HPLC (High Performance Liquid Chromatography) analysis method suitable for stearoyl sodium glutamate

Abstract

The invention belongs to the technical field of chemistry, and particularly provides an HPLC (High Performance Liquid Chromatography) analysis method applicable to sodium stearoyl glutamate, which is used for detecting the content of sodium stearoyl glutamate in other sodium acyl glutamate by using a two-dimensional chromatographic column switching HPLC analysis method. The two-dimensional column switching HPLC analysis method comprises the following steps: preparing a mobile phase, preparing a mixed sample solution, and detecting the prepared mixed sample solution by adopting the two-dimensional column switching HPLC analysis method to obtain the content of sodium stearoyl glutamate. According to the method provided by the invention, the content of the stearoyl sodium glutamate mixed in other acyl sodium glutamate can be accurately measured.

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, in particular to an HPLC analysis method suitable for sodium stearoyl glutamate. Background Art

[0002] Multidimensional liquid chromatography (MDLC) is a key development in liquid chromatography. It connects multiple chromatographic systems with different separation mechanisms, improving the separation capabilities of the chromatographic system to meet the separation requirements of complex samples. Among multidimensional chromatographic separation methods, 2D chromatography is the most commonly used. 2D-LC coupling typically involves connecting two independent chromatographic columns with different separation mechanisms in series. Switching valves enable connections between different columns, sharing multiple columns with detectors, and changing the flow direction of the mobile phase. Switching valves allow for flexible mobile phase changes and chromatographic mode switching, also known as column switching. 2D-LC can transfer several or all components separated by the first-dimensional column to the second-dimensional column for further analysis, processing and integrating the data from both analyses. Compared to conventional 1D-LC, 2D-LC offers double the peak capacity. Its online, automated operation significantly simplifies sample preparation, making it particularly suitable for the analysis of trace and complex samples.

[0003] Depending on whether the transferred components are directly transferred to the second dimension, methods can be categorized as either offline or online. Offline analysis involves manually collecting components separated by the first column and then injecting them into a second column for analysis. Online analysis involves directly transferring the target components from the first-dimension elution fraction to the second dimension, or using a collection and switching device to collect the first-dimension components for secondary analysis. The switching mode can be further categorized as partial or full switching, encompassing simple heart-cutting, multi-component heart-cutting, and comprehensive two-dimensional analysis.

[0004] Sodium acylglutamates are a versatile class of surfactants with excellent emulsifying, stabilizing, and moisturizing properties. They are widely used in food, cosmetics, and personal care products to enhance product stability and performance. By rationally selecting and utilizing sodium acylglutamates, product performance can be further optimized to meet the needs of diverse applications. Common sodium acylglutamates include sodium stearoyl glutamate, sodium lauroyl glutamate, sodium myristoyl glutamate, sodium palmitoyl glutamate, sodium oleoyl glutamate, sodium linoleoyl glutamate, and sodium α-linolenoyl glutamate.

[0005] Compared to other sodium acylglutamates, sodium stearoylglutamate is widely used and has a good safety profile. Therefore, it has been designated a "Generally Recognized as Safe" (GRAS) food additive by the U.S. Food and Drug Administration (FDA) and is suitable for use in the food industry. As sodium stearoylglutamate becomes increasingly popular as a daily chemical ingredient, its market demand is growing. Consequently, other sodium acylglutamates are being used to replace sodium stearoylglutamate. However, conventional liquid chromatography methods make it difficult to distinguish the characteristic peaks of these other sodium acylglutamates from those of sodium stearoylglutamate.

[0006] Under the premise that the sensitivity of conventional liquid chromatography cannot meet the detection requirements of sodium stearoyl glutamate, there is an urgent need for a method for detecting sodium stearoyl glutamate in a mixture containing sodium stearoyl glutamate and at least one other sodium acyl glutamate using multidimensional liquid chromatography. Summary of the Invention

[0007] In order to solve the problems in the prior art, the present invention aims to provide an HPLC analysis method suitable for sodium stearoyl glutamate.

[0008] In order to achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0009] According to a first aspect of the present invention, there is provided an HPLC analysis method for sodium stearoyl glutamate, wherein the HPLC analysis method is to detect the content of sodium stearoyl glutamate in a mixture comprising sodium stearoyl glutamate and at least one other sodium acyl glutamate using a two-dimensional chromatographic column switching HPLC analysis method, wherein the two-dimensional chromatographic column switching HPLC analysis method comprises the following steps:

[0010] Step 1: Prepare the first-dimension mobile phase and the second-dimension mobile phase;

[0011] Step 2: dissolving the sample to be tested in an aqueous solution containing 15%-20% methanol to obtain a mixed sample solution; the sample to be tested is a mixture of sodium stearoyl glutamate and at least one other sodium acyl glutamate, wherein the other sodium acyl glutamate is selected from at least one of sodium N-fatty acyl glutamate, sodium octanoyl glutamate, sodium hydrogenated tallow glutamate, sodium lauroyl glutamate, sodium cocoyl glutamate, sodium N-acetyl glutamate, sodium sebacoyl glutamate, and sodium N-benzoyl glutamate;

[0012] Step 3: using a two-dimensional column switching HPLC analysis method to detect the mixed sample solution prepared in step 2 to obtain the content of the sodium stearoyl glutamate;

[0013] Wherein, in the two-dimensional column switching HPLC analysis method, both the one-dimensional column and the two-dimensional column are C18 chromatographic columns.

[0014] In one aspect of the present invention, the first-dimensional mobile phase is composed of a first mixed organic solution containing a first additive and an aqueous potassium dihydrogen phosphate solution; the first mixed organic solution contains isopropanol and acetonitrile, and the volume ratio of the isopropanol and acetonitrile is selected from 1:4-8; the first additive is selected from ammonium trifluoroacetate.

[0015] In one aspect of the present invention, preferably, the first-dimensional mobile phase is composed of a first mixed organic solution containing a first additive and an aqueous potassium dihydrogen phosphate solution; the first mixed organic solution contains isopropanol and acetonitrile, and the volume ratio of isopropanol to acetonitrile is selected from 1:5-6; the first additive is selected from ammonium trifluoroacetate.

[0016] In one aspect of the present invention, specifically, the first-dimensional mobile phase is composed of a first mixed organic solution containing a first additive and an aqueous potassium dihydrogen phosphate solution; the first mixed organic solution contains isopropanol and acetonitrile, and the volume ratio of isopropanol to acetonitrile is selected from 1:5.5; the first additive is selected from ammonium trifluoroacetate.

[0017] In one aspect of the present invention, based on the total volume of the first-dimensional mobile phase, the volume of the potassium dihydrogen phosphate aqueous solution is 15%-30%; the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is selected from 15-25 mmol / L.

[0018] In one aspect of the present invention, specifically, based on the total volume of the first-dimensional mobile phase, the volume of the potassium dihydrogen phosphate aqueous solution is 25%; and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is selected from 20 mmol / L.

[0019] In one aspect of the present invention, based on the total mass of the first mixed organic solution, the mass percentage of the ammonium trifluoroacetate is selected from 2% to 8%.

[0020] In one aspect of the present invention, preferably, based on the total mass of the first mixed organic solution, the mass percentage of the ammonium trifluoroacetate is selected from 4% to 6%.

[0021] In one aspect of the present invention, specifically, based on the total mass of the first mixed organic solution, the mass percentage of the ammonium trifluoroacetate is selected from 5.5%.

[0022] In one aspect of the present invention, the second-dimensional mobile phase is a second mixed organic solution containing a second additive; the second mixed organic solution contains n-hexane and acetone, and the volume ratio of n-hexane to acetone is selected from 1:15-20; the additive is selected from tetrafluorosuccinic acid.

[0023] In one aspect of the present invention, specifically, the second-dimensional mobile phase is a second mixed organic solution containing a second additive; the second mixed organic solution contains n-hexane and acetone, and the volume ratio of n-hexane to acetone is selected from 1:16; the additive is selected from tetrafluorosuccinic acid.

[0024] In one aspect of the present invention, based on the total mass of the second mixed organic solution, the mass percentage of the tetrafluorosuccinic acid is selected from 5% to 10%.

[0025] In one aspect of the present invention, preferably, based on the total mass of the second mixed organic solution, the mass percentage of the tetrafluorosuccinic acid is selected from 7% to 9%.

[0026] In one aspect of the present invention, specifically, based on the total mass of the second mixed organic solution, the mass percentage of the tetrafluorosuccinic acid is selected from 8.5%.

[0027] In one aspect of the present invention, the other sodium acyl glutamate is at least one selected from sodium capryloyl glutamate, sodium hydrogenated tallow glutamate, sodium lauroyl glutamate and sodium cocoyl glutamate.

[0028] In one aspect of the present invention, preferably, the other sodium acyl glutamate is at least one selected from the group consisting of sodium hydrogenated tallow glutamate, sodium lauroyl glutamate and sodium cocoyl glutamate.

[0029] In one aspect of the present invention, further preferably, the other sodium acyl glutamate is selected from sodium lauroyl glutamate.

[0030] According to a second aspect of the present invention, there is provided an HPLC analysis method for sodium stearoyl glutamate, wherein the HPLC analysis method is to detect the content of sodium stearoyl glutamate in a mixture comprising sodium stearoyl glutamate and sodium lauroyl glutamate using a two-dimensional chromatographic column switching HPLC analysis method, wherein the two-dimensional chromatographic column switching HPLC analysis method comprises the following steps:

[0031] Step 1: Prepare the first-dimension mobile phase and the second-dimension mobile phase;

[0032] Step 2: dissolving the sample to be tested in an aqueous solution containing 15%-20% acetonitrile to obtain a mixed sample solution; the sample to be tested is a mixture of sodium stearoyl glutamate and sodium lauroyl glutamate;

[0033] Step 3: using a two-dimensional column switching HPLC analysis method to detect the mixed sample solution prepared in step 2 to obtain the content of the sodium stearoyl glutamate;

[0034] Wherein, in the two-dimensional column switching HPLC analysis method, the first-dimensional chromatographic column adopts a reverse-phase C8 silica gel chromatographic column, and the second-dimensional chromatographic column adopts a reverse-phase C18 silica gel chromatographic column.

[0035] In one aspect of the present invention, the two-dimensional column switching HPLC analysis method is implemented by a two-dimensional HPLC system.

[0036] In one aspect of the present invention, the two-dimensional HPLC system comprises a one-dimensional chromatographic column, a one-dimensional infusion pump, a two-dimensional chromatographic column, a two-dimensional infusion pump, an automatic sampler, an ultraviolet detector and a six-way valve; wherein the one-dimensional infusion pump is connected to the automatic sampler and the one-dimensional chromatographic column to form a flow path, and the two-dimensional infusion pump is connected to the ultraviolet detector and the two-dimensional chromatographic column to form another flow path; the two flow paths are respectively connected to two positions of the six-way valve.

[0037] In one aspect of the present invention, step 3 includes the following steps 3-1 to 3-3:

[0038] Step 3-1: passing the mixed sample solution prepared in step 2 through the automatic sample injector into the two-dimensional HPLC system;

[0039] Step 3-2: switching the six-way valve to obtain chromatographic peaks of other sodium acylglutamate, and then switching the six-way valve again;

[0040] Step 3-3: Switch the six-way valve to obtain the chromatographic peak of sodium stearoyl glutamate, and further obtain the content of sodium stearoyl glutamate through the calibration curve.

[0041] In one aspect of the present invention, the calibration curve of sodium stearoyl glutamate is Y=6.35×10 -1 X+8.14, r≥0.9990; r is the linear correlation coefficient; Y is the peak area of the corresponding peak in the chromatogram, in mAU·sec; X is the concentration of sodium stearoyl glutamate, in ng / mL.

[0042] In one aspect of the present invention, the detection conditions of the two-dimensional column switching HPLC analysis method are: a quantitative detection wavelength of 210 nm and a column temperature of 45°C.

[0043] The beneficial effects of the present invention are:

[0044] (1) The present invention provides a method for detecting sodium stearoyl glutamate in a mixture containing sodium stearoyl glutamate and at least one other sodium acyl glutamate using multidimensional liquid chromatography, which can accurately measure the content of sodium stearoyl glutamate in other sodium acyl glutamate.

[0045] (2) Compared with mass spectrometry, the two-dimensional column switching high performance liquid chromatography provided by the present invention requires lower cost of instruments and equipment, and lower cost of reagents and consumables.

[0046] (3) The two-dimensional column switching high performance liquid chromatography method provided by the present invention does not require repeated determination of the calibration curve, further saving time and reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the connection method of the two-dimensional high performance liquid chromatography system of the present invention. DETAILED DESCRIPTION

[0048] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following is merely an illustrative description of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0049] The present invention realizes a method for determining sodium stearoyl glutamate in serum by a two-dimensional column switching high performance liquid chromatography system; Figure 1 As shown in the figure, the two-dimensional high performance liquid chromatography system includes a one-dimensional chromatographic column 13, a one-dimensional infusion pump 11, a two-dimensional chromatographic column 14, a two-dimensional infusion pump 12, an automatic sampler 31, an ultraviolet detector 41 and a six-way valve 32; the one-dimensional chromatographic column 13 and the two-dimensional infusion pump 12 are located in a column oven 51, and the system also includes a solvent tray 61 for placing the test solvent and a waste liquid barrel 62 for placing the test waste liquid.

[0050] like Figure 1 As shown, the six-way valve 32 includes six positions 1-6. In the first state, position 1 of the six-way valve is connected to position 6, the two-dimensional infusion pump 12 is connected to position 1 of the six-way valve 32, and position 6 is connected to the two-dimensional chromatographic column 14 and the ultraviolet detector 41 in sequence; the one-dimensional infusion pump 11 is connected to the automatic sampler 31 and then connected to position 3 of the six-way valve 32, position 3 of the six-way valve 32 is connected to position 2, position 2 is connected to the inlet end of the one-dimensional chromatographic column 13, the outlet end of the one-dimensional chromatographic column 13 is connected to position 5 of the six-way valve 32, position 5 of the six-way valve 32 is connected to position 4, and position 4 is connected to the waste liquid bucket 62; the state at this time is as shown in FIG. Figure 1As shown; when the six-way valve 32 changes from the first state to the second state, position 1 of the six-way valve 32 is connected to position 2; the two-dimensional infusion pump 12 is connected to position 1 of the six-way valve 32, position 2 is connected to the inlet end of the one-dimensional chromatographic column 13, the outlet end of the one-dimensional chromatographic column 13 is connected to position 5 of the six-way valve 32, position 5 is connected to position 6, and position 6 is sequentially connected to the two-dimensional chromatographic column 14 and the ultraviolet detector 41. In the present invention, after the sample is concentrated by the one-dimensional chromatographic column 13, the six-way valve 32 is used to switch the columns from the first state to the second state, and the concentrated sample is transferred from the one-dimensional chromatographic column 13 to the two-dimensional chromatographic column 14 for further separation, and finally the detection is completed.

[0051] In the present invention, the sodium stearoyl glutamate stock solution and the sodium lauroyl glutamate stock solution can be obtained by the following steps:

[0052] Accurately weigh 10 mg of sodium lauroyl glutamate and transfer it to a 10 mL volumetric flask. Add acetonitrile until the sodium lauroyl glutamate is completely dissolved, then add acetonitrile to make the volume up to 10 mL to obtain a 1.0 mg / mL sodium lauroyl glutamate stock solution, which is stored at room temperature.

[0053] Accurately weigh 10 mg of sodium stearoyl glutamate and transfer it to a 10 mL volumetric flask. Add acetonitrile until the sodium stearoyl glutamate is completely dissolved, then add acetonitrile to make the volume up to 10 mL to obtain a 1.0 mg / mL sodium stearoyl glutamate stock solution, which is stored at room temperature.

[0054] In the present invention, the calibration curve can be obtained by the following steps:

[0055] Prepare mixed working solution 1 by taking 20 μL of the 1.0 mg / mL sodium stearoyl glutamate stock solution and 20 μL of the 1.0 mg / mL sodium lauroyl glutamate stock solution and adding 960 μL of sample.

[0056] Take 100 μL of mixed working solution 1 and add 900 μL of sample to prepare mixed working solution 2.

[0057] Take 160 μL of mixed working solution 2 and add 840 μL of sample to prepare calibrator point 6.

[0058] Take 500 μL of calibrator point 6 and add 500 μL of sample to prepare calibrator point 5.

[0059] Take 500 μL of calibrator point 5 and add 500 μL of sample to prepare calibrator point 4.

[0060] Take 500 μL of calibrator point 4 and add 500 μL of sample to prepare calibrator point 3.

[0061] Take 500 μL of calibrator point 3 and add 500 μL of sample to prepare calibrator point 2.

[0062] Take 500 μL of calibrator point 2 and add 500 μL of sample to prepare calibrator point 1.

[0063] For the above calibrators No. 1 to No. 6, the relationship between concentration and peak area is constructed to obtain a calibration curve.

[0064] In the present invention, a method for determining sodium stearoyl glutamate in serum by two-dimensional column switching high performance liquid chromatography using a two-dimensional high performance liquid chromatography system comprises the following steps:

[0065] Step 1: Prepare the first-dimension mobile phase solution and the second-dimension mobile phase solution;

[0066] Step 2: preparing a sodium stearoyl glutamate stock solution and a sodium lauroyl glutamate stock solution; and using the sodium stearoyl glutamate stock solution and the sodium lauroyl glutamate stock solution to prepare a mixed working solution;

[0067] Step 3: The mixed working solution prepared in Step 2 was tested using two-dimensional column switching high-performance liquid chromatography to generate a calibration curve, thereby determining the content of sodium stearoyl glutamate in the sample to be tested. Analysis of the acquired chromatograms revealed that sodium stearoyl glutamate and sodium lauroyl glutamate were completely separated, with no interference and symmetrical peaks.

[0068] The detection conditions of the above-mentioned two-dimensional column switching high-performance liquid chromatography method are: detection wavelength 210nm, column temperature 45°C; the first-dimensional chromatographic column adopts a reversed-phase C8 silica gel chromatographic column with a specification of 5μm and ID4.6×50mm; the second-dimensional chromatographic column adopts a reversed-phase C18 silica gel chromatographic column with a specification of 5μm and ID4.6×150mm.

[0069] The experimental conditions of the aforementioned two-dimensional column switching high-performance liquid chromatography method also include: the first-dimensional mobile phase is a mixed solution formed by 20 mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 20 mmol / L potassium dihydrogen phosphate aqueous solution accounts for ≥10%; the second-dimensional mobile phase is a mixed solution formed by 20 mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 20 mmol / L potassium dihydrogen phosphate aqueous solution accounts for ≥5%.

[0070] The present invention will be further described below by way of specific examples. The various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all by weight. Unless otherwise specified, it is understood that the experiments were conducted at room temperature.

[0071] Example:

[0072] Example 1:

[0073] Example 1 includes the following steps:

[0074] Prepare a first-dimensional mobile phase and a second-dimensional mobile phase: the first-dimensional mobile phase consists of a first mixed organic solution containing ammonium trifluoroacetate and a potassium dihydrogen phosphate aqueous solution; the first mixed organic solution contains isopropanol and acetonitrile, and the volume ratio of isopropanol to acetonitrile is selected from 1:5.5; based on the total volume of the first-dimensional mobile phase, the volume of the potassium dihydrogen phosphate aqueous solution is 25%; the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is selected from 20 mmol / L.

[0075] The second-dimensional mobile phase is a second mixed organic solution containing tetrafluorosuccinic acid; the second mixed organic solution contains n-hexane and acetone, and the volume ratio of n-hexane to acetone is selected from 1:16; based on the total mass of the second mixed organic solution, the mass percentage of tetrafluorosuccinic acid is selected from 8.5%.

[0076] Accurately weigh 10 mg of sodium lauroyl glutamate and transfer it to a 10 mL volumetric flask. Add acetonitrile until the sodium lauroyl glutamate is completely dissolved, then add acetonitrile to the volume to 10 mL to obtain a 1.0 mg / mL sodium lauroyl glutamate stock solution, which is stored at room temperature. Accurately weigh 10 mg of sodium stearoyl glutamate and transfer it to a 10 mL volumetric flask. Add acetonitrile until the sodium stearoyl glutamate is completely dissolved, then add acetonitrile to the volume to 10 mL to obtain a 1.0 mg / mL sodium stearoyl glutamate stock solution, which is stored at room temperature.

[0077] Prepare mixed working solution 1 by adding 20 μL of the 1.0 mg / mL sodium stearoyl glutamate stock solution and 20 μL of the 1.0 mg / mL sodium lauroyl glutamate stock solution. Add 960 μL of sample to prepare mixed working solution 1. Prepare mixed working solution 2 by adding 100 μL of mixed working solution 1 and 900 μL of sample. Prepare mixed working solution 2 by adding 160 μL of mixed working solution 2 and 840 μL of sample to prepare calibrator point 6. Prepare calibrator point 5 by adding 500 μL of sample to prepare calibrator point 5. Prepare calibrator point 4 by adding 500 μL of sample to prepare calibrator point 4. Prepare calibrator point 3 by adding 500 μL of sample to prepare calibrator point 3. Prepare calibrator point 2 by adding 500 μL of sample to prepare calibrator point 3. 500 μL of calibrator point 2 was taken and added to 500 μL of sample to prepare calibrator point 1. Calibrator points 1 to 6 were analyzed using a two-dimensional column switching HPLC analysis method: the prepared mixed sample solution passed through the autosampler and entered the two-dimensional HPLC system. The six-way valve was switched to obtain a chromatographic peak of sodium lauroyl glutamate. The six-way valve was then switched again to obtain a chromatographic peak of sodium stearoyl glutamate. A relationship between concentration and peak area was constructed, and a calibration curve was generated using the test data. The sodium stearoyl glutamate and sodium lauroyl glutamate were completely separated, with no interference and symmetrical peak shapes.

[0078] The calibration curve was obtained using the data from Tables 1 and 2 below:

[0079] Table 1

[0080]

[0081] Table 2

[0082]

[0083] 5) Using a two-dimensional liquid chromatography (2D-LC) heart-cutting mode, 400 μL of the sample to be tested was injected into the two-dimensional liquid chromatography system for detection; wherein the first-dimensional mobile phase consisted of a first mixed organic solution comprising ammonium trifluoroacetate and an aqueous potassium dihydrogen phosphate solution; the first mixed organic solution comprised isopropanol and acetonitrile, and the volume ratio of isopropanol to acetonitrile was selected from 1:5.5; the volume of the aqueous potassium dihydrogen phosphate solution was 25% based on the total volume of the first-dimensional mobile phase; the concentration of potassium dihydrogen phosphate in the aqueous potassium dihydrogen phosphate solution was selected from 20 mmol / L; the second-dimensional mobile phase was a second mixed organic solution comprising tetrafluorosuccinic acid; the second mixed organic solution comprised n-hexane and acetone, and the volume ratio of n-hexane to acetone was selected from 1:16; and the mass percentage of tetrafluorosuccinic acid was selected from 8.5% based on the total mass of the second mixed organic solution. The detection conditions were as follows: detection wavelength 210 nm, column temperature 45°C; the first-dimension chromatographic column used a reversed-phase C8 silica gel column (Supersil C8) with a specification of 5 μm and an ID of 4.6×50 mm; the second-dimension chromatographic column used a reversed-phase C18 silica gel column (Superesil ODS-B) with a specification of 5 μm and an ID of 4.6×150 mm.

[0084] The samples were tested 7 times to test the repeatability of the method provided by the present invention; the results are shown in Tables 3 and 4 below:

[0085] Table 3

[0086]

[0087]

[0088] Table 4

[0089]

[0090] It can be seen from Table 3 and Table 4 that the method provided by the present disclosure has good repeatability.

[0091] Comparative Example 1:

[0092] The steps of Comparative Example 1 are the same as those of Example 1, except that a mixed organic phase solution of methanol:acetonitrile = 9:1 (v / v) is used in Comparative Example 1.

[0093] Comparative Example 2:

[0094] The steps of Comparative Example 2 are the same as those of Example 1, except that the one-dimensional chromatographic column used in Comparative Example 2 is Supersil C8 5μm, ID4.6×150mm, and the two-dimensional chromatographic column is Superesil ODS-B 5μm, ID4.6×250mm; they are used to replace the one-dimensional chromatographic column Supersil C8 5μm, ID4.6×50mm, and the two-dimensional chromatographic column Superesil ODS-B 5μm, ID4.6×150mm used in Example 1.

[0095] Comparative Example 3:

[0096] The steps of Comparative Example 3 are the same as those of Example 1, except that the first-dimensional mobile phase used in Comparative Example 3 is a mixed solution of 10 mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 10 mmol / L potassium dihydrogen phosphate aqueous solution accounts for 10%; the second-dimensional mobile phase is a mixed solution of 10 mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 10 mmol / L potassium dihydrogen phosphate aqueous solution accounts for 5%.

[0097] Comparative Examples 1-3 were unable to completely separate sodium stearoyl glutamate and sodium lauroyl glutamate, and interference existed between the peaks.

[0098] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the disclosure disclosed herein. This disclosure is intended to cover any variations, uses, or adaptations of the present disclosure that follow the general principles of the present disclosure and include common knowledge or customary techniques in the art not disclosed herein.

Claims

1. A HPLC analysis method suitable for sodium stearoyl glutamate, characterized in that: The HPLC analysis method is to use a two-dimensional chromatographic column switching HPLC analysis method to detect the content of sodium stearoyl glutamate in a mixture containing sodium stearoyl glutamate and at least one other sodium acyl glutamate, and the two-dimensional chromatographic column switching HPLC analysis method comprises the following steps: Step 1: Prepare the first-dimension mobile phase and the second-dimension mobile phase; Step 2: dissolving the sample to be tested in an aqueous solution containing 15%-20% methanol to obtain a mixed sample solution; the sample to be tested is a mixture of sodium stearoyl glutamate and at least one other sodium acyl glutamate, wherein the other sodium acyl glutamate is selected from at least one of sodium N-fatty acyl glutamate, sodium octanoyl glutamate, sodium hydrogenated tallow glutamate, sodium lauroyl glutamate, sodium cocoyl glutamate, sodium N-acetyl glutamate, sodium sebacoyl glutamate, and sodium N-benzoyl glutamate; Step 3: using a two-dimensional column switching HPLC analysis method to detect the mixed sample solution prepared in step 2 to obtain the content of the sodium stearoyl glutamate; Wherein, in the two-dimensional column switching HPLC analysis method, both the one-dimensional column and the two-dimensional column are C18 chromatographic columns.

2. The HPLC analysis method according to claim 1, wherein The first-dimensional mobile phase is composed of a first mixed organic solution containing a first additive and a potassium dihydrogen phosphate aqueous solution; the first mixed organic solution contains isopropanol and acetonitrile, and the volume ratio of the isopropanol and acetonitrile is selected from 1:4-8; the first additive is selected from ammonium trifluoroacetate.

3. The HPLC analysis method according to claim 2, wherein Based on the total volume of the first-dimensional mobile phase, the volume of the potassium dihydrogen phosphate aqueous solution is 15%-30%; the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is selected from 15-25 mmol / L.

4. The HPLC analysis method according to claim 2, wherein Based on the total mass of the first mixed organic solution, the mass percentage of the ammonium trifluoroacetate is selected from 2% to 8%.

5. The HPLC analysis method according to claim 1, wherein The second-dimensional mobile phase is a second mixed organic solution containing a second additive; the second mixed organic solution contains n-hexane and acetone, and the volume ratio of n-hexane to acetone is selected from 1:15-20; the additive is selected from tetrafluorosuccinic acid.

6. The HPLC analysis method according to claim 5, wherein Based on the total mass of the second mixed organic solution, the mass percentage of the tetrafluorosuccinic acid is selected from 5% to 10%.

7. The HPLC analysis method according to claim 1, wherein The other sodium acyl glutamate is selected from at least one of sodium capryloyl glutamate, sodium hydrogenated tallow glutamate, sodium lauroyl glutamate and sodium cocoyl glutamate.

8. The HPLC analysis method according to claim 1, wherein The two-dimensional column switching HPLC analysis method is implemented by a two-dimensional HPLC system.

9. The HPLC analysis method according to claim 8, wherein The two-dimensional HPLC system includes a one-dimensional chromatographic column, a one-dimensional infusion pump, a two-dimensional chromatographic column, a two-dimensional infusion pump, an automatic sampler, an ultraviolet detector and a six-way valve; wherein, the one-dimensional infusion pump is connected to the automatic sampler and the one-dimensional chromatographic column to form a flow path, and the two-dimensional infusion pump is connected to the ultraviolet detector and the two-dimensional chromatographic column to form another flow path; the two flow paths are respectively connected to the two positions of the six-way valve.

10. The HPLC analysis method according to any one of claims 1, 8-9, characterized in that Step 3 includes the following steps 3-1 to 3-3: Step 3-1: passing the mixed sample solution prepared in step 2 through the automatic sample injector into the two-dimensional HPLC system; Step 3-2: switching the six-way valve to obtain chromatographic peaks of other sodium acylglutamate, and then switching the six-way valve again; Step 3-3: Switch the six-way valve to obtain the chromatographic peak of sodium stearoyl glutamate, and further obtain the content of sodium stearoyl glutamate through the calibration curve.

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