Method for detecting trace reducing sugar in mannitol based on ion chromatography

Abstract

The invention belongs to the field of reducing sugar detection, and particularly discloses a method for detecting trace reducing sugar in mannitol based on ion chromatography, which comprises the following steps: S1, dissolving a mannitol sample in high-purity water, fully and uniformly mixing to obtain a sample solution, and preparing a fructose standard solution with known concentration; s2, purifying the obtained sample solution and the fructose standard solution by using an extraction column, concentrating the purified sample solution and the fructose standard solution, and then filtering the concentrated sample solution and the fructose standard solution by using a filter membrane to remove insoluble impurities; according to the method, the reducing sugar, other sugar alcohols and interfering substances in the sample solution and the fructose standard solution are separated through ion chromatography, then the reducing sugar in the separation process is detected through an ampere detector, the content of the reducing sugar in the sample solution is calculated through a standard curve, the method has high precision, the trace reducing sugar can be detected, and the detection accuracy is high. The analysis requirements are met.

Description

Technical Field

[0001] The invention belongs to the field of reducing sugar detection, and particularly relates to a method for detecting trace reducing sugar in mannitol based on ion chromatography. Background Art

[0002] Mannitol is a sugar alcohol with the appearance of white crystalline powder, easily soluble in water, and has a cool taste. Due to its permeability, it can attract moisture, so it is very useful in pharmaceutical and food processing, mainly used as a pharmaceutical drug and food additive;

[0003] In the production process of food and medicine, in order to ensure that the ingredients of the product meet the standards, the composition of mannitol needs to be tested. Especially in sugar-free or low-sugar foods for diabetic patients, accurate detection of the content of reducing sugar can help provide effective information.

[0004] However, the traditional titration method is usually used to detect the reducing sugar content in mannitol, but the detection accuracy is not ideal and does not meet the trace analysis standard. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting trace reducing sugars in mannitol based on ion chromatography, so as to solve the problem that the detection accuracy of reducing sugar content in mannitol by conventional titration method is not ideal and cannot reach the trace analysis standard.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for detecting trace reducing sugars in mannitol based on ion chromatography, comprising:

[0008] S1. Dissolve the mannitol sample in high-purity water, mix thoroughly to obtain a sample solution, and prepare a fructose standard solution of known concentration;

[0009] S2. Purify the obtained sample solution and fructose standard solution using an extraction column, concentrate the purified sample solution and fructose standard solution, and then filter the concentrated sample solution and fructose standard solution using a filter membrane to remove insoluble impurities;

[0010] S3, flushing the chromatographic system with an eluent until the baseline is stable, injecting the treated sample solution into the chromatograph, recording the chromatogram, and using an anion exchange ion chromatography column to separate the sample solution and the fructose standard solution, respectively, to obtain sugar compounds in the sample solution and sugar compounds in the standard solution;

[0011] S4. Using an amperometric detector to detect the sugar compounds in the separation process, and establishing a standard working curve based on the signal output by the amperometric detector, the content of reducing sugar in the sample solution is calculated using the standard working curve.

[0012] Preferably, the extraction column in step S2 is a C18 SPE column, which is used to remove fat-soluble impurities in the sample solution and the standard solution.

[0013] Preferably, in step S3, the ion chromatography column is a CarboPac PA200 chromatography column, the eluent is a NaOH solution with a concentration of 100 to 200 mM, a pH of 13.0, a flow rate of 0.3 to 0.5 mL / min, a column temperature controlled at 30 to 40 degrees Celsius, and a linear gradient elution, with the eluent concentration changing time from 0% to 100% being 30 minutes.

[0014] Preferably, the concentration method in step S2 is at least one of rotary evaporation, freeze drying and nitrogen purging, and the mass of the sample before and after concentration is weighed, the concentration multiple is calculated, and the stability of the reducing sugar during the concentration process is verified.

[0015] Preferably, the step S4 uses principal component regression to correct the signal output by the ampere monitor.

[0016] Preferably, during the detection process of the amperometric detector in step S4, a blank solution or a standard solution is regularly injected, the baseline drift is recorded, and the baseline is corrected according to the baseline drift.

[0017] Preferably, the PAD potential parameter in step S4 is:

[0018] Detection potential: +0.1V;

[0019] Oxidation potential: +0.6V;

[0020] Regeneration potential: -0.8V;

[0021] The pulse period is 0.4 to 0.6 seconds, and the integration time is 0.3 to 0.5 seconds.

[0022] Preferably, a suppressor is used to reduce the NaOH background conductivity during the amperometric detector detection process in step S4.

[0023] Preferably, the standard working curve in step S4 is drawn with the peak area or peak height of the sugar compound in the standard solution as the ordinate and the concentration as the abscissa.

[0024] Preferably, the concentration range of the fructose standard solution in step S1 is 0.1 to 10 mg / L.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention removes fat-soluble impurities in a sample solution and a fructose standard solution through an extraction column, improves the cleanliness of the sample solution and the fructose standard solution, reduces matrix interference, then concentrates the sample solution and the fructose standard solution, increases the concentration of target reducing sugar, reduces the detection limit, and thus enhances signal strength, uses ion chromatography to separate reducing sugar and other sugar alcohols and interfering substances in the sample solution and the fructose standard solution, then uses an amperometric detector to detect the reducing sugar in the separation process, and calculates the content of reducing sugar in the sample solution through a standard curve, has high accuracy, can detect trace reducing sugar, and meets analysis requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0028] Figure 1 The figure is a flowchart of the method steps of the present invention. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0032] As attached Figure 1 As shown:

[0033] Embodiment 1: This embodiment provides a method for detecting trace reducing sugars in mannitol based on ion chromatography, comprising:

[0034] S1. Dissolve the mannitol sample in high-purity water, mix thoroughly to obtain a sample solution, and prepare a fructose standard solution of known concentration;

[0035] S2. Purify the obtained sample solution and fructose standard solution using an extraction column, concentrate the purified sample solution and fructose standard solution, and then filter the concentrated sample solution and fructose standard solution using a filter membrane to remove insoluble impurities;

[0036] S3, flushing the chromatographic system with an eluent until the baseline is stable, injecting the treated sample solution into the chromatograph, recording the chromatogram, and using an anion exchange ion chromatography column to separate the sample solution and the fructose standard solution, respectively, to obtain sugar compounds in the sample solution and sugar compounds in the standard solution;

[0037] S4. Using an amperometric detector to detect the sugar compounds in the separation process, and establishing a standard working curve based on the signal output by the amperometric detector, the content of reducing sugar in the sample solution is calculated using the standard working curve.

[0038] The fat-soluble impurities, such as protein, fat, etc., in the sample solution and the fructose standard solution are removed by an extraction column to avoid contamination of the ion chromatography column and the detector, thereby improving the cleanliness of the sample solution and the fructose standard solution, reducing matrix interference, and improving detection sensitivity and accuracy. The sample solution and the fructose standard solution are then concentrated to increase the concentration of the target reducing sugar, reduce the detection limit, thereby enhancing the signal strength, and further improving the detection sensitivity of reducing sugar. The sample solution and the fructose standard solution are then separated into reducing sugars and other sugar alcohols and interfering substances by ion chromatography, and the reducing sugars in the separation process are then detected by an amperometric detector. A standard curve is drawn with the peak area or peak height of the fructose standard solution as the ordinate and the concentration as the abscissa, and the content of reducing sugars in the sample solution is calculated by the standard curve based on the peak area or peak height of the sample solution.

[0039] Specifically, the extraction column in step S2 is a C18 SPE column, which is used to remove fat-soluble impurities in the sample solution and the standard solution.

[0040] Specifically, in step S3, the ion chromatography column is a CarboPac PA200 chromatography column, the eluent is a NaOH solution with a concentration of 100 to 200 mM, a pH value of 13.0, a flow rate of 0.3 to 0.5 mL / min, a column temperature of 30 to 40 degrees Celsius, a linear gradient elution, and a time of 30 minutes for the eluent concentration to change from 0% to 100%;

[0041] The CarboPac PA200 column is specially designed for efficient separation of sugars and sugar alcohols. It has high selectivity and high resolution, and can effectively separate reducing sugars in mannitol from other sugar alcohols and interfering substances.

[0042] The 100-200 mM NaOH solution as eluent can provide a high pH environment to dissociate sugar compounds into anions, thereby improving the separation of target reducing sugars from mannitol and other impurities.

[0043] A flow rate of 0.3 to 0.5 mL / min can extend the analysis time, improve the resolution, and reduce the detection limit;

[0044] Controlling the column temperature at 30-40 degrees Celsius can reduce the viscosity of the mobile phase, improve mass transfer efficiency, and increase sensitivity.

[0045] Specifically, the concentration method in step S2 is at least one of rotary evaporation, freeze drying and nitrogen purging, and the sample mass before and after concentration is weighed, the concentration multiple is calculated, and the stability of reducing sugars during the concentration process is verified. By concentrating agriculture, most of the solvent can be removed without losing the target reducing sugars.

[0046] Specifically, step S4 uses principal component regression to calibrate the signal output by the ampere monitor, and calibrates the test data by establishing a correction model to improve the reliability and accuracy of the test results. A strict quality control system can be established, including parallel sample analysis, spike recovery experiments, and blank experiments. By comparing the test results of different batches and different personnel, the stability and accuracy of the detection method are evaluated, and necessary adjustments and optimizations are made to the detection method based on the quality control results.

[0047] Specifically, in step S4, during the detection process of the amperometric detector, a blank solution or a standard solution is regularly injected, the baseline drift is recorded, and the baseline is corrected according to the baseline drift. The effect of the baseline correction is evaluated by comparing the baseline offset before and after the correction, and the stability and accuracy of the baseline correction are regularly monitored, thereby eliminating the influence of instrument errors and background noise on the detection results.

[0048] Specifically, the PAD potential parameter in step S4 is:

[0049] Detection potential: +0.1V;

[0050] Oxidation potential: +0.6V;

[0051] Regeneration potential: -0.8V;

[0052] The pulse period is 0.4 to 0.6 seconds, and the integration time is 0.3 to 0.5 seconds.

[0053] Specifically, in step S4, a suppressor is used to reduce the NaOH background conductivity during the amperometric detector detection process.

[0054] Specifically, in step S4, the standard working curve is drawn by taking the peak area or peak height of the sugar compound in the standard solution as the ordinate and the concentration as the abscissa.

[0055] Specifically, the concentration range of the fructose standard solution in step S1 is 0.1-10 mg / L.

[0056] Experimental example:

[0057] Weigh 1 g of mannitol sample and place it in a 100 mL volumetric flask;

[0058] Add about 80 mL of high-purity water, dissolve under ultrasonication for 15 minutes, make up to 100 mL with high-purity water, and mix well to obtain a sample solution;

[0059] Use C18 SPE cartridge for purification: activate the SPE cartridge with 5 mL of methanol; balance with 5 mL of high-purity water; load 10 mL of sample solution onto the SPE cartridge; elute with 5 mL of high-purity water and collect the eluate;

[0060] The purified sample solution was transferred to a nitrogen purge apparatus and purged with nitrogen at 40 °C until the sample volume was concentrated to 10 mL;

[0061] Weigh 100 mg of glucose and fructose respectively, place in a 100 mL volumetric flask, dissolve and dilute with high-purity water to obtain a 1000 mg / L standard stock solution, dilute the standard stock solution with high-purity water to prepare the following concentrations of standard working solutions: 0.1 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L;

[0062] Eluent: 150 mM NaOH solution, flow rate 0.3 mL / min;

[0063] Chromatographic column: CarboPac MA1 (4×250 mm), column temperature 40°C;

[0064] Suppressor: ASRS 300 anion suppressor;

[0065] PAD parameters: E1 is +0.1 V (0.40 s), E2 is +0.6 V (0.20 s), E3 is -0.8 V (0.40 s), pulse period is 0.5 s, integration time is 0.40 s, injection volume is 10 μL, and running time is 30 min;

[0066] Linear range: Glucose and fructose have good linear relationship in the concentration range of 0.1-10 mg / L, and the linear correlation coefficient (R 2 ) are all greater than 0.999;

[0067] Standard curve: The equation of glucose standard curve is y=125.6x+5.2, and the equation of fructose standard curve is y=118.3x+4.8;

[0068] Reducing sugar content in the sample: glucose content is 0.52 mg / L, fructose content is 0.31 mg / L;

[0069] The sample solution was injected six times, and the relative standard deviation (RSD) was calculated: the RSD for glucose was 3.2%, and the RSD for fructose was 3.8%;

[0070] Known amounts of glucose and fructose standards were added to the samples, and the recoveries were calculated: 98.5% for glucose and 97.8% for fructose;

[0071] Conclusion: The detection limits of glucose and fructose are both lower than 0.05 mg / L, which can meet the requirements of trace analysis. Trace reducing sugars can be detected, which meets the analysis requirements.

[0072] The results of repeatability experiments showed that the RSDs were all less than 4%, indicating that the method had good precision, and the spiked recoveries were between 97.8% and 98.5%, indicating that the method had high accuracy;

[0073] The standard curve has a good linear relationship and the sample test results are accurate and reliable.

[0074] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible (e.g., the size, scale, structure, shape and proportion of various elements, and parameter values ​​(e.g., temperature, pressure, etc.), installation arrangement, use of materials, color, directional changes, etc.) without substantially departing from the novel teachings and advantages of the subject matter described in the application. For example, the element shown as integrally formed can be composed of multiple parts or elements, the position of the element can be inverted or otherwise changed, and the nature or number or position of the discrete element can be changed or changed. Therefore, all such modifications are intended to be included in the scope of the present invention. The order or sequence of any process or method steps can be changed or reordered according to alternative embodiments. In the claims, any "device plus function" clause is intended to cover the structure of performing the function described herein, and is not only structurally equivalent but also equivalent structure. Without departing from the scope of the present invention, other replacements, modifications, changes and omissions can be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the invention is not limited to a specific embodiment, but extends to numerous modifications still falling within the scope of the appended claims.

[0075] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0076] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for detecting trace reducing sugars in mannitol based on ion chromatography, characterized in that: include: S1. Dissolve the mannitol sample in high-purity water, mix thoroughly to obtain a sample solution, and prepare a fructose standard solution of known concentration; S2. Purify the obtained sample solution and fructose standard solution using an extraction column, concentrate the purified sample solution and fructose standard solution, and then filter the concentrated sample solution and fructose standard solution using a filter membrane to remove insoluble impurities; S3, flushing the chromatographic system with an eluent until the baseline is stable, injecting the treated sample solution into the chromatograph, recording the chromatogram, and using an anion exchange ion chromatography column to separate the sample solution and the fructose standard solution, respectively, to obtain sugar compounds in the sample solution and sugar compounds in the standard solution; S4. Using an amperometric detector to detect the sugar compounds in the separation process, and establishing a standard working curve based on the signal output by the amperometric detector, the content of reducing sugar in the sample solution is calculated using the standard working curve.

2. A method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: The extraction column in step S2 is a C18 SPE column, which is used to remove fat-soluble impurities in the sample solution and the standard solution.

3. A method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: In step S3, the ion chromatography column is a CarboPac PA200 chromatography column, the eluent is a NaOH solution with a concentration of 100-200 mM, a pH value of 13.0, a flow rate of 0.3-0.5 mL / min, a column temperature controlled at 30-40 degrees Celsius, and a linear gradient elution, and the eluent concentration change time from 0% to 100% is 30 minutes.

4. The method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: The concentration method in step S2 is at least one of rotary evaporation, freeze drying and nitrogen purging, and the mass of the sample before and after concentration is weighed, the concentration multiple is calculated, and the stability of the reducing sugar during the concentration process is verified.

5. A method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: The step S4 uses principal component regression to correct the signal output by the amperometric detector.

6. The method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: In the step S4, during the detection process of the amperometric detector, blank solution or standard solution is regularly injected, the baseline drift is recorded, and the baseline is corrected according to the baseline drift.

7. The method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: The PAD potential parameter in step S4 is: Detection potential: +0.1V; Oxidation potential: +0.6V; Regeneration potential: -0.8V; The pulse period is 0.4 to 0.6 seconds, and the integration time is 0.3 to 0.5 seconds.

8. The method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: In the step S4, a suppressor is used to reduce the NaOH background conductivity during the amperometric detector detection process.

9. The method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: In step S4, the standard working curve is drawn by taking the peak area or peak height of the sugar compound in the standard solution as the ordinate and the concentration as the abscissa.

10. The method for detecting trace reducing sugars in mannitol based on ion chromatography according to claim 1, characterized in that: The concentration range of the fructose standard solution in step S1 is 0.1-10 mg / L.

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