Method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules and application of cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules

Abstract

The invention discloses a method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules and application, and relates to the technical field of traditional Chinese medicine identification. The detection method comprises the following steps: hydrolyzing and extracting cuttlebone medicinal materials or decoction pieces thereof, standard decoction and formula granules to obtain a test solution; dissolving the reference substance with a first hydrochloric acid solution with the concentration of 0.05 mol / L-1 mol / L to obtain a reference substance solution; and / or performing hydrolysis extraction on a cuttlebone reference medicinal material to obtain a reference medicinal material solution; determining by adopting a liquid chromatograph; a chromatographic column of the liquid chromatograph takes octadecylsilane chemically bonded silica as a stationary phase, a mixed solution of acetonitrile and a sodium acetate solution with the concentration of 0.05 mol / L-1 mol / L as a mobile phase A and a mixed solution of acetonitrile and water as a mobile phase B for gradient elution; in the mobile phase A, the volume ratio of the acetonitrile to the sodium acetate solution is 6: 94-8: 92; and in the mobile phase B, the volume ratio of acetonitrile to water is (3: 1)-(5: 1). The detection method disclosed by the invention can be used for determining various amino acid components in the cuttlebone, effectively identifying common similar and counterfeit products, and providing a scientific basis for formulating the quality standard of the cuttlebone.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and in particular to a method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules and an application thereof. Background Art

[0002] Cuttlebone is the dried inner shell of Sepia maindroni de Rochebrune or Sepiasculenta Hoyle, both members of the family Sepiidae. It is salty, astringent, and warm in nature, and enters the spleen and kidney meridians. It has anti-acid and analgesic properties, astringent and hemostatic properties, and astringent and anti-leukorrhea properties. It is one of the marine traditional Chinese medicines included in the 2020 edition of the Chinese Pharmacopoeia. Its primary component is calcium carbonate, along with chitin, protein, peptides, amino acids, polysaccharides, nucleosides, and other trace elements. Cuttlebone belongs to the mineral type of Chinese medicine, which is subdivided into shells. Shell-based Chinese medicine refers to a type of Chinese medicine that originates from the phylum Mollusca and whose medicinal part is shells. The 2020 edition of the Chinese Pharmacopoeia includes 6 types of shell-based Chinese medicines, namely oysters, cuttlebone, stone cassia, mother-of-pearl, corrugated shells and clam shells. Non-professional medicinal material identifiers can easily confuse various shell-based Chinese medicines, and the properties of shell fragments are similar. Therefore, distinguishing them through objective detection methods can make the use of medicines more accurate and avoid misuse.

[0003] Because the main component of shell-derived Chinese herbal medicines is calcium carbonate, identifying them solely based on calcium carbonate is difficult. One feasible approach is to test for amino acid components. Modern research uses methods such as Fourier transform near-infrared spectroscopy, formaldehyde enzymatic solution assays, PITC (phenyl isothiocyanate) pre-column derivatization, automatic amino acid analyzers, potentiometric titration, Folin phenol colorimetry, micellar electrokinetic capillary electrophoresis, and pH adjustment. These methods are relatively complex, while high-performance liquid chromatography is the most commonly used method for amino acid detection. Amino acid detection has been applied to cuttlebone. Existing literature uses methods such as ultrafast liquid chromatography-tandem mass spectrometry (UFLC-MS / MS) and automatic amino acid analyzers (ninhydrin post-column derivatization ion exchange chromatography). Automatic amino acid analyzers are complex to operate, and tandem mass spectrometry places high demands on both the instrument and the operator. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting the amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules, which can realize the determination of amino acid components in cuttlebone and identify common near-counterfeit products.

[0005] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned method for detecting the amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules in identifying cuttlebone and its near-counterfeit products.

[0006] The technical problem that the present invention also aims to solve is to provide a method for identifying cuttlebone, which can realize the identification of common cuttlebone counterfeits.

[0007] In order to solve the above technical problems, the present invention provides a method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules, which comprises:

[0008] The cuttlebone medicinal material or its decoction pieces, standard decoction, or formula granules are hydrolyzed and extracted to obtain a test solution;

[0009] The reference substance is dissolved in a first hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L to obtain a reference substance solution; and / or the cuttlebone reference medicinal material is hydrolyzed and extracted to obtain a reference medicinal material solution. Exemplarily, the concentration of the first hydrochloric acid solution is 0.08 mol / L, 0.1 mol / L, 0.16 mol / L, 0.25 mol / L, 0.36 mol / L, 0.5 mol / L, 0.66 mol / L, 0.78 mol / L, 0.85 mol / L, or 0.9 mol / L, but is not limited thereto. Preferably, it is 0.05 mol / L to 0.4 mol / L, and more preferably, it is 0.05 mol / L to 0.2 mol / L.

[0010] The test solution, reference solution and / or reference medicinal material solution are measured by liquid chromatography;

[0011] The chromatographic column of the liquid chromatograph is composed of octadecylsilane bonded silica gel as the stationary phase, a mixed solution of acetonitrile and sodium acetate solution as the mobile phase A, and a mixed solution of acetonitrile and water as the mobile phase B for gradient elution. The gradient elution curve is:

[0012] 0 min to 15 min, mobile phase A from 100% to 98%, mobile phase B from 0% to 2%;

[0013] 15-22 min, mobile phase A from 98% to 90%, mobile phase B from 2% to 10%;

[0014] 22-28 min, mobile phase A from 90% to 82%, mobile phase B from 10% to 18%;

[0015] 28-40 min, mobile phase A from 82% to 75%, mobile phase B from 18% to 25%;

[0016] 40-45 min, mobile phase A from 75% to 62%, mobile phase B from 25% to 38%;

[0017] 45-50 min, mobile phase A from 62% to 100%, mobile phase B from 38% to 0%;

[0018] The control substances include aspartic acid control substance, glutamic acid control substance, serine control substance, glycine control substance, threonine control substance, arginine control substance, alanine control substance, proline control substance, tyrosine control substance, valine control substance, isoleucine control substance, leucine control substance, phenylalanine control substance and L-lysine control substance;

[0019] In the mobile phase A, the volume ratio of acetonitrile to sodium acetate solution is 6:94 to 8:92, exemplified by, but not limited to, 6.5:93.5, 7:93, 7.5:92.5, or 8:92. Preferably, it is 6.5:93.5 to 7.5:92.5. The sodium acetate solution is an aqueous solution of sodium acetate, and its concentration is 0.05 mol / L to 1 mol / L, exemplified by, but not limited to, 0.08 mol / L, 0.13 mol / L, 0.25 mol / L, 0.33 mol / L, 0.46 mol / L, 0.57 mol / L, 0.6 mol / L, 0.8 mol / L, or 0.92 mol / L; preferably, it is 0.05 mol / L to 0.2 mol / L. The pH of the sodium acetate solution is 6 to 8, exemplified by, but not limited to, 6.3, 6.7, 7.2, 7.5, or 7.8.

[0020] In the mobile phase B, the volume ratio of acetonitrile to water is 3:1 to 5:1, exemplified by 3.1:1, 3.5:1, 3.9:1, 4.3:1, 4.7:1 or 4.9:1, but not limited thereto. Preferably, the volume ratio of acetonitrile to water is 3.5:1 to 5:1.

[0021] It should be noted that, in the present invention, only one of the control medicinal material solution or the reference substance solution can be used together with the test solution for determination, or the control medicinal material solution, the reference substance solution and the test solution can be used together for determination.

[0022] Preferably, in some embodiments of the present invention, in the mobile phase A, the volume ratio of acetonitrile to sodium acetate solution is 7:93; the concentration of the sodium acetate solution is 0.1 mol / L, and its pH is 6-7; and / or

[0023] In the mobile phase B, the volume ratio of acetonitrile to water is 4:1.

[0024] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 50 mm to 250 mm, exemplarily 100 mm, 150 mm, or 200 mm, but not limited thereto. The column diameter of the chromatographic column is 2.1 mm to 5 mm, exemplarily 3.0 mm, 3.9 mm, or 4.6 mm, but not limited thereto. The particle size of the stationary phase is 2.7 μm to 5 μm, exemplarily 3 μm or 5 μm, but not limited thereto.

[0025] The column temperature of the chromatographic column is 25° C. to 29° C., exemplified by 25.5° C., 26° C., 26.5° C., 27° C., 27.5° C., 28° C. or 28.5° C., but not limited thereto.

[0026] The flow rate of the liquid chromatograph is 0.75 mL / min to 0.85 mL / min, exemplified by, but not limited to, 0.77 mL / min, 0.79 mL / min, 0.81 mL / min, 0.83 mL / min, or 0.84 mL / min. The detection wavelength of the liquid chromatograph is 250 nm to 300 nm, exemplified by, but not limited to, 254 nm, 266 nm, 278 nm, 285 nm, 290 nm, or 296 nm.

[0027] Specifically, in some embodiments of the present invention, the injection volume of the test solution is 0.5 μL to 3 μL, exemplified by 0.8 μL, 1.2 μL, 1.6 μL, 2.3 μL, 2.5 μL, or 2.7 μL, but not limited thereto. The injection volume of the reference solution is 0.5 μL to 3 μL, exemplified by 0.8 μL, 1.2 μL, 1.6 μL, 2.3 μL, 2.5 μL, or 2.7 μL, but not limited thereto. The injection volume of the control medicinal material solution is 0.5 μL to 3 μL, exemplified by 0.8 μL, 1.2 μL, 1.6 μL, 2.3 μL, 2.5 μL, or 2.7 μL, but not limited thereto.

[0028] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 150 mm, a column diameter of 4.6 mm, and a particle size of the stationary phase of 3 μm; an exemplary chromatographic column may be a Shim-pack Scepter C18-120 column, but is not limited thereto.

[0029] The column temperature of the chromatographic column is 27°C;

[0030] The flow rate of the liquid chromatograph was 0.80 mL / min, the detection wavelength was 254 nm, the injection volume of the test solution was 1 μL, the injection volume of the reference solution was 1 μL, and the injection volume of the reference medicinal material solution was 1 μL.

[0031] Specifically, in some embodiments of the present invention, in the step of hydrolyzing and extracting the cuttlebone medicinal material or its decoction pieces, standard decoction, or formula granules to obtain the test solution, the cuttlebone medicinal material or its decoction pieces, standard decoction, or formula granules are hydrolyzed with a second hydrochloric acid solution having a concentration of 2 mol / L to 5 mol / L, and the solid phase obtained after solid-liquid separation is dissolved with a third hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L, and then derivatized with an acetonitrile solution of phenyl isothiocyanate having a concentration of 0.05 mol / L to 0.4 mol / L and an acetonitrile solution of triethylamine having a concentration of 0.5 mol / L to 4 mol / L, and finally extracted with n-hexane to obtain the test solution.

[0032] Exemplarily, the concentration of the second hydrochloric acid solution is 2.4 mol / L, 2.8 mol / L, 3.2 mol / L, 3.6 mol / L, 4.2 mol / L, 4.4 mol / L, or 4.8 mol / L, but is not limited thereto. Preferably, it is 2 mol / L to 4 mol / L, more preferably 3 mol / L to 4 mol / L, and even more preferably 4 mol / L.

[0033] Exemplarily, the concentration of the third hydrochloric acid solution is 0.08 mol / L, 0.1 mol / L, 0.16 mol / L, 0.25 mol / L, 0.36 mol / L, 0.5 mol / L, 0.66 mol / L, 0.78 mol / L, 0.85 mol / L, or 0.9 mol / L, but is not limited thereto. Preferably, it is 0.05 mol / L to 0.4 mol / L, more preferably 0.05 mol / L to 0.15 mol / L, and even more preferably 0.1 mol / L.

[0034] Exemplarily, the concentration of phenyl isothiocyanate in the acetonitrile solution of phenyl isothiocyanate is 0.06 mol / L, 0.15 mol / L, 0.19 mol / L, 0.23 mol / L, 0.28 mol / L, 0.31 mol / L, 0.35 mol / L or 0.38 mol / L, but is not limited thereto. Preferably, it is 0.05 mol / L to 0.2 mol / L, more preferably 0.05 mol / L to 0.15 mol / L, and even more preferably 0.1 mol / L.

[0035] Exemplarily, the concentration of triethylamine in the acetonitrile solution of triethylamine is 0.8 mol / L, 1.2 mol / L, 1.6 mol / L, 2.1 mol / L, 2.5 mol / L, 2.9 mol / L, 3.4 mol / L or 3.7 mol / L, but is not limited thereto. Preferably, it is 0.5 mol / L to 2 mol / L, more preferably 0.5 mol / L to 1.5 mol / L, and even more preferably 1 mol / L.

[0036] Preferably, in some embodiments of the present invention, the method for preparing the test solution comprises:

[0037] hydrolyzing the cuttlebone medicinal material, decoction piece, standard decoction or formula granule with a second hydrochloric acid solution having a concentration of 2 mol / L to 5 mol / L at 120° C. to 200° C. for 1.5 to 2.5 hours; dissolving the solid phase obtained after solid-liquid separation with a third hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L to obtain a first intermediate solution;

[0038] The first intermediate solution is derivatized with a 0.05 mol / L to 0.4 mol / L phenyl isothiocyanate in acetonitrile solution and a 0.5 mol / L to 4 mol / L triethylamine in acetonitrile solution at 15° C. to 40° C. for 1 to 5 hours; then a predetermined amount of a 40 vol% to 80 vol% acetonitrile aqueous solution is added to obtain a second intermediate solution;

[0039] The second intermediate solution is extracted with n-hexane, and the obtained lower layer solution is filtered to obtain the test solution;

[0040] Among them, the ratio of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules to the second hydrochloric acid solution is 0.1g~1g:15mL~30mL, exemplified by 0.2g:18mL, 0.4g:20mL, 0.6g:24mL, 0.8g:28mL or 0.9g:28mL, but not limited thereto, preferably 0.1g~0.8g:18mL~25mL.

[0041] Among them, the ratio of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules to the third hydrochloric acid solution is 0.1g~1g:20mL~30mL, exemplified by 0.2g:21mL, 0.4g:24mL, 0.5g:26mL, 0.7g:28mL or 0.9g:29mL, but not limited thereto, preferably 0.1g~0.5g:22mL~28mL.

[0042] Exemplarily, the hydrolysis temperature is 135° C., 150° C., 165° C., 180° C. or 190° C., but is not limited thereto. Preferably, the hydrolysis temperature is 130° C. to 160° C., more preferably 130° C. to 150° C., and even more preferably 140° C.

[0043] Exemplarily, the hydrolysis time is 1.8 h, 1.9 h, 2.2 h or 2.4 h, but is not limited thereto. Preferably, the hydrolysis time is 1.8 h to 2.4 h, more preferably 1.8 h to 2.2 h, and more preferably 2 h.

[0044] The volume ratio of the first intermediate solution to the acetonitrile solution of phenyl isothiocyanate is 10:1 to 10:4, exemplified by, but not limited to, 10:1.5, 10:1.8, 10:2.3, 10:2.7, 10:3.1, 10:3.5, or 10:3.8, preferably 10:2 to 10:3, and more preferably 10:2.5.

[0045] The volume ratio of the first intermediate solution to the triethylamine acetonitrile solution is 10:1 to 10:4, exemplified by, but not limited to, 10:1.5, 10:1.8, 10:2.3, 10:2.7, 10:3.1, 10:3.5, or 10:3.8, preferably 10:2 to 10:3, and more preferably 10:2.5.

[0046] The volume ratio of the first intermediate solution to the acetonitrile aqueous solution is 10:8 to 10:15, exemplified by 10:8.3, 10:8.7, 10:9.5, 10:10.3, 10:11.4, 10:12.5, 10:13.3 or 10:14.7, but not limited thereto.

[0047] Exemplarily, the concentration of the acetonitrile aqueous solution is 45 vol%, 52 vol%, 64 vol%, 71 vol% or 78 vol%, but is not limited thereto, preferably 40 vol% to 60 vol%, more preferably 40 vol% to 55 vol%, and more preferably 50 vol%.

[0048] The volume ratio of the second intermediate solution to n-hexane is 10:8 to 10:15, exemplified by, but not limited to, 10:8.5, 10:9, 10:9.5, 10:10, 10:11.5, 10:13, or 10:14.5, preferably 10:8 to 10:12, and more preferably 10:10.

[0049] Specifically, in some embodiments of the present invention, the method for preparing the control medicinal material solution includes:

[0050] The cuttlebone control medicinal material is hydrolyzed with a fourth hydrochloric acid solution having a concentration of 2 mol / L to 5 mol / L at 120° C. to 200° C. for 1.5 to 2.5 hours; the solid phase obtained after solid-liquid separation is dissolved with a fifth hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L to obtain a third intermediate solution; wherein the ratio of the cuttlebone control medicinal material to the fourth hydrochloric acid solution is 0.1 g to 1 g: 5 mL to 40 mL, and the ratio of the cuttlebone control medicinal material to the fifth hydrochloric acid solution is 0.1 g to 1 g: 5 mL to 50 mL;

[0051] The third intermediate solution is derivatized with a 0.05 mol / L to 0.4 mol / L acetonitrile solution of phenyl isothiocyanate and a 0.5 mol / L to 4 mol / L acetonitrile solution of triethylamine at 15° C. to 40° C. for 1 to 5 hours; then a predetermined amount of a 40 vol% to 80 vol% acetonitrile aqueous solution is added to obtain a fourth intermediate solution; wherein the volume ratio of the third intermediate solution to the acetonitrile solution of phenyl isothiocyanate is 10:1 to 10:4, the volume ratio of the third intermediate solution to the acetonitrile aqueous solution of triethylamine is 10:1 to 10:4, and the volume ratio of the third intermediate solution to the acetonitrile aqueous solution is 10:8 to 10:15;

[0052] The fourth intermediate solution is extracted with n-hexane, and the obtained lower layer solution is filtered to obtain a control medicinal material solution; wherein the volume ratio of the fourth intermediate solution to n-hexane is 10:8 to 10:15.

[0053] Specifically, in some embodiments of the present invention, the concentration of each reference substance in the reference substance solution is 5 μg / mL to 20 μg / mL, exemplified by, but not limited to, 8 μg / mL, 10.5 μg / mL, 12 μg / mL, 13.5 μg / mL, 15 μg / mL, 17.5 μg / mL, or 19 μg / mL. Preferably, the concentration is 5 μg / mL to 15 μg / mL, and more preferably, 10 μg / mL.

[0054] In addition, it should be noted that the solutions used in the present invention, unless otherwise specified, refer to solutions using water as a solvent.

[0055] Specifically, in some embodiments of the present invention, the chromatogram obtained by the measurement includes 16 common peaks, wherein peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 6 is threonine, peak 7 is arginine, peak 8 is alanine, peak 9 is proline, peak 10 is tyrosine, peak 11 is valine, peak 13 is isoleucine, peak 14 is leucine, peak 15 is phenylalanine, and peak 16 is L-lysine;

[0056] Taking peak 4 as S1 peak, calculate the relative retention time of peaks 1 to 3 and peaks 5 to 9. The relative retention time of each peak is within ±10% of the specified value. The specified values ​​of peaks 1 to 3 and peaks 5 to 9 are 0.35, 0.41, 0.90, 1.42, 1.50, 1.55, 1.64 and 1.73 respectively.

[0057] Taking peak 14 as the S2 peak, the relative retention times of peaks 10 to 13 and peaks 15 to 16 were calculated. The relative retention times of each peak were within ±10% of the specified value. Among them, the specified values ​​of peaks 10 to 13 and peaks 15 to 16 were 0.81, 0.83, 0.88, 0.99, 1.12, and 1.21, respectively.

[0058] Specifically, in some embodiments of the present invention, the cuttlebone slices may be cleaned cuttlebone (see page 307 of Part I of the 2020 edition of the Chinese Pharmacopoeia), or fried cuttlebone, or bran-processed cuttlebone, or vinegar-processed cuttlebone, but not limited thereto. Accordingly, the cuttlebone standard decoction refers to the preparation obtained by decocting, concentrating and freeze-drying the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules". The cuttlebone formula granules refer to the preparation obtained by extracting, separating, concentrating, drying and granulating the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", but not limited thereto.

[0059] Correspondingly, the present invention also discloses the use of the above-mentioned method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules in identifying cuttlebone and its near-counterfeit products.

[0060] Accordingly, the present invention also discloses a cuttlebone identification method for identifying cuttlebone and its near-imitations, comprising:

[0061] Provide the substance to be identified;

[0062] Detecting the substance to be identified using the above-mentioned method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules to obtain a chromatogram;

[0063] If the chromatogram shows peaks 1 to 16 at the same time, the substance to be identified includes or is cuttlebone; otherwise, it is a near-fake product.

[0064] For example, the near-fakes are oysters, cuttlebone, stone cassia and mother-of-pearl, but are not limited thereto. For example, the identification method of the present invention can also identify some other near-fakes, such as corrugated shells and clam shells.

[0065] The implementation of the present invention has the following beneficial effects:

[0066] The present invention establishes a method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions, or formula granules. The method can be simultaneously applied to the determination of different types of amino acids in Chinese medicine preparations such as cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules. This method not only provides an amino acid detection method for quality control of cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules, but can also be used to distinguish cuttlebone from different calcium-containing mineral drugs, greatly advancing the research progress of cuttlebone quality standards and promoting the identification research of mineral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 1 is a chromatogram of the cuttlebone standard decoction when different mobile phase systems are used in Example 1;

[0068] Figure 2 1 is a chromatogram of the cuttlebone standard decoction using different gradient conditions in Example 1;

[0069] Figure 3 1 is a chromatogram of the cuttlebone standard decoction at different column temperatures in Example 1;

[0070] Figure 4 1 is a chromatogram of the cuttlebone standard decoction at different flow rates in Example 1;

[0071] Figure 5 This is a graph showing the results of the specific property investigation in Example 1;

[0072] Figure 6 This is an overlay of chromatograms of 17 batches of cuttlebone medicinal materials in Example 1;

[0073] Figure 7 It is an overlay of chromatograms of 17 batches of cuttlebone slices in Example 1;

[0074] Figure 8 This is an overlay of chromatograms of 17 batches of cuttlebone standard decoction in Example 1;

[0075] Figure 9 This is an overlay of chromatograms of three batches of cuttlebone formula granules in Example 1;

[0076] Figure 10 This is a comparison chart of the chromatograms of cuttlebone, cassia bark, oyster, and mother-of-pearl in Example 2;

[0077] Among them, peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 6 is threonine, peak 7 is arginine, peak 8 is alanine, peak 9 is proline, peak 10 is tyrosine, peak 11 is valine, peak 13 is isoleucine, peak 14 is leucine, peak 15 is phenylalanine, peak 16 is L-lysine, and peak X is the derivatization reagent peak. DETAILED DESCRIPTION

[0078] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0079] Example 1 Establishment of a Detection Method for Cuttlebone Amino Acid Components

[0080] 1 Preparation of test solution

[0081] Medicinal Materials: Accurately weigh approximately 0.2g of fine cuttlebone powder and place it in a stoppered hydrolysis tube. Slowly and accurately add 20mL of 4mol / L hydrochloric acid solution, weigh the weight, stopper tightly, and hydrolyze at 150°C for 2 hours. Allow to cool, weigh again, and make up the loss with 4mol / L hydrochloric acid solution. Mix thoroughly, filter, and transfer to an evaporating dish. Wash the hydrolysis tube and filter paper with approximately 30mL of water in portions. Combine the washings in the evaporating dish and evaporate to dryness. Dissolve the residue in 0.1mol / L hydrochloric acid solution, transfer to a 25mL volumetric flask, and add 0.1mol / L hydrochloric acid solution to the mark. Shake well. Accurately measure 10mL of this solution and place it in a 25mL volumetric flask. Add 2.5mL of 0.1mol / L phenyl isothiocyanate (PITC) in acetonitrile and 2.5mL of 1mol / L triethylamine in acetonitrile. 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, let stand for 10 minutes, take the lower layer of solution, filter, and take the filtrate to obtain the product.

[0082] Decoction piece: Take about 0.2g of fine powder of cuttlebone decoction piece, accurately weigh it, place it in a stoppered hydrolysis tube, slowly and accurately add 20mL of 4mol / L hydrochloric acid solution, weigh it, stopper it, hydrolyze it at 150℃ for 2 hours, let it cool, weigh it again, make up the loss with 4mol / L hydrochloric acid solution, mix it, filter it, transfer it to an evaporating dish, wash the hydrolysis tube and filter paper with about 30mL of water in batches, add the washing liquid to the evaporating dish, evaporate it 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 scale, and shake it well. Accurately measure 10mL of the above solution, place it in a 25mL volumetric flask, add 2.5mL of 0.1mol / L phenyl isothiocyanate (PITC) acetonitrile solution, 2.5mL of 1mol / L triethylamine acetonitrile solution, shake it well, let it stand at room temperature for 2 hours, add 50% acetonitrile to the scale, and shake it well. Take 10 mL, add 10 mL of n-hexane, shake, let stand for 10 minutes, take the lower layer of solution, filter, and take the filtrate to obtain the product.

[0083] Standard decoction: Take 0.2 g of freeze-dried powder of cuttlebone standard decoction, accurately weigh it, place it in a stoppered hydrolysis tube, slowly and accurately add 20 mL of 4 mol / L hydrochloric acid solution, weigh it, stopper it tightly, hydrolyze it at 150°C for 2 hours, let it cool, weigh it again, make up the loss with 4 mol / L hydrochloric acid solution, mix it, filter it, transfer it to an evaporating dish, wash the hydrolysis tube and filter paper with about 30 mL of water in batches, combine the washings into the evaporating dish, evaporate it to dryness, dissolve the residue in 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, and shake it well. Accurately measure 10 mL of the above solution and place it in a 25 mL volumetric flask. Add 2.5 mL of a 0.1 mol / L phenyl isothiocyanate (PITC) solution in acetonitrile and 2.5 mL of a 1 mol / L triethylamine solution in acetonitrile. Shake well. After standing at room temperature for 2 hours, add 50% acetonitrile to the mark and shake well. Take 10 mL of the solution and add 10 mL of n-hexane. Shake well and let stand for 10 minutes. Remove the lower layer, filter, and collect the filtrate.

[0084] Formulated Granules: Accurately weigh approximately 0.5g of finely powdered cuttlebone formula granules and place them in a stoppered hydrolysis tube. Slowly and accurately add 20mL of 4mol / L hydrochloric acid solution, weigh the weight, stopper tightly, and hydrolyze at 150°C for 2 hours. Allow to cool, weigh again, and make up the loss with 4mol / L hydrochloric acid solution. Mix thoroughly, filter, and transfer to an evaporating dish. Wash the hydrolysis tube and filter paper with approximately 30mL of water in portions. Combine the washings in the evaporating dish and evaporate to dryness. Dissolve the residue in 0.1mol / L hydrochloric acid solution, transfer to a 25mL volumetric flask, and add 0.1mol / L hydrochloric acid solution to the mark. Shake well. Accurately measure 10mL of this solution and place it in a 25mL volumetric flask. Add 2.5mL of 0.1mol / L phenyl isothiocyanate (PITC) in acetonitrile and 2.5mL of 1mol / L triethylamine in acetonitrile. 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, let stand for 10 minutes, take the lower layer of solution, filter, and take the filtrate to obtain the product.

[0085] 2 Preparation of reference solution

[0086] Reference substance solution: Take appropriate amount of aspartic acid reference substance, glutamic acid reference substance, serine reference substance, glycine reference substance, threonine reference substance, arginine reference substance, alanine reference substance, proline reference substance, tyrosine reference substance, valine reference substance, isoleucine reference substance, leucine reference substance, phenylalanine reference substance and L-lysine reference substance, weigh them accurately, and add 0.1 mol / L hydrochloric acid solution to make a mixed solution containing 10 μg of each substance per 1 mL, which is used as the reference substance solution.

[0087] Reference Material Solution: Accurately weigh approximately 0.5 g of cuttlebone control material, place in a stoppered hydrolysis tube, accurately add 20 mL of 4 mol / L hydrochloric acid, and hydrolyze at 150°C for 2 hours. Cool, filter, and transfer to an evaporating dish. Wash the hydrolysis tube and filter paper with approximately 30 mL of water in portions. Combine the washings in the evaporating dish and evaporate to dryness. Dissolve the residue in 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. Accurately measure 10 mL of this solution and place in a 25 mL volumetric flask. 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. 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, let stand for 10 minutes, take the lower layer of solution, filter, and take the filtrate to obtain the product.

[0088] 3 Chromatographic conditions

[0089] Chromatographic column: Shim-pack Scepter C18-120 (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); mobile phase A: acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93), mobile phase B: acetonitrile-water (4:1), gradient elution as specified in Table 1; flow rate, 0.8 mL / min; column temperature, 27°C; detection wavelength, 254 nm. The number of theoretical plates, calculated based on the glycine peak, should be no less than 5000.

[0090] Table 1 Gradient elution program conditions

[0091]

[0092] 4 Determination

[0093] Accurately aspirate 1 μL of reference substance solution, 1 μL of control medicinal material reference substance solution, and 1 μL of test sample solution respectively, inject them into liquid chromatograph, and measure to obtain the result.

[0094] 5 Methodological Investigation

[0095] 5.1 Investigation of mobile phase system

[0096] This section examines the effect of the mobile phase system on the detection method of cuttlebone amino acid components. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then the test was carried out according to the following chromatographic conditions.

[0097] Chromatographic conditions: Shim-pack Scepter C18-120 (4.6 mm × 150 mm, 3 μm) column; acetonitrile as mobile phase A, phosphoric acid and sodium acetate as mobile phase B, respectively, at a flow rate of 0.6 mL / min; gradient elution according to the specifications in Table 2; column temperature, 30°C; detection wavelength, 254 nm; injection volume, 1 μL.

[0098] Table 2 Gradient elution program

[0099]

[0100] The experimental results are as follows Figure 1 As shown, the results show that when the mobile phase system is acetonitrile-sodium acetate, the chromatographic peak information in the obtained spectrum is more, so acetonitrile-sodium acetate is selected as the mobile phase system for the detection method of cuttlebone amino acid components.

[0101] 5.2 Gradient elution procedure investigation

[0102] This section examines the effect of the gradient elution procedure on the detection method of the amino acid components of cuttlebone. Specifically, a standard decoction of cuttlebone was taken and the test solution was prepared according to the method under "1", and then the test was carried out according to the following chromatographic conditions.

[0103] Chromatographic conditions: Shim-pack Scepter C18-120 (column length, 150 mm, inner diameter, 4.6 mm, particle size, 3 μm); acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93) as mobile phase A, acetonitrile-water (4:1) as mobile phase B, gradient elution according to the regulations in Table 3; flow rate, 0.8 mL / min; column temperature, 27°C; detection wavelength, 254 nm, injection volume, 1 μL.

[0104] Table 3 Gradient elution program

[0105]

[0106]

[0107] The experimental results are as follows Figure 2 As shown, the results show that when gradient condition 2 is adopted, the separation effect of each chromatographic peak is better, so gradient condition 2 is adopted.

[0108] 5.3 Column temperature investigation

[0109] This section examines the effect of column temperature on the detection method of cuttlebone amino acid components. Specifically, take cuttlebone standard decoction, prepare the test solution according to the method under "1", and then test according to the following chromatographic conditions.

[0110] Chromatographic conditions: Shim-pack Scepter C18-120 column (4.6 mm × 150 mm, 3 μm), mobile phase A: acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93), mobile phase B: acetonitrile-water (4:1), gradient elution as specified in Table 1; column temperature: 25°C, 27°C, and 29°C, flow rate: 0.8 mL / min, detection wavelength: 254 nm. Injection volume: 1 μL.

[0111] The experimental results are as follows Figure 3 As shown in the figure, when the column temperature is 25℃~29℃, the separation effect of each chromatographic peak is basically the same. When the column temperature is 27℃, the response value of each chromatographic peak is relatively higher, so 27℃ is the optimal column temperature.

[0112] 5.4 Flow rate investigation

[0113] This section examines the effect of flow rate on the detection method of cuttlebone amino acid components. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then tested under the following chromatographic conditions.

[0114] Chromatographic conditions: Shim-pack Scepter C18-120 column (4.6 mm × 150 mm, 3 μm), mobile phase A: acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93), mobile phase B: acetonitrile-water (4:1), gradient elution as specified in Table 1; column temperature: 27°C, 27°C, and 29°C, flow rates: 0.75 mL / min, 0.8 mL / min, and 0.85 mL / min, respectively; detection wavelength: 254 nm. Injection volume: 1 μL.

[0115] The experimental results are as follows Figure 4 As shown in the figure, when the flow rate is 0.75mL / min to 0.85mL / min, the separation effect of each chromatographic peak is basically the same. When the flow rate is 0.8mL / min, the response value of each chromatographic peak is relatively higher, so 0.8mL / min is the optimal flow rate.

[0116] 5.5 Instrument Inspection

[0117] This section examines the effects of different instruments on the detection method of cuttlebone amino acid components. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then tested under the following chromatographic conditions.

[0118] Chromatographic conditions: Shim-pack Scepter C18-120 column (4.6 mm × 150 mm, 3 μm), mobile phase A: acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93), mobile phase B: acetonitrile-water (4:1), gradient elution as specified in Table 1; column temperature: 27°C, flow rate: 0.8 mL / min, detection wavelength: 254 nm. Injection volume: 1 μL. Determination was performed on Waters and Shimadzu instruments.

[0119] The experimental results show that there is basically no difference in the separation effect of each chromatographic peak among different instruments, and the determination method has good reproducibility.

[0120] 5.6 Examination of test sample preparation methods

[0121] (1) Investigation of hydrochloric acid concentration

[0122] This section examines the effects of different hydrochloric acid concentrations on the detection of amino acid components in cuttlebone. By observing the peak shape and separation of 16 tentative chromatographic peaks and calculating the "total peak area / sample weight" of these 16 peaks, we compared the effects of different hydrochloric acid concentrations on the detection of amino acid components in cuttlebone and selected the optimal concentration. Specifically, an appropriate amount of standard cuttlebone decoction was taken, ground, and prepared into a test solution according to the method in "1." The hydrochloric acid concentrations used for hydrolysis were 2 mol / L, 3 mol / L, 4 mol / L, and 5 mol / L, respectively. The resulting test solutions were assayed according to the conditions in "3." The peak area of ​​each chromatographic peak was recorded, and the "total peak area / sample weight" was calculated. The results are shown in Table 4.

[0123] Table 4 Detection method of amino acid components in cuttlebone using hydrochloric acid solutions of different concentrations

[0124]

[0125] The above results show that there is no significant difference in the "total peak area / sample weight" ratio between the different hydrochloric acid concentrations. Combined with the assay results, 4 mol / L hydrochloric acid solution is the optimal choice as the hydrolysis reagent.

[0126] (2) Investigation of hydrolysis time

[0127] This section examines the effects of different hydrolysis times on the detection of amino acid components in cuttlebone. By observing the peak shape and separation of 16 tentative chromatographic peaks and calculating the "total peak area / sample weight" for each of the 16 peaks, the authors compared the effects of different hydrolysis times on the detection of amino acid components in cuttlebone and selected the optimal hydrolysis time. Specifically, an appropriate amount of standard cuttlebone decoction was taken, ground, and prepared into a test solution according to the method in "1." The hydrolysis times for the test solutions were 1.5 hours, 2 hours, and 2.5 hours, respectively. The resulting test solutions were assayed according to the conditions in "3." The peak area of ​​each chromatographic peak was recorded, and the "total peak area / sample weight" was calculated. The results are shown in Table 5.

[0128] Table 5 Results of the detection methods of amino acids in cuttlebone at different hydrolysis times

[0129]

[0130] From the above results, it can be seen that different hydrolysis times have no significant effect on the peak shape and separation of each chromatographic peak. Among them, when the hydrolysis time is 2h, the "total peak area / sample weight" is the highest, so 2h is selected as the optimal hydrolysis time.

[0131] 6 Methodological Validation

[0132] 6.1 Specificity Investigation

[0133] Take the standard decoction of cuttlebone and blank solvent to prepare the test solution and blank solvent solution according to the preparation method under "1". Take appropriate amount of aspartic acid reference substance, glutamic acid reference substance, serine reference substance, glycine reference substance, threonine reference substance, arginine reference substance, alanine reference substance, proline reference substance, tyrosine reference substance, valine reference substance, isoleucine reference substance, leucine reference substance, phenylalanine reference substance and L-lysine reference substance, weigh them accurately, and add 0.1m 100μg of aspartic acid, 150μg of glutamic acid, 200μg of serine, 110μg of glycine, 80μg of threonine, 100μg of arginine, 100μg of alanine, 100μg of proline, 150μg of tyrosine, 100μg of valine, 150μg of isoleucine, 80μg of leucine, 150μg of phenylalanine, and 90μg of L-lysine were prepared from 100μL / L hydrochloric acid solution per 1mL as a reference solution. Accurately pipette 1μL of each of the above solutions and inject it into the liquid chromatograph for determination according to the chromatographic conditions under item "3". The results are shown in the table. Figure 5 .

[0134] from Figure 5 It can be seen that the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0135] 6.2 Precision investigation

[0136] Take an appropriate amount of cuttlebone standard decoction, grind it into powder, take about 0.2 g, prepare the test solution according to the preparation method under item "1", and repeat the injection 6 times according to the chromatographic conditions determined under item "3". Taking the glycine peak as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 peak are between 0.02% and 0.13%, and the relative peak area RSD values ​​are between 0.26% and 1.70%; taking leucine as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 peak are between 0.01% and 0.15%, and the relative peak area RSD values ​​are between 0.36% and 2.30%. The RSD values ​​are all less than 3.0%.

[0137] 6.3 Repeatability Study

[0138] Take an appropriate amount of standard cuttlebone decoction, grind it into powder, take about 0.2 g, and make 6 parallel portions. Prepare 6 test sample solutions according to the preparation method under item "1". Perform chromatographic injection analysis according to the determination under item "3". The results showed that the same batch of samples were measured 6 times. Taking the glycine peak as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 peaks were between 0.04% and 0.24%, and the relative peak area RSD values ​​were between 0.73% and 3.96%; taking leucine as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 peaks were between 0.01% and 0.16%, and the relative peak area RSD values ​​were between 0.44% and 3.75%. The RSD values ​​were all less than 5.0%, indicating that the method had good repeatability.

[0139] 6.4 Stability investigation

[0140] Take an appropriate amount of cuttlebone standard decoction, grind it into powder, take about 0.2 g, prepare the test solution according to the preparation method under item "1", and according to the chromatographic conditions determined under item "3", inject and analyze at 0, 2, 4, 8, 12, and 24 hours, respectively. The results showed that the same test solution was analyzed at 0, 2, 4, 8, 12, and 24 hours, respectively. Taking the glycine peak as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 peak were 0.03% to 0.21%, and the relative peak area RSD values ​​were 0.81% to 3.23%; taking leucine as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 peak were 0.01% to 0.14%, and the relative peak area RSD values ​​were 0.63% to 2.31%. The RSD values ​​were all less than 5.0%, indicating that the test solution was relatively stable within 24 hours.

[0141] 7 Establishment of cuttlebone chromatogram

[0142] According to the established detection method, cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules were tested. Sample information is shown in Table 6. Using the glycine peak as reference peak S1, the relative retention times and peak areas of peaks 1 to 9 relative to S1 were calculated. Using the leucine peak as reference peak S2, the relative retention times and peak areas of peaks 10 to 16 relative to S2 were calculated, and the RSD values ​​were calculated. Common peaks were identified in the chromatograms of cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software."

[0143] Table 6 Sample information table

[0144]

[0145] 7.1 Test results of cuttlebone medicinal materials

[0146] The chromatogram overlay of 17 batches of cuttlebone medicinal materials is shown in the figure below. Figure 6 As shown in the figure, the chromatogram of the cuttlebone medicinal material has 16 common peaks. Taking the glycine peak as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 are between 0.16% and 1.06%, and the relative peak area RSD values ​​are between 15.13% and 62.18%. Taking leucine as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 are between 0.04% and 0.33%, and the relative peak area RSD values ​​are between 6.72% and 21.79%, indicating that each chromatographic peak can be stably measured in different batches of medicinal material samples.

[0147] 7.2 Test results of cuttlebone slices

[0148] The chromatogram overlay of 17 batches of cuttlebone slices is shown in the figure below. Figure 7 As shown in the figure, the chromatogram of the cuttlebone slices has 16 common peaks. Taking the glycine peak as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 are between 0.20% and 1.95%, and the relative peak area RSD values ​​are between 9.83% and 62.13%. Taking leucine as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 are between 0.02% and 0.41%, and the relative peak area RSD values ​​are between 6.79% and 14.61%, indicating that each chromatographic peak can be stably measured in different batches of slice samples.

[0149] 7.3 Test results of cuttlebone standard decoction

[0150] The chromatogram overlay of 17 batches of cuttlebone standard decoction is shown in the figure below. Figure 8As shown in the figure, the chromatogram of the cuttlebone standard decoction has 16 common peaks. Taking the glycine peak 4 as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 are between 0.02% and 0.35%, and the relative peak area RSD values ​​are between 5.68% and 62.11%. Taking the leucine peak 14 as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 are between 0.01% and 0.08%, and the relative peak area RSD values ​​are between 6.23% and 35.05%, indicating that each chromatographic peak can be stably measured in different batches of standard decoction samples.

[0151] 7.4 Test results of cuttlebone formula granules

[0152] The chromatograms of three batches of cuttlebone formula granules are overlaid as shown below: Figure 9 As shown in the figure, the chromatogram of the cuttlebone formula granules has 16 common peaks. Taking the glycine peak 4 as the reference peak S1, the relative retention time RSD values ​​of peaks 1 to 9 and S1 are between 0.00% and 0.04%, and the relative peak area RSD values ​​are between 0.16% and 3.44%. Taking the leucine peak 14 as the reference peak S2, the relative retention time RSD values ​​of peaks 10 to 16 and S2 are between 0.00% and 0.01%, and the relative peak area RSD values ​​are between 0.11% and 1.92%, indicating that each chromatographic peak can be stably measured in different batches of formula granule samples.

[0153] 7.5 Establishment of Cuttlebone Chromatographic Standards and Peak Identification

[0154] Based on the test results of the above-mentioned sample types, the chromatogram of cuttlebone was determined to meet the following standards: There were 16 chromatographic peaks in the sample chromatogram, and the retention times of the 16 chromatographic peaks in the chromatogram of the reference medicinal material should correspond to each other. Peaks 2, 4, 8, 9, 14, and 15 should correspond to the retention times of the corresponding reference material peaks, respectively. Taking the peak corresponding to the glycine reference peak (peak 4) as S1, the relative retention times of peaks 1 to 3 and peaks 5 to 9 were calculated. The relative retention times of each peak were within ±10% of the specified value. The specified values ​​for peaks 1 to 3 and peaks 5 to 9 were 0.35, 0.41, 0.90, 1.42, 1.50, 1.55, 1.64, and 1.73, respectively.

[0155] Taking peak 14 as the S2 peak, the relative retention times of peaks 10 to 13 and peaks 15 to 16 were calculated. The relative retention times of each peak were within ±10% of the specified value. Among them, the specified values ​​of peaks 10 to 13 and peaks 15 to 16 were 0.81, 0.83, 0.88, 0.99, 1.12, and 1.21, respectively.

[0156] Furthermore, the 16 chromatographic peaks of the cuttlebone test sample were identified, and it was confirmed that peak 1 was aspartic acid, peak 2 was glutamic acid, peak 3 was serine, peak 4 was glycine, peak 6 was threonine, peak 7 was arginine, peak 8 was alanine, peak 9 was proline, peak 10 was tyrosine, peak 11 was valine, peak 13 was isoleucine, peak 14 was leucine, peak 15 was phenylalanine, and peak 16 was L-lysine.

[0157] Example 2 Application of the Detection Method for Cuttlebone Amino Acid Components

[0158] This embodiment provides a method for identifying cuttlebone. Specifically, cuttlebone, oyster, stone cassia, and mother of pearl are taken and prepared into a test solution according to the conditions under item "1" in Example 1. The sample is injected and tested according to the chromatographic conditions under item "3". The results are as follows. Figure 10 shown.

[0159] The results showed significant differences between the chromatograms of oysters, stone cassia, and pearl shells, compared to cuttlebone. Specifically, stone cassia lacked Peak 5; oysters lacked Peaks 5-8, Peak 10, and Peak 12; and pearl shells lacked Peaks 5-8, Peak 10, and Peak 11. Furthermore, the response values ​​for the amino acid components measured also showed significant differences. This demonstrates that the method developed in this invention for detecting amino acid components in cuttlebone can effectively distinguish various near-counterfeit cuttlebone products.

[0160] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for detecting amino acid components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules, characterized in that: include: The cuttlebone medicinal material or its decoction pieces, standard decoction, or formula granules are hydrolyzed and extracted to obtain a test solution; Dissolving the reference substance in a first hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L to obtain a reference substance solution; and / or hydrolyzing and extracting the cuttlebone control medicinal material to obtain a control medicinal material solution; The test solution, reference solution and / or reference medicinal material solution are measured by liquid chromatography; The chromatographic column of the liquid chromatograph is composed of octadecylsilane bonded silica gel as the stationary phase, a mixed solution of acetonitrile and a sodium acetate solution with a concentration of 0.05 mol / L to 1 mol / L as the mobile phase A, and a mixed solution of acetonitrile and water as the mobile phase B for gradient elution. The gradient elution curve is: 0 min to 15 min, mobile phase A from 100% to 98%, mobile phase B from 0% to 2%; 15-22 min, mobile phase A from 98% to 90%, mobile phase B from 2% to 10%; 22-28 min, mobile phase A from 90% to 82%, mobile phase B from 10% to 18%; 28-40 min, mobile phase A from 82% to 75%, mobile phase B from 18% to 25%; 40-45 min, mobile phase A from 75% to 62%, mobile phase B from 25% to 38%; 45-50 min, mobile phase A from 62% to 100%, mobile phase B from 38% to 0%; The control substances include aspartic acid control substance, glutamic acid control substance, serine control substance, glycine control substance, threonine control substance, arginine control substance, alanine control substance, proline control substance, tyrosine control substance, valine control substance, isoleucine control substance, leucine control substance, phenylalanine control substance and L-lysine control substance; In the mobile phase A, the volume ratio of acetonitrile to sodium acetate solution is 6:94 to 8:92; in the mobile phase B, the volume ratio of acetonitrile to water is 3:1 to 5:

1.

2. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: In the mobile phase A, the volume ratio of acetonitrile to sodium acetate solution is 7:93; the concentration of the sodium acetate solution is 0.1 mol / L, and its pH is 6-7; and / or In the mobile phase B, the volume ratio of acetonitrile to water is 4:

1.

3. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: The column length of the chromatographic column is 50 mm to 250 mm, the column diameter is 2.1 mm to 5 mm, and the particle size of the stationary phase is 2.7 μm to 5 μm; The column temperature of the chromatographic column is 25°C to 29°C; The flow rate of the liquid chromatograph is 0.75 mL / min to 0.85 mL / min, and the detection wavelength is 250 nm to 300 nm; the injection volume of the test solution is 0.5 μL to 3 μL, the injection volume of the reference solution is 0.5 μL to 3 μL, and the injection volume of the reference medicinal material solution is 0.5 μL to 3 μL.

4. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1 or 3, characterized in that: The column length of the chromatographic column is 150 mm, the column diameter is 4.6 mm, and the particle size of the stationary phase is 3 μm; The column temperature of the chromatographic column is 27°C; The flow rate of the liquid chromatograph was 0.80 mL / min, the detection wavelength was 254 nm, the injection volume of the test solution was 1 μL, the injection volume of the reference solution was 1 μL, and the injection volume of the reference medicinal material solution was 1 μL.

5. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: In the step of hydrolyzing and extracting the cuttlebone medicinal material or its decoction pieces, standard decoction, or formula granules to obtain the test solution, the cuttlebone medicinal material or its decoction pieces, standard decoction, or formula granules are hydrolyzed with a second hydrochloric acid solution having a concentration of 2 mol / L to 5 mol / L, the solid phase obtained after solid-liquid separation is dissolved with a third hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L, and then derivatized with an acetonitrile solution of phenyl isothiocyanate having a concentration of 0.05 mol / L to 0.4 mol / L and an acetonitrile solution of triethylamine having a concentration of 0.5 mol / L to 4 mol / L, and finally extracted with n-hexane to obtain the test solution.

6. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1 or 5, characterized in that: The preparation method of the test solution comprises: hydrolyzing the cuttlebone medicinal material, decoction piece, standard decoction or formula granules with a second hydrochloric acid solution having a concentration of 2 mol / L to 5 mol / L at 120° C. to 200° C. for 1.5 to 2.5 hours; dissolving the solid phase obtained after solid-liquid separation with a third hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L to obtain a first intermediate solution; wherein the ratio of the cuttlebone medicinal material, decoction piece, standard decoction or formula granules to the second hydrochloric acid solution is 0.1 g to 1 g: 15 mL to 30 mL, and the ratio of the cuttlebone medicinal material, decoction piece, standard decoction or formula granules to the third hydrochloric acid solution is 0.1 g to 1 g: 20 mL to 30 mL; The first intermediate solution is derivatized with a 0.05 mol / L to 0.4 mol / L acetonitrile solution of phenyl isothiocyanate and a 0.5 mol / L to 4 mol / L acetonitrile solution of triethylamine at a temperature of 15° C. to 40° C. for 1 to 5 hours; then a predetermined amount of a 40 vol% to 80 vol% acetonitrile aqueous solution is added to obtain a second intermediate solution; wherein the volume ratio of the first intermediate solution to the acetonitrile solution of phenyl isothiocyanate is 10:1 to 10:4, the volume ratio of the first intermediate solution to the acetonitrile solution of triethylamine is 10:1 to 10:4, and the volume ratio of the first intermediate solution to the acetonitrile aqueous solution is 10:8 to 10:15; The second intermediate solution is extracted with n-hexane, and the obtained lower layer solution is filtered to obtain the test solution; wherein the volume ratio of the second intermediate solution to n-hexane is 10:8 to 10:

15.

7. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: The preparation method of the control medicinal material solution comprises: The cuttlebone control medicinal material is hydrolyzed with a fourth hydrochloric acid solution having a concentration of 2 mol / L to 5 mol / L at 120° C. to 200° C. for 1.5 to 2.5 hours; the solid phase obtained after solid-liquid separation is dissolved with a fifth hydrochloric acid solution having a concentration of 0.05 mol / L to 1 mol / L to obtain a third intermediate solution; wherein the ratio of the cuttlebone control medicinal material to the fourth hydrochloric acid solution is 0.1 g to 1 g: 5 mL to 40 mL, and the ratio of the cuttlebone control medicinal material to the fifth hydrochloric acid solution is 0.1 g to 1 g: 5 mL to 50 mL; The third intermediate solution is derivatized with a 0.05 mol / L to 0.4 mol / L acetonitrile solution of phenyl isothiocyanate and a 0.5 mol / L to 4 mol / L acetonitrile solution of triethylamine at 15° C. to 40° C. for 1 to 5 hours; then a predetermined amount of a 40 vol% to 80 vol% acetonitrile aqueous solution is added to obtain a fourth intermediate solution; wherein the volume ratio of the third intermediate solution to the acetonitrile solution of phenyl isothiocyanate is 10:1 to 10:4, the volume ratio of the third intermediate solution to the acetonitrile aqueous solution of triethylamine is 10:1 to 10:4, and the volume ratio of the third intermediate solution to the acetonitrile aqueous solution is 10:8 to 10:15; The fourth intermediate solution is extracted with n-hexane, and the obtained lower layer solution is filtered to obtain a control medicinal material solution; wherein the volume ratio of the fourth intermediate solution to n-hexane is 10:8 to 10:

15.

8. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: In the reference substance solution, the concentration of each reference substance is 5 μg / mL to 20 μg / mL.

9. The method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: The chromatogram obtained by the determination includes 16 common peaks, among which peak 1 is aspartic acid, peak 2 is glutamic acid, peak 3 is serine, peak 4 is glycine, peak 6 is threonine, peak 7 is arginine, peak 8 is alanine, peak 9 is proline, peak 10 is tyrosine, peak 11 is valine, peak 13 is isoleucine, peak 14 is leucine, peak 15 is phenylalanine, and peak 16 is L-lysine; Taking peak 4 as S1 peak, calculate the relative retention time of peaks 1 to 3 and peaks 5 to 9. The relative retention time of each peak is within ±10% of the specified value. The specified values ​​of peaks 1 to 3 and peaks 5 to 9 are 0.35, 0.41, 0.90, 1.42, 1.50, 1.55, 1.64 and 1.73 respectively. Taking peak 14 as the S2 peak, the relative retention times of peaks 10 to 13 and peaks 15 to 16 were calculated. The relative retention times of each peak were within ±10% of the specified value. Among them, the specified values ​​of peaks 10 to 13 and peaks 15 to 16 were 0.81, 0.83, 0.88, 0.99, 1.12, and 1.21, respectively.

10. Use of a method for detecting amino acid components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules for identifying cuttlebone and its near-counterfeit products.

11. A method for identifying cuttlebone, for distinguishing cuttlebone from its near-imitations, characterized in that: include: Provide the substance to be identified; Detecting the substance to be identified using the method for detecting amino acid components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to any one of claims 1 to 9 to obtain a chromatogram; If the chromatogram shows peaks 1 to 16 at the same time, the substance to be identified includes or is cuttlebone; otherwise, it is a near-fake product.

Images