CN115586286A - Method for measuring content of ammonium oxalate in fried stiff silkworm - Google Patents
Method for measuring content of ammonium oxalate in fried stiff silkworm Download PDFInfo
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Abstract
The invention provides a method for measuring the content of ammonium oxalate in fried stiff silkworm, which comprises the following steps: a) Dissolving a sample raw material by using a solvent to obtain a solution to be detected; b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured, wherein the chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution. The oxalic acid peak and the adjacent peak are well separated in the method, and other components in the liquid to be detected can be effectively eluted; the treatment of the liquid to be detected is simple and takes short time, the recovery rate of the liquid to be detected is high, and the cost of the diluent is low. Compared with the chromatographic separation conditions with stronger specificity in the existing literature, the method has the advantages of higher extraction efficiency of the liquid to be detected and simpler processing process.
Description
Technical Field
The invention relates to the technical field of detection methods, in particular to a method for determining the content of ammonium oxalate in fried stiff silkworm.
Background
Bombyx Batryticatus, name of Chinese medicine. A desiccated lethal larva of Bombyx mori Linnaeus, bombyx mori, a Bombyx mori, 4-5 instar, infected (or artificially inoculated) with Beauveria bassiana (Bals.) Vuillant. It is distributed in Jiangsu, zhejiang, sichuan and Guangdong provinces. Has the effects of calming endogenous wind, relieving spasm, dispelling pathogenic wind, relieving pain, eliminating phlegm and resolving masses. It is commonly used for treating crooked liver wind with phlegm, infantile convulsion, tetanus, wind stroke, headache due to wind-heat, conjunctival congestion, pharyngalgia, rubella pruritus, and parotitis.
The batryticated silkworm contains ammonium oxalate, the ammonium oxalate in the batryticated silkworm disclosed in the prior art is measured, but tailing exists, the separation degree of a main peak and an adjacent peak in a test sample is not good, and less polar components exist in the stir-fried batryticated silkworm and remain to the next needle; there may be interference in the quantification.
Therefore, it is necessary to develop a method for measuring the content of ammonium oxalate in the roasted stiff silkworm, which has better separation degree, stronger specificity, higher extraction efficiency of a sample and simpler treatment process.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a method for determining the content of ammonium oxalate in roasted Bombyx Batryticatus, which has better separation degree and stronger specificity.
The invention relates to a method for measuring the content of ammonium oxalate in stir-fried stiff silkworm, which comprises the following steps:
a) Dissolving a sample raw material by adopting a solvent to obtain a solution to be detected;
b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured, wherein the chromatographic conditions are as follows:
the chromatographic column is a C18 column; the mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution.
Preferably, the method further comprises preparing a control solution: dissolving an oxalic acid reference substance by adopting a solvent to obtain a reference substance solution;
measuring the reference substance solution by adopting high performance liquid chromatography to obtain a chromatogram of the reference substance; performing qualitative and quantitative analysis according to the concentration of the reference substance solution, the peak area of the reference substance in the chromatogram and the peak area of a component in the liquid to be detected, which corresponds to the reference substance, in the chromatogram;
and calculating according to the content of the oxalic acid to obtain the content of the ammonium oxalate.
Preferably, the gradient elution is specifically:
0-8min, phase A: 99% and phase B: 1 percent;
8-9min, phase A: 99-20%, phase B: 1% -80%;
9-15min, phase A: 20% -99%, phase B: 80% -1%;
15-30min, phase A: 99% and phase B: 1 percent.
Preferably, the chromatographic column is shim-pack GIST C18 XSelect GSS T3C 18, GL Sciences T3C 18, and the specification is 250 x 4.6mm 5 μm; the column temperature is 25 to 35 ℃.
Preferably, the flow rate of the mobile phase is 0.6 to 0.8mL/min.
Preferably, the detection wavelength is 214nm; the amount of sample was 10. Mu.L.
Preferably, the concentration of oxalic acid in the control solution is 0.042 mg/mL-0.833784 mg/mL.
Preferably, the linear equation for oxalic acid in the control solution is Y =8155010.6660x +60278.9960, the correlation coefficient is 0.998, the RSD of the response factor is 2.3%, and the absolute value of the Y-axis intercept accounts for 3.6% of the 100% response value.
Preferably, the solvent is 0.2% phosphoric acid aqueous solution; the extraction is ultrasonic extraction; the power of the ultrasonic wave is 500W, and the frequency is 40kHz; the ultrasonic treatment time is 30min.
Preferably, the ratio of the mass mg of the sample raw material to the volume mL of the solvent is 100.
Compared with the prior art, the invention provides a method for measuring the content of ammonium oxalate in fried stiff silkworm, which comprises the following steps: a) Dissolving a sample raw material by adopting a solvent to obtain a solution to be detected; b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured, wherein the chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution. The oxalic acid peak and the adjacent peak are well separated in the method, and other components in the liquid to be detected can be effectively eluted; the treatment of the liquid to be detected is simple and takes short time, the recovery rate of the liquid to be detected is high, and the cost of the diluent is low. Compared with the chromatographic separation conditions with stronger specificity in the existing literature, the method has the advantages of higher extraction efficiency of the liquid to be detected and simpler processing process.
Drawings
FIG. 1 is the HPLC detection spectrum of oxalic acid control in example 1;
FIG. 2 is HPLC detection spectrum of the test solution of Bombyx Batryticatus Preparata in example 1;
FIG. 3 is an HPLC detection profile of the sample solution in chromatographic condition 1 of example 3;
FIG. 4 is an HPLC detection profile of the sample solution of chromatographic condition 2 of example 3;
FIG. 5 is the chromatographic condition 3 of example 3 and HPLC detection profile of the test solution;
FIG. 6 is the HPLC detection profile of the sample solution of chromatographic condition 4 of example 3;
FIG. 7 is an HPLC detection profile of the sample solution of chromatographic condition 5 of example 3;
FIG. 8 is a HPLC detection profile of the test solution of chromatographic condition 6 of example 3;
FIG. 9 is the HPLC detection profile of the test solution of chromatographic condition 7 of example 3;
FIG. 10 is an HPLC detection profile of the sample solution under chromatographic conditions 8 of example 3;
FIG. 11 is a linear plot of oxalic acid over the 3.4 linear range of comparative example 1.
Detailed Description
The invention provides a method for measuring the content of ammonium oxalate in stir-fried stiff silkworm, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention relates to a method for measuring the content of ammonium oxalate in stir-fried stiff silkworm, which comprises the following steps:
a) Dissolving a sample raw material by using a solvent to obtain a solution to be detected;
b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured, wherein the chromatographic conditions are as follows:
the chromatographic column is a C18 column; the mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution.
The method for measuring the content of ammonium oxalate in the fried stiff silkworm provided by the invention comprises the steps of dissolving a sample raw material by using a solvent to obtain a solution to be measured.
The stir-fried stiff silkworm is not limited in the present invention, and may be known to those skilled in the art.
The ratio of the mass mg of the sample raw material to the volume mL of the solvent is 100:150, and the solvent is 0.2% phosphoric acid aqueous solution.
The extraction is ultrasonic extraction; the power of the ultrasonic wave is 500W, and the frequency is 40kHz; the ultrasonic treatment time is 30min.
The inventor finds that the solvent has better dissolving effect when being dissolved, and the solvent is adopted together with the extraction conditions, so that the extraction effect is good.
And measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured.
The invention also includes preparing a control solution: and dissolving the oxalic acid reference substance by adopting a solvent to obtain a reference substance solution.
Measuring the reference substance solution by adopting high performance liquid chromatography to obtain a chromatogram of the reference substance; and carrying out qualitative and quantitative analysis according to the concentration of the reference substance solution, the peak area of the reference substance in the chromatogram and the peak area of the component in the liquid to be detected, which corresponds to the reference substance, in the chromatogram.
The method preferably adopts an external standard method for calculation to obtain the content of oxalic acid.
Then, the content of the ammonium oxalate is calculated according to the content of the oxalic acid. The ammonium oxalate content of the present invention is equal to the oxalic acid content divided by 0.7255.
The chromatographic conditions of the invention are as follows:
the chromatographic column is a C18 column; preferably, the chromatographic column is shim-pack GIST C18, XSelect GSS T3C 18, GL Sciences T3C 18, 250X 4.6mm 5 μm; the column temperature is 25 to 35 ℃; more preferably 30 deg.c.
The chromatographic column has good separation effect.
The mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution.
Specifically, the gradient elution specifically comprises:
0-8min, phase A: 99% and phase B: 1 percent;
8-9min, phase A: 99-20%, phase B: 1% -80%;
9-15min, phase A: 20% -99%, phase B: 80% -1%;
15-30min, phase A: 99% and phase B: 1 percent.
The flow velocity of the mobile phase is preferably 0.6-0.8 mL/min; more preferably 0.7mL/min.
The invention adopts the mobile phase and the elution program, so that the separation degree of the component to be detected in the liquid to be detected is good.
The detection wavelength is 214nm; the amount of sample was 10. Mu.L.
The invention has good detection effect in the wavelength and more accurate result.
The concentration of oxalic acid in the reference solution is 0.042 mg/mL-0.833784 mg/mL. In the above concentration range, a good linear relationship is exhibited.
Specifically, the linear equation of oxalic acid in the control solution is Y =8155010.6660x +60278.9960, the correlation coefficient is 0.998, the RSD of the response factor is 2.3%, and the absolute value of the Y-axis intercept accounts for 3.6% of the 100% response value.
The invention provides a method for measuring the content of ammonium oxalate in fried stiff silkworm, which comprises the following steps: a) Dissolving a sample raw material by using a solvent to obtain a solution to be detected; b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured, wherein the chromatographic conditions are as follows: the chromatographic column is a C18 column; the mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution. The oxalic acid peak and the adjacent peak are well separated in the method, and other components in the liquid to be detected can be effectively eluted; the treatment of the liquid to be detected is simple and takes short time, the recovery rate of the liquid to be detected is high, and the cost of the diluent is low. Compared with the chromatographic separation conditions with stronger specificity in the existing literature, the method has the advantages of higher extraction efficiency of the liquid to be detected and simpler processing process.
In order to further illustrate the present invention, the following examples are provided to describe the method for determining the ammonium oxalate content in the parched Bombyx Batryticatus.
Example 1
Chromatographic conditions and system applicability test: octadecyl bonded silica gel was used as a packing agent (shim-pack GIST C18,4.6 mm. Times.250mm, 5um or equivalent performance column); taking 0.2% phosphoric acid water as a mobile phase A and methanol as a mobile phase B; gradient elution was performed as specified in the table below; the flow rate is 0.7mL/min, and the detection wavelength is 214nm; the column temperature was 30 ℃.
Preparation of a reference solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding 0.2% phosphoric acid water for dissolving, diluting to prepare 0.1mg/mL solution, shaking up, and filtering to obtain the product.
Preparing a test solution: taking 0.1g of fried stiff silkworm powder, placing in a conical flask with a suitable plug, adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, carrying out ultrasonic treatment (500W, 40KHZ) for 30min, cooling, weighing again, supplementing the weight loss with 0.2% phosphoric acid water, shaking up, and filtering to obtain the stiff silkworm powder.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results of the oxalic acid control are shown in FIG. 1, and the results of the test solution are shown in FIG. 2.
Comparative example 1
Preparation of a control solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding 50% ethanol water for dissolving, diluting to prepare 0.1mg/mL solution, shaking up, and filtering to obtain the final product.
Preparing a test solution: taking 0.1g of the powder, placing into a conical flask with a suitable plug, adding 100mL of 50% ethanol water, sealing the plug, weighing, ultrasonically treating (500W, 40KHZ) for 30min, cooling, weighing again, supplementing the lost weight with 50% ethanol water, shaking up, and filtering to obtain the final product.
Chromatographic conditions and system suitability test the same results as in example 1: the content of ammonium oxalate was 6.6%.
Comparative example 2
Preparation of a reference solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding water to dissolve and dilute the oxalic acid reference substance to prepare a solution of 0.1mg/mL, shaking up, and filtering to obtain the oxalic acid reference substance.
Preparing a test solution: taking 0.1g of the powder, placing into a conical flask with a suitable plug, adding 100mL of water, sealing, weighing, ultrasonically treating (500W, 40KHZ) for 30min, cooling, weighing again, supplementing the weight loss with water, shaking, and filtering to obtain the final product.
Chromatographic conditions and system suitability test as in example 1,
as a result: the content of ammonium oxalate was 7.3%.
Example 2
Preparation of a control solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding 0.2% phosphoric acid water for dissolving, diluting to prepare 0.1mg/mL solution, shaking up, and filtering to obtain the product.
Preparing a test solution: taking 0.1g of the product powder, placing in a conical flask with a suitable plug, adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, ultrasonically treating (500W, 40KHZ) for 30min, cooling, weighing again, supplementing the weight loss with 0.2% phosphoric acid water, shaking, and filtering to obtain the final product.
As a result: the content of ammonium oxalate is 8.9%.
Comparative example 3
Preparation of a control solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding 50% ethanol water for dissolving, diluting to prepare 0.1mg/mL solution, shaking up, and filtering to obtain the final product.
Preparing a test solution: taking 0.1g of the powder, placing in a conical flask with a suitable plug, adding 100mL of 50% ethanol water, sealing the plug, weighing, refluxing in water bath for 60min, cooling, weighing again, adding 50% ethanol water to make up the loss weight, shaking, and filtering to obtain the final product.
Chromatographic conditions and system applicability test the same results as in example 1 were obtained: the content of ammonium oxalate was 6.3%.
Comparative example 4
Preparation of a reference solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding water to dissolve and dilute the oxalic acid reference substance to prepare a solution of 0.1mg/mL, shaking up, and filtering to obtain the oxalic acid reference substance.
Preparing a test solution: taking 0.1g of the powder, placing in a conical flask with a suitable plug, adding 100mL of water, sealing the plug, weighing, refluxing in water bath for 60min, cooling, weighing again, supplementing the weight loss with water, shaking up, and filtering to obtain the final product.
Chromatographic conditions and system suitability test the same results as in example 1: the content of ammonium oxalate was 6.7%.
Comparative example 5
Preparation of a reference solution: taking a proper amount of oxalic acid reference substances, precisely weighing, adding 0.2% phosphoric acid water for dissolving, diluting to prepare a solution of 0.1mg/mL, shaking up, and filtering to obtain the oxalic acid reference substance.
Preparing a test solution: taking 0.1g of the powder, placing the powder into a conical flask with a suitable plug, adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, refluxing in water bath for 60min, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking up, and filtering to obtain the product.
Chromatographic conditions and system suitability test the same results as in example 1: the content of ammonium oxalate is 8.3%.
Example 3 (chromatographic Condition screening)
Preparation of a control solution: taking a proper amount of oxalic acid reference substance, precisely weighing, adding water to dissolve and dilute the oxalic acid reference substance to prepare a solution of 1.5mg/mL, shaking up, and filtering to obtain the oxalic acid reference substance.
Preparing a test solution: taking 0.1g of the powder, placing the powder into a conical flask with a suitable plug, adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, refluxing in water bath for 60min, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking up, and filtering to obtain the product.
Chromatographic conditions 1:
octadecyl bonded silica gel as filler (GL Sciences, 4.6 mm. Times.250mm, 5 um); acetonitrile-5 g/L diammonium phosphate water solution (10; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 3: the peak appearance was earlier and the peak pattern was worse.
Chromatographic conditions 2:
octadecyl bonded silica gel as filler (GL Sciences, 4.6 mm. Times.250mm, 5 um); acetonitrile-5 g/L diammonium phosphate aqueous solution (5; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 4: the peak appearance was early (< 3 min) and the peak pattern was poor.
Chromatographic conditions 3:
octadecyl bonded silica gel as filler (GL Sciences, 4.6 mm. Times.250mm, 5 um); acetonitrile-5 g/L diammonium hydrogen phosphate water solution (3; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 5: the peak appearance was early (< 3 min) and the peak pattern was poor.
Chromatographic conditions 4:
octadecyl bonded silica gel was used as a filler (GL Sciences, 4.6 mm. Times.250mm, 5 um); taking a diammonium hydrogen phosphate aqueous solution of 5g/L as a mobile phase; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 6: the peak appearance was early (< 3 min) and the peak pattern was poor.
Chromatographic conditions 5:
octadecyl bonded silica gel as filler (GL Sciences, 4.6 mm. Times.250mm, 5 um); methanol-5 g/L diammonium phosphate water solution (3; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 7: the peak pattern is poor, compared with the acetonitrile system, the retention time is delayed, but the main peak has interference.
Chromatographic conditions 6:
octadecyl bonded silica gel was used as filler (Atlantis T3, 4.6 mm. Times.250mm, 5 um); taking a diammonium phosphate aqueous solution with the concentration of 5g/L as a mobile phase; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 8: the peak appearance was earlier (< 3 min), the peak pattern was poorer, and there was no improvement compared to GL Sciences columns.
Chromatographic conditions 7:
octadecyl bonded silica gel was used as filler (Atlantis T3, 4.6 mm. Times.250mm, 5 um); acetonitrile-water (5; the flow rate was 1.0mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 9: the peak pattern was poor and there was no improvement compared to GL Sciences columns.
Chromatographic conditions 8:
octadecyl bonded silica gel as filler (GL Sciences, 4.6 mm. Times.250mm, 5um or equivalent performance column); taking 0.2% phosphoric acid water as a mobile phase A and methanol as a mobile phase B; gradient elution was performed as specified in the table below; the flow rate was 0.7mL/min and the detection wavelength was 214nm.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The results are shown in FIG. 10: the peak pattern is generally separated from the adjacent peaks by the baseline separation, but the durability is poor.
Verification example 1 methodology
1. Instrument/sample/control/reagent information
2. Content determination method
High performance liquid chromatography (0512 in the four-department general regulation of 2020 edition in Chinese pharmacopoeia).
Chromatographic conditions and System suitability test A chromatographic column with octadecylsilane bonded silica as a packing material (shim-pack GIST 250 mm. Times.4.6 mm,5 μm or equivalent performance); 0.2% phosphoric acid is used as a mobile phase A, and methanol is used as a mobile phase B; gradient elution was performed as specified in the table below; the detection wavelength was 214nm and the flow rate was 0.7mL per minute. The number of theoretical plates should not be less than 3000 in oxalic acid peak.
Preparation of reference solution an appropriate amount of oxalic acid reference was weighed precisely, and 0.2% phosphoric acid water was added to make a solution containing 0.25mg oxalic acid per 1 mL. (ammonium oxalate = oxalic acid/0.7255)
Preparation of test solution 0.1g is taken as the product powder, precisely weighed, placed in a suitable conical flask with a plug, precisely added with 100mL of 0.2% phosphoric acid, weighed, ultrasonically treated for 30 minutes (power 500W, frequency 40 kHZ), taken out, cooled, weighed again, complemented with 0.2% phosphoric acid, shaken, filtered, and a subsequent filtrate is taken, thus obtaining the test solution.
3 authenticating content
3.1 Wavelength selection
The detection wavelength is selected by carrying out ultraviolet scanning on oxalic acid at the wavelength of 190 nm-400 nm, and the oxalic acid has larger absorption at the wavelength of 214nm.
Solution preparation
Reference stock solution: taking about 20mg of oxalic acid reference substance, precisely weighing, placing in a 10mL measuring flask, dissolving with 0.2% phosphoric acid water, fixing the volume, and shaking up for later use. (about 2 mg/mL)
Control solution: precisely measuring 1mL of oxalic acid reference stock solution, placing the oxalic acid reference stock solution into a 10mL measuring flask, dissolving the oxalic acid reference stock solution by using 0.2% phosphoric acid water, fixing the volume, and shaking up for later use. (about 0.2 mg/mL)
Sample introduction procedure
TABLE 1 sample introduction sequence table for wavelength selection test
Measurement results
TABLE 2 test results of wavelength selection
3.2 System applicability
In the chromatogram obtained by continuously taking 6 reference solutions, the theoretical plate number is not lower than 3000 calculated according to the oxalic acid peak, the retention time RSD is not more than 1.0%, and the RSD of the peak area is not more than 2.0%.
Solution preparation
Blank solution: 0.2% phosphoric acid.
Control solution: the same control solution as under item 3.1.
Sample introduction procedure
TABLE 3 sample introduction sequence table for system applicability test
Measurement results
TABLE 4 System suitability test results
3.3 specificity
Blank should not interfere; in the chromatogram obtained from the reference solution, the theoretical plate number is not less than 3000 calculated according to the oxalic acid peak; the retention time of the oxalic acid peak detected in the chromatogram obtained from the test solution and the reference solution is consistent with the main peak retention time of the positioning solution, and the separation degree of the oxalic acid peak and the adjacent peak in the chromatogram obtained from the test solution is not less than 1.5.
Solution preparation
Blank solution: 0.2% phosphoric acid water.
Control solution: the same control solution as under item 3.1.
Test solution: weighing about 0.1g of the product, precisely weighing, placing in a conical flask with a suitable plug, precisely adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, performing ultrasonic treatment (power 500W; frequency 40 KHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking, filtering, and taking the subsequent filtrate to obtain the final product.
Sample introduction procedure
TABLE 5 specificity sample introduction sequence Listing
Measurement results
TABLE 6 results of the specificity test-Peak identification
3.4 Linear, range
Within the concentration range of 20-400% of the concentration of a reference solution, 5 concentration points are uniformly taken, the concentration is used as a horizontal coordinate, the peak area is used as a vertical coordinate to make a linear regression curve, the regression coefficient R of the obtained linear graph is not less than 0.990, the RSD of the response factor is not more than 10%, and the absolute value of the Y-axis intercept is within 10% of the 100% response value.
Solution preparation
Blank solution: 0.2% phosphoric acid water.
Control solution: the same as the control solution under item 3.1.
Linear stock solutions: namely, the control stock solution under item 3.1.
Linear solution 1 (20% concentration equivalent to control solution): precisely measuring 2mL of linear stock solution, placing the linear stock solution into a 100mL measuring flask, diluting the linear stock solution to a scale with 0.2% phosphoric acid water, and shaking up to obtain the product.
Linear solution 2 (corresponding to a control solution concentration of 50%): precisely measuring 1mL of linear stock solution, placing into a 20mL measuring flask, diluting with 0.2% phosphoric acid water to scale, and shaking up to obtain the final product.
Linear solution 3 (corresponding to 100% control solution concentration): precisely measuring 2mL of linear stock solution, placing into a 20mL measuring flask, diluting with 0.2% phosphoric acid water to scale, and shaking up to obtain the final product.
Linear solution 4 (corresponding to a control solution concentration of 200%): precisely measuring 2mL of linear stock solution, placing the linear stock solution into a 10mL measuring flask, diluting the linear stock solution to a scale with 0.2% phosphoric acid water, and shaking up to obtain the product.
Linear solution 5 (corresponding to a control solution concentration of 400%): precisely measuring 2mL of linear stock solution, placing the linear stock solution into a 5mL measuring flask, diluting the linear stock solution to a scale with 0.2% phosphoric acid water, and shaking up to obtain the product.
Sample introduction procedure
TABLE 7 sample introduction sequence table for linear and range tests
Measurement results
TABLE 8 Linear and Range test results
3.5 Accuracy of
The accuracy is realized by measuring the recovery rate between the actual detection amount and the addition amount by adding a certain amount of oxalic acid reference substance into the test sample to ensure that the total amount of oxalic acid in the test sample is respectively equivalent to 80% of the lower limit to 120% of the upper limit of the test sample. The recovery rate is required to be between 95 and 102 percent, and the RSD of 9 times of recovery rate is required to be not more than 10 percent.
Solution preparation
Blank solution: 0.2% phosphoric acid water.
Low isoconcentrate recovery solution (80% concentration): taking a proper amount (equivalent to 6.4mg of oxalic acid) of the product, precisely weighing, placing in a conical flask with a suitable plug, precisely adding 6.4mg of oxalic acid reference substance, precisely adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, ultrasonically treating (with the power of 500W and the frequency of 40 KHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. 3 parts are prepared in parallel.
Medium concentration recovery solution (100% concentration): taking a proper amount (equivalent to 11.5mg of oxalic acid) of the product, precisely weighing, placing in a conical flask with a suitable plug, precisely adding 11.5mg of oxalic acid reference substance, precisely adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, ultrasonically treating (with the power of 500W and the frequency of 40 KHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. 3 parts are prepared in parallel.
High concentration recovery solution (120% concentration): taking a proper amount (equivalent to 17.9mg of oxalic acid) of the product, accurately weighing, placing the product in a conical flask with a proper plug, accurately adding 17.9mg of oxalic acid reference substance, accurately adding 100mL of 0.2% phosphoric acid water, sealing the plug, weighing, carrying out ultrasonic treatment (power 500W and frequency 40 KHz) for 30 minutes, cooling, weighing again, complementing the lost weight with 0.2% phosphoric acid water, shaking up, filtering, and taking the subsequent filtrate to obtain the product. 3 parts are prepared in parallel.
Note: the content of oxalic acid in the recovery test solution is calculated according to the measurement result under the repeatability item.
Sample introduction procedure
Table 9 accuracy test sample introduction sequence table
Measurement results
TABLE 10 accuracy test results
3.6 precision
The precision refers to the degree of closeness between the results of multiple sampling measurements of the same uniform sample under specified test conditions, and the precision is judged by examining the repeatability and the intermediate precision.
3.6.1 Repeatability of
6 parts of repetitive test sample solution is prepared by an analyst A at the date A, and the oxalic acid content RSD in the 6 parts of repetitive test samples is not more than 10 percent when the analyst A measures the solution by using the instrument A.
Solution preparation
Blank solution: 0.2% phosphoric acid water.
Control solution: the same control solution as under item 3.1.
Repeated test solution: taking a proper amount of the product, taking about 0.1g, precisely weighing, placing in a conical flask with a stopper, precisely adding 100mL of 0.2% phosphoric acid water, sealing the stopper, weighing, carrying out ultrasonic treatment (power 500W; frequency 40 KHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product. 6 parts are prepared in parallel.
Sample introduction procedure
TABLE 11 repeatability test sample introduction sequence Listing
Test results
TABLE 12 repeatability test results for standard soup
3.6.2 intermediate precision
6 portions of the intermediate-precision test sample solution were prepared by the analyst B on the date B and measured by the instrument B.
In the chromatogram obtained by continuously taking 6 reference solutions, the theoretical plate number is not lower than 3000 calculated according to the oxalic acid peak, the retention time RSD is not more than 1.0 percent, and the RSD of the peak area is not more than 2.0 percent. The oxalic acid content RSD in 6 intermediate precision samples is not more than 10%.
The results of 12 times of measurement of repeatability and intermediate precision show that the oxalic acid content RSD is not more than 10%.
Solution preparation
Blank solution: 0.2% phosphoric acid water.
Control solution: prepared with the "control solution" under item 3.1.
Intermediate precision test solution: taking a proper amount of the product, taking about 0.1g, precisely weighing, placing in a conical flask with a stopper, precisely weighing 100mL of 0.2% phosphoric acid water, sealing the stopper, weighing, ultrasonically treating (with the power of 500W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. 6 parts are prepared in parallel.
Sample introduction procedure
TABLE 13 sample introduction sequence table for intermediate precision test
Test results
TABLE 14 intermediate precision System suitability results
TABLE 15 results of precision test
3.7 solution stability
Standing at room temperature for a period of time, injecting reference solution and test solution at different time points, and calculating oxalic acid peak of theoretical plate number of chromatogram obtained from reference solution to be not less than 3000; the oxalic acid peak area RSD of the reference solution and the test solution is not more than 10%, and impurities which interfere with oxalic acid detection are not required to be added.
Solution preparation
Blank solution: 0.2% phosphoric acid.
Control solution: the same control solution as under item 3.1.
Test solution: the same test solution as in item 3.3.
Sample injection program table
TABLE 16 sample introduction sequence table for solution stability test
Test results
TABLE 17 results of solution stability test
3.8 durability
The method is realized by investigating and changing chromatographic conditions (changing column temperature, flow rate, chromatographic column and instrument manufacturers) and testing the oxalic acid content in the test sample under normal chromatographic conditions. The theoretical plate number of the chromatogram obtained by the reference solution under the same chromatographic parameter and different conditions is required to be not less than 3000 calculated according to the oxalic acid peak, and the RSD of the oxalic acid content in the test sample is not more than 10%.
TABLE 18 durability test chromatographic Condition Change
Solution preparation
Blank solution: 0.2% phosphoric acid water.
Control solution: the test solution was prepared as the control solution under item 3.1.
Durability test article solution: taking a proper amount of the product, taking about 0.1g, precisely weighing, placing in a conical flask with a stopper, precisely weighing 100mL of 0.2% phosphoric acid water, sealing the stopper, weighing, ultrasonically treating (with the power of 500W and the frequency of 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 0.2% phosphoric acid water, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product. Two portions were prepared in parallel.
Sample introduction procedure
TABLE 19 sample introduction sequence table for durability test
Test results
TABLE 20 durability test-System suitability and results
TABLE 21 conclusion on durability
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A method for measuring the content of ammonium oxalate in stir-fried stiff silkworm comprises the following steps:
a) Dissolving a sample raw material by using a solvent to obtain a solution to be detected;
b) Measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain a chromatogram of the liquid to be measured, wherein the chromatographic conditions are as follows:
the chromatographic column is a C18 column; the mobile phase A is 0.2% phosphoric acid water solution, the mobile phase B is methanol, and the mobile phase adopts gradient elution.
2. The method of claim 1, further comprising preparing a control solution: dissolving an oxalic acid reference substance by adopting a solvent to obtain a reference substance solution;
measuring the reference substance solution by adopting high performance liquid chromatography to obtain a chromatogram of the reference substance; performing qualitative and quantitative analysis according to the concentration of the reference substance solution, the peak area of the reference substance in the chromatogram and the peak area of the component corresponding to the reference substance in the liquid to be detected in the chromatogram;
and calculating the content of the ammonium oxalate according to the content of the oxalic acid.
3. The method according to claim 1, characterized in that the gradient elution is in particular:
0-8min, phase A: 99% and phase B: 1 percent;
8-9min, phase A: 99-20%, phase B: 1% -80%;
9-15min, phase A: 20% -99%, phase B: 80% -1%;
15-30min, phase A: 99% and phase B: 1 percent.
4. The method of claim 1, wherein the chromatography column is shim-pack GIST C18, XSelect GSS T3C 18, GL Sciences T3C 18, 250 x 4.6mm 5 μ ι η in size; the column temperature is 25 to 35 ℃.
5. The method of claim 1, wherein the mobile phase flow rate is 0.6 to 0.8ml/min.
6. The method of claim 1, wherein the detection wavelength is 214nm; the amount of sample was 10. Mu.L.
7. The method of claim 2, wherein the concentration of oxalic acid in the control solution is from 0.042mg/mL to 0.833784mg/mL.
8. The method of claim 7 wherein the linear equation for oxalic acid in the control solution is Y =8155010.6660x +60278.9960, the correlation coefficient is 0.998, the RSD of the response factor is 2.3%, and the absolute value of the Y-intercept is 3.6% of the 100% response.
9. The method of claim 1, wherein the solvent is a 0.2% aqueous phosphoric acid solution; the extraction is ultrasonic extraction; the power of the ultrasonic wave is 500W, and the frequency is 40kHz; the ultrasonic treatment time is 30min.
10. The method according to claim 1, wherein the ratio of the mass mg of the sample raw material to the volume mL of the solvent is 100.
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| CN101591231A (en) * | 2009-06-23 | 2009-12-02 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of extraction process of Bombyx Batryticatus ammonium oxalate |
| CN110352920A (en) * | 2019-08-30 | 2019-10-22 | 江苏海洋大学 | A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine |
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