Method for determining content of amino acid in periplaneta americana by quantitative analysis of multi-components by single marker

Abstract

The invention relates to the technical field of content detection, in particular to a method for determining the content of amino acid in periplaneta americana by adopting a quantitative analysis of multi-components by single marker, which comprises the following steps: firstly, preparing a test solution from periplaneta americana powder and a hydrochloric acid solution; dissolving nine amino acids in a hydrochloric acid solution to obtain a mixed reference substance solution; taking the test solution, the blank control solution and the mixed control solution, respectively adding an acetonitrile solution of triethylamine and an acetonitrile solution of phenyl isothiocyanate, carrying out vortex mixing uniformly, then carrying out water bath in a dark place, carrying out normal hexane extraction, taking a lower-layer solution, and carrying out high performance liquid chromatography analysis; and by taking alanine as an internal standard substance, calculating relative correction factors of alanine and other eight amino acids, thereby determining the contents of nine amino acids in periplaneta americana. The method is high in analysis efficiency, stable and reliable, can be used for multi-component analysis of other medicinal materials, and is suitable for different analysis requirements, so that the application prospect is wider.

Description

Technical Field

[0001] The invention relates to the technical field of content detection, and in particular to a method for determining the amino acid content in American cockroaches by adopting a one-test-multiple-evaluation method. Background Art

[0002] The American cockroach was first recorded in the Shennong's Herbal Classic. It belongs to the genus Periplaneta of the family Blattaria of the order Blattaria of the class Insecta. It is also called cockroach or cockroach. It tastes salty and is cold in nature. It is listed as a medium-quality product. It has the effects of diuresis and swelling, detoxification and elimination of malnutrition, and blood circulation and blood stasis. Since the 1960s, Chinese researchers have conducted in-depth research on the extraction process, pharmacological effects and clinical applications of the American cockroach. It was found that the American cockroach contains amino acids, polysaccharides, nucleosides, polyols, steroids and terpenes. It has extremely high medicinal value and has pharmacological effects such as anti-fibrosis, tissue repair, anti-inflammatory and swelling, myocardial protection, and anti-tumor. It is clinically used to promote wound healing, treat tumors and ulcers, improve heart and kidney function, and enhance immunity. A series of high-quality Chinese medicine preparations such as Kangfuxin Liquid, Xinmailong Injection, Ganlong Capsules, and Xiaozheng Yigan Tablets have been successfully developed based on the American cockroach extract, so that the medicinal value of the American cockroach can be fully utilized. As a single Chinese medicine, the American cockroach has significant curative effects and is listed in the Yunnan Chinese Medicinal Materials Standard, Sichuan Chinese Medicinal Materials Standard, Fujian Chinese Medicinal Materials Standard, Hunan Chinese Medicinal Materials Standard, and Hunan Chinese Medicinal Pieces Processing Specification. At the same time, its body has a high protein content and can be used as high-grade feed; its body fluid is rich in fat and minerals and can be used to make nutritional health products and medicines; its shell can be used to extract antibacterial and antioxidant substances.

[0003] The current local standards all use UV / visible spectrophotometry to determine the total amino acid content in American cockroaches. This method has relatively low sensitivity and may not be sensitive enough for the detection of low-concentration amino acids. It cannot effectively detect the content of each component in the sample. In addition, there are problems such as cumbersome operation steps, high purchase cost of reference materials, large workload, and low analysis efficiency. The one-measurement-multiple-evaluation method is to determine the content of an easily available component (internal standard) and use the relative correction factor (f a / b ), a method for calculating the content of multiple components. This method does not have the problems of complicated operation steps, large content determination errors, high purchase cost of reference materials, large workload, and low analysis efficiency. Therefore, this method can be used to solve the problems of complicated operation steps, large content determination errors, and low analysis efficiency in the prior art.

[0004] Therefore, according to the above-mentioned related technologies, it is urgent to develop a method for determining the amino acid content in American cockroaches using a one-test-multiple-evaluation method. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a method for determining the amino acid content in American cockroaches using a one-measurement-multiple-evaluation method to solve the problems of complicated operating steps, large content determination errors and low analysis efficiency in the prior art.

[0006] Based on the above purpose, the present invention provides a method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method.

[0007] A method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method comprises the following steps:

[0008] Step S1: dissolving the American cockroach powder in a hydrochloric acid solution, filtering, evaporating to dryness, and then dissolving and fixing the volume with a hydrochloric acid solution to obtain a test solution;

[0009] Step S2: dissolving glycine, arginine, threonine, alanine, tyrosine, valine, isoleucine, leucine, and phenylalanine in a hydrochloric acid solution to obtain a mixed reference solution;

[0010] Step S3: Take 1 mL of the test solution, blank control solution and mixed reference solution, add 1 mL of triethylamine in acetonitrile solution and phenyl isothiocyanate in acetonitrile solution, vortex mix, place in a dark water bath, extract with n-hexane, take the lower solution and perform high performance liquid chromatography analysis. Alanine is used as the internal standard, and its relative correction factor with the other 8 amino acids is calculated to determine the content of 9 amino acids in American cockroaches.

[0011] Preferably, the amount of the American cockroach powder in step S1 is 0.1-10 g;

[0012] The concentration of the hydrochloric acid solution is 0.01-1.0 mol / L;

[0013] The volume of the test solution after the dilution is 1-250 mL.

[0014] Preferably, the concentration of glycine in the mixed reference solution in step S2 is 20.6-411.3 μg / mL;

[0015] The concentration of arginine in the mixed reference solution is 15.8-315.5 μg / mL;

[0016] The concentration of threonine in the mixed reference solution is 10.9-218.3 μg / mL;

[0017] The concentration of alanine in the mixed reference solution is 18.4-368.0 μg / mL;

[0018] The concentration of tyrosine in the mixed reference solution is 14.7-294.9 μg / mL;

[0019] The concentration of valine in the mixed reference solution is 15.5-309.0 μg / mL;

[0020] The concentration of isoleucine in the mixed reference solution is 8.0-159.3 μg / mL;

[0021] The concentration of leucine in the mixed reference solution is 18.5-370.1 μg / mL;

[0022] The concentration of phenylalanine in the mixed reference solution is 8.6-172.8 μg / mL;

[0023] The concentration of the hydrochloric acid solution is 0.01-1.0 mol / L.

[0024] Preferably, the blank control solution in step S3 is a 0.01-1.0 mol / L hydrochloric acid solution;

[0025] The concentration of the triethylamine acetonitrile solution is 1-2 mol / L;

[0026] The concentration of the acetonitrile solution of phenyl isothiocyanate is 1-2 mol / L.

[0027] Preferably, in step S3, the dosage ratio of the test solution, the acetonitrile solution of triethylamine, and the acetonitrile solution of phenyl isothiocyanate is 1 mL: 0.5-1 mL: 0.5-1 mL; the dosage ratio of the blank control solution, the acetonitrile solution of triethylamine, and the acetonitrile solution of phenyl isothiocyanate is 1 mL: 0.5-1 mL: 0.5-1 mL; the dosage ratio of the mixed reference solution, the acetonitrile solution of triethylamine, and the acetonitrile solution of phenyl isothiocyanate is 1 mL: 0.5-1 mL: 0.5-1 mL.

[0028] Preferably, the number of extractions during the n-hexane extraction in step S3 is 3-5 times.

[0029] Preferably, the chromatographic column used in the high performance liquid chromatography analysis in step S3 is a C18 chromatographic column;

[0030] The flow rate during the high performance liquid chromatography analysis is 0.3-1 mL / min;

[0031] The detection wavelength during the high performance liquid chromatography analysis is 254 nm;

[0032] The column temperature during the HPLC analysis is 20-40°C;

[0033] The injection volume used in the high performance liquid chromatography analysis is 1-20 μL.

[0034] Preferably, the preparation method of mobile phase A used in the high performance liquid chromatography analysis in step S3 is as follows:

[0035] Dissolve acetonitrile in a 0.1 mol / L sodium acetate solution, and then adjust the pH to 6.45-6.55 with 36% acetic acid to obtain mobile phase A;

[0036] The mass ratio of the acetonitrile to the sodium acetate solution is 1-10:90-99.

[0037] Preferably, the preparation method of mobile phase B used in the high performance liquid chromatography analysis in step S3 is as follows:

[0038] Mix acetonitrile and water evenly to obtain mobile phase B;

[0039] The mass ratio of acetonitrile to water is 1-6:1.

[0040] Preferably, when performing high performance liquid chromatography analysis, the mass ratio of mobile phase A and mobile phase B added at 0-8 min of elution is 97:3; the mass ratio of mobile phase A and mobile phase B added at 8-20 min of elution is 95:5; the mass ratio of mobile phase A and mobile phase B added at 20-35 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 35-40 min of elution is 93:7; the mass ratio of mobile phase A and mobile phase B added at 40-60 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 40-60 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 50-60 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 60-70 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 70-80 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 80-90 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 90-90 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 80-80 min of elution is 94.5:5.5; the mass ratio of mobile phase A and mobile phase B added at 80-9 ...80 min of elution is The mass ratio of mobile phase A and mobile phase B added at 60-63 min of elution was 78:22; the mass ratio of mobile phase A and mobile phase B added at 63-73 min of elution was 60:40; the mass ratio of mobile phase A and mobile phase B added at 73-83 min of elution was 55:45; the mass ratio of mobile phase A and mobile phase B added at 83-90 min of elution was 0:100; the mass ratio of mobile phase A and mobile phase B added at 90-100 min of elution was 97:3.

[0041] Preferably, the investigation method of the one-test-multiple-evaluation method of the present invention is:

[0042] (1) Linear relationship: By measuring the reference solution of different concentrations, a standard curve is established and the linear regression equation and correlation coefficient are calculated;

[0043] (2) Precision test: continuous sampling, calculation of relative standard deviation of peak area, and evaluation of intra-day and inter-day precision;

[0044] (3) Repeatability test: The same sample was measured multiple times, the relative standard deviation of the peak area was calculated, and the repeatability of the method was evaluated;

[0045] (4) Stability test: Samples were injected at different time points, and the relative standard deviation of the peak area was calculated to evaluate the stability of the test solution;

[0046] (5) Sample recovery test: Add a reference substance to a sample of known content, calculate the sample recovery, and evaluate the accuracy of the method;

[0047] (6) Relative correction factor investigation: Calculate the relative correction factor between alanine as the internal standard and other components.

[0048] Beneficial effects of the present invention:

[0049] The present invention provides a method for determining the amino acid content in American cockroaches by using a one-test multiple-evaluation method. The present invention adopts the one-test multiple-evaluation method to simplify the complex process of the traditional external standard method that requires each component to be determined separately. The demand for multiple reference substances is reduced, the cost of purchasing and using reference substances is reduced, and the consumption of experimental materials is reduced. The content information of multiple amino acids can be obtained through one measurement, the analysis efficiency is improved, and it is particularly suitable for occasions requiring rapid quality control. It has good linear relationship and recovery rate, ensures the accuracy and repeatability of the measurement results, and provides stable and reliable analysis data compared with the prior art.

[0050] This method is not only suitable for the determination of 9 kinds of amino acids in American cockroach medicinal materials, but can also be extended to the analysis of multiple components in other medicinal materials. The chromatographic conditions and parameters can be flexibly adjusted according to the specific conditions and needs of the laboratory to meet different analytical needs. A new quality control method is provided, which helps to improve the quality assurance of American cockroach medicinal materials and their extracts. This method provides technical support for the scientific research of American cockroach medicinal materials, and helps to in-depth study of their pharmacological effects and clinical applications. The use of chemical reagents and the generation of waste are reduced, which is in line with the concept of environmental protection and green chemistry. By comparing with the external standard method, the accuracy of the method is verified, making the obtained data more reliable and increasing the credibility of the research. Therefore, it has a wider application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1The HPLC chromatograms are of the test solution, blank solution and mixed reference solution prepared in Example 1 of the present invention, wherein Figure A is the HPLC chromatogram of the mixed reference solution, Figure B is the HPLC chromatogram of the test solution, and Figure C is the HPLC chromatogram of the blank solution; Peak 1 in Figure A is the characteristic peak of glycine, Peak 2 is the characteristic peak of arginine, Peak 3 is the characteristic peak of threonine, Peak 4 is the characteristic peak of alanine, Peak 5 is the characteristic peak of tyrosine, Peak 6 is the characteristic peak of valine, Peak 7 is the characteristic peak of isoleucine, Peak 8 is the characteristic peak of leucine, and Peak 9 is the characteristic peak of phenylalanine; Peak 1 in Figure B is the characteristic peak of glycine, Peak 2 is the characteristic peak of arginine, Peak 3 is the characteristic peak of threonine, Peak 4 is the characteristic peak of alanine, Peak 5 is the characteristic peak of tyrosine, Peak 6 is the characteristic peak of valine, Peak 7 is the characteristic peak of isoleucine, Peak 8 is the characteristic peak of leucine, and Peak 9 is the characteristic peak of phenylalanine. DETAILED DESCRIPTION

[0053] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0054] Example 1: A method for determining the amino acid content in American cockroaches using a one-measurement-multiple-evaluation method is as follows:

[0055] S1: Dissolve 0.1g of American cockroach powder in 1mL of 0.01mol / L hydrochloric acid solution, filter, evaporate to dryness, and then dissolve and dilute with hydrochloric acid solution to obtain the test solution;

[0056] S2: dissolving glycine, arginine, threonine, alanine, tyrosine, valine, isoleucine, leucine and phenylalanine in 0.01 mol / L hydrochloric acid solution to obtain a mixed reference solution; the concentration of glycine in the mixed reference solution is 20.6 μg / mL, the concentration of arginine is 15.8 μg / mL, the concentration of threonine is 10.9 μg / mL, the concentration of alanine is 18.4 μg / mL, the concentration of tyrosine is 14.7 μg / mL, the concentration of valine is 15.5 μg / mL, the concentration of isoleucine is 8.0 μg / mL, the concentration of leucine is 18.5 μg / mL, and the concentration of phenylalanine is 8.6 μg / mL;

[0057] S3: Dissolve 1 g of acetonitrile in 90 g of 0.1 mol / L sodium acetate solution, and then adjust the pH to 6.45 with 36% acetic acid to obtain mobile phase A; mix 1 g of acetonitrile and 1 g of water to obtain mobile phase B;

[0058] S4: Take 1 mL of the test solution, blank solution and mixed reference solution, add 0.5 mL of 1 mol / L triethylamine in acetonitrile solution and 0.5 mL of 1 mol / L phenyl isothiocyanate in acetonitrile solution, vortex mix, and place in a dark water bath. Extract with n-hexane 3 times, and take the lower layer of solution for high performance liquid chromatography analysis. Alanine is used as the internal standard to calculate its relative correction factor with the other 8 amino acids, so as to determine the content of 9 amino acids in American cockroaches; the blank solution is a 0.01 mol / L hydrochloric acid solution; the chromatographic column used for high performance liquid chromatography analysis is a Kromasil 100-5-C18 chromatographic column, the flow rate is 0.3 mL / min, the detection wavelength is 254 nm, and the column temperature is 20°C; the injection volume is 1 μL. When performing high performance liquid chromatography analysis, the mass ratio of mobile phase A to mobile phase B added at 0-8 min of elution is 97:3; the mass ratio of mobile phase A to mobile phase B added at 8-20 min of elution is 95:5; the mass ratio of mobile phase A to mobile phase B added at 20-35 min of elution is 94.5:5.5; the mass ratio of mobile phase A to mobile phase B added at 35-40 min of elution is 93:7; the mass ratio of mobile phase A to mobile phase B added at 40-60 min of elution is 7 8:22; the mass ratio of mobile phase A and mobile phase B added at 60-63min of elution was 77:23; the mass ratio of mobile phase A and mobile phase B added at 63-73min of elution was 60:40; the mass ratio of mobile phase A and mobile phase B added at 73-83min of elution was 55:45; the mass ratio of mobile phase A and mobile phase B added at 83-90min of elution was 0:100; the mass ratio of mobile phase A and mobile phase B added at 90-100min of elution was 97:3.

[0059] Example 2: A method for determining the amino acid content in American cockroaches using a one-measurement-multiple-evaluation method is as follows:

[0060] S1: Dissolve 5 g of American cockroach powder in 100 mL of 0.1 mol / L hydrochloric acid solution, filter, evaporate to dryness, and then dissolve and dilute with hydrochloric acid solution to obtain the test solution;

[0061] S2: dissolving glycine, arginine, threonine, alanine, tyrosine, valine, isoleucine, leucine and phenylalanine in 0.1 mol / L hydrochloric acid solution to obtain a mixed reference solution; the concentration of glycine in the mixed reference solution is 200 μg / mL, the concentration of arginine is 150 μg / mL, the concentration of threonine is 110 μg / mL, the concentration of alanine is 180 μg / mL, the concentration of tyrosine is 150 μg / mL, the concentration of valine is 150 μg / mL, the concentration of isoleucine is 83 μg / mL, the concentration of leucine is 181 μg / mL, and the concentration of phenylalanine is 88 μg / mL;

[0062] S3: Dissolve 5 g of acetonitrile in 95 g of 0.1 mol / L sodium acetate solution, and then adjust the pH to 6.5 with 36% acetic acid to obtain mobile phase A; mix 4 g of acetonitrile with 1 g of water to obtain mobile phase B;

[0063] S4: Take 1 mL of the test solution, blank solution and mixed reference solution, add 0.8 mL of 1.5 mol / L triethylamine in acetonitrile solution and 0.8 mL of 1.5 mol / L phenyl isothiocyanate in acetonitrile solution, vortex mix, and place in a dark water bath. Extract with n-hexane 4 times, and take the lower layer of solution for high performance liquid chromatography analysis. Alanine is used as the internal standard to calculate its relative correction factor with the other 8 amino acids, so as to determine the content of 9 amino acids in American cockroaches; the blank solution is a 0.1 mol / L hydrochloric acid solution; the chromatographic column used for high performance liquid chromatography analysis is a Kromasil100-5-C18 chromatographic column, the flow rate is 0.8 mL / min, the detection wavelength is 254 nm, and the column temperature is 30 ° C; the injection volume is 10 μL. When performing high performance liquid chromatography analysis, the mass ratio of mobile phase A to mobile phase B added at 0-8 min of elution is 97:3; the mass ratio of mobile phase A to mobile phase B added at 8-20 min of elution is 95:5; the mass ratio of mobile phase A to mobile phase B added at 20-35 min of elution is 94.5:5.5; the mass ratio of mobile phase A to mobile phase B added at 35-40 min of elution is 93:7; the mass ratio of mobile phase A to mobile phase B added at 40-60 min of elution is 7 8:22; the mass ratio of mobile phase A and mobile phase B added at 60-63min of elution was 77:23; the mass ratio of mobile phase A and mobile phase B added at 63-73min of elution was 60:40; the mass ratio of mobile phase A and mobile phase B added at 73-83min of elution was 55:45; the mass ratio of mobile phase A and mobile phase B added at 83-90min of elution was 0:100; the mass ratio of mobile phase A and mobile phase B added at 90-100min of elution was 97:3.

[0064] Example 3: A method for determining the amino acid content in American cockroaches using a one-measurement-multiple-evaluation method is as follows:

[0065] S1: Dissolve 10 g of American cockroach powder in 250 mL of 1.0 mol / L hydrochloric acid solution, filter, evaporate to dryness, and then dissolve and dilute with hydrochloric acid solution to obtain the test solution;

[0066] S2: dissolving glycine, arginine, threonine, alanine, tyrosine, valine, isoleucine, leucine and phenylalanine in a 1.0 mol / L hydrochloric acid solution to obtain a mixed reference solution; the concentration of glycine in the mixed reference solution is 411.3 μg / mL, the concentration of arginine is 315.5 μg / mL, the concentration of threonine is 218.3 μg / mL, the concentration of alanine is 368.0 μg / mL, the concentration of tyrosine is 294.9 μg / mL, the concentration of valine is 309.0 μg / mL, the concentration of isoleucine is 159.3 μg / mL, the concentration of leucine is 370.1 μg / mL, and the concentration of phenylalanine is 172.8 μg / mL;

[0067] S3: Dissolve 10 g of acetonitrile in 99 g of 0.1 mol / L sodium acetate solution, and then adjust the pH to 6.55 with 36% acetic acid to obtain mobile phase A; mix 6 g of acetonitrile with 1 g of water to obtain mobile phase B;

[0068] S4: Take 1 mL of the test solution, blank control solution and mixed reference solution, add 1 mL of 2 mol / L triethylamine in acetonitrile solution and 1 mL of 2 mol / L phenyl isothiocyanate in acetonitrile solution, vortex mix, place in a dark water bath, extract with n-hexane 5 times, and take the lower layer of solution for high performance liquid chromatography analysis. Alanine is used as the internal standard to calculate its relative correction factor with the other 8 amino acids, so as to determine the content of 9 amino acids in American cockroaches; the blank control solution is a 1.0 mol / L hydrochloric acid solution; the chromatographic column used for high performance liquid chromatography analysis is an Agilent ZORBAX Eclipse plus C18 chromatographic column, the flow rate is 1 mL / min, the detection wavelength is 254 nm, and the column temperature is 40°C; the injection volume is 20 μL. When performing high performance liquid chromatography analysis, the mass ratio of mobile phase A to mobile phase B added at 0-8 min of elution is 97:3; the mass ratio of mobile phase A to mobile phase B added at 8-20 min of elution is 95:5; the mass ratio of mobile phase A to mobile phase B added at 20-35 min of elution is 94.5:5.5; the mass ratio of mobile phase A to mobile phase B added at 35-40 min of elution is 93:7; the mass ratio of mobile phase A to mobile phase B added at 40-60 min of elution is 7 8:22; the mass ratio of mobile phase A and mobile phase B added at 60-63min of elution was 77:23; the mass ratio of mobile phase A and mobile phase B added at 63-73min of elution was 60:40; the mass ratio of mobile phase A and mobile phase B added at 73-83min of elution was 55:45; the mass ratio of mobile phase A and mobile phase B added at 83-90min of elution was 0:100; the mass ratio of mobile phase A and mobile phase B added at 90-100min of elution was 97:3.

[0069] Performance Testing:

[0070] Linear relationship:

[0071] By measuring the reference solution of different concentrations, a standard curve was established, and the linear regression equation and correlation coefficient were calculated; the linear regression equation of glycine was y=7351.9x-22.335, the correlation coefficient was 1.0000, and the linear range was 20.6-411.3μg·mL -1 The linear regression equation of arginine is y=3165.2x-6.1728, the correlation coefficient is 1.0000, and the linear range is 15.8-315.5μg·mL -1 The linear regression equation of threonine is y=4271.7x-5.9732, the correlation coefficient is 1.0000, and the linear range is 10.9-218.3μg·mL -1The linear regression equation of alanine is y=6173.9x-11.873, the correlation coefficient is 1.0000, and the linear range is 18.4-368.0μg·mL -1 The linear regression equation of tyrosine was y=3415.4x-8.000, the correlation coefficient was 1.0000, and the linear range was 14.7-294.9 μg·mL -1 The linear regression equation of valine is y=4995.5x-7.0636, the correlation coefficient is 1.0000, and the linear range is 15.5-309.0μg·mL -1 The linear regression equation of isoleucine is y=4587.3x-4.9989, the correlation coefficient is 1.0000, and the linear range is 8.0-159.3μg·mL -1 The linear regression equation of leucine is y=4431.8x-10.944, the correlation coefficient is 1.0000, and the linear range is 18.5-370.1μg·mL -1 The linear regression equation of phenylalanine is y=3825.7x-8.0663, the correlation coefficient is 0.9999, and the linear range is 8.6-172.8μg·mL -1 .

[0072] Precision test:

[0073] Continuous sampling was performed to calculate the relative standard deviation of the peak area and evaluate the intra-day and inter-day precision. The results showed that the test solution had good intra-day and inter-day precision.

[0074] Repeatability test:

[0075] The same sample was measured multiple times, the relative standard deviation of the peak area was calculated, and the repeatability of the method was evaluated; the measurement results showed that the test solution had good repeatability;

[0076] Stability test:

[0077] Samples were injected and measured at different time points, and the relative standard deviation of the peak area was calculated to evaluate the stability of the test solution. The test results showed that the test solution had good stability.

[0078] Sample recovery test:

[0079] The reference substance was added to the samples with known content, and the recovery rate was calculated to evaluate the accuracy of the method. The results showed that the average recovery rate was between 98.70% and 101.93%.

[0080] Relative correction factor investigation:

[0081] The relative correction factors of alanine as the internal standard and other components were calculated, and the calculated relative correction factors are shown in Table 1-2; then the influence of the instrument and the chromatographic column on the relative correction factor was determined, and the results are shown in Table 3-4; then the influence of the flow rate on the relative correction factor was determined, and the results are shown in Table 5-6; finally, the influence of the column temperature on the relative correction factor was determined, and the results are shown in Table 7-8;

[0082] Determination of the content of 9 amino acids in American cockroaches:

[0083] Combining the linear relationship with the relative correction factor, the contents of 9 amino acids in American cockroach were calculated, and the results are shown in Tables 9-11;

[0084] Table 1

[0085]

[0086] Table 2

[0087]

[0088] Table 3

[0089]

[0090] Table 4

[0091]

[0092]

[0093] Table 5

[0094]

[0095] Table 6

[0096]

[0097] Table 7

[0098]

[0099] Table 8

[0100]

[0101]

[0102] Table 9

[0103]

[0104] Table 10

[0105]

[0106]

[0107] Table 11

[0108]

[0109] Data Analysis:

[0110] It can be seen from the various tests and test results that the method for determining the amino acid content in American cockroaches of the present invention determines the content of an easily available component (internal standard) and uses the relative correction factor between the internal standard and other components to calculate the content of multiple components; this method reduces the dependence on a separate reference substance for each component and simplifies the analysis process. The present invention uses high performance liquid chromatography, selects a specific chromatographic column and mobile phase, and combines a gradient elution program to ensure the effective separation and determination of 9 kinds of amino acids. The present invention selects alanine as an internal standard and calculates its relative correction factor with other 8 kinds of amino acids, which is the key to the accuracy of the method. After that, the accuracy and comparability of the determination results between different components are ensured by accurately calculating the relative correction factor. It can also be seen from the test results that the method of the present invention shows a good linear relationship, and the average sample recovery rate is between 98.70% and 101.93%, indicating the accuracy and repeatability of the method. The method of the present invention also provides a theoretical reference and practical application for the quality control of amino acids in American cockroach medicinal materials and extracts thereof, which helps to ensure the quality and consistency of medicinal materials. Compared with the traditional external standard method, the one-test-multiple-evaluation method reduces the operation steps and the purchase cost of the reference substance, and improves the analysis efficiency. The ability to simultaneously determine the 9 kinds of amino acids in the American cockroach medicinal material is of great significance for the comprehensive evaluation and quality control of the medicinal material. Therefore, the determination method of the present invention is not only accurate, stable, and cost-effective, but can also be used for the quantitative analysis of multiple components in the American cockroach medicinal material, especially the content determination of the 9 kinds of amino acids, and the method can also be used in quality control.

[0111] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0112] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining the amino acid content in American cockroaches using a one-measurement-multiple-evaluation method, characterized in that: The following steps are involved: Step S1: hydrolyzing the American cockroach powder in a hydrochloric acid solution, filtering, evaporating to dryness, and then dissolving and fixing the volume with a hydrochloric acid solution to obtain a test solution; Step S2: dissolving glycine, arginine, threonine, alanine, tyrosine, valine, isoleucine, leucine, and phenylalanine in a hydrochloric acid solution to obtain a mixed reference solution; Step S3: Take 1 mL of the test solution, blank control solution and mixed reference solution, add 1 mL of triethylamine in acetonitrile solution and phenyl isothiocyanate in acetonitrile solution respectively, vortex mix well and place in a light-proof water bath, extract with n-hexane, and take the lower layer of solution for HPLC analysis.

2. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: The amount of the American cockroach powder in step S1 is 0.1-10 g; The concentration of the hydrochloric acid solution is 0.01-1.0 mol / L; The volume of the test solution after the dilution is 1-250 mL.

3. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: In step S2 The concentration of glycine in the mixed reference solution is 20.6-411.3 μg / mL; The concentration of arginine in the mixed reference solution is 15.8-315.5 μg / mL; The concentration of threonine in the mixed reference solution is 10.9-218.3 μg / mL; The concentration of alanine in the mixed reference solution is 18.4-368.0 μg / mL; The concentration of tyrosine in the mixed reference solution is 14.7-294.9 μg / mL; The concentration of valine in the mixed reference solution is 15.5-309.0 μg / mL; The concentration of isoleucine in the mixed reference solution is 8.0-159.3 μg / mL; The concentration of leucine in the mixed reference solution is 18.5-370.1 μg / mL; The concentration of phenylalanine in the mixed reference solution is 8.6-172.8 μg / mL; The concentration of the hydrochloric acid solution is 0.01-1.0 mol / L.

4. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: The blank control solution in step S3 is a 0.01-1.0 mol / L hydrochloric acid solution; The concentration of the triethylamine acetonitrile solution is 1-2 mol / L; The concentration of the acetonitrile solution of phenyl isothiocyanate is 1-2 mol / L.

5. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: In step S3, the dosage ratio of the test solution, the acetonitrile solution of triethylamine, and the acetonitrile solution of phenyl isothiocyanate is 1 mL: 0.5-1 mL: 0.5-1 mL; the dosage ratio of the blank control solution, the acetonitrile solution of triethylamine, and the acetonitrile solution of phenyl isothiocyanate is 1 mL: 0.5-1 mL: 0.5-1 mL; the dosage ratio of the mixed reference solution, the acetonitrile solution of triethylamine, and the acetonitrile solution of phenyl isothiocyanate is 1 mL: 0.5-1 mL: 0.5-1 mL.

6. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: The number of extractions during the n-hexane extraction in step S3 is 3-5 times.

7. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: The chromatographic column used in the high performance liquid chromatography analysis in step S3 is a C18 chromatographic column; The flow rate during the high performance liquid chromatography analysis is 0.3-1 mL / min; The detection wavelength during the high performance liquid chromatography analysis is 254 nm; The column temperature during the HPLC analysis is 20-40°C; The injection volume used in the high performance liquid chromatography analysis is 1-20 μL.

8. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: The preparation method of mobile phase A used in the high performance liquid chromatography analysis described in step S3 is as follows: Dissolve acetonitrile in a 0.1 mol / L sodium acetate solution, and then adjust the pH to 6.45-6.55 with 36% acetic acid to obtain mobile phase A; The mass ratio of the acetonitrile to the sodium acetate solution is 1-10:90-99.

9. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: The preparation method of mobile phase B used in the high performance liquid chromatography analysis described in step S3 is as follows: Mix acetonitrile and water evenly to obtain mobile phase B; The mass ratio of acetonitrile to water is 1-6:

1.

10. The method for determining the amino acid content in American cockroaches by using a one-measurement-multiple-evaluation method according to claim 1, characterized in that: When performing the high performance liquid chromatography analysis, the mass ratio of the mobile phase A and the mobile phase B added at 0-8 min of elution is 97:3; the mass ratio of the mobile phase A and the mobile phase B added at 8-20 min of elution is 95:5; the mass ratio of the mobile phase A and the mobile phase B added at 20-35 min of elution is 94.5:5.5; the mass ratio of the mobile phase A and the mobile phase B added at 35-40 min of elution is 93:7; the mass ratio of the mobile phase A and the mobile phase B added at 40-60 min of elution is 78:22; the mass ratio of the mobile phase A and the mobile phase B added at 60-63 min of elution is 77:23; The mass ratio of mobile phase A and mobile phase B added at 63-73 minutes of elution was 60:40; the mass ratio of mobile phase A and mobile phase B added at 73-83 minutes of elution was 55:45; the mass ratio of mobile phase A and mobile phase B added at 83-90 minutes of elution was 0:100; the mass ratio of mobile phase A and mobile phase B added at 90-100 minutes of elution was 97:3.

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