CN115792000A - Method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets - Google Patents
Method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets Download PDFInfo
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Abstract
The invention discloses a method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets, which comprises the steps of preparing a standard solution, drawing a standard curve, preparing a test solution and measuring a sample. The invention takes glucosamine hydrochloride tablets as a research object, adopts high performance liquid chromatography to measure the content of 5-hydroxymethylfurfural, establishes a kinetic model through the data fitting of the content of 5-hydroxymethylfurfural in different temperature thermal reaction systems, reveals the formed kinetic characteristics of the kinetic model, aims to effectively control and reduce the content of 5-hydroxymethylfurfural in the production and storage processes of the glucosamine hydrochloride tablets, and provides a reference for improving the safety and stability of related products.
Description
Technical Field
The invention belongs to the technical field of detection correlation, and particularly relates to a method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets.
Background
Glucosamine is a marine biological preparation, is hexosamine hydrolyzed and extracted from natural chitin, can effectively promote synthesis of mucopolysaccharide of human body, improve viscosity of joint synovial fluid, improve metabolism of joint cartilage, is helpful for repair capability of chondrocyte, and has important significance for preventing and treating osteoporosis. Glucosamine is widely used in most areas as a drug for the treatment and prevention of systemic osteoarthritis, and the FDA in the united states classifies glucosamine as a dietary supplement for osteoarthritis patients. Currently, glucosamine compounds mainly exist in two forms of hydrochloride and sulfate, and glucosamine hydrochloride tablets (capsules) are the most commonly used over-the-counter medicines in clinic, and have higher stability, purity and curative effect than glucosamine sulfate.
5-hydroxymethylfurfural (5-HMF) is a furan ring-containing small molecular compound widely existing in foods and medicines containing saccharides and mainly derived from Maillard reaction and thermal decomposition of hexose. The Maillard reaction is a complex reaction between an amino compound and a carbonyl compound, the self structure of glucosamine contains amino and carbonyl, a single substance has the condition of the Maillard reaction, and the formation mechanism of the Maillard reaction is reported in documents to be that the glucosamine is subjected to a series of reactions such as condensation, rearrangement, enolization, dehydration, cyclization and the like under certain conditions to form 5-HMF. The glucosamine can generate obvious browning phenomenon under the heating condition, the phenomenon characteristics of the Maillard reaction are met, 5-HMF is taken as a common component related to the quality of food and medicines, and the safety research of the 5-HMF is always a focus of attention. Zhuqingfen et al predict 5-HMF genotoxicity by the widely internationally recognized toxicological prediction software, and the international harmonization conference (ICH) M7 classification mode required by the human drug registration technology shows that 5-HMF is a 2 nd genotoxic impurity in the drug, has genetic toxicity to human body and may have potential carcinogenicity. Therefore, it is necessary to quantitatively determine the 5-HMF formed in glucosamine-related products and study the change law thereof by analysis of the formation kinetics thereof.
At present, the determination of 5-HMF in glucosamine hydrochloride raw materials is reported in documents, and a plurality of documents are also researched on a formation kinetic model of 5-HMF in a Maillard reaction system of monosaccharide and sugar ammonia in food, but the determination of the content of 5-HMF in glucosamine hydrochloride preparations (tablets and capsules) and the formation kinetic research thereof are not reported. Because the glucosamine hydrochloride capsules are prepared by directly filling capsules after raw materials are sieved, the process is simple, the production process of the tablets is more complex, and the process characteristics and process parameters adopted by different manufacturers are different, so that the formation of Maillard reaction products in the product is greatly influenced.
Disclosure of Invention
In order to solve the problems, the glucosamine hydrochloride tablet is taken as a research object, the content of 5-HMF is measured by adopting a high performance liquid chromatography, a kinetic model is established by fitting 5-HMF content data in thermal reaction systems at different temperatures, and formed kinetic characteristics of the kinetic model are revealed, so that the content of 5-HMF in the production and storage processes of the glucosamine hydrochloride tablet is effectively controlled and reduced, and reference is provided for improving the safety and stability of related products.
In order to achieve the purpose, the invention adopts the following technical scheme:
a detection method of 5-hydroxymethylfurfural in glucosamine hydrochloride tablets comprises the following steps:
1) Preparation of standard solution: weighing a 5-HMF standard substance in a volumetric flask, adding pure water to a constant volume to prepare a standard stock solution with the mass concentration of 0.142mg/mL, and storing at 4 ℃; sucking the standard stock solution, and diluting with pure water to obtain standard working solution with mass concentration of 5.698 mug/mL for later use;
2) Drawing a standard curve: sucking the standard working solution obtained in the step 1), diluting the standard working solution into a series of standard solutions with mass concentrations of 5.698 mu g/mL, 2.849 mu g/mL, 1.425 mu g/mL, 0.712 mu g/mL, 0.285 mu g/mL and 0.057 mu g/mL by using pure water, detecting the standard solutions by using a high performance liquid chromatograph, recording peak areas, and drawing a standard curve by using the mass concentration of 5-HMF as a horizontal coordinate and the corresponding peak areas as a vertical coordinate;
3) Preparing a test solution: taking 10 glucosamine hydrochloride tablets, removing the coating, grinding, precisely weighing an appropriate amount of the glucosamine hydrochloride tablets, placing the glucosamine hydrochloride tablets into a volumetric flask, adding pure water, performing ultrasonic treatment, fixing the volume to a scale by using the pure water, shaking up, standing, taking supernatant, and filtering by using a filter membrane to obtain a test solution;
4) And (3) sample determination: and 3) detecting the sample solution obtained in the step 3) by using a high performance liquid chromatograph, calculating the mass of 5-HMF contained in the sample according to a standard curve, and converting the mass into the content of the labeled amount of the glucosamine hydrochloride.
As a preferable scheme of the invention, the conditions of the high performance liquid chromatography are as follows: and (3) chromatographic column: shim-pack GIST C 18 -aq4.6mm × 150mm,3 μm; mobile phase: 0.1% phosphoric acid solution-methanol, volume ratio 90; or 0.1% formic acid solution-methanol, in a volume ratio of 90:10; flow rate: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 284nm; sample injection amount: 20 μ L.
As a preferable scheme of the invention, the mobile phase is 0.1% phosphoric acid solution-methanol, and the volume ratio is 90.
As a preferable mode of the present invention, in the step 3), the volume of the volumetric flask is 50mL, the amount of pure water added is 40mL, and the ultrasonic treatment time is 10min.
As a preferable mode of the present invention, in the step 3), the pore size of the filter is 0.22 μm.
As a preferred scheme of the invention, the method further comprises the step 5) of designing a thermal reaction system: taking 4 parts of the sample fine powder obtained in the step 3), placing the sample fine powder in a weighing bottle, flatly paving the sample fine powder, covering and sealing the sample fine powder, placing the sample fine powder in a constant temperature box with the temperature of 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, placing the sample fine powder in the constant temperature box for 7 days under each temperature condition, precisely weighing an appropriate amount of fine powder in three parallel parts every 1 day, preparing a sample solution according to the step 3), carrying out sample injection measurement according to the step 4), calculating the average content of 5-HMF, and finally carrying out data processing.
As a preferred scheme of the invention, the data processing is to analyze by using a zero-order kinetic model or a first-order kinetic model, to obtain reaction rates at different temperatures by fitting, and to calculate the apparent activation energy of the 5-HMF formation reaction by using an Arrhenius formula.
As a preferred embodiment of the present invention, the zero order kinetic model: c t ＝C 0 +k 0 ·t (1)；
A first-order kinetic model: c t ＝C 0 exp(k 1 ·t) (2)；
In the formula: c t 5-HMF content (mg/kg) at time t; c 0 Is the initial content of 5-HMF (mg/kg); t is heat treatment time (h); k is a radical of 0 And k 1 Respectively, rate constants;
arrhenius formula k = k f exp(-Ea/RT) (3)；
In the formula, k f Is the rate factor (1/d); ea is the apparent activation energy/(kJ/mol); r is a gas constant (8.314J/(k.mol)); t is the thermodynamic temperature (K).
As a preferred scheme of the invention, the model established after fitting is based on root mean square error RMSE and regression coefficient R 2 Precision factor A f And a deviation factor B f And four parameters are used as quantitative methods for model evaluation.
As a preferred embodiment of the present invention, the formula is as follows:
A f ＝10 (Σ | lg (predicted value/measured value) |)/n (5)；
B f ＝10 (Σlg (predicted value/measured value))/n (6)；
n is the number of measured values, usually A f 、B f And regression coefficient (R) 2 ) Values of (A) were close to 1,RMSE, indicating better model fitness, analysis of variance, regression and kinetic calculations were plotted using Origin 2021 software, and ANOVA tests were performed on all experiments to determine significance with a confidence interval of 95%, the results being expressed as mean values and the significance level being P<0.05。
Compared with the prior art, the invention has the following beneficial effects:
the invention takes glucosamine hydrochloride tablets as a research object, adopts high performance liquid chromatography to measure the content of 5-HMF, establishes a kinetic model through the data fitting of the content of 5-HMF in thermal reaction systems with different temperatures, reveals the formed kinetic characteristics of the kinetic model, aims to effectively control and reduce the content of 5-HMF in the production and storage processes of the glucosamine hydrochloride tablets, and provides a reference for improving the safety and stability of related products.
Drawings
FIG. 1 is a chromatogram of a standard solution of the present invention.
FIG. 2 is a chromatogram of a test solution of the present invention.
FIG. 3 is a chromatogram of a blank solution of the invention.
FIG. 4 is the result of measuring the content of 5-HMF in different batches of samples according to the invention and a trend chart.
FIG. 5 is a graph showing the variation of 5-HMF content in a thermal reaction model at different temperatures and times in accordance with the present invention.
Detailed Description
In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further described with reference to the following specific embodiments, but the following embodiments are only the preferred embodiments of the invention, and not all embodiments. Other embodiments obtained by persons skilled in the art without making creative efforts based on the embodiments in the implementation belong to the protection scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Materials and reagents used in the present invention:
glucosamine hydrochloride tablets are commercially available and purchased on the Internet, 8 batches of samples (serial numbers S1 to S8) are all from the same manufacturer, and the specifications are as follows: 0.75 g/tablet; lot number (date of manufacture): 201257 (20.12.30), 210213 (21.02.18), 210503 (21.05.07), 210914 (21.09.30), 211102 (21.11.01), 211121 (21.11.12), 220137 (22.01.21) and 220326 (22.03.15).
5-hydroxymethylfurfural (batch No. 111626-201912, 99.2% pure, institute for the verification of food and drug, china), methanol (chromato-graphic, merck, usa); phosphoric acid (super pure, national chemical group, chemical reagents limited); water (ultrapure water, 18.2M Ω Millipore corporation, usa).
The apparatus and equipment used in the present invention:
agilent 1260 high performance liquid chromatograph (Agilent corporation, usa); XSE205DU electronic balance [ Mettler-Torledo instruments (Shanghai) Co., ltd ], DHG-9071A electric heating constant temperature drying oven (Shanghai sperm macro experiment equipment Co., ltd.).
Chromatographic conditions are as follows: and (3) chromatographic column: shim-pack GIST C 18 -AQ (4.6 mm. Times.150mm, 3 μm); mobile phase: 0.1% phosphoric acid solution-methanol (volume ratio, 90; flow rate: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 284nm; sample introduction amount: 20 μ L.
Example 1
The embodiment provides a method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets, which comprises the following steps:
(1) Preparation of Standard solutions
Precisely weighing 7.18mg of the 5-HMF standard substance in a 50mL volumetric flask, adding pure water to dissolve and fix the volume to prepare a standard stock solution with the mass concentration of 0.142mg/mL, and storing at 4 ℃. Precisely sucking a proper amount of stock solution, and diluting with pure water to prepare standard working solution with mass concentration of 5.698 mug/mL.
(2) Drawing of standard curve
A proper amount of standard stock solution is precisely absorbed and diluted into a series of standard solutions containing 5-HMF 5.698 mu g/mL, 2.849 mu g/mL, 1.425 mu g/mL, 0.712 mu g/mL, 0.285 mu g/mL and 0.057 mu g/mL by pure water. Detecting with high performance liquid chromatograph, and recording peak area. And (3) taking the mass concentration (X, mu g/mL) of the 5-HMF as an abscissa and taking the corresponding peak area (Y) as an ordinate to draw a standard curve.
(3) Preparation of test solution
Taking 10 glucosamine hydrochloride tablets, removing coatings, grinding, precisely weighing a proper amount (about 500mg of glucosamine hydrochloride) and placing the proper amount into a 50mL measuring flask, adding about 40mL of pure water, carrying out ultrasonic treatment for 10min, fixing the volume to a scale with the pure water, shaking up, standing for 5min, taking supernatant, and filtering through a 0.22 mu m filter membrane to obtain a test solution.
(4) Determination of 5-HMF content in a sample
And (3) carrying out sample injection measurement according to chromatographic conditions, calculating the mass of the 5-HMF contained in the sample according to a standard curve, and converting the mass into the content of the labeled amount of the glucosamine hydrochloride.
Example 2
This example provides a kinetic experiment of 5-HMF formation:
(1) Thermal reaction system design
Taking 4 parts of the same batch of sample fine powder, placing the sample fine powder in a weighing bottle, spreading the sample fine powder in a flat way, covering and sealing the bottle, placing the bottle in a constant temperature box with the temperature of 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, placing the bottle for 7 days at each temperature, precisely weighing a proper amount of fine powder (about equivalent to 500mg of glucosamine hydrochloride) every 1 day, placing the bottle in a 50mL measuring bottle, adding about 40mL of pure water, carrying out ultrasonic treatment for 10min, fixing the volume to a scale by using the pure water, shaking the bottle uniformly, standing the bottle for 5min, taking supernatant, and filtering the supernatant through a 0.22 mu m filter membrane to obtain a thermal reaction sample solution, wherein the three parts are in parallel. And (3) carrying out sample injection measurement according to chromatographic conditions, calculating the mass of the 5-HMF in the thermal reaction sample according to a standard curve, and converting the mass into the content of the labeled amount of the glucosamine hydrochloride.
(2) Data processing
Analyzing the change rule of the 5-HMF content in the system along with time by using a zero-order kinetic model or a first-order kinetic model, fitting to obtain reaction rates at different temperatures, and calculating the apparent activation energy of the 5-HMF forming reaction by using an Arrhenius formula, wherein the formula is as follows:
zero order kinetic model: c t ＝C 0 +k 0 ·t (1)；
A first-order kinetic model: c t ＝C 0 exp(k 1 ·t) (2)；
In the formula: c t 5-HMF content (mg/kg) at time t; c 0 Is the initial content (mg/kg) of 5-HMF; t is heat treatment time (h); k is a radical of 0 And k 1 Respectively, rate constants.
Arrhenius formula k = k f exp(-Ea/RT) (3)；
In the formula, k f Is the rate factor (1/d); ea is the apparent activation energy/(kJ/mol); r is a gas constant (8.314J/(k.mol)); t is the thermodynamic temperature (K).
The model built after fitting is based on Root Mean Square Error (RMSE) and regression coefficient (R) 2 ) Precision factor (a) f ) And a bias factor (B) f ) Four parameters were used as a quantitative methodAnd (5) evaluating a line model. The formula is as follows:
A f ＝10 (Σ | lg (predicted value/measured value) |)/n (5)；
B f ＝10 (Σlg (predicted value/measured value))/n (6)；
n is the number of measured values, usually A f 、B f And regression coefficient (R) 2 ) Values of (A) close to 1,RMSE are lower, indicating better model fit. Analysis of variance (ANOVA), regression and kinetic calculations were plotted using Origin 2021 software, and ANOVA tests were performed on all experiments to determine significance with a confidence interval of 95%, the results being expressed as the mean (standard deviation (SD)) and the significance level being P<0.05。
Example 3
This example provides a methodological validation experiment
(1) Specialization inspection
The method comprises the steps of weighing a proper amount of 5-HMF standard solution, glucosamine hydrochloride tablet sample solution and blank sample solution (weighing a proper amount of 5-HMF negative sample, operating according to a preparation method of a test solution, injecting the 5-HMF negative sample into a liquid chromatograph, and under an optimized chromatographic condition, well separating 5-HMF from adjacent peaks in the glucosamine hydrochloride tablet sample, wherein no interference peak exists in the blank sample solution at the same retention time as 5-HMF, which shows that the method has good specificity, and the chromatogram of the standard solution, the sample solution and the blank sample solution is shown in figure 1, and figure 2 and figure 3.
(2) Linear range, quantitative limit and detection limit
And (3) carrying out sample injection determination on the series of standard solutions under the optimized chromatographic condition, and carrying out linear regression analysis by taking the mass concentration of the 5-HMF as a horizontal ordinate (X) and the peak area as a vertical ordinate (Y), wherein the result shows that the linear relation of the response value of the 5-HMF in the mass concentration range of 0.057-5.698 mu g/mL is good. The regression equation is: y =151.69X-1.389, linear correlation coefficient r 2 =1.000. Taking the solution with the lowest mass concentration in the linear range for further dilution,the signal-to-noise ratio S/N =10 was used as a quantitative limiting solution, and the signal-to-noise ratio S/N =3 was used as a detection limiting solution. The measured limit of quantitation is 17.09ng/mL, and the detection limit is 5.70ng/mL, which indicates that the method has higher sensitivity.
(3) Precision survey
Sample introduction precision test
And respectively taking a standard solution and a sample solution for continuous sampling for 6 needles, measuring the peak area of 5-HMF, and calculating Relative Standard Deviations (RSDs) of the peak areas of the standard solution and the sample solution to be 0.03 percent and 0.21 percent respectively, thereby indicating that the precision of the instrument is good.
Repeatability test
6 parts of the same sample fine powder are taken to prepare a sample solution, sample injection measurement is carried out according to chromatographic conditions, and the average content of 5-HMF of 6 samples is calculated to be 30.44 mu g/g, and the RSDs is 0.51 percent, which shows that the method has good repeatability.
(4) Stability survey
And (3) taking the same sample solution, respectively injecting samples for 0 hour, 4 hours, 8 hours, 12 hours, 18 hours and 24 hours, determining the peak area of 5-HMF, wherein the peak area RSDs of 6 time points is 0.26 percent, which indicates that the sample solution is stable within 24 hours.
(5) Recovery rate experiment
Precisely weighing 9 parts of sample fine powder with known content, respectively placing the sample fine powder into 50mL measuring bottles, precisely adding three levels of standard solutions of a low sample, a middle sample and a high sample to prepare a sample standard solution, carrying out sample injection measurement according to chromatographic conditions, and calculating the standard recovery rate, wherein the results are shown in Table 1. The average standard recovery rates of 5-HMF are respectively 99.73%,99.63% and 99.38%, and the average standard recovery rates of RSDs are respectively 1.30%,0.56% and 0.30%, which shows that the method is accurate and reliable.
TABLE 1 recovery test results
(6) Sample assay
A proper amount of sample fine powder is precisely weighed, three samples are parallelly weighed, the 5-HMF content of 8 batches of samples is measured according to the method, and the measurement result and the content change trend chart are shown in figure 4. The content of 5-HMF in samples of different batches is greatly different, and from analysis of the change trend, the content of 5-HMF in the samples has a large positive correlation with the storage time of the product, and the 5-HMF in the glucosamine hydrochloride tablets at room temperature shows an obvious dynamic change trend.
Example 4
This example provides an assay of the kinetics of 5-HMF formation
From the appearance of the sample, the color of the sample under each temperature condition deepens along with the prolonging of the standing time, the color is darker as the temperature is higher, the color is gradually changed from initial white to light brown, the browning phenomenon is obvious, and the phenomenon characteristic of the Maillard reaction is met. The 5-HMF content of the samples of the thermal reaction system at different temperatures and times is shown in Table 2. The reaction kinetics curves were fitted using Origin Pro 2021 as shown in figure 5.
Table 2. The 5-HMF content in the thermal reaction system samples at different temperatures and times (μ g/g,
n＝3)
as can be seen from Table 2 and FIG. 5, the content of 5-HMF in the system gradually increased with the increase of the heating temperature, and the formation amount of 5-HMF was positively correlated with the heating time at the same temperature. Comparing the results of the measurements on day 1 and the initial day (day 0) at different temperatures, it was found that the 5-HMF content rapidly increased on day 1 at a temperature higher than 70 ℃ and 80 ℃ by more than 10 times as compared with the increase on day 0. The increase is less than 5 times at 50 ℃ and 60 ℃, and the increase is uniform at different time points. The obvious rapid induction period exists when the heating temperature exceeds 60 ℃, and the finding has guiding significance on the selection and control of the drying temperature of the drug granules in the production flow of the glucosamine hydrochloride tablet adopting the wet granulation process.
The kinetic analysis of 5-HMF formation in glucosamine hydrochloride tablets is shown in Table 3. As can be seen from Table 3, the formation amount of 5-HMF and the heating time are in linear relationship under the conditions of 50, 60, 70 and 80 ℃, which accords with the zero order kinetic model, and in the equation obtained by linear fitting, the fitting curve R is in 2 Are all made of>0.96, model evaluation results show that different reaction temperature models have high data fitting degree and good accuracy, and can well describe the rule of the 5-HMF content changing along with time. The temperature dependence of 5-HMF was estimated using the Arrhenius equation and Ea for 5-HMF was calculated to be 16.00kJ/mol, indicating that glucosamine hydrochloride tablets can accelerate the reaction rate at higher temperatures and the kinetic rate constant increases with increasing temperature. The results of the kinetic analysis of 5-HMF formation show that temperature and time are important factors affecting the formation of 5-HMF in glucosamine hydrochloride tablets, and that the higher the temperature, the higher the formation rate, and especially the sharper the initial formation rate.
TABLE 3.5 kinetic analysis of HMF formation
The invention establishes an HPLC (high performance liquid chromatography) content determination method for a Maillard reaction product 5-HMF in a glucosamine hydrochloride tablet, and after the method is adopted to determine the 5-HMF in different batches of samples, the obvious positive correlation between the storage time and the 5-HMF content is found. And (3) fitting the measured data of the 5-HMF in the thermal reaction system samples at different heating temperatures and different time to form a kinetic model, showing that the change rule of the content of the 5-HMF at different temperatures along with time follows a zero-order kinetic model, and the kinetic reaction rate constant is increased along with the rise of the temperature, which shows that the reaction temperature and the reaction time have important influence on the formation of the 5-HMF in the glucosamine hydrochloride tablet.
5-HMF is a potential health hazard to users as an endogenous toxic degradation product produced during the production and storage of glucosamine hydrochloride tablets. The glucosamine hydrochloride tablet can be continuously administered for 6 weeks or more in the treatment and prevention of arthritis. Referring to the requirements related to the ICH guidelines, the Threshold of Toxicological Concern (TTC) for class 2 genotoxic impurities, calculated as 20, is 13.33. Mu.g/g 5-HMF acceptability in glucosamine hydrochloride tablets. As can be seen from Table 2, 4 of the 8 lots exceeded this limit, and the overrun samples were earlier in the batch number, indicating that although the 5-HMF content of the product was lower when shipped from the factory, 5-HMF in the glucosamine hydrochloride tablets tended to increase dynamically as the storage time was extended at room temperature. At the same time, kinetic studies further demonstrate that temperature and time have a large influence on the formation of 5-HMF. Therefore, in order to effectively control the formation of 5-HMF in the glucosamine hydrochloride tablet and reduce the content of the 5-HMF, a production enterprise needs to further reduce the drying temperature and time of wet granulation in the production process, and a reasonable storage temperature needs to be regulated for the products on the market.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any equivalent changes, modifications and evolutions of the above embodiments according to the essential technology of the present invention are still within the scope of the technical solution of the present invention.
Claims (10)
1. A detection method of 5-hydroxymethylfurfural in glucosamine hydrochloride tablets is characterized by comprising the following steps:
1) Preparation of standard solution: weighing a 5-HMF standard substance in a volumetric flask, adding pure water to a constant volume to prepare a standard stock solution with the mass concentration of 0.142mg/mL, and storing at 4 ℃; weighing the standard stock solution, and diluting with pure water to obtain standard working solution with mass concentration of 5.698 mu g/mL for later use;
2) Drawing a standard curve: weighing the standard working solution obtained in the step 1), diluting the standard working solution into a series of standard solutions with mass concentrations of 5.698 mu g/mL, 2.849 mu g/mL, 1.425 mu g/mL, 0.712 mu g/mL, 0.285 mu g/mL and 0.057 mu g/mL by using pure water, detecting by using a high performance liquid chromatography, recording peak areas, and drawing a standard curve by using the mass concentration of 5-HMF as a horizontal coordinate and the corresponding peak areas as a vertical coordinate;
3) Preparing a test solution: taking 10 glucosamine hydrochloride tablets, removing coatings, grinding, precisely weighing a proper amount, placing in a volumetric flask, adding pure water, performing ultrasonic treatment, fixing the volume to a scale with the pure water, shaking up, standing, taking supernatant, and filtering with a filter membrane to obtain a test solution;
4) And (3) sample determination: and (4) detecting the test solution obtained in the step 3) in a high performance liquid chromatograph, calculating the mass of the 5-HMF contained in the sample according to a standard curve, and converting into the content of the labeled amount of the glucosamine hydrochloride.
2. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets according to claim 1, wherein the conditions of the high performance liquid chromatography are as follows: and (3) chromatographic column: shim-pack GIST C 18 AQ4.6 mm. Times.150mm, 3 μm; mobile phase: 0.1% phosphoric acid solution-methanol, volume ratio 90; or 0.1% formic acid solution-methanol, in a volume ratio of 90:10; flow rate: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 284nm; sample injection amount: 20 μ L.
3. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets as claimed in claim 2, wherein the mobile phase is 0.1% phosphoric acid solution-methanol in a volume ratio of 90.
4. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets as claimed in claim 1, wherein in the step 3), the volumetric flask is 50mL, the added pure water is 40mL, and the ultrasonic treatment time is 10min.
5. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets as claimed in claim 1, wherein the pore size of the filter membrane in the step 3) is 0.22 μm.
6. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets according to claim 1, characterized by further comprising the step 5) of designing a thermal reaction system: taking 4 parts of the sample fine powder obtained in the step 3), placing the sample fine powder in a weighing bottle, flatly paving the sample fine powder, covering and sealing the sample fine powder, placing the sample fine powder in a constant temperature box with the temperature of 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, placing the sample fine powder in the constant temperature box for 7 days under each temperature condition, precisely weighing an appropriate amount of fine powder in three parallel parts every 1 day, preparing a sample solution according to the step 3), carrying out sample injection measurement according to the step 4), calculating the average content of 5-HMF, and finally carrying out data processing.
7. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets as claimed in claim 6, wherein the data processing comprises analyzing by a zero-order kinetic model or a first-order kinetic model, fitting to obtain reaction rates at different temperatures, and calculating the apparent activation energy of the 5-HMF formation reaction by using an Arrhenius formula.
8. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets as claimed in claim 7, wherein the zero order kinetic model is as follows: c t ＝C 0 +k 0 ·t (1)；
A first-order kinetic model: c t ＝C 0 exp(k 1 ·t) (2)；
In the formula: c t 5-HMF content (mg/kg) at time t; c 0 Is the initial content of 5-HMF (mg/kg); t is heat treatment time (h); k is a radical of 0 And k 1 Respectively, rate constants;
arrhenius formula k = k f exp(-Ea/RT) (3)；
In the formula, k f Is the rate factor (1/d); ea is tableApparent activation energy/(kJ/mol); r is a gas constant (8.314J/(k. Mol)); t is the thermodynamic temperature (K).
9. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets as claimed in claim 6, wherein the model established after fitting is based on Root Mean Square Error (RMSE) and regression coefficient R 2 Precision factor A f And a deviation factor B f And the four parameters are used as a quantitative method for model evaluation.
10. The method for detecting 5-hydroxymethylfurfural in glucosamine hydrochloride tablets according to claim 9, characterized by comprising the following formula:
A f ＝10 (Σ | lg (predicted value/measured value) |)/n (5)；
B f ＝10 (Σlg (predicted value/measured value))/n (6)；
n is the number of measured values, usually A f 、B f And regression coefficient (R) 2 ) Values of (A) were close to 1,RMSE, indicating better model fitness, analysis of variance, regression and kinetic calculations were plotted using Origin 2021 software, and ANOVA tests were performed on all experiments to determine significance with a confidence interval of 95%, the results being expressed as mean values and the significance level being P<0.05。
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