Method for measuring sodium cyclamate content by HPLC-RID

Abstract

The invention discloses a method for determining the content of sodium cyclamate by utilizing HPLC-RID (High Performance Liquid Chromatography-Resistance Identifier), and belongs to the technical field of quantitative detection of substances. The method comprises the following steps: (1) respectively preparing a sodium cyclamate standard working solution and a to-be-detected sample solution; (2) sequentially carrying out chromatographic analysis and determination on the sodium cyclamate standard working solution by adopting HPLC-RID (High Performance Liquid Chromatography-Relative Identifier), and obtaining a standard curve of sodium cyclamate; (3) carrying out chromatographic analysis determination on the sample solution to be detected by adopting HPLC-RID, carrying out multi-point correction according to the standard curve, and carrying out quantitative analysis on sodium cyclamate in the sample solution to be detected according to the retention time and peak area of sodium cyclamate in a chromatogram to obtain a detection result; according to the method, the sodium cyclamate can be directly and quantitatively detected through the differential refraction detector by specifically adjusting the mobile phase, the chromatographic column type and the column temperature of the liquid chromatography, and derivation treatment on the sodium cyclamate is not needed, so that the determination steps are simplified, the detection period is shortened, and the detection accuracy is improved; the method is suitable for large-scale popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of quantitative detection of substances, specifically to the quantitative detection of sodium cyclamate, and particularly to a method for determining the content of sodium cyclamate by HPLC-RID. Background Art

[0002] Sodium cyclamate, with the chemical name sodium cyclohexanesulfamate, foreign name Sodium cyclamate, alias sodium cyclohexylsulfamate, sodium cyclamate, is an organic compound with the chemical formula C 6 H 12 NNaO 3 S, molecular weight of 201.219, CAS registry number 139-05-9, EINECS registry number 205-348-9, melting point 265 °C, density 1.32 g / cm 3 , appearance is white powder or white crystal, is a commonly used sweetener, and its sweetness is 30-40 times that of sucrose. In 1969, it was found that rats fed with saccharin-sodium cyclohexanesulfamate had bladder cancer, so in 1970, the United States and Japan successively prohibited the use of saccharin-sodium cyclohexanesulfamate. However, in subsequent continuous research, no carcinogenic effect of sodium cyclohexanesulfamate was found. After oral administration of sodium cyclohexanesulfamate by humans, 40% is excreted in urine and 60% is excreted in feces, without accumulation. Even so, in China's "Hygienic Standards for the Use of Food Additives" and "Standards for the Use of Food Additives" (GB 2760-2024), the dosage of sodium cyclamate in food processing is strictly restricted.

[0003] In existing detection standards for the content of sodium cyclamate, several conventional determination methods are given, including: the method for detecting this substance in the national standard SN / T 1948—2007 "Detection Method for Sodium Cyclohexanesulfamate in Import and Export Foods" is liquid chromatography-mass spectrometry-mass spectrometry; the method for detecting this substance in the national standard GB 1886.37—2015 "Food Additive Sodium Cyclohexanesulfamate (Also Known as Sodium Cyclamate)" is titration method; the method for detecting this substance in the national standard NY / T3473-2019 "Determination of Neotame, Alitame, Aspartame, Sodium Cyclamate, Acesulfame Potassium, Saccharin Sodium in Feeds" is liquid chromatography-tandem mass spectrometry.

[0004] The existing patent technologies also involve many methods for determining cyclamate, such as: the method used in Chinese patent CN111983011B "Method for detecting cyclamate in steamed bread based on four-way equidistant induction potential with different phases" is equidistant induction potential detection; the methods used in Chinese patent CN101799461B "Method for determining the content of sodium cyclohexylaminosulfonate in tobacco additives" and Chinese patent CN105259263B "Method for determining the content of sodium cyclohexylaminosulfonate in food" are gas chromatography; the method used in Chinese patent CN107884509B "Method for detecting compound Hedyotis diffusa granules" is thin layer chromatography; The method used in the Chinese patent CN105606757B "Method for simultaneous determination of sweeteners and preservatives in tobacco flavors" is ion chromatography; Chinese patent CN101634648B "Method for simultaneous determination of six trace sweeteners in liquor by ultra-high pressure liquid chromatography-time-of-flight mass spectrometry", Chinese patent CN103293240B "Method for simultaneous detection of salicylic acid, aristolochic acid A, cyclamate and β-naphthol", Chinese patent CN115266991B "Method for simultaneous detection of multiple sweeteners in rice wine", Chinese patent CN104634895B "A method for simultaneous detection of liquor by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry" The instruments used in these 9 patents are: "A method for detecting 6 kinds of sweeteners in milk or dairy products", "A method for detecting sweeteners in milk or dairy products" by Chinese patent CN113092651B, "A method for detecting cyclamate in liquor" by Chinese patent CN108508128A, "A method for detecting cyclamate in liquor" by Chinese patent CN108872448B, "A method for detecting 5 kinds of sweeteners in tobacco flavors by ultra-high performance liquid chromatography-tandem mass spectrometry", "A method for simultaneously detecting 6 kinds of synthetic sweeteners in tea" by Chinese patent CN109490452B, "A method for simultaneously detecting 6 kinds of synthetic sweeteners in tea" by Chinese patent CN110824064B, "A method for determining the main sweeteners in edible flavors and spices by HPLC-MS / MS", all of which use the same method. It is a high-performance liquid chromatography coupled with various mass spectrometers. The instrument is expensive, and the detector used is also an ultraviolet detector. Chinese patent CN105628818B "Method for simultaneous detection of six sweeteners in feed additives by high-performance liquid chromatography" discloses a method for detecting six sweeteners in feed additives by high-performance liquid chromatography. This method is cheap and fast, but has poor specificity and sensitivity, and is also detected by ultraviolet detector. Chinese patent CN111141855B "A method for detecting sodium cyclamate in acetylcysteine ​​oral solution" uses high-performance liquid chromatography, which not only requires gradient elution, but also uses an ultraviolet detector. It can be seen that in the existing quantitative detection methods of sodium cyclamate liquid chromatography, ultraviolet detectors are used for detection.

[0005] However, sodium cyclamate itself has weak absorption of ultraviolet light, so it cannot be directly detected by using the absorption characteristics of ultraviolet or visible light, that is, the content of sodium cyclamate cannot be directly determined by liquid chromatography-ultraviolet detector. Instead, through appropriate derivatization treatment, sodium cyclamate has good ultraviolet or visible light absorption characteristics, and then indirect determination can be carried out by using liquid chromatography-ultraviolet detector. However, when carrying out derivatization treatment, not only does it increase the complexity of the determination method, prolong the detection time, but also introduces more influencing factors, resulting in a decrease in the accuracy of the detection result. Obviously, it is not conducive to accurately and quickly determining the content of sodium cyclamate. Therefore, there is an urgent need to develop a simple, effective, and highly operable method for directly determining sodium cyclamate in feed flavorants, which can avoid the limitations of instrument detectors and is more suitable for large-scale popularization and application. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the existing quantitative detection methods for sodium cyclamate, such as complex process, low accuracy, and inability to directly measure, and provide a method for determining the content of sodium cyclamate by using HPLC-RID.

[0007] A method for determining the content of sodium cyclamate by using HPLC-RID includes the following steps:

[0008] (1) Prepare a sodium cyclamate standard working solution and a sample solution to be measured respectively;

[0009] (2) Use HPLC-RID to conduct chromatographic analysis and determination on the sodium cyclamate standard working solution in sequence, and perform linear regression processing on the peak area of sodium cyclamate in the determination result and its corresponding mass concentration to obtain a linear regression equation; use the curve corresponding to this linear regression equation as the standard curve of sodium cyclamate;

[0010] (3) Use HPLC-RID to conduct chromatographic analysis and determination on the sample solution to be measured, and perform multi-point calibration according to the standard curve, and conduct quantitative analysis on the sodium cyclamate in the sample solution to be measured based on the retention time and peak area of sodium cyclamate in the chromatogram to obtain the detection result.

[0011] The method for determining the content of sodium cyclamate by using HPLC-RID in the present invention enables sodium cyclamate to be directly quantitatively detected by a differential refractive index detector through targeted adjustment of the mobile phase, chromatographic column type, and column temperature of liquid chromatography, without the need for further derivatization treatment of sodium cyclamate. Thus, it simplifies the determination steps, shortens the detection cycle, improves the detection accuracy, and effectively avoids the limitations of instrument detectors; this determination method is simple, effective, highly operable, and can directly determine sodium cyclamate in feed flavorants, and the results are accurate, making it suitable for large-scale popularization and application.

[0012] Among them, the HPLC-RID (High Performance Liquid Chromatography-Refractive Index Detection) is a liquid chromatography analysis technology based on a refractive index detector (RID), belonging to a general detection technology, which detects the target substance by measuring the change in refractive index between the sample flow path and the reference flow path. Its principle is based on the refractive index difference of light in different substances. When the sample flows out of the chromatographic column, its refractive index is different from that of the mobile phase, and this difference is captured by the detector and converted into an electrical signal.

[0013] Among them, preferably, in step (1), the preparation method of the sodium cyclamate standard working solution is as follows: first, prepare a sodium cyclamate standard stock solution using a sodium cyclamate standard product, and then prepare a sodium cyclamate standard working solution using the sodium cyclamate standard stock solution.

[0014] Preferably, the purity of the sodium cyclamate standard product is not less than 98%.

[0015] Preferably, the concentration of the sodium cyclamate standard stock solution is 1.0 mg / mL; the storage time is not less than 1 month, and the storage temperature is not higher than 4°C.

[0016] Among them, preferably, in step (1), the concentrations of the sodium cyclamate standard working solutions are 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, 100.0 μg / mL, and 200.0 μg / mL respectively.

[0017] Among them, preferably, in step (1), the preparation method of the sample solution to be tested includes: ultrasonically assisting in dissolving an appropriate amount of the sample to be tested with distilled water, and after forming a solution, filtering it with a hydrophilic filter membrane to obtain the sample solution to be tested.

[0018] Preferably, the ultrasonic-assisted dissolution time is 10 - 20 min; this makes the dissolution of the sample to be tested better and the dissolution effect of sodium cyclamate better.

[0019] Preferably, the pore size of the hydrophilic filter membrane is not greater than 0.22 μm; more preferably, the pore size of the hydrophilic filter membrane is 0.1 - 0.2 μm; the filtering effect is better, reducing the influence of impurities on the results.

[0020] Among them, preferably, in steps (2) and (3), the chromatographic conditions of the HPLC-RID are as follows:

[0021] Chromatographic column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm);

[0022] Mobile phase: Methanol aqueous solution with a volume fraction of 10%;

[0023] Flow rate: 1.0 mL / min;

[0024] Detector temperature: 40 °C.

[0025] Column temperature: 30 ± 0.8 °C.

[0026] Using 10% methanol as the mobile phase is more cost-effective than acetonitrile, with good repeatability and accuracy. It can achieve accurate and rapid detection of sodium cyclamate, better meet the actual detection needs, and the unique mobile phase ratio makes the separation effect of sodium cyclamate more excellent.

[0027] Among them, preferably, in step (3), the content of sodium cyclamate in the sample solution to be measured is calculated by the following formula:

[0028] X = (C × V) / (m × 1000) × 100%;

[0029] In the formula, X is the content of sodium cyclamate in the sample solution to be measured (%); C is the concentration of sodium cyclamate in the sample obtained from the standard curve, mg / mL; V is the total volume of the sample solution to be measured (mL); m is the mass of the sample to be measured (g).

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] The method for determining the content of sodium cyclamate in the present invention enables sodium cyclamate to be directly quantitatively detected by a differential refractive index detector through targeted adjustment of the mobile phase, column type, and column temperature of liquid chromatography, without the need for derivatization of sodium cyclamate. This simplifies the determination steps, shortens the detection cycle, improves the detection accuracy, and effectively avoids the limitations of the instrument detector; this determination method is simple, effective, easy to operate, and can directly determine the sodium cyclamate in feed flavorings, with accurate results and suitable for large-scale popularization and application. Description of the Drawings

[0032] Figure 1 It is the chromatogram of the sodium cyclamate standard working solution in Example 1 of the present invention;

[0033] Figure 2 It is the standard curve graph of sodium cyclamate obtained by measuring the sodium cyclamate standard working solution in Example 1 of the present invention;

[0034] Figure 3 It is the chromatogram of the sample solution 1 to be measured in Example 1 of the present invention;

[0035] Figure 4 It is the chromatogram of the sample solution 2 to be measured in Example 1 of the present invention;

[0036] Figure 5 It is the chromatogram of the sample solution 3 to be measured in Example 1 of the present invention;

[0037] Figure 6 It is the chromatogram of the sample solution 4 to be measured in Example 1 of the present invention;

[0038] Figure 7 It is the chromatogram of the sample solution 5 to be measured in Example 1 of the present invention;

[0039] Figure 8 It is the chromatogram of the sample solution 6 to be measured in Example 1 of the present invention;

[0040] Figure 9 It is the chromatogram of the sample solution 7 to be measured in Example 1 of the present invention;

[0041] Figure 10 It is the chromatogram of the sample solution 1 to be measured in Comparative Example 1 of the present invention;

[0042] Figure 11 It is the chromatogram of the sample solution 1 to be measured in Comparative Example 2 of the present invention;

[0043] Figure 12 It is the chromatogram of the sample solution 1 to be measured in Comparative Example 3 of the present invention;

[0044] Figure 13 It is the chromatogram of the sample solution 1 to be measured in Comparative Example 4 of the present invention;

[0045] Figure 14 It is the chromatogram of the sample solution 1 to be measured in Comparative Example 5 of the present invention. Detailed implementation manners

[0046] The present invention will be further described in detail below in conjunction with test examples and specific implementation manners. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0047] Example 1

[0048] The content of sodium cyclamate in feed additives was determined by HPLC-RID.

[0049] Reagents and materials: Methanol: Chromatographically pure; Water: Grade 1 water specified in GB / T6682; Sodium cyclamate standard: Purity > 98%;

[0050] Instruments and equipment: Commonly used laboratory instruments and equipment, such as volumetric flasks, pipettes, measuring cups, etc.; Analytical balance: Sensitivity 0.0001 g; High-performance liquid chromatograph equipped with an RID detector; Ultrasonic cleaner.

[0051] The specific detection steps are as follows:

[0052] (1) Preparation of standard stock solution: Accurately weigh 0.1000 g of sodium cyclamate into a 100 mL volumetric flask, add distilled water, ultrasonically dissolve until completely dissolved, then cool and make up to the mark. This sample is the standard stock solution with a concentration of 1.0 mg / mL. This solution is stored at 4 °C with a shelf life of 1 month.

[0053] Preparation of standard working solutions: Accurately measure 0.1 mL, 0.2 mL, 0.4 mL, 1.0 mL, 2.0 mL, 5 mL, 10 mL, and 20 mL of the standard stock solution into separate 100 mL volumetric flasks, dilute to the mark with distilled water, and mix well to obtain standard working solutions with concentrations of 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, 100.0 μg / mL, and 200.0 μg / mL respectively. These solutions are prepared freshly before use.

[0054] Preparation of sample solution to be tested: Accurately weigh 2.0 g (accurate to 0.0001 g) of the sample (7 samples) into a 100 mL volumetric flask, add 50 mL of distilled water, ultrasonically treat for 20 minutes, cool and make up to 100 mL, shake well, and filter through a 0.22 μm aqueous filter membrane to obtain the sample solution to be tested.

[0055] (2) Perform chromatographic analysis and determination on the standard working solutions of sodium cyclamate in sequence using HPLC-RID, and perform linear regression on the peak area of sodium cyclamate in the measured chromatogram ( Figure 1 ) and its corresponding mass concentration to obtain a linear regression equation. Use the curve corresponding to this linear regression equation as the standard curve of sodium cyclamate (Table 1, Figure 2 ). Accurately measure 20 μL of 1.0 μg / mL and 2.0 μg / mL sodium cyclamate standard working solutions and inject them into the liquid chromatograph, inject continuously for 6 times, calculate the measured concentration of sodium cyclamate according to the standard curve, and then calculate the standard deviation of the 6 measured concentrations. Three times the standard deviation is the detection limit of sodium cyclamate in this method.

[0056] Table 1 Linear regression equation (standard curve), correlation coefficient, and detection limit of sodium cyclamate

[0057]

[0058]

[0059] Note: In the linear equation, y is the peak area corresponding to sodium cyclamate in the standard working solution, and x is the mass concentration of sodium cyclamate in the standard working solution.

[0060] The chromatographic conditions of the above HPLC-RID are as follows:

[0061] Chromatographic column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm);

[0062] Mobile phase: Methanol aqueous solution with a volume fraction of 10%;

[0063] Flow rate: 1.0 mL / min;

[0064] Detector temperature: 40 °C.

[0065] Column temperature: 30 ± 0.8 °C;

[0066] (3) Use HPLC-RID to perform chromatographic analysis and determination on the sample solution to be measured (7 samples to be measured, each sample is repeated 3 times), obtain the corresponding chromatograms (see Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 in turn), and perform multi-point calibration according to the standard curve. Use the retention time and peak area of sodium cyclamate in the chromatogram to perform quantitative analysis on sodium cyclamate in the sample solution to be measured, and obtain the test results (see Table 2, Table 3, Table 4);

[0067] Among them, the content of sodium cyclamate in the sample solution to be measured is calculated by the following formula:

[0068] X = (C × V) / (m × 1000) × 100%;

[0069] In the formula, X is the content of sodium cyclamate in the sample solution to be measured (%); C is the concentration of sodium cyclamate in the sample obtained from the standard curve, mg / mL; V is the total volume of the sample solution to be measured (mL); m is the mass of the sample to be measured (g).

[0070] Table 2 Content of sodium cyclamate in the sample solution to be measured (the 1st time)

[0071]

[0072]

[0073] Table 3 Content of sodium cyclamate in the sample solution to be measured (the 2nd time)

[0074] Name Sample Weight (g) Concentration (μg / mL) Peak Area Content of Sodium Cyclamate Retention Time Sample 1 0.7781 57.336 87529.7 7.37％ 4.259min Sample 2 0.8361 47.052 71552.3 5.63％ 4.242min Sample 3 0.9031 10.926 15429.7 1.21％ 4.149min Sample 4 0.8379 7.89 10712.1 0.94％ 4.146min Sample 5 0.8837 118.035 181828.5 13.36％ 4.321min Sample 6 0.5569 12.889 18479.7 2.31％ 4.150min Sample 7 0.6495 3.352 3663.4 0.52％ 4.159min

[0075] Table 4 Content of sodium cyclamate in the sample solution to be measured (the 3rd time)

[0076] Name Sample Weight (g) Concentration (μg / mL) Peak Area Content of Sodium Cyclamate Retention Time Sample 1 0.7543 56.768 85820.9 7.53％ 4.244min Sample 2 0.8015 44.856 67369.2 5.60％ 4.231min Sample 3 0.7721 7.42 9384.4 0.96％ 4.133min Sample 4 0.8379 9.027 11874 1.08％ 4.149min Sample 5 0.9266 123.608 189349.3 13.34％ 4.309min Sample 6 0.6560 16.642 23669.1 2.54％ 4.142min Sample 7 0.5331 2.434 1661.6 0.46％ 4.201min

[0077] Example 2

[0078] Repeatability experiment of the method for determining the content of sodium cyclamate in the present invention:

[0079] Using the same method and parameters as in Example 1, the test sample solution 5 was detected 3 times, with 6 consecutive injections each time, and the relative standard deviation (RSD) of the measured sodium cyclamate content was calculated. The results are shown in Table 5, Table 6, and Table 7;

[0080] Table 5. Relative standard deviation of sodium cyclamate content in test sample solution 5 (the 1st time)

[0081]

[0082]

[0083] Table 6. Relative standard deviation of sodium cyclamate content in test sample solution 5 (the 2nd time)

[0084]

[0085] Table 7. Relative standard deviation of sodium cyclamate content in test sample solution 5 (the 3rd time)

[0086]

[0087] The results show that the relative standard deviations of sodium cyclamate in sample 5 are 1.94%, 2.33%, and 1.93% respectively, all less than 5%. It can be seen that the measurement results of the method of the present invention have good repeatability.

[0088] Example 3

[0089] Precision experiment of the method for determining sodium cyclamate content of the present invention:

[0090] Using the same method and parameters as in Example 1, 6 parallel determinations were carried out on test sample solution 5 and test sample solution 7, and the results are shown in Table 8 and Table 9;

[0091] Table 8. Results of precision test of sodium cyclamate method for test sample solution 5

[0092]

[0093] Table 9. Results of precision test of sodium cyclamate method for test sample solution 7

[0094]

[0095] The results show that according to the above method and conditions, the relative standard deviations of sodium cyclamate in low-content and high-content flavoring agent samples are 1.23% and 4.46% respectively, both less than 5%, and the precision test has good results.

[0096] Example 4

[0097] Recovery experiment of the method for determining sodium cyclamate content of the present invention:

[0098] Using the same method and parameters as in Example 1, compounds such as blank silica, sodium sulfate, and glucose were used as carriers to conduct a spike recovery test on the sample. Six parallel determinations were carried out on 2 spiked samples and repeated 3 times. The spike recovery rate and average recovery rate were calculated, and the results are shown in Table 10;

[0099] Table 10. Results of the recovery test

[0100]

[0101]

[0102]

[0103] The results show that according to the above method and conditions, the average recovery rate of sodium cyclamate is between 95.68% and 108.61%. This indicates that this method has good precision and accuracy for samples of different types, different contents, and determinations at different times.

[0104] Comparative Example 1:

[0105] The method for determining the sodium cyclamate content in Example 1 of the present invention was used to detect the sample solution 1 to be tested, with the only difference being that an aqueous acetonitrile solution with a volume fraction of 10% was used as the mobile phase;

[0106] The chromatographic results are as Figure 10 , analysis Figure 10 and Figure 3 show that due to the change in the chromatographic mobile phase, the differential refractive index detector cannot detect sodium cyclamate, and there is no characteristic peak of sodium cyclamate in the chromatogram, so the quantitative detection of sodium cyclamate cannot be achieved.

[0107] Comparative Example 2:

[0108] The method for determining the sodium cyclamate content in Example 1 of the present invention was used to detect the sample solution 1 to be tested, with the only difference being that the column temperature was 25 °C.

[0109] The chromatographic results are as Figure 11 , analysis Figure 11 and Figure 3 show that due to the too low column temperature, the capture effect of the differential refractive index detector on sodium cyclamate is significantly deteriorated, and 2 characteristic peaks appear in the chromatogram, resulting in a significant reduction in the accuracy of the quantitative detection result of sodium cyclamate.

[0110] Comparative Example 3:

[0111] The method for determining the sodium cyclamate content in Example 1 of the present invention was used to detect the sample solution 1 to be tested, with the only difference being that the column temperature was 35 °C.

[0112] The chromatographic results are asFigure 12 , analysis Figure 12 and Figure 3 it can be seen that due to the too high temperature of the chromatographic column, the capture effect of the differential refractive index detector on sodium cyclamate has significantly deteriorated, and no characteristic peak appears in the chromatogram, resulting in the inability to quantitatively detect sodium cyclamate.

[0113] Comparative Example 4:

[0114] The method for determining the content of sodium cyclamate in Example 1 of the present invention was used to detect the sample solution 1 to be tested, with the only difference being that an aqueous methanol solution with a volume fraction of 8% was used as the mobile phase.

[0115] The chromatographic results are as Figure 13 , analysis Figure 13 and Figure 3 it can be seen that due to the too low methanol concentration in the mobile phase, the capture effect of the differential refractive index detector on sodium cyclamate has significantly deteriorated. Although 1 characteristic peak appears in the chromatogram, the area of the characteristic peak has significantly decreased, resulting in a significant decrease in the accuracy of the quantitative detection result of sodium cyclamate.

[0116] Comparative Example 5:

[0117] The method for determining the content of sodium cyclamate in Example 1 of the present invention was used to detect the sample solution 1 to be tested, with the only difference being that an aqueous methanol solution with a volume fraction of 12% was used as the mobile phase.

[0118] The chromatographic results are as Figure 14 , analysis Figure 14 and Figure 3 it can be seen that due to the too high methanol concentration in the mobile phase, the capture effect of the differential refractive index detector on sodium cyclamate has significantly deteriorated. Two characteristic peaks appear in the chromatogram, resulting in a significant decrease in the accuracy of the quantitative detection result of sodium cyclamate.

[0119] According to the detection results of Comparative Examples 1-5, it can be seen that the present invention enables the direct quantitative detection of sodium cyclamate through a differential refractive index detector by specifically adjusting the mobile phase, the type of chromatographic column, and the column temperature of the liquid chromatography, without the need for further derivatization of sodium cyclamate. Arbitrary adjustment of the chromatographic parameters may lead to a significant decrease in the accuracy of the detection results or the inability to detect sodium cyclamate.

Claims

1. A method for determining the content of sodium cyclamate using HPLC-RID, characterized in that: The following steps are involved: (1) Prepare the sodium cyclamate standard working solution and the sample solution to be tested respectively; (2) using HPLC-RID to perform chromatographic analysis on the standard working solution of cyclamate in turn, and performing linear regression processing on the peak area of ​​cyclamate in the measurement results and its corresponding mass concentration to obtain a linear regression equation; and using the curve corresponding to the linear regression equation as the standard curve of cyclamate; (3) using HPLC-RID to perform chromatographic analysis on the sample solution to be tested, and performing multi-point calibration according to the standard curve, and performing quantitative analysis on the cyclamate in the sample solution to be tested based on the retention time and peak area of ​​cyclamate in the chromatogram to obtain the test result; Wherein, in steps (2) and (3), the chromatographic conditions of the HPLC-RID are: Chromatographic column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: 10% methanol-water solution; Flow rate: 1.0 mL / min; Detector temperature: 40°C. Column temperature: 30±0.8℃.

2. The method for measuring sodium cyclamate content according to claim 1, characterized in that: In step (1), the preparation method of the cyclamate standard working solution is: first use the cyclamate standard product to prepare the cyclamate standard reserve solution, and then use the cyclamate standard reserve solution to prepare the cyclamate standard working solution.

3. The method for measuring sodium cyclamate content according to claim 2, characterized in that: The purity of the cyclamate standard product is not less than 98%.

4. The method for measuring sodium cyclamate content according to claim 2, characterized in that: The concentration of the cyclamate standard stock solution is 1.0 mg / mL; the storage time is not less than 1 month, and the storage temperature is not higher than 4°C.

5. The method for measuring sodium cyclamate content according to claim 1, characterized in that: In step (1), the concentrations of the cyclamate standard working solutions are 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, 100.0 μg / mL, and 200.0 μg / mL, respectively.

6. The method for measuring sodium cyclamate content according to claim 1, characterized in that: The method for preparing the test sample solution comprises: dissolving an appropriate amount of the test sample with distilled water by ultrasonic assistance, and after forming a solution, filtering with a water filter membrane to obtain the test sample solution.

7. The method for measuring sodium cyclamate content according to claim 6, characterized in that: The pore size of the water filter membrane is no greater than 0.22 μm.

8. The method for measuring sodium cyclamate content according to claim 7, characterized in that: The pore size of the water filter membrane is 0.1-0.2 μm.

9. The method for measuring sodium cyclamate content according to claim 1, characterized in that: In step (3), the content of sodium cyclamate in the sample solution to be tested is calculated by the following formula: X=(C×V) / (m×1000)×100%; In the formula, X is the content of cyclamate in the sample solution to be tested (%); C is the concentration of cyclamate in the sample obtained by the standard curve, mg / mL; V is the total volume of the sample solution to be tested (mL); and m is the mass of the sample to be tested (g).

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