CN112816587A

CN112816587A - Method for detecting glucosamine content in glucosamine chondroitin preparation containing active peptide and organic calcium

Info

Publication number: CN112816587A
Application number: CN202011624757.XA
Authority: CN
Inventors: 陆文岐; 伍彪; 常弘杰; 张业敏; 余孝云; 邓颜伟; 何水杰
Original assignee: Joincare Pharmaceutical Industry Group Co ltd; Shenzhen Haibin Pharmaceutical Co ltd; SHENZHEN TAITAI PHARMACEUTICAL INDUSTRY Ltd
Current assignee: Joincare Pharmaceutical Industry Group Co ltd; Shenzhen Haibin Pharmaceutical Co ltd; SHENZHEN TAITAI PHARMACEUTICAL INDUSTRY Ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-05-18

Abstract

The invention provides a method for detecting the glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium, which is a high performance liquid chromatography and is carried out by adopting a high performance liquid chromatograph with an ultraviolet detector, wherein a chromatographic column adopted in the high performance liquid chromatography is a C18 chromatographic column; the adopted mobile phase consists of a first mobile phase and a second mobile phase, wherein the first mobile phase is methanol, and the second mobile phase is acetic acid aqueous solution. The detection method provided by the invention is suitable for quality control of glucosamine in the glucosamine chondroitin preparation containing active peptide and organic calcium, solves the interference of functional components and excipients on the detection of the glucosamine, improves the specificity and the accuracy of detection, and improves the accuracy of detection of the glucosamine in the glucosamine chondroitin preparation containing the active peptide and the organic calcium and the reliability of quality control.

Description

Method for detecting glucosamine content in glucosamine chondroitin preparation containing active peptide and organic calcium

Technical Field

The invention relates to the technical field of functional food detection, in particular to a method for detecting the glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium.

Background

Glucosamine, also known as glucosamine, is called glucosamine for short, and is an important hexosamine which is formed by replacing one hydroxyl group of glucose with an amino group, is easily soluble in water and hydrophilic solvents, and is a substance necessary for synthesizing proteoglycan in the matrix of human articular cartilage. Studies have shown that glucosamine can help repair and maintain cartilage and stimulate the growth of chondrocytes, and currently, glucosamine and chondroitin sulfate have become the most widely used dietary supplements for the improvement of osteoarticular diseases.

In the prior art, a High Performance Liquid Chromatography (HPLC) method has been widely used for glucosamine content determination and quality control because of its characteristics of simplicity, sensitivity, rapidity, accuracy, good reproducibility, etc., such as:

huangxin, Chenqingsong, the university of Fujian university journal (Nature science edition) 5 months in 2009, Vol 25, No. 3, p80-83, discloses a method for measuring glucosamine sulfate by reversed-phase high performance liquid chromatography, which adopts isocratic elution, has a flow rate of 0.6ml/min and a detection wavelength of 254nm, and can be used for detecting the glucosamine sulfate; the method for measuring the content of the chondroitin sulfate and the glucosamine hydrochloride by using the liquid chromatography is partially specified in GB/T20365-2006 determination of the content of the chondroitin sulfate and the glucosamine hydrochloride-liquid chromatography.

However, in order to better improve osteoporosis, stiffness and other symptoms in bone joint diseases and improve product efficacy, in the practical application process, functional components such as bioactive peptides (such as collagen peptides, casein phosphopeptides and the like) and organic calcium (such as calcium citrate malate) need to be matched on the basis of glucosamine and chondroitin sulfate, and food additives such as microcrystalline cellulose and lactose or pharmaceutical excipients are also selected to be added as excipients. However, the combination of functional components and the use of excipients can cause difficulty in quality control of glucosamine, for example, in the detection process of glucosamine, the functional components and excipients combined with glucosamine can interfere, thereby reducing the detection accuracy of glucosamine. Therefore, the method for detecting glucosamine disclosed in the prior art is not suitable for detecting the content of glucosamine in compound preparation.

Therefore, the development of a method suitable for detecting the glucosamine content in a preparation containing bioactive peptides, organic calcium and other excipients and chondroitin sulfate is an urgent problem to be solved in the field.

Disclosure of Invention

The invention aims to provide a method for detecting the glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium. The method provided by the invention utilizes the high performance liquid chromatography to detect the glucosamine content in the glucosamine chondroitin preparation containing active peptide and organic calcium, solves the interference of other functional components compounded with glucosamine and excipients on the detection of the glucosamine, improves the specificity and accuracy of the detection, and has accurate and reliable detection result.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows.

In one aspect, the invention provides a method for detecting the glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium, which is performed by high performance liquid chromatography and adopting a high performance liquid chromatograph with an ultraviolet detector, wherein in the high performance liquid chromatography, a chromatographic column adopted is a C18 chromatographic column; the adopted mobile phase consists of a first mobile phase and a second mobile phase, wherein the first mobile phase is methanol, and the second mobile phase is acetic acid aqueous solution;

preferably, the particle size of the packing in the chromatographic column is 5 μm;

preferably, the temperature of the chromatographic column is 20-30 ℃; more preferably 25 ℃;

preferably, the aqueous acetic acid solution is a 0.04% aqueous acetic acid solution;

preferably, the detection method is eluted according to the following procedure:

when 0min is reached, the volume ratio of the first mobile phase to the second mobile phase is 10-25: 90-75; continuing for 7 min;

beginning at 7.01min, adjusting the volume ratio of the first mobile phase to the second mobile phase to be 100:0, and continuing until 15 min;

starting at 15.01min, adjusting the volume of the first mobile phase and the second mobile phase to be 10-25: 90-75, and stopping at 25 min;

more preferably, the detection method is eluted according to the following procedure:

at 0min, the volume ratio of the first mobile phase to the second mobile phase is 20: 80; continuing for 7 min;

beginning at 15.01min, adjusting the volume of the first mobile phase and the second mobile phase to be 20:80, and stopping at 25 min;

preferably, in the high performance liquid chromatography, the flow rate for elution is 1.0 mL/min;

preferably, the detection wavelength of the ultraviolet detector is 240-260 nm; more preferably 254 nm.

In some embodiments of the present invention, there is provided a method for detecting glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium, wherein the method is high performance liquid chromatography, and the method is performed by using a high performance liquid chromatograph with an ultraviolet detector, and comprises the following steps:

(1) preparation of reagent I: accurately transferring 5mL of phenyl isothiocyanate into a 100mL volumetric flask, dissolving with methanol and fixing the volume to obtain a reagent I;

(2) preparation of reagent II: adding 0.2mol/L NaH₂PO₄Mixing the aqueous solution with 0.2mol/L Na₂HPO₄Mixing the water solutions according to the volume ratio of 5:95(V/V) to obtain a reagent II;

(3) preparing a blank solution: putting 1mL of ionized water into a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, putting the test tube in a water bath at the temperature of 60-80 ℃, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking a lower layer solution, and filtering with a filter membrane to obtain an empty solution;

(4) preparing a reference substance solution: weighing a glucosamine hydrochloride reference substance 12.5mg to 100mL in a volumetric flask, dissolving with deionized water to a constant volume to obtain a solution III, precisely absorbing 1mL of the solution III, placing the solution III in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of a reagent I, shaking up, placing the solution I in a water bath at the temperature of 60-80 ℃, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, performing oscillation extraction, standing for 20min, taking a lower layer solution, and filtering with a filter membrane to obtain a reference substance solution;

(5) preparing a test solution: taking 10 minimum preparation units of glucosamine chondroitin preparation containing active peptide and organic calcium, crushing into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing in a 50mL measuring flask, adding a proper amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant fluid to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing in a water bath at 60-80 ℃, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillatory extraction, standing for 20min, taking supernatant fluid, and filtering by using a filter membrane to obtain a sample solution;

(6) preparing a bone collagen peptide sample solution: weighing 50mg of collagen peptide, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to a scale, shaking up, centrifuging for 10min at 4000 rpm, taking supernatant to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing in a water bath at 60-80 ℃, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillation extraction, standing for 20min, taking subnatant, and filtering with a 0.45 mu m filter membrane to obtain a collagen peptide sample solution;

(7) setting detection conditions of a high performance liquid chromatograph:

chromatographic column parameters: c18, 250mm × 4.6mm, 5 μm;

column temperature: 20-30 ℃;

detection wavelength of ultraviolet detector: 240-260 nm;

mobile phase: the first mobile phase is methanol, and the second mobile phase is 0.04% acetic acid water solution;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

(8) measurement: precisely measuring blank solution, reference solution, sample solution and ossein protein peptide sample solution by 10 μ L, injecting into liquid chromatograph, recording peak area, and calculating by external standard method according to the following formula:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

conversion factor: 2, one molecule of glucosamine potassium sulfate corresponds to two molecules of glucosamine hydrochloride.

In other embodiments of the present invention, there is provided a method for detecting glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium, the method being high performance liquid chromatography using high performance liquid chromatography with an ultraviolet detector, comprising the steps of:

(3) preparing a blank solution: putting 1mL of ionized water into a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, heating in a water bath at 80 ℃ for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking a subnatant, and filtering with a filter membrane to obtain an empty solution;

(4) preparing a reference substance solution: weighing a glucosamine hydrochloride reference substance 12.5mg to 100mL in a volumetric flask, dissolving with deionized water to a constant volume to obtain a solution III, precisely absorbing 1mL of the solution III, placing the solution III in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing the solution I in a water bath at 80 ℃ for heating for 20min, cooling to room temperature, adding 1mL of n-hexane, performing oscillation extraction, standing for 20min, taking a lower layer liquid, and filtering with a filter membrane to obtain a reference substance solution;

(5) preparing a test solution: taking 10 minimum preparation units of glucosamine chondroitin preparation containing active peptide and organic calcium, crushing into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing into a 50mL measuring flask, adding a proper amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant fluid to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing into a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing into a 80 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillating extraction, standing for 20min, taking supernatant fluid, and filtering with a filter membrane to obtain a sample solution;

(6) preparing a bone collagen peptide sample solution: weighing 50mg of bone collagen peptide, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to a scale, shaking up, centrifuging for 10min at 4000 rpm, taking supernatant to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing in a water bath at 80 ℃ for heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking supernatant, and filtering with a 0.45-micrometer filter membrane to obtain a bone collagen peptide sample solution;

(7) setting detection conditions of a high performance liquid chromatograph:

chromatographic column parameters: c18, 250mm × 4.6mm, 5 μm;

column temperature: 25 ℃;

detection wavelength of ultraviolet detector: 254 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

(8) and (3) detection: precisely measuring blank solution, reference solution, sample solution and ossein protein peptide sample solution by 10 μ L, injecting into liquid chromatograph, recording peak area, and calculating by external standard method according to the following formula:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

In some embodiments of the present invention, the glucosamine chondroitin preparation containing active peptide and organic calcium used in the preparation of the test solution may be a tablet, granule, powder containing glucosamine chondroitin containing active peptide and organic calcium according to the present invention; preferably, the glucosamine chondroitin tablet containing active peptide and organic calcium of the invention, and more preferably, the glucosamine chondroitin tablet containing active peptide and organic calcium has the following formula:

220g of glucosamine potassium sulfate, 160g of sodium chondroitin sulfate, 100g of collagen peptide powder (meeting the standard requirement of Q/BDBS 0002S), 30g of casein phosphopeptide, 50g of lactose, 50g of copovidone, 40g of microcrystalline cellulose, 5g of silicon dioxide, 5g of magnesium stearate and 29.7g of film coating premix; wherein the film coating premix is prepared from the following components in a weight ratio of 0.5: 5: 2: 1: 1.5 talc powder: hydroxypropyl methylcellulose: polyvinyl alcohol: polyethylene glycol: titanium dioxide.

The advantages of the detection method provided by the invention over the prior art at least comprise the following aspects:

1) according to the detection method provided by the invention, the content of glucosamine in the glucosamine chondroitin preparation containing active peptide and organic calcium is detected by taking a methanol-acetic acid aqueous solution as a mobile phase, so that the interference of other compound components in the detection process is effectively eliminated, and the accuracy of the detection result is high;

2) the detection method disclosed by the invention adopts a common C18 column, the detection temperature is 20-30 ℃, the condition is mild, the influence of the environment on the method is reduced, the detection cost is reduced, and the industrial application is facilitated;

3) the detection method realizes the effective separation of the glucosamine and other components, is suitable for detecting the glucosamine content in the glucosamine chondroitin preparation containing active peptide and organic calcium, and fills the blank of the technical field;

4) the detection method provided by the invention is simple and convenient to operate and calculate, and has high detection accuracy and good reproducibility.

Drawings

Fig. 1 is an HPLC profile of each sample in example 1, wherein fig. 1A is a detection profile of a glucosamine hydrochloride control, fig. 1B is a detection profile of a test sample, fig. 1C is a detection profile of a collagen peptide powder sample, and fig. 1D is a detection profile of a blank solution.

Fig. 2 is an HPLC profile of each sample in example 2, wherein fig. 2A is a detection profile of a glucosamine hydrochloride control, fig. 2B is a detection profile of a test sample, fig. 2C is a detection profile of a collagen peptide powder sample, and fig. 2D is a detection profile of a blank solution.

Fig. 3 is an HPLC profile of each sample in example 3, wherein fig. 3A is a detection profile of a glucosamine hydrochloride control, fig. 3B is a detection profile of a test sample, fig. 3C is a detection profile of a collagen peptide powder sample, and fig. 3D is a detection profile of a blank solution.

Fig. 4 is an HPLC profile of each sample in example 4, wherein fig. 4A is a detection profile of a glucosamine hydrochloride control, fig. 4B is a detection profile of a test sample, fig. 4C is a detection profile of a collagen peptide powder sample, and fig. 4D is a detection profile of a blank solution.

Fig. 5 is an HPLC profile of each sample in example 5, wherein fig. 5A is a detection profile of a glucosamine hydrochloride control, fig. 5B is a detection profile of a test sample, fig. 5C is a detection profile of a collagen peptide powder sample, and fig. 5D is a detection profile of a blank solution.

Fig. 6 is an HPLC profile of each sample in example 6, wherein fig. 6A is a detection profile of a glucosamine hydrochloride control, fig. 6B is a detection profile of a test sample, fig. 6C is a detection profile of a collagen peptide powder sample, and fig. 6D is a detection profile of a blank solution.

FIG. 7 is an HPLC chromatogram of samples of example 7 at different column temperatures, wherein FIG. 7A1 is a detection chromatogram of a glucosamine hydrochloride control at a column temperature of 20 ℃; FIG. 7B1 is a detection spectrum of the sample at a column temperature of 20 deg.C; FIG. 7C1 is the detection spectrum of the collagen peptide powder sample at a column temperature of 20 ℃; FIG. 7D1 is a detection spectrum of a blank solution at a column temperature of 20 ℃; FIG. 7A2 is a graph showing the detection of glucosamine hydrochloride control at a column temperature of 25 deg.C; FIG. 7B2 is a detection spectrum of the sample at a column temperature of 25 deg.C; FIG. 7C2 is the detection spectrum of the collagen peptide powder sample at a column temperature of 25 ℃; FIG. 7D2 is a detection spectrum of a blank solution at a column temperature of 25 ℃; FIG. 7A3 is a detection spectrum of glucosamine hydrochloride control at a column temperature of 30 deg.C; FIG. 7B3 is a detection spectrum of the sample at a column temperature of 30 ℃; FIG. 7C3 is the detection spectrum of the collagen peptide powder sample at a column temperature of 30 ℃; FIG. 7D3 is the detection spectrum of the blank solution at a column temperature of 30 ℃.

FIG. 8 is an HPLC chromatogram of each sample at different detection wavelengths in example 8, wherein FIG. 8B1 is a detection HPLC chromatogram of a test sample at a detection wavelength of 230 nm; FIG. 8B2 is a HPLC chromatogram for detecting a sample at a detection wavelength of 240 nm; FIG. 8B3 is a HPLC chromatogram for detecting a sample at a detection wavelength of 254 nm; FIG. 8B4 is a HPLC chromatogram for detecting a sample with a detection wavelength of 260 nm; FIG. 8B5 is a HPLC chromatogram for detecting a sample at a detection wavelength of 270 nm; FIG. 8B6 is a HPLC chromatogram for detecting a sample with a detection wavelength of 280 nm; 8D1 is an HPLC (high performance liquid chromatography) spectrum detected by a blank solution when the detection wavelength is 230 nm; FIG. 8D2 is an HPLC chromatogram of blank solution detection at a detection wavelength of 240 nm; FIG. 8D3 is an HPLC chromatogram of blank solution detection at a detection wavelength of 254 nm; FIG. 8D4 is an HPLC chromatogram of blank solution detection at a detection wavelength of 260 nm; FIG. 8D5 is an HPLC chromatogram of blank solution detection at a detection wavelength of 270 nm; FIG. 8D6 is an HPLC chromatogram of detection of a blank solution at a detection wavelength of 280 nm.

FIG. 9 is an HPLC chromatogram of each sample under different elution procedures in example 9, where 9A1 is an elution procedure-glucosamine hydrochloride control detection chromatogram, 9B1 is an elution procedure-test sample detection chromatogram, 9C1 is an elution procedure-collagen peptide powder sample detection chromatogram, and 9D1 is an elution procedure-blank solution detection chromatogram; 9A2 is a detection map of a glucosamine hydrochloride reference substance in an elution procedure, 9B2 is a detection map of a test substance in an elution procedure II, 9C2 is a detection map of a collagen peptide powder sample in an elution procedure II, and 9D2 is a blank solution detection map in an elution procedure II; 9A3 is a glucosamine hydrochloride reference substance detection map of an elution procedure, 9B3 is a three-sample detection map of the elution procedure, 9C3 is a three-bone collagen peptide powder sample detection map of the elution procedure, and 9D3 is a three-blank solution detection map of the elution procedure; 9A4 is a detection map of a tetrammine glucose hydrochloride reference substance in an elution procedure, 9B4 is a detection map of a four-sample in the elution procedure, 9C4 is a detection map of a four-bone collagen peptide powder sample in the elution procedure, and 9D4 is a four-blank solution detection map in the elution procedure; 9A5 is a detection map of a five-glucosamine hydrochloride reference substance in an elution procedure, 9B5 is a detection map of a five-sample in the elution procedure, 9C5 is a detection map of a five-sample ossein protein peptide powder in the elution procedure, and 9D5 is a five-blank solution detection map in the elution procedure; 9A6 is a detection map of a hexa-glucosamine hydrochloride reference substance in an elution procedure, 9B6 is a detection map of six test samples in an elution procedure, 9C6 is a detection map of a hexa-ossein protein peptide powder sample in an elution procedure, and 9D6 is a detection map of a six blank solution in an elution procedure.

Fig. 10 is an HPLC profile of each sample in comparative example 1, in which fig. 10A is a detection profile of glucosamine hydrochloride control, fig. 10B is a detection profile of experimental sample 2, and fig. 10C is a detection profile of collagen powder sample.

Fig. 11 is an HPLC profile of each sample in comparative example 2, in which fig. 11A is a detection profile of glucosamine hydrochloride control, fig. 11B is a detection profile of experimental sample 2, and fig. 11C is a detection profile of collagen powder sample.

Fig. 12 is an HPLC profile of each sample in comparative example 3, in which fig. 12A is a detection profile of glucosamine hydrochloride control, fig. 12B is a detection profile of experimental sample 2, fig. 12C is a detection profile of collagen powder sample, and fig. 12D is a detection profile of blank solution.

FIG. 13 is a glucosamine standard curve chart of test example 1.

Detailed description of the preferred embodiments

The present invention will be further described with reference to specific embodiments, and the raw materials, reagent materials and the like used in the following examples and test examples are commercially available products unless otherwise specified.

The glucosamine hydrochloride control used in the following examples and test examples was purchased from the institute for food and drug testing, China, and the content thereof was 100%.

The filters used in the following examples and test examples were each 0.45 μm filters.

The following examples are intended only to illustrate the invention, wherein the experimental procedures, without specific definition, are conventional.

Preparation of experimental sample 1:glucosamine chondroitin tablet containing active peptide and organic calcium

1. The formula is as follows: 330g of citric acid calcium malate, 220g of glucosamine potassium sulfate, 160g of chondroitin sulfate sodium, 100g of collagen peptide powder (meeting the requirement of Q/BDBS 0002S standard), 30g of casein phosphopeptide, 50g of lactose, 50g of copovidone, 40g of microcrystalline cellulose, 5g of silicon dioxide and 5g of magnesium stearate.

2. The preparation method comprises the following steps:

(1) measuring the water content of each raw material and auxiliary material in the formula, and controlling the water content to be less than or equal to 5.0 percent;

(2) sieving calcium citrate malate, collagen peptide powder, casein phosphopeptide, lactose, copovidone and microcrystalline cellulose with 60 mesh sieve; sieving chondroitin sulfate sodium and magnesium stearate with a 80-mesh sieve, and weighing the raw and auxiliary materials according to the formula;

(3) mixing the glucosamine potassium sulfate salt and the silicon dioxide for 5 minutes, sieving by a 80-mesh sieve, and mixing for 5 minutes; mixing with other sieved materials for 30 minutes by using an automatic lifting hopper mixer, then loading the materials into a turnover container and weighing, and controlling the water content of the materials to be less than or equal to 5.0%;

(4) preparing the material obtained in the step (3) into 1000 tablets, controlling the hardness to be 10-23 kg, controlling the tablet weight to be within the range of 0.99g +/-5%, and controlling the water content to be less than or equal to 5.0%.

Preparation of experimental sample 2:glucosamine chondroitin tablet containing active peptide and organic calcium

2. The preparation method comprises the following steps:

(4) tabletting the material obtained in the step (3) to prepare 1000 tablets, wherein the hardness is controlled to be 10-25 kg, the weight of the tablets is controlled to be within the range of 0.99g +/-5%, and the water content is controlled to be less than or equal to 5.0%;

(5) weighing 29.7g of film coating premix (consisting of talcum powder, hydroxypropyl methylcellulose, polyvinyl alcohol, polyethylene glycol and titanium dioxide in a weight ratio of 0.5: 5: 2: 1: 1.5), adding 198g of purified water as a coating solvent to prepare a coating solution, controlling the air inlet temperature during coating to be about 75 ℃, the bed temperature to be about 45 ℃ and the air outlet temperature to be about 55 ℃ and the atomization pressure to be 0.1-0.3 Mpa, and coating the tablet core prepared in the step (4), wherein each coated tablet is about 1.02 +/-5 percent g.

Preparation of experimental sample 3:containing activityAmino sugar chondroitin granules of peptide and organic calcium

1. The formula is as follows: 330g of citric acid calcium malate, 220g of glucosamine potassium sulfate, 160g of chondroitin sulfate sodium, 100g of collagen peptide powder (meeting the requirement of Q/BDBS 0002S standard), 30g of casein phosphopeptide, 60g of lactose, 50g of maltodextrin, 40g of pregelatinized starch, 9g of sodium carboxymethyl starch, 0.95g of silicon dioxide and 0.05g of sucralose.

2. The preparation method comprises the following steps:

(2) sieving calcium citrate malate, ossein peptide powder, casein phosphopeptide, lactose, maltodextrin and pregelatinized starch with 60 mesh sieve; sieving chondroitin sulfate sodium, carboxymethyl starch sodium, silicon dioxide and sucralose by a 80-mesh sieve, and weighing the raw and auxiliary materials according to the formula;

(4) and (4) performing dry granulation on the material obtained in the step (3) to prepare 200 bags of granules, wherein the filling amount difference is controlled within the range of 5g +/-5%, and the water content is controlled to be less than or equal to 5.0%.

Preparation of experimental sample 4:glucosamine chondroitin powder containing active peptide and organic calcium

1. The formula is as follows: 330g of citric acid calcium malate, 220g of glucosamine potassium sulfate, 160g of chondroitin sulfate sodium, 100g of collagen peptide powder (meeting the requirement of Q/BDBS 0002S standard), 30g of casein phosphopeptide, 40g of lactose, 70g of xylitol, 45g of galactooligosaccharide, 4g of Arabic gum, 0.95g of silicon dioxide and 0.05g of stevioside.

2. The preparation method comprises the following steps:

(2) sieving calcium citrate malate, ossein protein peptide powder, casein phosphopeptide, lactose, xylitol, galactooligosaccharide and acacia gum with 60 mesh sieve; sieving chondroitin sulfate sodium, silicon dioxide and stevioside by a 80-mesh sieve, and weighing the raw and auxiliary materials according to the formula;

(4) preparing the powder obtained in the step (3) into 200 bags of powder, wherein the filling amount difference is controlled within the range of 5g +/-5%, and the water content is controlled to be less than or equal to 5.0%.

Example 1

Detecting a sample: experimental sample 1

The method comprises the following operation steps:

(3) preparing a blank solution: putting 1mL of ionized water into a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, heating in a water bath at 60 ℃ for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking a subnatant, and filtering with a filter membrane to obtain an empty solution;

(4) preparing a reference substance solution: weighing a glucosamine hydrochloride reference substance 12.5mg to 100mL in a volumetric flask, dissolving with deionized water to a constant volume to obtain a solution III, precisely sucking 1mL of the solution III, placing the solution III in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing the solution I in a water bath at 60 ℃ for heating for 20min, cooling to room temperature, adding 1mL of n-hexane, performing oscillation extraction, standing for 20min, taking a lower layer solution, and filtering with a 0.45-micrometer filter membrane to obtain a reference substance solution;

(5) preparing a test solution: taking 10 pieces (experiment sample 1) of glucosamine chondroitin tablets containing active peptide and organic calcium, crushing into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing in a 50mL measuring flask, adding a proper amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant fluid to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing in a 60 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillating extraction, standing for 20min, taking subnatant, and filtering with a filter membrane to obtain a sample solution;

(6) preparing a bone collagen peptide sample solution: weighing 50mg of bone collagen peptide, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to a scale, shaking up, centrifuging for 10min at 4000 rpm, taking supernatant to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing in a 60 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking supernatant, and filtering with a 0.45 mu m filter membrane to obtain a bone collagen peptide sample solution;

(7) setting detection conditions of a high performance liquid chromatograph:

the instrument comprises the following steps: waters liquid chromatograph

Chromatographic column parameters: agilent ZORBAX Eclipse XDB-C18 column, 250mm × 4.6mm, 5 μm, No. LC-151;

column temperature: 25 ℃;

detector wavelength: 252 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

And (3) detection results: the calculated glucosamine content is 21.33g/100g, the HPLC chromatogram of each sample is shown in figure 1, wherein the detection result of glucosamine hydrochloride is shown in figure 1A, the detection result of the test sample is shown in figure 1B, the detection result of the collagen peptide powder sample is shown in figure 1C, the detection result of the blank solution is shown in figure 1D, and the chromatogram peak data of the test sample is shown in Table 1:

TABLE 1 chromatographic Peak data for test articles

Serial number	Retention time	Area of	Peak height	Degree of separation
					1	4.499	1580332	158197
2	5.265	192803	17507	2.81

As shown in FIG. 1, the glucosamine hydrochloride control in FIG. 1A has peak times of 4.495min and 5.262 min; the test article in FIG. 1B had a peak time of 4.499min and 5.265min for glucosamine, and a resolution of 2.81; FIG. 1C shows no interference at a retention time of 4.495min, from which it can be seen that the glucosamine separation in FIG. 1B is good and the specificity is good.

Example 2

Detecting a sample: experimental sample 2

The method comprises the following operation steps:

(4) preparing a reference substance solution: weighing a glucosamine hydrochloride reference substance 12.5mg to 100mL in a volumetric flask, dissolving with deionized water to a constant volume to obtain a solution III, precisely sucking 1mL of the solution III, placing the solution III in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing the solution I in a water bath at 80 ℃ for heating for 20min, cooling to room temperature, adding 1mL of n-hexane, performing oscillation extraction, standing for 20min, taking a lower layer solution, and filtering with a 0.45-micrometer filter membrane to obtain a reference substance solution;

(5) preparing a test solution: taking 10 pieces (experiment sample 2) of glucosamine chondroitin film-coated tablet containing active peptide and organic calcium, grinding into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing in a 50mL measuring flask, adding a proper amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant fluid to obtain solution IV, precisely sucking 1mL of solution IV, placing in a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing in a 80 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillation extraction, standing for 20min, taking supernatant fluid, and filtering through a 0.45-micron filter membrane to obtain a test solution;

(7) setting detection conditions of a high performance liquid chromatograph:

the instrument comprises the following steps: waters liquid chromatograph

column temperature: 25 ℃;

detector wavelength: 254 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

And (3) detection results: the calculated glucosamine content is 21.54g/100g, the HPLC chromatogram of each sample is shown in figure 2, wherein the detection result of glucosamine hydrochloride is shown in figure 2A, the detection result of the test sample is shown in figure 2B, the detection result of the collagen peptide powder sample is shown in figure 2C, the detection result of the blank solution is shown in figure 2D, and the chromatogram peak data of the test sample is shown in Table 2:

TABLE 2 chromatographic peak data for test articles

Serial number	Retention time (min)	Area of	Peak height	Degree of separation
					1	4.493	1585981	158446
2	5.258	193432	17531	2.80

As shown in FIG. 2, the glucosamine hydrochloride control of FIG. 2A showed peak times of 4.498min and 5.265 min; the test article in FIG. 2B had a peak time of 4.493min and 5.258min for glucosamine, and a resolution of 2.80; FIG. 2C shows no interference at a retention time of 4.498min, from which it can be seen that the glucosamine separation in FIG. 2B is good and the specificity is good.

Example 3

Detecting a sample: experimental sample 3

The method comprises the following operation steps:

(3) preparing a blank solution: putting 1mL of ionized water into a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, heating in a 70 ℃ water bath for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking a subnatant, and filtering with a filter membrane to obtain an empty solution;

(4) preparing a reference substance solution: weighing a glucosamine hydrochloride reference substance 12.5mg to 100mL in a volumetric flask, dissolving with deionized water to a constant volume to obtain a solution III, precisely sucking 1mL of the solution III, placing the solution III in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing the solution I in a 70 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, performing oscillation extraction, standing for 20min, taking a lower layer solution, and filtering with a 0.45-micrometer filter membrane to obtain a reference substance solution;

(5) preparing a test solution: taking 10 bags (experimental sample 3) of glucosamine chondroitin granules containing active peptide and organic calcium, crushing into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing in a 50mL measuring flask, adding a proper amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant to obtain solution IV, precisely absorbing 1mL of solution IV, placing in a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing in a 70 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillatory extraction, standing for 20min, taking supernatant, and filtering through a 0.45-micrometer filter membrane to obtain a test sample solution;

(6) preparing a bone collagen peptide sample solution: weighing 50mg of bone collagen peptide, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to a scale, shaking up, centrifuging for 10min at 4000 rpm, taking supernatant to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing in a 70 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking supernatant, and filtering with a 0.45 mu m filter membrane to obtain a bone collagen peptide sample solution; (ii) a

(7) Setting detection conditions of a high performance liquid chromatograph:

the instrument comprises the following steps: waters liquid chromatograph

column temperature: 25 ℃;

detector wavelength: 256 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

And (3) detection results: the calculated glucosamine content is 21.35g/100g, the HPLC chromatogram of each sample is shown in figure 3, wherein the detection result of glucosamine hydrochloride is shown in figure 3A, the detection result of the test sample is shown in figure 3B, the detection result of the collagen peptide powder sample is shown in figure 3C, the detection result of the blank solution is shown in figure 3D, and the chromatogram peak data of the test sample is shown in Table 3:

TABLE 3 chromatographic peak data of test article

Serial number	Retention time	Area of	Peak height	Degree of separation
					1	4.504	1592219	158835
2	5.274	199846	17684	2.75

As shown in FIG. 3, the glucosamine hydrochloride control in FIG. 3A has peak times of 4.499min and 5.268 min; the test article in FIG. 3B had a peak time of 4.504min and 5.274min for glucosamine, and a resolution of 2.75; FIG. 3C shows no interference at a retention time of 4.499min, from which it can be seen that the glucosamine separation in FIG. 3B is good and the specificity is good.

Example 4

Detecting a sample: experimental sample 4

The method comprises the following operation steps:

(5) preparing a test solution: taking 10 bags (experiment sample 4) of glucosamine chondroitin powder containing active peptide and organic calcium, crushing into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant fluid to obtain solution IV, precisely sucking 1mL of solution IV, placing in a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing in a 60 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillating extraction, standing for 20min, taking supernatant fluid, and filtering through a 0.45-micrometer filter membrane to obtain a sample solution;

(6) preparing a bone collagen peptide sample solution: preparing a bone collagen peptide sample solution: weighing 50mg of bone collagen peptide, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to a scale, shaking up, centrifuging at 4000 rpm for 10min, taking supernatant to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing in a 60 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillation extraction, standing for 20min, taking subnatant, and filtering with a 0.45 mu m filter membrane to obtain a bone collagen peptide sample solution;

(7) setting detection conditions of a high performance liquid chromatograph:

the instrument comprises the following steps: waters liquid chromatograph

column temperature: 25 ℃;

detector wavelength: 254 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

And (3) detection results: the calculated glucosamine content is 21.28g/100g, the HPLC chromatogram of each sample is shown in fig. 4, wherein the detection result of glucosamine hydrochloride reference is shown in fig. 4A, the detection result of the test sample is shown in fig. 4B, the detection result of the collagen peptide powder sample is shown in fig. 4C, the detection result of the blank solution is shown in fig. 4D, and the chromatographic peak data of the test sample is shown in table 4:

TABLE 4 chromatographic peak data of test article

Serial number	Retention time (min)	Area of	Peak height	Degree of separation
					1	4.497	1591331	158370
2	5.266	200370	17634	2.79

As shown in FIG. 4, the glucosamine hydrochloride control of FIG. 4A showed peak times of 4.497min and 5.266 min; the test article in FIG. 4B had a peak time of 4.497min and 5.266min for glucosamine, and a degree of separation of 2.79; FIG. 4C shows no interference at a retention time of 4.497min, from which it can be seen that the glucosamine separation in FIG. 4B is good and the specificity is good.

Example 5

Detecting a sample: experimental sample 2.

The method comprises the following operation steps: the detection was performed by an active liquid chromatograph, and the other steps were the same as in example 2.

And (3) detection results: the calculated glucosamine content is 21.44g/100g, as shown in fig. 5, wherein the detection result of glucosamine hydrochloride is shown in fig. 5A, the detection result of the test sample is shown in fig. 5B, the result of the collagen peptide powder sample is shown in fig. 5C, the blank spectrum is shown in fig. 5D, wherein the chromatographic peak data of the test sample is shown in table 5:

TABLE 5 sample chromatographic Peak data

As shown in FIG. 5, the glucosamine hydrochloride control of FIG. 5A showed peak times of 6.384min and 7.455 min; the test article in FIG. 5B had a peak time of 6.375min and 7.445min for glucosamine, and a degree of separation of 3.6; FIG. 5C shows no interference at a retention time of 6.384min, from which it can be seen that the glucosamine separation in FIG. 5B is good and the specificity is good.

Example 6

Detecting a sample: experimental sample 2.

The method comprises the following operation steps: the same procedure as in example 5 was followed using an Inertsil ODS-3C18 column, numbered LC-156.

And (3) detection results: the calculated glucosamine content is 21.42g/100g, as shown in fig. 6, wherein the detection result of glucosamine hydrochloride is shown in fig. 6A, the detection result of the test sample is shown in fig. 6B, the detection result of the collagen peptide powder sample is shown in fig. 6C, the blank spectrum is shown in fig. 6D, and the chromatographic peak data of the test sample is shown in table 6:

TABLE 6 chromatographic peak data of test article

As shown in FIG. 6, the glucosamine hydrochloride control in FIG. 6A showed peak times of 6.386min and 7.460 min; the test article in FIG. 6B had a peak time of 6.371min and 7.440min for glucosamine, and a degree of separation of 3.6; no interference was observed at a retention time of 6.386min as shown in FIG. 6C, from which it can be seen that the glucosamine separation in FIG. 6B was good and specific.

EXAMPLE 7 selection of column temperature

Detecting a sample: experimental sample 2.

The method comprises the following operation steps: to verify the effect of different column temperatures on the separation effect, detection was performed using an active liquid chromatograph, column number LC-151 was used, different column temperatures were selected, and other steps were the same as in example 2, and a glucosamine chondroitin tablet containing active peptide and organic calcium (experimental sample 2) was detected.

And (3) detection results: as shown in fig. 7, wherein, when the column temperature is 20 ℃, the detection result of the glucosamine hydrochloride control is shown in fig. 7a1, the detection result of the test sample is shown in fig. 7B1, the detection result of the collagen peptide powder sample is shown in fig. 7C1, and the detection result of the blank solution is shown in fig. 7D 1; when the column temperature is 25 ℃, the detection result of the glucosamine hydrochloride control is shown in fig. 7a2, the detection result of the test sample is shown in fig. 7B2, the detection result of the collagen peptide powder sample is shown in fig. 7C2, and the detection result of the blank solution is shown in fig. 7D 2; when the column temperature is 30 ℃, the detection result of the glucosamine hydrochloride reference substance is shown in fig. 7a3, the detection result of the test sample is shown in fig. 7B3, the detection result of the collagen peptide powder sample is shown in fig. 7C3, and the detection result of the blank solution is shown in fig. 7D 3; the chromatographic peak data of the sample to be tested are shown in the table 7:

TABLE 7 chromatographic peak data of samples obtained at different column temperatures

As shown in FIG. 7, when the column temperatures are 20 deg.C, 25 deg.C, and 30 deg.C, respectively, no interference peak appears near the glucosamine retention time as shown by HPLC (FIGS. 7C1, 7C2, and 7C3) of the collagen peptide powder sample; HPLC (high performance liquid chromatography) spectrums (figures 7A1, 7A2 and 7A3) obtained by detecting glucosamine hydrochloride reference substances and HPLC spectrums (figures 7B1, 7B2 and 7B3) obtained by detecting test samples show that glucosamine is well separated, wherein the retention time is moderate when the column temperature is 25 ℃, the separation effect is proper, the separation degree is 3.7, therefore, the column temperature is selected to be 20-30 ℃, and the preferred column temperature is 25 ℃.

EXAMPLE 8 selection of wavelength

Detecting a sample: experimental sample 2.

The method comprises the following operation steps: to verify the effect of different detection wavelengths on the separation effect, detection was performed using an Agilent liquid chromatograph, using column number LC-152 (column parameters: Agilent ZORBAX Eclipse XDB-C18 column, 250 mm. times.4.6 mm, 5 μm.), selecting different detection wavelengths, and other procedures were the same as in example 2, and the glucosamine chondroitin tablets (Experimental sample 2) containing active peptides and organic calcium were detected.

And (3) detection results: as shown in fig. 8, 8B1 is an HPLC profile of the test sample at a detection wavelength of 230 nm; FIG. 8B2 is an HPLC chromatogram of the test sample at a detection wavelength of 240 nm; FIG. 8B3 is an HPLC chromatogram of the test sample at a detection wavelength of 254 nm; FIG. 8B4 is an HPLC chromatogram of the test sample at a detection wavelength of 260 nm; FIG. 8B5 is an HPLC chromatogram of the test sample at a detection wavelength of 270 nm; FIG. 8B6 is an HPLC chromatogram of the test sample at a detection wavelength of 280 nm; 8D1 is an HPLC (high performance liquid chromatography) spectrum detected by a blank solution when the detection wavelength is 230 nm; FIG. 8D2 is an HPLC chromatogram of blank solution detection at a detection wavelength of 240 nm; FIG. 8D3 is an HPLC chromatogram of blank solution detection at a detection wavelength of 254 nm; FIG. 8D4 is an HPLC chromatogram of blank solution detection at a detection wavelength of 260 nm; FIG. 8D5 is an HPLC chromatogram of blank solution detection at a detection wavelength of 270 nm; FIG. 8D6 is an HPLC chromatogram of blank solution detection at a detection wavelength of 280 nm; the chromatographic peak data of the test sample obtained by detection at different detection wavelengths are shown in Table 8:

TABLE 8 chromatographic peak data of test sample obtained by detection at different detection wavelengths

As shown in fig. 8, with the increase of the detection wavelength, the peak height of the sample tends to increase first and then decrease, when the detection wavelength is 240 to 260nm, the baseline is stable, the sample peak is obvious, and the resolution is good, wherein when the wavelength is 254nm, the comprehensive baseline stability, peak absorption and resolution are best, so the detection wavelength is selected to be 240 to 260nm, and 254nm is preferred.

Examples9 selection of elution procedure

Detecting a sample: experimental sample 2.

The method comprises the following operation steps: to verify the effect of different elution procedures on the separation effect, the detection was performed using an active liquid chromatograph, different elution procedures were selected, and other procedures were the same as in example 2, and the detection was performed on the aminoglycoside cartilage tablet containing active peptide and organic calcium (experimental sample 2).

And (3) elution procedure I:

and (3) detection results: as shown in fig. 9, the results of the glucosamine hydrochloride control are shown in fig. 9a1, the results of the test sample are shown in fig. 9B1, the results of the collagen peptide powder sample are shown in fig. 9C1, the results of the blank solution are shown in fig. 9D1, and the chromatographic peak data of the test sample are shown in table 9-1:

TABLE 9-1 chromatographic Peak data for test articles

As shown in FIG. 9, the glucosamine hydrochloride control sample in FIG. 9A1 has a chromatographic peak number 1 showing peak appearance time of 6.414min and a chromatographic peak number 2 lacking; the elution procedure was not applicable because the peak time of the glucosamine sample in FIG. 9B1 was 6.408min for chromatographic peak number 1 and missing for chromatographic peak number 2.

And (2) elution procedure II:

and (3) detection results: the calculated glucosamine content is 21.30g/100g, the HPLC chromatogram of each sample is shown in fig. 9, wherein the detection result of glucosamine hydrochloride control is shown in fig. 9A2, the detection result of the test sample is shown in fig. 9B2, the detection result of the collagen peptide powder sample is shown in fig. 9C2, the detection result of the blank solution is shown in fig. 9D2, and the chromatographic peak data of the test sample is shown in Table 9-2:

TABLE 9-2 chromatographic Peak data of test articles

As shown in FIG. 9, the glucosamine hydrochloride control of FIG. 9A2 showed peak times of 6.823min and 8.680 min; the test article in FIG. 9B2 showed a peak time of 6.822min and 8.683min for glucosamine, and a degree of separation of 3.7; no interference at retention time 6.823min is shown in fig. 9C 2.

And (3) elution procedure III:

and (3) detection results: the calculated glucosamine content is 21.46g/100g, as shown in fig. 9, wherein the detection result of glucosamine hydrochloride control is shown in fig. 9a3, the detection result of the test sample is shown in fig. 9B3, the result of the collagen peptide powder sample is shown in fig. 9C3, the detection result of the blank solution is shown in fig. 9D3, wherein the chromatographic peak data of the test sample is shown in table 9-3:

TABLE 9-3 sample chromatographic Peak data

As shown in FIG. 9, the glucosamine hydrochloride control of FIG. 9A3 showed peak times of 6.385min and 7.459 min; the test article in fig. 9B3 had a peak time of 6.375min and 7.446min for glucosamine, and a degree of separation of 3.6; fig. 9C3 shows no interference at retention time 6.385min, from which it can be seen that the glucosamine separation degree is good in fig. 9B 3.

And (4) elution procedure IV:

and (3) detection results: the calculated glucosamine content is 21.32g/100g, the HPLC chromatogram of each sample is shown in fig. 9, wherein the detection result of glucosamine hydrochloride control is shown in fig. 9A4, the detection result of the test sample is shown in fig. 9B4, the detection result of the collagen peptide powder sample is shown in fig. 9C4, the detection result of the blank solution is shown in fig. 9D4, and the chromatographic peak data of the test sample is shown in tables 9-4:

TABLE 9-4 chromatographic peak data of test article

As shown in FIG. 9, the glucosamine hydrochloride control of FIG. 9A4 showed peak times of 6.197min and 7.087 min; the test article in FIG. 9B4 showed a peak time of 6.193min and 7.083min for glucosamine, and a degree of separation of 3.4; no interference at retention time 6.197min is shown in fig. 9C 4.

And (5) an elution procedure five:

and (3) detection results: the HPLC profile of each sample is shown in fig. 9, wherein the detection result of glucosamine hydrochloride control is shown in fig. 9a5, the detection result of the test sample is shown in fig. 9B5, the detection result of the collagen peptide powder sample is shown in fig. 9C5, the detection result of the blank solution is shown in fig. 9D5, and the chromatographic peak data of the test sample is shown in tables 9-5:

TABLE 9-5 chromatographic peak data of test article

As shown in FIG. 9, the glucosamine hydrochloride control of FIG. 9A5 peaked at 5.588min and 6.065 min; the test article in FIG. 9B5 showed a peak time of 5.583min and 6.060min for glucosamine, and a degree of separation of 2.8; interference at a retention time of 5.588min is shown in FIG. 9C 5.

And (5) elution procedure six:

and (3) detection results: the HPLC profile of each sample is shown in fig. 9, wherein the detection result of glucosamine hydrochloride control is shown in fig. 9a6, the detection result of the test sample is shown in fig. 9B6, the detection result of the collagen peptide powder sample is shown in fig. 9C6, the blank profile is shown in fig. 9D6, and the chromatographic peak data of the test sample is shown in tables 9-6:

TABLE 9-6 chromatographic peak data of test article

As shown in FIG. 9, the glucosamine hydrochloride control sample in FIG. 9A6 has a chromatographic peak number 1 showing peak appearance time of 7.155min and a chromatographic peak number 2 lacking; the elution procedure was not applicable because the peak time of the glucosamine sample in FIG. 9B6 was 7.144min for chromatographic peak number 1 and missing for chromatographic peak number 2.

As can be seen from the above experimental results, neither the first elution procedure nor the sixth elution procedure can detect the chromatographic peak of the sequence number 2; adopting an elution procedure V, interference exists at the retention time of the glucosamine; the second to the fourth elution procedures can effectively separate the components, wherein the separation degrees of the second elution procedure and the third elution procedure reach 3.6 and 3.7 respectively, and the third elution procedure is preferred due to the fact that the peak time of the second elution procedure is slightly late and the detection time and the actual production detection need are comprehensively considered.

EXAMPLE 10 selection of treatment temperature for test articles

The method comprises the following operation steps: in order to verify the influence of the test solution prepared at different water bath temperatures (step (5)) on the peak appearance of the test solution, an Aglient liquid chromatograph is used for detection, and in step (5), the water bath temperatures of 55 ℃, 60 ℃, 70 ℃ and 85 ℃ are respectively selected to process the test solution. Respectively and precisely weighing 2 six parts of 0.1g of experimental sample at each water bath temperature, putting the experimental samples into a 100ml volumetric flask, adding a proper amount of pure water, carrying out ultrasonic extraction for 15min, cooling, and then fixing the volume and shaking up. 2ml of the glucosamine standard solution and 2ml of the test sample solution are mixed, and 1ml of the mixture is measured in the step (5) from "placing in a 15ml test tube with a stopper".

And (3) detection results:

table 10-155 ℃ water bath temperature recovery is shown in the table below:

table 10-285 ℃ water bath temperature recovery is shown in the table below:

table 10-360 ℃ water bath temperature recovery is shown in the table below:

table 10-470 ℃ water bath temperature recovery is shown in the table below:

as can be seen from the above experimental results, in the step (5), the water bath temperatures of 55 ℃ and 85 ℃ are respectively selected to process the test sample, the average recovery rates are 86.2% and 114.3% respectively, the RSD values are 8.1% and 5.6% respectively, and the requirement of Chinese pharmacopoeia on the recovery rate is not met; the water bath temperature is respectively selected to be 60 ℃ and 70 ℃ to process the test sample, the average recovery rate is respectively 97.7 percent and 98.9 percent, the RSD value is 0.9 percent, and the detection requirement is met.

Comparative example 1

Detecting a sample: experimental sample 2.

The method comprises the following operation steps: according to the measurement (liquid chromatography) operation of the contents of chondroitin sulfate and glucosamine hydrochloride in GB/T20365-2006, sample solutions are respectively prepared from an experimental sample 2 (glucosamine chondroitin tablets containing active peptide and organic calcium) and collagen peptide powder according to the preparation method of the sample solutions in GB/T20365-2006.

And (3) detection results: as shown in fig. 10, the results of the glucosamine hydrochloride control are shown in fig. 10A, and the results of the sample detection are shown in fig. 10B (experimental sample 2) and fig. 10C (collagen powder). In FIG. 10A, the peak appearance time of the target glucosamine hydrochloride is 2.161min for the chromatographic peak number 1, 2.304min for the chromatographic peak number 2, in FIG. 10B the peak appearance time of the target glucosamine hydrochloride number 1 is 2.154min, in FIG. 2 the peak appearance time of the chromatographic peak number 2 is 2.300min, and in FIG. 10C the interfering peaks with retention times of 2.164 and 2.308min are shown.

Therefore, at the peak time of the glucosamine chromatographic peak, the active peptide ossein peptide interferes with the detection of the glucosamine, and the baseline is unstable under the method, so that the method cannot be used for detecting the content of the glucosamine in the glucosamine chondroitin preparation containing the active peptide and organic calcium.

Comparative example 2

Detecting a sample: experimental sample 2.

The method comprises the following operation steps:

preparation of a standard stock solution: precisely weighing a proper amount of glucosamine hydrochloride reference substance, placing the reference substance in a volumetric flask, adding 0.1mol/L sodium acetate solution, and ultrasonically dissolving. Diluting to the scale with 0.1mol/L sodium acetate solution, and mixing to obtain standard stock solution with concentration of about 1.0 mg/ml.

Preparation of standard working solution: sucking a proper amount of the standard stock solution into another volumetric flask, adding 350 μ l of Phenylisothiocyanate (PITC), adding 10ml of methanol, ultrasonically dissolving, adding methanol water solution (methanol/water: 65/35; V/V) to scale, mixing, heating in 80 deg.C water bath for 10min, and cooling to room temperature; transfer about 5ml of the above solution to a centrifuge tube, add about 5ml heptane, shake for 2min, transfer the methanol layer to an HPLC vial and inject.

Preparing a bone collagen peptide sample solution: weighing 50mg of bone collagen peptide, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to a scale, shaking up, centrifuging for 10min at 4000 rpm, taking supernatant to obtain a solution IV, precisely absorbing 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing in a 60 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out shaking extraction, standing for 20min, taking supernatant, and filtering with a 0.45 mu m filter membrane to obtain a bone collagen peptide sample solution;

preparing a test sample: a certain amount of the test sample (sample 2) was weighed into a volumetric flask, and about 0.1mol/L sodium acetate solution was added thereto for ultrasonic dissolution. Diluting to scale with 0.1mol/L sodium acetate solution, and mixing; sucking the above solution into another volumetric flask, adding 350 μ l Phenyl Isothiocyanate (PITC), adding 10ml methanol, ultrasonic dissolving, adding methanol water solution (methanol/water: 65/35; V/V) to scale, mixing, heating in 80 deg.C water bath for 10min, and cooling to room temperature; transfer 5ml of the above solution to a centrifuge tube, add 5ml of heptane, shake for 2min, transfer the methanol layer to an HPLC vial and inject.

Liquid chromatography conditions:

the instrument comprises the following steps: an agent high performance liquid chromatograph and an ultraviolet detector;

a chromatographic column: c18 column, 150mm X4.6 mm, 5 μm number LC-012

Flow rate: 1.0mL/min

Detection wavelength: UV240nm

Column temperature: 30 deg.C

Sample introduction amount: 2 μ L

A first mobile phase: phosphoric acid solution; a second mobile phase: acetonitrile;

elution was carried out according to the procedure shown in the following table:

time (min)	First mobile phase (%)	Second mobile phase (%)
			0	85	15
1.5	85	15
			2.0	0	100
3.5	0	100
			5.0	85	15
7.0	85	15

And (3) detection results: as shown in fig. 11, the results of the glucosamine hydrochloride control and the test sample are shown in fig. 11A and fig. 11B, respectively, and the results of the collagen peptide powder sample and the test sample are shown in fig. 10C, respectively, and the chromatographic peak data of the test sample are shown in table 11:

TABLE 11 chromatographic peak data of test articles

Among them, the peak-off time of the target glucosamine hydrochloride in fig. 11A is 2.902min and 3.287min, the peak-off time of the target glucosamine hydrochloride in fig. 11B is 2.901min and 3.287min, and the presence of an interference peak with a retention time of 2.898min is shown in fig. 11C. Therefore, at the peak time of the glucosamine chromatogram, the active peptide ossein peptide interferes with the detection of the glucosamine, and the peptide cannot be used for detecting the content of the glucosamine in the glucosamine chondroitin preparation containing the active peptide and the organic calcium.

Comparative example 3

Detecting a sample: experimental sample 2.

The method comprises the following operation steps:

(7) setting detection conditions of a high performance liquid chromatograph:

the instrument comprises the following steps: waters hplc, uv detector;

column temperature: 25 ℃;

detector wavelength: 254 nm;

mobile phase: methanol/water/acetic acid 20/79.96/0.04 (V/V);

flow rate: 0.6 mL/min;

and (3) an elution mode: isocratic elution.

(8) Measurement: precisely measuring blank solution, reference solution, sample solution and ossein protein peptide sample solution 10 μ L each, injecting into liquid chromatograph, and isocratic eluting.

And (3) detection results: as shown in fig. 12, the results of the glucosamine hydrochloride control are shown in fig. 12A, the results of the test sample are shown in fig. 12B, the results of the collagen peptide powder sample are shown in fig. 12C, the results of the blank solution are shown in fig. 12D, and the data of the chromatographic peaks of the test sample are shown in table 12:

TABLE 12 chromatographic peak data of test article

Among them, the peak-off time of the target glucosamine hydrochloride in fig. 12A is 6.532min and 7.147min, the peak-off time of the target glucosamine hydrochloride in fig. 12B is 6.545min and 7.216min, and the presence of interference peaks with retention times of 6.255min and 7.297min is shown in fig. 12C. Therefore, at the peak time of the glucosamine chromatographic peak, the active peptide ossein protein peptide interferes with the detection of the glucosamine, and the method cannot be accurately used for detecting the content of the glucosamine in the glucosamine chondroitin preparation containing the active peptide and organic calcium.

Test example 1Verification method for content determination method of glucosamine potassium sulfate

1. Instrument and reagent preparation

1.1 instruments

HPLC-1260, Agilent, USA;

CPA225D, Sidoris scientific instruments (Beijing) Inc

Ultrasonic extractor, Kunshan ultrasonic Instrument Co., Ltd

HWS-26 constant temperature water bath, Shanghai Tantake Technology GmbH

XW-80A vortex mixer, Shanghai Jingke industries Ltd

1.2 reagents and reagent preparation

1.2.1 methanol (chromatographically pure); d, glucosamine hydrochloride (China institute for food and drug testing, purity: 100%); high purity water;

1.2.2 formulation of reagent I: accurately transferring 5mL of phenyl isothiocyanate into a 100mL volumetric flask, dissolving with methanol and fixing the volume to obtain a reagent I;

1.2.3 preparation of reagent II: adding 0.2mol/L NaH₂PO₄Mixing the aqueous solution with 0.2mol/L Na₂HPO₄Mixing the water solutions according to the volume ratio of 5:95(V/V) to obtain a reagent II;

1.2.4 preparation of control solutions: weighing a glucosamine hydrochloride reference substance 12.5mg to 100mL in a volumetric flask, dissolving with deionized water to a constant volume to obtain a solution III, precisely sucking 1mL of the solution III, placing the solution III in a 15mL test tube with a plug, adding 1mL of a reagent II, shaking up, adding 1mL of the reagent I, shaking up, placing the solution I in a 70 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, performing oscillation extraction, standing for 20min, taking a lower layer solution, and filtering with a 0.45-micrometer filter membrane to obtain a reference substance solution;

1.2.5 preparation of test solution: taking 10 bags (experimental sample 2) of glucosamine chondroitin powder containing active peptide and organic calcium, crushing into 100-mesh powder, uniformly mixing, weighing about 50mg, precisely weighing, placing in a 50mL measuring flask, adding an appropriate amount of deionized water, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume to the scale, shaking up, centrifuging at 4000 rpm for 10min, taking the supernatant to obtain a solution IV, precisely sucking 1mL of the solution IV, placing in a 15mL test tube with a plug, adding 1mL of reagent II, shaking up, adding 1mL of reagent I, shaking up, placing in a 60 ℃ water bath, heating for 20min, cooling to room temperature, adding 1mL of n-hexane, carrying out oscillating extraction, standing for 20min, taking the lower layer solution, and filtering through a 0.45-micron filter membrane to obtain a test sample solution;

2. chromatographic conditions

A chromatographic column: agilent ZORBAX Eclipse XDB-C18, 4.6 x 250, 5 μm; detection wavelength: 254 nm; flow rate: 1.0 ml/min; sample introduction amount: 10 mu l of the mixture; column temperature: room temperature; mobile phase see table a 1:

TABLE A1 flow phase comparison example

3. Inspection of the Linear Range

3.1 preparation of Standard Curve

3.1.1 weighing 12.5mg glucosamine hydrochloride into a 50ml volumetric flask, diluting and dissolving with pure water, wherein the concentration is 0.2088mg glucosamine. Taking 1, 2, 4, 8 and 10ml of the solution to a 10ml volumetric flask, diluting the solution with pure water to a constant volume, wherein the prepared concentrations are respectively as follows: 20.88, 41.76, 83.53, 167.05 and 208.82 mu g/ml. Precisely sucking 1ml of reference substance solution (1 ml of deionized water is taken as a blank and is followed) and placing the reference substance solution in a 15ml test tube with a plug, adding 1ml of buffer solution and 1ml of phenylisothiocyanate methanol solution, uniformly mixing by vortex oscillation, heating in a water bath at 80 ℃ for 20min, and cooling in cold water to room temperature. Adding 1ml n-hexane respectively, shaking thoroughly, standing for 20 min. Taking the supernatant, and filtering with 0.45 μ l microporous membrane.

3.2 Experimental data

Standard serial number	Concentration of μ g/ml	Peak area
			std-1	20.88	236.0
std-2	41.76	474.6
			std-3	83.53	928.0
std-4	167.05	1814.7
			std-5	208.82	2332.6

The glucosamine standard curve is shown in figure 13.

3.3 conclusion of the experiment

Coefficient of correlation R²The linear relationship is good when the concentration of glucosamine is 0.9994, and the measured glucosamine concentration is between 20.88 and 208.82 mu g/ml, which shows good current relationship.

4. Precision test

4.1 Experimental methods

Precisely sucking 10 μ l of the control solution, and injecting into liquid chromatograph in parallel with 6 needles.

4.2 Experimental data

4.3 conclusion of the experiment

The sample was continuously injected through 6 needles, and the RSD (%) of the peak area was 0.1%, indicating that the precision of the instrument was good.

5. Stability test

5.1 Experimental methods

10. mu.l of the control solution was injected into a liquid chromatograph for 0, 1, 2, 3, and 4 hours, respectively, and the peak area was measured.

5.2 Experimental data

5.3 conclusion of the experiment

The peak area RSD (%) of the sample solution after being placed for 1, 2, 3 and 4 hours is 0.4%, which shows that the sample solution has good stability after being placed for 4 hours.

6. Reproducibility test

6.1 Experimental methods

Six test sample solutions are prepared in parallel, and the content of glucosamine potassium sulfate is measured.

6.2 Experimental data

6.3 conclusion of the experiment

According to the experimental results, the RSD was 1.2%, and the reproducibility was good.

7. Recovery rate experiment

7.1 Experimental methods

Accurately weighing 6 parts of 2 samples of 0.1g of experimental sample, placing the samples in a volumetric flask of 100ml, adding a proper amount of pure water, carrying out ultrasonic extraction for 15min, cooling, fixing the volume and shaking up. 2ml of the glucosamine standard solution and 2ml of the sample solution are mixed, and 1ml of the mixed solution is measured from "placing in a 15ml test tube with a stopper" in the preparation of the test solution.

7.2 Experimental data

7.3 conclusion of the experiment

According to the result, the RSD is 0.6%, the method refers to the data requirement of the recovery rate in Chinese pharmacopoeia, the test sample with the same concentration is measured, 6 measurement results are used for evaluation, and the experimental result meets the requirement.

8. Sample assay

8.1 Experimental methods

Taking 10 minimum preparation units of glucosamine chondroitin preparation containing active peptide and organic calcium, grinding into 100 mesh powder, mixing, weighing 50mg, precisely weighing into 50ml volumetric flask, adding appropriate amount of pure water, ultrasonic extracting for 15min, cooling, fixing volume, and shaking. About 5ml of the suspension was centrifuged at high speed for 10min (4000 rpm), and the supernatant was used as a test solution. The preparation of the test sample solution was carried out from "1 ml precise pipetting". A trailing blank.

8.2 Experimental data

Calculating the formula:

c: glucosamine Potassium sulfate, g/100g

A sample A: peak area of sample

And C, marking: concentration of glucosamine hydrochloride, mg/ml

Marking A: peak area of standard

m: sample weighing, mg

605.52: glucosamine potassium sulfate molecular weight;

215.63: molecular weight of glucosamine hydrochloride.

2: conversion factor (two molecules of glucosamine hydrochloride out of one molecule of glucosamine potassium sulfate)

8.3 conclusion of the experiment

The glucosamine potassium sulfate content in the glucosamine chondroitin preparation containing active peptide and organic calcium meets the requirement.

9. Summary of the invention

Experiments such as linearity, stability, precision, reproducibility, recovery rate and the like are carried out on the method for measuring the content of glucosamine potassium sulfate in the glucosamine chondroitin preparation containing active peptide and organic calcium, and the method is proved to be reasonable and feasible and can achieve the purpose of quality control on the content of glucosamine potassium sulfate in the glucosamine chondroitin preparation containing active peptide and organic calcium.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A method for detecting the glucosamine content in a glucosamine chondroitin preparation containing active peptide and organic calcium is carried out by adopting a high performance liquid chromatography with an ultraviolet detector, wherein a chromatographic column in the high performance liquid chromatography is a C18 chromatographic column; the adopted mobile phase consists of a first mobile phase and a second mobile phase, wherein the first mobile phase is methanol, and the second mobile phase is acetic acid aqueous solution.

2. The detection method according to claim 1, wherein the particle size of the packing in the chromatographic column is 5 μm.

3. The detection method according to claim 1 or 2, wherein the column temperature of the chromatographic column is 20 to 30 ℃; preferably 25 deg.c.

4. The detection method according to any one of claims 1 to 3, wherein the aqueous acetic acid solution is a 0.04% aqueous acetic acid solution.

5. The detection method according to any one of claims 1 to 4, wherein elution is performed according to the following procedure:

preferably, elution is carried out according to the following procedure:

beginning at 7.01min, adjusting the volume of the first mobile phase and the second mobile phase to be 100:0, and continuing until 15 min;

beginning at 15.01min, the volumes of the first mobile phase and the second mobile phase were adjusted to 20:80, and ending at 25 min.

6. The detection method according to any one of claims 1 to 5, wherein, in the high performance liquid chromatography, elution is performed at a flow rate of 1.0 mL/min.

7. The detection method according to any one of claims 1 to 6, wherein the detection wavelength of the ultraviolet detector is 240 to 260 nm; preferably 254 nm.

8. The detection method according to any one of claims 1 to 7, wherein the detection method comprises the steps of:

(7) setting detection conditions of a high performance liquid chromatograph:

chromatographic column parameters: c18, 250mm × 4.6mm, 5 μm;

column temperature: 20-30 ℃;

detection wavelength of ultraviolet detector: 240-260 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;

9. The detection method according to any one of claims 1 to 8, wherein the detection method comprises the steps of:

(7) setting detection conditions of a high performance liquid chromatograph:

chromatographic column parameters: c18, 250mm × 4.6mm, 5 μm;

column temperature: 25 ℃;

detection wavelength of ultraviolet detector: 254 nm;

flow rate: 1.0 mL/min;

elution was performed according to the following procedure:

wherein, sample C: percent content of sample, g/100 g;

a sample A: peak area of the sample;

and C, marking: concentration of glucosamine hydrochloride, mg/ml;

marking A: peak area of control;

and (5) M sample: weighing the sample, and mg;

glucosamine potassium sulfate molecular weight: 605.52, respectively;

molecular weight of glucosamine hydrochloride: 215.63, respectively;