Method for measuring content of amino acid in periplaneta americana

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicinal materials, and particularly relates to a method for determining the content of amino acid in periplaneta americana. The method comprises the following steps: a, preparation of a test solution: taking American cockroach medicinal material powder, adding water, carrying out ultrasonic treatment, carrying out solid-liquid separation to obtain a separation solution, enabling the separation solution to pass through a cation exchange resin column, soaking, eluting, collecting the eluent, drying by distillation, adding a hydrochloric acid solution, dissolving, filtering, and taking a subsequent filtrate to obtain the test solution; b, preparing a reference substance solution; and c, determination: respectively and precisely sucking the reference substance solution and the test solution, injecting into a high performance liquid chromatograph, determining, and calculating the content by using an external standard two-point method logarithmic equation. The method provided by the invention has good linearity, repeatability, stability and recovery rate, and can effectively monitor the content of various amino acids in periplaneta americana.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a method for determining the content of amino acids in American cockroaches. Background Art

[0002] The American cockroach was first recorded in the Shennong's Herbal Classic. It is a traditional Chinese medicine with cold and salty properties. It has the functions of promoting blood circulation and dispersing blood stasis, eliminating malnutrition, promoting diuresis and reducing swelling, and detoxifying. It is mainly used to treat accumulation of masses, malnutrition in children, beriberi edema, furunculosis poisoning and various injuries. It is currently reported that the American cockroach contains polysaccharides, peptides and amino acids, nucleosides, oils and fats, polyols, alkaloids, isocoumarins, flavonoids, steroids and terpenoid compounds. Its main pharmacological activities are anti-tumor, anti-liver fibrosis, anti-inflammatory and detumescence, tissue repair and myocardial protection.

[0003] Amino acid is one of the active ingredients in American cockroaches. The detection methods of amino acids in American cockroaches include visible spectrophotometry, amino acid analyzer method, and derivatization method. The visible spectrophotometry method is used to determine the content of total amino acids with alanine as the reference substance. This method has poor specificity, cannot truly reflect the composition of each amino acid in American cockroaches, and has poor safety. The amino acid analyzer method is expensive and has high operating costs; the derivatization method is cumbersome to operate, has poor separation, has interference, and has poor stability of the test sample.

[0004] At present, there is an urgent need to develop a method that is easy to operate and can accurately detect the amino acid content in American cockroaches. Summary of the invention

[0005] The technical problem to be solved by the invention is to provide a method which is easy to operate and can accurately detect the contents of multiple amino acids in American cockroaches.

[0006] The technical solution adopted by the present invention to solve the above technical problem is to provide a method for determining the amino acid content in American cockroaches. The determination method comprises the following steps:

[0007] a. Preparation of the test solution: Take the American cockroach powder, add water and ultrasonicate, separate the solid and liquid to obtain a separated solution, take the separated solution, pass it through a cation exchange resin column, soak, elute, collect the eluate, evaporate to dryness, add hydrochloric acid solution to dissolve, filter, and take the filtrate to obtain;

[0008] b. Preparation of reference solution: accurately weigh glycine reference, serine reference, aspartic acid reference, glutamic acid reference, threonine reference, alanine reference, cystine reference, proline reference, lysine hydrochloride reference, valine reference, methionine reference, histidine reference, tyrosine reference, arginine reference, isoleucine reference, leucine reference, and phenylalanine reference, and add hydrochloric acid solution to prepare;

[0009] c. Determination: Accurately pipette the reference solution and the test solution into high performance liquid chromatography, and calculate the content using the external standard two-point method logarithmic equation.

[0010] Furthermore, in step a, the preparation of the test solution is as follows: 0.5-2 g of American cockroach medicinal material powder is placed in a stoppered conical flask, 50 mL of water is added, the stopper is sealed, the weight is weighed, ultrasonicated for 15-120 min, cooled, weighed again, the lost weight is supplemented with water, shaken well, solid-liquid separation is performed to obtain a separated liquid, 10 mL of the separated liquid is passed through a 732 type cation exchange resin column, soaked for 10 minutes, eluted with 200 mL of water, the water is discarded, and then eluted with 150 mL of 4 mol / L ammonia solution, the eluate is collected, evaporated to dryness, 2 mL of 0.1 mol / L hydrochloric acid solution is added to the residue to dissolve it, filtered, and the filtrate is obtained.

[0011] Furthermore, 1 g of the American cockroach medicinal material powder is placed in a stoppered conical bottle.

[0012] Preferably, the ultrasonication time is 60 min.

[0013] Furthermore, the power of the ultrasound is 200-800W, and the frequency of the ultrasound is 20-80kHz.

[0014] Preferably, the power of the ultrasound is 500W and the frequency is 40kHz.

[0015] Furthermore, the solid-liquid separation is centrifugation or filtration.

[0016] Furthermore, the inner diameter of the cation exchange resin column is 1 to 2 cm, and the column height is 10 to 20 cm.

[0017] Preferably, the inner diameter of the cation exchange resin column is 1.3 cm and the column height is 15 cm.

[0018] Further, in step b, the reference solution is prepared as follows: accurately weigh glycine reference, serine reference, aspartic acid reference, glutamic acid reference, threonine reference, alanine reference, cystine reference, proline reference, lysine hydrochloride reference, valine reference, methionine reference, histidine reference, tyrosine reference, arginine reference, isoleucine reference, leucine reference, and phenylalanine reference, and add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 2.5 μmol of each in 1 mL.

[0019] Furthermore, in step c, the determination is to accurately aspirate 1 to 20 μL and 1 to 20 μL of the reference solution, and inject 1 to 20 μL of the test solution into a high performance liquid chromatograph for determination, and calculate the content using an external standard two-point method logarithmic equation to obtain the result.

[0020] Preferably, the determination is to accurately aspirate 5 μL and 10 μL of the reference solution, respectively, and inject 10 μL of the test solution into a high performance liquid chromatograph for determination, and calculate the content using an external standard two-point method logarithmic equation to obtain the result.

[0021] Furthermore, in step c, the detection conditions of the high performance liquid chromatograph are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; 1% heptafluorobutyric acid solution and methanol are used as mobile phases for gradient elution; the column temperature is 25-40°C; evaporative light scattering detector is used for detection; the drift tube temperature is 50-80°C; the gas pressure is 20-40psi; the sprayer mode is cooling; the gain value is 100-1000; and the theoretical plate number calculated based on the phenylalanine peak should be no less than 5000.

[0022] Furthermore, in step c, the elution procedure of the mobile phase is: from 0 to 10 min, the volume percentage of the 1% heptafluorobutyric acid solution is 100%, and the volume percentage of methanol is 0%; from 10 to 50 min, the volume percentage of the 1% heptafluorobutyric acid solution decreases from 100% to 40%, and the volume percentage of methanol increases from 0 to 60%.

[0023] Preferably, in step c, the drift tube temperature is 60° C.; the gas pressure is 30 psi; the nebulizer mode is cooling; and the gain value is 500.

[0024] Preferably, in step c, the chromatographic column is a Yuexu LP-C18 chromatographic column, 4.6 mm×250 mm, 5 μm.

[0025] Preferably, in step c, the column temperature is 25°C.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a method for measuring the content of various amino acids in American cockroaches. The present invention obtains a detection method by preparing a test solution and improving detection parameters, and can accurately measure the total amount of 17 kinds of amino acids, including glycine, serine, aspartic acid, glutamic acid, threonine, alanine, cystine, proline, lysine, valine, methionine, histidine, tyrosine, arginine, isoleucine, leucine, and phenylalanine, in American cockroach medicinal materials, and can truly reflect the composition of each amino acid and accurately quantify. The peak shape of each peak of the sample measured by the method of the present invention is good, the separation between the peaks is good, the precision is high, the repeatability is good, the linearity, repeatability, stability and recovery rate are good, and the content of various amino acids in American cockroaches can be effectively monitored. The whole detection process of the method of the present invention is short in time, which greatly saves time and cost, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1It is the HPLC chromatogram of the reference substance solution in Example 1 (in order of peak elution time, glycine, serine, aspartic acid, glutamic acid, threonine, alanine, cystine, proline, lysine, valine, methionine, histidine, tyrosine, arginine, isoleucine, leucine, and phenylalanine).

[0029] Figure 2 It is the HPLC chromatogram of the test solution in Example 1 (in order of peak elution time, glycine, serine, aspartic acid, glutamic acid, threonine, alanine, proline, lysine, valine, histidine, tyrosine, arginine, isoleucine, leucine, and phenylalanine).

[0030] Figure 3 This is the chromatogram of the Atlantis T3 C18 chromatographic column in the selection of chromatographic column in Example 1.

[0031] Figure 4 This is the chromatogram of the CAPCELLAQ chromatographic column in the selection of the chromatographic column in Example 1.

[0032] Figure 5 This is the chromatogram of the Symmetryshield RP 18 chromatographic column in the selection of chromatographic column in Example 1.

[0033] Figure 6 This is the chromatogram of the Phenomenex Fusion-RP chromatographic column in the selection of chromatographic column in Example 1.

[0034] Figure 7 This is the chromatogram of the Yuexu LP-C18 chromatographic column selected in Example 1.

[0035] Figure 8 This is a chromatogram of the mobile phase selected in Example 1 using methanol and nonafluoropentanoic acid-heptafluorobutyric acid-trifluoroacetic acid-water (1:0.5:0.6:1000) as the mobile phase.

[0036] Fig. 9 This is a chromatogram of the mobile phase selected in Example 1 using methanol-1% nonafluorovaleric acid aqueous solution as the mobile phase.

[0037] Fig.10 This is a chromatogram of the mobile phase selected in Example 1 using methanol-1% pentafluoropropionic acid aqueous solution as the mobile phase.

[0038] Fig.11 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and acetonitrile as the mobile phase (time 0-10-60 min, acetonitrile 0-1-20%).

[0039] Fig.12This is a chromatogram of the mobile phase selection in Example 1 using 1% heptafluorobutyric acid and methanol as the mobile phase (time 0-10-30 min, methanol 0-0-20%).

[0040] Fig.13 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and methanol as the mobile phase (time 0-10-30 min, methanol 0-0-40%).

[0041] Fig.14 This is a chromatogram of the mobile phase selection in Example 1 using 1% heptafluorobutyric acid and methanol as the mobile phase (time 0-10-11 min, methanol 0-0-20%).

[0042] Fig.15 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and methanol as the mobile phase (time 0-10-60 min, methanol 0-0-50%).

[0043] Fig.16 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and acetonitrile as the mobile phase (time 0-10-30 min, acetonitrile 0-0-20%).

[0044] Fig.17 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and acetonitrile as the mobile phase (time 0-10-11 min, acetonitrile 0-0-10%).

[0045] Fig.18 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and acetonitrile as the mobile phase (time 0-10-50-51-60 min, acetonitrile 0-0-20-0-0%).

[0046] Fig.19 This is a chromatogram of the mobile phase selected in Example 1 using 1% heptafluorobutyric acid and acetonitrile as the mobile phase (time 0-10-60 min, acetonitrile 0-0-20%).

[0047] Fig. 20 This is the chromatogram for selecting the column temperature in Example 1.

[0048] Fig.21 This is the selected chromatogram of the detector parameter drift tube temperature in Example 1.

[0049] Fig. 22 Selected chromatograms of different gas pressures for detector parameters in Example 1.

[0050] Fig.23 These are selected chromatograms of different detector gains in Example 1.

[0051] Fig.24This is the standard curve of glycine for linear investigation in Example 1.

[0052] Fig.25 This is the standard curve of serine for linear investigation in Example 1.

[0053] Fig.26 This is the standard curve of aspartic acid for linear investigation in Example 1.

[0054] Fig. 27 This is the standard curve of glutamate for linear investigation in Example 1.

[0055] Fig.28 This is the standard curve for linearity testing of threonine in Example 1.

[0056] Fig.29 This is the standard curve of alanine for linear investigation in Example 1.

[0057] Fig.30 This is the standard curve for the linearity test of cystine in Example 1.

[0058] Fig.31 This is the standard curve for the linearity test of proline in Example 1.

[0059] Fig.32 This is the standard curve for the linearity test of lysine in Example 1.

[0060] Fig.33 This is the standard curve for the linear investigation of valine in Example 1.

[0061] Fig.34 This is the standard curve for the linearity test of methionine in Example 1.

[0062] Fig.35 This is the standard curve of histidine for linear investigation in Example 1.

[0063] Fig.36 This is the standard curve for linearity testing of tyrosine in Example 1.

[0064] Fig.37 This is the standard curve of arginine for linear investigation in Example 1.

[0065] Fig.38 This is the standard curve of isoleucine for linear investigation in Example 1.

[0066] Fig.39 This is the standard curve of leucine for linearity investigation in Example 1.

[0067] Fig.40 This is the standard curve for linear investigation of phenylalanine in Example 1.

[0068] Fig.41 This is a chromatogram for the specificity investigation of Example 1. DETAILED DESCRIPTION

[0069] The present invention will be further described in detail below through specific examples. The raw materials and reagents involved in the specific implementation methods are all commercially available.

[0070] Example 1

[0071] 1. Instruments and test drugs

[0072] 1.1 Instrument

[0073] Waters e2695 high performance liquid chromatograph, 2424 evaporative light scattering detector, Empower 2 workstation; Mettler-Toledo XS105D electronic analytical balance; KQ 5200DB CNC ultrasonic cleaner from Kunshan Ultrasonic Instrument Co., Ltd.; TG 16-W micro high-speed centrifuge from Changsha Xiangyi Centrifuge Co., Ltd.; 1L filtration device from Tianjin Jinteng Experimental Equipment Co., Ltd.; 0.2μm water system microporous filtration membrane from Tianjin Jinteng Experimental Equipment Co., Ltd.

[0074] 1.2 Reagents and Reference Materials

[0075] Chromatographic grade methanol and acetonitrile were from Fisher Company, USA; water was Yibao purified water; and the other reagents were of analytical grade.

[0076] 17 kinds of amino acid mixed standard solution (H type, batch number: DSP2558, Wako Pure Chemical Industries, Ltd., Japan).

[0077] 17 kinds of amino acid mixed standard solution (H type, batch number: DSE4882, Wako Pure Chemical Industries, Ltd., Japan).

[0078] 17 kinds of amino acid mixed solution (16001, GBW(E)100062, China National Institute of Metrology).

[0079] Glycine (C 2 H 5 NO 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0080] Serine (C 3 H 7 NO 3 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0081] Aspartic acid (C 4 H 7 NO 4), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0082] Glutamate (C 5 H 9 NO 4 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0083] Threonine (C 4 H 9 NO 3 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0084] Alanine (C 3 H 7 NO 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0085] Cystine (C 6 H 12 N 2 O 4 S 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.8%).

[0086] Proline (C 5 H 9 NO 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0087] Lysine hydrochloride (C 6 H 14 N 2 O 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 100.0%).

[0088] Valine (C 5 H 11 NO 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0089] Methionine (C5 H 11 NO 2 S), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0090] Histidine (C 6 H 9 N 3 O2), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0091] Tyrosine (C 9 H 11 NO 3 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.8%).

[0092] Arginine (C 6 H 14 N 4 O 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content is 99.9%).

[0093] Isoleucine (C 6 H 13 NO 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0094] Leucine (C 6 H 13 NO 2 ), China Food and Drug Inspection Institute batch number: 140624-201506, chemical reference substance for content determination (content 99.9%).

[0095] Phenylalanine (C 9 H 11 NO 2 ), China Food and Drug Inspection Institute, batch number: 140624-201506, chemical reference substance for content determination (content 100.0%).

[0096] American cockroach, batch number 161002, Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.

[0097] 2. Methods and Results

[0098] 2.1 Chromatographic conditions

[0099] Octadecylsilane bonded silica gel as filler (LP-C 18 Chromatographic column, 4.6 mm × 250 mm, 5 μm); 1% heptafluorobutyric acid solution as mobile phase A, methanol as mobile phase B, elution according to the gradient in Table 1; column temperature 25°C; evaporative light scattering detector detection. The theoretical plate number calculated based on the phenylalanine peak should not be less than 5000.

[0100] Table 1 Mobile phase gradient elution program

[0101] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 100 0 10～60 100→50 0→50

[0102] 2.2 Preparation of test solution

[0103] Accurately weigh 1 g of American cockroach medicinal material (batch number 161002) powder, place it in a stoppered conical flask, add 50 mL of water, weigh it, extract it by ultrasound (power 500 W, frequency 40 kHz) for 60 min, let it cool, weigh it again, make up the lost weight with water, shake it well, centrifuge it (8000 r / min) for 30 min, accurately transfer 10 mL of the centrifuge liquid, pass it through a 732 type cation exchange resin column (inner diameter 1.3 cm, column height 15 cm), soak it for 10 min, elute it with 200 mL of water, discard the water, and then elute it with 150 mL of 4 mol / L ammonia water at a flow rate of 5 mL / min, collect the eluate, evaporate it to dryness, accurately add 2 mL of 0.1 mol / L hydrochloric acid solution to dissolve the residue, mix it well, filter it, and take the filtrate to obtain the product.

[0104] 2.3 Preparation of reference solution

[0105] Referring to the provisions of GB5009.124-2016 "Determination of Amino Acids in Foods", 17 kinds of amino acid mixed standard solutions were selected.

[0106] Alternatively, accurately weigh glycine reference substance, serine reference substance, aspartic acid reference substance, glutamic acid reference substance, threonine reference substance, alanine reference substance, cystine reference substance, proline reference substance, lysine hydrochloride reference substance, valine reference substance, methionine reference substance, histidine reference substance, tyrosine reference substance, arginine reference substance, isoleucine reference substance, leucine reference substance, and phenylalanine reference substance respectively, place them in a 50 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve them into a mixed reference solution containing 2.5 μmol of each individual reference substance per 1 mL, and shake well to obtain the mixed reference solution.

[0107] 2.4 Determination method

[0108] Accurately pipette 5μL and 10μL of the reference solution, and inject 10μL of the test solution into the high performance liquid chromatograph for determination. Calculate using the external standard two-point method logarithmic equation to obtain the product. This product contains glycine (C 2 H5 NO 2 ), serine (C 3 H 7 NO 3 ), aspartic acid (C 4 H 7 NO 4 ), Glutamate (C 5 H 9 NO 4 ), threonine (C 4 H 9 NO 3 ), alanine (C 3 H 7 NO 2 ), cystine (C 6 H 12 N 2 O 4 S 2 ), proline (C 5 H 9 NO 2 ), lysine (C 6 H 14 N 2 O 2 ), valine (C 5 H 11 NO 2 ), methionine (C 5 H 11 NO 2 S), histidine (C 6 H 9 N 3 O 2 ), tyrosine (C 9 H 11 NO 3 ), arginine (C 6 H 14 N 4 O 2 ), isoleucine (C 6 H 13 NO 2 ), leucine (C 6 H 13 NO 2 ), phenylalanine (C 9 H 11 NO 2 ) shall not be less than 2.0%. The spectra of the reference solution and the test solution are shown in Figure 1-2 .

[0109] 2.5 Screening of chromatographic conditions

[0110] 2.5.1 Selection of chromatographic columns

[0111] The present invention investigated the Atlantis T3 C18 chromatographic column (4.6 mm × 250 mm, 5 μm), and 17 amino acids were able to achieve baseline separation (see Figure 3 ); The separation of the CAPCELLAQ column (4.6 mm × 150 mm, 5 μm) was poor, and the two amino acid reference substances with retention times of 3.168 min and 3.266 min were not completely separated. (See Figure 4 ); when the Symmetryshield RP 18 column (4.6 mm × 250 mm, 5 μm) was used for separation, the first and second peaks completely overlapped and were not separated (see Figure 5 ); The separation of Phenomenex Fusion-RP (4.6 mm × 150 mm, 4 μm) column is even worse, with the 1st and 2nd peaks, and the 4th and 5th peaks overlapping each other (see Figure 6 ); Yuexu LP-C18 (4.6mm×250mm, 5μm) has better separation, and 17 peaks are completely separated (see Figure 7 ).

[0112] Comparing the chromatographic columns, only Atlantis T3 C18 (4.6mm×250mm, 5μm) and Yuexu LP-C18 (4.6mm×250mm, 5μm) columns can achieve baseline separation of the target peaks. Because the pH value of the mobile phase is <2, which exceeds the pH range of 2 to 8 of the T3 column, Yuexu LP-C18 column (4.6mm×250mm, 5μm) was selected.

[0113] 2.5.2 Selection of mobile phase

[0114] The present invention investigated the use of nonafluoropentanoic acid-heptafluorobutyric acid-trifluoroacetic acid-water (1:0.5:0.6:1000) as mobile phase A and methanol as mobile phase B for gradient elution. As a result, multiple peaks were not completely separated between 30min and 40min (see Figure 8 ); methanol (B)-1% nonafluoropentanoic acid aqueous solution (A) as the mobile phase, gradient elution, the results of the first and second peaks completely overlapped together (see Fig. 9 ); and the mobile phase was methanol (B)-1% pentafluoropropionic acid aqueous solution (A) for gradient elution, and the results showed that the 4th and 5th peaks completely overlapped and were not separated (see Fig.10 ); using methanol (B)-1% heptafluorobutyric acid aqueous solution (A) as the mobile phase, gradient elution was performed, and the separation was good, and 17 peaks were completely separated (see Figure 7 ). The above gradient elution procedures are all carried out according to Table 1.

[0115] By comparing several different mobile phases, the results showed that when methanol-1% heptafluorobutyric acid aqueous solution was used as the mobile phase, the separation was the best and the 17 amino acids could be completely separated.

[0116] The present invention also conducted investigations on different gradients of 1% heptafluorobutyric acid aqueous solution (A) and organic phase (methanol, acetonitrile):

[0117] From 0 to 10 minutes, the volume percentage of acetonitrile increased from 0 to 1%, and from 10 to 60 minutes, the volume percentage of acetonitrile increased from 1% to 20%. Fig.11 .

[0118] From 0 to 10 minutes, the volume percentage of methanol remains unchanged at 0%; from 10 to 30 minutes, the volume percentage of methanol increases from 0 to 20%. Fig.12 .

[0119] From 0 to 10 minutes, the volume percentage of methanol remained unchanged at 0%; from 10 to 30 minutes, the volume percentage of methanol increased from 0 to 40%. Fig.13 .

[0120] From 0 to 10 minutes, the volume percentage of methanol remained unchanged at 0%; from 10 to 11 minutes, the volume percentage of methanol increased from 0 to 20%. Fig.14 .

[0121] From 0 to 10 minutes, the volume percentage of methanol remains unchanged at 0%; from 10 to 60 minutes, the volume percentage of methanol increases from 0 to 50%. Fig.15 .

[0122] From 0 to 10 minutes, the volume percentage of acetonitrile remained unchanged at 0%; from 10 to 30 minutes, the volume percentage of acetonitrile increased from 0 to 20%. Fig.16 .

[0123] From 0 to 10 minutes, the volume percentage of acetonitrile remained unchanged at 0%; from 10 to 11 minutes, the volume percentage of acetonitrile increased from 0 to 10%. Fig.17 .

[0124] From 0 to 10 minutes, the volume percentage of acetonitrile remained unchanged at 0%; from 10 to 50 minutes, the volume percentage of acetonitrile increased from 0 to 20%, from 50 to 51 minutes, the volume percentage of acetonitrile decreased from 20% to 0%, and from 51 to 60 minutes, the volume percentage of acetonitrile remained at 0%. Fig.18 .

[0125] From 0 to 10 minutes, the volume percentage of acetonitrile remained unchanged at 0%; from 10 to 60 minutes, the volume percentage of acetonitrile increased from 0 to 20%. Fig.19 .

[0126] 2.5.3 Selection of column temperature

[0127] The effects of 25℃, 30℃ and 40℃ on separation were investigated. The results showed that the peaks were best separated when the column temperature was 25℃, so the column temperature was finally determined to be 25℃. Fig. 20 .

[0128] 2.5.4 Selection of detector parameters

[0129] The present invention investigated the signal response of drift tube temperatures of 40°C, 50°C, 60°C, 70°C, and 80°C. The result showed that the temperature of 40°C was too low and the baseline noise was too large; there was no significant difference at other temperatures. Considering the sample stability and the influence of the solvent evaporation temperature, the drift tube temperature was finally selected to be 60°C. Fig.21 .

[0130] When the drift tube temperature was 60℃, the effects of gas pressures of 20psi, 25psi, 30psi, 35psi, and 40psi on separation were investigated. The results showed that gas pressure was inversely proportional to peak area. The lower the gas pressure, the larger the peak area, and the separation of each peak slightly worsened. When the gas pressure was too high, the peak area was too small, affecting the integration. After comprehensive consideration, 30psi was selected as the gas pressure. Fig. 22 .

[0131] The sprayer mode examined both cooling and heating, and the results showed no significant difference, so cooling was ultimately chosen.

[0132] The effect of gain on separation was also investigated. When the gain value was 100, the signal was weakened and the peak area was reduced; when the gain was 1000, the peak area increased and the separation was slightly worse; when the gain was 500, the peak area was appropriate and the baseline was also better, so the detector gain value was selected as 500. Fig.23 .

[0133] 2.6 Investigation of test solution preparation

[0134] 2.6.1 Selection of extraction solvent

[0135] The present invention investigates different solvents of water, 30% ethanol and 70% ethanol. 1g of American cockroach medicinal material (batch number 161002) powder is accurately weighed, placed in a stoppered conical bottle, 50mL of solvent is accurately added, the weight is weighed, ultrasonic treatment (power 500W, frequency 40kHz) is performed for 60min, cooled, weighed again, the lost weight is supplemented, shaken, allowed to stand, centrifuged (8000r / min) for 30min, 10mL of supernatant is accurately transferred to an evaporating dish, evaporated to dryness on a water bath, 1mL of 0.1mol / L HCl solution is accurately added, ultrasonically dissolved, passed through a 0.2μm microporous filter membrane, and the filtrate is taken for determination, and the results are shown in Table 2. The water extract has the highest content and is safe, non-toxic and pollution-free, so water is selected as the extraction solvent.

[0136] Table 2 Extraction solvent investigation results table (n = 3)

[0137] Extraction solvent water 30% ethanol 70% ethanol Total amount (mg / g) 27.27 20.72 20.31

[0138] 2.6.2 Selection of extraction method

[0139] Accurately weigh 1 g of American cockroach powder (batch number 161002), place in a stoppered conical flask, accurately add 50 mL of water, weigh the weight, heat under reflux for 60 min, cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate as the test solution.

[0140] Separately, accurately weigh 1 g of American cockroach powder (batch number 161002), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of water, ultrasonically treat (power 500 W, frequency 40 kHz) for 60 min, let cool, weigh it again, make up the lost weight with water, shake well, centrifuge (8000 r / min) for 30 min, accurately transfer 10 mL of the centrifuge liquid to an evaporating dish, evaporate it to dryness on a water bath, accurately add 1 mL of 0.1 mol / L HCl solution, ultrasonically dissolve it, pass it through a 0.2 μm microporous filter membrane, and take the filtrate as the test solution.

[0141] The test solutions prepared by the two different methods were tested, and the results are shown in Table 3. The ultrasonic extraction method was selected because the sample solution had a higher content.

[0142] Table 3 Extraction method investigation results table (n = 3)

[0143] Extraction method Water Ultrasound Water Reflux Total amount (mg / g) 25.13 22.21

[0144] 2.6.3 Selection of extraction time

[0145] 1 g of American cockroach medicinal material (batch number 161002) powder was accurately weighed and placed in a stoppered conical bottle. 50 mL of water was accurately added and weighed. The mixture was extracted by ultrasound (power 500 W, frequency 40 kHz) for 15 min, 30 min, 60 min, 90 min, and 120 min, respectively. The mixture was cooled and weighed again. The weight lost was supplemented with water. The mixture was shaken and centrifuged (8000 r / min) for 30 min. 10 mL of the centrifuge was accurately transferred to an evaporating dish and evaporated to dryness on a water bath. 1 mL of 0.1 mol / L HCl solution was accurately added and dissolved by ultrasound. The mixture was filtered through a 0.2 μm microporous filter membrane and the filtrate was taken for determination. The results are shown in Table 4. The amino acid content gradually increased with the increase of ultrasound time, and there was no significant change after 60 min. The amino acids in the test sample could be completely extracted in 60 min, and the extraction time was selected to be 60 min.

[0146] Table 4 Extraction time investigation results table (n = 3)

[0147] Extraction time 15min 30min 60min 90min 120min Total amount (mg / g) 24.23 25.69 28.67 28.64 28.70

[0148] 2.6.4 Selection of purification method for test solution

[0149] The test sample solution prepared by direct sampling and evaporation after ultrasonic extraction has poor separation, and the interference peak before 6 minutes is too large, resulting in the baseline of the target peak not being completely lowered, and the interference of glutamic acid is relatively large; the present invention investigates the 10% sulfosalicylic acid precipitation method and the 732 type cation exchange resin method to purify the American cockroach medicinal material extract, and the result is that the 10% sulfosalicylic acid precipitation does not improve the interference and baseline, but introduces an impurity peak; after the test sample extract passes through the cation exchange resin, the large impurity peak before 6 minutes is improved, the baseline of the target peak becomes better, the interference of glutamic acid is also reduced, and the total amount of amino acids before and after the resin treatment is only 1.43%, and there is no obvious difference, so the purification method is selected as the cation exchange resin treatment, and the results are shown in Table 5.

[0150] Table 5 Purification method investigation results

[0151]

[0152]

[0153] 2.6.5 Selection of column elution volume

[0154] 1 g of American cockroach powder (batch number 161201) was accurately weighed and placed in a stoppered conical flask. 50 mL of water was accurately added and weighed. Ultrasonic extraction (power 500 W, frequency 40 kHz) was performed for 60 min. The mixture was cooled and weighed again. The lost weight was supplemented with water. The mixture was shaken and centrifuged (8000 r / min) for 30 min. 10 ml of the centrifuge solution was accurately drawn and passed through a 732 cation exchange resin column (inner diameter 1.3 cm, column height 15 cm). The mixture was then soaked for 10 min. n, eluted with 200mL of water, discarded the water, eluted with 4mol / L ammonia water, collected 80mL, 20mL, and 20mL in sections, evaporated to dryness respectively, accurately added 2mL of 0.1mol / L hydrochloric acid solution to the residue to dissolve, mixed, filtered, and took the filtrate for detection. The results showed that 80mL elution volume can elute more than 98% of the amino acids; 120mL elution can completely elute all the amino acids. In order to ensure the complete elution of amino acids, the elution volume was determined to be 150mL. The results are shown in Table 6.

[0155] Table 6 Column elution volume investigation results (n = 3)

[0156] Elution volume (ml) 80 20 20 Total amount (mg / g) 23.03 0.29 Not detected

[0157] 3 Methodological investigation

[0158] 3.1 Linear investigation

[0159] 1 μL, 2 μL, 5 μL, 10 μL, and 20 μL of the 17 kinds of amino acid mixed standard solutions were injected into the liquid chromatograph for determination. The logarithm of the concentration (mg / mL) of the reference substance was used as the abscissa, and the logarithm of the peak area of ​​the reference substance was used as the ordinate to calculate the regression equation, correlation coefficient, and linear range. The results are shown in Tables 7 to 23. Figures 24-40 .

[0160] Table 7 Investigation of linear relationship of glycine

[0161] serial number X(mg / mL) Response value 1 0.018780 32610.280101 2 0.037560 82056.112175 3 0.093900 393515.247804 4 0.187800 1210533.421076 5 0.375600 3115790.552982

[0162] Glycine regression equation: Y = 1.56e + 000X + 7.18e + 000, correlation coefficient r = 999136. The results show that in the concentration range of 0.01878 ~ 0.3756 mg / mL, when the injection volume is 10 μL, the logarithm of the glycine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0163] Table 8 Investigation of linear relationship of serine

[0164]

[0165]

[0166] Serine regression equation: Y = 1.58e + 000X + 7.03e + 000, correlation coefficient r = 999809. The results showed that in the concentration range of 0.02628 ~ 0.5256 mg / mL, when the injection volume was 10 μl, the logarithm of the serine peak area had a good linear relationship with the logarithm of the reference substance concentration.

[0167] Table 9 Investigation of linear relationship of aspartic acid

[0168] serial number X(mg / mL) Response value 1 0.033280 27367.288142 2 0.066560 115426.833085 3 0.166400 541568.210820 4 0.332800 1616244.197193 5 0.665600 4458440.708935

[0169] Aspartic acid regression equation: Y = 1.69e + 000X + 7.00e + 000, correlation coefficient r = 998029. The results show that: in the concentration range of 0.03328 ~ 0.6656 mg / mL, when the injection volume is 10μl, the logarithm of the aspartic acid peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0170] Table 10 Investigation of linear relationship of glutamate

[0171] serial number X(mg / mL) Response value 1 0.036780 37381.914097 2 0.073560 129183.527965 3 0.183900 506621.950591 4 0.367800 1421686.674600 5 0.735600 3270201.456674

[0172] Glutamic acid regression equation: Y = 1.46e + 000X + 6.77e + 000, correlation coefficient r = 998077. The results show that: in the concentration range of 0.03678 ~ 0.7356 mg / mL, when the injection volume is 10μl, the logarithm of the glutamate peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0173] Table 11 Investigation of linear relationship of threonine

[0174] serial number X(mg / mL) Response value 1 0.029780 35272.189921 2 0.059560 104542.156477 3 0.148900 466109.941590 4 0.297800 1337069.850539 5 0.595600 3331214.994108

[0175] Threonine regression equation: Y = 1.54e + 000X + 6.91e + 000, correlation coefficient r = 999341. The results show that: in the concentration range of 0.02978 ~ 0.5956 mg / mL, when the injection volume is 10μl, the logarithm of the threonine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0176] Table 12 Investigation of linear relationship of alanine

[0177] serial number X(mg / mL) Response value 1 0.022280 26904.576427 2 0.044560 95201.821188 3 0.111400 426758.824899 4 0.222800 1303683.810489 5 0.445600 3628343.930741

[0178] Alanine regression equation: Y = 1.64e + 000X + 7.17e + 000, correlation coefficient r = 999407. The results show that: in the concentration range of 0.02228 ~ 0.4456 mg / mL, when the injection volume is 10μl, the logarithm of the alanine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0179] Table 13 Investigation of the linear relationship of cystine

[0180] serial number X(mg / mL) Response value 1 0.060080 75015.526133 2 0.120160 293980.689988 3 0.300400 1464705.175875 4 0.600800 4561189.657533 5 1.201600 12051161.444604

[0181] Cystine regression equation: Y = 1.70e + 000X + 7.00e + 000, correlation coefficient r = 998345. The results show that: in the concentration range of 0.06008 ~ 1.2016mg / mL, when the injection volume is 10μl, the logarithm of the cystine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0182] Table 14 Proline linear relationship investigation

[0183] serial number X(mg / mL) Response value 1 0.028780 38113.724890 2 0.057560 119939.097694 3 0.143900 544538.561417 4 0.287800 1719026.900069 5 0.575600 4952297.268348

[0184] Proline regression equation: Y = 1.63e + 000X + 7.10e + 000, correlation coefficient r = 999896. The results show that: in the concentration range of 0.02878 ~ 0.5756 mg / mL, when the injection volume is 10μl, the logarithm of the proline peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0185] Table 15 Investigation of the linear relationship of lysine

[0186]

[0187]

[0188] The regression equation of lysine is: Y = 1.61e + 000X + 7.24e + 000, and the correlation coefficient is r = 999176. The results show that in the concentration range of 0.03655-0.7310 mg / mL, when the injection volume is 10 μL, the logarithm of the lysine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0189] Table 16 Investigation of linear relationship of valine

[0190] serial number X(mg / mL) Response value 1 0.029300 41915.019537 2 0.058600 128530.867962 3 0.146500 561212.764807 4 0.293000 1710661.025046 5 0.586000 4975596.506264

[0191] Valine regression equation: Y = 1.60e + 000X + 7.08e + 000, correlation coefficient r = 999964. The results show that: in the concentration range of 0.0293 ~ 0.586 mg / mL, when the injection volume is 10μl, the logarithm of the valine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0192] Table 17 Investigation of the linear relationship of methionine

[0193] serial number X(mg / mL) Response value 1 0.037300 51247.405626 2 0.074600 161328.095703 3 0.186500 723255.962924 4 0.373000 2168363.105696 5 0.746000 6257833.479421

[0194] Methionine regression equation: Y = 1.61e + 000X + 7.02e + 000, correlation coefficient r = 999882. The results show that: in the concentration range of 0.0373 ~ 0.746 mg / mL, when the injection volume is 10 μ L, the logarithm of the methionine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0195] Table 18 Histidine linear relationship investigation

[0196] serial number X(mg / mL) Response value 1 0.038800 73829.973779 2 0.077600 264755.059080 3 0.194000 1203836.175676 4 0.388000 3573314.195724 5 0.776000 9755536.451013

[0197] Histidine regression equation: Y = 1.63e + 000X + 7.10e + 000, correlation coefficient r = 999131. The results show that in the concentration range of 0.0388-0.776 mg / mL, when the injection volume is 10 μL, the logarithm of the histidine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0198] Table 19 Tyrosine linear relationship investigation

[0199] serial number X(mg / mL) Response value 1 0.045300 64788.328606 2 0.090600 228828.265144 3 0.226500 1075200.990526 4 0.453000 3253359.197950 5 0.906000 9222140.522799

[0200] Tyrosine regression equation: Y = 1.66e + 000X + 7.07e + 000, correlation coefficient r = 999412. The results show that: in the concentration range of 0.0453 ~ 0.906 mg / mL, when the injection volume is 10 μ L, the logarithm of the tyrosine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0201] Table 20 Arginine linear relationship investigation

[0202] serial number X(mg / mL) Response value 1 0.043550 81651.189962 2 0.087100 294982.895462 3 0.217750 1370494.960737 4 0.435500 4128058.689118 5 0.871000 10870244.430304

[0203] The regression equation of arginine is: Y = 1.64e + 000X + 7.18e + 000, and the correlation coefficient is r = 998863. The results show that in the concentration range of 0.04355-0.871 mg / mL, when the injection volume is 10 μL, the logarithm of the arginine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0204] Table 21 Isoleucine linear relationship investigation

[0205] serial number X(mg / mL) Response value 1 0.032800 55937.931548 2 0.065600 164587.670933 3 0.164000 739219.218876 4 0.328000 2202999.852933 5 0.656000 6256753.367305

[0206] Isoleucine regression equation: Y = 1.58e + 000X + 7.10e + 000, correlation coefficient r = 999903. The results show that: in the concentration range of 0.0328 ~ 0.656 mg / mL, when the injection volume is 10 μ L, the logarithm of the isoleucine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0207] Table 22 Leucine linear relationship investigation

[0208]

[0209]

[0210] The regression equation of leucine is: Y = 1.61e + 000X + 7.09e + 000, and the correlation coefficient is r = 999763. The results show that in the concentration range of 0.0328 to 0.656 mg / mL, when the injection volume is 10 μL, the logarithm of the leucine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0211] Table 23 Investigation of linear relationship of phenylalanine

[0212] serial number X(mg / mL) Response value 1 0.041300 65350.417587 2 0.082600 221986.072795 3 0.206500 991004.798968 4 0.413000 3122222.538058 5 0.826000 8858480.686262

[0213] Phenylalanine regression equation: Y = 1.64e + 000X + 7.11e + 000, correlation coefficient r = 999706. The results show that: in the concentration range of 0.0413 ~ 0.826 mg / mL, when the injection volume is 10 μ L, the logarithm of the phenylalanine peak area has a good linear relationship with the logarithm of the reference substance concentration.

[0214] 3.2 Investigation of injection precision

[0215] According to the proposed method, 17 kinds of amino acid mixed standard solutions were injected continuously for 6 times, each injection was 10 μL, the peak area was recorded, and the RSD value was calculated. The results are shown in Table 24.

[0216] Table 24 Results of injection precision investigation

[0217]

[0218]

[0219] 3.3 Exclusivity Investigation

[0220] Take 0.1mol / L HCl solution as negative blank and measure according to the law. The result is at the retention time position corresponding to the chromatographic peak of the reference substance, and there is no interference peak, indicating that the method has good specificity. Fig.41 .

[0221] 3.4 Stability study of test solution

[0222] The powder of American cockroach medicinal material (batch number 161002) was taken, and a test solution was prepared according to the test solution preparation method of the present invention. The test was performed at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h, and the peak area was recorded. Since there were too many single amino acid data, they were uniformly calculated as the total amino acid content in the test solution for the convenience of statistics, and the RSD value was calculated. The results are shown in Table 25. The test solution was stable within 10 h.

[0223] Table 25 Stability study of test solution

[0224]

[0225] 3.5 Repeatability study

[0226] The powder of American cockroach medicinal material (batch number 161002) was taken, and 6 test solutions were prepared according to the method of the present invention for determination, and the total content and RSD value of 17 kinds of amino acids in the medicinal material were calculated. The results are shown in Table 26. It can be seen that the method has good repeatability.

[0227] Table 26 Repeatability test results (n=6)

[0228] serial number 1 2 3 4 5 6 average value RSD% Total amount (mg / g) 28.62 28.51 28.59 28.86 29.54 29.12 28.87 1.37

[0229] 3.6 Sample recovery rate

[0230] Take the powder of American cockroach medicinal material (batch number 161002) with known content, take 6 portions of the test sample of 1 / 2 amount according to the method of the present invention, respectively put them in 100mL conical flasks with stoppers, accurately add 50mL of an aqueous solution containing 17 kinds of amino acid mixed standard solutions (25mL→500mL), weigh the weight, extract by ultrasound (power 500W, frequency 40kHz) for 60min, let cool, weigh the weight again, make up the lost weight with water, shake well, centrifuge (8000r / min) for 30min, accurately measure 10mL of the centrifuge, pass it through a 732 type cation exchange resin column (inner diameter 1.3cm, column height 15cm), soak for 10min, elute with 200mL of water, discard the water, elute with 150mL of 4mol / L ammonia water, collect the eluate, evaporate to dryness, accurately add 2mL of 0.1mol / L hydrochloric acid solution to the residue to dissolve, shake well, filter, and take the filtrate to obtain. The results are shown in Table 27.

[0231] Table 27 Recovery results of 17 kinds of amino acids (n=6)

[0232]

[0233] As can be seen from Table 27, the average recovery rate of the total amount of 17 amino acids was 103.5%, RSD = 1.01%.

[0234] 3.7 Content Limit

[0235] According to the method of the present invention, the content of 17 kinds of amino acids in 10 batches of medicinal materials was determined, and the results showed that the amino acid content was 2.5% to 3.2%, with an average value of 2.8%. The original medicinal material standard stipulates that the total amino acid content shall not be less than 0.45%. The total amount of 17 kinds of amino acids determined by the current method is much higher than the original standard, and is more accurate and specific. The results are shown in Table 28.

[0236] Table 28 Determination results of total amino acid content in 10 samples of American cockroach (n=3)

[0237] batch number Total amount of 17 amino acids (mg / g) Total amount (mg / g, based on dry product) 160401 24.49 26.16 160501 29.58 31.50 160601 27.43 29.53 160901 27.97 29.92 160902 24.28 26.22 161002 28.87 31.44 161101 27.12 29.48 161201 23.39 25.07 170101 25.01 27.24 170201 25.40 27.05

[0238] In summary, the present invention can accurately determine the total amount of 17 amino acids in American cockroach medicinal materials by preparing the test solution and improving the detection parameters, and can more truly and accurately reflect the composition of each amino acid and accurately quantify. The peak shape of each peak of the sample measured by the method of the present invention is good, the separation between the peaks is good, the precision is high, the repeatability is good, it has good linearity, repeatability, stability and recovery rate, can effectively monitor the content of multiple amino acids in American cockroaches, and has good application prospects.

Claims

1. The method for determining the amino acid content in American cockroach is characterized in that: The steps include: a. Preparation of the test solution: Take the American cockroach powder, add water and ultrasonicate, separate the solid and liquid to obtain a separated solution, take the separated solution, pass it through a cation exchange resin column, soak, elute, collect the eluate, evaporate to dryness, add hydrochloric acid solution to dissolve, filter, and take the filtrate to obtain; b. Preparation of reference solution: accurately weigh glycine reference, serine reference, aspartic acid reference, glutamic acid reference, threonine reference, alanine reference, cystine reference, proline reference, lysine hydrochloride reference, valine reference, methionine reference, histidine reference, tyrosine reference, arginine reference, isoleucine reference, leucine reference, and phenylalanine reference, and add hydrochloric acid solution to prepare; c. Determination: Accurately pipette the reference solution and the test solution into high performance liquid chromatography, and calculate the content using the external standard two-point method logarithmic equation.

2. The measuring method according to claim 1, characterized in that: In step a, the preparation of the test solution is as follows: 0.5-2 g of American cockroach medicinal material powder is placed in a stoppered conical flask, 50 mL of water is added, the stopper is sealed, the weight is weighed, ultrasonicated for 15-120 min, cooled, weighed again, the lost weight is supplemented with water, shaken well, solid-liquid separation is performed to obtain a separated liquid, 10 mL of the separated liquid is passed through a 732 type cation exchange resin column, soaked for 10 minutes, eluted with 200 mL of water, the water is discarded, and then eluted with 150 mL of 4 mol / L ammonia solution, the eluate is collected, evaporated to dryness, 2 mL of 0.1 mol / L hydrochloric acid solution is added to the residue to dissolve it, filtered, and the filtrate is obtained.

3. The assay method according to claim 2, characterized in that: In step a, The ultrasound time is 60 min; The power of the ultrasound is 200-800W, and the frequency is 20-80kHz; Preferably, the power of ultrasound is 500W and the frequency is 40kHz; The solid-liquid separation is centrifugation or filtration; The inner diameter of the cation exchange resin column is 1 to 2 cm, and the column height is 10 to 20 cm; Preferably, the inner diameter of the cation exchange resin column is 1.3 cm and the column height is 15 cm.

4. The measuring method according to claim 1, characterized in that: In step b, the reference solution is prepared as follows: accurately weigh glycine reference, serine reference, aspartic acid reference, glutamic acid reference, threonine reference, alanine reference, cystine reference, proline reference, lysine hydrochloride reference, valine reference, methionine reference, histidine reference, tyrosine reference, arginine reference, isoleucine reference, leucine reference, and phenylalanine reference, add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 2.5 μmol of each in 1 mL, and obtain.

5. The assay method according to claim 1, wherein: In step c, the determination is to accurately aspirate 1 to 20 μL and 1 to 20 μL of the reference solution, and inject 1 to 20 μL of the test solution into a high performance liquid chromatograph for determination, and calculate the content using an external standard two-point method logarithmic equation to obtain the result.

6. The assay method according to claim 1, characterized in that: In step c, the detection conditions of the high performance liquid chromatograph are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; 1% heptafluorobutyric acid solution and methanol are used as mobile phases for gradient elution; the column temperature is 25-40° C.; an evaporative light scattering detector is used for detection; the drift tube temperature is 50-80° C.; the gas pressure is 20-40 psi; the sprayer mode is cooling; the gain value is 100-1000; and the number of theoretical plates calculated based on the phenylalanine peak should be no less than 5000.

7. The assay method according to claim 6, characterized in that: In step c, the elution procedure of the mobile phase is: from 0 to 10 min, the volume percentage of the 1% heptafluorobutyric acid solution is 100%, and the volume percentage of methanol is 0%; from 10 to 50 min, the volume percentage of the 1% heptafluorobutyric acid solution decreases from 100% to 40%, and the volume percentage of methanol increases from 0 to 60%.

8. The assay method according to claim 6, characterized in that: In step c, the drift tube temperature was 60°C; the gas pressure was 30 psi; the nebulizer mode was cooling; and the gain value was 500.

9. The assay method according to claim 6, characterized in that: In step c, the chromatographic column is a Yuexu LP-C18 chromatographic column, 4.6 mm×250 mm, 5 μm.

10. The determination method according to claim 6, characterized in that: In step c, the column temperature was 25°C.

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