about 4.5 with 5 mol / L sodium hydroxide, and filter to obtain the glucoheptonic acid solution.

[0042] The test results obtained by using sodium dihydrogen phosphate to decomplex are as follows Figure 5 As shown, compared Figure 1 The polysaccharide acid or polysaccharide salt obtained by further hydrolysis of the solution decomplexed by potassium dihydrogen phosphate has a much higher detection response value than that when sodium dihydrogen phosphate is used.

[0043] Solution preparation

[0044] System suitability solution: Place 5 mg of glucose, 1 mg of sodium gluconate, and 1 mg of sodium glucoheptonate in the same injection vial and dissolve in 1 mL of blank solvent. (Glucose positioning solution: Take an appropriate amount of glucose reference substance and blank solvent (mobile phase, 10 mmol / L disodium hydrogen phosphate (pH adjusted to 2.8 with phosphoric acid)-acetonitrile (20:80)) to prepare a solution containing 5 mg of glucose per mL; Sodium gluconate positioning solution: Take an appropriate amount of sodium gluconate reference substance and blank solvent to prepare a solution containing 1 mg of sodium gluconate per mL; Sodium glucoheptonate positioning solution: Take an appropriate amount of sodium glucoheptonate reference substance and blank solvent to prepare a solution containing 1 mg of sodium glucoheptonate per mL.)

[0045] Chromatographic conditions

[0046] A XAmi de column (5m 100A 4.6*250nm) was used; the mobile phase was 10mmol / L disodium hydrogen phosphate (pH adjusted to 2.8 with phosphoric acid)-acetonitrile (20:80), the flow rate was 1.0ml / min, and the column temperature was 35℃; an ultraviolet detector was used with a detection wavelength of 200nm.

[0047] Accurately draw 10 μl of blank solvent, system suitability solution, and hydrolysis solution respectively, and inject them into the liquid chromatograph to record the chromatogram. The chromatograms of blank solvent, glucose, sodium gluconate, sodium glucoheptonic acid, and sample are shown in the table. Figure 1 The results showed that blank solvent, glucose and gluconic acid did not interfere with the determination of glucoheptonic acid.

[0048] System precision test

[0049] Accurately pipette 10 μl of the system suitability solution and inject it six times continuously. The RSD% value of the retention time of the main peak of the system suitability solution was calculated. The results are shown in Table 1. The results show that the RSD% of the main peak retention time in the six injections of the system suitability solution were 0.3%, 0.2%, and 0.2%, respectively, indicating that the system precision of this verification method is good.

[0050] Table 1 System precision test results

[0051]

[0052]

[0053] Repeatability test

[0054] Six parallel sample solutions were prepared, and 10 μl of each solution was injected for analysis. The RSD% values for the retention time of the glucoheptonic acid peak were calculated, as shown in Table 2. The results showed that all six sample injections contained a glucoheptonic acid peak, with an RSD% of 0.1%, demonstrating good reproducibility of this validation method.

[0055] Table 2 Repeatability test

[0056]

[0057] Durability test

[0058] To investigate the impact of minor changes in test conditions on the assay results, we performed small variations in flow rate, pH, column temperature, detection wavelength, and mobile phase ratio, and examined their impact on the results. The ranges of variation for each factor are shown in Table 3. Test solutions, glucose, sodium gluconate, and sodium glucoheptonate were prepared, and 10 μl injections were performed for measurement. Retention time results for each solution are shown in Table 3. These results demonstrate that glucose and gluconic acid do not interfere with the glucoheptonate determination when minor changes in test conditions are made.

[0059] Table 3 Durability test

[0060]

[0061] By using high-performance liquid chromatography, a unique identification method for the hydrolysis product of polysaccharide iron with CAS number: 57680-55-4 was established, and glucose, sodium gluconate, and sodium glucoheptonic acid were used as controls. The method has high precision, good repeatability, and good durability, laying the foundation for subsequent quality research on polysaccharide iron with CAS number: 57680-55-4.

[0062] 2) Method for determining the molecular weight of polysaccharides in polysaccharide iron. This method uses a molecular exclusion method to determine the molecular weight of polysaccharides, comprising the following steps:

[0063] Chromatographic conditions

[0064] A hydrophilic spherical polymer was used as the filler, Shodex OHpak or other suitable chromatographic columns were selected, 0.71% sodium sulfate solution was used as the mobile phase, and the pH was adjusted to 4.5 (±0.3) with phosphoric acid; the column temperature was 30°C; the flow rate was 1.0 ml per minute; the differential refractive index detector temperature was 40°C; and the injection volume was 20 μl.

[0065] Solution preparation

[0066] Test solution: Take about 100 mg of polysaccharide iron, add 4 ml of about 2 mol / L sodium dihydrogen phosphate or potassium dihydrogen phosphate, decomplex in a water bath at 50°C until it turns off-white, take out and cool, dilute to the scale with purified water, filter, and take the filtrate for sampling and detection.

[0067] Molecular weight reference solution: Take glucose molecular weight reference substances with molecular weights of 1030, 2700, 5250, 9750, and 13050 and prepare them into about 5 mg / ml as molecular weight reference solutions.

[0068] Establishment of standard curve

[0069] Take the molecular weight reference solution and inject it, record the chromatogram, and use GPC software to draw the standard curve. The correlation coefficient R should not be less than 0.998. Figure 2 , where R is 0.999.

[0070] Determination of molecular weight of test sample

[0071] Take the test sample solution for testing, record the chromatogram, and calculate the weight-average molecular weight of the test sample using the molecular weight standard curve.

[0072] Example 2

[0073] Polysaccharide iron, CAS number: 9004-66-4

[0074] 1) Polysaccharide terminal group detection in polysaccharide iron:

[0075] An Agilent high performance liquid chromatograph was used for detection, and a mixed solution of glucose and the corresponding sodium gluconate was used as a reference substance to detect whether the retention time of the polysaccharide acid in the polysaccharide acid solution was consistent with that of the sodium gluconate in the reference substance, so as to determine the polysaccharide acid category of the polysaccharide iron;

[0076] Polysaccharide iron decomplexation and polysaccharide hydrolysis

[0077] Take 10 g of polysaccharide iron with CAS number: 9004-66-4, add 150 ml of water and heat at 80 ° C to dissolve, add about 20 g of potassium dihydrogen phosphate (0.146 mol), heat at 80 ° C to decomplex, centrifuge and precipitate, take the supernatant, adjust the pH value to about 1.5 with concentrated hydrochloric acid, seal, and place at 180 ° C for hydrolysis reaction for 60 minutes. Take out and cool, adjust the pH to about 4.5 with 5 mol / L sodium hydroxide, and filter to obtain gluconic acid solution.

[0078] Solution preparation

[0079] System suitability solution: Take 5 mg of glucose and 1 mg of sodium gluconate and place them in the same injection vial. Add 1 mL of blank solvent to dissolve them. (Glucose positioning solution: Take an appropriate amount of glucose reference substance and prepare a solution containing 5 mg of glucose per 1 mL of blank solvent. Sodium gluconate positioning solution: Take an appropriate amount of sodium gluconate reference substance and prepare a solution containing 1 mg of sodium gluconate per 1 mL of blank solvent.)

[0080] Chromatographic conditions: A XAmi de column (5m 100A 4.6*250nm) was used; the mobile phase was 10mmol / L disodium hydrogen phosphate (pH adjusted to 2.8 with phosphoric acid)-acetonitrile (20:80), the flow rate was 1.0ml / min, and the column temperature was 35°C; an ultraviolet detector was used with a detection wavelength of 200nm.

[0081] Accurately pipette 10 μl of blank solvent, system suitability solution, and hydrolysis solution respectively, inject them into the liquid chromatograph, and record the chromatograms. The chromatograms of blank solvent, glucose, and sodium gluconate samples are shown in Figure 3 The results showed that blank solvent and glucose did not interfere with the determination of gluconic acid.

[0082] By using high-performance liquid chromatography, a unique identification method for the polysaccharide iron hydrolysis product with CAS number: 9004-66-4 was established, and glucose and sodium gluconate were used as controls. The method has high precision, good repeatability and good durability, laying the foundation for subsequent quality research on polysaccharide iron 9004-66-4.

[0083] 2) Method for determining the molecular weight of polysaccharides in polysaccharide iron. This method uses a molecular exclusion method to determine the molecular weight of polysaccharides, comprising the following steps:

[0084] Chromatographic conditions

[0085] A hydrophilic spherical polymer was used as the filler, Shodex OHpak or other suitable chromatographic columns were selected, 0.71% sodium sulfate solution was used as the mobile phase, and the pH was adjusted to 4.5 (±0.3) with phosphoric acid; the column temperature was 30°C; the flow rate was 1.0 ml per minute; the differential refractive index detector temperature was 40°C; and the injection volume was 20 μl.

[0086] Solution preparation

[0087] Test solution: Take about 100 mg of polysaccharide iron, add 4 ml of about 2 mol / L sodium dihydrogen phosphate or potassium dihydrogen phosphate, decomplex in a water bath at 50°C until it turns off-white, take out and cool, dilute to the scale with purified water, filter, and take the filtrate for sampling and detection.

[0088] Molecular weight reference solution: Take glucose molecular weight reference substances with molecular weights of 1030, 2700, 5250, 9750, and 13050 and prepare them into about 5 mg / ml as molecular weight reference solutions.

[0089] a. Establishment of standard curve

[0090] Take the molecular weight reference solution and inject it, record the chromatogram, and use GPC software to draw the standard curve. The correlation coefficient R should not be less than 0.998. Figure 4 , where R is 0.999.

[0091] The above technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention. Any changes that may be made to certain parts thereof by those skilled in the art all reflect the principles of the present invention and fall within the scope of protection of the present invention.

Claims

1. A polysaccharide iron identification method comprising the following steps: 1) Decomplexation: dissolve the polysaccharide iron sample in water, add phosphate to decomplex, precipitate, and collect the supernatant; the phosphate is potassium dihydrogen phosphate, and the polysaccharide iron is glucoheptose iron or dextran iron; 2) Hydrolysis: take the supernatant from step 1), adjust the pH to acidic with acid, heat and hydrolyze, cool after hydrolysis is complete, adjust the pH to 4-6 with alkali, and filter to obtain a polysaccharide acid solution; 3) Detection of polysaccharide end groups in the polysaccharide iron using an Agilent high-performance liquid chromatograph, using a mixed solution of glucose and sodium polysaccharide acid corresponding to the polysaccharide iron as a reference substance. The relative retention times of the polysaccharide acid in the hydrolysis solution in step 2) and the sodium polysaccharide acid in the reference substance were tested to determine the polysaccharide acid type of the polysaccharide iron. When the polysaccharide iron is glucoheptose iron, the preparation of the mixed solution of glucose and the corresponding polysaccharide sodium of the polysaccharide iron is as follows: 5 mg of glucose, 1 mg of sodium gluconate, and 1 mg of sodium glucoheptose are placed in the same injection vial, 1 ml of blank solvent is added to dissolve, the blank solvent is the mobile phase, and 10 mmol / L of disodium hydrogen phosphate and acetonitrile adjusted to pH 2.8 with phosphoric acid are prepared at a ratio of 20: 80; When the polysaccharide iron is dextran iron, the preparation of the mixed solution of glucose and sodium gluconate corresponding to the polysaccharide iron is as follows: 5 mg of glucose and 1 mg of sodium gluconate are placed in the same injection vial, and 1 ml of blank solvent is added to dissolve the solution; The detection conditions were as follows: XAmide column; mobile phase: 10 mmol / L sodium hydrogen phosphate (20:80)-acetonitrile (10 mmol / L sodium hydrogen phosphate) adjusted to pH 2.8 with phosphoric acid; flow rate: 1.0 ml / min; column temperature: 35°C; UV detector: 200 nm; 4) Determination of the molecular weight of polysaccharides in polysaccharide iron: The molecular weight of the polysaccharide is determined by the size exclusion method; polysaccharide iron is taken, phosphate is added to decomplex it until it turns off-white, the polysaccharide iron is removed and cooled, and the polysaccharide iron is diluted to the mark with purified water. The polysaccharide iron is filtered, and the filtrate is sampled for testing.

2. The polysaccharide iron identification method according to claim 1, wherein: Step 1) When decomplexing, the ratio of the amount of water added to the weight of the test sample is 8-15 ml / g.

3. The polysaccharide iron identification method according to claim 2, wherein: The mass ratio of the added amount of the phosphate to the test sample is 0.005-0.08 mol / g.

4. The polysaccharide iron identification method according to claim 3, wherein: The decomplexation temperature in step 1) is above 80°C.

5. The polysaccharide iron identification method according to any one of claims 1 to 4, characterized in that: In step 4), the phosphate for decomplexation is sodium dihydrogen phosphate or potassium dihydrogen phosphate, and the mass ratio of the added amount of phosphate to the test sample is 0.005-0.08 mol / g.

6. The polysaccharide iron identification method according to claim 5, characterized in that: In the hydrolysis step, acid is used to adjust the pH to 1-2, the hydrolysis temperature is controlled at 140-180° C., and the hydrolysis time is 60-100 minutes.

7. The polysaccharide iron identification method according to claim 6, characterized in that The molecular weight determination of polysaccharides in polysaccharide iron includes the following steps: 1) Chromatographic conditions A hydrophilic spherical polymer was used as a filler, a Shodex OHpak column was selected, a 0.71% sodium sulfate solution was used as the mobile phase, and the pH was adjusted to 4.5±0.3 with phosphoric acid; the column temperature was 30°C; the flow rate was 1.0 ml per minute; the differential refractive index detector temperature was 40°C; and the injection volume was 20 μl. 2) Preparation of test solution After the decomplexation is completed, the reaction solution is taken out, cooled, diluted to the scale with purified water, filtered, and the filtrate is sampled for detection; 3) Molecular weight reference solution: Take 5-6 glucose molecular weight references and prepare a 5 mg / ml solution as the molecular weight reference solution. 4) Establishment of standard curve Take the molecular weight reference solution and inject it, record the chromatogram, and use GPC software to draw the standard curve. The correlation coefficient R must not be less than 0.998; 5) Determination of molecular weight of test sample Take the test sample solution for testing, record the chromatogram, and calculate the weight-average molecular weight of the test sample using the molecular weight standard curve.

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