Glucosamine purity detection method and impurity qualitative method

Abstract

The invention relates to the technical field of glucosamine purity detection, in particular to a glucosamine purity detection method and an impurity qualitative method. The glucosamine purity detection method adopts ultra performance convergence chromatography for detection, and comprises the following steps: providing a sample solution; fructosazine, deoxyfructosazine and pyrrole-2-formaldehyde are used as reference substances to prepare a reference substance solution; the chromatographic column is Waters Torus DEA (Diethyl Ether Anhydride); a mobile phase A is CO2, a mobile phase B is methanol, and gradient elution is carried out. The invention provides a method for detecting the purity of glucosamine by adopting ultra performance convergence chromatography, the method adopts supercritical CO2 and methanol as mobile phases to separate impurities in the glucosamine, and the analysis time is shortened to 6.5 minutes. The method can effectively separate the impurities in the glucosamine, and the purity of the glucosamine is detected by adopting the ultra performance convergence chromatography except several conventionally controlled impurities. Two unknown impurities are also separated out.

Description

Technical Field

[0001] The invention relates to the technical field of glucosamine purity detection, and in particular to a glucosamine purity detection method and an impurity qualitative method. Background Art

[0002] Regarding the determination of glucosamine-related substances, reversed-phase high-performance liquid chromatography is currently used for control, and the mobile phase uses sodium heptane sulfonate or phosphate aqueous solution, acetonitrile solution for isocratic or gradient elution. The main impurities controlled include fructose zine, 2,5-deoxyfructose zine, 5-hydroxymethylfurfural, 2-methylpyrazine, etc. However, since the above methods contain salt in the mobile phase, they cannot be directly connected to the mass spectrometer to qualitatively identify impurities; and they are time-consuming, and the analysis time of various glucosamine reversed-phase high-performance liquid chromatography ranges from 25 to 64 minutes; their separation mechanism is reversed-phase chromatography, which is a single method, and other separation mechanisms should be developed to control potential impurities that cannot be separated by reversed-phase chromatography.

[0003] Ultra-high performance convergence chromatography (UPCC) chromatography uses supercritical CO2 as the mobile phase, and the separation principle is essentially different from that of traditional liquid chromatography. The traditional method uses liquid as the mobile phase, and the compounds are separated by distribution, adsorption / desorption, binding / elution or other types of physical and chemical effects between the stationary phase and the liquid mobile phase of the chromatographic column. UPCC uses supercritical CO2 as the mobile phase, solid adsorbents or polymers bonded to the carrier as the stationary phase, and uses the solvent and capacity of the mobile phase for analysis and separation. The UPCC method combines the advantages of gas chromatography and high-performance liquid chromatography. It can analyze low-volatility, high-boiling-point, and poorly stable polymer and biomacromolecule samples that cannot be separated by gas chromatography. It has higher chromatographic column efficiency and analysis speed than liquid chromatography, can be used in conjunction with a variety of detectors, and has more advantages than traditional liquid chromatography in preparation. At the same time, CO2 is cheap and easy to obtain, simple post-processing, economical, and environmentally friendly. The method has a fast separation speed and high analysis efficiency, especially in the analysis of chiral compounds. It has great advantages, and is less used in the separation of sugar substances. Summary of the invention

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

[0005] The glucosamine purity detection method adopts ultra-high performance convergence chromatography for detection, and the detection method comprises: providing a sample solution; the sample solution is a glucosamine solution, or the sample solution is a thermal degradation solution of glucosamine sulfate; using fructosazine, deoxyfructosazine, and pyrrole-2-carboxaldehyde as reference substances to prepare a reference substance solution; The chromatographic column was Waters Torus DEA; mobile phase A was CO2, mobile phase B was methanol, and elution was performed as follows:

[0006] 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 in a volume ratio of 10:45:45.

[0007] Since the back pressure is high when the UPCC instrument is running, water is easy to freeze in the instrument, and it is necessary to avoid containing more water in the sample solution. In solvents with lower water phase, the solubility of the sample is low. To improve the solubility, the inventors tried other solvent systems such as water: acetonitrile = 10:90 and water: methanol: acetonitrile = 10:22.5:67.5. In these two solvents, the I6 separation or peak shape is poor. In some embodiments of the present invention, water: isopropanol: n-hexane = 10:45:45 is selected as the sample solvent, and the sample has a higher response in this solvent.

[0008] In some embodiments, the sample solution is a glucosamine solution, and the content of glucosamine sulfate is 5-15 mg / mL, preferably 10 mg / mL.

[0009] 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 the glucosamine sulfate before degradation.

[0010] Furthermore, the method for preparing the glucosamine sulfate thermal degradation solution comprises heating the glucosamine sulfate solution in a 75-85° C. water bath for 1-1.5 hours.

[0011] Furthermore, the preparation method of the sample solution includes: thermally degrading the glucosamine sulfate solution (for example, 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 a sample solution.

[0012] Specifically, the concentration of the glucosamine sulfate solution is 80-120 mg / mL, for example, 100 mg / mL.

[0013] The study found that using the above-mentioned water-isopropanol-n-hexane as the sample solvent, the sample has a higher response in this solvent and can significantly improve the accuracy of the detection results.

[0014] The present invention finds that when the chromatographic column Waters Torus DEA is selected to analyze the sample solution, the separation degree of I6 and 5-hydroxymethylfurfural is the best, and the peak shape of I6 is the best, which is significantly better than other chromatographic columns.

[0015] In some specific embodiments, the chromatographic column is a Waters Torus DEA Column, 3.0 mm×100 mm, 1.7 μm.

[0016] The present invention finds that selecting mobile phase A as CO2 and mobile phase B as methanol 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.

[0017] Although ammonia or diethylamine is usually used as an additive to improve the peak shape in ultra-high performance convergence chromatography (UPCC) chromatography, the present invention found that adding a base (ammonia or diethylamine) did not improve the detection of glucosamine purity in the present method. On the contrary, when diethylamine was added, the baseline fluctuated more, affecting the calculation of the peak area. Therefore, ammonia or diethylamine was not added to the mobile phase used in the present method.

[0018] In addition, the method of the present invention can be detected in a relatively wide temperature range, such as 30-45°C, and there is no obvious difference in the separation effect of the components. In some specific embodiments, the detection column temperature can be 30°C, 35°C, 40°C or 45°C, and ideal detection results can be obtained.

[0019] In some specific embodiments, elution is performed as follows:

[0020] Specifically, the flow rate is 1.0-1.5 mL / min, for example 1.2 mL / min.

[0021] Specifically, the back pressure is 1950-2020 psi, for example 2000 psi.

[0022] Specifically, the detector is a diode array detector.

[0023] Specifically, the detection wavelength range is 260-275 nm and 215-225 nm, for example 268 nm and 220 nm.

[0024] Specifically, the injection volume was 5 μL.

[0025] Specifically, the detection method also includes using fructosazine, deoxyfructosazine, pyrrole-2-carboxaldehyde, and 5-hydroxymethylfurfural as reference substances to prepare reference substance liquid.

[0026] Specifically, the detection method also includes preparing a standard curve with the above-mentioned reference solution, injecting the sample solution, the reference solution and the blank solution into an ultra-high performance convergence chromatography (UPCC) chromatograph under the above-mentioned chromatographic conditions, recording the chromatogram, and calculating the content of impurities in the sample by an external standard method based on the peak areas of the reference solution and the sample chromatograms.

[0027] 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 in a volume ratio of 10:45:45.

[0028] Specifically, the glucosamine purity detection method comprises: Prepare sample solution; The content of glucosamine sulfate in the sample solution is 10 mg / mL; Alternatively, the sample solution is prepared by heating a 100 mg / mL glucosamine sulfate solution in an 80° C. water bath for 1 hour to obtain a thermally degraded glucosamine sulfate solution; cooling, mixing with isopropanol and n-hexane, filtering, and preparing a sample solution; the content of the sample solution is 10 mg / mL based on the glucosamine sulfate before degradation; The solvent of the sample solution is composed of water, isopropanol and n-hexane in a volume ratio of 10:45:45; Fructosazine, deoxyfructosazine, pyrrole-2-carboxaldehyde, and 5-hydroxymethylfurfural are used as reference substances to prepare reference substance solutions; wherein the concentrations of the fructosazine reference substance solution, the deoxyfructosazine reference substance solution, and the pyrrole-2-carboxaldehyde reference substance solution are all 0.05 to 0.2 mg / mL; the concentration of the 5-hydroxymethylfurfural reference substance solution is 0.03 to 0.1 mg / mL; more specifically, fructosazine, deoxyfructosazine, pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, and the like can be used as reference substances. Hydroxymethylfurfural is used as a reference substance to prepare a reference substance stock solution; wherein the concentrations of the fructosazine reference substance stock solution, the deoxyfructosazine reference substance stock solution and the pyrrole-2-carboxaldehyde reference substance stock solution are all 10 mg / mL; the concentration of the 5-hydroxymethylfurfural reference substance stock solution is 5 mg / mL; an appropriate amount of the reference substance stock solution is taken and diluted to prepare the fructosazine reference substance solution, the deoxyfructosazine reference substance solution, the pyrrole-2-carboxaldehyde reference substance solution and the 5-hydroxymethylfurfural reference substance solution; A blank solution was prepared by taking ultrapure water, isopropanol and n-hexane in a volume ratio of 10:45:45; Chromatographic conditions: The chromatographic column was Waters Torus DEA; mobile phase A was CO2, mobile phase B was methanol, and elution was performed as follows: Elution was performed as follows:

[0029] Flow rate: 1.2 mL / min; Back pressure is 2000psi; The detector is a diode array detector; The detection wavelength range is 268nm and 220nm; The injection volume was 5 μL.

[0030] Under the above chromatographic conditions, the sample solution, reference solution and blank solution were respectively injected into the ultra-high performance convergence chromatography (UPCC) chromatograph, and the chromatograms were recorded. The impurity content in the sample was calculated by the external standard method based on the peak areas of the reference solution and sample chromatograms.

[0031] Specifically, the impurities include fructosazine, deoxyfructosazine, pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, C6H5NO2, 1-tolyl-3-pentanone, and 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-trans-butenyl)pyrazine and its cis isomers.

[0032] The ultra-high performance convergence chromatography separation method of the present invention is combined with a high-resolution mass spectrometer to carry out qualitative detection of two unknown impurities in a thermal degradation solution of glucosamine.

[0033] The present invention also provides a method for qualitative determination of glucosamine impurities, comprising: The above-mentioned ultra-high performance convergence chromatography method was used for detection; Then mass spectrometry detection was performed.

[0034] Specifically, mass spectrometry conditions: positive ion scanning mode, ESI ion source parameters are as follows: ion source temperature is 120°C, capillary voltage is 1kV, desolvation gas flow rate is 800L / Hr, curtain gas flow rate is 50L / Hr, compensation pump mobile phase is 0.1% formic acid-methanol, flow rate is 0.3mL / min. Scan type is Full MS, MS / MS, scan time is 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, the m / z of each unknown impurity parent ion is shown in the table below.

[0035] UPCC-MS impurity parent ion m / z

[0036] After analysis, I2 and I3 were 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-trans-butenyl)pyrazine and its cis isomer, respectively; I5 was C6H5NO2; and I6 was 1-tolyl-3-pentanone.

[0037] The commonly used methods for quality control of glucosamine can separate impurities that are easily degraded during storage and transportation, but the mobile phases are all salt solutions, which are not compatible with mass spectrometry. The UPCC method has fast separation, good separation effect and response for impurities with higher polarity (such as 5-hydroxymethylfurfural, etc.), and has a special separation effect. It can effectively separate impurities that are difficult to separate by other chromatographic methods; it can be well adapted to mass spectrometry and is suitable for the study of unknown impurities, providing a new idea for the analysis of related substances of glucosamine. The UPCC method has a better separation effect on polar impurities with cytotoxicity and genotoxicity in glucosamine (such as 5-hydroxymethylfurfural, etc.) than reversed-phase liquid chromatography (see Figure 8 ), with a good peak shape and high response. At the same time, the UPCC method itself has the advantages of fast equilibrium and high separation efficiency, which greatly shortens the analysis time. Through this method, unknown impurities I5 and I6 that were not separated in HPLC were found, and the structure of I6 was analyzed and speculated to be 1-tolyl-3-pentanone. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 UPCC chromatograms of the blank solution and the glucosamine thermal degradation sample solution of Example 2.

[0039] Figure 2 This is the high-resolution mass spectrum of I2 in Example 2.

[0040] Figure 3 This is the high-resolution mass spectrum of I3 in Example 2.

[0041] Figure 4 This is the high-resolution mass spectrum of I5 in Example 2.

[0042] Figure 5 This is the high-resolution mass spectrum of I6 in Example 2.

[0043] Figure 6 It is the ultraviolet spectra of I2 and I3 in Example 2.

[0044] Figure 7 This is the structural analysis result of I2 and I3 in Example 2.

[0045] Figure 8 This is the structural analysis result of I6 in Example 2.

[0046] Fig. 9 This is the fragmentation process of each daughter ion of I6 in Example 2.

[0047] Fig.10 This is a diagram of the separation effect of different column temperatures in Example 3.

[0048] Fig.11 and Fig.12 This is the chromatogram of the thermal degradation solution of glucosamine by reverse phase liquid chromatography of comparative example 1, wherein: Fig.11 This is a partial enlarged view.

[0049] Fig.13 This is a chromatographic comparison of the thermal degradation solutions of glucosamine separated by different chromatographic columns in Comparative Example 2.

[0050] Fig.14 This is a chromatogram comparison of the thermal degradation solutions of glucosamine in different solvents in Comparative Example 3.

[0051] Fig.15 This is a chromatogram comparison of the thermal degradation solution of glucosamine in different mobile phases in Comparative Example 4.

[0052] Fig.16 This is a chromatogram comparison of the thermal degradation solution of glucosamine with different mobile phases in Comparative Example 5. DETAILED DESCRIPTION

[0053] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0054] Example 1 Glucosamine Sulfate Purity Detection Method Weigh glucosamine sulfate, add water to dissolve it and prepare a solution with a concentration of about 100 mg / mL. Take 100 μL, mix it with 450 μL of isopropanol and 450 μL of n-hexane, vortex mix, and filter to prepare the sample solution.

[0055] Weigh about 10 mg each of fructosazine, deoxyfructosazine, and pyrrole-2-carboxaldehyde reference substances and about 5 mg of 5-hydroxymethylfurfural, add ultrapure water to dissolve them and make up to 100 mL as reference substance stock solutions.

[0056] Take 100 μL of the reference stock solution, mix it with 450 μL of isopropanol and 450 μL of n-hexane, vortex mix, and filter to prepare the reference solution.

[0057] Take 100 μL of ultrapure water, mix it with 450 μL of isopropanol and 450 μL of n-hexane to prepare a blank solution.

[0058] Inject the reference solution, sample solution and blank solution separately.

[0059] 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, with gradient elution according to 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; and the injection volume was 5 μL.

[0060] Table 1 Gradient elution table

[0061] The determination results of related substances in glucosamine sulfate capsules and tablets from different manufacturers are shown in Table 2.

[0062] Table 2 Determination results of related substances in glucosamine sulfate capsules and tablets from different manufacturers

[0063] Example 2 Methodological Investigation and Results In the early stage, the stability of glucosamine was investigated by light degradation, acid degradation, alkali degradation, oxidative degradation and thermal degradation. Among them, glucosamine has the worst thermal stability and produces the most types of impurities. Therefore, the thermal degradation solution was used to investigate the impurity separation and accuracy of the method when establishing this method.

[0064] (1) Exclusivity Weigh glucosamine sulfate, add water to dissolve and prepare a solution with a concentration of about 100 mg / mL, heat in a water bath at 80°C for 1 hour to obtain a thermal degradation solution of glucosamine sulfate, cool it down, take 100 μL, mix it with 450 μL of isopropanol and 450 μL of n-hexane, vortex mix well, and filter it as a thermal degradation solution (heat destruction to examine the method's ability to separate degradation impurities).

[0065] Weigh about 10 mg each of fructosazine, deoxyfructosazine, and pyrrole-2-carboxaldehyde reference substances and about 5 mg of 5-hydroxymethylfurfural, add ultrapure water to dissolve them and make up to 1 mL as reference substance stock solutions.

[0066] Take ultrapure water and prepare a blank solution in the same way, and inject the sample solution and the blank solution.

[0067] The results showed that the blank solution had no interference with the determination of the components in the sample. Except for the separation degree of I2 and deoxyfructose succinate of 1.12, the separation degrees of the other components were all above 1.5 ( Figure 1 ), this method has good specificity.

[0068] (2) Detection limit and quantification limit Take an appropriate amount of reference stock solution, prepare mixed standard solutions of different concentrations, take 100 μL and mix with 450 μL of isopropanol and 450 μL of n-hexane, filter after mixing, and inject. The detection limit and quantification limit of each impurity were obtained according to the signal-to-noise ratio S / N=3 and S / N=10, respectively. The most toxic 5-hydroxymethylfurfural and pyrrole-2-carboxaldehyde with a close retention time were selected as the objects of investigation, and the calculation results of the detection limit are shown in Table 3.

[0069] Table 3 Detection limit and quantification limit

[0070] (3) Linear Take an appropriate amount of reference stock solution to prepare a series of concentration mixed standard solutions. Take 100 μL and mix it with 450 μL of isopropanol and 450 μL of n-hexane as the linear sample solution. Inject the samples from low to high concentrations, and draw a standard curve with the peak area of ​​each component (A) as the ordinate and the reference concentration (C, μg / mL) as the abscissa. The linear results are shown in Table 4, and the fit R 2 All of them were greater than 0.9990, indicating that pyrrole-2-carboxaldehyde and 5-hydroxymethylfurfural had good linearity in the ranges of 58.6~1172.0μg / mL and 39.2~784.0μg / mL, respectively.

[0071] Table 4 Calculation results of linear equations

[0072] (4) Precision Take the thermal degradation solution of sodium glucosamine sulfate, add an appropriate amount of reference substance stock solution and quantify it, so that the concentration of the added reference substance in the quantified solution is about 100μg / mL, take 100μL of the spiked solution and mix it with 450μL of isopropanol and 450μL of n-hexane, filter it after mixing, and make 6 parallel copies as the repeatable sample solution. After injection, calculate the peak area RSD. After 1 day, prepare the intermediate precision sample solution according to the preparation method of the repeatable sample solution, and combine it with the repeatable sample after injection to calculate the peak area RSD. The calculation results are shown in Table 5, indicating that the method has good precision.

[0073] Table 5 Precision calculation results

[0074] (5) Recovery rate Add an appropriate amount of reference substance stock solution to the sample solution and quantify it, prepare three concentrations of spiked solutions of low, medium and high concentrations, so that the concentrations of each reference substance added in the solution after quantification are about 80 (low), 100 (medium) and 120 (high) μg / mL, respectively. Take 100 μL of the spiked solution and mix it with 450 μL of isopropanol and 450 μL of n-hexane, mix well and filter, and make 6 parallel copies as the recovery solution. Calculate the recovery rate and peak area RSD of each concentration solution. The results are shown in Table 6. The recovery rate of each component is between 90.42% and 105.26%, indicating that the recovery rate is good.

[0075] Table 6 Recovery calculation results

[0076] (6) Stability After the sample solution was injected, it was left for 8 hours and injected again to calculate the peak area RSD. 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%, which proved that the sample solution was stable within 8 hours.

[0077] Mass spectrometry methods Mass spectrometry conditions: positive ion scanning mode, ESI ion source parameters are as follows: ion source temperature is 120℃, capillary voltage is 1kV, desolvation gas flow rate is 800L / Hr, curtain gas flow rate is 50L / Hr, compensation pump mobile phase is 0.1% formic acid-methanol, flow rate is 0.3mL / min. Scan type is Full MS, MS / MS, scan time is 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 unknown impurity parent ion is shown in Table 7.

[0078] Table 7 m / z of each impurity parent ion in UPCC-MS

[0079] Impurity analysis In the thermal degradation solution of glucosamine sulfate sodium salt, there are 6 impurities including pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, deoxyfructazine, fructazine, I2, I3, I5 and I6, as well as 2 unknown impurities I5 and I6 newly separated from the purity separation system, with retention times of 1.994min and 1.586min, respectively ( Figure 1 ). The high-resolution mass spectrometry results of I2, I3, I5 and I6 are shown in Figures 2 to 5 ; I2, I3 UV spectra see Figure 6 .

[0080] The molecular weight of I2 and I3 is 286 (see Figure 2 and Figure 3 ), combined with its spectral information (see Figure 6 ) The inferred structures are respectively similar to 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-trans-butenyl)pyrazine and its cis isomer ( Figure 7 The molecular weight of I5 is 123, and its molecular formula is inferred to be C6H5NO2 through its daughter ions; the molecular weight of I6 is 176, and its molecular formula is inferred to be C 12 H 16 O is 1-tolyl-3-pentanone, the fragment information of I5 and I6 is shown in Table 8, and the possible structure is shown in Figure 8 , the fragmentation process of each ion can be inferred from Fig. 9 .

[0081] Table 8 Mass spectrum information of each ion of I6

[0082] Example 3 Compared with Example 1, the only difference is that the column temperature is replaced with 35°C, 40°C, and 45°C respectively. Fig.10 . Fig.10 In the figure, from bottom to top, the column temperatures are: 30℃, 35℃, 40℃, 45℃.

[0083] The results showed that changing the column temperature did not significantly change the separation effect of the components, but after adjusting the column temperature to 35℃, 40℃, and 45℃, the baseline fluctuated greatly.

[0084] Comparative Example 1 This comparative example adopts reversed-phase liquid chromatography for detection. The reversed-phase high-performance liquid phase conditions are as follows: the chromatographic column is AvantorAlltima C18 (250×4.6mm, 5μm), the mobile phase is sodium heptane sulfonate buffer (take 0.5g of sodium heptane sulfonate and add appropriate amount of water to dissolve it, add 4mL of 56g / L potassium hydroxide solution and 0.5mL of phosphoric acid, and then dilute to 1000mL with water)-acetonitrile (1000:50), the flow rate is 1mL / min, the ultraviolet detector has a detection wavelength of 260nm, and the column temperature is 30°C.

[0085] Weigh glucosamine sulfate, dissolve it in water and prepare a solution with a concentration of about 100 mg / mL. Heat it in a water bath at 80°C for 1 hour to obtain a thermal degradation stock solution of glucosamine sulfate. Take 100 μL, add 400 μL of water, vortex and mix to obtain a thermal degradation test solution. The injection volume is 20 μL, inject it into the liquid chromatograph, record the chromatogram, and the results are shown in the table. Fig.11 and Fig.12 , Fig.11 This is a partial enlarged view. Fig.11 and Fig.12In the analysis, the glucosamine salt types 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.

[0086] Compared with the method proposed in this paper, the reversed-phase high performance liquid chromatography method can only effectively separate five impurities, namely fructosazine, deoxyfructosazine, I2, I3, and 5-hydroxymethylfurfural, in the thermal degradation solution of glucosamine. The two degradation impurities I5 and I6 and pyrrole-2-carboxaldehyde were not separated. The chromatographic peak width of 5-hydroxymethylfurfural was larger, and the sensitivity was lower than that of the method proposed in this paper.

[0087] By comparison, it can be seen that the UPCC method has a better effect on the detection of polar impurities with cytotoxicity and genotoxicity (such as 5-hydroxymethylfurfural) in glucosamine than the reversed phase liquid chromatography ( Fig.11 ) better separation, better peak shape, and higher response.

[0088] Comparative Example 2 Chromatographic column screening Compared with Example 1, the only difference is that the chromatographic columns are replaced with Waters Torus 2-PIC chromatographic columns and Waters Torus DIOL chromatographic columns respectively.

[0089] Results Fig.13 . Fig.13 In the figure, the columns used from bottom to top are Waters Torus 2-PIC, Waters Torus DIOL and Waters Torus DEA. like Fig.13 As shown, 5-HMF and I6 impurities cannot be effectively separated using the Waters Torus 2-PIC column, while 5-HMF and I6 can be effectively separated using the Waters Torus DEA column.

[0090] Using the Waters Torus DIOL column, although the separation of strongly polar impurities was good, 5-hydroxymethylfurfural and I6 could not be effectively separated and appeared as one chromatographic peak, and the peak shapes of fructosazine and deoxyfructosazine were poor.

[0091] Comparative Example 3 Sample Solvent Screening Compared with Example 1, the only difference is that the sample solvents are replaced with water:acetonitrile = 10:90 (volume ratio) and water:methanol:acetonitrile = 10:22.5:67.5 (volume ratio) respectively.

[0092] Results Fig.14 . Fig.14In the experiment, the solvents used from bottom to top were 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).

[0093] like Fig.14 As shown in the figure, when the sample solvent is replaced with water: acetonitrile = 10:90 (volume ratio), the signals of each chromatographic peak decrease to varying degrees when the concentration of the sample solution remains unchanged, and the peak signals of impurities such as I6, deoxyfructosazine, and fructosazine decrease significantly. When the sample solvent is replaced with water: methanol: acetonitrile = 10:22.5:67.5 (volume ratio), the peak signals of impurities such as I6 and fructosazine decrease significantly when the concentration of the sample solution remains unchanged.

[0094] Comparative Example 4 Mobile Phase Screening Compared with Example 1, the only difference is that the mobile phase B is replaced with ethanol.

[0095] Results Fig.15 . Fig.15 In the experiment, the mobile phases B used from bottom to top were methanol and ethanol respectively.

[0096] like Fig.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 fructosazine were no longer visible.

[0097] Comparative Example 5 Mobile Phase Screening Compared with Example 1, the only difference is that the mobile phase B is replaced by methanol added with 0.2% ammonia water (volume ratio) and methanol added with 0.2% diethylamine (volume ratio).

[0098] Results Fig.16 . Fig.16 In the experiment, the mobile phases B used from bottom to top were methanol (containing 0.2% ammonia water), methanol, and methanol (containing 0.2% diethylamine).

[0099] like Fig.16 As shown in the figure, after adding 0.2% ammonia modifier to mobile phase B, the peak shape did not improve significantly, and the response of deoxyfructazine decreased, affecting the quantification of the impurity. After adding 0.2% diethylamine modifier to mobile phase B, the peak shape did not improve significantly, and the baseline fluctuation increased, the solvent peak became larger, affecting the quantification of the small impurity peak.

[0100] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the purity of glucosamine, characterized in that: The detection is performed by ultra-high performance convergence chromatography, and the detection method comprises: providing a sample solution; the sample solution is a glucosamine solution, or the sample solution is a thermal degradation solution of glucosamine sulfate; using fructosazine, deoxyfructosazine, and pyrrole-2-carboxaldehyde as reference substances to prepare a reference substance solution; the chromatographic column is Waters Torus DEA; the mobile phase A is CO2, and the mobile phase B is methanol, and the elution is performed as follows:

2. The method for detecting the purity of glucosamine according to claim 1, characterized in that: The preparation method of the glucosamine sulfate thermal degradation solution comprises heating the glucosamine sulfate solution in a water bath at 75-85° C. for 1-1.5 hours.

3. The method for detecting the purity of glucosamine according to claim 1 or 2, characterized in that: 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).

4. The method for detecting the glucosamine purity according to any one of claims 1 to 3, characterized in that: The column temperature is 30~50℃.

5. The method for detecting the purity of glucosamine according to any one of claims 1 to 4, characterized in that: Elution was performed as follows:

6. The method for detecting the purity of glucosamine according to any one of claims 1 to 5, characterized in that: The flow rate is 1.0~1.5mL / min; and / or, Back pressure is 1950~2020psi; and / or, The detector is a diode array detector; and / or, The detection wavelength range is 260~275nm and 215~225nm; and / or, The injection volume was 5 μL.

7. The method for detecting the purity of glucosamine according to any one of claims 1 to 6, characterized in that: The glucosamine purity detection method comprises: Prepare sample solution; The content of glucosamine sulfate in the sample solution is 10 mg / mL; Alternatively, the sample solution is prepared by heating a 100 mg / mL glucosamine sulfate solution in an 80° C. water bath for 1 hour to obtain a thermally degraded glucosamine sulfate solution; cooling, mixing with isopropanol and n-hexane, filtering, and preparing a sample solution; the content of the sample solution is 10 mg / mL based on the glucosamine sulfate before degradation; The solvent of the sample solution is composed of water, isopropanol and n-hexane in a volume ratio of 10:45:45; Fructosazine, deoxyfructosazine, pyrrole-2-carboxaldehyde, and 5-hydroxymethylfurfural were used as reference substances to prepare reference substance solutions; wherein the concentrations of the fructosazine reference substance solution, the deoxyfructosazine reference substance solution, and the pyrrole-2-carboxaldehyde reference substance solution were all 0.05-0.2 mg / mL; the concentration of the 5-hydroxymethylfurfural reference substance solution was 0.03-0.1 mg / mL; A blank solution was prepared by taking ultrapure water, isopropanol and n-hexane in a volume ratio of 10:45:45; Chromatographic conditions: The chromatographic column was Waters Torus DEA; mobile phase A was CO2, mobile phase B was methanol, and elution was performed as follows: Elution was performed as follows: Flow rate: 1.2 mL / min; Back pressure is 2000psi; The detector is a diode array detector; The detection wavelength range is 268nm and 220nm; The injection volume was 5 μL; Under the above chromatographic conditions, the sample solution, reference solution and blank solution are respectively injected into the ultra-high performance convergence chromatograph, the chromatograms are recorded, and the impurity content in the sample is calculated by the external standard method based on the peak areas of the reference solution and sample chromatograms.

8. The method for detecting the purity of glucosamine according to any one of claims 1 to 7, characterized in that: Impurities include fructosazine, deoxyfructosazine, pyrrole-2-carboxaldehyde, 5-hydroxymethylfurfural, C6H5NO2, 1-tolyl-3-pentanone, and 2-(tetrahydroxybutyl)-5-(3′,4′-dihydroxy-1′-trans-butenyl)pyrazine and its cis isomer.

9. A method for qualitative determination of glucosamine impurities, characterized in that: include: Detection is performed using the ultra-high performance convergence chromatography method described in any one of claims 1 to 8; Then mass spectrometry detection was performed.

10. The method for qualitative determination of glucosamine impurities according to claim 9, characterized in that: Mass spectrometry conditions: positive ion scanning mode, ESI ion source parameters are as follows: ion source temperature is 120 ° C, capillary voltage is 1 kV, desolvation gas flow rate is 800 L / Hr, curtain gas flow rate is 50 L / Hr, compensation pump mobile phase is 0.1% formic acid-methanol, flow rate is 0.3 mL / min; scan type is Full MS, MS / MS, scan time is 0~6 min; Full MS parameters: collision energy 10~45 V, scan range 50~1200 m / z; MS / MS parameters: collision energy 30 V, scan range 5~600 m / z, each unknown impurity parent ion m / z is as follows:

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