Method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for detecting glucosamine sulfate by using a liquid chromatography-mass spectrometry technology. The method comprises the following steps: adopting an octadecyl silane bonded silica gel column as a chromatographic column; an acidic aqueous solution is used as a mobile phase A; and carrying out gradient elution by using an organic solution as a mobile phase B. The detection method has the advantages of high sensitivity, strong specificity, high precision, strong accuracy and simple and rapid operation, and can greatly shorten the experiment time and realize rapid detection of the dissolution curves of the glucosamine sulfate capsule imitation medicine and the reference preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis. Specifically, the present invention relates to a detection method for detecting glucosamine sulfate capsules by using a liquid chromatography-mass spectrometry instrument. Background Art

[0002] Glucosamine is a natural amino monosaccharide, which is an essential component for synthesizing proteoglycans in the matrix of human articular cartilage. It is a specific substance recognized by the international medical community for repairing bone joints, and has a positive effect on the bone joint problems of middle-aged and elderly people in particular. Glucosamine is a physiologically active substance, so it has good tolerance, a very low incidence of adverse reactions, and is safe to use. In the 2018 edition of the Guidelines for the Diagnosis and Treatment of Osteoarthritis in China, glucosamine is recommended as a slow-acting drug for relieving the symptoms of osteoarthritis, and it is easy to promote in the market.

[0003] Its pharmacological mechanism of action is that it is a normal component of cartilage matrix polysaccharide chains and synovial fluid polyglucosamine, which can stimulate chondrocytes to synthesize physiological polyglucosamine and proteoglycans, and stimulate synovial cells to synthesize hyaluronic acid. In addition, this product can also inhibit the enzymes that damage cartilage, such as the activities of collagenase and phospholipase A2, prevent the generation of superoxide free radicals in damaged tissues, and inhibit the activity of lysosomal enzymes. Therefore, this product shows a mild anti-inflammatory effect.

[0004] Osteoarthritis is a chronic degenerative disease with a slow and progressive development of the lesion. It is the most common arthritis, mainly manifested by the wear and tear of articular cartilage. The clinical manifestations are joint pain, swelling, joint deformity and limited movement. The prevalence of osteoarthropathy in the 40-year-old population in China is relatively high. Glucosamine has a long history of being used for the prevention and treatment of osteoarthritis. Glucosamine can be used as a treatment option for early and mid-stage osteoarthritis, but it has poor efficacy for patients with end-stage osteoarthritis with severe wear of articular cartilage.

[0005] Therefore, the market value of glucosamine sulfate capsules is extremely important. At present, the reference preparation of this variety announced by the state was developed and launched by Rotta Pharmaceutical Factory in Italy in the 1970s, with the trade name of Viscosupplement, and the specification is 0.25 g (calculated as glucosamine sulfate). According to the requirements of national regulations, generic drugs need to be consistent with the reference preparation in terms of quality, efficacy and safety.

[0006] Glucosamine sulfate belongs to carbohydrate compounds, and its ultraviolet absorption wavelength is end absorption. When using conventional high-performance liquid chromatography, there will be significant baseline background noise interference. Therefore, through literature retrieval, we found that other content detection methods mainly include high-performance liquid chromatography - pre-column derivation method, high-performance liquid chromatography - evaporative light scattering detector method (HPLC-ELSD), high-performance liquid chromatography - refractive index detector method (HPLC-RID), etc. However, the above methods have low detection efficiency, poor specificity, require a long gradient elution time for separation, low sensitivity, complex experimental operation process, resulting in large detection errors. Moreover, the amount of samples for dissolution curve detection is huge, and the dissolution at each time point needs to be controlled within 5 minutes as much as possible to ensure the detection efficiency. Based on this, there is an urgent need to develop a dissolution curve detection method that is convenient, fast, highly sensitive, specific, precise, and accurate. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide a high-performance liquid chromatography - mass spectrometry method for dissolution curves that is convenient, fast, highly sensitive, specific, precise, and accurate, thereby realizing the rapid detection and comparison of the dissolution curves of generic glucosamine sulfate capsules and reference preparations.

[0008] In one aspect of the present invention, the present invention provides a method for detecting the dissolution curve of glucosamine sulfate capsules using a liquid chromatography - mass spectrometry instrument.

[0009] The determination method described in the present invention can be realized according to the following method:

[0010] (1) Take an appropriate amount of the test sample, dissolve it in water, and prepare a test sample solution with a certain concentration.

[0011] (2) Set the flow rate of the mobile phase for gradient elution to 0.6 mL / min; the fragmentation voltage to 325 V; the column temperature of the chromatographic column to 30 °C; the temperature of the drying gas of the ion source to 325 °C, and the flow rate to 10 mL / min; the capillary voltage to 4000 V.

[0012] (3) Take 2.5 - 7.5 μL, preferably 5 μL, of the test sample solution in step (1), inject it into the liquid chromatography - mass spectrometry instrument, and complete the determination of glucosamine.

[0013] The liquid chromatography - mass spectrometry detection method: Use HILIC 3μm (50*2.1mm) as the chromatographic column (hydrophilic chromatographic columns such as TitankHILIC, Agilent HILIC Z, waters T3, etc. can all be used); use an acidic aqueous solution as the mobile phase for elution; use a Fragment voltage of 60 - 180 V.

[0014] Using this method, the content of glucosamine in the sample to be tested can be quickly detected, thereby effectively controlling the quality of the bulk drug and its preparations. Moreover, this detection method has high sensitivity, strong specificity, high precision, high accuracy, and is easy to operate, and can effectively control the quality of drugs.

[0015] The method for determining the content of glucosamine in the bulk drug and its preparations by using a liquid chromatography-mass spectrometry instrument according to an embodiment of the present invention may further have the following additional technical features:

[0016] According to an embodiment of the present invention, the chromatographic column is 50 mm. Thus, the resolution between various impurities can be further improved.

[0017] According to an embodiment of the present invention, the particle size of the filler in the chromatographic column is 1.8 - 5 μm, preferably 3 μm. Thus, the resolution can be further improved.

[0018] The best separation effect and the best peak shape are obtained thereby.

[0019] According to an embodiment of the present invention, the first organic solvent is at least one selected from methanol and acetonitrile, preferably acetonitrile. The best peak shape and separation effect are obtained thereby, and the retention time is moderate.

[0020] According to an embodiment of the present invention, the acidic aqueous solution is an aqueous solution of 0.2% by volume of formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution. The best baseline is obtained thereby, and it can ensure that the blank solvent has no interference with the detection of impurities.

[0021] According to an embodiment of the present invention, the concentration of the acidic aqueous solution is 0.1% - 0.3% by volume, preferably 0.2% by volume. Thus, the resolution can be further improved, and the ionization degree of each impurity can be increased under the condition of obtaining a good peak shape and separation effect, improving the detection sensitivity.

[0022] According to an embodiment of the present invention, a sample solution is prepared by using a mixed solution of the second organic solvent and water for the sample to be tested; the sample solution is injected into a high-performance liquid chromatograph to complete the determination of glucosamine in the bulk drug and its preparations.

[0023] According to an embodiment of the present invention, in the mixed solution, the volume ratio of the second organic solvent to the water is 15:85. Thus, the symmetry of the peak can be further improved, and the solvent effect can be prevented.

[0024] According to an embodiment of the present invention, the fragmentation voltage is 60 - 180 V, preferably 130 V. Thus, the detection sensitivity can be further improved.

[0025] According to an embodiment of the present invention, the detection conditions are as follows: the flow rate of the mobile phase is 0.5 to 0.7 mL / min, preferably 0.6 mL / min. Thus, the resolution can be further improved while the peak shape is improved.

[0026] According to an embodiment of the present invention, the column temperature of the chromatographic column is 25°C to 35°C, preferably 30°C. Thus, the separation of each component can be further improved.

[0027] According to an embodiment of the present invention, the temperature of the drying gas of the ion source is 300°C to 350°C, preferably 325°C. Thus, the ionization rate of each component can be further increased, and the detection sensitivity can be improved.

[0028] According to an embodiment of the present invention, the flow rate of the drying gas of the ion source is 8 to 12 mL / min, preferably 10 mL / min. Thus, the interference of charged droplets can be further reduced, the ionization rate of each component can be increased, and the detection sensitivity can be improved.

[0029] According to an embodiment of the present invention, the capillary voltage is 3000 V to 5000 V, preferably 4000 V. Thus, the maximum ionization efficiency can be achieved, fragmentation in the ion source can be prevented, and the detection sensitivity can be improved.

[0030] According to an embodiment of the present invention, the volume of the sample solution injected into the liquid chromatography-mass spectrometry instrument is 2.5 to 7.5 μL, preferably 5 μL.

[0031] The elution and separation effect of the method of the present invention is good, the detection sensitivity is high, and the specificity is strong. It can separate glucosamine in the sample under the same chromatographic conditions and has high sensitivity. The content of glucosamine in the sample can be detected quickly, accurately and reliably. The present invention effectively controls the quality of the raw material drug and its preparations by detecting glucosamine in the raw material drug and its preparations by using the LCMS method.

[0032] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above and other purposes, features and advantages of the present invention more obvious and understandable, the following is described in detail with reference to the preferred embodiments. Brief Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly introduced below.

[0034] Figure 1 : Chromatogram of the blank solution detected under the conditions of Example 1;

[0035] Figure 2 : Chromatogram of Linear 1 detected under the conditions of Example 1;

[0036] Figure 3 : Chromatogram of Linear 2 detected under the conditions of Example 1;

[0037] Figure 4 : Chromatogram of Linear 3 detected under the conditions of Example 1;

[0038] Figure 5 : Chromatogram of Linear 4 detected under the conditions of Example 1;

[0039] Figure 6 : Chromatogram of Linear 5 detected under the conditions of Example 1;

[0040] Figure 7 : Chromatogram of 100% sample addition recovery solution detected under the conditions of Example 1;

[0041] Figure 8 : Chromatogram of the test solution of the reference preparation detected under the conditions of Example 1;

[0042] Figure 9 : Chromatogram of the test solution of the generic preparation detected under the conditions of Example 1. Detailed Description of the Embodiment

[0043] The following details the specific embodiments of the present invention. As part of this specification, the principles of the present invention are illustrated through examples. Other aspects, features, and advantages of the present invention will become apparent through this detailed description.

[0044] The following details the embodiments of the present invention. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the invention. For those embodiments where specific techniques or conditions are not indicated, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0045] In the embodiments of the present invention, the generic preparation used is self-made by the inventor, and the reference preparation is purchased externally.

[0046] Reference preparation (ROTTAPHARM S.P.A, specification: 0.25 g, batch number: 8176874)

[0047] Generic preparation (self-made, specification: 0.25 g, batch number: 24100801)

[0048] All the liquid chromatography - mass spectrometry instruments used in the following example tests are Agilent 1260 - MSD liquid chromatography - mass spectrometry instruments.

[0049] Example 1

[0050] Chromatographic column: Titank HILIC 3μm (50*2.1mm) was used as the chromatographic column;

[0051] Mobile phase: An aqueous solution of 0.2% (v / v) formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution;

[0052] Column temperature: 30 °C;

[0053] Flow rate: 0.6 mL / min;

[0054] Fragmentation voltage: 130 V;

[0055] Ion source drying gas temperature: 325 °C;

[0056] Ion source drying gas flow rate: 10 mL / min;

[0057] Capillary voltage: 4000 V;

[0058] Sample injection volume: 5 μL

[0059] Test procedure:

[0060] Diluent: Water

[0061] Blank solution: Diluent

[0062] Reference preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute it to the mark with water, shake well. Take 1 mL of the above solution and transfer it to a 10 mL volumetric flask, dissolve and dilute it to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0063] Self - made preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute it to the mark with water, shake well. Take 1 mL of the above solution and transfer it to a 10 mL volumetric flask, dissolve and dilute it to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0064] Reference stock solution: Take about 30 mg of the reference substance of this product, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve and dilute it to the mark, shake well. Accurately pipette 1 mL of the above solution into a 100 mL volumetric flask, dilute it to the mark with water, shake well. (3 μg / mL)

[0065] Linear solution 1 (120%): Accurately measure 6 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.36 μg / mL)

[0066] Linear solution 2 (100%): Accurately measure 5 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0067] Linear solution 3 (50%): Accurately measure 2.5 ml of the reference stock solution, transfer it to a 50-ml volumetric flask, dilute it to the mark with the diluent, and shake well. That is the solution obtained. (0.15 μg / ml)

[0068] Linear solution 4 (20%): Accurately measure 1 ml of the reference stock solution, transfer it to a 50-ml volumetric flask, dilute it to the mark with the diluent, and shake well. That is the solution obtained. (0.06 μg / ml)

[0069] Linear solution 5 (5%): Accurately measure 5 ml of linear solution 3, transfer it to a 50-ml volumetric flask, dilute it to the mark with the diluent, and shake well. That is the solution obtained. (0.015 μg / ml)

[0070] 100% spiked recovery test sample solution: Take an appropriate amount of the blank excipient of this product, dissolve it and dilute it to the mark with 100% linear solution, and shake well. Filter it to obtain the solution.

[0071] Respectively measure the above solutions, conduct analysis under the above liquid chromatography-mass spectrometry conditions, record the chromatogram, and the results are shown in Appendix Figure 1 Appendix Figure 2 Appendix Figure 3 Appendix Figure 4 Appendix Figure 5 Appendix Figure 6 Appendix Figure 7 Appendix Figure 8 Appendix Figure 9 .

[0072] Conclusion: Figure 1 It shows that the blank solution has no interference in the detection of each impurity and has good specificity; Figures 2 to 6 It shows that each linear relationship is good; Figure 7 It shows that the accuracy of each impurity is good under this detection condition; Figures 8 to 9 It shows that the content of the self-made preparation and the reference preparation meets the regulations.

[0073] Example 2

[0074] Chromatographic column: Use Titank HILIC 3μm (50*2.1mm) as the chromatographic column;

[0075] Mobile phase: An aqueous solution of 0.2% (v / v) formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution;

[0076] Column temperature: 30°C;

[0077] Flow rate: 0.5 mL / min;

[0078] Fragmentation voltage: 130 V;

[0079] Ion source drying gas temperature: 325°C;

[0080] Ion source drying gas flow rate: 10 mL / min;

[0081] Capillary voltage: 4000 V;

[0082] Sample injection volume: 5 μL

[0083] Test procedure:

[0084] Diluent: water

[0085] Blank solution: diluent

[0086] Reference preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute it to the mark with water, shake well, take 1 mL of the above solution to a 10 mL volumetric flask, dissolve and dilute it to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0087] Self-made preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute it to the mark with water, shake well, take 1 mL of the above solution to a 10 mL volumetric flask, dissolve and dilute it to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0088] Reference substance stock solution: Take about 30 mg of the reference substance of this product, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve and dilute it to the mark, shake well. Accurately pipette 1 mL of the above solution into a 100 mL volumetric flask, dilute it to the mark with water, and shake well. (3 μg / mL)

[0089] Linear solution 1 (120%): Accurately measure 6 mL of the reference substance stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.36 μg / mL)

[0090] Linear solution 2 (100%): Accurately measure 5 mL of the reference substance stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0091] Linear solution 3 (50%): Accurately measure 2.5 mL of the reference substance stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.15 μg / mL)

[0092] Linear solution 4 (20%): Accurately measure 1 mL of the reference substance stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.06 μg / mL)

[0093] Linear solution 5 (5%): Accurately measure 5 mL of linear solution 3, place it in a 50 mL volumetric flask, dilute it to the mark with the diluent, shake well, and you will get it. (0.015 μg / mL)

[0094] 100% spiked recovery test sample solution: Take an appropriate amount of the blank excipient of this product, dissolve it with 100% linear solution and dilute to the mark, shake well. Filter to obtain.

[0095] Measure the above solutions respectively, analyze them under the above liquid chromatography - mass spectrometry conditions, and record the chromatograms.

[0096] Conclusion: Under these detection conditions, the blank has no interference with the detection, the linear relationship is good, and the contents of the self - made preparation and the reference preparation meet the requirements.

[0097] Example 3

[0098] Chromatographic column: Use Titank HILIC 3μm (50 * 2.1mm) as the chromatographic column;

[0099] Mobile phase: An aqueous solution of 0.2% (v / v) formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution;

[0100] Column temperature: 30°C;

[0101] Flow rate: 0.7 mL / min;

[0102] Fragmentation voltage: 130 V;

[0103] Ion source drying gas temperature: 325°C;

[0104] Ion source drying gas flow rate: 10 mL / min;

[0105] Capillary voltage: 4000 V;

[0106] Injection volume: 5 μL

[0107] Test procedure:

[0108] Diluent: Acetonitrile - water (80:20) (v / v)

[0109] Blank solution: Diluent

[0110] Reference preparation test sample solution: Take 1 mL of the dissolution solution of this product, place it in a 100 - mL volumetric flask, dilute to the mark with water, shake well, take 1 mL of the above solution to a 10 - mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well to obtain. (0.3 μg / mL)

[0111] Self - made preparation test sample solution: Take 1 mL of the dissolution solution of this product, place it in a 100 - mL volumetric flask, dilute to the mark with water, shake well, take 1 mL of the above solution to a 10 - mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well to obtain. (0.3 μg / mL)

[0112] Reference stock solution: Weigh accurately about 30 mg of the reference substance of this product, place it in a 100-ml volumetric flask, add water to dissolve and dilute to the mark, and shake well. Accurately pipette 1 ml of the above solution into a 100-ml volumetric flask, add water to dilute to the mark, and shake well. (3 μg / ml)

[0113] Linear solution 1 (120%): Accurately measure 6 ml of the reference stock solution, place it in a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well. (0.36 μg / ml)

[0114] Linear solution 2 (100%): Accurately measure 5 ml of the reference stock solution, place it in a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well. (0.3 μg / ml)

[0115] Linear solution 3 (50%): Accurately measure 2.5 ml of the reference stock solution, place it in a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well. (0.15 μg / ml)

[0116] Linear solution 4 (20%): Accurately measure 1 ml of the reference stock solution, place it in a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well. (0.06 μg / ml)

[0117] Linear solution 5 (5%): Accurately measure 5 ml of linear solution 3, place it in a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well. (0.015 μg / ml)

[0118] 100% spiked recovery test sample solution: Take an appropriate amount of the blank excipient of this product, dissolve and dilute to the mark with the 100% linear solution, and shake well. Filter to obtain the solution.

[0119] Respectively pipette the above solutions, analyze under the above liquid chromatography-mass spectrometry conditions, and record the chromatogram.

[0120] Conclusion: Under this detection condition, the blank has no interference with the detection, the linear relationship is good, and the contents of the self-made preparation and the reference preparation meet the requirements.

[0121] Example 4

[0122] Chromatographic column: Use Titank HILIC 3 μm (50 * 2.1 mm) as the chromatographic column;

[0123] Mobile phase: An aqueous solution of 0.2% (v / v) formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution;

[0124] Column temperature: 30 °C;

[0125] Flow rate: 0.6 mL / min;

[0126] Fragmentation voltage: 132 V;

[0127] Ion source drying gas temperature: 345 °C;

[0128] Ion source drying gas flow rate: 10 mL / min;

[0129] Capillary voltage: 4000 V;

[0130] Sample injection volume: 5 μL

[0131] Test procedure:

[0132] Diluent: water

[0133] Blank solution: diluent

[0134] Reference preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute to the mark with water, shake well. Take 1 mL of the above solution and transfer it to a 10 mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0135] Self-made preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute to the mark with water, shake well. Take 1 mL of the above solution and transfer it to a 10 mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0136] Reference stock solution: Take about 30 mg of the reference substance of this product, weigh it accurately, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well. Accurately pipette 1 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with water, shake well. (3 μg / mL)

[0137] Linear solution 1 (120%): Accurately measure 6 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well, and you will get it. (0.36 μg / mL)

[0138] Linear solution 2 (100%): Accurately measure 5 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well, and you will get it. (0.3 μg / mL)

[0139] Linear solution 3 (50%): Accurately measure 2.5 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well, and you will get it. (0.15 μg / mL)

[0140] Linear solution 4 (20%): Accurately measure 1 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well, and you will get it. (0.06 μg / mL)

[0141] Linear solution 5 (5%): Accurately measure 5 ml of linear solution 3, transfer it to a 50-ml volumetric flask, dilute it to the mark with the diluent, and shake well. That is the solution obtained. (0.015 μg / ml)

[0142] 100% spiked recovery test sample solution: Take an appropriate amount of the blank excipient of this product, dissolve it and dilute it to the mark with 100% linear solution, and shake well. Filter it to obtain the solution.

[0143] Respectively measure the above solutions, analyze them under the above liquid chromatography-mass spectrometry conditions, and record the chromatogram.

[0144] Conclusion: Under this detection condition, the blank has no interference with the detection, the linear relationship is good, and the contents of the self-made preparation and the reference preparation meet the requirements.

[0145] Example 5

[0146] Chromatographic column: Use Titank HILIC 3μm (50 * 2.1 mm) as the chromatographic column;

[0147] Mobile phase: An aqueous solution of 0.2% (v / v) formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution;

[0148] Column temperature: 30 °C;

[0149] Flow rate: 0.6 mL / min;

[0150] Fragmentation voltage: 130 V;

[0151] Ion source drying gas temperature: 325 °C;

[0152] Ion source drying gas flow rate: 10 mL / min;

[0153] Capillary voltage: 4000 V;

[0154] Injection volume: 5 μL

[0155] Test procedure:

[0156] Diluent: Water

[0157] Blank solution: Diluent

[0158] Reference preparation test sample solution: Take 1 ml of the dissolution solution of this product, transfer it to a 100-ml volumetric flask, dilute it to the mark with water, shake well, take 1 ml of the above solution to a 10-ml volumetric flask, dissolve it and dilute it to the mark with the diluent, and shake well. That is the solution obtained. (0.3 μg / ml)

[0159] Self - made preparation test solution: Take 1 ml of the dissolution solution of this product, place it in a 100 - ml volumetric flask, dilute it to the mark with water, shake well. Take 1 ml of the above solution and transfer it to a 10 - ml volumetric flask, dissolve and dilute it to the mark with the dilution solution, shake well to obtain. (0.3 μg / ml)

[0160] Reference stock solution: Take about 30 mg of the reference substance of this product, accurately weigh it, place it in a 100 - ml volumetric flask, add water to dissolve and dilute it to the mark, shake well. Accurately transfer 1 ml of the above solution to a 100 - ml volumetric flask, dilute it to the mark with water, shake well. (3 μg / ml)

[0161] Linear solution 1 (120%): Accurately measure 6 ml of the reference stock solution, place it in a 50 - ml volumetric flask, dilute it to the mark with the dilution solution, shake well to obtain. (0.36 μg / ml)

[0162] Linear solution 2 (100%): Accurately measure 5 ml of the reference stock solution, place it in a 50 - ml volumetric flask, dilute it to the mark with the dilution solution, shake well to obtain. (0.3 μg / ml)

[0163] Linear solution 3 (50%): Accurately measure 2.5 ml of the reference stock solution, place it in a 50 - ml volumetric flask, dilute it to the mark with the dilution solution, shake well to obtain. (0.15 μg / ml)

[0164] Linear solution 4 (20%): Accurately measure 1 ml of the reference stock solution, place it in a 50 - ml volumetric flask, dilute it to the mark with the dilution solution, shake well to obtain. (0.06 μg / ml)

[0165] Linear solution 5 (5%): Accurately measure 5 ml of linear solution 3, place it in a 50 - ml volumetric flask, dilute it to the mark with the dilution solution, shake well to obtain. (0.015 μg / ml)

[0166] 100% spiked recovery test solution: Take an appropriate amount of the blank excipients of this product, dissolve and dilute it to the mark with the 100% linear solution, shake well. Filter to obtain.

[0167] Respectively measure the above solutions, analyze them under the above liquid - phase mass spectrometry conditions, and record the chromatogram. Conclusion: Under this detection condition, the blank has no interference with the detection, the linear relationship is good, and the contents of the self - made preparation and the reference preparation meet the requirements.

[0168] Comparative Example 1

[0169] Chromatographic column: Inertsustain Phenyl (250 * 4.0 mm, 5 μm)

[0170] Mobile phase: An aqueous solution of 0.2% (v / v) formic acid and 2.00 mM ammonium acetate - acetonitrile (15:85) solution;

[0171] Column temperature: 30 °C;

[0172] Flow rate: 0.6 mL / min;

[0173] Fragmentation voltage: 130 V;

[0174] Ion source drying gas temperature: 350 °C;

[0175] Ion source drying gas flow rate: 10 mL / min;

[0176] Capillary voltage: 4000 V;

[0177] Sample injection volume: 5 μL

[0178] Test procedure:

[0179] Diluent: water

[0180] Blank solution: diluent

[0181] Reference preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute to the mark with water, shake well. Take 1 mL of the above solution and transfer it to a 10 mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well to obtain the solution. (0.3 μg / mL)

[0182] Self - made preparation test solution: Take 1 mL of the dissolution solution of this product, place it in a 100 mL volumetric flask, dilute to the mark with water, shake well. Take 1 mL of the above solution and transfer it to a 10 mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well to obtain the solution. (0.3 μg / mL)

[0183] Reference stock solution: Take about 30 mg of the reference substance of this product, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well. Accurately pipette 1 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with water, shake well. (3 μg / mL)

[0184] Linear solution 1 (120%): Accurately measure 6 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well to obtain the solution. (0.36 μg / mL)

[0185] Linear solution 2 (100%): Accurately measure 5 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well to obtain the solution. (0.3 μg / mL)

[0186] Linear solution 3 (50%): Accurately measure 2.5 mL of the reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, shake well to obtain the solution. (0.15 μg / mL)

[0187] Linear solution 4 (20%): Accurately measure 1 ml of the reference stock solution, place it in a 50-ml volumetric flask, dilute it to the mark with the diluent, and shake well to obtain. (0.06 μg / ml)

[0188] Linear solution 5 (5%): Accurately measure 5 ml of linear solution 3, place it in a 50-ml volumetric flask, dilute it to the mark with the diluent, and shake well to obtain. (0.015 μg / ml)

[0189] 100% spiked recovery test sample solution: Take an appropriate amount of the blank excipient of this product, dissolve and dilute it to the mark with 100% linear solution, and shake well. Filter to obtain.

[0190] Respectively measure the above solutions, analyze them under the above liquid chromatography - mass spectrometry conditions, and record the chromatograms.

[0191] Conclusion: Under this detection condition, the blank has no interference with the detection, the linear relationship is good, and the contents of the self - made preparation and the reference preparation meet the requirements.

[0192] Each raw material listed in the present invention, as well as the upper and lower limits and interval values of each raw material of the present invention, and the upper and lower limits and interval values of the process parameters (such as temperature, time, etc.) can all implement the present invention, and the examples are not listed one by one here.

[0193] The above is the preferred implementation manner of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and changes can be made, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A method for detecting glucosamine sulfate by liquid chromatography - mass spectrometry, characterized in that, It includes the following steps: (1) Take an appropriate amount of the sample to be tested, dissolve it in water, and prepare a sample solution to be tested with a certain concentration; (2) The target molecular weight for mass spectrometry quantitative detection is 180.2; The chromatographic conditions during liquid chromatography testing are as follows: column temperature: 25°C to 35°C; injection volume: 2.5 to 7.5 μL; flow rate: 0.5 to 0.7 mL / min; the column length of the chromatographic column is 50 mm, the inner diameter is 2.1 mm, the chromatographic column is a hydrophilic chromatographic column, the particle size of the packing in the chromatographic column is 1.8 to 5 μm; the chromatographic column packing is organic hybrid silica gel; the mobile phase is a homogeneous acidic solution after mixing an organic solvent and an acidic aqueous solution; The mass spectrometry conditions are as follows: ion source temperature: 300°C to 350°C; the flow rate of the drying gas of the ion source is 8 to 12 mL / min; the capillary voltage is 3000 V to 5000 V, and the fragmentation voltage is 60 to 180 V; (3) Inject the sample solution to be tested in step (1) into the liquid chromatography-mass spectrometry instrument to complete the determination of glucosamine.

2. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The acidic aqueous solution is an aqueous solution - acetonitrile (15:85, V:V) solution of 0.1% to 0.3% (by volume) formic acid and 2.00 mM ammonium acetate.

3. The method for detecting glucosamine sulfate by using liquid chromatography-mass spectrometry technology according to claim 1, wherein: The particle size of the packing in the chromatographic column is 3 μm.

4. The method for detecting glucosamine sulfate by using liquid chromatography - mass spectrometry technology according to claim 1, characterized in that: The chromatographic column selected is HILIC 3μm (50*2.1mm).

5. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 1, wherein: The sample to be tested is glucosamine sulfate bulk drug or glucosamine sulfate preparation.

6. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 5, characterized in that: The glucosamine sulfate preparation is glucosamine sulfate capsules.

7. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The fragmentation voltage is 130 V.

8. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 1, wherein: The flow rate of the mobile phase is 0.6 mL / min; the column temperature of the chromatographic column is 30°C.

9. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The temperature of the drying gas of the ion source is 325°C; the flow rate of the drying gas of the ion source is 10 mL / min; the capillary voltage is 4000 V.

10. The method for detecting glucosamine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The injection volume is 5 μL.

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