Amino acid HPLC detection method for oyster medicinal materials, processed products and their standard decoction or formula granules

Abstract

The application discloses an amino acid HPLC detection method for oyster medicinal materials, processed products and standard decoctions or formula granules, and comprises the following steps: preparing a control solution; taking oyster medicinal materials, oyster processed products, oyster standard decoctions, oyster formula granules, oyster processed product standard decoctions or oyster processed product formula granules to prepare a test solution; respectively taking a preset amount of the test solution and the control solution, and injecting into a liquid chromatograph for detection, wherein the liquid chromatograph uses octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and a phosphoric acid solution as a mobile phase B for gradient elution to obtain an amino acid HPLC detection curve atlas. The application has good reproducibility, is accurate and reliable, and can provide an amino acid detection control means for oyster medicinal materials, oyster processed products and standard decoctions or formula granules.

Description

TECHNICAL FIELD

[0001] The present application relates to the quality analysis and detection technology field of traditional Chinese medicine, and particularly relates to an amino acid HPLC detection method for oyster medicinal materials, processed products, and standard decoctions or formula granules thereof. BACKGROUND

[0002] Oyster is the shell of Ostrea gigas Thunberg, Ostrea rivularis Gould or Ostrea talienwhanensis Crosse of Ostreidae, is a commonly used traditional Chinese medicine in clinical practice recorded in various editions of Chinese Pharmacopoeia, belongs to minerals in the ocean, is distributed in the coastal areas of China and can be collected throughout the year, and is used raw or after calcination, and is ground when used. Oyster tastes salty, astringent and slightly cold, and is attributed to the liver and kidney channels. It has the effects of calming the mind, suppressing yang and tonifying yin, softening and resolving masses, and is used to treat palpitation and insomnia, dizziness and tinnitus, scrofula and phlegm nodes, and masses and lumps. Calcined oyster has the effects of astringing and tonifying, and is often used to treat spontaneous and night sweating, spermatorrhea, leukorrhagia, stomachache and acid regurgitation. Oyster was first recorded in Shennong's Herbal Classic, and was listed as the top grade, "Oyster, salty and flat, is used to treat cold and heat, warm and wet, shock, anger and gas, remove the cramped and mouse fistula, and treat leukorrhagia in women. Long-term use can strengthen the bone joints, kill evil spirits and prolong life". In the Han Dynasty, oyster was used in a large number of prescriptions in Treatise on Cold Pathogenic Diseases and Synopsis of Golden Chamber, and the number of prescriptions reached 11. The above all reflect the important position of oyster in traditional Chinese medicine clinical practice.

[0003] Modern pharmacological studies have shown that oyster has protective effect on experimental liver injury, can significantly reduce the content of serum ALT and AST in mice with acute liver injury caused by CCl4, and reduce the degree of liver cell damage; oyster polysaccharide can significantly reduce and inhibit the degree of blood droplet of influenza virus cultured in dog kidney cells, and has a certain therapeutic effect on mice infected with type I herpes simplex virus, that is, it can enhance immunity; oyster natural active peptide (BPO) can inhibit the proliferation of human gastric cancer BGC-823 cells, and has significant antitumor effect; oyster water extract can delay the brain aging of ovariectomized rats, increase the thickness of molecular layer of striate cortex of rats, reduce the ratio of thickness of molecular layer to total thickness of cortex, increase the number of large pyramidal cells per unit area in hippocampal CA2 area, and enhance the activity of superoxide dismutase (SOD) and reduce the content of malondialdehyde (MDA); oyster extract can also reduce the increase of blood glucose in mice caused by alloxan, without affecting the blood glucose of normal mice; oyster glycosaminoglycan has 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 damage. In clinic, oyster can also treat insomnia, chronic otitis media, infantile hyperhidrosis, uterine fibroids, breast hyperplasia and other diseases. In summary, in recent years, the pharmacological and clinical effects of oyster have broken through its basic functions in ancient times, and the medicinal value of oyster has been greatly improved.

[0004] Oyster contains a variety of essential amino acids for the human body. For the determination of amino acids, modern research adopts Fourier transform near-infrared spectroscopy, formaldehyde method enzyme hydrolysate determination, PITC (phenyl isothiocyanate) pre-column derivatization 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. High performance liquid chromatography is the most commonly used method for detecting amino acids.

[0005] Oyster and its processed products, mineral traditional Chinese medicines and formula granules are commonly used in clinical practice of traditional Chinese medicine. However, there is no detection method for amino acids in oyster and its processed products in the current standard, which is a defect in quality control. At the same time, establishing an amino acid detection method for each of oyster and its processed products, standard decoction and formula granules will increase the detection cost of traditional Chinese medicine standard research. Moreover, there is no amino acid determination item and HPLC determination item for oyster and its processed products in the 2020 edition of Chinese Pharmacopoeia (Volume 1). SUMMARY

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

[0007] In order to solve the above technical problems, the present application provides an amino acid HPLC detection method for oyster medicinal materials, processed products and standard decoction or formula granules, comprising the following steps:

[0008] A control solution is prepared, which comprises serine control, glycine control, glutamic acid control, aspartic acid control, threonine control, proline control, alanine control, valine control, lysine control, leucine control and phenylalanine control;

[0009] An oyster medicinal material, oyster processed product, oyster standard decoction, oyster formula granule, oyster processed product standard decoction or oyster processed product formula granule is taken to prepare a test solution;

[0010] A preset amount of the test solution and the control solution are taken respectively and injected into a liquid chromatograph for determination, wherein the liquid chromatograph uses octadecylsilane bonded silica gel as a 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 atlas.

[0011] In an embodiment, the gradient elution is performed according to the following procedure:

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

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

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

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

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

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

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

[0019] The liquid chromatograph uses octadecylsilane bonded silica gel as a 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] The flow rate of the mobile phase is 0.25 mL / min to 0.29 mL / min during the gradient elution.

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

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

[0023] The oyster medicinal material powder, the oyster processed product powder, the oyster standard decoction, the oyster formula granules, the oyster processed product standard decoction or the oyster processed product formula granules are subjected to acid hydrolysis treatment to obtain the test sample 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 control sample solution is prepared by the following method:

[0026] The serine control sample, the glycine control sample, the glutamic acid control sample, the aspartic acid control sample, the threonine control sample, the proline control sample, the alanine control sample, the valine control sample, the lysine control sample, the leucine control sample and the phenylalanine control sample are added to 0.05 to 0.2 mol / L hydrochloric acid to form a mixed solution as the control sample solution.

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

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

[0029] The peak of valine is taken as a reference peak S2, the relative retention times of the peak No. 9 to the peak No. 11 to the reference peak S2 meet the following conditions: the peak No. 9 is 1.18, the peak No. 10 is 1.26, and the peak No. 11 is 1.29, and the relative standard deviations are within ±10%.

[0030] The application of the amino acid HPLC detection method of the oyster medicinal material, processed product and standard decoction or formula granules in (1) or (2):

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

[0032] (2) Identification and quality control of the oyster medicinal material, processed product and standard decoction or formula granules.

[0033] The application has the following beneficial effects:

[0034] The application establishes a method for determining the amino acid content of the oyster medicinal material, processed product and standard decoction or formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a chromatogram of a different elution gradient in Example 4 of the application;

[0036] Figure 2 is a chromatogram of a different flow rate in Example 4 of the application;

[0037] Figure 3 is a chromatogram of a different column temperature in Example 4 of the application;

[0038] Figure 4 is a chromatogram of a different column temperature in Example 4 of the application;

[0039] Figure 5 is a characteristic chromatogram superimposition of 18 batches of oyster medicinal materials in Example 5 of the application;

[0040] Figure 6 is a characteristic chromatogram superimposition of 18 batches of oyster decoction pieces in Example 5 of the application;

[0041] Figure 7 is a characteristic chromatogram superimposition of 18 batches of oyster standard decoction in Example 5 of the application;

[0042] Figure 8 is a characteristic chromatogram superimposition of 3 batches of oyster (Ostrea giga) formula granules in Example 5 of the application;

[0043] Figure 9is the characteristic chromatogram superimposition of 16 batches of calcined oyster medicinal materials in Example 5 of the present application;

[0044] Figure 10 is the characteristic chromatogram superimposition of 16 batches of calcined oyster medicinal materials in Example 5 of the present application;

[0045] Figure 11 is the characteristic chromatogram superimposition of 16 batches of calcined oyster medicinal materials in Example 5 of the present application;

[0046] Figure 12 is the characteristic chromatogram superimposition of 3 batches of calcined oyster (Ostrea gigas) formula granules in Example 5 of the present application. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0048] Example 1: Investigation of amino acid hydrolysis mode of oyster (Ostrea gigas) formula granules

[0049] The oyster (Ostrea gigas) formula granules were subjected to direct acid hydrolysis, hydrolysis after extraction with different solvents (hydrochloric acid, different concentrations of ethanol), and hydrolysis by different extraction methods (water extraction and alcohol precipitation, solid-phase extraction, ion exchange), respectively, to investigate the influence of different hydrolysis modes on the HPLC determination results of amino acids.

[0050] (1) Direct acid hydrolysis

[0051] An appropriate amount of oyster (Ostrea gigas) formula granules was finely ground, about 2.0 g was accurately weighed and placed in a 50 mL centrifuge tube, 10 mL of dilute hydrochloric acid solution was added to dissolve it, centrifuged, the supernatant was discarded, and the precipitate was placed in a stoppered hydrolysis tube. 10 mL of 9 mol / L hydrochloric acid was accurately added, and hydrolysis was carried out at 150℃ for 3 hours. After cooling, the lost weight was made up with 9 mol / L hydrochloric acid solution, mixed well, and the hydrolysis tube and filter paper were washed with a small amount of water several times, and the filtrate was combined and evaporated to dryness. The residue was dissolved in 0.1 mol / L hydrochloric acid solution and transferred to a 20 mL volumetric flask. 0.1 mol / L hydrochloric acid solution was added to the mark, and shaken well to obtain the product. 10 mL of the above test sample solution was accurately measured and placed in a 25 mL volumetric flask. 2.5 mL of 0.1 mol / L phenylisothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution were added, shaken well, and placed at room temperature for 2 hours. Then 50% acetonitrile was added to the mark and shaken well. 10 mL was taken and 10 mL of n-hexane was added, shaken, and placed for 10 minutes. The lower layer solution was taken and filtered.

[0052] The results show that 11 characteristic peaks are obtained, the distribution of each characteristic peak is relatively uniform, the peak shape is good, and the extraction effect of each amino acid is good.

[0053] (2)70% ethanol extraction followed by acid hydrolysis

[0054] Take oyster (Kino oyster) formula granules 10 g, add 70% ethanol 200 mL, reflux extraction for 1 hour, cool, centrifuge, take supernatant, evaporate to dryness, add 9 mol / L hydrochloric acid hydrolysis.

[0055] The results show that: oyster (Kino oyster) formula granules 10 g, add 70% ethanol extraction, supernatant evaporation to dryness, and then acid hydrolysis, compared with direct acid hydrolysis, 70% ethanol extraction will increase the response value of chromatographic peak, but the baseline noise also increased significantly, the baseline is not smooth, and missing chromatographic peak 8, the overall extraction effect is not good.

[0056] (3)40% ethanol extraction followed by acid hydrolysis

[0057] Take oyster (Kino oyster) formula granules 10 g, add 40% ethanol 200 mL, reflux extraction for 1 hour, cool, centrifuge, take supernatant, evaporate to dryness, add 9 mol / L hydrochloric acid hydrolysis.

[0058] The results show that: oyster (Kino oyster) formula granules 10 g, add 40% ethanol extraction, supernatant evaporation to dryness, and then acid hydrolysis, compared with direct acid hydrolysis, 40% ethanol extracted ingredients concentrated in the first half, chromatogram does not appear the required detection of amino acid composition.

[0059] (4) water extraction and different concentration of alcohol precipitation followed by acid hydrolysis

[0060] Take oyster (Kino oyster) formula granules 10 g, add water 200 mL, reflux extraction for 1 hour, cool, centrifuge, take supernatant, gradually add ethanol, so that its concentration is 10% ethanol, 20% ethanol, 50% ethanol, respectively, centrifuge, take the precipitate, add 9 mol / L acid hydrolysis.

[0061] The results show that: oyster (Kino oyster) formula granules 10 g, add water extraction, then add ethanol, adjust the concentration of ethanol in the solvent, respectively 10% ethanol, 20% ethanol, 50% ethanol, the precipitate obtained by alcohol precipitation contains a small amount of amino acids, the enrichment effect is not good.

[0062] (5) macroporous resin D101 column

[0063] Oyster (Kino oyster) formula granules 1 g, acid dissolution, centrifuge, take supernatant, column separation.

[0064] The results show that: through D101 type macroporous resin, respectively, with water, low concentration of alcohol to ethanol elution, the enrichment effect is not good.

[0065] (6) macroporous resin HP20 column

[0066] Oyster (Oyster) formula granules 1 g, acid lysis, centrifugation, take supernatant, column separation.

[0067] The results show that: through HP20 type macroporous resin, respectively with water, low concentration of alcohol to ethanol elution, 50% ethanol elution, part of the chromatographic peak missing, but compared with other solvent elution, the baseline is smooth, amino acids have a certain residue, but the enrichment effect is not good.

[0068] (7) macroporous resin LD20 column

[0069] Take oyster (Oyster) formula granules 6g, add water 20ml, heat reflux dissolution, cool, centrifugal take supernatant, column.

[0070] The results show that: through LD20 type macroporous resin, respectively with 10% methanol, 20% methanol, 50% methanol elution, under each elution solvent, the separation effect of each compound is not good.

[0071] (8) gel LH-20 column

[0072] Take oyster (Oyster) formula granules 5-6g, add water 20ml, heat reflux dissolution, cool, centrifugal take supernatant, column.

[0073] The results show that: through gel LH-20, there is no amino acid enrichment with water elution, and gel LH-20 column is very slow, so this method is abandoned.

[0074] (9) ion exchange resin (strong acid cation) column

[0075] Take oyster (Oyster) formula granules 5-6g, add water 20ml, heat reflux dissolution, cool, centrifugal take supernatant, column.

[0076] The results show that: after ion exchange resin pretreatment, 10% ammonia water-100% ammonia water 3-5 column volume elution, ammonia water elution has part of amino acid residue, but the last amino acid enrichment effect is not good.

[0077] (10) salt fractionation

[0078] Take oyster (Oyster) formula granules 5-8g, add water 20ml, heat reflux dissolution, cool, centrifugal take supernatant, add sodium chloride reagent step by step salting out. Combine the precipitate, hydrolysis.

[0079] The results show that: the salt fractionation results can not reflect the protein / amino acid enrichment.

[0080] Example 2: Oyster (Oyster) formula granules amino acid preparation method

[0081] The hydrochloric acid concentration, hydrochloric acid dosage, hydrolysis temperature, hydrolysis time, derivatization reagent dosage and derivatization time of oyster (Ostrea gigas) formula granules were investigated.

[0082] (1) Hydrochloric acid concentration investigation

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

[0084] Accurately take 10 mL of the above test sample solution and control sample solution, respectively, and place them in 25 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) in acetonitrile and 2.5 mL of 1 mol / L triethylamine in acetonitrile, shake well, and let stand at room temperature for 2 hours. Add 50% acetonitrile to the mark, 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 filtrate. Accurately take 1 μl of the test sample solution, inject it into the liquid chromatograph, and determine it. The results show that when different hydrochloric acid concentrations are used for extraction, 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 Investigation results of oyster (Ostrea gigas) formula granules characteristic map hydrochloric acid concentration

[0086]

[0087]

[0088] (2) Acid dosage investigation

[0089] Take oyster (Ostrea gigas Thunb) formula granules, grind finely, take about 2.0 g, accurately weigh, 5 groups in parallel, 2 portions each, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a hydrolysis tube with a plug, accurately add 5 mL, 10 mL, 15 mL, 20 mL and 25 mL of 9 mol / L hydrochloric acid respectively, tightly plug, hydrolyze at 150℃ for 2 hours, take out, cool, make up the weight loss 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 filtrate, 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 mark, shake well, and get it.

[0090] Accurately take 10 mL of the above test solution and control solution respectively, and place them in 25 mL volumetric flasks, add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and stand at room temperature for 2 hours, then add 50% acetonitrile to the mark, 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 filtrate. Accurately take 10 μl, inject into the liquid chromatograph, and determine. The results show that there is no obvious difference in the number of characteristic peaks and the separation degree in the characteristic chromatogram when different amounts of acid are used for extraction. 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 smaller. Considering the difference between samples and the parallelism between double samples, the amount of hydrochloric acid is selected as 10 mL. The experimental results are shown in Table 2.

[0091] Table 2 Oyster (Ostrea gigas Thunb) formula granules characteristic chromatogram different acid amount investigation results table

[0092]

[0093] (3) Hydrolysis temperature investigation

[0094] Take oyster (Ostrea gigas Thunb) formula granules, grind finely, take about 2.0 g, accurately weigh, 4 groups in parallel, 2 portions each, add 15 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and place it in a hydrolysis tube with a plug, accurately add 10 mL of 9 mol / L hydrochloric acid, and hydrolyze at 110℃, 130℃, 150℃ and 180℃ respectively for 2 hours, take out, cool, make up the weight loss 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 filtrate, 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 mark, shake well, and get it.

[0095] Precisely take 10 mL of the above test sample solution and control sample solution respectively, and place them in 25 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and stand at room temperature for 2 hours. Then add 50% acetonitrile to the mark, 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. Precisely take 10 μl, inject into the liquid chromatograph, and determine. The results show that, under different hydrolysis temperatures, except that the hydrolysis is not complete at 110℃, the number of characteristic peaks and the separation degree in the characteristic chromatogram at 130℃, 150℃ and 180℃ have no obvious difference, and the response of each characteristic peak at 150℃ is not significantly different from that at 180℃. Considering the safety of experimental operation, the hydrolysis temperature is selected as 150℃. The experimental results are shown in Table 3.

[0096] Table 3 Investigation results of characteristic chromatogram of oyster (Ostrea rivularis Gould) formula granules under different hydrolysis temperatures

[0097]

[0098] (4) Hydrolysis time investigation

[0099] Take oyster (Ostrea rivularis Gould) formula granules, grind finely, take about 2.0 g, accurately weigh, 5 groups in parallel, 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, take the precipitate and place it in a hydrolysis tube with a stopper, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150℃ for 1 h, 2 h, 3 h, 4 h and 5 h respectively, take out, cool down, make up the weight loss 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 filtrate, 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 mark, shake well, and obtain.

[0100] Precisely take 10 mL of the above test sample solution and control sample solution respectively, and place them in 25 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and stand at room temperature for 2 hours. Then add 50% acetonitrile to the mark, 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. Precisely take 10 μl, inject into the liquid chromatograph, and determine. The results show that, under different hydrolysis temperatures, except that the hydrolysis is not complete at 110℃, the number of characteristic peaks and the separation degree in the characteristic chromatogram at 130℃, 150℃ and 180℃ have no obvious difference, and the response of each characteristic peak at 150℃ is not significantly different from that at 180℃. Considering the safety of experimental operation, the hydrolysis temperature is selected as 150℃. The experimental results are shown in Table 3. Precisely take 10 mL of the above test sample solution and control sample solution respectively, and place them in 25 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and stand at room temperature for 2 hours. Then add 50% acetonitrile to the mark, 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. Precisely take 10 μl, inject into the liquid chromatograph, and determine. The results show that, under different hydrolysis temperatures, except that the hydrolysis is not complete at 110℃, the number of characteristic peaks and the separation degree in the characteristic chromatogram at 130℃, 150℃ and 180℃ have no obvious difference, and the response of each characteristic peak at 150℃ is not significantly different from that at 180℃. Considering the safety of experimental operation, the hydrolysis temperature is selected as 150℃. The experimental results are shown in Table 3.

[0101] Table 4 Oyster (Ostrea gigas) formula granules characteristic map different hydrolysis time observation results table

[0102]

[0103] (5) Derivatization time observation

[0104] Take oyster (Ostrea gigas) formula granules, grind finely, take about 2.0 g, accurately weigh, parallel 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 hydrolysis tube with a stopper, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150°C for 3 hours, take out, cool, make up the weight lost with 9 mol / L hydrochloric acid solution, mix well, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrate, evaporate to dryness, add 0.1 mol / L hydrochloric acid solution to dissolve, transfer to a 20 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, shake well, and obtain.

[0105] Accurately take 10 mL of the above test solution and control solution, respectively, and place them in a 25 mL volumetric flask, add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and respectively stand at room temperature for 1 h, 2 h and 3 h, then add 50% acetonitrile to the mark, 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 filtrate. Accurately take 10 μl, inject into the liquid chromatograph, and determine. The results show that there is no significant difference in the number of characteristic peaks and the separation degree in the characteristic map when the derivatization time is 2 h and 3 h, and the response value of each characteristic peak and the "total peak area / sample weight" difference is not large, and the "total peak area / sample weight" is the largest when the derivatization time is 2 h, so the derivatization time is selected as 2 h. The experimental results are shown in Table 5.

[0106] Table 5 Oyster (Ostrea gigas) formula granules characteristic map different derivatization time observation results table

[0107]

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

[0109] According to the experimental results, the oyster (near Jiang oyster) formula granules characteristic map sample pretreatment method is determined as follows: taking oyster (near Jiang oyster) formula granules, grinding, taking about 2.0g, accurately weighing, placing in a 50mL centrifuge tube, adding 10mL of dilute hydrochloric acid solution to dissolve, centrifuging, discarding the supernatant, taking the precipitate and placing it in a hydrolysis tube with a plug, accurately adding 10mL of 9mol / L hydrochloric acid, hydrolyzing at 150℃ for 3 hours, taking out, cooling, supplementing the lost weight with 9mol / L hydrochloric acid solution, mixing, filtering, washing the hydrolysis tube and filter paper with a small amount of water for several times, combining the filtrate, evaporating to dryness, dissolving the residue with 0.1mol / L hydrochloric acid solution, transferring to a 20mL volumetric flask, adding 0.1mol / L hydrochloric acid solution to the mark, shaking to obtain.

[0110] Precisely take 10mL of the above test solution and control solution respectively, and place them in a 25mL volumetric flask. Add 2.5mL of 0.1mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5mL of 1mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 2 hours. Then add 50% acetonitrile to the mark and shake well. Take 10mL and add 10mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, and take the filtrate to obtain.

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

[0112] (1) Hydrochloric acid concentration investigation

[0113] This experiment investigates the influence of different hydrochloric acid concentrations on the characteristic map of calcined oyster (near Jiang oyster) formula granules. Select 6mol / L, 9mol / L, and 12mol / L hydrochloric acid concentrations. Observe the peak shape and separation effect of the 11 tentative characteristic peaks, calculate the "total peak area / sample weight" of the 11 characteristic peaks, and compare the influence of different hydrochloric acid concentrations on the characteristic map of calcined oyster (near Jiang oyster) formula granules to select the best hydrochloric acid concentration.

[0114] Take calcined oyster (near Jiang oyster) formula granules, grind, take about 2.0g, accurately weigh, divide into 3 groups, 2 samples in each group, place in a 50mL centrifuge tube, add 15mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, place the precipitate in a hydrolysis tube with a plug, accurately add 25mL of 6mol / L, 9mol / L, and 12mol / L hydrochloric acid, hydrolyze at 150℃ for 2 hours, take out, cool, supplement the lost weight with the corresponding acid, mix, filter, wash the hydrolysis tube and filter paper with a small amount of water for several times, combine the filtrate, evaporate to dryness, dissolve the residue with 0.1mol / L hydrochloric acid solution, transfer to a 25mL volumetric flask, add 0.1mol / L hydrochloric acid solution to the mark, shake well to obtain.

[0115] Accurately 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 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 2 hours. Then, add 50% acetonitrile to the mark and shake well. Take 10 mL of the solution, add 10 mL of n-hexane, shake, let stand for 10 minutes, collect the lower layer solution, filter, and collect the filtrate. Accurately pipette 1 μL of the test solution and inject it into the liquid chromatograph for determination. The results show that when using different hydrochloric acid concentrations for extraction, the "total peak area / sample weight" value of calcined oyster shell particles is the largest when the hydrochloric acid concentration is 12 mol / L. However, there are certain response differences between samples, and the experiment poses a safety hazard if the hydrochloric acid concentration is too high. Therefore, hydrochloric acid with a hydrolysis concentration of 9 mol / L was selected. The experimental results are shown in Table 6.

[0116] Table 6. Characteristic spectrum of calcined oyster (Oyster oyster) formulation granules and results of hydrochloric acid concentration investigation.

[0117]

[0118]

[0119] (2) Investigation of acid dosage

[0120] Take an appropriate amount of calcined oyster (Oyster muscaria) granules, grind them finely, and accurately weigh about 2.0g. Divide the mixture into three parallel groups, two portions per group, and place each group in a 50mL centrifuge tube. Add 15mL of dilute hydrochloric acid solution to dissolve the granules, centrifuge, discard the supernatant, and place the precipitate in a stoppered hydrolysis tube. Accurately add 5mL, 15mL, and 25mL of 9mol / L hydrochloric acid to each tube, respectively, and seal tightly. Hydrolyze at 150℃ for 2 hours. Remove the tube, cool it, and replenish the lost weight with 9mol / L hydrochloric acid solution. Mix well, filter, and 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 0.1mol / L hydrochloric acid solution, transfer it to a 25mL volumetric flask, add 0.1mol / L hydrochloric acid solution to the mark, and shake well to obtain the final product.

[0121] Accurately 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 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 2 hours. Then, add 50% acetonitrile to the mark and shake well. Take 10 mL of the solution, add 10 mL of n-hexane, shake, let stand for 10 minutes, collect the lower layer solution, filter, and collect the filtrate. Accurately pipette 10 μL and inject it into the liquid chromatograph for determination. The results show that there is no significant difference in the number of characteristic peaks and resolution in the characteristic chromatograms when different amounts of acid are used for extraction. To ensure sufficient extraction, 25 mL of 9 mol / L hydrochloric acid solution was selected. The experimental results are shown in Table 7.

[0122] Table 7 Calcined oyster (Ostrea gigas) formula granules characteristic map different acid dosage observation results table

[0123]

[0124] (3) Hydrolysis temperature investigation

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

[0126] Precisely take 10mL of the above test sample solution and control sample solution, respectively, in 25mL volumetric flask, add 2.5mL of 0.1mol / L phenyl isothiocyanate (PITC) acetonitrile solution, 2.5mL of 1mol / L triethylamine acetonitrile solution, shake well, and place at room temperature for 2 hours, then add 50% acetonitrile to the mark, shake well. Take 10mL, add 10mL of n-hexane, shake, stand for 10 minutes, take the lower layer solution, filter, and take the filtrate. Precisely take 10μl, inject into liquid chromatograph, determine, and get it. The results show that there is no significant difference in the separation degree of each characteristic peak and the "total peak area / sample weight" in the characteristic map of different hydrolysis temperatures. It is shown that the calcined oyster granules are completely hydrolyzed at 150℃, so the hydrolysis temperature is selected as 150℃. The experimental results are shown in Table 8.

[0127] Table 8 Calcined oyster (Ostrea gigas) formula granules characteristic map different hydrolysis temperature observation results table

[0128]

[0129] (4) Hydrolysis time investigation

[0130] Take the right amount of calcined oyster (near oyster) formula granules, grind finely, take about 2.0g, accurately weigh, parallel 5 groups, 2 groups each, put in 50mL centrifuge tube, add dilute hydrochloric acid solution 15mL to dissolve, centrifuge, discard the supernatant, take the precipitate and put it in a hydrolysis tube with a plug, accurately add 9mol / L hydrochloric acid 10mL, 150℃ respectively hydrolysis 1h, 2h, 3h, 4h, 5h, take out, cool, make up the weight lost with 9mol / L hydrochloric acid solution, mix evenly, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrate, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve, transfer to a 25mL volumetric flask, add 0.1mol / L hydrochloric acid solution to the mark, shake evenly, and get it.

[0131] Precisely take 10mL of the above test solution and control solution respectively, and place them in 25mL volumetric flasks. Add 2.5mL of 0.1mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5mL of 1mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 2 hours. Then add 50% acetonitrile to the mark and shake well. Take 10mL and add 10mL of n-hexane, shake, and let stand for 10 minutes. Take the lower layer solution, filter, and take the filtrate. Precisely take 10μl and inject it into the liquid chromatograph for determination. The results show that the extraction time is different, and the hydrolysis is complete at 3 hours. There is no significant difference in the number of characteristic peaks and the separation degree in the characteristic chromatograms of 3 hours, 4 hours and 5 hours of hydrolysis. In order to save time and cost, the hydrolysis time is finally selected as 3 hours. The experimental results are shown in Table 9.

[0132] Table 9 Different hydrolysis time investigation results of calcined oyster (near oyster) formula granules characteristic chromatogram

[0133]

[0134] (5) Derivation time investigation

[0135] Take the right amount of calcined oyster (near oyster) formula granules, grind finely, take about 2.0g, accurately weigh, parallel 3 groups, 2 groups each, put in 50mL centrifuge tube, add dilute hydrochloric acid solution 15mL to dissolve, centrifuge, discard the supernatant, take the precipitate and put it in a hydrolysis tube with a plug, accurately add 9mol / L hydrochloric acid 10mL, 150℃ hydrolysis 3 hours, take out, cool, make up the weight lost with 9mol / L hydrochloric acid solution, mix evenly, filter, wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrate, evaporate to dryness, add 0.1mol / L hydrochloric acid solution to dissolve, transfer to a 25mL volumetric flask, add 0.1mol / L hydrochloric acid solution to the mark, shake evenly, and get it.

[0136] Accurately take 10 mL of the above test solution and the control solution, respectively, and place them in 25 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 1 h, 2 h, and 3 h, respectively. Then add 50% acetonitrile to the mark, shake well. Take 10 mL, add 10 mL of n-hexane, shake, and let stand for 10 min. Take the lower layer solution, filter, and take the subsequent filtrate, which is obtained. Accurately take 10 μl, inject into the liquid chromatograph, and determine, which is obtained. The results show that, for the investigation of different derivatization times, the derivatization reaction is complete at 2 h. The "total peak area / sample weight" is the largest at 2 h of derivatization. The derivatization time is finally selected to be 2 h. The experimental results are shown in Table 10.

[0137] Table 10 Investigation results of different derivatization times of the characteristic chromatogram of calcified oyster (Ostrea gigas) formula granules

[0138]

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

[0140] According to the above experimental results, the sample pretreatment method of the characteristic chromatogram of calcified oyster (Ostrea gigas) formula granules is determined as follows: take calcified oyster (Ostrea gigas) formula granules, grind finely, take about 2.0 g, accurately 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, accurately add 25 mL of 9 mol / L hydrochloric acid, hydrolyze at 150 ℃ for 3 h, take out, cool, add 9 mol / L hydrochloric acid solution to make up the weight loss, 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 to a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to the mark, shake well, and obtain.

[0141] Accurately take 10 mL of the above test solution and the control solution, respectively, and place them in 25 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 1 h, 2 h, and 3 h, respectively. Then add 50% acetonitrile to the mark, shake well. Take 10 mL, add 10 mL of n-hexane, shake, and let stand for 10 min. Take the lower layer solution, filter, and take the subsequent filtrate, which is obtained.

[0142] Example 4: Investigation of chromatographic conditions

[0143] (1) Gradient optimization

[0144] Chromatographic conditions: acetonitrile as mobile phase A, 0.4% phosphoric acid as mobile phase B, gradient elution according to the provisions in Table 11; flow rate was 0.27 mL / min; column temperature was 25°C; detection wavelength was 254 nm; injection volume was 1 μL.

[0145] Table 11 Four gradient elution method tables

[0146]

[0147] The results are shown in Table 11. It was found that the gradient condition of method 4 had a relatively uniform chromatographic peak distribution and good peak shape, and was selected as the analysis condition of the oyster standard decoction characteristic chromatogram. Figure 1

[0148] (2) Chromatographic condition robustness investigation

[0149] 1) Flow rate optimization

[0150] Chromatographic conditions: YMC Triart chromatographic column (2.1 mm x 150 mm, 1.9 μm); acetonitrile as mobile phase A, 0.4% phosphoric acid as mobile phase B, gradient elution according to the provisions in method 4; flow rate was 0.25 mL / min, 0.27 mL / min, 0.29 mL / min; column temperature was 27°C; detection wavelength was 254 nm; injection volume was 1 μL.

[0151] The results are shown in Table 11. It was found that the gradient condition of method 4 had a relatively uniform chromatographic peak distribution and good peak shape, and was selected as the analysis condition of the oyster standard decoction characteristic chromatogram. Figure 2

[0152] 2) Column temperature optimization

[0153] Chromatographic conditions: YMC Triart chromatographic column (2.1 mm x 150 mm, 1.9 μm); acetonitrile as mobile phase A, 0.4% phosphoric acid as mobile phase B, gradient elution according to the provisions in method 4; flow rate was 0.27 mL / min; column temperature was 25°C, 27°C, 29°C; detection wavelength was 254 nm; injection volume was 1 μL.

[0154] The results are shown in Table 11. It was found that the gradient condition of method 4 had a relatively uniform chromatographic peak distribution and good peak shape, and was selected as the analysis condition of the oyster standard decoction characteristic chromatogram. Figure 3

[0155] 3) Chromatographic column investigation

[0156] ​​​There are certain differences in the analysis results of different specifications of chromatographic columns. According to the properties of the separated substances, the change of the chromatographic column can significantly affect the analysis results, so four kinds of chromatographic columns are selected in this study, which are YMC Triart (250mmx4.6mm, 5um) chromatographic column; Kromasil 100-5-C18 (250mmx4.6mm, 5um); Wartes HSS T3 (250mmx4.6mm, 5um) chromatographic column; Phenomenex Luna Omega C18 (250mmx4.6mm, 5um) chromatographic column.

[0157] Chromatographic conditions: acetonitrile as mobile phase A; 0.4% phosphoric acid as mobile phase B, gradient elution according to the provisions in "Method 4" above; flow rate is 0.9mL per minute; column temperature is 30℃; detection wavelength is 254nm; injection volume is 10uL.

[0158] Results are shown in Table 1 Figure 4 It is shown that the replacement of chromatographic column, mobile phase and column temperature and flow rate, etc. cannot achieve ideal separation results, so the ultra-high chromatographic column YMC Triart (2.1mmx150mm, 1.9um) is used for separation optimization.

[0159] Example 5: sample determination

[0160] Chromatographic conditions: YMC Triart C18 chromatographic column (2.1mmx150mm, 1.9um) chromatographic column is selected; acetonitrile as mobile phase A, 0.4% phosphoric acid as mobile phase B, gradient elution according to the provisions in Table 12; flow rate is 0.27mL per minute; column temperature is 27℃; detection wavelength is 254nm; injection volume is 1uL.

[0161] Table 12 Gradient elution table

[0162]

[0163] Test sample preparation method:

[0164] Take the sample powder, finely grind, take about 2.0 g, accurately weigh, put in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to dissolve, centrifuge, discard the supernatant, take the precipitate and put it in a hydrolysis tube with a plug, accurately add 10 mL of 9 mol / L hydrochloric acid, hydrolyze at 150℃ for 3 hours, take out, cool, make up the weight loss 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 filtrate, 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 mark, shake well, and obtain. Accurately measure 10 mL of the above test solution, respectively, and put it in a 25 mL volumetric flask, add 2.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 2.5 mL of 1 mol / L triethylamine acetonitrile solution, shake well, and stand at room temperature for 2 hours. Add 50% acetonitrile to the mark, 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 filtrate.

[0165] 1) Oyster medicinal materials

[0166] The characteristic chromatograms of 18 batches of oyster medicinal materials were analyzed. The proline chromatographic peak was taken as the reference peak S1, and the relative retention time and relative peak area of peaks 1-7 to peak S1 were calculated. The valine peak was taken as the reference peak S2, and the relative retention time and relative peak area of peaks 9-11 to peak S2 were calculated, and the RSD value was calculated. The experimental results are shown in Tables 13 and 14 below, and the superimposed chromatogram of the characteristic chromatogram of 18 batches of oyster medicinal materials is shown in Figure 5 .

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

[0168]

[0169]

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

[0171]

[0172]

[0173] 2) Oyster decoction pieces

[0174] The characteristic chromatograms of 18 batches of oyster decoction pieces were analyzed. The proline chromatographic peak was taken as the reference peak S1, and the relative retention time and relative peak area of peaks 1-7 to peak S1 were calculated. The valine peak was taken as the reference peak S2, and the relative retention time and relative peak area of peaks 9-11 to peak S2 were calculated, and the RSD value was calculated. The experimental results are shown in Tables 15 and 16 below, and the superimposed chromatogram of the characteristic chromatogram of 18 batches of oyster decoction pieces is shown in Figure 6.

[0175] Table 15 Characteristic chromatogram of 18 batches of oyster slices (relative retention time)

[0176]

[0177]

[0178] Table 16 Characteristic chromatogram of 18 batches of oyster slices (relative peak area)

[0179]

[0180]

[0181] 3) Standard oyster decoction

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

[0183] Table 17 Relative retention time of the characteristic chromatogram of 18 batches of standard oyster decoction

[0184]

[0185]

[0186] Table 18 Relative peak area of the characteristic chromatogram of 18 batches of standard oyster decoction

[0187]

[0188] 4) Oyster (Ostrea gigas Thunberg) formula granules

[0189] Take 3 batches of oyster (Ostrea gigas Thunberg) formula granules respectively to prepare test sample solution; according to the chromatographic conditions, sample injection determination, the results are shown in Tables 19 and 20 and Figure 8 .

[0190] Table 19 Characteristic chromatogram of oyster (Ostrea gigas Thunberg) formula granules (relative retention time)

[0191]

[0192] Table 20 Characteristic chromatogram of oyster (Ostrea gigas Thunberg) formula granules (relative peak area)

[0193]

[0194] 5) Calcined concha mallei

[0195] According to the determination method of characteristic spectrum of calcined concha mallei medicinal materials, the characteristic spectrum of 16 batches of calcined concha mallei medicinal material samples was determined, taking proline peak as reference peak S1, the relative retention time and relative peak area of peaks 1-7 and S1 peak were calculated; taking valine peak as reference peak S2, the relative retention time and relative peak area of peaks 8-11 and S2 peak were calculated, and the RSD value was calculated, the results are shown in Tables 21 and 22 below, and the superimposed chromatogram of the characteristic spectrum of 16 batches of calcined concha mallei medicinal materials is shown in Figure 9 .

[0196] Table 21 Characteristic spectrum of 16 batches of calcined concha mallei medicinal materials (relative retention time)

[0197]

[0198]

[0199] Table 22 Characteristic spectrum of 16 batches of calcined concha mallei medicinal materials (relative peak area)

[0200]

[0201] 6) Calcined concha mallei decoction pieces

[0202] According to the determination method of characteristic spectrum of calcined concha mallei decoction pieces, the characteristic spectrum of 16 batches of calcined concha mallei decoction pieces was determined, taking proline peak as reference peak S1, the relative retention time and relative peak area of peaks 1-7 and S1 peak were calculated; taking valine peak as reference peak S2, the relative retention time and relative peak area of peaks 8-11 and S2 peak were calculated, and the RSD value was calculated, the results are shown in Tables 23 and 24 below, and the superimposed chromatogram of the characteristic spectrum of 16 batches of calcined concha mallei decoction pieces is shown in Figure 10 .

[0203] Table 23 Characteristic spectrum of 16 batches of calcined concha mallei decoction pieces (relative retention time)

[0204]

[0205] Table 24 Characteristic spectrum of 16 batches of calcined concha mallei decoction pieces (relative peak area)

[0206]

[0207]

[0208] 7) Calcined concha mallei standard decoction

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

[0210] Table 25 Characteristic spectrum of 16 batches of standard decoction of calcined oyster (relative retention time)

[0211]

[0212]

[0213] Table 26 Characteristic spectrum of 16 batches of standard decoction of calcined oyster (relative peak area)

[0214]

[0215]

[0216] 8) Calcined oyster (Ostrea gigas Thunberg) formula granules

[0217] Prepare sample solutions from 3 batches of calcined oyster (Ostrea gigas Thunberg) formula granules, respectively; according to the chromatographic conditions, the sample is determined, and the results are shown in Tables 27 and 28 and Figure 12 .

[0218] Table 27 Characteristic spectrum of calcined oyster (Ostrea gigas Thunberg) formula granules (relative retention time)

[0219]

[0220] Table 28 Characteristic spectrum of calcined oyster (Ostrea gigas Thunberg) formula granules (relative peak area)

[0221]

[0222] From the experimental results, it can be seen that the characteristic spectrum of oyster medicinal materials and oyster decoction pieces, oyster standard decoction, oyster (near Jiang oyster) formula granules, calcined oyster medicinal materials and calcined oyster decoction pieces, calcined oyster standard decoction, calcined oyster (near Jiang oyster) formula granules can be stably presented, and correspond to the retention time of 11 characteristic peaks in the reference chromatogram of the control medicinal materials. The method for determining amino acids of oyster medicinal materials and its standard decoction or formula granules established by the present application can be simultaneously applied to the determination of different types of amino acid contents in oyster medicinal materials, oyster processed products, oyster standard decoction, oyster formula granules, oyster processed product standard decoction, oyster processed product formula granules. Not only does it provide a control means for amino acid determination for oyster and its processed products, greatly saving the cost of quality standard research process and future sample inspection, but also provides a reference for the determination of amino acids in other mineral traditional Chinese medicines.

[0223] The above is the preferred embodiment of the application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements are also considered to be within the scope of protection of the present application.

Claims

1. A method for constructing a characteristic spectrum of oyster medicinal material, standard decoction, or formulated granules, characterized in that, Includes the following steps: Prepare a reference solution, wherein the reference solution 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, oyster standard decoction or oyster formula granules, add hydrochloric acid solution to dissolve and perform acid hydrolysis treatment, then filter, evaporate the obtained filtrate to dryness, and dissolve the residue in hydrochloric acid solution to obtain the test solution; Take the pre-set amounts of the test solution and reference solution, respectively, and mix them thoroughly with phenyl isothiocyanate and triethylamine in acetonitrile solution. Add n-hexane, filter the lower layer solution, and collect the filtrate. Inject the filtrate into a liquid chromatograph for determination. The liquid chromatograph uses a YMC Triart C18 column with dimensions of 2.1 mm × 150 mm and 1.9 μm. Gradient elution is performed with acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B to obtain the characteristic chromatograms of oyster medicinal materials, standard decoctions, or formula granules. The gradient elution is performed according to the following procedure: From 0 to 14 minutes, mobile phase A decreased from 15% to 20%, and mobile phase B decreased from 85% to 80%. Between 14 and 17 minutes, mobile phase A decreased from 20% to 36%, and mobile phase B decreased from 80% to 64%. From 17 to 22 minutes, mobile phase A was maintained at 36% and mobile phase B was maintained at 64%. From 22 to 40 minutes, mobile phase A decreased from 36% to 38%, and mobile phase B decreased from 64% to 62%. Over 40-45 minutes, mobile phase A decreased from 38% to 70%, and mobile phase B decreased from 62% to 30%. The concentration of the phosphoric acid solution is 0.25%~0.65%; The ultraviolet detection wavelength of the liquid chromatograph is 250nm~260nm.

2. The method for constructing the characteristic spectrum of oyster medicinal materials, standard decoctions, or formulated granules as described in claim 1, characterized in that, Column temperature: 25℃~29℃; During gradient elution, the flow rate of the mobile phase is 0.25 mL / min to 0.29 mL / min.

3. The method for constructing the characteristic spectrum of oyster medicinal materials, standard decoctions, or formulated granules as described in claim 1, characterized in that, In the acid hydrolysis treatment, hydrochloric acid with a concentration of 8 mol / L to 10 mol / L is used to hydrolyze the solution at 130℃ to 180℃ for 2 to 5 hours.

4. The method for constructing the characteristic spectrum of oyster medicinal material, standard decoction, or formula granules as described in claim 1, characterized in that, The reference solution was prepared according to the following method: Take serine reference standards, glycine reference standards, glutamic acid reference standards, aspartic acid reference standards, threonine reference standards, proline reference standards, alanine reference standards, valine reference standards, lysine reference standards, leucine reference standards, and phenylalanine reference standards, add 0.05~0.2 mol / L hydrochloric acid to prepare a mixed solution, which is used as the reference solution.

5. The method for constructing the characteristic spectrum of oyster medicinal materials, standard decoctions, or formulated granules as described in claim 1, characterized in that, The characteristic spectrum of the oyster medicinal material, standard decoction, or formula granules includes 11 common peaks, of which peak 1 corresponds to serine, peak 2 corresponds to glycine, peak 3 corresponds to glutamic acid, peak 4 corresponds to aspartic acid, peak 5 corresponds to threonine, peak 6 corresponds to proline, peak 7 corresponds to alanine, peak 8 corresponds to valine, peak 9 corresponds to leucine, peak 10 corresponds to lysine, and peak 11 corresponds to phenylalanine.

6. The method for constructing the characteristic spectrum of oyster medicinal material, standard decoction, or formula granules as described in claim 5, characterized in that, In the characteristic chromatograms of the oyster medicinal materials, standard decoctions, or formula granules, with the proline peak as the reference peak S1, the relative retention times of peaks 1 to 5 and peak 7 with the reference peak S1 are as follows: peak 1 0.61, peak 2 0.65, peak 3 0.8, peak 4 0.83, peak 5 0.9, peak 7 1.09, with a relative standard deviation within ±10%. Using the valine peak as the reference peak S2, the relative retention times of peaks 9 to 11 with the reference peak S2 are consistent: peak 9 1.18, peak 10 1.26, peak 11 1.29, with relative standard deviations within ±10%.

7. The application of the method for constructing the characteristic spectrum of oyster medicinal material, standard decoction or formula granules as described in any one of claims 1 to 6 in (1): (1) Quality control of oyster medicinal materials, standard decoctions, and formula granules.

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