Analysis method of glucosamine hydrochloride

Abstract

The invention relates to the technical field of analysis, in particular to an analysis method of glucosamine hydrochloride. The invention provides an analysis method of glucosamine hydrochloride, which can accurately, sensitively and quickly detect the content of glucosamine hydrochloride in a product (such as oral liquid), and can be used for realizing quality control on the product containing glucosamine hydrochloride.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and more specifically to an analytical method for glucosamine hydrochloride. Background Technology

[0002] Glucosamine hydrochloride is a widely used ingredient in dietary supplements and pharmaceuticals, primarily used to support joint health, relieve joint pain, promote cartilage repair, and reduce inflammation. Currently, commercially available glucosamine hydrochloride products are mainly in tablet, capsule, and granule forms. For these solid dosage forms, high-performance liquid chromatography (HPLC) is typically used for detection, as this method is simple, rapid, and highly specific. However, existing literature reports very little on how to determine and analyze the content of glucosamine hydrochloride in liquid dosage forms (such as oral solutions containing glucosamine hydrochloride).

[0003] Furthermore, existing methods are all based on solid samples. Liquid formulations (such as oral liquids) have different matrices than solids, resulting in significant differences in chromatographic separation and sample extraction. Therefore, existing detection methods for solid dosage forms cannot be directly applied. Thus, there is a need to develop an analytical method suitable for determining the glucosamine hydrochloride content in liquid formulations containing glucosamine hydrochloride. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide an analytical method for glucosamine hydrochloride. Using this method, the content of glucosamine hydrochloride in products (such as oral liquids) can be accurately, sensitively, and rapidly detected, and can be used for quality control of products containing glucosamine hydrochloride.

[0005] Therefore, the first aspect of the present invention provides a method for analyzing glucosamine hydrochloride. In some embodiments of the present invention, the method includes:

[0006] High performance liquid chromatography was used to detect glucosamine hydrochloride in the sample.

[0007] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0008] The chromatographic column uses a stationary phase with hydrophilic properties;

[0009] The mobile phase consists of ammonium acetate solution and acetonitrile, and isocratic elution is performed.

[0010] The volume ratio of the ammonium acetate solution to acetonitrile is (5:95) to (8:92).

[0011] The analytical method for glucosamine hydrochloride provided by this invention can accurately, sensitively, and rapidly detect the content of glucosamine hydrochloride in products (such as oral liquids). This method can be used to achieve quality control of products containing glucosamine hydrochloride.

[0012] In some embodiments of the present invention, the concentration of the ammonium acetate solution is 18-22 mmol / L.

[0013] In some embodiments of the present invention, the ammonium acetate solution needs to be adjusted to pH 3.9-4.1 with acetic acid.

[0014] In some embodiments of the present invention, the isocratic elution flow rate is 0.7-0.9 ml / min.

[0015] In some embodiments of the present invention, the column temperature of the chromatographic column is 25-35°C.

[0016] In some embodiments of the present invention, the detection wavelength of the high-performance liquid chromatography is 192 nm.

[0017] In some embodiments of the present invention, the high performance liquid chromatography method employs an ultra-high performance liquid chromatograph (UHPLC), wherein the detector of the UHPLC is an ultraviolet detector or a diode array detector.

[0018] In some embodiments of the present invention, the high-performance liquid chromatography method uses a WatersAcquity BEH Amide column.

[0019] In some embodiments of the present invention, the injection volume for detection by high performance liquid chromatography is 5-10 μl.

[0020] In some embodiments of the present invention, the sample to be tested is prepared as a solution, wherein the solution contains water and acetonitrile, and the volume ratio of water to acetonitrile is (7:43)-(10:40). If the sample solution contains less water, the amount of glucosamine hydrochloride dissolved in the sample will be less, resulting in a lower detection result.

[0021] In some embodiments of the present invention, when preparing the solution of the sample to be tested, it is necessary to mix the sample to be tested with water and perform ultrasonic treatment, and then mix the ultrasonically treated solution with acetonitrile.

[0022] In some embodiments of the present invention, the ultrasonic treatment time is 5 min to 30 min.

[0023] The analytical method for glucosamine hydrochloride of the present invention has the following advantages compared with the national standard method: faster peak elution time, improved injection efficiency; and no ion-pairing reagents are used, reducing damage to the chromatographic column.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0026] Figure 1 The results of a full-wavelength UV scan of the glucosamine hydrochloride reference standard in Example 2 are shown, where the horizontal axis represents wavelength (in nanometers) and the vertical axis represents absorption intensity (in AU).

[0027] Figures 2a-2d The chromatograms of the blank solution, blank preparation, reference solution and test solution in Example 3 are shown respectively, where the horizontal axis is time (in minutes) and the vertical axis is absorption intensity (in AU);

[0028] Figure 3 The standard curve plot generated using the standard working solution in Example 4 is shown, where the horizontal axis represents the concentration of the reference standard (in mg / ml) and the vertical axis represents the peak area. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0034] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0035] According to a specific embodiment of the present invention, the present invention provides an analytical method for glucosamine hydrochloride, comprising:

[0036] High performance liquid chromatography was used to detect glucosamine hydrochloride in the sample.

[0037] The chromatographic conditions for the high-performance liquid chromatography method are as follows:

[0038] The chromatographic column uses a stationary phase with hydrophilic properties;

[0039] The mobile phase consists of ammonium acetate solution and acetonitrile, and isocratic elution is performed.

[0040] The volume ratio of the ammonium acetate solution to acetonitrile is (5:95) to (8:92).

[0041] According to a specific embodiment of the present invention, the volume ratio of the ammonium acetate solution to acetonitrile can be, for example, any ratio within the range of (5:95) to (8:92), such as 5:95, 6:94, 7:93, 8:92, etc.

[0042] According to a specific embodiment of the present invention, the concentration of the ammonium acetate solution is 18-22 mmol / L, for example, it can be 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, etc.

[0043] According to a preferred embodiment of the present invention, in the analytical method for glucosamine hydrochloride of the present invention, when glucosamine hydrochloride in the sample is detected by high performance liquid chromatography, the concentration of ammonium acetate solution in the mobile phase is 20 mmol / L.

[0044] According to a specific embodiment of the present invention, the pH of the ammonium acetate solution needs to be adjusted to 3.9-4.1 with acetic acid. For example, the pH of the ammonium acetate solution needs to be adjusted to 3.9, 4.0, 4.1, etc. with acetic acid.

[0045] According to a specific embodiment of the present invention, the isocratic elution flow rate is 0.7-0.9 ml / min. For example, it can be 0.7 ml / min, 0.8 ml / min, or 0.9 ml / min, etc.

[0046] According to a preferred embodiment of the present invention, the flow rate of the isocratic elution is 0.8 ml / min.

[0047] According to a specific embodiment of the present invention, the column temperature of the chromatographic column is 25-35℃. For example, the column temperature of the chromatographic column is 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, etc.

[0048] According to a preferred embodiment of the present invention, the column temperature of the chromatographic column is 30°C.

[0049] According to a specific embodiment of the present invention, the detection wavelength of the high performance liquid chromatography is 192 nm.

[0050] According to a specific embodiment of the present invention, the high performance liquid chromatography method employs an ultra-high performance liquid chromatograph for detection, wherein the detector of the ultra-high performance liquid chromatograph is an ultraviolet detector or a diode array detector.

[0051] According to a specific embodiment of the present invention, the chromatographic column used in the high-performance liquid chromatography is a WatersAcquity BEH Amide. There are no particular limitations on the column specifications; for example, it can be 1.7 μm 2.1 x 50 mm.

[0052] According to a specific embodiment of the present invention, the injection volume for detection by high-performance liquid chromatography is 5-10 μl. For example, it can be 10 μl.

[0053] According to a specific embodiment of the present invention, the sample to be tested is prepared into a solution, wherein the solution contains water and acetonitrile, and the volume ratio of water to acetonitrile is (7:43)-(10:40). The volume ratio of water to acetonitrile can be, for example, any ratio within the range of (7:43)-(10:40), such as 7:43, 8:42, 9:41, 10:40, etc.

[0054] According to a specific embodiment of the present invention, when preparing the test sample solution, the test sample needs to be mixed with water and subjected to ultrasonic treatment, and the ultrasonically treated solution is then mixed with acetonitrile.

[0055] According to a specific embodiment of the present invention, the ultrasonic treatment time is 5 min to 30 min.

[0056] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1: Detection method for glucosamine hydrochloride

[0058] The following conditions were used to detect glucosamine hydrochloride:

[0059] Instrument: Ultra-high performance liquid chromatograph with ultraviolet detector or diode array detector

[0060] Chromatographic column: Waters Acquity BEH Amide 1.7μm 2.1*50mm

[0061] Mobile phase: 20 mmol / L ammonium acetate solution (adjusted to pH 4.0 with acetic acid) - acetonitrile = 5:95

[0062] Flow rate: 0.8 ml / min; Column temperature: 30℃; Injection volume: 10 μl; Wavelength: 192 nm; Run time: 7 min

[0063] Preparation of reference solution (0.2 mg / ml): Accurately weigh 10 mg of D-glucosamine hydrochloride reference standard (China National Institutes for Food and Drug Control), place it in a 50 ml volumetric flask, add 7 ml of water, sonicate for 10 minutes, cool to room temperature, add acetonitrile to the mark, shake well, filter through a 0.2 μm PTFE filter membrane, discard 3 ml of the initial filtrate, and the solution is ready.

[0064] Preparation of test solution (3.6 mg / ml): Weigh 0.18 g of the test sample accurately, place it in a 50 ml volumetric flask, add 7 ml of water, sonicate for 10 minutes, cool to room temperature, add acetonitrile to make up to the mark, shake well, filter through a 0.2 μm PTFE filter membrane, discard 3 ml of the initial filtrate, and the test solution is ready.

[0065] Example 2: Determination of Detection Wavelength

[0066] A full-wavelength ultraviolet scan of glucosamine hydrochloride reference standard was performed, and the results are as follows: Figure 1 As shown. Its optimal absorption is determined to be at the very bottom of the ultraviolet range; the lower the ultraviolet wavelength, the higher the absorption. Therefore, the detection method of this invention sets the ultraviolet detection wavelength to 192 nm.

[0067] Example 3

[0068] Using the method described in Example 1, the blank solution, blank preparation, reference solution, and test solution were tested respectively, and the chromatograms are shown below. Figures 2a-2d As shown in the figure. The results indicate that there was no interference at the elution point of the main peak in the blank solution and blank preparation. In the reference standard and test solution, the resolution between the main peak and the other chromatographic peaks met the requirements, indicating that the detection method of this invention has good specificity.

[0069] Blank solution composition: water-acetonitrile = 7:43, V / V

[0070] Composition of blank preparation: Weigh 0.18g of blank preparation (preparation sample without glucosamine hydrochloride) accurately, place it in a 50ml volumetric flask, add 7ml of water, sonicate for 10 minutes, cool to room temperature, add acetonitrile to make up to the mark, shake well, filter through a 0.2μm filter membrane, discard 3ml of the initial filtrate, and the blank preparation is obtained.

[0071] The reference standard and test solution are the same as in Example 1.

[0072] Example 4

[0073] 1. Limit of detection, limit of quantitation

[0074] The detection limit was evaluated according to the signal-to-noise ratio method in GB / T27417-2017. The signal-to-noise ratio of the analyzed sample was verified to evaluate and confirm the method's detection limit and quantitation limit. The detection limit was calculated using the formula Lod = 3 × C / SN, and the quantitation limit was calculated using the formula Lod = 10 × C / SN. The method's detection limit and quantitation limit were then evaluated. Low-concentration samples were processed according to the method in Example 1, and the concentration and signal-to-noise ratio (S / N) were measured on the instrument. The verification results of the detection limit and quantitation limit are shown in Table 1 below:

[0075] Table 1

[0076] name Concentration (mg / ml) S / N Detection limit 0.653 5.1 Limit of Quantification 2.178 12.6

[0077] The aforementioned low-concentration samples refer to the addition of reference solutions of different concentrations to a blank formulation. The limit of quantitation is obtained through the signal-to-noise ratio result, and the limit of detection is obtained by dilution based on the limit of quantitation.

[0078] Based on the experimental analysis results of the above low-concentration samples, the detection limit of this method can reach 0.653 mg / ml, and the quantitation limit can reach 2.178 mg / ml.

[0079] 2. Linearity and Range

[0080] A standard curve was constructed using standard working solutions, with concentrations of 0.100 mg / ml, 0.160 mg / ml, 0.200 mg / ml, 0.300 mg / ml, and 0.400 mg / ml (named L1-L5, respectively). A curve was fitted with peak area on the ordinate and glucosamine hydrochloride concentration on the abscissa. The correlation coefficient of the fitted curve was greater than 0.99. Specific data and curves are shown in Table 2 and below, respectively. Figure 3 :

[0081] Table 2

[0082]

[0083] 3. Accuracy

[0084] To determine the accuracy of the method for detecting glucosamine hydrochloride of the present invention, samples at three concentration levels were tested, approximately 0.160 mg / ml, 0.200 mg / ml, and 0.300 mg / ml (referred to as 80%, 100%, and 150% in Table 3, respectively). Specific data are shown in Table 3 below:

[0085] Table 3

[0086]

[0087] The results in Table 3 show that the calculated recovery rates for each spiking recovery rate solution should be in the range of 90%-105%, and the RSD calculated from the nine spiking recovery rates is less than 10%.

[0088] 4. Precision

[0089] Six reproducible samples (S1-S6) were tested for glucosamine hydrochloride according to the method in Example 1 to evaluate the precision of the method for detecting glucosamine hydrochloride of the present invention. Precision includes repeatability and reproducibility; specific results are shown in Tables 4 and 5 below.

[0090] Table 4

[0091]

[0092]

[0093] Table 5

[0094]

[0095] The results in Tables 4 and 5 show that the RSD of the content in the 6 repeatable samples is less than 2%, and the RSD of the content in the 6 reproducible samples is less than 4%, indicating that the detection method of the present invention has good repeatability and reproducibility.

[0096] 5. Durability

[0097] The membrane adsorption experiment of glucosamine hydrochloride (i.e., filtration with a 0.2 μm PTFE membrane as in Example 1) involved discarding different volumes of the same solution and comparing the peak areas with the centrifuged solution. After discarding 3 ml, the ratio was between 98.0% and 102.0%, which met the requirements. The results are shown in Table 6 below:

[0098] Table 6

[0099] Filter membrane adsorption Compared with centrifuged solutions Filter and discard 0ml 97.1％ Filter and discard 1ml 97.9％ Filter and discard 2ml 97.5％ Filter and discard 3ml 100.7％ Filter and discard 4ml 99.9％ Filter and discard 5ml 100.3％

[0100] Example 5

[0101] By changing different experimental parameters, we examined whether there was any interference between the blank solution and the blank preparation solution, and whether the resolution of the main peak in the reference solution and the test solution met the requirements. We compared the results with the content results of the method of this invention, and whether the ratio was between 98.0% and 102.0%. The specific results are shown in Table 7 below:

[0102] Table 7

[0103]

[0104] The results in Table 7 above show that the method of the present invention can achieve better separation and accuracy under the following conditions:

[0105] (1) When preparing the sample, add 7ml to 10ml of water (correspondingly, add 40 to 43ml of acetonitrile) and sonicate for 5min to 30min.

[0106] (2) In the chromatographic parameters, the flow rate was 0.7 ml / min to 0.9 ml / min, the column temperature was 25℃ to 35℃, the concentration of ammonium acetate in the mobile phase was 18 mmol / L to 22 mmol / L, the ratio of the mobile phase was 20 mmol / L ammonium acetate solution (adjusted to pH 4.0 with acetic acid) - acetonitrile = (5:95) to (8:92), the salt solution was 20 mmol / L ammonium acetate solution (adjusted to pH 3.9 to 4.1 with acetic acid), the injection volume was 5 μl to 10 μl, and the chromatographic column was Waters Acquity BEH Amide 1.7 μm 2.1*50 mm.

[0107] Example 6

[0108] The results of the small-scale batch were determined using the method in Example 1. The actual values ​​and theoretical values ​​were both between 98% and 102%, which met the requirements. The results are shown in Table 8 below:

[0109] Table 8

[0110]

[0111] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0112] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for analyzing glucosamine hydrochloride, characterized in that, include: High performance liquid chromatography was used to detect glucosamine hydrochloride in the sample. The chromatographic conditions for the high-performance liquid chromatography method are as follows: The chromatographic column uses a stationary phase with hydrophilic properties; The mobile phase consists of ammonium acetate solution and acetonitrile, and isocratic elution is performed. The volume ratio of the ammonium acetate solution to acetonitrile is (5:95) to (8:92).

2. The analytical method according to claim 1, characterized in that, The concentration of the ammonium acetate solution is 18-22 mmol / L.

3. The analytical method according to claim 1, characterized in that, The ammonium acetate solution needs to be adjusted to pH 3.9-4.1 with acetic acid.

4. The analytical method according to claim 1, characterized in that, The isocratic elution flow rate is 0.7-0.9 ml / min.

5. The analytical method according to claim 1, characterized in that, The column temperature of the chromatographic column is 25-35℃.

6. The analytical method according to claim 1, characterized in that, The detection wavelength of the high-performance liquid chromatography method is 192 nm.

7. The analytical method according to claim 1, characterized in that, The high performance liquid chromatography method uses an ultra-high performance liquid chromatograph for detection, and the detector of the ultra-high performance liquid chromatograph is an ultraviolet detector or a diode array detector.

8. The analytical method according to claim 1, characterized in that, The high-performance liquid chromatography method used a Waters Acquity BEH Amide column.

9. The analytical method according to claim 1, characterized in that, When performing detection using the high-performance liquid chromatography method, the injection volume is 5-10 μl.

10. The analytical method according to claim 1, characterized in that, The sample to be tested is prepared into a solution, wherein the solution contains water and acetonitrile, and the volume ratio of water to acetonitrile is (7:43)-(10:40).

11. The analytical method according to claim 10, characterized in that, When preparing the solution of the sample to be tested, the sample to be tested needs to be mixed with water and subjected to ultrasonic treatment, and the ultrasonically treated solution is then mixed with acetonitrile.

12. The analytical method according to claim 11, characterized in that, The ultrasonic treatment time is 5 min to 30 min.

Images