CN113325101A

CN113325101A - Method for detecting dissolution rates of glucosamine hydrochloride preparation in different media

Info

Publication number: CN113325101A
Application number: CN202110586677.8A
Authority: CN
Inventors: 陈永红; 云邱; 王兰花; 薛亚军; 王华娟
Original assignee: Nanjing Healthnice Pharmaceutical Co ltd; Nanjing Yinuo Medicine Technology Co ltd; Nanjing Healthnice Pharmaceutical Technology Co ltd
Current assignee: Nanjing Healthnice Pharmaceutical Co ltd; Nanjing Yinuo Medicine Technology Co ltd; Nanjing Healthnice Pharmaceutical Technology Co ltd
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2021-08-31
Anticipated expiration: 2041-05-27
Also published as: CN113325101B

Abstract

The invention relates to a method for detecting dissolution rate of a glucosamine hydrochloride preparation, which uses potassium dihydrogen phosphate buffer solution-acetonitrile containing a positive ion pair reagent (tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide) as a mixed mobile phase for isocratic elution, optimizes the proportion of the mixed mobile phase, prolongs the retention time of a main peak of the glucosamine hydrochloride on a chromatographic column (such as a C8 column) during chromatographic analysis, avoids the interference of the main peak by dissolution media and auxiliary material solutions, and has good reproducibility and high sensitivity. The dissolution rate detection method provided by the invention has low requirements on a chromatographic column in the chromatographic analysis process, does not need to adopt an amino column and does not need pre-column derivatization reaction, can adapt to the accurate determination of the dissolution rate of the glucosamine hydrochloride preparation in different dissolution media, discharges the interference of blank solvents and auxiliary materials, and has the advantages of simple operation, high sensitivity, low cost, good reproducibility, high accuracy and wide application range.

Description

Method for detecting dissolution rates of glucosamine hydrochloride preparation in different media

Technical Field

The invention belongs to the field of analysis and detection of pharmaceutical preparations, and particularly relates to a method for detecting dissolution rates of a glucosamine hydrochloride preparation in different media.

Background

Glucosamine is a natural amino monosaccharide extracted from crustacean shells, and is an important structural component of glycosaminoglycan and hyaluronic acid. It is a very special medicine, belonging to both medicine and health-care product. Because the glucosamine belongs to biological extraction, has small side effect, and can be directly sold as food or health care products abroad; and because of good therapeutic effect on osteoarthritis, the medicine is declared in Italy for the first time in the 80 th of the 20 th century. In china, glucosamine is reported as a basic drug in the form of hydrochloride or sulfate.

In the quality control of oral tablets for pharmaceutical use, the examination of the dissolution profile of oral tablets is an indispensable examination item. The in-vitro release condition of the drug tablet under different dissolution medium conditions (simulating the environment of dissolving the drug tablet in the gastrointestinal tract of a human body) can be examined by measuring the dissolution rate of the oral tablet, and the in-vivo release pattern of the drug tablet can be indirectly evaluated.

Accurate detection of dissolution rates of different drug tablets is the basis for ensuring the accuracy and reliability of dissolution rate experiments. The classic detection method of glucosamine hydrochloride content is an ultraviolet spectrophotometry after an Elson-Morgan reaction, has complex operation and poor reproducibility, and has the methods of derivatization high performance liquid chromatography, high performance liquid-evaporative light scattering detector method, high performance liquid-differential refraction detector method, amino column method of high performance liquid-ultraviolet detector and the like reported in literature, or has complex operation or higher cost or weak specificity. Currently, the united states pharmacopoeia (USP43) contains a dissolution rate detection method for glucosamine tablets, that is, a high performance liquid chromatography without derivatization, in which a chromatographic column (250 × 4.6mm, 5 μm) using octyl silane-bonded silica gel as a filler, 0.05% phosphoric acid solution (potassium hydroxide adjusted to pH 3.0) -acetonitrile (60:40) as a mobile phase, a detection wavelength of 195nm, and a flow rate of 0.6 ml/min. However, in this assay method, when the dissolution rates are measured using a sodium acetate solution at pH4.5 and a phosphate solution at pH6.8 as dissolution media, respectively, the blank solvent seriously interferes with the detection of the glucosamine hydrochloride peak.

In order to overcome the defects of the existing analysis method, it is very important to search a detection method for detecting the dissolution rate of the glucosamine hydrochloride preparation under the condition of various dissolution media.

Disclosure of Invention

The invention aims to provide a method for detecting the dissolution rate of a glucosamine hydrochloride preparation in different media on the basis of the prior art.

The technical scheme of the invention is as follows:

a method for detecting dissolution rate of glucosamine hydrochloride preparation comprises the following steps:

(1) preparing a test solution: dissolving glucosamine hydrochloride preparation in dissolution medium, quantitatively diluting, taking out dissolution liquid at time t, and filtering to obtain filtrate as test solution at time t;

(2) preparing a reference substance solution: taking a glucosamine hydrochloride reference substance, adding a dissolution medium for dissolving, quantitatively diluting, and shaking up to obtain a reference substance solution;

(3) blank solution: a dissolution medium;

(4) and (3) detecting by high performance liquid chromatography: detecting the test solution, the reference solution and the blank solution by adopting a high performance liquid chromatography, recording a chromatogram, and calculating the concentration and/or the content of glucosamine hydrochloride in the glucosamine hydrochloride preparation according to a peak area so as to obtain the dissolution rate of the glucosamine hydrochloride preparation at time t;

wherein, the chromatographic conditions detected by the high performance liquid chromatography are as follows: the chromatographic column takes octyl silane bonded silica gel as a filling agent; performing isocratic elution by taking the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the volume ratio of the mobile phase A to the mobile phase B in the isocratic elution process is 75-85: 25-15; the mobile phase A is potassium dihydrogen phosphate buffer solution containing 1-10 mmol/L of positive ion pairing reagent, the pH value of the buffer solution is adjusted to 3.0-4.0 by phosphoric acid, and the positive ion pairing reagent in the mobile phase A is tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide; mobile phase B was acetonitrile.

In step (1), a time point at time t can be selected according to actual needs, for example, if time t is 5min, that is, in step (4), the dissolution rate of the glucosamine hydrochloride preparation at 5min is determined.

In the step (4), the isocratic elution time is 6min, and the tailing factor of the glucosamine hydrochloride peak is not more than 2.0, which meets the requirement.

The method for detecting the dissolution rate can be used for detecting the dissolution rate of the glucosamine hydrochloride preparation in different media, and the glucosamine hydrochloride preparation can be but is not limited to a glucosamine hydrochloride tablet, a glucosamine hydrochloride capsule, a glucosamine hydrochloride granule or a glucosamine hydrochloride dispersing agent, wherein the glucosamine hydrochloride tablet has the best effect.

Further, the dissolution medium mentioned in the present invention can be, but is not limited to, acetate buffer solution with pH of 3.8-5.8, phosphate buffer solution with pH of 4.5-8.0, or water. For example, the dissolution medium is a pH4.5 sodium acetate solution, a pH6.8 phosphate solution or water, and the dissolution is measured with reference to the second method of 0931, the four general rules of the chinese pharmacopoeia 2020 edition.

The method for detecting the dissolution rate provided by the invention uses a potassium dihydrogen phosphate buffer solution (mobile phase A) -acetonitrile (mobile phase B) containing a positive ion pair reagent (tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide) as a mixed mobile phase for isocratic elution, and limits the concentration of the positive ion pair reagent and the proportion of the mobile phase A and the mobile phase B in the mixed mobile phase in the isocratic elution process, so that the retention time of a main peak of glucosamine hydrochloride in a chromatographic column is prolonged during chromatographic analysis, and the interference of a dissolution medium and an auxiliary material solution on the main peak under low wavelength is avoided; in the sample treatment process, the pre-column derivatization reaction is not needed, the operation is simple, the reproducibility is good, and the accuracy is high; the detector adopted in the detection process is the most common ultraviolet absorption detector which is high in sensitivity and insensitive to the environment, and an evaporation light scattering detector which is expensive or a differential refraction detector which is low in sensitivity and sensitive to the environment temperature is avoided, so that the cost is low, the sensitivity is high, and the operation is simple and convenient; the dissolution rate of the glucosamine hydrochloride preparation can be accurately measured in different media, the interference caused by blank solvents and auxiliary materials is eliminated, the reproducibility is good, the sensitivity is high, and the application range is wide.

The method for detecting the dissolution rate of the glucosamine hydrochloride preparation provided in the prior art generally has the problems of complex operation, high cost, low specificity or serious interference of blank solvents on the detection of a glucosamine hydrochloride peak and the like. Aiming at the problems, the invention tries to adjust the composition of the mobile phase and the proportion of the mixed mobile phase during chromatographic analysis, and as a result, the invention discovers that the retention time of the main peak of the glucosamine hydrochloride in a chromatographic column can be prolonged by adding tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide serving as a positive ion pair reagent into potassium dihydrogen phosphate buffer solution and using the mixture of the tetrabutylammonium hydroxide, the tetrabutylammonium hydrogen sulfate or the tetrabutylammonium bromide and acetonitrile serving as a mixed mobile phase, and adjusting the concentration of the positive ion pair reagent and the proportion of the mixed mobile phase in the isocratic elution process, so that the interference of a dissolution medium and an auxiliary material solution on the main peak at low wavelength is avoided, and the invention has the advantages of low cost, high sensitivity and simple operation. By adopting the detection method of the invention and adopting other similar positive ion pair reagents, such as dodecyl trimethyl ammonium chloride or hexadecyl trimethyl ammonium bromide, even if the concentration of the positive ion pair reagents is adjusted and the proportion of the mixed mobile phase is optimized, the problem of interference of a blank solvent on the main peak of glucosamine hydrochloride when the dissolution rate is measured by respectively taking a sodium acetate solution with pH4.5 and a phosphate solution with pH6.8 or water as dissolution media cannot be solved, the separation efficiency is poor, and the accuracy is low.

In the method for detecting the dissolution rate, even if tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide is added into the mobile phase A as a positive ion pairing reagent in the isocratic elution process, the concentration of the positive ion pairing reagent in the mobile phase A is strict, and the measurement of the dissolution rate of the glucosamine hydrochloride preparation is interfered when the concentration is too high or too low: when the concentration of the positive ion pair reagent is too low (less than 1mmol/L), the theoretical plate number of a glucosamine hydrochloride peak in a phosphate solution with the medium pH6.8 is reduced, a tailing factor is greater than 2.0, a main peak and a subsequent impurity peak cannot be separated from a base line, and the dissolution rate in the medium cannot be accurately measured; when the concentration of the positive ion pair reagent is too high, the mixture of buffer salt and the organic phase can occur, and the separated out or dissolution medium interferes with the determination of the dissolution peak of the glucosamine hydrochloride, for example, when the tetrabutylammonium bromide is 18.6mmol/L, the potassium dihydrogen phosphate buffer solution is separated out when the potassium dihydrogen phosphate buffer solution is mixed with the organic phase; when the tetrabutylammonium hydroxide is (16mmol/L), the retention time of an impurity peak in a sodium acetate solution with the medium pH of 4.5 is also delayed, the separation degree of the impurity peak and a main peak of glucosamine hydrochloride is reduced, and the detection of the main peak is easy to interfere.

In the step (4), the concentration of the positive ion pairing reagent (tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide) in the mobile phase A is 1-10 mmol/L, can be but is not limited to 1mmol/L, 3mmol/L, 3.5mmol/L, 3.85mmol/L, 4.65mmol/L, 5mmol/L, 5.5mmol/L, 6mmol/L, 8mmol/L or 1mmol/L, in a preferred embodiment, the concentration of the cation pairing agent (tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide) in the mobile phase A is 3-5 mmol/L, for example, the concentration of tetrabutylammonium hydroxide in mobile phase A is 3.85mmol/L or 5mmol/L, the concentration of tetrabutylammonium hydrogen sulfate in mobile phase A is 3.85mmol/L, and the concentration of tetrabutylammonium bromide in mobile phase A is 4.65 mmol/L.

In step (4), the concentration of the potassium dihydrogen phosphate in the mobile phase A is 10-60mmol/L, but can be, but is not limited to, 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L or 60mmol/L, and preferably, the concentration of the potassium dihydrogen phosphate in the mobile phase A is 50 mmol/L.

In step (4), the mobile phase a is formulated with phosphoric acid to a pH of 3.0-4.0, and the pH may be, but is not limited to, 3.0, 3.2, 3.5, 3.7, or 4.0, and preferably, the mobile phase a is formulated with phosphoric acid to a pH of 3.5.

In a preferred embodiment, the mobile phase A is potassium dihydrogen phosphate buffer containing tetrabutylammonium hydroxide of 3.85mmol/L, and the preparation method comprises the following steps: taking 6.8g of monopotassium phosphate to be added into a proper amount of water, then adding 10ml of 10% tetrabutyl ammonium hydroxide aqueous solution, stirring to dissolve, adding water to dilute to 1000ml, and adjusting the pH value to 3.5 by using phosphoric acid.

In a preferred scheme, the mobile phase A is a potassium dihydrogen phosphate buffer solution containing 3.85mmol/L tetrabutylammonium hydrogen sulfate, and the preparation method specifically comprises the following steps: taking 6.8g of monopotassium phosphate to be added into a proper amount of water, then adding 1.3g of tetrabutylammonium hydrogen sulfate, stirring and dissolving, adding water to dilute to 1000ml, and adjusting the pH value to 3.5 by using phosphoric acid.

In a preferred embodiment, the mobile phase A is potassium dihydrogen phosphate buffer containing 4.65mmol/L tetrabutylammonium bromide, and the preparation method comprises the following steps: taking 6.8g of monopotassium phosphate to be added into a proper amount of water, then adding 1.5g of tetrabutylammonium bromide, stirring and dissolving, adding water to dilute to 1000ml, and adjusting the pH value to 3.5 by using phosphoric acid.

In a preferred embodiment, the mobile phase A is a potassium dihydrogen phosphate buffer containing 5mmol/L tetrabutylammonium hydroxide, and is specifically prepared by the following steps: taking 6.8g of monopotassium phosphate to be added into a proper amount of water, adding 13ml of 10% tetrabutyl ammonium hydroxide aqueous solution, stirring to dissolve, adding water to dilute to 1000ml, and adjusting the pH value to 3.5 by using phosphoric acid.

In the step (4), the volume ratio of the mobile phase A to the mobile phase B in the isocratic elution process is 75-85: 25-15, but not limited to 75:25, 78:22, 80:20, 82:18 or 85:15, in a preferred embodiment, the volume ratio of the mobile phase A to the mobile phase B in the isocratic elution process is 78-82: 22-18, and further preferably, the volume ratio of the mobile phase A to the mobile phase B in the isocratic elution process is 80: 20.

When the high performance liquid chromatography is adopted for detection, octyl silane bonded silica gel is used as a filler for a chromatographic column, a reversed phase chromatographic column (C8 column) can be generally adopted as the chromatographic column, and the chromatographic column can be, but is not limited to Inertsutatin C8 or Kromasil 100-5C 8; preferably, the column has a length of 250mm, a diameter of 4.6mm and a packing particle size of 5 μm. For example, the column is InertsutainC 8 (250X 4.6mm, 5 μm) or Kromasil 100-5C8 (250X 4.6mm, 5 μm).

In the step (4), the chromatographic conditions detected by the high performance liquid chromatography further comprise: the detection wavelength is 190-200 nm, preferably 195 nm; the flow rate is 0.8-1.2ml/min, preferably 1.0 ml/min; the column temperature is 30-40 ℃, and preferably 35 ℃; the injection volume is 5-50 mul, preferably 10 mul.

According to the method for detecting the dissolution rate of the glucosamine hydrochloride preparation, 500-1000 ml of dissolution medium is used as a solvent, the rotation speed is 50-100 revolutions per minute, samples are taken after 5-60 minutes (or other planned time points), and the dissolution rate is measured according to the second method of 0931 of the four-part general rules of 2020 edition of Chinese pharmacopoeia.

In a preferred scheme, the method for detecting the dissolution rate of the glucosamine hydrochloride preparation comprises the following steps:

(1) preparing a test solution: taking a glucosamine hydrochloride tablet, taking 500-1000 ml of dissolution medium as a solvent, taking the dissolution liquid as a test solution after 5-60 minutes at a rotation speed of 50-100 revolutions per minute;

(2) preparing a reference substance solution: taking a glucosamine hydrochloride reference substance, adding a dissolution medium to dissolve the glucosamine hydrochloride reference substance, quantitatively diluting the glucosamine hydrochloride reference substance to prepare a solution containing 0.2-1.5 mg of glucosamine hydrochloride per ml, and shaking up the solution to be used as a reference substance solution;

(3) blank solution: dissolving out medium, wherein the dissolving medium is sodium acetate solution with pH of 4.5, phosphate solution with pH of 6.8 or water;

(4) precisely measuring a test solution, a reference solution and a blank solution for detection, recording a chromatogram, and calculating the dissolution rate of glucosamine hydrochloride in the glucosamine hydrochloride tablet according to an external standard method by using a peak area;

wherein, the chromatographic conditions detected by the high performance liquid chromatography are as follows: the chromatographic column is Inertsutatin C8 or Kromasil 100-5C 8; performing isocratic elution by taking the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the volume ratio of the mobile phase A to the mobile phase B is 80:20 in the isocratic elution process; the mobile phase A is potassium dihydrogen phosphate buffer solution containing 3-5 mmol/L tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide, and the pH value of the buffer solution is adjusted to 3.0-4.0 by phosphoric acid; the mobile phase B is acetonitrile; the detection wavelength is 195 nm; the flow rate is 1.0 ml/min; the column temperature was 35 ℃; the injection volume was 10. mu.l.

In a more preferred embodiment, the method for detecting the dissolution rate of the glucosamine hydrochloride preparation comprises the following steps:

(1) preparing a test solution: taking a glucosamine hydrochloride tablet, taking 900ml of dissolution medium as a solvent, taking the dissolution liquid as a test solution after 5-60 minutes at a rotation speed of 50 revolutions per minute;

(2) preparing a reference substance solution: taking a glucosamine hydrochloride reference substance, adding a dissolution medium to dissolve, quantitatively diluting to prepare a solution containing 0.8mg of glucosamine hydrochloride per ml, and shaking up to obtain a reference substance solution;

wherein, the chromatographic conditions detected by the high performance liquid chromatography are as follows: the chromatographic column is InertsutatinC 8; performing isocratic elution by taking the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the volume ratio of the mobile phase A to the mobile phase B is 80:20 in the isocratic elution process; the mobile phase A is potassium dihydrogen phosphate buffer solution containing 3.85mmol/L tetrabutylammonium hydroxide, and the pH value of the buffer solution is adjusted to 3.5 by phosphoric acid; the mobile phase B is acetonitrile; the detection wavelength is 195 nm; the flow rate is 1.0 ml/min; the column temperature was 35 ℃; the injection volume was 10. mu.l.

By adopting the technical scheme of the invention, the advantages are as follows:

(1) the invention provides a method for detecting dissolution rate of a glucosamine hydrochloride preparation, which comprises the steps of taking monopotassium phosphate buffer solution-acetonitrile containing a positive ion pair reagent (tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide) as a mixed mobile phase for isocratic elution, optimizing the proportion of the mixed mobile phase, prolonging the retention time of a main peak of the glucosamine hydrochloride on a chromatographic column (such as a C8 column) during chromatographic analysis, avoiding the interference of a main peak by a dissolution medium and an auxiliary material solution under low wavelength (such as 195nm), leading tailing factors to be 1.00-1.50, leading the peak purity to be more than 990, leading the retention time of the glucosamine hydrochloride peak to be 3.5-5 min, and having the advantages of quick detection time, good accuracy, good reproducibility and high sensitivity.

(2) The invention provides a method for detecting dissolution of glucosamine hydrochloride preparation, wherein the peak areas and the concentrations of glucosamine hydrochloride in a medium pH4.5 sodium acetate solution, a pH6.8 phosphate solution and water are in good linear relation, the linear correlation coefficients are all larger than 0.999, the recovery rate of each medium is within the range of 98-102%, the RSD is less than 10%, and the accuracy is good.

(3) Compared with a high-efficiency liquid phase-evaporative light scattering detector method, a high-efficiency liquid phase-differential refraction detector method or an amino column method of a high-efficiency liquid phase-ultraviolet detector in the prior art, the method has the advantages that the requirement on a chromatographic column is low in the process of chromatographic analysis, the amino column is not required, the pre-column derivatization reaction is not required, the method can be suitable for accurately measuring the dissolution rate of the glucosamine hydrochloride preparation in different dissolution media, the interference of blank solvents and auxiliary materials is eliminated, the operation is simple, the sensitivity is high, the cost is low, the reproducibility is good, the accuracy is high, and the application range is wide.

Drawings

FIG. 1 is a blank chromatogram of a solution from example 1 using a pH4.5 sodium acetate solution as the dissolution medium;

FIG. 2 is a chromatogram of an excipient solution in example 1, using a pH4.5 sodium acetate solution as the dissolution medium;

FIG. 3 is a chromatogram of a control solution in example 1, using a pH4.5 sodium acetate solution as the dissolution medium;

FIG. 4 is a chromatogram of the dissolution rate of glucosamine hydrochloride tablets in example 1 using a sodium acetate solution of pH4.5 as the dissolution medium;

FIG. 5 is a blank chromatogram of a phosphate solution at pH6.8 in example 1;

FIG. 6 is a chromatogram of an adjuvant solution from example 1 using a phosphate solution of pH6.8 as the medium;

FIG. 7 is a chromatogram of a control solution in example 1 in the presence of a phosphate solution at pH 6.8;

FIG. 8 is a dissolution chromatogram of glucosamine hydrochloride tablets in example 1 with a phosphate solution of pH6.8 as the medium;

FIG. 9 is a blank chromatogram of the dissolution medium water in example 1;

FIG. 10 is a chromatogram of an excipient solution using water as the dissolution medium in example 1;

FIG. 11 is a chromatogram of a control solution in example 1 using water as the dissolution medium;

FIG. 12 is a chromatogram showing the dissolution rate of glucosamine hydrochloride tablets in example 1 using water as the dissolution medium;

FIG. 13 is a blank chromatogram of a solution (medium pH4.5 sodium acetate solution) of comparative example 1, measured according to the USP dissolution test method;

FIG. 14 chromatogram of a control solution (medium pH4.5 sodium acetate solution) of comparative example 1, tested according to the USP dissolution test method;

FIG. 15 shows a blank solution chromatogram (medium pH6.8 phosphate solution) of comparative example 1, measured according to the USP dissolution test method;

FIG. 16 chromatogram of control solution (medium pH6.8 phosphate solution) of comparative example 1, which is determined by the dissolution detection method in United states pharmacopoeia

FIG. 17 is a standard curve of the dissolution medium of example 2, sodium acetate solution at pH 4.5;

FIG. 18 is a standard curve chart of the medium pH6.8 sodium phosphate solution in example 2;

FIG. 19 is a standard curve of water as dissolution medium in example 2;

FIG. 20 is a chromatogram of a test solution of comparative example 2 using a phosphate solution of pH6.8 as a medium;

FIG. 21 is a chromatogram of an adjuvant solution of comparative example 3 using a pH4.5 sodium acetate solution as the dissolution medium;

FIG. 22 is a chromatogram of a test solution in comparative example 3 using a pH4.5 sodium acetate solution as a dissolution medium;

FIG. 23 is a dissolution profile of example 8 using a pH4.5 sodium acetate solution as the dissolution medium;

FIG. 24 is a graph of the dissolution profile of example 8 in the presence of a phosphate solution at pH 6.8;

FIG. 25 is a graph showing the elution profile in example 8 using water as a medium.

Detailed Description

The present invention will be described in further detail with reference to specific examples for better understanding of the technical solutions of the present invention, but it should not be construed that the present invention is limited to the examples, and modifications and variations that are made by ordinary skill in the art or by conventional means without departing from the scope of the present invention are included in the present invention. The reagents and equipment used in the invention are all the conventional reagents and equipment in the technical field unless otherwise specified.

1. Instrument for measuring the position of a moving object

Name of instrument	Numbering	Source
			High performance liquid chromatograph	Agilent 1100	Agilent Technologies (China) Co.,Ltd.
Electronic balance	AUW120D	Shimadzu instruments Ltd
			Electronic balance	SQP	SARTORIUS (SHANGHAI) TRADING Co.,Ltd.
Electronic balance	FA124	SHANGHAI SUNNY HENGPING SCIENTIFIC INSTRUMENT Co.,Ltd.
			Electronic balance	XPE204	Mettler
Ultrasonic cleaner	KH-500DE	Kunshan Selenma ultrasonic Instrument Co Ltd
			Ultrasonic cleaner	KQ-3200DE	Kunshan Selenma ultrasonic Instrument Co Ltd
Digital acidimeter	PHS-3C	Shanghai Jingke
			Electric heating blowing dry box	GZX-9240MBE	Shanghai Bowen realty Co Ltd medical equipment factory
Refrigerator with a door	BCD-2372E3CS	Mitsubishi
			Dissolution instrument	FADT-800RC	SHANGHAI FOCS ANALYTICAL INSTRUMENTS Co.,Ltd.
Dissolution instrument	FADT-1200RC	SHANGHAI FOCS ANALYTICAL INSTRUMENTS Co.,Ltd.
			Intelligent vacuum degassing instrument	FAVD-25	SHANGHAI FOCS ANALYTICAL INSTRUMENTS Co.,Ltd.

2. Reagent

Name of reagent	Rank of	Source
			Acetonitrile	HPLC	Shanghai Shaoxing high purity reagents Co Ltd
Potassium dihydrogen phosphate	AR	SINOPHARM CHEMICAL REAGENT Co.,Ltd.
			Phosphoric acid	HPLC	Aladdin
Tetrabutylammonium hydroxide (10% aqueous solution)	/	Adamas
			Tetrabutylammonium hydrogen sulfate	/	Adamas
Tetrabutylammonium bromide	/	Adamas
			Sodium hydroxide	AR	SINOPHARM CHEMICAL REAGENT Co.,Ltd.
Anhydrous sodium acetate	AR	GENERAL REAGENT
			Acetic acid	AR	NANJING CHEMICAL REAGENT Co.,Ltd.
Purified water	/	Nanjing HainaPharmaceutical Co Ltd
			Ultrapure water	/	Millipore

3. Reference, sample and adjuvant information

Name (R)	Source	Batch number	Content (%)
				Glucosamine hydrochloride reference substance	China Institute for food and drug control	140649-201606	100.0％
Glucosamine hydrochloride tablet	NANJING HEALTHNICE PHARMACEUTICAL Co.,Ltd.	200529121	/
				Glucosamine hydrochloride	YANGZHOU RIXING BIO-TECH Co.,Ltd.	20181002	99.72
Auxiliary materials	NANJING HEALTHNICE PHARMACEUTICAL TECHNOLOGY Co.,Ltd.	2020060301/	/

Example 1: determination of conditions for high performance liquid chromatography

(1) Taking glucosamine hydrochloride tablets, taking 900ml of sodium acetate solution with pH of 4.5 as a solvent according to a determination method of dissolution rate and release rate (determination method of 0931 in the four-part general rule of China pharmacopoeia 2020 edition), rotating at 50 r/min, taking a dissolution liquid at 45min according to the method operation, filtering by a 0.45 mu m filter membrane, and taking a subsequent filtrate as a test solution; accurately weighing appropriate amount of glucosamine hydrochloride reference substance, dissolving in dissolved medium, diluting to obtain 0.8mg glucosamine hydrochloride solution per 1ml, and shaking to obtain reference substance solution; according to the formula of the auxiliary materials in the glucosamine hydrochloride tablet, taking 900ml of sodium acetate solution with pH of 4.5 as a solvent, rotating at 50 r/min, taking a dissolution liquid at 45min, filtering with a 0.45 mu m filter membrane, and taking a subsequent filtrate as an auxiliary material solution; the blank solution was a sodium acetate solution with a dissolution medium ph 4.5.

The dissolution rate was measured using the following high performance liquid chromatography conditions:

a chromatographic column: InertsusatinC 8, 250X 4.6mm, 5 μm; octyl silane bonded silica gel is used as a filling agent;

mobile phase A: potassium dihydrogen phosphate buffer solution containing 3.85mmol/L tetrabutylammonium hydroxide (taking 6.8g potassium dihydrogen phosphate to appropriate amount of water, adding 10ml tetrabutylammonium hydroxide aqueous solution 10%, stirring to dissolve, adding water to dilute to 1000ml, adjusting pH to 3.5 with phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 6 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out. System applicability requirements: the tailing factor of the glucosamine hydrochloride peak is less than 2.0.

The determination method comprises the following steps: precisely measuring the blank solution, the adjuvant solution, the reference solution and the sample solution respectively at a volume of 10 μ l, injecting into a liquid chromatograph, and recording chromatogram as shown in fig. 1-4.

As can be seen from the graphs 1-4, the blank solution and the auxiliary material solution have no interference to the main peak of glucosamine hydrochloride by taking the sodium acetate solution with pH4.5 as the dissolution medium; glucosamine hydrochloride peak tailing factors in the reference solution and the glucosamine hydrochloride tablet dissolution test solution meet the regulations, and the detection results are shown in table 1.

(2) Taking glucosamine hydrochloride tablets, and preparing a test solution, a reference solution and an auxiliary material solution according to the steps in the step (1) by taking 900ml of phosphate solution with pH of 6.8 as a solvent according to a dissolution and release determination method (determination of 0931 second method of the four general rules of the 2020 edition of Chinese pharmacopoeia). The phosphate solution with a pH of 6.8 as a blank solution. Precisely measuring the blank solution, the adjuvant solution, the reference solution and the sample solution by 10 μ l each, injecting into a liquid chromatograph, detecting according to the chromatographic conditions in step (1), and recording chromatogram, as shown in fig. 5-8.

As can be seen from FIGS. 5 to 8, the phosphate solution with pH of 6.8 is used as the dissolution medium, and the blank solution and the auxiliary material solution have no interference on the main peak of glucosamine hydrochloride; glucosamine hydrochloride peak tailing factors in the reference solution and the glucosamine hydrochloride tablet dissolution test solution meet the regulations, and the detection results are shown in table 1.

(3) Taking glucosamine hydrochloride tablets, and preparing a test solution, a reference solution and an auxiliary material solution according to the steps in the step (1) by taking 900ml of water as a solvent according to a dissolution and release determination method (determination of 0931 second method of the general rule of four parts in 2020 edition of Chinese pharmacopoeia). Taking water as a blank solution. Precisely measuring the blank solution, the adjuvant solution, the reference solution and the sample solution by 10 μ l each, injecting into a liquid chromatograph, detecting according to the chromatographic conditions in step (1), and recording chromatogram, as shown in FIGS. 9-12.

As can be seen from FIGS. 9 to 12, the blank solution and the adjuvant solution both have no interference to the main peak of glucosamine hydrochloride by using water as the dissolution medium; glucosamine hydrochloride peak tailing factors in the reference solution and the glucosamine hydrochloride tablet dissolution test solution meet the regulations, and the detection results are shown in table 1.

TABLE 1 chromatographic data

Comparative example 1:

referring to a dissolution rate detection method of glucosamine tablets in United states pharmacopoeia (USP43), the specific chromatographic conditions are as follows: performing isocratic elution with a chromatographic column (Inertsutatin C8, 250X 4.6mm, 5 μm) using octyl silane-bonded silica gel as a filler, and 0.05% phosphoric acid solution (pH adjusted to 3.0 with potassium hydroxide) -acetonitrile (60:40) as a mixed mobile phase; detection wavelength is 195nm, flow rate is 0.6ml/min, sample injection volume is 10 μ l, column temperature: 35 ℃ is carried out.

The blank solution and the reference solution in the step (1) of the example 1 are respectively precisely measured by 10 mu l, injected into a liquid chromatograph, detected according to the chromatographic conditions, and recorded with chromatogram, and the chromatogram is shown in figures 13-14.

As can be seen from fig. 13 to 14, when the dissolution rate of the glucosamine hydrochloride tablet is detected by isocratic elution using a mixed mobile phase of phosphoric acid solution and acetonitrile containing no tetrabutylammonium hydroxide as a reagent of positive ion pair, the dissolution medium ph4.5 sodium acetate solution interferes with the detection of the glucosamine hydrochloride peak, and the dissolution rate cannot be accurately measured, resulting in low accuracy.

The blank solution and the reference solution in the step (1) of the example 1 are respectively precisely measured by 10 mu l, injected into a liquid chromatograph, detected according to the chromatographic conditions, and chromatogram maps are recorded, wherein the chromatogram maps are shown in fig. 15-16.

As can be seen from fig. 15 to 16, when the dissolution rate of the glucosamine hydrochloride tablet is detected by isocratic elution using a mixed mobile phase of acetonitrile and a phosphoric acid solution containing no tetrabutylammonium hydroxide as a reagent containing a positive ion pair, the phosphate solution with a dissolution ph of 6.8 also interferes with the detection of the glucosamine hydrochloride peak, and the dissolution rate cannot be accurately measured, resulting in low accuracy.

Aiming at the problems, the optimal chromatographic conditions are expected to be screened out by optimizing the proportion of a mobile phase in the isocratic elution process, trying different chromatographic columns, adjusting the column temperature and the like, so that the problem of interference of a dissolution medium on a glucosamine hydrochloride peak is solved, and the results fail. For example, the ratio of phosphoric acid solution to acetonitrile in the mixed mobile phase during isocratic elution is adjusted to 95:5, 90:10, or 80: 20; the temperature of the chromatographic column is 25 ℃ or 35 ℃ or 40 ℃, and different brands of chromatographic columns Kromasil 100-5C8(250 x 4.6mm, 5 μm), SHIMADZU Shim GIST C8(250 x 4.6mm, 5 μm), Inertsutain C8(250 x 4.6mm, 5 μm), Inertsutain C8(150 x 4.6mm, 5 μm), Inertsil C8-4(4.6 x 250mm, 3 μm) are screened, so that the interference of the dissolved medium on the glucosamine hydrochloride peak cannot be improved.

Example 2: linearity of glucosamine hydrochloride in sodium acetate solution at pH4.5, phosphate solution at pH6.8 and water

42mg (parallel 3 parts) of glucosamine hydrochloride reference substance is precisely weighed, sodium acetate solution with medium pH4.5, phosphate solution with pH6.8 and water are respectively added to dissolve and dilute to 10ml, and the linear stock solution of the sodium acetate solution with medium pH4.5, phosphate solution with pH6.8 and water is obtained after shaking up. Precisely transferring linear stock solutions of each medium into different 10ml measuring bottles with the volume of 0.16ml, 0.4ml, 1ml, 1.6ml, 2ml and 2.4ml respectively, adding the medium to dilute to scale, and shaking up; to obtain a linear solution of medium pH4.5 sodium acetate, pH6.8 phosphate solution and water.

Analytical detection method the chromatographic detection was carried out using the analytical method described in step (1) in example 1.

Precisely measuring 10 μ l of the linear solutions with the above concentrations, injecting into a liquid chromatograph, recording chromatogram, performing linear regression with the sample concentration (mg/ml) as abscissa (X-axis) and peak area as ordinate (Y-axis), and drawing standard curve, wherein the results are shown in Table 2, and the line graphs are shown in FIGS. 17-19.

TABLE 2 Linear relationship survey data

Medium	Concentration Range (ug/ml)	Regression equation	Correlation coefficient
				pH4.5 sodium acetate solution	67.408～1011.120	y＝0.4709x+4.4096	0.9999
phosphate solution of pH6.8	67.872～1018.080	y＝0.4711x+2.1895	0.9999
				Water (W)	68.032～1020.480	y＝0.4501x+4.8383	0.9998

As can be seen from table 2: the medium pH4.5 sodium acetate solution of the glucosamine hydrochloride is in the concentration range of 67.408-1011.120 ug/ml, the peak area and the concentration have good linear relation, and the correlation coefficient is 0.9999; the medium pH6.8 phosphate solution of the glucosamine hydrochloride is in the concentration range of 67.872-1018.080 ug/ml, the peak area and the concentration have good linear relation, and the correlation coefficient is 0.9999; the glucosamine hydrochloride medium water has a peak area and concentration in a good linear relation within the concentration range of 68.032-1020.480 ug/ml, and the correlation coefficient is 0.9998.

Example 3: the recovery rate of glucosamine hydrochloride in sodium acetate solution with pH4.5, phosphate solution with pH6.8 and water

Taking a proper amount (parallel 6 parts) of glucosamine hydrochloride reference substance, precisely weighing, respectively adding medium pH4.5 sodium acetate, pH6.8 phosphate solution and water to dissolve, and diluting to prepare a solution containing about 0.8mg of glucosamine hydrochloride in each 1ml, wherein each medium is parallel 2 parts.

About 1042mg (parallel 3 parts) of glucosamine hydrochloride is precisely weighed, and is dissolved and diluted to 50ml by adding sodium acetate with the medium pH4.5 and phosphate solution with the medium pH6.8 respectively and water to be used as stock solutions of each medium.

The 10% limit: weighing appropriate amount of adjuvants, placing in 50ml measuring flask, precisely transferring 0.2ml of stock solution, adding medium to dissolve and dilute to scale, shaking, filtering, and collecting the subsequent filtrate; each medium was in parallel 3 parts.

The 50% limit: weighing appropriate amount of adjuvants, placing in 50ml measuring flask, precisely transferring 1.0ml stock solution, adding medium to dissolve and dilute to scale, shaking, filtering, and collecting the subsequent filtrate; each medium was in parallel 3 parts.

100% Limit: weighing appropriate amount of adjuvants, placing in 50ml measuring flask, precisely transferring 2.0ml of stock solution, adding medium to dissolve and dilute to scale, shaking, filtering, and collecting the subsequent filtrate; each medium was in parallel 3 parts.

The 120% limit: weighing appropriate amount of adjuvants, placing in 50ml measuring flask, precisely transferring 2.4ml of stock solution, adding medium to dissolve and dilute to scale, shaking, filtering, and collecting the subsequent filtrate; each medium was in parallel 3 parts.

The control solution and recovery solution were measured accurately at 10. mu.l each, and chromatograms were recorded, and the results are shown in Table 3.

TABLE 3 recovery test data

Medium	Average recovery (%)	RSD(％)
			pH4.5 sodium acetate solution	100.8	2.53
phosphate solution of pH6.8	101.8	1.53
			Water (W)	101.1	1.06

As can be seen from table 3: the dissolution rate recovery rates of the medium pH4.5 sodium acetate solution, the pH6.8 phosphate solution and water are in the range of 98-102%, the RSD is less than 10%, and the accuracy is good.

Example 4:

(2) And (3) replacing the dissolution medium in the step (1) with a phosphate solution with pH6.8, and preparing a test solution, a reference solution and an auxiliary solution according to the steps in the step (1). The phosphate solution with a pH of 6.8 as a blank solution.

(3) Replacing the dissolution medium in the step (1) with water, and preparing a test solution, a reference solution and an auxiliary solution according to the step (1). Taking water as a blank solution.

And (3) respectively adopting the following high performance liquid chromatography conditions to carry out dissolution rate detection on the solutions prepared in the steps (1) to (3):

mobile phase A: potassium dihydrogen phosphate buffer solution containing 3.85mmol/L tetrabutylammonium hydrogen sulfate (taking 6.8g potassium dihydrogen phosphate to appropriate amount of water, adding 1.3g tetrabutylammonium hydrogen sulfate, stirring to dissolve, adding water to dilute to 1000ml, and adjusting pH to 3.5 with phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 6 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out. System applicability requirements: the tailing factor of the glucosamine hydrochloride peak is less than 2.0.

The experimental results are as follows: when the dissolution rate of the glucosamine hydrochloride tablet is detected by selecting potassium dihydrogen phosphate buffer solution containing 3.85mmol/L tetrabutylammonium hydrogen sulfate and acetonitrile as mixed mobile phases, sodium acetate solution with the medium pH of 4.5, phosphate solution with the medium pH of 6.8, water and auxiliary material solution thereof have no interference on the main peak of the glucosamine hydrochloride, tailing factors of the glucosamine hydrochloride peak in a reference solution and a dissolution rate test sample solution of the glucosamine hydrochloride tablet are in accordance with the regulations, and the dissolution rate of the glucosamine hydrochloride tablet in three different media can be accurately determined.

Example 5:

mobile phase A: potassium dihydrogen phosphate buffer solution containing 4.65mmol/L tetrabutylammonium bromide (taking 6.8g potassium dihydrogen phosphate to appropriate amount of water, adding 1.5g tetrabutylammonium bromide, stirring for dissolving, adding water for diluting to 1000ml, and adjusting pH to 3.5 with phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 6 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out. System applicability requirements: the tailing factor of the glucosamine hydrochloride peak is less than 2.0.

The experimental results are as follows: when the dissolution rate of the glucosamine hydrochloride tablet is detected by selecting potassium dihydrogen phosphate buffer solution containing 4.65mmol/L tetrabutylammonium bromide and acetonitrile as a mixed mobile phase, sodium acetate solution with the medium pH of 4.5, phosphate solution with the medium pH of 6.8, water and auxiliary material solution thereof have no interference on the main peak of the glucosamine hydrochloride, tailing factors of the glucosamine hydrochloride peak in a reference solution and a dissolution rate test solution of the glucosamine hydrochloride tablet conform to the regulations, and the dissolution rate of the glucosamine hydrochloride tablet in three different media can be accurately determined.

Example 6:

a chromatographic column: octyl silane bonded silica gel (InertsutainC 8, 250X 4.6mm, 5 μm) was used as a filler;

mobile phase A: a potassium dihydrogen phosphate buffer solution containing 5mmol/L tetrabutylammonium hydroxide (6.8 g potassium dihydrogen phosphate is added into a proper amount of water, 13ml tetrabutylammonium hydroxide aqueous solution of 10% is added, water is added for dilution to 1000ml after stirring and dissolution, and the pH is adjusted to 3.5 by phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 6 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out. System applicability requirements: the tailing factor of the glucosamine hydrochloride peak is less than 2.0.

The experimental results are as follows: when the dissolution rate of the glucosamine hydrochloride tablet is detected by selecting a potassium dihydrogen phosphate buffer solution containing 5mmol/L tetrabutylammonium hydroxide and acetonitrile as a mixed mobile phase, a sodium acetate solution with a medium pH of 4.5, a phosphate solution with a pH of 6.8, water and an auxiliary material solution thereof do not interfere with a main peak of the glucosamine hydrochloride, tailing factors of the glucosamine hydrochloride peak in a reference solution and a dissolution rate test solution of the glucosamine hydrochloride tablet conform to the regulation, and the dissolution rate of the glucosamine hydrochloride tablet in three different media can be accurately determined.

Comparative example 2:

precisely measuring 10 mul of blank solution and sample solution of pH6.8 medium in step (2) of example 6, respectively, and performing dissolution rate detection by using the following high performance liquid chromatography conditions:

mobile phase A: a potassium dihydrogen phosphate buffer solution containing 0.6mmol/L tetrabutylammonium hydrogen sulfate (taking 6.8g to 1000ml of water of potassium dihydrogen phosphate, stirring and dissolving, then adding 0.2g tetrabutylammonium hydrogen sulfate, stirring and dissolving, and adjusting pH to 3.5 by using phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 10 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out.

The results are shown in Table 4 and the chromatogram is shown in FIG. 20.

TABLE 4 summary of chromatographic data

Dissolution media	Sample name	Retention time (min)	Number of theoretical plates	Tailing factor
					phosphate solution of pH6.8	Test solution	6.312	7830	2.22

As can be seen from table 4 and fig. 20 above: when the concentration of the positive ion-pairing reagent tetrabutylammonium hydrogen sulfate in the mobile phase is about 0.6mmol/L, the tailing factor of the glucosamine hydrochloride peak is increased (>2.0) and the number of theoretical plates is reduced (< 10000); the results show that when the concentration of the positive ion pair reagent is too low, the main glucosamine hydrochloride peak has a serious tailing effect, and the subsequent impurity peaks cannot be separated from the base line, so that the dissolution rate in a phosphate solution medium with pH of 6.8 cannot be accurately measured.

Comparative example 3:

taking a dissolution medium pH4.5 sodium acetate solution as a blank solution, preparing an auxiliary material solution, a test solution and a reference solution according to the step (1) in the example 6, respectively and precisely measuring 10 mu l of each of the blank solution, the auxiliary material solution, the test solution and the reference solution, and performing dissolution rate detection by adopting the following high performance liquid chromatography conditions:

mobile phase A: potassium dihydrogen phosphate buffer solution containing 16mmol/L tetrabutylammonium hydroxide (taking 6.8g to 1000ml of water of potassium dihydrogen phosphate, stirring for dissolving, adding 40ml of 10% tetrabutylammonium hydroxide, stirring for dissolving, and adjusting pH to 3.5 with phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 10 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out.

The detection results are shown in Table 5, and the chromatograms are shown in FIGS. 21 to 22.

TABLE 5 summary of chromatographic data

As can be seen from table 5 and fig. 21 to 22: when the concentration of positive ions to the reagent in the mobile phase is 16mmol/L, the retention time of an impurity peak in a sodium acetate solution with a medium pH4.5 is also delayed, and the separation degree of the impurity peak and a main peak of glucosamine hydrochloride is less than 2.0; it is demonstrated that when the concentration of the positive ion pair reagent is too high, the separation degree of the impurity peak and the main peak of the sodium acetate solution with the medium pH of 4.5 is reduced, and the detection of the main peak is easy to interfere.

Comparative example 4:

mobile phase A: potassium dihydrogen phosphate buffer solution containing 18.6mmol/L tetrabutylammonium bromide (taking 6.8g to 1000ml water of potassium dihydrogen phosphate, stirring to dissolve, then adding 6g tetrabutylammonium bromide, stirring to dissolve, and adjusting pH to 3.5 with phosphoric acid); the mobile phase B is acetonitrile; mobile phase a and mobile phase B were cloudy at a volume ratio of 80: 20. The concentration of the positive ion pair reagent tetrabutylammonium bromide cannot be too high, otherwise, the positive ion pair reagent tetrabutylammonium bromide is separated out when being mixed with an organic phase and cannot be used as a mobile phase of a high-efficiency liquid phase.

By screening the concentration (1mmol/L, 3mmol/L, 3.5mmol/L, 3.85mmol/L, 4.65mmol/L, 5mmol/L, 5.5mmol/L, 6mmol/L, 8mmol/L or 10mmol/L) of the positive ion pair reagent (tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide) in the mobile phase A, the concentration is 1-10 mmol/L, preferably 3-5 mmol/L, so that when the potassium dihydrogen phosphate buffer solution of the positive ion pair reagent is mixed with the organic phase according to the ratio of 80:20, the mixture is clarified and does not precipitate; the impurity peak in the chromatogram can achieve baseline separation with the main peak, has good specificity, and can accurately determine the dissolution rate in each medium (medium pH4.5 sodium acetate solution, pH6.8 phosphate solution, and water).

Example 7: determination of dissolution rate of glucosamine hydrochloride tablets

Taking 6 tablets from three batches of the product, taking 900ml of water as a solvent according to a dissolution and release rate measuring method (measured by 0931 second method of the general rule of four departments of the 2020 edition of Chinese pharmacopoeia), rotating at 75 r/min, operating according to the method, taking a proper amount of solution after 30 minutes, filtering with a 0.45 μm filter membrane, and taking the subsequent filtrate as a test solution. And precisely weighing a proper amount of reference substance dried to constant weight, adding a dissolving medium for dissolving, diluting to prepare a solution containing about 0.8mg of glucosamine hydrochloride in each 1ml, and shaking up to obtain the reference substance solution. Taking medium water as blank solution. The dissolution rate was measured by the following high performance liquid chromatography: a chromatographic column: octyl silane bonded silica gel is a filling agent (Inertsutatin C8, 250X 4.6mm, 5 μm; mobile phase: 3.85mmol/L tetrabutylammonium hydroxide buffer solution (potassium dihydrogen phosphate 6.8g is taken to be added to water 1000ml, 10% tetrabutylammonium hydroxide aqueous solution 10ml is added, pH is adjusted to 3.5) -acetonitrile (80: 20) by phosphoric acid, flow rate: 1.0ml/min, sample amount: 10 μ L, column temperature: 35 ℃, detection wavelength: 195nm, isocratic elution, running time 6min, system applicability requires that tailing factors of glucosamine hydrochloride peak do not exceed 2.0. precisely measure the above blank solution, sample solution and control solution 10 μ L each, inject into liquid chromatograph, record chromatogram, calculate dissolution rate of each tablet by peak area according to external standard method, see Table 6.

TABLE 6 dissolution rate measurement results

The results show that: the dissolution rate RSD of each batch is less than 2.0 percent, and the uniformity is good.

Example 8: determination of dissolution curves of glucosamine hydrochloride tablets in different media

(1) Taking 12 tablets of the product, taking three batches of the product, taking 900ml of sodium acetate solution with pH of 4.5 as a solvent according to a method for measuring dissolution rate and release rate (according to a second method of 0931 in the general rule of four parts of the 2020 edition of Chinese pharmacopoeia), rotating at 50 r/min, operating according to the method, taking 5ml of dissolution liquid (simultaneously adding medium with the same volume) when 5, 10, 15, 20, 30 and 45min, filtering by using a 0.45 mu m filter membrane, and taking the subsequent filtrate as a test solution; accurately weighing appropriate amount of glucosamine hydrochloride reference substance, dissolving in dissolved medium, diluting to obtain 0.8mg glucosamine hydrochloride solution per 1ml, and shaking to obtain reference substance solution; the blank solution was a sodium acetate solution with a dissolution medium ph 4.5.

(2) And (3) replacing the dissolution medium in the step (1) with a phosphate solution with pH of 6.8, and preparing a test solution and a reference solution according to the steps in the step (1). The phosphate solution with a pH of 6.8 as a blank solution.

(3) Replacing the dissolution medium in the step (1) with water, and preparing a test solution and a reference solution according to the step in the step (1). Taking water as a blank solution.

a chromatographic column: kromasil 100-5C8 (250X 4.6mm, 5 μm); mobile phase A: a potassium dihydrogen phosphate buffer solution containing 5mmol/L tetrabutylammonium hydroxide (6.8 g potassium dihydrogen phosphate is added into a proper amount of water, 13ml tetrabutylammonium hydroxide aqueous solution of 10% is added, water is added for dilution to 1000ml after stirring and dissolution, and the pH is adjusted to 3.5 by phosphoric acid); the mobile phase B is acetonitrile; isocratic elution is carried out on the mobile phase A and the mobile phase B according to the volume ratio of 80:20, and the running time is 6 min; detection wavelength: 195 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: 35 ℃ is carried out. System applicability requirements: the tailing factor of the glucosamine hydrochloride peak is less than 2.0. The dissolution rate of each tablet at different time is calculated by peak area according to an external standard method. The experimental results are shown in tables 7-9, and the dissolution curves are shown in tables 23-25.

TABLE 7 dissolution rate measurement results

Table 8 dissolution rate measurement results

TABLE 9 dissolution rate measurement results

The experimental results show that: the dissolution rate of glucosamine hydrochloride tablets in three different media (medium pH4.5 sodium acetate solution, pH6.8 phosphate solution and water) can be accurately determined by selecting potassium dihydrogen phosphate buffer solution containing 5mmol/L tetrabutylammonium hydroxide-acetonitrile as a mixed mobile phase and C8 (250X 4.6mm, 5 mu m) of different brands as chromatographic columns.

To sum up, in the embodiments 1 to 7, the method for detecting the dissolution rate of glucosamine hydrochloride tablets provided by the present invention has good specificity in the dissolution rate test of glucosamine hydrochloride tablets in which the dissolution media are respectively a sodium acetate solution with ph4.5, a phosphate solution with ph6.8, and water, and the media and the auxiliary material solution have no interference to the main peak of glucosamine hydrochloride; tailing factors are all between 1.00 and 1.50, peak purity is more than 990, and glucosamine hydrochloride peak retention time is 3.5 to 5min and is very quick.

The peak areas and the concentrations of the glucosamine hydrochloride in the medium pH4.5 sodium acetate solution, pH6.8 phosphate solution and water are in good linear relation, the linear correlation coefficients are all larger than 0.999, the recovery rate of each medium is also in the range of 98-102%, the RSD is smaller than 10%, and the accuracy is good.

Therefore, the detection method provided by the invention is convenient, efficient, accurate and good in specificity.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting dissolution rate of a glucosamine hydrochloride preparation is characterized by comprising the following steps:

(3) blank solution: a dissolution medium;

the chromatographic conditions detected by the high performance liquid chromatography are as follows: the chromatographic column takes octyl silane bonded silica gel as a filling agent; performing isocratic elution by taking the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the volume ratio of the mobile phase A to the mobile phase B in the isocratic elution process is 75-85: 25-15; the mobile phase A is a potassium dihydrogen phosphate buffer solution containing 1-10 mmol/L of a positive ion pairing reagent, the pH value of the buffer solution is adjusted to 3.0-4.0 by phosphoric acid, and the positive ion pairing reagent is tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide; the mobile phase B is acetonitrile.

2. The method for detecting dissolution rate of glucosamine hydrochloride preparation according to claim 1, wherein in the step (4), the concentration of the positive ion pair reagent in the mobile phase A is 3-5 mmol/L, preferably 3.85 mmol/L.

3. The method for detecting dissolution rate of glucosamine hydrochloride preparation according to claim 2, wherein in the step (4), the concentration of potassium dihydrogen phosphate in the mobile phase A is 10-60mmol/L, preferably the concentration of potassium dihydrogen phosphate is 10-50 mmol/L; in formulating the mobile phase a, the pH was adjusted to 3.5 with phosphoric acid.

4. The method for detecting the dissolution rate of the glucosamine hydrochloride preparation according to claim 1, wherein in the step (4), the volume ratio of the mobile phase A to the mobile phase B in the isocratic elution process is 78-82: 22-18, preferably 80: 20.

5. The method for detecting the dissolution rate of glucosamine hydrochloride formulation according to claim 1, wherein in the step (1), the glucosamine hydrochloride formulation is glucosamine hydrochloride tablet, glucosamine hydrochloride capsule, glucosamine hydrochloride granule or glucosamine hydrochloride dispersion agent; glucosamine hydrochloride tablets are preferred.

6. The method for detecting the dissolution rate of glucosamine hydrochloride preparation according to claim 1, wherein the dissolution medium is acetate buffer solution with pH of 3.8-5.8, phosphate buffer solution with pH of 4.5-8.0, or water; preferably, the dissolution medium is a sodium acetate solution at pH4.5, a phosphate solution at pH6.8 or water.

7. The method for detecting dissolution rate of glucosamine hydrochloride formulation according to claim 1, wherein in the step (4), the chromatographic column is Inertsutatin C8 or Kromasil 100-5C 8; preferably, the chromatographic column has a length of 250mm, a diameter of 4.6mm and a packing particle size of 5 μm.

8. The method for detecting dissolution rate of glucosamine hydrochloride preparation according to claim 1, wherein in the step (4), the chromatographic conditions of the HPLC detection comprise: the detection wavelength is 190-200 nm, preferably 195 nm; the flow rate is 0.8-1.2ml/min, preferably 1.0 ml/min; the column temperature is 30-40 ℃, and preferably 35 ℃; the injection volume is 5-50 mul, preferably 10 mul.

9. The method for detecting the dissolution rate of glucosamine hydrochloride formulation according to any one of claims 1 to 8, comprising the steps of:

(3) blank solution: dissolving out a medium, wherein the dissolving medium is a sodium acetate solution with pH4.5, a phosphate solution with pH6.8 or water;

the chromatographic conditions detected by the high performance liquid chromatography are as follows: the chromatographic column is Inertsutatin C8 or Kromasil 100-5C 8; performing isocratic elution by taking the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the volume ratio of the mobile phase A to the mobile phase B is 80:20 in the isocratic elution process; the mobile phase A is potassium dihydrogen phosphate buffer solution containing 3-5 mmol/L tetrabutylammonium hydroxide, tetrabutylammonium hydrogen sulfate or tetrabutylammonium bromide, and the pH value of the buffer solution is adjusted to 3.0-4.0 by phosphoric acid; the mobile phase B is acetonitrile; the detection wavelength is 195 nm; the flow rate is 1.0 ml/min; the column temperature was 35 ℃; the injection volume was 10. mu.l.

10. The method for detecting the dissolution rate of glucosamine hydrochloride formulation according to claim 1, comprising the steps of:

the chromatographic conditions detected by the high performance liquid chromatography are as follows: the chromatographic column is InertsutatinC 8; performing isocratic elution by taking the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the volume ratio of the mobile phase A to the mobile phase B is 80:20 in the isocratic elution process; the mobile phase A is potassium dihydrogen phosphate buffer solution containing 3.85mmol/L tetrabutylammonium hydroxide, and the pH value of the buffer solution is adjusted to 3.5 by phosphoric acid; the mobile phase B is acetonitrile; the detection wavelength is 195 nm; the flow rate is 1.0 ml/min; the column temperature was 35 ℃; the injection volume was 10. mu.l.