Method for detecting purity of glucosamine and method for qualitatively determining impurities

Abstract

The present application relates to the technical field of glucosamine purity detection, and particularly relates to a glucosamine purity detection method and an impurity qualitative method. The glucosamine purity detection method is detected by using super high performance combined phase chromatography, and the detection method comprises the following steps: providing a sample solution; taking fructosazone, deoxyfructosazone and pyrrole-2-methyl formaldehyde as control samples to prepare a control sample solution; a chromatographic column is a Waters Torus DEA; a mobile phase A is CO2, and a mobile phase B is methanol, and gradient elution is performed. The present application provides a method for detecting the purity of glucosamine by using super high performance combined phase chromatography, the method uses supercritical CO2 and methanol as the mobile phase, separates the impurities in glucosamine, shortens the analysis time to 6.5 minutes, and the method can effectively separate the impurities in glucosamine, and in addition to several impurities controlled regularly, two unknown impurities are also separated.

Description

Technical Field

[0001] This invention relates to the field of glucosamine purity detection technology, specifically to methods for glucosamine purity detection and methods for impurity characterization. Background Technology

[0002] Currently, reversed-phase high-performance liquid chromatography (RP-HPLC) is commonly used for the determination of related substances in glucosamine. The mobile phase employs isocratic or gradient elution with sodium heptanesulfonate or phosphate aqueous solution and acetonitrile solution. The main impurities controlled include fructosamine, 2,5-deoxyfructosamine, 5-hydroxymethylfurfural, and 2-methylpyrazine. However, these methods, due to the presence of salts in the mobile phase, cannot be directly coupled with mass spectrometry for qualitative analysis of impurities. Furthermore, they are time-consuming, with analysis times for various glucosamine R-HPLC methods ranging from 25 to 64 minutes. The separation mechanism is also limited to R-phase chromatography, resulting in a single method. Therefore, it is necessary to develop alternative separation mechanisms to control potential impurities that cannot be separated by R-phase chromatography.

[0003] Ultra-high performance phase chromatography (UPCC) uses supercritical CO2 as the mobile phase, and its separation principle differs fundamentally from traditional liquid chromatography (HPLC). Traditional methods use liquids as the mobile phase, where compounds are separated through partitioning, adsorption / desorption, binding / elution, or other types of physical and chemical reactions between the stationary phase and the liquid mobile phase. UPCC uses supercritical CO2 as the mobile phase and a solid adsorbent or a polymer bonded to a support as the stationary phase, utilizing the solvent and molecular capacity of the mobile phase for analysis and separation. UPCC combines the advantages of gas chromatography and high performance liquid chromatography (HPLC). It can analyze low-volatility, high-boiling-point, and unstable polymers and biomolecules that are difficult to separate with gas chromatography. Compared to HPLC, it offers higher column efficiency and analytical speed, can be coupled with various detectors, and has advantages in preparative analysis. Furthermore, CO2 is inexpensive and readily available, post-processing is simple, and the method is economical and environmentally friendly. It offers fast separation speed and high analytical efficiency, especially in the analysis of chiral compounds, although it is less commonly used in the separation of carbohydrates. Summary of the Invention

[0004] This invention provides a method for detecting the purity of glucosamine using ultra-high performance phase chromatography. This method uses supercritical CO2 and methanol as the mobile phase to separate impurities in glucosamine, shortening the analysis time to 6.5 minutes. This method can effectively separate impurities in glucosamine, and in addition to several impurities that are conventionally controlled, it also separates two unknown degradation impurities.

[0005] A method for detecting the purity of glucosamine is adopted, which uses ultra-high performance phase chromatography. The detection method includes: providing a sample solution; the sample solution is a glucosamine solution, or the sample solution is a thermal degradation solution of glucosamine sulfate; and preparing a reference solution using fructosine, deoxyfructosine, and pyrrole-2-carboxaldehyde as reference standards.

[0006] The chromatographic column was a Waters Torus DEA column; mobile phase A was CO2, and mobile phase B was methanol. Elution was performed as follows:

[0007]

[0008] Furthermore, the solvent of the sample solution is composed of water, isopropanol and n-hexane in a volume ratio of (8~10):(40~50):(40~50), preferably 10:45:45.

[0009] Because the UPCC instrument operates under high back pressure, water easily freezes within the instrument, necessitating the avoidance of excessive water content in the sample solution. However, the sample solubility is low in solvents with a low aqueous phase. To improve solubility, the inventors experimented with other solvent systems such as water:acetonitrile = 10:90 and water:methanol:acetonitrile = 10:22.5:67.5. In these two solvents, I6 resolution or peak shape was poor. In some embodiments of this invention, water:isopropanol:n-hexane = 10:45:45 was selected as the sample solvent, in which the sample exhibited a higher response.

[0010] In some embodiments, the sample solution is an glucosamine solution, and the content of glucosamine sulfate is 5~15 mg / mL, preferably 10 mg / mL.

[0011] In some embodiments, the sample solution is a thermal degradation solution of glucosamine sulfate, and its content is 5-15 mg / mL, preferably 10 mg / mL, based on glucosamine sulfate before degradation.

[0012] Furthermore, the preparation method of the glucosamine sulfate thermal degradation solution includes heating the glucosamine sulfate solution in a water bath at 75~85℃ for 1~1.5 hours.

[0013] Further, the method for preparing the sample solution includes: thermally degrading the glucosamine sulfate solution (e.g., heating in a water bath at 75-85°C for 1-1.5 hours) to obtain a thermally degraded glucosamine sulfate solution; cooling (usually cooling to room temperature), mixing with isopropanol and n-hexane, filtering, and preparing the sample solution.

[0014] Specifically, the concentration of the glucosamine sulfate solution is 80~120 mg / mL, for example 100 mg / mL.

[0015] The study found that using the above-mentioned water-isopropanol-n-hexane as the sample solvent resulted in a higher sample response, which significantly improved the accuracy of the detection results.

[0016] This invention has found that when using the Waters Torus DEA column to analyze sample solutions, the separation of I6 and 5-hydroxymethylfurfural is the best, and the peak shape of I6 is also the best, which is significantly superior to other chromatographic columns.

[0017] In some specific embodiments, the chromatographic column is a Waters Torus DEA column, 3.0 mm × 100 mm, 1.7 μm.

[0018] This invention has found that using CO2 as mobile phase A and methanol as mobile phase B can improve the separation degree; if ethanol is used as mobile phase B, the elution ability of ethanol is slightly weaker than that of methanol, which can slightly improve the separation degree, but when ethanol is used, the I6 peak disappears.

[0019] Although ammonia or diethylamine is commonly used as an additive in ultra-high performance combined phase (UPCC) chromatography to improve peak shape, this invention found that adding alkali (ammonia or diethylamine) does not improve the determination of glucosamine purity in this method. In fact, adding diethylamine leads to greater baseline fluctuations, affecting peak area calculation. Therefore, this method does not add ammonia or diethylamine to the mobile phase.

[0020] Furthermore, the method of this invention can be used for detection within a relatively wide temperature range, such as 30~45℃, without significant difference in the separation effect of the components. In some specific embodiments, the detection column temperature can be 30℃, 35℃, 40℃, or 45℃, all of which can yield ideal detection results.

[0021] In some specific embodiments, the following elution is performed:

[0022]

[0023] Specifically, the flow rate is 1.0~1.5 mL / min, for example 1.2 mL / min.

[0024] Specifically, the back pressure is 1950~2020 psi, for example 2000 psi.

[0025] Specifically, the detector is a diode array detector.

[0026] Specifically, the detection wavelength range is 260~275nm and 215~225nm, for example 268nm and 220nm.

[0027] Specifically, the injection volume was 5 μL.

[0028] Specifically, the detection method further includes preparing a reference solution using fructosine, deoxyfructosine, pyrrole-2-carboxaldehyde, and 5-hydroxymethylfurfural as reference standards.

[0029] Specifically, the detection method further includes preparing a standard curve using the above-mentioned reference solution, injecting the sample solution, reference solution, and blank solution into an ultra-high performance combined phase (UPCC) chromatograph under the above-mentioned chromatographic conditions, recording the chromatograms, and calculating the content of impurities in the sample using the external standard method based on the peak areas of the reference solution and sample chromatograms.

[0030] Specifically, the blank solution is composed of ultrapure water, isopropanol and n-hexane in a volume ratio of (8~10):(40~50):(40~50), preferably 10:45:45.

[0031] Specifically, the method for detecting the purity of glucosamine includes:

[0032] Prepare sample solution;

[0033] The content of glucosamine sulfate in the sample solution was 10 mg / mL;

[0034] Alternatively, the sample solution can be prepared as follows: a 100 mg / mL glucosamine sulfate solution is heated in an 80°C water bath for 1 hour to obtain a thermally degraded glucosamine sulfate solution; after cooling, it is mixed with isopropanol and n-hexane, filtered, and the sample solution is prepared; the content of glucosamine sulfate before degradation is 10 mg / mL.

[0035] The solvent of the sample solution is composed of water, isopropanol and n-hexane in a volume ratio of 10:45:45;

[0036] Reference solutions were prepared using fructosamine, deoxyfructosamine, pyrrole-2-carboxaldehyde, and 5-hydroxymethylfurfural as reference standards, respectively. The concentrations of the fructosamine, deoxyfructosamine, and pyrrole-2-carboxaldehyde reference solutions were all 0.05–0.2 mg / mL; the concentration of the 5-hydroxymethylfurfural reference solution was 0.03–0.1 mg / mL. More specifically, reference solutions could be prepared using fructosamine, deoxyfructosamine, pyrrole-2-carboxaldehyde, and 5-hydroxymethylfurfural as reference standards, respectively. Hydroxymethylfurfural was used as a reference standard, and a reference standard stock solution was prepared. The concentrations of the fructosamine reference standard stock solution, deoxyfructosamine reference standard stock solution, and pyrrole-2-carboxaldehyde reference standard stock solution were all 10 mg / mL; the concentration of the 5-hydroxymethylfurfural reference standard stock solution was 5 mg / mL. An appropriate amount of the reference standard stock solution was taken and diluted to prepare the fructosamine reference standard solution, deoxyfructosamine reference standard solution, pyrrole-2-carboxaldehyde reference standard solution, and 5-hydroxymethylfurfural reference standard solution.

[0037] Prepare a blank solution by mixing ultrapure water, isopropanol, and n-hexane in a volume ratio of 10:45:45.

[0038] Chromatographic conditions:

[0039] The chromatographic column was a Waters Torus DEA column; mobile phase A was CO2, and mobile phase B was methanol. Elution was performed as follows:

[0040] Perform the following elution:

[0041]

[0042] Flow rate 1.2 mL / min;

[0043] Back pressure is 2000 psi;

[0044] The detector is a diode array detector;

[0045] The detection wavelength range is 268nm and 220nm;

[0046] The injection volume was 5 μL.

[0047] Under the above chromatographic conditions, the sample solution, reference solution, and blank solution were injected into an ultra-high performance combined phase (UPCC) chromatograph, and the chromatograms were recorded. Based on the peak areas in the chromatograms of the reference and sample, the content of impurities in the sample was calculated using the external standard method.

[0048] Specifically, the impurities include fructosine, deoxyfructosine, pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, C6H5NO2, 1-tolyl-3-pentanone, and 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-transbutenyl)pyrazine and its cis isomers.

[0049] The present invention utilizes an ultra-high efficiency phase chromatography separation method coupled with high resolution mass spectrometry to qualitatively detect two unknown impurities in a glucosamine thermal degradation solution.

[0050] This invention also provides a method for qualitative analysis of glucosamine impurities, comprising:

[0051] The above-described ultra-high performance phase chromatography method was used for detection;

[0052] Then mass spectrometry detection is performed.

[0053] Specifically, the mass spectrometry conditions were as follows: positive ion scanning mode, and the ESI ion source parameters were as follows: ion source temperature 120℃, capillary voltage 1kV, desolvation gas flow rate 800L / Hr, curtain gas flow rate 50L / Hr, and the mobile phase of the compensation pump was 0.1% formic acid-methanol at a flow rate of 0.3mL / min. The scan types were Full MS and MS / MS, with scan times ranging from 0 to 6 min. Full MS parameters: collision energy 10–45V, scan range 50–1200 m / z. MS / MS parameters: collision energy 30V, scan range 5–600 m / z. The m / z values ​​of the parent ions of each unknown impurity are shown in the table below.

[0054] m / z of each impurity precursor ion in UPCC-MS

[0055]

[0056] Analysis revealed that I2 and I3 were 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-transbutenyl)pyrazine and its cis isomer, respectively; I5 was C6H5NO2; and I6 was 1-tolyl-3-pentanone.

[0057] Current methods for glucosamine quality control can separate impurities that are easily degraded during storage and transportation, but the mobile phases are all salt solutions, making them incompatible with mass spectrometry. The UPCC method offers rapid separation, good separation efficiency and response for highly polar impurities (such as 5-hydroxymethylfurfural), and unique separation properties, effectively separating impurities that are difficult to separate using other chromatographic methods. It is well-suited for mass spectrometry and suitable for the study of unknown impurities, providing a new approach for the analysis of related substances in glucosamine. The UPCC method exhibits better separation performance than reversed-phase liquid chromatography for cytotoxic and genotoxic polar impurities in glucosamine (such as 5-hydroxymethylfurfural) (see [link to relevant analysis]). Figure 8 The peak shape was good and the response was high. Meanwhile, the UPCC method itself has the advantages of rapid equilibration and high separation efficiency, greatly shortening the analysis time. Using this method, unknown impurities I5 and I6, which were not separated in HPLC, were discovered. The structure of I6 was analyzed and it was speculated to be 1-tolyl-3-pentanone. Attached Figure Description

[0058] Figure 1 The UPCC chromatograms are of the blank solution and the glucosamine thermal degradation sample solution in Example 2.

[0059] Figure 2 This is the high-resolution mass spectrum of I2 in Example 2.

[0060] Figure 3 This is the high-resolution mass spectrum of I3 in Example 2.

[0061] Figure 4This is the I5 high-resolution mass spectrum from Example 2.

[0062] Figure 5 This is the high-resolution mass spectrum of I6 in Example 2.

[0063] Figure 6 The images show the ultraviolet spectra of I2 and I3 in Example 2.

[0064] Figure 7 The structural analysis results of I2 and I3 in Example 2 are shown.

[0065] Figure 8 The structural analysis results of I6 in Example 2 are shown.

[0066] Figure 9 This refers to the fragmentation process of each daughter ion of I6 in Example 2.

[0067] Figure 10 This is a diagram showing the separation effect at different column temperatures in Example 3.

[0068] Figure 11 and Figure 12 The chromatogram of the glucosamine thermal degradation solution by reversed-phase liquid chromatography in Comparative Example 1 is shown below. Figure 11 This is a magnified view of a portion of the image.

[0069] Figure 13 This is a comparative chromatogram of glucosamine thermal degradation solutions separated by different chromatographic columns in Comparative Example 2.

[0070] Figure 14 This is a comparative chromatogram of glucosamine thermal degradation solutions in different solvents, as shown in Comparative Example 3.

[0071] Figure 15 This is a comparative chromatogram of glucosamine thermal degradation solutions with different mobile phases in Comparative Example 4.

[0072] Figure 16 This is a comparative chromatogram of glucosamine thermal degradation solutions with different mobile phases in Comparative Example 5. Detailed Implementation

[0073] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0074] Example 1: Method for Determining the Purity of Glucosamine Sulfate

[0075] Weigh out glucosamine sulfate, dissolve it in water to prepare a solution with a concentration of approximately 100 mg / mL, take 100 μL, mix it with 450 μL isopropanol and 450 μL n-hexane, vortex to mix, filter, and use as the sample solution.

[0076] Weigh approximately 10 mg each of fructosine, deoxyfructosine, and pyrrole-2-carboxaldehyde reference standards, and approximately 5 mg of 5-hydroxymethylfurfural. Dissolve each in ultrapure water and bring the volume to 100 mL to prepare stock solutions of the reference standards.

[0077] Take 100 μL of the reference stock solution, mix it with 450 μL of isopropanol and 450 μL of n-hexane, vortex to mix, filter, and use as the reference solution.

[0078] Take 100 μL of ultrapure water and mix it with 450 μL of isopropanol and 450 μL of n-hexane to prepare a blank solution.

[0079] Inject the reference solution, sample solution, and blank solution separately.

[0080] The chromatographic column was a Waters Torus DEA column (3.0 mm × 100 mm, 1.7 μm); the mobile phase A was CO2 and B was methanol, eluted according to the gradient in Table 1; the flow rate was 1.2 mL / min; the column temperature was 30 °C; the back pressure was 2000 psi; the detector was a diode array detector with a detection wavelength range of 268 nm and 220 nm; the injection volume was 5 μL.

[0081] Table 1 Gradient elution table

[0082]

[0083] Table 2 shows the results of related substance determination for glucosamine sulfate capsules and tablets from different manufacturers.

[0084] Table 2. Results of related substance determination for glucosamine sulfate capsules and tablets from different manufacturers.

[0085]

[0086] Example 2: Methodological Investigation and Results

[0087] The stability of glucosamine was previously investigated using photodegradation, acid degradation, alkali degradation, oxidative degradation, and thermal degradation. Among these methods, glucosamine showed the worst thermal stability and generated the most types of impurities. Therefore, this method was developed using thermal degradation solutions to examine the impurity separation accuracy and performance.

[0088] (1) Exclusivity

[0089] Weigh out glucosamine sulfate, dissolve it in water to prepare a solution with a concentration of approximately 100 mg / mL, heat it in an 80°C water bath for 1 hour to obtain a glucosamine sulfate thermal degradation solution. After cooling, take 100 μL of the solution and mix it with 450 μL of isopropanol and 450 μL of n-hexane. Vortex the mixture, filter it, and use it as the thermal degradation solution (heating destroys the degradation, and the method is used to examine its ability to separate degradation impurities).

[0090] Weigh approximately 10 mg each of fructosine, deoxyfructosine, and pyrrole-2-carboxaldehyde reference standards, and approximately 5 mg of 5-hydroxymethylfurfural. Dissolve each in ultrapure water and bring the volume to 1 mL to prepare stock solutions of the reference standards.

[0091] Prepare a blank solution using ultrapure water in the same manner, and then inject the sample solution and the blank solution together.

[0092] The results showed that the blank solution did not interfere with the determination of the components in the sample. Except for I2 and deoxyfructazine, whose resolution was 1.12, the resolutions of the other components were all above 1.5. Figure 1 This method has good specificity.

[0093] (2) Limit of detection and limit of quantitation

[0094] Take an appropriate amount of the reference stock solution and prepare mixed standard solutions of different concentrations. Take 100 μL of the mixed standard solution and mix it with 450 μL of isopropanol and 450 μL of n-hexane. After mixing well, filter and inject the solution. The detection limit and quantitation limit of each impurity were obtained according to the signal-to-noise ratios S / N=3 and S / N=10, respectively. 5-hydroxymethylfurfural, which has the highest toxicity, and pyrrole-2-carboxaldehyde, which has a similar retention time, were selected as the impurities for investigation. The calculation results of the detection limit are shown in Table 3.

[0095] Table 3 Limit of Detection and Limit of Quantification

[0096]

[0097] (3) Linear

[0098] Take an appropriate amount of the reference standard stock solution and prepare a series of mixed standard solutions of varying concentrations. Mix 100 μL of each solution with 450 μL of isopropanol and 450 μL of n-hexane to prepare the linear sample solution. Inject the solutions in ascending order of concentration. Plot a standard curve with the peak area (A) of each component as the ordinate and the concentration of the reference standard (C, μg / mL) as the abscissa. The linearity results are shown in Table 4, and the goodness of fit R0 is... 2 Both values ​​were greater than 0.9990, indicating that pyrrole-2-carboxaldehyde and 5-hydroxymethylfurfural exhibited good linearity in the ranges of 58.6–1172.0 μg / mL and 39.2–784.0 μg / mL, respectively.

[0099] Table 4 Calculation results of linear equations

[0100]

[0101] (4) Precision

[0102] Take the thermal degradation solution of sodium glucosamine sulfate, add an appropriate amount of reference standard stock solution and quantify it to ensure that the concentration of the added reference standard in the quantified solution is approximately 100 μg / mL. Take 100 μL of this spiked solution and mix it with 450 μL of isopropanol and 450 μL of n-hexane. After mixing and filtering, prepare six parallel aliquots as repeatable sample solutions. Calculate the peak area RSD after injection. One day later, prepare intermediate precision sample solutions according to the repeatable sample solution preparation method. After injection, combine the intermediate precision sample solutions with the repeatable sample solutions and calculate the peak area RSD. The calculation results are shown in Table 5, indicating that the method has good precision.

[0103] Table 5 Precision Calculation Results

[0104]

[0105] (5) Recovery rate

[0106] An appropriate amount of reference stock solution was added to the sample solution and quantified to prepare three spiking solutions with low, medium, and high concentrations, so that the concentrations of each reference standard in the quantified solution were approximately 80 (low), 100 (medium), and 120 (high) μg / mL, respectively. 100 μL of this spiking solution was mixed with 450 μL of isopropanol and 450 μL of n-hexane, filtered, and the mixture was prepared in six parallel aliquots as recovery solutions. The recovery rate and peak area RSD of each concentration solution were calculated. The results are shown in Table 6. The recovery rates of each component ranged from 90.42% to 105.26%, indicating good recovery.

[0107] Table 6 Recovery Rate Calculation Results

[0108]

[0109] (6) Stability

[0110] After the sample solution was injected and left to stand for 8 hours, it was injected again, and the peak area RSD was calculated. The RSD of the peak area of ​​pyrrole-2-carboxaldehyde was 1.32%, and the RSD of the peak area of ​​5-hydroxymethylfurfural was 1.50%, proving that the sample solution was stable within 8 hours.

[0111] Mass spectrometry

[0112] Mass spectrometry conditions: Positive ion scanning mode. ESI ion source parameters were as follows: ion source temperature 120℃, capillary voltage 1kV, desolvation gas flow rate 800L / Hr, curtain gas flow rate 50L / Hr, mobile phase of compensation pump 0.1% formic acid-methanol, flow rate 0.3mL / min. Scan types were Full MS and MS / MS, with scan times ranging from 0 to 6 min. Full MS parameters: collision energy 10–45V, scan range 50–1200 m / z. MS / MS parameters: collision energy 30V, scan range 5–600 m / z. The m / z values ​​of the parent ions of each unknown impurity are shown in Table 7.

[0113] Table 7. m / z of each impurity precursor ion in UPCC-MS

[0114]

[0115] Impurity Analysis

[0116] In the thermal degradation solution of sodium glucosamine sulfate, six impurities were found: pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, deoxyfructazine, fructosamine, I2, I3, I5, and I6. Two newly isolated unknown impurities, I5 and I6, were also found in this purity separation system, with retention times of 1.994 min and 1.586 min, respectively. Figure 1 The high-resolution mass spectrometry results for I2, I3, I5, and I6 are shown in [reference needed]. Figures 2 to 5 The UV spectra of I2 and I3 are shown below. Figure 6 .

[0117] The molecular weights of I2 and I3 are 286 (see [reference]). Figure 2 and Figure 3 ), combined with its spectral information (see Figure 6 The inferred structures are related to 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-transbutenyl)pyrazine and its cis isomers. Figure 7 I5 has a molecular weight of 123, and its molecular formula can be deduced as C6H5NO2 based on its daughter ions; I6 has a molecular weight of 176, and its molecular formula can be deduced as C6H5NO2 based on its daughter ions. 12 H 16 O is 1-tolyl-3-pentanone. Information on fragments I5 and I6 is shown in Table 8. Possible structures are shown below. Figure 8 The fragmentation process of each ion is inferred from the following: Figure 9 .

[0118] Table 8 Mass Spectrometry Information for I6 Ions

[0119]

[0120] Example 3

[0121] Compared to Example 1, the only difference is that the column temperatures were replaced with 35°C, 40°C, and 45°C, respectively. The test results are shown below. Figure 10 . Figure 10 In the middle, from bottom to top, the column temperatures are 30℃, 35℃, 40℃, and 45℃ respectively.

[0122] The results showed that changing the column temperature did not significantly alter the separation effect of the components, but the baseline fluctuated considerably after adjusting the column temperature to 35℃, 40℃, and 45℃.

[0123] Comparative Example 1

[0124] This comparative example uses reversed-phase liquid chromatography (RP-HPLC). The HPLC conditions are as follows: the column is an Avantor Alltima C18 (250×4.6 mm, 5 μm); the mobile phase is sodium heptanesulfonate buffer (0.5 g of sodium heptanesulfonate dissolved in an appropriate amount of water, then 4 mL of 56 g / L potassium hydroxide solution and 0.5 mL of phosphoric acid are added, and then diluted with water to 1000 mL) - acetonitrile (1000:50); the flow rate is 1 mL / min; the UV detector is used; the detection wavelength is 260 nm; and the column temperature is 30 °C.

[0125] Weigh out glucosamine sulfate, dissolve it in water to prepare a solution with a concentration of approximately 100 mg / mL, and heat it in an 80°C water bath for 1 hour to obtain a stock solution of glucosamine sulfate for thermal degradation. Take 100 μL of this stock solution, add 400 μL of water, and vortex to mix to obtain the thermal degradation test solution. Inject 20 μL of this solution into the liquid chromatograph and record the chromatogram. The results are shown in the figure. Figure 11 and Figure 12 , Figure 11 This is a magnified view of a portion of the image. Figure 11 and Figure 12 The glucosamine salts used from bottom to top are: glucosamine sulfate, glucosamine sulfate sodium chloride sample 1, glucosamine sulfate sodium chloride sample 2, and glucosamine sulfate potassium chloride.

[0126] Compared with the method described in this paper, reversed-phase high-performance liquid chromatography can only effectively separate five impurities in the glucosamine thermal degradation solution: fructosine, deoxyfructosine, I2, I3, and 5-hydroxymethylfurfural. It does not separate two degradation impurities, I5 and I6, or pyrrole-2-carboxaldehyde. Furthermore, the chromatographic peak of 5-hydroxymethylfurfural is relatively broad, and its sensitivity is lower than that of the method described in this paper.

[0127] Comparison shows that the UPCC method is superior to reversed-phase liquid chromatography (RPLC) in removing cytotoxic and genotoxic polar impurities in glucosamine (such as 5-hydroxymethylfurfural). Figure 11 It achieves better separation, better peak shape, and higher response.

[0128] Comparative Example 2: Column Screening

[0129] Compared with Example 1, the only difference is that the chromatographic columns are replaced with Waters Torus 2-PIC columns and Waters Torus DIOL columns, respectively.

[0130] See results Figure 13 . Figure 13 In the image, the chromatographic columns used from bottom to top are Waters Torus 2-PIC, Waters Torus DIOL, and Waters Torus DEA, respectively.

[0131] like Figure 13 As shown, using the Waters Torus 2-PIC column, 5-hydroxymethylfurfural and I6 impurities cannot be effectively separated, while the Waters Torus DEA column can effectively separate 5-hydroxymethylfurfural and I6.

[0132] Using a Waters Torus DIOL column, although the separation of highly polar impurities was good, 5-hydroxymethylfurfural and I6 could not be effectively separated, appearing as a single chromatographic peak, while the peak shapes of fructosamine and deoxyfructosamine were poor.

[0133] Comparative Example 3: Solvent Screening

[0134] Compared with Example 1, the only difference is that the sample solvents were replaced with water:acetonitrile = 10:90 (volume ratio) and water:methanol:acetonitrile = 10:22.5:67.5 (volume ratio), respectively.

[0135] See results Figure 14 . Figure 14 In the mixture, the solvents used from bottom to top are water:acetonitrile = 10:90 (volume ratio), water:isopropanol:n-hexane = 10:45:45 (volume ratio), and water:methanol:acetonitrile = 10:22.5:67.5 (volume ratio).

[0136] like Figure 14 As shown, when the sample solvent was replaced with water:acetonitrile = 10:90 (volume ratio), the signals of all chromatographic peaks decreased to varying degrees while the sample solution concentration remained unchanged. The signals of impurity peaks such as I6, deoxyfructidine, and fructidine decreased significantly. When the sample solvent was replaced with water:methanol:acetonitrile = 10:22.5:67.5 (volume ratio), the signals of impurity peaks such as I6 and fructidine decreased significantly while the sample solution concentration remained unchanged.

[0137] Comparative Example 4: Screening of Mobile Phase

[0138] Compared with Example 1, the only difference is that mobile phase B is replaced with ethanol.

[0139] See results Figure 15 . Figure 15 In the diagram, the mobile phases B used from bottom to top are methanol and ethanol, respectively.

[0140] like Figure 15 As shown, after replacing mobile phase B with ethanol, the detected impurity peaks were significantly reduced, and the impurity peaks of pyrrole-2-carboxaldehyde, I6, and fructosine were no longer visible.

[0141] Comparative Example 5: Mobile Phase Screening

[0142] Compared with Example 1, the only difference is that the mobile phase B is replaced with methanol with 0.2% ammonia (volume ratio) or methanol with 0.2% diethylamine (volume ratio).

[0143] See results Figure 16 . Figure 16 In the middle, the mobile phase B used from bottom to top are methanol (containing 0.2% ammonia), methanol, and methanol (containing 0.2% diethylamine).

[0144] like Figure 16 As shown, adding 0.2% ammonia as a modifier to mobile phase B did not significantly improve the peak shape, and the deoxyfructose response decreased, affecting the quantification of this impurity. Adding 0.2% diethylamine as a modifier to mobile phase B also did not significantly improve the peak shape, and the baseline fluctuation increased, with the solvent peak becoming larger, affecting the quantification of small impurity peaks.

[0145] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting glucosamine, characterized in that, Ultra-high performance phase chromatography (UHPLC) was used for detection, followed by mass spectrometry for qualitative analysis of the impurities. The detection method includes: providing a sample solution; The sample solution is an glucosamine solution, or the sample solution is a glucosamine sulfate thermal degradation solution; The preparation method of the glucosamine solution is as follows: weigh glucosamine sulfate, dissolve it in water and prepare a solution with a concentration of 100 mg / mL, take 100 μL, mix it with 450 μL isopropanol and 450 μL n-hexane, vortex mix, filter, and use it as the sample solution. The preparation method of the glucosamine sulfate thermal degradation solution is as follows: weigh glucosamine sulfate, dissolve it in water and prepare a solution with a concentration of 100 mg / mL, heat it in an 80°C water bath for 1 hour to obtain the glucosamine sulfate thermal degradation solution, cool it and take 100 μL, mix it with 450 μL isopropanol and 450 μL n-hexane, vortex mix it, filter it, and use it as the sample solution; the content is 10 mg / mL based on glucosamine sulfate before degradation. A reference solution was prepared using fructosine, deoxyfructosine, and pyrrole-2-carboxaldehyde as reference standards. The impurities include fructosine, deoxyfructosine, pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, C6H5NO2, 1-tolyl-3-pentanone, and 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-transbutenyl)pyrazine and its cis isomers; The chromatographic column was a Waters Torus DEA column; Mobile phase A is CO2, mobile phase B is methanol, and the following elution is performed: Mass spectrometry conditions: positive ion scanning mode; ESI ion source parameters as follows: ion source temperature 120℃, capillary voltage 1kV, desolvation gas flow rate 800L / Hr, curtain gas flow rate 50L / Hr, mobile phase of compensation pump 0.1% formic acid-methanol, flow rate 0.3mL / min; scan type: Full MS, MS / MS, scan time 0~6min; Full MS parameters: collision energy 10~45V, scan range 50~1200m / z; MS / MS parameters: collision energy 30V, scan range 5~600m / z, m / z of each impurity precursor ion included: 309.1067, 124.0403 and 177.1290.

2. The method for detecting glucosamine according to claim 1, characterized in that, The column temperature is 30℃.

3. The method for detecting glucosamine according to claim 1 or 2, characterized in that, Perform the following elution:

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