CN115166102A - UPLC-PDA (ultra-performance liquid chromatography-personal digital assistant) determination method for chondroitin sulfate, glucosamine and derivatives thereof - Google Patents
UPLC-PDA (ultra-performance liquid chromatography-personal digital assistant) determination method for chondroitin sulfate, glucosamine and derivatives thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a UPLC-PDA (ultra performance liquid chromatography-personal digital assistant) determination method for chondroitin sulfate, glucosamine and derivatives thereof, which comprises the following steps: (1) preparing a sample solution; (2) preparing a standard solution; (3) measuring: the measurement was carried out using UPLC-PDA under the following instrument conditions: a chromatographic column: waters Xbridge C18 (5 μm, 4.6X 150 mm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample introduction amount: 5 mu L of the solution; detection wavelength: 192nm; mobile phase A: acetonitrile, mobile phase B: pentane sodium sulfonate water solution with the concentration of 10 mmol/L; gradient elution. The UPLC-PDA determination method of the chondroitin sulfate, the glucosamine and the derivatives thereof has the advantages of tolerance and good reproducibility, and the chondroitin sulfate, the glucosamine and the derivatives thereof are completely separated.
Description
Technical Field
The invention belongs to the technical field of medicine content determination, and particularly relates to a UPLC-PDA (ultra performance liquid chromatography-personal digital assistant) determination method for chondroitin sulfate, glucosamine and derivatives thereof.
Background
Chondroitin sulfate is generally derived from cartilage of cattle, chicken, pigs and sharks, so that the chondroitin sulfate belongs to a biological medicine, is generally considered to be beneficial to treating joint diseases, is matched with some glucosamine to relieve pain and promote the regeneration of cartilage, so that the chondroitin sulfate can reduce the pain of osteoarthritis, reduce joint swelling, improve joint functions, prevent effusion from appearing or gaps between knee joints and hand joints from being narrow, provide some padding effects, buffer impact and friction, have a protective effect on some corneal collagen fibers, enhance permeability and improve blood circulation, and therefore, the bone joint diseases are more commonly used.
Glucosamine hydrochloride, glucosamine potassium sulfate, glucosamine sulfate and the like are derivatives of glucosamine, and the main function is to be applied to the treatment of arthritis. Glucosamine is a monosaccharide widely existing in the nature, can be extracted from the nature and also can be generated by the human body, has the main functions of inhibiting the damage of the cartilage surface, participating in the synthesis of cartilage and inhibiting the damage of aseptic inflammation to the cartilage surface, is widely applied to the treatment and prevention of osteoarthritis of various joints clinically, and has important significance for the conservative treatment of hip joints, knee joints and ankle joints.
For the measurement of the content of chondroitin sulfate and glucosamine, the tolerance and the reproducibility of the national standard (GB/T20365-2006) method are poor, and the separation degree of the target object is poor (< 1.2). Especially, the sample with complex components can not be detected by using the national standard method.
Therefore, the development of a UPLC-PDA determination method with tolerance and good reproducibility and capable of completely separating chondroitin sulfate, glucosamine and derivatives thereof is a technical problem which needs to be solved by the technical personnel in the field.
Disclosure of Invention
In view of this, the invention provides a UPLC-PDA determination method for chondroitin sulfate, glucosamine and derivatives thereof, which has good tolerance, good reproducibility and good separation degree.
In order to achieve the purpose, the invention adopts the following technical scheme:
the UPLC-PDA determination method of chondroitin sulfate, glucosamine and derivatives thereof comprises the following steps:
(1) Preparing a sample solution: precisely weighing 0.02g of sample powder, placing the sample powder in a 50mL triangular flask with a plug, soaking the sample powder in 5mL acetonitrile solution, fixing the volume to 50mL by water, carrying out ultrasonic extraction for 15min, and filtering the supernatant with a 0.22-micron filter membrane for later use;
(2) Preparation of standard solutions: accurately weighing 5-10mg of chondroitin sulfate and glucosamine or glucosamine derivatives, soaking with 1mL of acetonitrile, fixing the volume to 10mL with water, uniformly mixing, preparing mother liquor with the concentration of 1mg/mL, respectively taking two kinds of mother liquor of the reference, placing in a same 10mL volumetric flask, adding water to fix the volume to a scale to obtain a solution, diluting the solution with a mixed solution of acetonitrile and water with the volume ratio of 10 to 90 to obtain a series of reference solutions, taking the appropriate amount of the series of reference solutions, and filtering with a 0.22 mu m microporous filter membrane for later use;
(3) The determination method comprises the following steps: the measurement was carried out using UPLC-PDA under the following instrument conditions: a chromatographic column: waters Xbridge C18 (5 μm, 4.6X 150 mm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample introduction amount: 5 mu L of the solution; detection wavelength: 192nm; mobile phase A: acetonitrile, mobile phase B: pentane sodium sulfonate water solution with the concentration of 10 mmol/L; gradient elution.
The invention also provides application of the UPLC-PDA determination method in simultaneous determination of chondroitin sulfate, glucosamine and derivatives thereof.
Further, the glucosamine derivative is glucosamine potassium sulfate, glucosamine hydrochloride, or glucosamine sulfate.
The invention has the beneficial effects that: the UPLC-PDA determination method of the chondroitin sulfate, the glucosamine and the derivatives thereof has the advantages of tolerance and good reproducibility, and the chondroitin sulfate, the glucosamine and the derivatives thereof are completely separated.
Drawings
FIG. 1 is a UPLC-PDA chromatogram of chondroitin sulfate and glucosamine potassium sulfate, (1. Chondroitin sulfate; 2. Glucosamine potassium sulfate);
FIG. 2 is a UPLC-PDA chromatogram of chondroitin sulfate and glucosamine hydrochloride, (1. Chondroitin sulfate; 2. Glucosamine hydrochloride);
FIG. 3 is a UPLC-PDA chromatogram of chondroitin sulfate and glucosamine sulfate, (1. Chondroitin sulfate; 2. Glucosamine sulfate);
FIG. 4 is a UPLC-PDA chromatogram of the sample solution of example 1, (1. Chondroitin sulfate; 2. Potassium glucosamine sulfate);
FIG. 5 is a UPLC-PDA chromatogram of the sample solution of example 2, (1. Chondroitin sulfate; 2. Glucosamine hydrochloride);
FIG. 6 is UPLC-PDA chromatogram of sample solution of example 2, (2. Glucosamine sulfate)
FIG. 7 is UPLC-PDA chromatogram of national standard (GB/T20365-2006) method, (1. Chondroitin sulfate; 2. Glucosamine potassium sulfate)
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
UPLC-PDA determination of chondroitin sulfate and glucosamine potassium sulfate in Tianhuang tablets
The sample is mainly prepared by laboratories according to a formula of a Tianhuang tablet (patent application number: 201811403920.2) developed in the early stage, and mainly comprises calcium carbonate, chondroitin sulfate, glucosamine potassium sulfate, morinda officinalis extract, turmeric extract and the like.
(1) Preparing a sample solution: precisely weighing 0.02g of sample powder, placing the sample powder in a 50mL triangular flask with a plug, dispersing the sample powder in 5mL acetonitrile solution, adding water to a constant volume of 50mL, carrying out ultrasonic extraction for 15min, cooling to room temperature, taking supernatant, and filtering with a 0.22 mu m filter membrane for later use.
(2) Preparation of standard solutions: respectively and precisely weighing 10mg of chondroitin sulfate and glucosamine potassium sulfate contrast products, infiltrating the chondroitin sulfate and the glucosamine potassium sulfate contrast products by using 1mL of acetonitrile, fixing the volume to 10mL by using water, uniformly mixing to prepare mother liquor with the concentration of 1mg/mL, respectively taking 2mL of the mother liquor of the two contrast products, placing the mother liquor in a same 10mL volumetric flask, adding water to fix the volume to scale to obtain a solution, diluting the solution by using acetonitrile-water (volume ratio is 10;
(3) And (3) determination: the measurement was carried out using UPLC-PDA under the following instrument conditions: and (3) chromatographic column: waters Xbridge C18 (5 μm, 4.6X 150 mm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample introduction amount: 5 mu L of the solution; detection wavelength: 192nm; a mobile phase A: acetonitrile, mobile phase B: pentane sodium sulfonate water solution with the concentration of 10 mmol/L; gradient elution, gradient elution procedure is shown in table 1.
TABLE 1 gradient elution schedule
| Time (min) | Flow rate (mL/min) | A(％) | B(％) |
| 0 | 0.3 | 5 | 95 |
| 6 | 0.3 | 5 | 95 |
| 10 | 0.3 | 95 | 5 |
| 12 | 0.3 | 5 | 95 |
| 14 | 0.3 | 5 | 95 |
The UPLC-PDA chromatograms of the chondroitin sulfate, glucosamine potassium sulfate control and the sample solution of example 1 are shown in figures 1 and 4.
Comparative experiment
The main differences between the method of the invention and the national standard (GB/T20365-2006) are shown in Table 2;
TABLE 2 comparison of the method with national standards
Comparative experiments show that the national standard method has poor tolerance and reproducibility, and the target object has poor separation degree (< 1.2), which is shown in figure 7;
the method completely separates chondroitin sulfate and glucosamine potassium sulfate by optimizing experimental conditions (the separation degree is more than 1.5), and is shown in attached drawings 1 and 4, particularly, the sample of the embodiment 1 is added with two traditional Chinese medicines, namely morinda officinalis and curcuma longa, the sample is complex, other substances exist between two target objects, the content of chondroitin sulfate and glucosamine potassium sulfate in the radix rehmanniae tablets cannot be detected by using a national standard method.
Verification experiment
1. Overview of the method
Weighing a proper amount of sample, dispersing with acetonitrile, adding aqueous solution for ultrasonic extraction, performing ultra-high performance liquid chromatography separation, detecting at a wavelength of 192nm, and quantitatively determining the content of chondroitin sulfate and glucosamine potassium sulfate in the sample by an external standard method.
2. Analytical procedure
2.1 Standard solution preparation
2.1.1 preparation of control solutions: 10mg of chondroitin sulfate and glucosamine potassium sulfate contrast product is accurately weighed, dissolved in 1mL of acetonitrile, added with water to reach a constant volume of 10.0mL, and mixed uniformly to prepare a solution of chondroitin sulfate and glucosamine potassium sulfate with the concentration of 1 mg/mL.
3.1.2 preparation of control use solution: respectively putting 2mL of the stock solutions of the chondroitin sulfate and the glucosamine potassium sulfate into the same 10mL volumetric flask, adding water to a constant volume to a scale, and shaking up to obtain the use solutions with the concentrations respectively as follows: chondroitin sulfate 200. Mu.g/mL, glucosamine potassium sulfate 200. Mu.g/mL.
3.1.3 Standard working solution
Accurately sucking a proper amount of standard use solution into a 1.0mL volumetric flask, diluting with acetonitrile-water (10).
3.2 sample preparation
Precisely weighing about 0.02g of sample, placing the sample in a 50mL triangular flask with a plug, dispersing the sample in 5mL acetonitrile solution, adding water to a constant volume of 50mL, ultrasonically extracting for 15min, cooling to room temperature, and filtering the sample solution through a 0.22-micron filter membrane for later use.
3.3 determination of
3.3.1 Instrument reference conditions
And (3) chromatographic column: waters Xbridge C18 (150 mm. Times.4.6 mm, μm 5 pm).
Mobile phase: 10mmol/L sodium pentanesulfonate water solution and acetonitrile
Flow rate: 0.3mL/min
Column temperature: 35 deg.C
Sample introduction amount: 5 μ L
4. Analysis of results
4.1 Standard Curve and Linear Range
4.1.1 precise uptake of the concentrations of the standard working solutions of the series, operating under the above-mentioned liquid phase conditions, and determination of the peak areas. Plotting linear equation curve by peak area and concentration of standard solution. Chondroitin sulfate and glucosamine potassium sulfate show good linear relation (R) in corresponding concentration range and peak area 2 >0.999 See table 3).
TABLE 3 Linear regression equation and Linear Range for chondroitin sulfate and glucosamine Potassium sulfate salts
4.2 detection Limit
Diluting the sample solution step by step, repeatedly injecting sample, and recording chromatogram. The concentration when the signal-to-noise ratio is 3 is taken as a detection limit, the detection limits of the chondroitin sulfate and the glucosamine potassium sulfate are respectively 0.5 mu g/mL (1.25 mg/g) and 0.2 mu g/mL (0.5 mg/g), and the sensitivity of the method meets the requirement.
4.3 stability test
The sample solutions were placed at room temperature for 0h, 2h,4h, 6h, 8h, 10h, 12h, respectively, at time points (n = 3), peak areas were recorded, and the contents of chondroitin sulfate and glucosamine potassium sulfate in the samples were calculated, with RSD values of 0.99% and 0.73% for the two components, indicating that the sample solutions were stable within 12 h.
TABLE 4 stability test results
4.4 repeatability test
Precisely measuring 6 parts of samples, preparing a sample solution according to the method 3.2, detecting according to a 3.3 chromatographic method, recording peak areas, wherein the RSD of the contents of chondroitin sulfate and glucosamine potassium sulfate in the sample is 1.05 percent and 0.77 percent respectively, which shows that the method has good repeatability.
TABLE 5 results of the repeatability tests
4.5 accuracy test
The method has the advantages that the accuracy of the method is high, the same batch of samples (0.01 g) with known content are accurately measured, 1.0 time of chondroitin sulfate and glucosamine potassium sulfate standard solution is added respectively, the sample solution is prepared according to the preparation method of the test solution, the sample solution is measured under the chromatographic conditions, the calculation and the recovery rate measurement are carried out, the results show that the recovery rates of the chondroitin sulfate and the glucosamine potassium sulfate are 99.39 percent and 99.43 percent respectively, and the RSD is 1.21 percent and 1.27 percent respectively, and the method has high accuracy.
TABLE 6 results of the recovery test with addition of standard
4.6 determination of the sample content
And (3) preparing sample solutions by taking three batches of self-made samples according to the preparation method of the samples, and detecting under the chromatographic condition, wherein the average content of the chondroitin sulfate and glucosamine potassium sulfate salt in the three batches of samples is 54.8mg/g and 85.8mg/g, and the RSD is 1.04 percent and 1.57 percent respectively.
Example 2
And (3) measuring chondroitin sulfate and glucosamine hydrochloride in the Move Free Yijieguan chondroitin calcium tablet.
The product is purchased from the great wall of the Jingdong city, and comprises the following main components: chonfroitin Sulfate Sodium (chondroitin Sulfate), glucosamine Hydrochloride (Glucosamine Hydrochloride), hyaluronic Acid (Hyaluronic Acid), calcium front borate (calcium fructose borate).
(1) Preparing a sample solution: precisely weighing 0.02g of sample powder, placing the sample powder in a 50mL triangular flask with a plug, dispersing the sample powder in 5mL acetonitrile solution, adding water to a constant volume of 50mL, carrying out ultrasonic extraction for 15min, cooling to room temperature, taking supernatant, and filtering with a 0.22 mu m filter membrane for later use.
(2) Preparation of standard solutions: respectively and precisely weighing 10mg of chondroitin sulfate and glucosamine hydrochloride reference substances, infiltrating the chondroitin sulfate and glucosamine hydrochloride reference substances with 1mL of acetonitrile, fixing the volume to 10mL with water, uniformly mixing to prepare mother liquor with the concentration of 1mg/mL, respectively taking 2mL and 4mL of the mother liquor of the chondroitin sulfate and glucosamine hydrochloride reference substances, putting the mother liquor into the same 10mL volumetric flask, adding water to fix the volume to scale to obtain a solution, diluting the solution with acetonitrile-water (10);
(3) And (3) determination: the measurement was carried out using UPLC-PDA under the following instrument conditions: a chromatographic column: waters Xbridge C18 (5 μm, 4.6X 150 mm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample introduction amount: 5 mu L of the solution; detection wavelength: 192nm; a mobile phase A: acetonitrile, mobile phase B: pentane sodium sulfonate water solution with the concentration of 10 mmol/L; gradient elution, gradient elution procedure is shown in table 7.
TABLE 7 gradient elution schedule
| Time (min) | Flow rate (mL/min) | A(％) | B(％) |
| 0 | 0.3 | 5 | 95 |
| 6 | 0.3 | 5 | 95 |
| 10 | 0.3 | 95 | 5 |
| 12 | 0.3 | 5 | 95 |
| 14 | 0.3 | 5 | 95 |
Verification test
1. Overview of the method
Weighing a proper amount of sample, dispersing with acetonitrile, adding a water solution, performing ultrasonic extraction, performing ultra-high performance liquid chromatography separation, detecting at a wavelength of 192nm, and quantitatively determining the content of chondroitin sulfate and glucosamine hydrochloride in the sample by an external standard method.
2. Analytical procedure
2.1 Standard solution preparation
2.1.1 preparation of control solutions: 10mg of chondroitin sulfate and glucosamine hydrochloride reference substances are accurately weighed, dissolved by 1mL of acetonitrile, added with water to be constant volume of 10.0mL and uniformly mixed to prepare the solution of chondroitin sulfate and glucosamine hydrochloride with the concentration of 1 mg/mL.
3.1.2 preparation of control use solution: respectively putting the chondroitin sulfate 2mL and the glucosamine hydrochloride 4mL into the same 10mL volumetric flask, adding water to a constant volume to a scale, and shaking up to obtain the use solutions with the concentrations respectively as follows: chondroitin sulfate 200. Mu.g/mL, glucosamine hydrochloride 400. Mu.g/mL.
3.1.3 Standard working solution
Accurately sucking a proper amount of standard use solution into a 1.0mL volumetric flask, diluting with acetonitrile-water (5.
3.2 sample preparation
Precisely weighing about 0.02g of sample, placing the sample into a 50mL volumetric flask, dispersing the sample with 5mL of acetonitrile solution, adding water to a constant volume of 50mL, ultrasonically extracting for 15min, cooling to room temperature, and filtering the sample solution with a 0.22 mu m filter membrane for later use.
3.3 determination of
3.3.1 Instrument reference conditions (same as example 1)
4. Analysis of results
4.1 Standard Curve and Linear Range
4.1.1 precisely sucking the concentration of the series of standard working solutions, operating under the liquid phase condition, and measuring the peak areas of the working solutions. And (5) drawing a linear equation curve by using the peak area and the concentration of the standard solution. Chondroitin sulfate and glucosamine hydrochloride show good linear relation (R) in corresponding concentration range and peak area 2 >0.999 See table 8).
TABLE 8 Linear regression equation, linear Range for chondroitin sulfate and glucosamine hydrochloride
| Compound (I) | Linear regression equation | Linear Range (μ g/mL) | R 2 |
| Chondroitin sulfate | Y＝13.487X-5.6618 | 5～200 | 1.0000 |
| Glucosamine hydrochloride | Y＝2.0864X+3.7519 | 5～400 | 0.9996 |
4.2 detection Limit
Diluting the sample solution step by step, repeatedly injecting sample, and recording chromatogram. The concentration when the signal-to-noise ratio is 3 is taken as a detection limit, the detection limits of chondroitin sulfate and glucosamine hydrochloride are 0.5 mu g/mL (1.25 mg/g) and 1.0 mu g/mL (2.5 mg/g) respectively, and the sensitivity of the method meets the requirement.
4.3 stability test
The sample solutions were placed at room temperature for 0h, 2h,4h, 6h, 8h, 10h, 12h, respectively, at time points (n = 3), peak areas were recorded, and the contents of chondroitin sulfate and glucosamine hydrochloride in the samples were calculated, with RSD values of 1.07% and 1.40% for the contents of both components, indicating that the sample solutions were stable within 12 h.
TABLE 9 stability test results
4.4 repeatability test
Precisely measuring 6 parts of sample, preparing a sample solution according to the method of 3.2, detecting according to a 3.3 chromatographic method, recording peak areas, wherein RSD of the contents of chondroitin sulfate and glucosamine hydrochloride in the sample is 1.16% and 1.21% respectively, and the method is good in repeatability.
TABLE 10 results of the repeatability tests
4.5 accuracy test
The method has the advantages that the accuracy of the method is improved by accurately measuring the same batch of samples (0.01 g) with known content, adding about 1.0 time of chondroitin sulfate and glucosamine hydrochloride reference substances respectively, preparing sample solutions according to the preparation method of the test solution, measuring under the chromatographic conditions, calculating and measuring the recovery rate, and the result shows that the recovery rates of the chondroitin sulfate and the glucosamine hydrochloride are 99.10 percent and 99.43 percent respectively, and the RSD is 0.69 percent and 1.27 percent respectively, thus the method has good accuracy.
TABLE 11 sample recovery test results
4.6 determination of the sample content
The preparation method of the sample is adopted to prepare a sample solution, and the average content of chondroitin sulfate and glucosamine hydrochloride in the Move Free Yijieguo calcium chondroitin sulfate tablet is 79.88mg/g,600.24mg/g and RSD is 1.76% and 1.49% respectively by detecting under the chromatographic conditions (the chromatogram is shown in the attached figure 2 and the attached figure 5).
Example 3
UPLC-PDA method determination of glucosamine sulfate in glucosamine sulfate tablet
Glucosamine sulfate tablets were purchased from the kyotong mall and had the following main ingredients: glucosamine sulfate sodium chloride double salt (each glucosamine sulfate tablet is 0.25g in content determination), and auxiliary materials: lactose, povidone K30, sodium carboxymethylcellulose, etc.
(1) Preparing a sample solution: precisely weighing 0.02g of sample powder, placing the sample powder in a 50mL triangular flask with a plug, dispersing the sample powder in 5mL acetonitrile solution, adding water to a constant volume of 50mL, carrying out ultrasonic extraction for 15min, cooling to room temperature, taking supernatant, and filtering with a 0.22 mu m filter membrane for later use.
(2) Preparation of standard solutions: respectively and precisely weighing 10mg of chondroitin sulfate and glucosamine sulfate reference substances, soaking the chondroitin sulfate and glucosamine sulfate reference substances by using 1mL of acetonitrile, fixing the volume to 10mL by using water, uniformly mixing to prepare mother liquor with the concentration of 1mg/mL, respectively taking 2mL and 4mL of the mother liquor of the two reference substances, placing the mother liquor in a same 10mL volumetric flask, adding water to fix the volume to a scale to obtain a solution, diluting the solution by using acetonitrile-water (10);
(3) And (3) determination: the measurement was carried out using UPLC-PDA under the following instrument conditions: and (3) chromatographic column: waters Xbridge C18 (5 μm, 4.6X 150 mm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample injection amount: 5 mu L of the solution; detection wavelength: 192nm; mobile phase A: acetonitrile, mobile phase B: pentane sodium sulfonate water solution with the concentration of 10 mmol/L; gradient elution, gradient elution procedure is shown in table 12.
TABLE 12 gradient elution schedule
Verification test
1. Overview of the method
Weighing a proper amount of sample, dispersing with acetonitrile, adding a water solution, performing ultrasonic extraction, performing ultra-high performance liquid chromatography separation, detecting at a wavelength of 192nm, and quantitatively determining the glucosamine sulfate content in the sample by an external standard method.
2. Analytical procedure
2.1 Standard solution preparation
2.1.1 control solution preparation: 10mg of chondroitin sulfate and glucosamine sulfate reference substances are accurately weighed, dissolved in 1mL of acetonitrile, added with water to reach a constant volume of 10.0mL, and mixed uniformly to prepare a solution of chondroitin sulfate and glucosamine sulfate with the concentration of 1 mg/mL.
3.1.2 preparation of control use solution: respectively putting the chondroitin sulfate 2mL and the glucosamine sulfate 4mL into the same 10mL volumetric flask, adding water to a constant volume to a scale, and shaking up to obtain the use solutions with the concentrations respectively as follows: chondroitin sulfate 200. Mu.g/mL, glucosamine sulfate 400. Mu.g/mL.
3.1.3 Standard working solution
Accurately sucking a proper amount of standard use solution into a 1.0mL volumetric flask, diluting with acetonitrile-water (5.
3.2 sample preparation
Precisely weighing about 0.02g of sample, placing the sample into a 50mL triangular flask with a plug, dispersing the sample in 5mL acetonitrile solution, adding water to a constant volume of 50mL, carrying out ultrasonic extraction for 15min, cooling to room temperature, and filtering the sample solution through a 0.22-micrometer filter membrane for later use.
3.3 determination of
3.3.1 Instrument reference conditions (same as example 1)
4. Analysis of results
4.1 Standard Curve and Linear Range
4.1.1 precise uptake of the concentrations of the standard working solutions of the series, operating under the above-mentioned liquid phase conditions, and determination of the peak areas. And (5) drawing a linear equation curve by using the peak area and the concentration of the standard solution. Chondroitin sulfate and glucosamine sulfate show good linear relation (R) in corresponding concentration range and peak area 2 >0.999 See table 13).
TABLE 13 Linear regression equation, linear Range for chondroitin sulfate and glucosamine sulfate
4.2 detection Limit
Diluting the sample solution step by step, repeatedly injecting sample, and recording chromatogram. The concentration with the signal-to-noise ratio of 3 is taken as the detection limit, the detection limits of chondroitin sulfate and glucosamine sulfate are respectively 0.5 mu g/mL (1.25 mg/g) and 1.0 mu g/mL (2.5 mg/g), and the sensitivity of the method meets the requirement.
4.3 stability test
The sample solutions are respectively placed at room temperature, the samples are respectively injected at time points of 0h, 2h,4h, 6h, 8h, 10h and 12h, peak areas are recorded (the product does not contain chondroitin sulfate as can be seen from sample components and chromatograms, so that only glucosamine sulfate methodology is considered in stability experiments), the glucosamine sulfate content in the samples is calculated, the RSD of the content is 0.84%, and the sample solutions are stable within 12 h.
TABLE 14 stability test results
4.4 repeatability test
6 parts of samples are precisely measured, a sample solution is prepared according to the method 3.2, the peak area is recorded according to the detection of a 3.3 chromatographic method, and the RSD of the glucosamine sulfate content in the sample is 0.78%, which indicates that the method has good repeatability.
TABLE 15 results of the repeatability tests
4.5 accuracy test
The accuracy of the method for measuring the glucosamine sulfate by the sample adding recovery method is that the same batch of samples (0.01 g) with known content are accurately measured, about 1.0 time of glucosamine sulfate reference substances are respectively added, sample solutions are prepared according to the preparation method of the sample solutions, the sample solutions are measured under the chromatographic conditions, the calculation and the measurement of the recovery rate are carried out, the result shows that the recovery rate of the glucosamine sulfate is 0.69 percent and 1.27 percent respectively, and the method has good accuracy.
TABLE 16 sample recovery test results
4.6 determination of sample content
The sample solution is prepared by the preparation method of the sample, and the average value of the glucosamine sulfate content in the glucosamine sulfate tablet is 542.85mg/g and the RSD is 1.82 percent respectively by detecting under the chromatographic conditions (the chromatogram is shown in the attached figures 3 and 6).
Summary of the invention
Under the established method, the data of the chondroitin sulfate, glucosamine sulfate potassium sulfate, glucosamine hydrochloride, glucosamine sulfate and the like determined by the method are accurate and the result is credible through the verification of methodology (including linear range, detection limit, repeatability, accuracy and the like) and the detection of different actual samples.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. The UPLC-PDA determination method of chondroitin sulfate, glucosamine and derivatives thereof is characterized by comprising the following steps:
(1) Preparing a sample solution: precisely weighing 0.02g of sample powder, placing the sample powder in a 50mL triangular flask with a plug, soaking the sample powder in 5mL acetonitrile solution, fixing the volume to 50mL by water, carrying out ultrasonic extraction for 15min, and filtering the supernatant with a 0.22-micron filter membrane for later use;
(2) Preparation of standard solutions: accurately weighing 5-10mg of chondroitin sulfate and glucosamine or glucosamine derivatives, soaking with 1mL of acetonitrile, fixing the volume to 10mL with water, uniformly mixing, preparing mother liquor with the concentration of 1mg/mL, respectively taking appropriate amount of the mother liquor of the two references, placing in the same 10mL volumetric flask, adding water to fix the volume to a scale to obtain a solution, diluting the solution with a mixed solution of acetonitrile and water with the volume ratio of 10 to 90 to obtain a series of reference solutions, taking the appropriate amount of the series of reference solutions, and passing through a 0.22 mu m microporous filter membrane for later use;
(3) The determination method comprises the following steps: the measurement was carried out using UPLC-PDA under the following instrument conditions: a chromatographic column: waters Xbridge C18 (5 μm, 4.6X 150 mm); flow rate: 0.3mL/min; column temperature: 35 ℃; sample introduction amount: 5 mu L of the solution; detection wavelength: 192nm; mobile phase A: acetonitrile, mobile phase B: pentane sodium sulfonate water solution with the concentration of 10 mmol/L; gradient elution.
2. Use of the UPLC-PDA assay method according to claim 1 for the simultaneous assay of chondroitin sulfate and glucosamine and derivatives thereof.
3. The use of the UPLC-PDA assay method according to claim 2 for the simultaneous assay of chondroitin sulfate and glucosamine and derivatives thereof, wherein said glucosamine derivative is glucosamine potassium sulfate, glucosamine hydrochloride, or glucosamine sulfate.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101271093A (en) * | 2008-05-05 | 2008-09-24 | 北京诚创康韵医药科技有限公司 | Analytical method of glucosamine |
| CN107941963A (en) * | 2017-12-29 | 2018-04-20 | 珍奥集团股份有限公司 | The content assaying method of aminoglucose hydrochloride and chondroitin sulfate in a kind of increase bone density health food |
| CN108872441A (en) * | 2018-08-14 | 2018-11-23 | 江苏康缘药业股份有限公司 | A method of measurement Glucosamine and chondroitin sulfate |
| CN110715986A (en) * | 2019-09-06 | 2020-01-21 | 深圳市荣格保健品有限公司 | Method for determining glucosamine hydrochloride in health food |
| WO2021011538A1 (en) * | 2019-07-15 | 2021-01-21 | R.P. Scherer Technologies, Llc | Capsule dosage forms, methods of preparation and methods of use thereof |
-
2022
- 2022-08-11 CN CN202210963850.6A patent/CN115166102A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101271093A (en) * | 2008-05-05 | 2008-09-24 | 北京诚创康韵医药科技有限公司 | Analytical method of glucosamine |
| CN107941963A (en) * | 2017-12-29 | 2018-04-20 | 珍奥集团股份有限公司 | The content assaying method of aminoglucose hydrochloride and chondroitin sulfate in a kind of increase bone density health food |
| CN108872441A (en) * | 2018-08-14 | 2018-11-23 | 江苏康缘药业股份有限公司 | A method of measurement Glucosamine and chondroitin sulfate |
| WO2021011538A1 (en) * | 2019-07-15 | 2021-01-21 | R.P. Scherer Technologies, Llc | Capsule dosage forms, methods of preparation and methods of use thereof |
| CN110715986A (en) * | 2019-09-06 | 2020-01-21 | 深圳市荣格保健品有限公司 | Method for determining glucosamine hydrochloride in health food |
Non-Patent Citations (2)
| Title |
|---|
| 牛增元;袁玲玲;叶曦雯;孙忠松;王境堂;纪雷;: "高效液相色谱法同时测定硫酸软骨素和盐酸氨基葡萄糖的含量", 药物分析杂志, vol. 26, no. 06, pages 802 - 804 * |
| 王敏娟等: "亲水作用色谱法同时测定保健品中的硫酸软骨素和氨基葡萄糖", 《中国卫生检验杂志》, vol. 27, no. 17, pages 2475 - 2477 * |
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