CN115902036A - Method for determining urea content in allantoin aluminum - Google Patents
Method for determining urea content in allantoin aluminum Download PDFInfo
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Abstract
The invention relates to a method for measuring urea content in allantoin aluminum, which comprises the following steps: step 1: taking 1000mg of an allantoin aluminum raw material medicine, precisely weighing, placing in a 100mL measuring flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 30min, placing at room temperature, fixing the volume to a scale, and shaking up; step 2: accurately weighing 10mg of allantoin reference substance, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing to room temperature, metering to desired volume, and shaking to obtain allantoin reference substance solution; and step 3: taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, adding methanol for ultrasonic dissolution, cooling and fixing the volume to obtain a urea reference substance solution (0.4 mg/mL); and 4, step 4: accurately sucking 10 μ L of each of the reference solution and the test solution, injecting into high performance liquid chromatograph, and recording chromatogram; and 5: and calculating the content of urea in the test sample according to the chromatogram.
Description
Technical Field
The invention belongs to the field of analytical chemistry methods, and particularly relates to a method for measuring the content of urea which is a main relevant impurity by using an HPLC-UV method. Because the urea content in the raw material medicine allantoin aluminum is lower than 1%, the content of related substance urea is determined by utilizing the solubility difference between urea and allantoin aluminum, the allantoin aluminum is difficult to dissolve in an organic solvent, and the urea is easy to dissolve in the organic solvent in an organic solvent enrichment mode.
Technical Field
Allantoin aluminum (Aldioxa), whose chemical name is dihydroxy- (5-oxo-4-ureido-2-imidazolinyl) oxyaluminum, is white crystalline powder, odorless, tasteless, practically insoluble in water, ethanol, diethyl ether, slightly soluble in dilute hydrochloric acid or dilute nitric acid, and often cloudy due to partial hydrolysis. The raw material medicine allantoin aluminum is a reaction product obtained by melting, condensing and hydrolyzing allantoin and aluminum isopropoxide, and is mainly used for treating gastric and duodenal ulcers clinically.
The content determination item of the allantoin aluminum bulk drug by the existing drug standard loading titration method has no related substance examination and research. The product contains allantoin (C) calculated according to dry product specified in current pharmacopoeia standard 4 H 6 N 4 O 3 ) 65.3 to 74.3 percent of aluminum (Al) and 11.1 to 13.0 percent of aluminum (Al).
The experiment adopts an HPLC-UV method according to the relevant requirements of the four guiding principles of Chinese pharmacopoeia 2015 edition, the standardization process technology guiding principle established by chemical medicine quality standards, the chemical medicine impurity research technology guiding principle and the like, establishes a method for measuring the content of the allantoin aluminum raw material medicine and checking related substances, carries out methodology investigation, perfects and improves the quality standard specified by the pharmacopoeia, and provides a theoretical basis for quality control and production process improvement of the allantoin aluminum raw material medicine.
Disclosure of Invention
The invention aims to measure the content of urea which is a main impurity by an HPLC-UV method. Because the urea content in the raw material medicine allantoin aluminum is lower than 1%, the content of related substance urea is determined by utilizing the solubility difference between urea and allantoin aluminum, the allantoin aluminum is difficult to dissolve in an organic solvent, and the urea is easy to dissolve in the organic solvent in an organic solvent enrichment mode.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:
a method for measuring the urea content in allantoin aluminum comprises the following steps:
step 1: taking 1000mg of an allantoin aluminum raw material medicine, precisely weighing, placing in a 100mL measuring flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 30min, placing at room temperature, fixing the volume to a scale, and shaking up;
and 2, step: taking 10mg of allantoin reference substance, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing at room temperature, metering to desired volume, and shaking to obtain allantoin reference substance solution;
and step 3: taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, adding methanol for ultrasonic dissolution, cooling and fixing the volume to obtain a urea reference substance solution (0.4 mg/mL);
and 4, step 4: precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and recording chromatogram;
and 5: calculating the content of allantoin and urea in the test sample according to the chromatogram;
the chromatographic conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: TSK gel Amide-80 (250X 4.6mm,5 μm); mobile phase: acetonitrile-water (90; detection wavelength: 210nm; flow rate: 0.8mL/min; column temperature: 30 ℃; sample introduction amount: 10 μ L.
The determination method is obtained by screening, and the screening process is as follows:
specificity test
The blank solvent (water) and 10. Mu.L of each of the reference and test solutions were measured precisely and injected into a liquid chromatograph, and the results are shown in FIG. 1.
The result shows that the blank solvent has no interference, the retention time of the main peak of the test solution is consistent with that of the reference solution, and the specificity of the method is strong.
System applicability
Respectively and precisely weighing an appropriate amount of allantoin reference substance and urea reference substance, adding water to dissolve and dilute to obtain a mixed solution containing about 80 μ g of allantoin and 50 μ g of urea per 1mL as a system applicability solution. In addition, a proper amount of allantoin reference substance and urea reference substance are precisely weighed, and water is respectively added to prepare solutions with the concentration of about 100 mu g/mL and 50 mu g/mL to serve as positioning solutions. Water and the above solutions were each measured precisely at 10. Mu.L, and the solutions were injected into a liquid chromatograph, and chromatograms were recorded, and the results are shown in Table 1 and FIG. 2.
TABLE 1 System suitability test results
The results show that: the order of the appearance of the peaks is urea and allantoin, the separation degree is 8.46, the theoretical plate number of the main peak is 6934, and the system has good applicability.
Linear relation and detection limit investigation
Accurately weighing allantoin reference substance 10.03mg, placing in 100mL measuring flask, adding water to dissolve and dilute to scale, and shaking to obtain reference substance stock solution. Precisely sucking the stock solutions of 0.5mL, 1.0mL, 2.0mL, 4.0mL, 6.0mL and 8.0mL into a 10mL measuring flask respectively, adding water to dilute to a scale, and shaking up; precisely sucking 0.5ml and 1ml to 2ml of linear solution of 5 mu g/ml into a volumetric flask, adding water to dilute to a scale, and shaking up; accurately sucking 1ml of linear solution with the concentration of 1.25 mu g/ml and 2.5 mu g/ml to 2ml of volumetric flasks respectively, adding water to dilute the linear solution to a scale mark, and shaking up; precisely sucking 1.25 mu g/ml linear solution 1ml to 2ml volumetric flask, adding water to dilute to the scale, and shaking up.
Precisely measuring 10 μ L of each of the solution and the stock solution, injecting into a liquid chromatograph, recording chromatogram, performing linear regression with reference concentration as abscissa and peak area as ordinate, and calculating regression equation and correlation coefficient, the result is shown in Table 2 and FIG. 3.
TABLE 2
The results show that: allantoin is in the range of 0.6269 mu g/mL-100.3 mu g/mL, the peak area is in direct proportion to the concentration, the linear regression equation is y =12.36x +1.2869, and the correlation coefficient R 2 Is 1, the linear relationship is good.
The allantoin detection limits and quantitation limits are shown in Table 3, FIGS. 4-5.
TABLE 3 content determination detection and quantitation limits
The results show that: the method has high sensitivity.
Sample introduction precision test
Preparing a reference solution, precisely measuring 10 mu L of the reference solution as a sample injection precision solution, injecting the reference solution into a liquid chromatograph, repeatedly injecting the sample for 6 times, recording a chromatogram, calculating RSD (red fluorescence) of the peak area and retention time of a main peak of the sample for 6 times, and inspecting the sample injection precision, wherein the result is shown in a table 4.
TABLE 4 sample introduction precision test results
The results show that: and continuous 6 times of sample injection, wherein the retention time RSD of the main peak is 0.20 percent, the peak area RSD is 0.52 percent, the peak areas RSD are less than 2.0 percent, and the sample injection precision of the instrument is better.
Stability of solution
(1) Stability of test solution
Preparing a test solution, standing at room temperature for 0h, 2h, 4h, 6h, 8h, 12h and 24h, precisely measuring 10 mu L of the test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, and observing the peak area change condition of a main peak, wherein the result is shown in Table 5.
TABLE 5 assay sample solution stability results
The results show that: the sample solution is placed at room temperature for 24h, the sample peak area has no obvious change, the RSD is 1.21 percent (less than 2.0 percent), and the sample solution is stable at room temperature for at least 24 h.
(2) Stability of control solutions
The reference solution is placed at room temperature for 0h, 2h, 4h, 6h, 8h, 12h and 24h, 10 mu L of the reference solution is precisely measured and injected into a liquid chromatograph, a chromatogram is recorded, the result is shown in Table 6, and the peak area change condition of the main peak is examined.
TABLE 6 control solution stability results
The results show that: the control solution is placed at room temperature for 24h, the peak area of the main peak has no obvious change, the RSD is 1.20 percent (less than 2.0 percent), and the control solution is stable at room temperature for at least 24 h.
Precision test
(1) Repeatability test
6 parts of test solution; precisely measuring 10 mu L of the solution, injecting the solution into a liquid chromatograph, and recording a chromatogram; taking another appropriate amount of allantoin reference substance, precisely weighing, preparing into solution containing 100 μ g of allantoin per 1mL with water, using as reference substance solution, measuring by the same method, and calculating the content of allantoin as hydrolysate in the sample by peak area according to external standard method, the result is shown in Table 7.
TABLE 7 repeatability results of the assay
The results show that: the average content is 62.66%, the RSD is 0.35% (n = 6), and the method has good repeatability.
(2) Intermediate precision
The content was measured by another analyst according to the method under "repeatability test" in different instruments at different times, and the results are shown in tables 8 to 9.
TABLE 8 measurement of intermediate precision results
TABLE 9 summary of precision test data
As can be seen from the above table, the average content of the product is 63.02% (n = 12) and RSD is 0.76%, which are measured by different experimenters and different instruments at different times in the same laboratory. The method has good precision.
In conclusion, the content determination method has the advantages of good system applicability, linearity, good sample injection precision, strong specificity, good precision and good durability, and is suitable for content determination of the allantoin aluminum bulk drug.
HPLC analysis of Urea in Alallantoin
Taking 1000mg of allantoin aluminum raw material medicine, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing to room temperature, fixing the volume to the scale, and shaking uniformly. Precisely weighing 10mg of allantoin reference substance, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing at room temperature, metering to desired volume, and shaking to obtain allantoin reference substance solution. Taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, adding methanol for ultrasonic dissolution, cooling and fixing the volume to obtain a urea reference substance solution (0.4 mg/mL). Respectively and precisely absorbing 10 μ L of each of the reference solution and the test solution, injecting into a liquid chromatograph, measuring, recording chromatogram, and comparing retention time and ultraviolet absorption characteristics of impurity peak and urea reference peak in the allantoin aluminum raw material drug, the results are shown in FIGS. 6-10.
As a result: the retention time of HPLC detection is consistent with that of a urea reference substance, and the ultraviolet absorption characteristic is consistent, so that the impurity is further determined to be urea.
Chromatographic conditions and System suitability test
And (3) chromatographic column: TSK gel Amide-80 (250X 4.6mm,5 μm); mobile phase: acetonitrile-water (90; detection wavelength: 210nm; flow rate: 0.8mL/min; column temperature: 30 ℃; sample introduction amount: 10 μ L. The content of related substances is calculated by urea content, and the theoretical plate number is not less than 5000 calculated by allantoin peak.
Investigation of enrichment of urea, a relevant substance, in different organic solvents
Taking three parts of raw material medicine allantoin Aluminum (ADX), each part is about 1000mg, precisely weighing, placing in a 100mL measuring flask, respectively adding appropriate amount of acetonitrile, methanol and anhydrous ethanol, performing ultrasonic treatment for 30min, standing to room temperature, fixing volume to scale, and shaking up. Taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, respectively adding acetonitrile, methanol and ethanol, ultrasonically dissolving, cooling, and fixing volume to obtain reference substance solution (0.4 mg/mL). Precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, recording chromatogram, and calculating the content of related substance urea, the results are shown in Table 10.
TABLE 10 determination of urea content in various organic solvent-enriched substances
The results show that: and (3) enriching related substances of the Adx bulk drug by respectively adopting acetonitrile, methanol and absolute ethyl alcohol, and measuring the urea content: methanol, acetonitrile and absolute ethyl alcohol, so that the methanol solvent is adopted to investigate related substances of the Adx bulk drug.
Preparation of test solution
Taking about 1000mg of allantoin aluminum, precisely weighing, placing in a 100mL measuring flask, adding methanol for ultrasonic dissolution, placing to room temperature, adding methanol for dilution, diluting to constant volume to scale, and shaking up.
Preparation of control solutions
Taking 10mg of urea reference substance, precisely weighing, placing in a 10mL measuring flask, adding methanol for ultrasonic dissolution, placing to room temperature, adding methanol for dilution to scale, and shaking up to obtain impurity stock solution. Precisely absorbing 5ml to 100ml of urea stock solution in a volumetric flask, adding methanol to dilute and fix the volume to a scale, and shaking up. (0.05 mg/ml)
Assay method
Precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. If a urea peak exists in a chromatogram of the test solution, the area of the peak is calculated according to an external standard method and is not more than 2.0 percent.
System suitability test
Respectively precisely weighing appropriate amount of allantoin reference substance and urea reference substance, adding methanol to dissolve and dilute to obtain mixed solution containing about 80 μ g of allantoin (because allantoin is difficult to dissolve in organic solvent, the allantoin reference substance solution is suspension dilution) and 250 μ g of urea per 1mL as system applicability solution. Methanol was added to prepare solutions having concentrations of about 100. Mu.g/mL and 250. Mu.g/mL, respectively, as the positioning solutions. Precisely measuring methanol and the above solutions respectively by 10 μ L, injecting into liquid chromatograph, and recording chromatogram. The results are shown in Table 11 and FIG. 11.
TABLE 11 results of the System suitability test
The results show that: the peak emergence sequence is urea and allantoin in turn, the separation degree of the urea and the main peak is 10.81, the theoretical plate number of the main peak is 9914, and the system has good applicability.
Specificity test
(1) Acid breakdown test
Precisely weighing about 10mg of the product, placing the product in a 100mL measuring flask, adding 5mL of 1mol/L hydrochloric acid, heating at 60 ℃ for 30min, adding 1mol/L equivalent sodium hydroxide for neutralization, adding about 90mL of water, performing ultrasonic treatment for 30min, fixing the volume to scale, and shaking up to obtain an acid destruction solution.
(2) Alkali breakdown test
Taking about 10mg of the product, precisely weighing, placing in a 100mL measuring flask, adding 5mL of 0.1mol/L sodium hydroxide, adding 0.1mol/L hydrochloric acid with the same amount for neutralization, adding about 90mL of water, performing ultrasonic treatment for 30min, fixing the volume to the scale, and shaking up to obtain an alkali destruction solution.
(3) Oxidative destruction test
Precisely weighing about 10mg of the product, placing the product in a 100mL measuring flask, adding 5mL of 30% hydrogen peroxide, heating at 60 ℃ for 30min, adding about 90mL of water, performing ultrasonic treatment for 30min, fixing the volume to a scale, and shaking up to obtain an oxidative heating destruction solution.
(4) High temperature solid failure test
Placing 1g of the product in a beaker, placing the beaker in an oven at 105 ℃ for 1h, taking out, placing the beaker to room temperature, taking about 10mg, precisely weighing, placing the beaker in a 100mL measuring flask, adding about 90mL of water, carrying out ultrasonic treatment for 30min, fixing the volume to a scale, and shaking up to obtain a high-temperature destruction solution.
(5) Non-destructive solution
Taking about 10mg of the product, precisely weighing, placing in a 100mL measuring flask, adding about 90mL of water, performing ultrasonic treatment for 30min, metering to a certain volume, and shaking up to obtain an undamaged solution.
Precisely absorbing 10 μ L of blank solvent and the above solution, respectively, injecting into a liquid chromatograph, and recording chromatogram. The results are shown in Table 12 and FIG. 12.
TABLE 12 results of the specificity test
The results show that: the blank solvent does not interfere the detection of related substances of the product, the product is completely separated from the main peak under the conditions, the separation degree between impurities is better, and the purity factor of the main peak is larger than the threshold value under each test condition. According to A General assembly C (wherein A) General (1) Representing the total peak area of the test solution, C representing the concentration of the test solution) to calculate the response value of unit concentration, taking the response value of the undamaged solution as a base number (the material recovery is 100%), calculating the material recovery rate of the damaged solution, and obtaining the result of acid and alkali oxidation heating damage and basic balance under the condition of high-temperature solid damage, wherein the specificity of the method is strong; the crude drug is unstable and easy to be degraded under alkaline conditionIn the technological process, the pH value of the reaction system should be strictly controlled.
Precision of sample introduction
A proper amount of urea reference substance is precisely weighed and diluted by adding methanol to prepare a solution containing about 100 mu g/mL of urea. Respectively measuring 10 μ L precisely, injecting into liquid chromatograph, and continuously sampling for 6 times, the results are shown in Table 13.
TABLE 13 urea sample introduction precision test
The results show that: the peak area RSD of related substance urea is less than 2.0%, and the sample injection precision of the instrument is good.
Detection limit and quantification limit
Weighing 10mg of urea reference substance, precisely weighing, adding into a 25ml volumetric flask, adding methanol, ultrasonically dissolving, fixing volume, and shaking up to obtain urea reference substance stock solution (400 μ g/ml); accurately sucking 5ml of stock solution into a 10ml volumetric flask, adding methanol to a constant volume, and shaking up (200 mug/ml); diluting with the same method to obtain control solutions (100 μ g/ml, 50 μ g/ml); and sequentially sucking 0.5, 1, 2, 5ml to 10ml of reference substance solution of 50 mu g/ml into volumetric flasks, adding methanol to a constant volume to scale, and shaking up to obtain the product (2.5, 5, 10, 25 mu g/ml). Determining a detection limit by using the corresponding concentration or the quantity injected into the instrument when the signal-to-noise ratio (S/N) is 3, wherein the detection limit represents the lowest concentration or quantity of the detected object which can be reliably detected; and determining a limit of quantitation by using the corresponding concentration or the amount injected into the instrument when the S/N is 10, wherein the limit of quantitation represents the lowest concentration or amount of the measured object which can be quantitatively measured. 10. Mu.L of the sample was precisely measured, and the sample was injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in Table 14 and FIGS. 13 to 14.
TABLE 14 detection and quantitation limits for the relevant substances Urea
The results show that: the method has high sensitivity.
Investigation of linear relationships
Weighing 10mg of urea reference substance, precisely weighing, adding into a 25ml volumetric flask, adding methanol, ultrasonically dissolving, fixing the volume, and shaking up to obtain a urea reference substance stock solution (400 mu g/ml); accurately sucking 5ml of stock solution into a 10ml volumetric flask, adding methanol to a constant volume, and shaking up (200 mug/ml); diluting with the same method to obtain control linear solution (100, 50, 25 μ g/ml); precisely measuring 10 μ L, injecting into a liquid chromatograph, recording chromatogram, and performing linear regression with concentration as abscissa and peak area as ordinate. The results are shown in Table 15 and FIG. 15.
TABLE 15 Urea Linear test results
The results show that: the urea is in the range of 25.43-406.80 mug/mL, the peak area is in direct proportion to the concentration, the linear regression equation is y =1553.4x-12.408, the correlation coefficient r is 0.9972, and the linear relation is good.
Precision test
(1) Repeatability test
And precisely measuring 1 part of the reference solution and 6 parts of the test solution, respectively measuring 10 mu L of each of the reference solution and the test solution, injecting into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of urea impurities. The results are shown in Table 16.
TABLE 16 results of the repeatability tests (run by shenwei)
The results show that: the RSD of the urea impurity content in 6 parts of test solution is not more than 2.0% (n = 6), and the method has good repeatability.
(2) Intermediate precision
The other analyst carries out related substance detection on different instruments at different times according to the method under the item of 'repeatability test'. The results are shown in tables 17 to 18.
TABLE 17 results of intermediate precision tests (by lisiyuan)
TABLE 18 summary of precision test results
Note: the testing instrument used by shenwei was Agilent HPLC 1260 (DEAD 016640);
the testing instrument used by lisiyuan was Agilent HPLC 1100 (DE 60400285)
The results show that: through detection of different persons in different instruments at different times, the RSD of the urea impurity content in 12 parts of test sample solution is not more than 2.0% (n = 12), and the method has good precision.
Stability of solution
(1) Stability of urea control solution to standing
Accurately weighing a 20 mg-10 mL volumetric flask of a urea reference substance, adding methanol, ultrasonically dissolving, diluting to a constant volume to a scale, and shaking up to obtain a urea reference substance stock solution; precisely absorbing a proper amount of stock solution, and diluting with methanol to obtain urea reference substance solution containing 0.25mg of urea reference substance per 1 mL; precisely measuring urea reference substance solutions (standing at room temperature for 0h, 2h, 4h, 6h, 8h, and 12 h) each 10 μ L, injecting into liquid chromatograph, and recording chromatogram. The results are shown in Table 19.
TABLE 19 standing stability test of urea control solutions
The results show that: the peak area RSD of the main peak of the solution in the urea control bottle is 0.851 percent and less than 2.0 percent, the peak area of the impurity control sample at each time point has no obvious change, and the stability of the solution placed at room temperature for 12 hours is good.
(2) The allantoin control solution is stable on standing.
Because the allantoin is difficult to dissolve in the organic solvent, the allantoin reference substance solution prepared by the method is suspension, and the prepared system applicability solution is prepared by mixing the urea reference substance solution and the allantoin reference substance solution according to a proportion. Accurately weighing a 10mg to 100mL volumetric flask of an allantoin reference substance, adding methanol, performing ultrasonic treatment, fixing the volume to a scale, and shaking up to obtain a urea reference substance solution; precisely measuring allantoin reference substance solutions (standing at room temperature for 0h, 2h, 4h, 6h, 8h, and 12 h) each 10 μ L, injecting into liquid chromatograph, and recording chromatogram. The results are shown in Table 20.
TABLE 20 allantoin control solution standing stability test
The results show that: the RSD of the main peak area of the allantoin contrast bottle solution is 0.534 percent and is less than 2.0 percent, the main peak area is stable when the allantoin contrast bottle solution is placed at room temperature for 12 hours, no new impurities are generated, and the stability is good.
(3) System applicability solution placement stability
Accurately weighing appropriate amount of allantoin reference substance and urea reference substance, respectively, adding methanol to dissolve and dilute to obtain mixed solution containing about 80 μ g of allantoin and 250 μ g of urea per 1mL as system applicability solution. Precisely measuring system applicability solutions (standing at room temperature for 0h, 2h, 4h, 6h, 8h and 12 h) each 10 μ L, injecting into liquid chromatograph, and recording chromatogram. The results are shown in Table 21.
TABLE 21 suitability for System solution stability test
The results show that: the separation degree of the system applicability solution at each time point within 24 hours after the solution is placed at room temperature meets the requirement, and the proportion of each component has no obvious change.
Accuracy of
Accurately weighing appropriate amount of urea reference substance, adding methanol to dilute to obtain solution containing about 1mg urea per 1mL, and using as urea reference substance stock solution. Precisely sucking a proper amount of stock solution, adding methanol to dilute to obtain a solution containing 0.05mg of urea per 1mL, and using the solution as an impurity reference solution.
9 parts of test solution is prepared according to the method under the item 3.1.2, and urea reference stock solution is respectively added for sample adding and recovery rate investigation. Preparing test solution with three concentrations according to the addition amount of 80%, 100% and 120%, detecting each three parts according to related substance examination method, precisely measuring reference solution and each accuracy solution 10 μ L, injecting into liquid chromatograph, recording chromatogram, and calculating with peak area by external standard method. The results are shown in Table 22.
TABLE 22 measurement of Urea recovery
The results show that: the average recovery rate of the urea in the limit concentration range of 80-120% is 94.72%, and the RSD is 2.90%; the recovery rate is between 90% and 105%, the RSD is less than 5.0%, and the method has good accuracy.
And (4) conclusion: in conclusion, the system has good applicability; the specificity is strong; the sensitivity is higher; the peak area and the concentration of the relevant substance urea in a certain concentration range form a good linear relation; the precision is better; the recovery rate of the impurity sample is between 90 and 105 percent, and the accuracy is high; the durability is good, and the method can be used for checking related substances of allantoin aluminum bulk drugs.
Drawings
FIG. 1 HPLC chromatogram of content determination specificity test
FIG. 2 HPLC chromatogram for the suitability test of the content determination System
FIG. 3 allantoin Standard Curve
FIG. 4 HPLC chromatogram of detection limit of content measurement
FIG. 5 content determination quantitation limit HPLC profile
FIG. 6 Urea control HPLC chromatogram
FIG. 7 allantoin control HPLC chromatogram
FIG. 8 HPLC chromatogram of raw material medicine after allantoin aluminum impurity enrichment
FIG. 9 ultraviolet absorption spectrum of urea reference substance
FIG. 10 ultraviolet absorption spectrum of allantoin aluminum as raw material drug
FIG. 11 HPLC chromatogram for substance system applicability test
FIG. 12 HPLC chromatogram of alkali Destruction experiment
FIG. 13 HPLC chromatogram of detection limit of related substance urea
FIG. 14 related substance urea quantitative limit HPLC chromatogram
FIG. 15 Urea Standard Curve
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Example 1
1. A method for measuring the urea content in allantoin aluminum is characterized by comprising the following steps: step 1: taking 1000mg of an allantoin aluminum raw material medicine, precisely weighing, placing in a 100mL measuring flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 30min, placing at room temperature, fixing the volume to a scale, and shaking up;
step 2: taking 10mg of allantoin reference substance, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing at room temperature, metering to desired volume, and shaking to obtain allantoin reference substance solution;
and step 3: taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, adding methanol for ultrasonic dissolution, cooling and fixing the volume to obtain a urea reference substance solution (0.4 mg/mL);
and 4, step 4: precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and recording chromatogram;
and 5: calculating the content of allantoin and urea in the test sample according to the chromatogram;
wherein the chromatographic conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: TSK gel Amide-80 (250X 4.6mm,5 μm); mobile phase: acetonitrile-water (90; detection wavelength: 210nm; flow rate: 0.8mL/min; column temperature: 30 ℃; sample introduction amount: 10 μ L.
Claims (3)
1. A method for measuring the urea content in allantoin aluminum, which is characterized by comprising the following steps:
step 1: taking an allantoin aluminum raw material medicine, placing into a measuring flask, adding a proper amount of methanol, carrying out ultrasonic treatment, placing to room temperature, fixing the volume to a scale, and shaking up;
step 2: taking a urea reference substance, placing the urea reference substance in a measuring flask, adding methanol for ultrasonic dissolution, and cooling to a constant volume to obtain a urea reference substance solution;
and step 3: precisely absorbing 5-20 μ L of reference solution and sample solution respectively, injecting into high performance liquid chromatograph, and recording chromatogram;
and 4, step 4: calculating the content of urea in the test sample according to the chromatogram;
wherein the chromatographic conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: TSK gelAmide-80 (250X 4.6mm,5 μm); mobile phase: acetonitrile-water (90; detection wavelength: 210nm; flow rate: 0.8mL/min; column temperature: 30 ℃; sample introduction amount: 10 μ L.
2. The assay method according to claim 1, wherein the method comprises the steps of: step 1: taking 1000mg of allantoin aluminum raw material medicine, accurately weighing, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing to room temperature, metering to a certain volume, and shaking uniformly;
and 2, step: taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, adding methanol for ultrasonic dissolution, cooling and fixing the volume to obtain a urea reference substance solution (0.4 mg/mL);
and step 3: precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and recording chromatogram;
and 4, step 4: calculating the content of allantoin and urea in the test sample according to the chromatogram;
wherein the chromatographic conditions of the high performance liquid chromatograph are as follows:
and (3) chromatographic column: TSK gelAmide-80 (250X 4.6mm,5 μm); mobile phase: acetonitrile-water (90; detection wavelength: 210nm; flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10 μ L.
3. A method for determining the content of allantoin and urea in aluminum allantoinate, which is characterized by comprising the following steps:
step 1: taking 1000mg of an allantoin aluminum raw material medicine, precisely weighing, placing in a 100mL measuring flask, adding an appropriate amount of methanol, carrying out ultrasonic treatment for 30min, placing at room temperature, fixing the volume to a scale, and shaking up;
and 2, step: taking 10mg of allantoin reference substance, precisely weighing, placing in a 100mL measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment for 30min, standing at room temperature, metering to desired volume, and shaking to obtain allantoin reference substance solution;
and step 3: taking 10mg of urea reference substance, precisely weighing, placing in a 25mL measuring flask, adding methanol for ultrasonic dissolution, cooling and fixing the volume to obtain a urea reference substance solution (0.4 mg/mL);
and 4, step 4: precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and recording chromatogram;
and 5: calculating the content of allantoin and urea in the test sample according to the chromatogram;
wherein the chromatographic conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: TSK gel Amide-80 (250X 4.6mm,5 μm); mobile phase: acetonitrile-water (90; detection wavelength: 210nm; flow rate: 0.8mL/min; column temperature: 30 ℃; sample injection amount: 10 μ L.
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