Amino acid HPLC (high performance liquid chromatography) detection method for oyster medicinal material, processed product and standard decoction or formula granules thereof

Abstract

The invention discloses an amino acid HPLC (High Performance Liquid Chromatography) detection method for an oyster medicinal material, a processed product and a standard decoction or formula granules thereof. The method comprises the following steps: preparing a reference solution; taking an oyster medicinal material, an oyster processed product, an oyster standard decoction, oyster formula granules, an oyster processed product standard decoction or oyster processed product formula granules, and preparing a test solution; a preset amount of the test solution and the reference solution are taken respectively and injected into a liquid chromatograph for determination, the liquid chromatograph takes octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a phosphoric acid solution as a mobile phase B for gradient elution, and an amino acid HPLC determination curve map is obtained. The method is good in reproducibility, accurate and reliable, and a control means for amino acid determination can be provided for oyster medicinal materials, oyster processed products and standard decoction or formula granules thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and particularly relates to an amino acid HPLC detection method for oyster medicinal materials, processed products, and their standard decoctions or formula granules. Background Art

[0002] Oyster is the shell of the animals Ostrea gigas Thunberg, Ostrea rivularis Gould, or Ostrea talienwhanensis Crosse of the family Ostreidae. It is a commonly used traditional Chinese medicine in clinical practice included in Chinese Pharmacopoeia in previous editions. It belongs to the mineral traditional Chinese medicine in the ocean and is distributed along the coast of China. It can be harvested throughout the year and used raw or after calcination, and crushed before use. Oyster tastes salty, astringent, and slightly cold, and belongs to the liver and kidney meridians. It has the effects of calming the mind with heavy substances, suppressing yang to nourish yin, and softening hardness and dissipating nodules, and is mainly used for treating palpitations and insomnia, dizziness and tinnitus, scrofula and phlegm nodes, abdominal masses, etc. Calcined oyster has the effects of astringing and consolidating, and relieving acid and pain, and is commonly used for spontaneous sweating and night sweating, spermatorrhea and emission, metrorrhagia and leukorrhea, and stomachache with acid regurgitation. Oyster was first recorded in Shennong Ben Cao Jing and listed as a superior grade. "Oyster tastes salty and flat, and is mainly used for treating typhoid fever and chills, warm malaria with shivering, fright and anger, relieving convulsion and scrofula, and red and white leukorrhea in women. Taking it for a long time can strengthen the bones and joints, eliminate pathogenic factors, and prolong life." In the Han Dynasty, Zhang Zhongjing used oyster in a large number of prescriptions in Treatise on Febrile Diseases and Synopsis of Prescriptions of the Golden Chamber to treat diseases, and the number reached 11. All of the above reflect the important position of oyster in traditional Chinese clinical practice.

[0003] Modern pharmacological studies have shown that oysters have a protective effect on experimental liver injury, can significantly reduce the serum ALT and AST levels in mice with acute liver injury induced by CCl4, and alleviate the degree of hepatocyte injury; oyster polysaccharide can significantly reduce and inhibit the hemagglutination degree of influenza virus in dog kidney cell culture, and has a certain therapeutic effect on mice infected with herpes simplex virus type I, that is, it can enhance immunity; oyster natural active peptide (BPO) can inhibit the proliferation of human gastric cancer BGC-823 cells and has a significant anti-tumor effect; the aqueous extract of oyster can delay brain aging in ovariectomized rats, increase the thickness of the molecular layer of the striate cortex, decrease the ratio of the thickness of the molecular layer to the total thickness of the cortex, increase the number of large pyramidal cells per unit area in the CA2 region of the hippocampus, enhance the activity of superoxide dismutase (SOD), and decrease the content of malondialdehyde (MDA); the oyster extract can also reduce the elevated blood glucose in mice induced by alloxan without affecting the blood glucose of normal mice; oyster glycosaminoglycan has a protective effect on oxidative damage of vascular endothelial cells induced by hydrogen peroxide and can effectively prevent the occurrence of various cardiovascular diseases such as hypertension, arteriosclerosis, and stroke caused by vascular endothelial injury. Clinically, oysters can also treat diseases such as insomnia, chronic otitis media, hyperhidrosis in children, uterine fibroids, and breast hyperplasia. In summary, in recent years, the pharmacological and clinical effects of oysters have exceeded their basic efficacy in ancient times, and the medicinal value of oysters has been greatly improved.

[0004] Oysters contain a variety of essential amino acids for the human body. For the determination of amino acids, modern research uses Fourier transform near-infrared spectroscopy, formaldehyde method for enzyme hydrolysate determination, PITC (phenylisothiocyanate) pre-column derivation method, amino acid automatic analyzer, potentiometric titration method, Folin phenol colorimetric method, micellar electrokinetic capillary electrophoresis method, pH adjustment method, etc. The methods are relatively complex. In addition, high-performance liquid chromatography is the most commonly used amino acid detection method.

[0005] Oysters, their processed products of mineral Chinese medicines, and their formula granules are commonly used medicines in traditional Chinese medicine clinics. However, there is no detection method for amino acids in the current standards for oysters and their processed products, which is a defect in quality control. At the same time, establishing an amino acid detection method for each of the medicinal materials, standard decoctions, and formula granules of oysters and their processed products will increase the detection cost of traditional Chinese medicine standard research. Moreover, in the first part of the Chinese Pharmacopoeia (2020 Edition), there is no item for amino acid determination and no HPLC determination item for oysters and their processed products. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an HPLC detection method for amino acids in oyster medicinal materials, processed products, and their standard decoctions or formula granules. This method has good reproducibility, is accurate and reliable, and can provide a control means for amino acid determination for oysters, calcined oysters, and their standard decoctions or formula granules.

[0007] To solve the above technical problems, the present invention provides an amino acid HPLC detection method for oyster medicinal materials, processed products, and their standard decoctions or formula granules, comprising the following steps:

[0008] Prepare a reference solution, which includes serine reference substance, glycine reference substance, glutamic acid reference substance, aspartic acid reference substance, threonine reference substance, proline reference substance, alanine reference substance, valine reference substance, lysine reference substance, leucine reference substance, and phenylalanine reference substance.

[0009] Take oyster medicinal materials, processed oyster products, oyster standard decoctions, oyster formula granules, processed oyster product standard decoctions, or processed oyster product formula granules to prepare a test solution.

[0010] Respectively take a preset amount of the test solution and the reference solution, inject them into a liquid chromatograph for determination. The liquid chromatograph uses octadecylsilane-bonded silica gel as the filler, acetonitrile as mobile phase A, and a phosphoric acid solution as mobile phase B for gradient elution to obtain an amino acid HPLC determination curve spectrum.

[0011] In one embodiment, the gradient elution is carried out according to the following procedure:

[0012] 0 - 14 min, mobile phase A changes from 15% → 20%, and mobile phase B changes from 85% → 80%;

[0013] 14 min - 17 min, mobile phase A changes from 20% → 36%, and mobile phase B changes from 80% → 64%;

[0014] 17 min - 22 min, mobile phase A remains at 36%, and mobile phase B remains at 64%;

[0015] 22 min - 40 min, mobile phase A changes from 36% → 38%, and mobile phase B changes from 64% → 62%;

[0016] 40 min - 45 min, mobile phase A changes from 38% → 70%, and mobile phase B changes from 62% → 30%.

[0017] In one embodiment, the concentration of the phosphoric acid solution is 0.25% - 0.65%.

[0018] In one embodiment, the preset amount of the test solution or the reference solution is 0.5 μL - 1.5 μL;

[0019] The liquid chromatograph uses octadecylsilane-bonded silica gel as the filler, with a column length of 150 mm, an inner diameter of 2.1 mm, a particle size of 1.6 μm - 2.1 μm, and a column temperature of 25°C - 29°C;

[0020] When gradient elution is performed, the flow rate of the mobile phase is 0.25 mL / min to 0.29 mL / min;

[0021] The ultraviolet detection wavelength of the liquid chromatograph is 250 nm to 260 nm.

[0022] In one embodiment, before the test solution and the reference solution are injected into the liquid chromatograph for determination, the test solution is prepared by the following method:

[0023] Take oyster medicinal material powder, processed oyster powder, oyster standard decoction, oyster formula granule, processed oyster standard decoction or processed oyster formula granule, perform acid hydrolysis treatment to obtain the test solution.

[0024] In one embodiment, in the acid hydrolysis treatment, 8 mol / L to 10 mol / L hydrochloric acid is used for hydrolysis at 130 °C to 180 °C for 2 h to 5 h.

[0025] In one embodiment, the reference solution is prepared by the following method:

[0026] Take serine reference substance, glycine reference substance, glutamic acid reference substance, aspartic acid reference substance, threonine reference substance, proline reference substance, alanine reference substance, valine reference substance, lysine reference substance, leucine reference substance, phenylalanine reference substance, add 0.05 to 0.2 mol / L hydrochloric acid to make a mixed solution as the reference solution.

[0027] In one embodiment, the amino acid HPLC determination curve map includes 11 common peaks. Among them, the peak corresponding to serine is peak No. 1, the peak corresponding to glycine is peak No. 2, the peak corresponding to glutamic acid is peak No. 3, the peak corresponding to aspartic acid is peak No. 4, the peak corresponding to threonine is peak No. 5, the peak corresponding to proline is peak No. 6, the peak corresponding to alanine is peak No. 7, the peak corresponding to valine is peak No. 8, the peak corresponding to leucine is peak No. 9, the peak corresponding to lysine is peak No. 10, and the peak corresponding to phenylalanine is peak No. 11.

[0028] In one embodiment, in the amino acid HPLC determination curve map, taking the proline peak as the reference peak S1, the relative retention times of peak No. 1 to peak No. 5 and peak No. 7 with the reference peak S1 meet the following: peak No. 1 is 0.61, peak No. 2 is 0.65, peak No. 3 is 0.8, peak No. 4 is 0.83, peak No. 5 is 0.9, peak No. 7 is 1.09, and the relative standard deviation is within ±10%;

[0029] Taking the valine peak as the reference peak S2, the relative retention times of peak No. 9 to peak No. 11 with the reference peak S2 meet the following: peak No. 9 is 1.18, peak No. 10 is 1.26, peak No. 11 is 1.29, and the relative standard deviation is within ±10%.

[0030] Use of the amino acid HPLC detection method for the oyster medicinal material, its processed product, and its standard decoction or formula granule in (1) or (2):

[0031] (1) Determination of the amino acid content of the oyster medicinal material, its processed product, its standard decoction, and formula granule;

[0032] (2) Authenticity identification and quality control of the oyster medicinal material, its processed product, its medicinal material, standard decoction, and formula granule.

[0033] Implementing the present invention has the following beneficial effects:

[0034] The present invention has established a method for determining amino acids in the oyster medicinal material, its processed product, and its standard decoction or formula granule. This method can be simultaneously applied to the determination of the contents of different types of amino acids in the oyster medicinal material, processed oyster product, oyster standard decoction, oyster formula granule, standard decoction of processed oyster product, and formula granule of processed oyster product. It not only provides a control means for amino acid determination for the quality control of oysters, their processed products, and formula granules, greatly saving the cost of quality standard research and future sample inspection, but also provides a reference for the amino acid determination method of other mineral Chinese medicines. Brief Description of the Drawings

[0035] Figure 1 is the chromatogram of different elution gradients in Example 4 of the present invention;

[0036] Figure 2 is the chromatogram of different flow rates in Example 4 of the present invention;

[0037] Figure 3 is the chromatogram of different column temperatures in Example 4 of the present invention;

[0038] Figure 4 is the chromatogram of different chromatographic columns in Example 4 of the present invention;

[0039] Figure 5 is the superimposed characteristic chromatogram of 18 batches of oyster medicinal materials in Example 5 of the present invention;

[0040] Figure 6 is the superimposed characteristic chromatogram of 18 batches of oyster cut pieces in Example 5 of the present invention;

[0041] Figure 7 is the superimposed characteristic chromatogram of 18 batches of oyster standard decoctions in Example 5 of the present invention;

[0042] Figure 8 is the superimposed characteristic chromatogram of 3 batches of oyster (Crassostrea rivularis) formula granules in Example 5 of the present invention;

[0043] Figure 9It is the superimposed chromatogram of the characteristic fingerprints of 16 batches of calcined oyster medicinal materials in Example 5 of the present invention;

[0044] Figure 10 It is the superimposed chromatogram of the characteristic fingerprints of 16 batches of calcined oyster decoction pieces in Example 5 of the present invention;

[0045] Figure 11 It is the superimposed chromatogram of the characteristic fingerprints of 16 batches of standard decoctions of calcined oysters in Example 5 of the present invention;

[0046] Figure 12 It is the superimposed chromatogram of the characteristic fingerprints of 3 batches of calcined oyster (Crassostrea rivularis) formula granules in Example 5 of the present invention. Detailed implementation manners

[0047] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0048] Example 1: Investigation on the amino acid hydrolysis method of oyster (Crassostrea rivularis) formula granules

[0049] The oyster (Crassostrea rivularis) formula granules were respectively subjected to direct acid hydrolysis, hydrolysis after extraction with different solvents (hydrochloric acid, ethanol with different concentrations) before hydrolysis, and hydrolysis with different extraction methods (water extraction and alcohol precipitation, solid phase extraction, ion exchange), and the effects of different hydrolysis methods on the HPLC determination results of amino acids were investigated.

[0050] (1) Direct acid hydrolysis

[0051] Take an appropriate amount of oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, place them in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 3 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrates, evaporate to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid solution, transfer it to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it. Accurately measure 10 mL of each of the above test solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenylisothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, let stand at room temperature for 2 hours, then add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, let stand for 10 minutes, take the lower layer solution, filter, and take the continuous filtrate.

[0052] The results showed that: 11 characteristic peaks were obtained, the distribution of each characteristic peak was relatively uniform, the peak shape was good, and the extraction effect of each amino acid was better.

[0053] (2) Acid hydrolysis after extraction with 70% ethanol

[0054] Take 10 g of oyster (Crassostrea rivularis) formula granules, add 200 mL of 70% ethanol, reflux for 1 hour, cool, centrifuge, take the supernatant, evaporate to dryness, and hydrolyze with 9 mol / L hydrochloric acid.

[0055] The results showed that: for 10 g of oyster (Crassostrea rivularis) formula granules, after extraction with 70% ethanol, the supernatant was evaporated to dryness and then acid hydrolyzed. Compared with direct acid hydrolysis, the response value of chromatographic peaks increased after extraction with 70% ethanol, but the baseline noise also increased significantly, the baseline was unstable, and chromatographic peak 8 was missing, with overall poor extraction effect.

[0056] (3) Acid hydrolysis after extraction with 40% ethanol

[0057] Take 10 g of oyster (Crassostrea rivularis) formula granules, add 200 mL of 40% ethanol, reflux for 1 hour, cool, centrifuge, take the supernatant, evaporate to dryness, and hydrolyze with 9 mol / L hydrochloric acid.

[0058] The results showed that: for 10 g of oyster (Crassostrea rivularis) formula granules, after extraction with 40% ethanol, the supernatant was evaporated to dryness and then acid hydrolyzed. Compared with direct acid hydrolysis, the components extracted by 40% ethanol were concentrated in the first half, and the amino acid components to be detected did not appear in the chromatogram.

[0059] (4) Acid hydrolysis after water extraction and alcohol precipitation at different concentrations

[0060] Take 10 g of oyster (Crassostrea rivularis) formula granules, add 200 mL of water, reflux for 1 hour, cool, centrifuge, take the supernatant, gradually add ethanol to make the concentration 10% ethanol, 20% ethanol, 50% ethanol, centrifuge respectively, take the precipitate, and hydrolyze with 9 mol / L acid.

[0061] The results showed that: for 10 g of oyster (Crassostrea rivularis) formula granules, after water extraction and then adding ethanol to adjust the ethanol concentration in the solvent to 10% ethanol, 20% ethanol, 50% ethanol respectively, the precipitate obtained by alcohol precipitation contained very little amino acid, with poor enrichment effect.

[0062] (5) Column chromatography with macroporous resin D101

[0063] Dissolve 1 g of oyster (Crassostrea rivularis) formula granules in acid, centrifuge, take the supernatant, and separate by column chromatography.

[0064] The results showed that: through D101 macroporous resin, eluting with water, low-concentration alcohol to ethanol respectively, the enrichment effect was not good.

[0065] (6) Column chromatography with macroporous resin HP20

[0066] 1 g of oyster (Crassostrea rivularis) formula granules was dissolved in acid, centrifuged, and the supernatant was taken and separated by column chromatography.

[0067] The results showed that by using HP20 macroporous resin, elution was carried out with water, low-concentration alcohol to ethanol respectively. After elution with 50% ethanol, some chromatographic peaks were missing in the chromatogram. However, compared with elution with other solvents, the baseline was stable, there was a certain residue of amino acids, but the enrichment effect was not good.

[0068] (7) Column chromatography through macroporous resin LD20

[0069] 6 g of oyster (Crassostrea rivularis) formula granules were taken, 20 mL of water was added, heated under reflux to dissolve, cooled, centrifuged, and the supernatant was taken and passed through the column.

[0070] The results showed that by using LD20 macroporous resin, elution was carried out with 10% methanol, 20% methanol, and 50% methanol respectively. Under each elution solvent, the separation effect of each compound was not good.

[0071] (8) Column chromatography through gel LH-20

[0072] 5 - 6 g of oyster (Crassostrea rivularis) formula granules were taken, 20 mL of water was added, heated under reflux to dissolve, cooled, centrifuged, and the supernatant was taken and passed through the column.

[0073] The results showed that by using gel LH-20, elution with water was carried out, and there was no enrichment of amino acids. Moreover, the column chromatography through gel LH-20 was very slow, so this method was abandoned.

[0074] (9) Column chromatography through ion exchange resin (strong acidic cation)

[0075] 5 - 6 g of oyster (Crassostrea rivularis) formula granules were taken, 20 mL of water was added, heated under reflux to dissolve, cooled, centrifuged, and the supernatant was taken and passed through the column.

[0076] The results showed that after pretreatment of the ion exchange resin, the sample was loaded, and elution was carried out with 10% ammonia water - 100% ammonia water for 3 - 5 column volumes. There was some residue of amino acids after ammonia water elution, but the final enrichment effect of amino acids was not good.

[0077] (10) Salting-out treatment

[0078] 5 - 8 g of oyster (Crassostrea rivularis) formula granules were taken, 20 mL of water was added, heated under reflux to dissolve, cooled, centrifuged, and the supernatant was taken. Sodium chloride reagent was added for stepwise salting-out. The precipitate was combined and hydrolyzed.

[0079] The results showed that the salting-out result did not show protein / amino acid enrichment.

[0080] Example 2: Investigation on the preparation method of amino acids from oyster (Crassostrea rivularis) formula granules

[0081] The hydrolysis hydrochloric acid concentration, hydrochloric acid dosage, hydrolysis temperature, hydrolysis time, derivatization reagent dosage and derivatization time of the amino acid characteristic chromatogram of oyster (Crassostrea rivularis) formula granules were investigated.

[0082] (1) Investigation of hydrochloric acid concentration

[0083] Take an appropriate amount of oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 3 groups, 2 portions in each group, place them in a 50 mL centrifuge tube, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, place the precipitate in a stoppered hydrolysis tube, accurately add 20 mL of 6 mol / L, 9 mol / L, 12 mol / L hydrochloric acid, hydrolyze at 150 °C for 2 hours, take out, cool, make up the lost weight with the corresponding acid, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water for multiple times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and obtain the solution.

[0084] Accurately measure 10 mL of each of the above-mentioned test solution and reference solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, after standing at room temperature for 2 hours, add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, take the continuous filtrate, and obtain the solution. Accurately absorb 1 μL of the test solution and inject it into the liquid chromatograph for determination. The results show that when extracting with different hydrochloric acid concentrations, the "total peak area / sample weight" is the largest when the hydrochloric acid concentration is 9 mol / L. To ensure complete extraction, the hydrolysis hydrochloric acid concentration is selected as 9 mol / L. The experimental results are shown in Table 1.

[0085] Table 1 Results of the investigation of hydrochloric acid concentration of the characteristic chromatogram of oyster (Crassostrea rivularis) formula granules

[0086]

[0087]

[0088] (2) Investigation of acid dosage

[0089] Take an appropriate amount of oyster (Crassostrea rivularis Gould) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 5 groups, 2 portions in each group, place them in 50 mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 5 mL, 10 mL, 15 mL, 20 mL, 25 mL of 9 mol / L hydrochloric acid respectively, stopper tightly, hydrolyze at 150 °C for 2 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water for several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it.

[0090] Precisely measure 10 mL of each of the above-mentioned test solution and reference solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, after standing at room temperature for 2 hours, add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, take the continuous filtrate, and you will get it. Precisely absorb 10 μL and inject it into the liquid chromatograph for determination, and you will get it. The results show that when extracting with different amounts of acid, there are no obvious differences in the number and resolution of characteristic peaks in the characteristic chromatogram. When the amount of 9 mol / L hydrochloric acid solution is 10 mL and 15 mL, the difference in "total peak area / sample weight" is relatively small. Considering the sample difference and the parallelism between the two samples, the amount of hydrochloric acid used is selected as 10 mL. The experimental results are shown in Table 2.

[0091] Table 2 Investigation results of different amounts of acid used in the characteristic chromatogram of oyster (Crassostrea rivularis Gould) formula granules

[0092]

[0093] (3) Investigation of hydrolysis temperature

[0094] Take an appropriate amount of oyster (Crassostrea rivularis Gould) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 4 groups, 2 portions in each group, place them in 50 mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 110 °C, 130 °C, 150 °C, 180 °C for 2 hours respectively, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water for several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it.

[0095] Precisely measure 10 mL each of the above-mentioned test solution and reference solution, and place them separately in 25-mL volumetric flasks. Add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine. Shake well. After standing at room temperature for 2 hours, add 50% acetonitrile to the mark and shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower-layer solution, filter, and take the consecutive filtrate to obtain the solution. Precisely pipette 10 μL and inject it into the liquid chromatograph for determination to obtain the result. The results show that when investigating the influence of different hydrolysis temperatures on the characteristic chromatogram, except that the hydrolysis is incomplete at 110 °C, there are no obvious differences in the number and resolution of the characteristic peaks in the characteristic chromatograms at 130 °C, 150 °C, and 180 °C, and the responses of the characteristic peaks at 150 °C are not very different from those at 180 °C. Considering the safety of the experimental operation comprehensively, the hydrolysis temperature is selected as 150 °C. The experimental results are shown in Table 3.

[0096] Table 3 Results of Investigating Different Hydrolysis Temperatures of the Characteristic Chromatogram of Oyster (Crassostrea rivularis) Formula Granules

[0097]

[0098] (4) Investigation of hydrolysis time

[0099] Take an appropriate amount of oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 5 groups, 2 portions in each group, place them in 50-mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, and hydrolyze at 150 °C for 1 h, 2 h, 3 h, 4 h, and 5 h respectively. Take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and the filter paper with a small amount of water several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 20-mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, and shake well to obtain the solution.

[0100] Precisely measure 10 mL each of the above-mentioned test solution and reference solution, and place them separately in 25-mL volumetric flasks. Add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine. Shake well. After standing at room temperature for 2 hours, add 50% acetonitrile to the mark and shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower-layer solution, filter, and take the consecutive filtrate to obtain the solution. Precisely pipette 10 μL and inject it into the liquid chromatograph for determination to obtain the result. The results show that when extracting with different hydrolysis temperatures, except that the hydrolysis is incomplete at 1 h, there are no obvious differences in the number and resolution of the characteristic peaks in the characteristic chromatograms at 2 h, 3 h, 4 h, and 5 h, and the difference in "total peak area / sample weight" is not significant. The hydrolysis time is selected as 3 h. The experimental results are shown in Table 4.

[0101] Table 4 Investigation results of the characteristic chromatograms of oyster (Crassostrea rivularis) formula granules at different hydrolysis times

[0102]

[0103] (5) Investigation of derivatization time

[0104] Take an appropriate amount of oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 3 groups, 2 portions in each group, place in a 50 mL centrifuge tube, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 3 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrates, evaporate to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid solution, transfer to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it.

[0105] Accurately measure 10 mL of each of the above-mentioned test solution and reference solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, after standing at room temperature for 1 h, 2 h, and 3 h respectively, add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, take the continued filtrate, and you will get it. Accurately absorb 10 μL and inject it into the liquid chromatograph for determination, and you will get it. The results show that when the derivatization time is 2 h and 3 h, there is no obvious difference in the number and resolution of the characteristic peaks in the characteristic chromatogram, and there is little difference in the response values of each characteristic peak and "total peak area / sample weight", and the "total peak area / sample weight" is the largest when the derivatization time is 2 h, so the derivatization time of 2 h is selected. The experimental results are shown in Table 5.

[0106] Table 5 Investigation results of the characteristic chromatograms of oyster (Crassostrea rivularis) formula granules at different derivatization times

[0107]

[0108] (6) Determination of the preparation method of the test solution

[0109] According to the above experimental results, the sample pretreatment method for the characteristic chromatogram of oyster (Crassostrea rivularis) formula granules was determined as follows: Take an appropriate amount of oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, place in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 3 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water multiple times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get the solution.

[0110] Precisely measure 10 mL each of the above test solution and reference solution, place them respectively in 25 mL volumetric flasks, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, let stand at room temperature for 2 hours, then add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, let stand for 10 minutes, take the lower layer solution, filter, and take the continuous filtrate, and you will get the solution.

[0111] Example 3: Investigation on the preparation method of amino acids in calcined oyster (Crassostrea rivularis) formula granules

[0112] (1) Investigation on hydrochloric acid concentration

[0113] This experiment investigated the influence of different hydrochloric acid concentrations on the characteristic chromatogram of calcined oyster (Crassostrea rivularis) formula granules. Hydrochloric acid concentrations of 6 mol / L, 9 mol / L, and 12 mol / L were selected. By observing the peak shapes and separation effects of the tentatively determined 11 characteristic peaks, and calculating the "total peak area / sample weight" of the 11 characteristic peaks to compare the influence of different hydrochloric acid concentrations on the characteristic chromatogram of calcined oyster (Crassostrea rivularis) formula granules, and select the optimal hydrochloric acid concentration.

[0114] Take an appropriate amount of calcined oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 3 groups, 2 portions in each group, place in 50 mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, place the precipitate in a stoppered hydrolysis tube, accurately add 25 mL of 6 mol / L, 9 mol / L, and 12 mol / L hydrochloric acid, hydrolyze at 150 °C for 2 hours, take out, cool, make up the lost weight with the corresponding acid, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water multiple times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer to a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get the solution.

[0115] Precisely measure 10 mL each of the above-mentioned test solution and reference solution, and place them respectively in 25-mL volumetric flasks. Add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well. After standing at room temperature for 2 hours, add 50% acetonitrile to the mark, and shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower-layer solution, filter, and take the subsequent filtrate to obtain the solution. Precisely absorb 1 μL of the test solution and inject it into the liquid chromatograph for determination to obtain the result. The results show that when extracting with different hydrochloric acid concentrations, the value of "total peak area / sample weight" of calcined oyster granules is the largest when the hydrochloric acid concentration is 12 mol / L, but there are certain response differences among samples, and there are safety hazards in the experiment with too high hydrochloric acid concentration. Therefore, hydrochloric acid with a hydrolysis concentration of 9 mol / L is selected. The experimental results are shown in Table 6.

[0116] Table 6 Results of the investigation on hydrochloric acid concentration of the characteristic chromatogram of formula granules of calcined oyster (Crassostrea rivularis)

[0117]

[0118]

[0119] (2) Investigation on the amount of acid used

[0120] Take an appropriate amount of formula granules of calcined oyster (Crassostrea rivularis), grind them finely, take about 2.0 g, precisely weigh, in parallel with 3 groups, 2 portions in each group, place them in 50-mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, precisely add 5 mL, 15 mL, and 25 mL of 9 mol / L hydrochloric acid respectively, tightly stopper, hydrolyze at 150 °C for 2 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water for several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 25-mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, and shake well to obtain the solution.

[0121] Precisely measure 10 mL each of the above-mentioned test solution and reference solution, and place them respectively in 25-mL volumetric flasks. Add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well. After standing at room temperature for 2 hours, add 50% acetonitrile to the mark, and shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower-layer solution, filter, and take the subsequent filtrate to obtain the solution. Precisely absorb 10 μL and inject it into the liquid chromatograph for determination to obtain the result. The results show that when extracting with different amounts of acid used, there are no obvious differences in the number and resolution of characteristic peaks in the characteristic chromatogram. To ensure sufficient extraction, the amount of 9 mol / L hydrochloric acid solution used is selected as 25 mL. The experimental results are shown in Table 7.

[0122] Table 7 Investigation results of characteristic chromatograms of calcined oyster (Crassostrea rivularis) formula granules with different amounts of acid

[0123]

[0124] (3) Investigation of hydrolysis temperature

[0125] Take an appropriate amount of calcined oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, weigh accurately, parallel into 4 groups, 2 portions in each group, place in a 50 mL centrifuge tube, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 25 mL of 9 mol / L hydrochloric acid, hydrolyze at 110 °C, 130 °C, 150 °C, and 180 °C for 2 hours respectively, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer to a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you get it.

[0126] Precisely measure 10 mL of each of the above-mentioned test solution and reference solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, after standing at room temperature for 2 hours, add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, take the subsequent filtrate, and you get it. Precisely absorb 10 μL and inject it into the liquid chromatograph for determination, and you get it. The results show that when extracting with different hydrolysis temperatures, there is no obvious difference in the resolution of each characteristic peak and "total peak area / sample weight" in the characteristic chromatograms at hydrolysis temperatures of 150 °C and 180 °C, indicating that the calcined oyster granules are completely hydrolyzed at 150 °C. Therefore, the hydrolysis temperature of 150 °C is selected. The experimental results are shown in Table 8.

[0127] Table 8 Investigation results of characteristic chromatograms of calcined oyster (Crassostrea rivularis) formula granules with different hydrolysis temperatures

[0128]

[0129] (4) Investigation of hydrolysis time

[0130] Take an appropriate amount of calcined oyster (Crassostrea rivularis Gould) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 5 groups, 2 portions in each group, place them in 50 mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 1 h, 2 h, 3 h, 4 h, 5 h respectively, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water for several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it.

[0131] Precisely measure 10 mL of each of the above-mentioned test solution and reference solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, after standing at room temperature for 2 hours, add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, take the continuous filtrate, and you will get it. Precisely absorb 10 μL and inject it into the liquid chromatograph for determination, and you will get it. The results show that when extracting with different hydrolysis times, it has been completely hydrolyzed at 3 hours of hydrolysis. There are no obvious differences in the number and resolution of characteristic peaks in the characteristic chromatograms of 3 hours, 4 hours, and 5 hours of hydrolysis. In order to save time cost, the hydrolysis time of 3 hours is finally selected. The experimental results are shown in Table 9.

[0132] Table 9 Results of investigation on different hydrolysis times of the characteristic chromatogram of calcined oyster (Crassostrea rivularis Gould) formula granules

[0133]

[0134] (5) Investigation on derivatization time

[0135] Take an appropriate amount of calcined oyster (Crassostrea rivularis Gould) formula granules, grind them finely, take about 2.0 g, weigh accurately, in parallel for 3 groups, 2 portions in each group, place them in 50 mL centrifuge tubes, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 3 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water for several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it.

[0136] Precisely measure 10 mL each of the above-mentioned test solution and reference solution, and place them separately in 25-mL volumetric flasks. Add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well. After standing at room temperature for 1 h, 2 h, and 3 h respectively, add 50% acetonitrile to the mark, and shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower-layer solution, filter, and take the consecutive filtrate to obtain the solution. Precisely absorb 10 μL and inject it into the liquid chromatograph for determination to obtain the result. The results show that when investigating different derivatization times, the derivatization reaction is complete in 2 hours, and the "total peak area / sample weight" is the largest at 2 hours of derivatization. Finally, the derivatization time of 2 hours is selected. The experimental results are shown in Table 10.

[0137] Table 10 Results of investigating different derivatization times for the characteristic chromatogram of calcined oyster (Crassostrea rivularis) formula granules

[0138]

[0139] (6) Determination of the preparation method of the test solution

[0140] According to the above experimental results, the sample pretreatment method for the characteristic chromatogram of calcined oyster (Crassostrea rivularis) formula granules is determined as follows: Take an appropriate amount of calcined oyster (Crassostrea rivularis) formula granules, grind them finely, take about 2.0 g, precisely weigh, place in a 50-mL centrifuge tube, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, precisely add 25 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 3 hours, take out, cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer to a 25-mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, and shake well to obtain the solution.

[0141] Precisely measure 10 mL each of the above-mentioned test solution and reference solution, and place them separately in 25-mL volumetric flasks. Add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well. After standing at room temperature for 2 hours, add 50% acetonitrile to the mark, and shake well. Take 10 mL, add 10 mL of n-hexane, shake, stand for 10 minutes, take the lower-layer solution, filter, and take the consecutive filtrate to obtain the solution.

[0142] Example 4: Investigation of chromatographic conditions

[0143] (1) Gradient optimization

[0144] Chromatographic conditions: Acetonitrile was used as mobile phase A, and 0.4% phosphoric acid was used as mobile phase B. Gradient elution was carried out according to the regulations in Table 11; the flow rate was 0.27 mL per minute; the column temperature was 25 °C; the detection wavelength was 254 nm; the injection volume was 1 μL.

[0145] Table 11 Four gradient elution methods

[0146]

[0147] The results were as Figure 1 shown. By comparing the above four different gradient conditions, it was found that the chromatographic peaks in the chromatogram obtained under the gradient conditions of Method 4 were more evenly distributed and had good peak shapes. It was selected as the analysis conditions for the characteristic chromatogram of oyster standard decoction.

[0148] (2) Investigation on the durability of chromatographic conditions

[0149] 1) Optimization of flow rate

[0150] Chromatographic conditions: YMC Triart chromatographic column (2.1 mm × 150 mm, 1.9 μm); Acetonitrile was used as mobile phase A, and 0.4% phosphoric acid was used as mobile phase B. Gradient elution was carried out according to the regulations in "Method 4" above; the flow rates were 0.25 mL, 0.27 mL, and 0.29 mL per minute; the column temperature was 27 °C; the detection wavelength was 254 nm; the injection volume was 1 μL.

[0151] The results were as Figure 2 shown. When the flow rates were 0.25 mL, 0.27 mL, and 0.29 mL, the resolution of each chromatographic peak in the chromatogram was good, and the change in flow rate had little effect on the separation of each characteristic peak, indicating that the analysis method had good durability in the range of flow rate 0.27 ± 0.02 mL / min.

[0152] 2) Optimization of column temperature

[0153] Chromatographic conditions: YMC Triart chromatographic column (2.1 mm × 150 mm, 1.9 μm); Acetonitrile was used as mobile phase A; 0.4% phosphoric acid was used as mobile phase B. Gradient elution was carried out according to the regulations in "Method 4" above; the flow rate was 0.27 mL per minute; the column temperatures were 25 °C, 27 °C, and 29 °C; the detection wavelength was 254 nm; the injection volume was 1 μL.

[0154] The results were as Figure 3 shown. It was found that when the column temperature was 27 ± 2 °C, the change in column temperature had little effect on the separation of each characteristic peak, indicating that the analysis method had good durability in the range of column temperature 27 ± 2 °C.

[0155] 3) Investigation of chromatographic column

[0156] There are certain differences in the analysis effects of chromatographic columns with different specifications. According to the properties of the substances to be separated, the change of chromatographic columns can have a significant impact on the analysis results. Therefore, in this study, 4 chromatographic columns were selected, namely: YMC Triart (250mm×4.6mm, 5μm) chromatographic column; Kromasil 100-5-C18 (250mm×4.6mm, 5μm); Wartes HSS T3 (250mm×4.6mm, 5μm) chromatographic column; Phenomenex Luna Omega C18 (250mm×4.6mm, 5μm) chromatographic column.

[0157] Chromatographic conditions: Acetonitrile was used as mobile phase A; 0.4% phosphoric acid was used as mobile phase B, and gradient elution was carried out according to the regulations in "Method 4" above; the flow rate was 0.9 mL per minute; the column temperature was 30 °C; the detection wavelength was 254 nm; the injection volume was 10 μL.

[0158] The results are as Figure 4 shown that ideal separation results have not been achieved by changing the chromatographic column, mobile phase, column temperature and flow rate, etc. Therefore, an ultra-high chromatographic column YMC Triart (2.1mm×150mm, 1.9μm) was used for separation optimization.

[0159] Example 5: Sample determination

[0160] Chromatographic conditions: YMC Triart C18 chromatographic column (2.1mm×150mm, 1.9μm) was selected; acetonitrile was used as mobile phase A, and 0.4% phosphoric acid was used as mobile phase B, and gradient elution was carried out according to the regulations in Table 12; the flow rate was 0.27 mL per minute; the column temperature was 27 °C; the detection wavelength was 254 nm; the injection volume was 1 μL.

[0161] Table 12 Gradient elution table

[0162]

[0163] Preparation method of test samples:

[0164] Take an appropriate amount of the sample powder, grind it finely, take about 2.0 g, weigh it accurately, place it in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to dissolve it, centrifuge, discard the supernatant, take the precipitate and place it in a stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 °C for 3 hours, take it out, let it cool, make up the lost weight with 9 mol / L hydrochloric acid solution, mix well, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrates, evaporate to dryness, dissolve the residue with 0.1 mol / L hydrochloric acid solution, transfer it to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and you will get it. Accurately measure 10 mL of each of the above test solution, place them in 25 mL volumetric flasks respectively, add 2.5 mL of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate (PITC) and 2.5 mL of acetonitrile solution of 1 mol / L triethylamine, shake well, let it stand at room temperature for 2 hours, then add 50% acetonitrile to the scale, shake well. Take 10 mL, add 10 mL of n-hexane, shake, let it stand for 10 minutes, take the lower layer solution, filter, and take the subsequent filtrate.

[0165] 1) Oyster medicinal materials

[0166] Analyze the characteristic fingerprints of 18 batches of oyster medicinal materials. Using the proline chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area of peaks 1 - 7 and the S1 peak; using the valine peak as the reference peak S2, calculate the relative retention time and relative peak area of peaks 9 - 11 and the S2 peak, and calculate the RSD value. The experimental results are shown in Tables 13 and 14 below, and the superimposed diagram of the characteristic fingerprints of 18 batches of oyster medicinal materials is shown in Figure 5 .

[0167] Table 13 Characteristic fingerprints of 18 batches of oyster medicinal materials (relative retention time)

[0168]

[0169]

[0170] Table 14 Characteristic fingerprints of 18 batches of oyster medicinal materials (relative peak area)

[0171]

[0172]

[0173] 2) Oyster decoction pieces

[0174] Analyze the characteristic fingerprints of 18 batches of oyster decoction pieces. Using the proline chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area of peaks 1 - 7 and the S1 peak; using the valine peak as the reference peak S2, calculate the relative retention time and relative peak area of peaks 9 - 11 and the S2 peak, and calculate the RSD value. The experimental results are shown in Tables 15 and 16 below, and the superimposed diagram of the characteristic fingerprints of 18 batches of oyster decoction pieces is shown in Figure 6。

[0175] Table 15 Characteristic Chromatograms of 18 Batches of Oyster Decoction Slices (Relative Retention Time)

[0176]

[0177]

[0178] Table 16 Characteristic Chromatograms of 18 Batches of Oyster Decoction Slices (Relative Peak Area)

[0179]

[0180]

[0181] 3) Standard Decoction of Oyster

[0182] According to the determination method of the characteristic chromatogram of the standard decoction of oyster, the characteristic chromatograms of 18 batches of standard decoction of oyster samples were determined. Using the proline peak as the reference peak S1, calculate the relative retention time and relative peak area of peaks 1 - 7 and the S1 peak; using the valine peak as the reference peak S2, calculate the relative retention time and relative peak area of peaks 8 - 11 and the S2 peak, and calculate the RSD value. The results are shown in Table 17 and Table 18 below. The superimposed chromatogram of the characteristic chromatograms of 18 batches of standard decoction of oyster is shown in Figure 7 。

[0183] Table 17 Relative Retention Time of the Characteristic Chromatograms of 18 Batches of Standard Decoction of Oyster

[0184]

[0185]

[0186] Table 18 Relative Peak Area of the Characteristic Chromatograms of 18 Batches of Standard Decoction of Oyster

[0187]

[0188] 4) Formula Granules of Oyster (Crassostrea rivularis)

[0189] Take 3 batches of formula granules of oyster (Crassostrea rivularis) respectively to prepare the test solution; according to the chromatographic conditions, inject the sample for determination. The results are shown in Table 19 and Table 20 below and Figure 8 。

[0190] Table 19 Characteristic Chromatograms of Formula Granules of Oyster (Crassostrea rivularis) (Relative Retention Time)

[0191]

[0192] Table 20 Characteristic Chromatograms of Formula Granules of Oyster (Crassostrea rivularis) (Relative Peak Area)

[0193]

[0194] 5) Calcined oyster herbal medicine

[0195] According to the determination method of the characteristic spectrum of calcined oyster medicinal materials, the characteristic spectrum of 16 batches of calcined oyster medicinal materials samples was determined. The proline peak was used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 7 with respect to the S1 peak were calculated; the valine peak was used as the reference peak S2, and the relative retention time and relative peak area of ​​peaks 8 to 11 with respect to the S2 peak were calculated, and the RSD value was calculated. The results are shown in Tables 21 and 22 below, and the superposition diagram of the characteristic spectrum of 16 batches of calcined oyster medicinal materials is shown in Figure 9 .

[0196] Table 21 Characteristic spectra of 16 batches of calcined oyster medicinal materials (relative retention time)

[0197]

[0198]

[0199] Table 22 Characteristic spectra of 16 batches of calcined oyster medicinal materials (relative peak area)

[0200]

[0201] 6) Calcined Oyster Pieces

[0202] According to the determination method of the characteristic spectrum of calcined oyster slices, the characteristic spectrum of 16 batches of calcined oyster slices samples was determined. The proline peak was used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 7 with respect to peak S1 were calculated; the valine peak was used as the reference peak S2, and the relative retention time and relative peak area of ​​peaks 8 to 11 with respect to peak S2 were calculated, and the RSD value was calculated. The results are shown in Tables 23 and 24 below, and the characteristic spectrum overlay of 16 batches of calcined oyster slices is shown in Figure 10 .

[0203] Table 23 Characteristic spectra of 16 batches of calcined oyster slices (relative retention time)

[0204]

[0205] Table 24 Characteristic spectra of 16 batches of calcined oyster slices (relative peak area)

[0206]

[0207]

[0208] 7) Calcined Oyster Standard Decoction

[0209] According to the determination method of the characteristic chromatogram of the standard decoction of calcined oyster, the characteristic chromatograms of 16 batches of the standard decoction of calcined oyster were determined. Using the proline peak as the reference peak S1, calculate the relative retention time and relative peak area of peaks 1 - 7 with respect to the S1 peak; using the valine peak as the reference peak S2, calculate the relative retention time and relative peak area of peaks 8 - 11 with respect to the S2 peak, and calculate the RSD value. The results are shown in Tables 25 and 26 below, and the superimposed chromatograms of the characteristic chromatograms of 16 batches of the standard decoction of calcined oyster are shown in Figure 11 .

[0210] Table 25 Characteristic Chromatograms of 16 Batches of the Standard Decoction of Calcined Oyster (Relative Retention Time)

[0211]

[0212]

[0213] Table 26 Characteristic Chromatograms of 16 Batches of the Standard Decoction of Calcined Oyster (Relative Peak Area)

[0214]

[0215]

[0216] 8) Formula Granules of Calcined Oyster (Ostrea rivularis Gould)

[0217] Take 3 batches of formula granules of calcined oyster (Ostrea rivularis Gould) respectively, prepare the test solution; according to the chromatographic conditions, inject samples for determination. The results are shown in Tables 27 and 28 below and Figure 12 .

[0218] Table 27 Characteristic Chromatograms of Formula Granules of Calcined Oyster (Ostrea rivularis Gould) (Relative Retention Time)

[0219]

[0220] Table 28 Characteristic Chromatograms of Formula Granules of Calcined Oyster (Ostrea rivularis Gould) (Relative Peak Area)

[0221]

[0222] It can be seen from the experimental results that the characteristic chromatograms of oyster medicinal materials, oyster decoction pieces, oyster standard decoctions, oyster (Crassostrea rivularis) formula granules, calcined oyster medicinal materials, calcined oyster decoction pieces, calcined oyster standard decoctions, and calcined oyster (Crassostrea rivularis) formula granules can be stably presented and correspond to the retention times of 11 characteristic peaks in the reference chromatogram of the control medicinal materials. The method for determining amino acids in oyster medicinal materials and their standard decoctions or formula granules established by the present invention can be simultaneously applied to the determination of the contents of different types of amino acids in oyster medicinal materials, processed oyster products, oyster standard decoctions, oyster formula granules, processed oyster standard decoctions, and processed oyster formula granules. It not only provides a control means for amino acid determination for the quality control of oysters and their processed products, greatly saving the cost of quality standard research and future sample inspection, but also provides a reference for the amino acid determination method of other mineral Chinese medicines.

[0223] The above are the preferred embodiments of the invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. An amino acid HPLC detection method for oyster medicinal materials, processed products, their standard decoctions or formula granules, characterized in that, It includes the following steps: Prepare a reference solution, which includes serine reference, glycine reference, glutamic acid reference, aspartic acid reference, threonine reference, proline reference, alanine reference, valine reference, lysine reference, leucine reference, and phenylalanine reference; Take oyster medicinal materials, processed oyster products, oyster standard decoctions, oyster formula granules, processed oyster product standard decoctions, or processed oyster product formula granules to prepare a test solution; Respectively take a preset amount of the test solution and the reference solution, inject them into a liquid chromatograph for determination. The liquid chromatograph uses octadecylsilane-bonded silica gel as the filler, acetonitrile as mobile phase A, and a phosphoric acid solution as mobile phase B for gradient elution to obtain an amino acid HPLC determination curve map.

2. The amino acid HPLC detection method for oyster medicinal materials, processed products, their standard decoctions or formula granules as described in claim 1, characterized in that, The gradient elution is carried out according to the following procedure: From 0 to 14 min, mobile phase A changes from 15% → 20%, and mobile phase B changes from 85% → 80%; From 14 min to 17 min, mobile phase A changes from 20% → 36%, and mobile phase B changes from 80% → 64%; From 17 min to 22 min, mobile phase A remains at 36%, and mobile phase B remains at 64%; From 22 min to 40 min, mobile phase A changes from 36% → 38%, and mobile phase B changes from 64% → 62%; From 40 min to 45 min, mobile phase A changes from 38% → 70%, and mobile phase B changes from 62% → 30%.

3. The amino acid HPLC detection method for the oyster medicinal materials, processed products, their standard decoctions or formula granules as described in claim 1, characterized in that, The concentration of the phosphoric acid solution is 0.25% - 0.65%.

4. The amino acid HPLC detection method for oyster medicinal materials, processed products, their standard decoctions or formula granules as described in claim 1, characterized in that, The preset amount of the test solution or the reference solution is 0.5 μL - 1.5 μL; The liquid chromatograph uses octadecylsilane-bonded silica gel as the filler, with a column length of 150 mm, an inner diameter of 2.1 mm, a particle size of 1.6 μm - 2.1 μm, and a column temperature of 25°C - 29°C; When performing gradient elution, the flow rate of the mobile phase is 0.25 mL / min - 0.29 mL / min; The ultraviolet detection wavelength of the liquid chromatograph is 250 nm - 260 nm.

5. The amino acid HPLC detection method for oyster medicinal materials, processed products, their standard decoctions or formula granules as described in claim 1, characterized in that, The test solution is prepared by the following method: Take oyster medicinal material powder, processed oyster product powder, oyster standard decoction, oyster formula granule, processed oyster product standard decoction, or processed oyster product formula granule, and perform acid hydrolysis treatment to obtain the test solution.

6. The amino acid HPLC detection method for oyster medicinal materials, processed products, and their standard decoctions or formula granules as described in claim 5, characterized in that, In the acid hydrolysis treatment, 8 mol / L - 10 mol / L hydrochloric acid is used for hydrolysis at 130°C - 180°C for 2 h - 5 h.

7. The amino acid HPLC detection method for the oyster medicinal materials, processed products, their standard decoctions or formula granules as described in claim 1, characterized in that, The reference solution is prepared according to the following method: Take serine reference, glycine reference, glutamic acid reference, aspartic acid reference, threonine reference, proline reference, alanine reference, valine reference, lysine reference, leucine reference, and phenylalanine reference, and add 0.05 - 0.2 mol / L hydrochloric acid to make a mixed solution as the reference solution.

8. The amino acid HPLC detection method for oyster medicinal materials, processed products, their standard decoctions or formula granules as claimed in claim 1, characterized in that, The amino acid HPLC determination curve map includes 11 common peaks. Among them, the peak corresponding to serine is Peak 1, the peak corresponding to glycine is Peak 2, the peak corresponding to glutamic acid is Peak 3, the peak corresponding to aspartic acid is Peak 4, the peak corresponding to threonine is Peak 5, the peak corresponding to proline is Peak 6, the peak corresponding to alanine is Peak 7, the peak corresponding to valine is Peak 8, the peak corresponding to leucine is Peak 9, the peak corresponding to lysine is Peak 10, and the peak corresponding to phenylalanine is Peak 11.

9. The amino acid HPLC detection method for oyster medicinal materials, processed products, their standard decoctions or formula granules as described in claim 8, characterized in that, In the amino acid HPLC determination curve map, taking the proline peak as the reference peak S1, the relative retention times of Peak 1 to Peak 5 and Peak 7 with respect to the reference peak S1 are as follows: Peak 1 is 0.61, Peak 2 is 0.65, Peak 3 is 0.8, Peak 4 is 0.83, Peak 5 is 0.9, and Peak 7 is 1.09, and the relative standard deviation is within ±10%; Taking the valine peak as the reference peak S2, the relative retention times of Peak 9 to Peak 11 with respect to the reference peak S2 are as follows: Peak 9 is 1.18, Peak 10 is 1.26, and Peak 11 is 1.29, and the relative standard deviation is within ±10%.

10. Application of the amino acid HPLC detection method for oyster medicinal materials, processed products, and their standard decoctions or formula granules as described in any one of claims 1 to 9 in (1) or (2): (1) Determination of the amino acid content of oyster medicinal materials, processed products, and their standard decoctions and formula granules; (2) Authenticity identification and quality control of oyster medicinal materials, processed products, and their standard decoctions and formula granules.

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