CN112444567A - Method for determining glucose and gluconic acid in oxidized dextrin - Google Patents

Method for determining glucose and gluconic acid in oxidized dextrin Download PDF

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CN112444567A
CN112444567A CN201910793268.8A CN201910793268A CN112444567A CN 112444567 A CN112444567 A CN 112444567A CN 201910793268 A CN201910793268 A CN 201910793268A CN 112444567 A CN112444567 A CN 112444567A
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assay
mobile phase
glucose
measuring
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陈庆财
赵骞
杨瑞峰
刘霞
王玲昀
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Shanghai Institute of Materia Medica of CAS
Jiangsu Aosaikang Pharmaceutical Co Ltd
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention provides a method for determining glucose and gluconic acid in oxidized dextrin, which comprises the following steps: preparing a sample solution, preparing a mobile phase solution, preparing a standard curve solution and detecting by an integral pulse amperometry. The determination method of the invention adopts the ion chromatography and the electrochemical detector, can simultaneously complete the detection of the glucose and the gluconic acid, has high sensitivity, and the detection limit can reach ppb level.

Description

Method for determining glucose and gluconic acid in oxidized dextrin
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for determining glucose and gluconic acid in oxidized dextrin.
Background
Dextrins are widely used in the adhesive industry, paper industry, pharmaceutical industry, mining industry, food industry and textile industry. In the pharmaceutical industry, pyrolytic dextrin is widely used as excipient and adhesive for granule, and after micromolecular pigment and other substances are removed by separation of pyrolytic dextrin, relatively single dextrin is obtained, has stable property and good viscosity, can bond the active ingredients of the medicine together, and has excellent solubility, so that the granule can be quickly dissolved in water. The pyrolytic dextrin added to the tablet can also play a certain role in disintegration because of being rapidly dissolved in water.
Oxidized dextrin is widely applied to the pharmaceutical and food industries, generally, the content of glucose and gluconic acid in the oxidized dextrin needs to be measured in the application process, the ratio of the glucose and the gluconic acid in a sample is accurately measured, the oxidation degree of the reaction is favorably monitored and inferred, and the content of the dextrin in the sample can be indirectly calculated.
The ultraviolet spectrophotometer method can only be used for measuring the glucose content in a sample, and the gluconic acid content is not legal. A High Performance Liquid Chromatography (HPLC) method is an analysis means widely applied at present, wherein an ion chromatography method is used for separation by ion exclusion, but the peak position of a reactant glucose and an oxidation product sodium gluconate coincide, a serial ultraviolet detector and a differential detector are needed for detection, the method has relatively low sensitivity and an unsatisfactory detection result, while in the existing anion column IonPac AS11-HC detection method by utilizing integral pulse ampere, 5mmol/L NaOH is used AS an eluent, the elution capacity of the glucose is weak, and meanwhile, a 250mm ion chromatography column is used, the peak time of the glucose is about 3min, and the peak time is also not suitable.
Therefore, it is desirable to provide a method for simultaneously detecting glucose and gluconic acid with high sensitivity.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a method for measuring glucose and gluconic acid in oxidized dextrin with high sensitivity.
The technical scheme of the invention is as follows:
the method for measuring glucose and gluconic acid in oxidized dextrin adopts a Dionex CarboPac PA20 BioLC chromatographic column: in mobile phase H2Performing gradient elution by taking O, a mobile phase NaOH solution and a mobile phase NaOAc solution as eluent; the conditions of the gradient elution include (volume percent):
time (min) H2O(％) NaOH solution (%) NaOAc solution (%)
0-8 M1 N1 100-M1-N1
8.1-20 M2 N2 100-M2-N2
20.1-25 M2 N3 100-M2-N3
25.1-30 M2 N2 100-M2-N2
30.1-45 M1 N1 100-M1-N1
Wherein M is more than or equal to 921≤94，0≤M2≤1，3≤N1≤5，96≤N2≤98，0≤N3≤1。
Preferably, the concentration of the NaOH solution is 150-250 mmol/L, and the concentration of the NaOAc solution is 450-550 mmol/L.
Preferably, the concentration of the NaOH solution is 200mmol/L, and the concentration of the NaOAc solution is 500 mmol/L.
Preferably, the conditions of the gradient elution are in particular:
time (min) H2O(％) NaOH solution (%) NaOAc solution (%)
0-8 93 5 2
8.1-20 0 98 2
20.1-25 0 0 100
25.1-30 0 98 2
30.1-45 93 5 2
Further, the chromatographic conditions of the assay method are:
the column temperature of the chromatographic column is 28-32 ℃, and/or
The flow rate of the mobile phase is 0.2-0.6 ml/min.
Further, the chromatographic conditions are specifically: the column temperature of the column was 30 ℃ and the mobile phase flow rate was 0.4 ml/min.
Still further, the assay method comprises: preparing a sample solution, preparing a mobile phase solution, preparing a standard curve solution and detecting by an integral pulse amperometry.
Preferably, the preparation of the sample solution comprises:
(1) weighing 0.1g of oxidized dextrin sample, adding 8-12% by mass of sulfuric acid, destroying at 100 ℃ for 1.0-1.5 h, cooling, and diluting with water to 10 ml;
(2) measuring 0.2ml of the solution obtained in the step (1), diluting to 100ml with water, uniformly mixing, and taking the solution as a sample solution after passing through a 0.22 micron water film.
Preferably, 1-2 ml of sulfuric acid with the mass percentage of 8-12% is added in the step (1).
Preferably, the step (1) is carried out by adding 10% by mass of sulfuric acid.
Preferably, the destruction at 100 ℃ in step (1) is carried out by means of oil bath heating.
Preferably, the preparation of the standard curve solution comprises:
(1) weighing 20mg of glucose and 5.2mg of sodium gluconate into a measuring flask, adding water to a constant volume of 1000ml, and shaking up to obtain std5 solution;
(2) respectively measuring 8ml, 6ml, 4ml and 2ml of the solution obtained in the step (1) into measuring bottles, respectively adding water to a constant volume of 10ml, shaking up for later use, and respectively naming as std4, std3, std2 and std 1;
(3) and (3) respectively passing the solutions obtained in the step (2) through 0.22 micron water films to obtain standard curve solutions.
Oxidized dextrin samples in the present invention include, but are not limited to: oxidized dextrins, physical mixtures of oxidized dextrins with other substances, complexes of oxidized dextrins with metals.
Has the advantages that:
for the oxidized dextrin sample, dilute sulfuric acid is added, and dextrin in the sample is destroyed under the heating condition to generate glucose and gluconic acid, so that the content of the glucose and the gluconic acid is measured by adopting an integral pulse ampere detection method, and the content of the dextrin in the sample can be indirectly calculated.
The method for determining the glucose and the gluconic acid adopts the ion chromatography and the electrochemical detector, can simultaneously complete the detection of the glucose and the gluconic acid, and has high sensitivity and detection limit reaching ppb level.
Drawings
FIG. 1 is a blank water spectrum;
FIG. 2 is a spectrum of a standard solution of glucose and gluconic acid (std 3);
FIG. 3 is a spectrum of a sample solution prepared in example 1;
fig. 4 is an enlarged spectrum of fig. 3.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1
Preparing a sample solution:
(1) weighing 0.1g of oxidized dextrin sample, adding 1ml of sulfuric acid with the mass percentage of 10%, destroying for 1h at 100 ℃ in an oil bath, cooling and diluting to 10ml with water;
(2) measuring 0.2ml of the solution obtained in the step (1), diluting to 100ml with water, uniformly mixing, and taking the solution as a sample solution after passing through a 0.22 micron water film.
Preparation of mobile phase solution:
(1) preparing 200mmol/L NaOH solution: measuring 16ml of NaOH solution with the mass percentage of 50% into a beaker, adding 1000ml of water, uniformly stirring, and passing through a 0.22 micron water film to a mobile phase bottle;
(2) preparing 500mmol/L NaOAc solution: 41g of NaOAc was weighed into a beaker containing 10001ml of water, dissolved with stirring and passed over a 0.22 micron water film into a mobile phase vial.
Preparation of a standard curve solution:
(1) weighing 20mg of glucose and 5.2mg of sodium gluconate to 1000ml of measuring flask, adding water to a constant volume to reach a scale, and shaking up to obtain std5 solution;
(2) respectively measuring 8ml, 6ml, 4ml and 2ml to 10ml of the solution obtained in the step (1) in measuring bottles, respectively adding water to fix the volume to a scale, and shaking up for later use, wherein the solution is named as std4, std3, std2 and std 1;
(3) and (3) respectively enabling the solution obtained in the step (2) to pass through a 0.22 micron water film to a liquid phase vial to be used as a standard curve solution.
Example 2
Selecting an ion chromatography instrument and setting parameters:
the instrument model is as follows: Thermo-Fisher ICS-5000+
Name of the protection column: dionex CarboPacTM PA20 BioLCTM 3 x 30mm Guard
Analytical column name: dionex CarboPacTM PA20 BioLCTM 3 x 150mm Analytical
Column temperature: 30 deg.C
Electrochemical detector parameter setting:
amperometric parameter (amperometric detector) electrode material: gold electrode
Reference electrode: Ag/AgCl
Potential waveform: sugar four potential waveform
Detector temperature: 28-32 DEG C
In mobile phase H2Performing gradient elution by using O, mobile phase NaOH solution (200mmol/L) and mobile phase NaOAc solution (500mmol/L) as eluent under the conditions shown in Table 1, setting the flow rate at 0.4ml/min and the sample injection volume at 5 μ L, and performing sample injection analysis.
TABLE 1
Time (min) H2O(％) NaOH solution (%) NaOAc solution (%)
0-8 93 5 2
8.1-20 0 98 2
20.1-25 0 0 100
25.1-30 0 98 2
30.1-45 93 5 2
Example 3
The standard curve solution prepared in example 1 was injected in a volume of 5. mu.L, and a standard curve was plotted, and the correlation coefficient of the standard curve is shown in Table 2.
TABLE 2
Peak Name Cal.Type Coefficient of linear correlation Intercept of a beam Slope of Linear Range (mg/L)
Glucose LOff 0.9998 1.7420 2.8074 4～20
Gluconic acid LOff 0.9999 0.0008 0.3987 1～5
The sample solution prepared in example 1 was injected into a sample volume of 5 μ L, and the results of the contents of glucose and gluconic acid in the sample solution were calculated and shown in table 3.
TABLE 3
Figure BDA0002180112030000051
Example 4
The accuracy of the determination method is verified, the oxidized dextrin sample No. 1-4 is additionally taken for determination, and then the content of glucose is determined by an ultraviolet spectrophotometer method for verification, and specific results are shown in Table 4.
TABLE 4
Figure BDA0002180112030000061
The verification result in table 4 shows that the determination method of the present invention has good accuracy, thereby proving that the method can simultaneously determine the contents of glucose and gluconic acid, and has good accuracy and high sensitivity.
In the determination method, the NaOH solution is selected as the mobile phase, and the dissociation constant PKa values of the glucose and the gluconic acid in the NaOH solution are greatly different, so that the glucose and the gluconic acid can be completely separated by using a Dionex CarboPac PA20 BioLC chromatographic column under the proper mobile phase condition, and the problem of co-elution is avoided.
In addition, in the conventional anion column IonPac AS11-HC method, 5mmol/L NaOH is used AS eluent, and compared with the gradient elution (200mmol/L NaOH solution and 500mmol/L NaOAc solution) in the method (sugar analysis column CarboPac PA20) of the invention, the elution capacity for glucose is weaker; meanwhile, the ion chromatographic column with the thickness of 250mm is used, the peak-off time of glucose is about 3min, the peak-off time is relatively early, and the peak-off time is also not suitable because the column has dead volume.
The method for determining the glucose and the gluconic acid adopts the ion chromatography and the electrochemical detector, can simultaneously complete the detection of the glucose and the gluconic acid, and has high sensitivity and detection limit reaching ppb level.

Claims (10)

1. The method for measuring glucose and gluconic acid in oxidized dextrin is characterized by adopting a Dionex CarboPac PA20 BioLC chromatographic column: in mobile phase H2Performing gradient elution by taking O, a mobile phase NaOH solution and a mobile phase NaOAc solution as eluent; the conditions for the gradient elution include:
time (min) H2O(％) NaOH solution (%) NaOAc solution (%) 0-8 M1 N1 100-M1-N1 8.1-20 M2 N2 100-M2-N2 20.1-25 M2 N3 100-M2-N3 25.1-30 M2 N2 100-M2-N2 30.1-45 M1 N1 100-M1-N1
Wherein M is more than or equal to 921≤94，0≤M2≤1，3≤N1≤5，96≤N2≤98，0≤N3≤1。
2. The method according to claim 1, wherein the NaOH solution has a concentration of 150 to 250mmol/L and the NaOAc solution has a concentration of 450 to 550 mmol/L.
3. The assay of claim 2, wherein the NaOH solution has a concentration of 200mmol/L and the NaOAc solution has a concentration of 500 mmol/L.
4. The assay method according to claim 1, wherein the conditions of the gradient elution are in particular:
time (min) H2O(％) NaOH solution (%) NaOAc solution (%) 0-8 93 5 2 8.1-20 0 98 2 20.1-25 0 0 100 25.1-30 0 98 2 30.1-45 93 5 2
。
5. The assay of claim 1, wherein the chromatographic conditions of the assay are:
the column temperature of the chromatographic column is 28-32 ℃, and/or
The flow rate of the mobile phase is 0.2-0.6 ml/min.
6. The assay method according to claim 5, wherein the chromatographic conditions are in particular: the column temperature of the column was 30 ℃ and the mobile phase flow rate was 0.4 ml/min.
7. The assay of any one of claims 1 to 6, wherein the assay comprises: preparing a sample solution, preparing a mobile phase solution, preparing a standard curve solution and detecting by an integral pulse amperometry.
8. The assay method of claim 7, wherein the sample solution is formulated to comprise:
(1) weighing 0.1g of oxidized dextrin sample, adding 8-12% by mass of sulfuric acid, destroying at 100 ℃ for 1.0-1.5 h, cooling, and diluting with water to 10 ml;
(2) measuring 0.2ml of the solution obtained in the step (1), diluting to 100ml with water, uniformly mixing, and taking the solution as a sample solution after passing through a 0.22 micron water film.
9. The method according to claim 8, wherein 1 to 2ml of sulfuric acid having a mass percentage of 8 to 12% is added in the step (1).
10. The assay of claim 7, wherein the standard curve solution is formulated by:
(1) weighing 20mg of glucose and 5.2mg of sodium gluconate into a measuring flask, adding water to a constant volume of 1000ml, and shaking up to obtain std5 solution;
(2) respectively measuring 8ml, 6ml, 4ml and 2ml of the solution obtained in the step (1) into measuring bottles, respectively adding water to a constant volume of 10ml, shaking up for later use, and respectively naming as std4, std3, std2 and std 1;
(3) and (3) respectively passing the solutions obtained in the step (2) through 0.22 micron water films to obtain standard curve solutions.
CN201910793268.8A 2019-08-27 2019-08-27 Method for determining glucose and gluconic acid in oxidized dextrin Pending CN112444567A (en)

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CN102590370A (en) * 2012-01-16 2012-07-18 南京林业大学 Method for synchronously determining monosaccharide, uronic acid and saccharic acid in wood fiber material reaction system
CN103323562A (en) * 2013-05-27 2013-09-25 广东中烟工业有限责任公司 Method for detecting water-soluble sugar content in main stream smoke
CN105319307A (en) * 2014-06-12 2016-02-10 饶君凤 Analytic method of detecting monosaccharide composition in crocus sativus polysaccharide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102590370A (en) * 2012-01-16 2012-07-18 南京林业大学 Method for synchronously determining monosaccharide, uronic acid and saccharic acid in wood fiber material reaction system
CN103323562A (en) * 2013-05-27 2013-09-25 广东中烟工业有限责任公司 Method for detecting water-soluble sugar content in main stream smoke
CN105319307A (en) * 2014-06-12 2016-02-10 饶君凤 Analytic method of detecting monosaccharide composition in crocus sativus polysaccharide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
梁立娜等: "高效阴离子交换-脉冲安培检测同时分析单糖和糖醛酸", 《分析化学》 *
王鑫等: "离子交换色谱法测定葡萄糖氧化反应液中的葡萄糖和葡萄糖酸", 《化学分析计量》 *

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