A method for constructing a characteristic chromatogram of oyster and its pharmaceutical preparation and application thereof

Abstract

This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a characteristic chromatogram of oyster and its pharmaceutical preparations, and further disclosing a method for constructing the characteristic chromatogram and a quality testing method for oyster and its pharmaceutical preparations. The method for constructing the characteristic chromatogram of oyster and its pharmaceutical preparations of this invention uses oyster medicinal materials and oyster pharmaceutical preparations as the detection objects, and establishes a characteristic chromatogram method for the pharmaceutical preparations based on high-performance liquid chromatography. This method achieves good separation effect, improves analytical efficiency, and can clearly distinguish oyster from common shellfish drugs, ensuring accurate medication.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a characteristic spectrum of oyster and its pharmaceutical preparations, and further disclosing a method for constructing the characteristic spectrum and a method for quality testing of oyster and its pharmaceutical preparations. Background Technology

[0002] Oysters, belonging to the order Anisomyaria and family Ostreidae, are a highly nutritious seafood. Studies have found that oysters are rich in amino acids, with arginine being the most abundant, and lysine also present in relatively high amounts. Therefore, deep processing technologies based on oyster products have attracted widespread attention in the industry. Oysters are a commonly used marine medicinal herb, with the shell being the medicinal part. Compared to other shell medicines, it possesses the unique effects of "sedative and calming, nourishing yin and suppressing yang." However, previous research on the characteristic mapping methods of oysters is limited, and research on its pharmacodynamic material basis is also relatively weak.

[0003] Therefore, the field expects to establish a method for the comprehensive and systematic detection of oysters and their pharmaceutical preparations, especially a method for establishing characteristic spectra of oysters and their pharmaceutical preparations, which is of great significance for their comprehensive quality detection and overall quality control. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing characteristic spectra of oysters and their pharmaceutical preparations, so as to comprehensively reflect the intrinsic quality and medication safety of oysters and their pharmaceutical preparations.

[0005] The second technical problem to be solved by the present invention is to provide a method for quality testing of oysters and their pharmaceutical preparations.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for constructing characteristic spectra of oysters, especially Pacific oysters and their pharmaceutical preparations, including the step of performing high-performance liquid chromatography detection on the test solution of oysters and / or oyster pharmaceutical preparations;

[0007] The chromatographic conditions included: using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.08-0.12% phosphoric acid solution as mobile phase B, with gradient elution performed according to the following program:

[0008] 0-7 min, A:B ratio was 17%:83%;

[0009] 7-10 min, A:B ratio changed from 17% to 22%; A:B ratio changed from 83% to 78%.

[0010] 10-15 min, A:B ratio is 22%:78%;

[0011] 15-16 min, A:B ratio changed from 22% to 35%; 78%:B ratio changed from 65%.

[0012] 16-20 min, A:B ratio 35%:65%;

[0013] Over 20-30 minutes, the ratio of A to B changed from 35% to 36% and from 65% to 64%.

[0014] Preferably, in the chromatographic conditions, acetonitrile is used as mobile phase A and 0.10% phosphoric acid solution is used as mobile phase B.

[0015] Specifically, in the method for constructing the characteristic chromatograms of oysters and their pharmaceutical preparations, the chromatographic conditions in the high-performance liquid chromatography detection step further include: a flow rate of 0.25-0.30 ml / min, a column temperature of 30-40℃, and a detection wavelength of 254±2 nm.

[0016] Preferably, the chromatographic conditions include: a flow rate of 0.25 ml / min, a column temperature of 35 °C, and a detection wavelength of 254 nm.

[0017] Specifically, the method for constructing the characteristic spectrum of oyster and its pharmaceutical preparation includes the following method for preparing the test sample solution: taking the test sample and accurately adding it to the first acid solution to remove calcium carbonate, and continuing to add the second acid solution for hydrolysis, collecting the hydrolysate, washing it and evaporating it to dryness, and adding the residue to the third acid solution for redissolution to obtain the final product;

[0018] Preferably, the method further includes a step of derivatizing the test sample solution, comprising: taking the test sample solution and adding it to an acetonitrile solution containing phenyl isothiocyanate and an acetonitrile solution containing triethylamine, mixing them together, continuing to add n-hexane and mixing, and filtering the lower layer solution to obtain the final product.

[0019] Specifically, the method for constructing the characteristic spectrum of oyster and its pharmaceutical preparation further includes the step of preparing a reference solution of control medicinal material, which specifically includes: taking oyster reference medicinal material and accurately adding a first acid solution to mix and remove calcium carbonate, and continuing to add a second acid solution for hydrolysis, collecting the hydrolysate, washing and evaporating it to dryness, and adding the residue to a third acid solution for redissolution to obtain the final product;

[0020] Preferably, the method further includes a step of derivatizing the reference drug solution, comprising: taking the reference drug solution and adding it to an acetonitrile solution containing phenyl isothiocyanate and an acetonitrile solution containing triethylamine, mixing them together, adding hexane and mixing, and then filtering the lower layer solution to obtain the final product.

[0021] Specifically, the method for constructing the characteristic chromatogram of oyster and its pharmaceutical preparations further includes the step of preparing a reference solution and the step of constructing the characteristic chromatogram of the reference solution based on the high performance liquid chromatography method;

[0022] The reference standards include glycine, proline, alanine, and / or lysine hydrochloride;

[0023] The method for preparing the reference solution includes: precisely adding the reference standard to a third acid solution and mixing to obtain the solution;

[0024] Preferably, the method further includes a step of derivatizing the reference solution, comprising: taking the reference solution and adding it to an acetonitrile solution containing phenyl isothiocyanate and an acetonitrile solution containing triethylamine, mixing them, adding n-hexane and mixing, and filtering the lower layer solution to obtain the final product.

[0025] Specifically, the method for constructing the characteristic maps of oysters and their pharmaceutical preparations:

[0026] The first acid solution comprises dilute hydrochloric acid with a concentration of 9.5-10.5%; and / or,

[0027] The second acid solution comprises concentrated hydrochloric acid with a concentration of 35-37%; and / or,

[0028] The third acid solution comprises a hydrochloric acid solution with a concentration of 0.08-0.12 mol / L, preferably with a concentration of 0.1 mol / L; and / or,

[0029] The temperature of the hydrolysis step is 120-180℃; and / or,

[0030] The concentration of the acetonitrile solution containing phenyl isothiocyanate is 0.08-0.12 mol / L, preferably 0.1 mol / L; and / or,

[0031] The concentration of the triethylamine-containing acetonitrile solution is 0.08-0.12 mol / L, preferably 0.1 mol / L.

[0032] Specifically, the method for constructing the characteristic spectrum of oysters and their pharmaceutical preparations includes oyster pharmaceuticals that directly include oyster formula granules, oyster slices, or oyster standard decoctions.

[0033] The present invention also discloses a characteristic spectrum and / or control characteristic spectrum of oyster, especially the Pacific oyster and its pharmaceutical preparations, wherein the characteristic spectrum or control characteristic spectrum of oyster and its pharmaceutical preparations is constructed by the method described above;

[0034] Preferably, the characteristic spectrum has 9 characteristic peaks, with the peak corresponding to the glycine reference standard peak being the S peak, and the relative retention times of the other characteristic peaks relative to the S peak being within ±10% of a specified value. The specified values ​​for each characteristic peak are: peak 1: 0.62, peak 2: 0.90, peak 4: 1.19, peak 5: 1.26, and peak 9: 3.78.

[0035] This invention also discloses the method for constructing characteristic spectra of oysters and their pharmaceutical preparations and / or the application of the characteristic spectra of oysters and their pharmaceutical preparations and / or control characteristic spectra in the field of quality testing of oysters, especially Pacific oysters and their pharmaceutical preparations.

[0036] The present invention also discloses a quality detection method for oysters, especially Pacific oysters and their pharmaceutical preparations, including the steps of constructing the characteristic spectrum and the control characteristic spectrum according to the method, and the step of comparing the characteristic spectrum with the control characteristic spectrum.

[0037] The present invention describes a method for constructing characteristic chromatograms of oysters, especially the Pacific oyster, and its pharmaceutical preparations. Using oyster medicinal materials and oyster pharmaceutical preparations as the detection objects, a characteristic chromatogram method for these preparations is established based on high-performance liquid chromatography (HPLC). The chromatogram of the test sample should show nine characteristic peaks, among which peaks 3, 6, 7, and 8 should correspond to the retention times of reference peaks of glycine, proline, alanine, and lysine, respectively. The peak corresponding to the glycine reference peak is designated as the S peak. The relative retention times of peaks 1-2, 4-5, and 9 with the S peak are calculated, and their relative retention times should be within ±10% of the specified values, which are 0.62 (peak 1), 0.90 (peak 2), 1.19 (peak 4), 1.26 (peak 5), and 3.78 (peak 9). This invention establishes characteristic chromatograms of oysters and their preparations, achieving good separation effects, improving analytical efficiency, and clearly distinguishing oysters from common shellfish drugs, ensuring accurate medication use.

[0038] The method for constructing characteristic maps of oysters and their pharmaceutical preparations described in this invention aims to establish a method for constructing characteristic maps of oyster raw materials, especially Pacific oysters, and oyster pharmaceutical preparations. This method distinguishes oysters from other commonly used shellfish drugs such as oyster shells, clam shells, mother-of-pearl, and abalone shells, ensuring accurate origin and accurate clinical medication. Attached Figure Description

[0039] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0040] Figure 1 The chromatogram of the test sample in Example 1;

[0041] Figure 2 The results of the chromatographic conditions and system suitability study in Example 2;

[0042] Figure 3 The results are shown under different chromatographic conditions in Example 3;

[0043] Figure 4 The results of the investigation at different detection wavelengths in Example 3;

[0044] Figure 5 The results are from the investigation at different flow rates in Example 3;

[0045] Figure 6 The results of the investigation at different column temperatures in Example 3;

[0046] Figure 7 The results are from the investigation under different mobile phase systems in Example 3;

[0047] Figure 8 The results are from the investigation of different phosphoric acid concentrations in the mobile phase system in Example 3;

[0048] Figure 9 The results of the investigation under different chromatographic columns in Example 3;

[0049] Figure 10 The results of the precision experiment in Example 6;

[0050] Figure 11 The results of the repeatability test of the method in Example 6;

[0051] Figure 12 The results of the intermediate precision (different operators) assessment in Example 6;

[0052] Figure 13 The results of the specificity assessment in Example 6;

[0053] Figure 14 The results of the stability test in Example 6;

[0054] Figure 15 The results of the durability test using different instruments are shown in Example 6.

[0055] Figure 16 The reference chromatograms of oyster (Ostrea gigas) in Example 8, the reference chromatograms of 14 batches of standard decoction (freeze-dried powder) of oyster (Ostrea gigas) slices, and the reference chromatograms of oyster (Ostrea gigas) formula granules are shown.

[0056] Figure 17 The chromatograms are of different types of adulterants in Example 9. Detailed Implementation

[0057] In the following embodiments of the present invention, referring to the methods in the literature, a method for determining the characteristic chromatogram of standard decoction (lyophilized powder) of oyster (Ostrea gigas) slices was established. Based on the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules" and the "Technical Requirements for Research and Formulation of Quality Standards of Traditional Chinese Medicine in the Chinese Pharmacopoeia", the method for determining the characteristic chromatogram of standard decoction of oyster (Ostrea gigas) slices was established by examining the sample processing methods and methodologies (system adaptability and specificity, precision and repeatability, solution stability).

[0058] The instruments, equipment, and reagents involved in the following embodiments of the present invention are shown in Tables 1-2 below.

[0059] Table 1. Statistics on Instruments and Equipment

[0060]

[0061]

[0062] Table 2 Reagents and Test Chemicals

[0063] reagents and reagents Grade / Purity batch number factory hydrochloric acid AR P2275219 Shanghai Titan Technology Co., Ltd. water purified water 20221218C / 20230701R Guangzhou Watsons Food & Beverage Co., Ltd. Acetonitrile Chromatographic purity 0114230602 Shanghai Xingke High Purity Solvent Co., Ltd. Phosphoric acid Chromatographic purity F6130152 Thermo Fisher Scientific (China) Co., Ltd. phenyl isothiocyanate 5g / bottle P2271812 Shanghai Titan Technology Co., Ltd. glycine 20mg / ≥98.0% 16296 Shanghai Shidander Standard Technical Service Co., Ltd. L-Isoleucine 20mg / ≥98.0% 12924 Shanghai Shidander Standard Technical Service Co., Ltd. L-Leucine 20mg / ≥98.0% 6572 Shanghai Shidander Standard Technical Service Co., Ltd. L-serine 20mg / ≥98.0% 13821 Shanghai Shidander Standard Technical Service Co., Ltd. Tyrosine 100mg / 99.9% 140609-202215 China National Institutes for Food and Drug Control L-phenylalanine 100mg / ≥98.0% 6134 Shanghai Shidander Standard Technical Service Co., Ltd. L-Aspartic acid 20mg / ≥98.0% 11002 Shanghai Shidander Standard Technical Service Co., Ltd. L-threonine 20mg / ≥98.0% 3884 Shanghai Shidander Standard Technical Service Co., Ltd. L-glutamic acid 20mg / ≥98.0% 2189 Shanghai Shidander Standard Technical Service Co., Ltd. alanine 100mg / 99.9% 140680-202206 China National Institutes for Food and Drug Control Valine 20mg / ≥98.0% 2989 Shanghai Shidander Standard Technical Service Co., Ltd. Lysine hydrochloride 100mg / 100% 140673-202211 China National Institutes for Food and Drug Control L-histidine 100mg / ≥98.0% 14341 Shanghai Shidander Standard Technical Service Co., Ltd. L-arginine 20mg / ≥98.0% 8721 Shanghai Shidander Standard Technical Service Co., Ltd. L-proline 20mg / ≥98.0% 9300 Shanghai Shidander Standard Technical Service Co., Ltd. L-hydroxyproline 20mg / ≥98.0% 7457 Shanghai Shidander Standard Technical Service Co., Ltd.

[0064] Example 1

[0065] The method for detecting the characteristic chromatogram of the *Ostrea gigas* pharmaceutical preparation described in this embodiment uses ultra-high performance liquid chromatography (UHPLC) to detect the test sample solution. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material (column length 150 mm, column inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, with gradient elution as specified in Table 3; the flow rate is 0.25 ml / min, the column temperature is 35 °C, and the detection wavelength is 254 nm. The theoretical plate number, calculated based on the glycine peak, should not be less than 5000.

[0066] Table 3 Gradient elution program

[0067] Time (min) Mobile phase A (%) Mobile phase B (%) 0～7 17 83 7～10 17～22 83～78 10～15 22 78 15～16 22～35 78～65 16～20 35 65 20～30 35～36 65～64

[0068] Preparation of reference solutions: Accurately weigh 0.4 g of oyster (Ostrea gigas) reference material, place it in a hydrolysis tube, slowly add 10 ml of dilute hydrochloric acid solution, then add 3 ml of hydrochloric acid, hydrolyze at 150℃ for 1 hour, cool, transfer to an evaporating dish, wash 3 times with water (2-3 ml each time), combine the washings in the evaporating dish, evaporate to dryness, dissolve the residue with an appropriate amount of 0.1 mol / L hydrochloric acid solution, transfer to a 20 ml volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid solution, and shake well. This is the reference solution for the reference material. Separately, accurately weigh appropriate amounts of glycine, proline, alanine, and lysine hydrochloride reference standards, add 0.1 mol / L hydrochloric acid solution to prepare a solution containing 10 μg per ml, i.e. (lysine weight = lysine hydrochloride weight / 1.2455), which is used as the reference solution for the reference standards.

[0069] Preparation of the test solution: Weigh approximately 0.5 g of powder accurately and place it in a hydrolysis tube. Slowly add 10 ml of dilute hydrochloric acid solution, then add 3 ml of hydrochloric acid. Hydrolyze at 150°C for 1 hour. Cool the solution and transfer it to an evaporating dish. Wash the solution three times with water (2-3 ml each time). Combine the washings with the evaporating dish and evaporate to dryness. Dissolve the residue in an appropriate amount of 0.1 mol / L hydrochloric acid solution and transfer it to a 20 ml volumetric flask. Dilute to the mark with 0.1 mol / L hydrochloric acid solution and shake well to obtain the test solution.

[0070] Accurately measure 10 ml each of the above-mentioned reference drug solution, test solution (centrifuge if necessary and take the supernatant), and reference solution, and place them in a 50 ml centrifuge tube. Add 2.5 ml of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate and 2.5 ml of acetonitrile solution of 1 mol / L triethylamine to each tube, shake well, let stand at room temperature for 1 hour, shake well again, add 15 ml of n-hexane, shake, let stand for 10 minutes, take the lower layer solution, filtrate, and collect the subsequent filtrate to obtain the final product.

[0071] Assay: Accurately pipette 2 μl each of the derivatized reference solution and the test solution into the liquid chromatograph and determine the result. See attached chromatogram for the determination. Figure 1 As shown.

[0072] It is evident that the chromatogram of the test sample should exhibit 9 characteristic peaks, which should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 6, 7, and 8 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the glycine reference peak is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.62 (peak 1), 0.90 (peak 2), 1.19 (peak 4), 1.26 (peak 5), and 3.78 (peak 9). The relative peak areas of peaks 2 and 3, 4 and 5, and 6 and 7 should be calculated, and the specified values ​​are: not greater than 0.19 (peak 2 / peak 3), not less than 0.65 (peak 4 / peak 5), and not less than 0.61 (peak 6 / peak 7).

[0073] The results are as follows: Peak 2: Serine; Peak 3: Glycine; Peak 4: Glutamic acid; Peak 5: Aspartic acid; Peak 6: Proline; Peak 7: Alanine; Peak 8: Lysine; Peak 9: Phenylalanine.

[0074] Example 2

[0075] This embodiment verifies the chromatographic conditions and system suitability based on the method in Example 1.

[0076] The test solution and reference solution were prepared according to the preparation methods for the test solution and reference solution in Example 1. UPLC analysis was performed under the chromatographic conditions described in the "Characteristic Chromatography" section. The chromatograms of the reference solution, test solution, and negative control are shown in the attached figures. Figure 2 As shown in (a)-(c), the chromatographic data of the reference standard and the test sample are shown in Tables 4-5 below.

[0077] Table 4. Chromatographic data of the reference standard

[0078] Serial Number name Retention time Peak area Peak height Theoretical number of plates Tail factor Resolution 1 glycine 7.687 556516 56503 14729 1.27 11.16 2 proline 12.616 348923 38149 45983 1.27 15.86 3 alanine 13.262 353219 37504 47597 1.27 2.70 4 Lysine 27.581 367938 31320 128920 1.24 4.56

[0079] Table 5 Chromatographic data of the test samples

[0080] Serial Number name Retention time Peak area Peak height Theoretical number of plates Resolution Tail Factor 1 4.786 923710 165507 20371 2.77 1.72 2 6.949 116592 15415 19701 3.46 1.42 3 glycine 7.690 1519704 177498 20281 3.48 1.44 4 9.132 191098 19491 20487 1.47 1.31 5 9.734 223962 20319 19411 2.19 1.18 6 proline 12.651 318325 37851 56922 5.56 1.37 7 alanine 13.280 258300 30023 58450 2.83 1.35 8 Lysine 27.569 168581 14167 128443 2.83 1.28 9 28.828 102056 7106 104374 1.28 1.74

[0081] It is evident that the chromatographic method of the present invention has good system adaptability for both the reference solution and the test solution, and can be used as a method for detecting the characteristic chromatograms of standard decoctions of oyster (Ostrea gigas) slices.

[0082] Example 3

[0083] This embodiment optimizes the feature map method based on the method in Embodiment 1.

[0084] Oyster shells are formed through biomineralization of organic matter, using a small amount of organic macromolecules (proteins, glycoproteins, or polysaccharides) as a framework and calcium carbonate as the unit for molecular manipulation, resulting in a highly ordered multilayered microstructure. The organic matter in oyster shells is further divided into soluble and insoluble organic matter. Research on similar varieties revealed certain differences in the types and proportions of amino acids; therefore, a characteristic chromatogram of amino acid components is proposed. Referring to relevant literature and methods in standards for similar varieties, the test solution was initially prepared according to the following method, and the method was preliminarily developed under the following chromatographic conditions.

[0085] Preparation of the test solution: Take 0.5g of oyster decoction standard (lyophilized powder) or 1g of oyster decoction powder, place it in a hydrolysis tube, slowly add 10ml of 6mol / L hydrochloric acid solution, hydrolyze at 150℃ for 1 hour, cool, transfer to an evaporating dish, wash 3 times with water (2-3ml each time), add the washings to the evaporating dish, evaporate to dryness, add an appropriate amount of 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 20ml volumetric flask, dilute to the mark with 0.1mol / L hydrochloric acid solution, shake well, and the test solution is ready.

[0086] Accurately measure 10 ml of the above test solution and place it in a 50 ml centrifuge tube. Add 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 1 hour. Add 15 ml of n-hexane, shake, and let stand for 10 minutes. Take the lower layer solution, filter, and collect the filtrate to obtain the final product.

[0087] (1) Selection and optimization of mobile phase gradient

[0088] The gradient elution procedures involved in each of the following conditions in this embodiment are shown in Table 6 below.

[0089] Table 6 Gradient elution program

[0090]

[0091]

[0092] Condition 1: Waters 2695 instrument was used, with acetonitrile as mobile phase A and 0.02% phosphoric acid solution as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to program 1 in Table 6; the flow rate was 1 ml / min; the column temperature was 25℃; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 (a) The results show that there are many chromatographic peaks with retention times of 7-18 minutes in the chromatogram, indicating poor separation. Therefore, the elution gradient was optimized based on condition 1.

[0093] Condition 2: Waters 2695 instrument was used, with acetonitrile as mobile phase A and 0.02% phosphoric acid solution as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to procedure 2 in Table 6; the flow rate was 1 ml / min; the column temperature was 25℃; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 (b) The results show that the separation effect of each chromatographic peak is relatively good under this condition, but most of the chromatographic peaks are concentrated in the first 15 minutes, which can be optimized by adjusting other conditions.

[0094] Condition 3: Waters 2695 instrument was used, with acetonitrile-water (80:20) as mobile phase A and 50 mmol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid)-acetonitrile (93:7) as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to procedure 6 in Table 6; the flow rate was 1 ml / min; the column temperature was 40 °C; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 (c) It can be seen that the resolution of the chromatographic peaks at 4-6 minutes was not improved, so the pH of mobile phase B was adjusted as an attempt.

[0095] Condition 4: Waters 2695 instrument was used, with acetonitrile-water (80:20) as mobile phase A and 50 mmol / L sodium acetate solution (pH adjusted to 5.5 with acetic acid)-acetonitrile (93:7) as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to procedure 7 in Table 6; the flow rate was 1 ml / min; the column temperature was 40 °C; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 (d)

[0096] Condition 5: Waters 2695 instrument was used, with acetonitrile-water (80:20) as mobile phase A and 50 mmol / L sodium acetate solution (pH adjusted to 4.5 with acetic acid)-acetonitrile (93:7) as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to procedure 8 in Table 6; the flow rate was 1 ml / min; the column temperature was 40 °C; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 Middle (e).

[0097] Condition 6: Waters 2695 instrument was used, with acetonitrile-water (80:20) as mobile phase A and 50 mmol / L sodium acetate solution (pH adjusted to 5.8 by acetic acid)-acetonitrile (93:7) as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to the specifications in Table 6; the flow rate was 1 ml / min; the column temperature was 40 °C; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 (f)

[0098] Based on the results of conditions 4-6 above, it can be seen that the separation effect of mobile phase B is better when the pH is 5.5 and 5.8. Therefore, gradient optimization was carried out under the condition of pH=5.8, and after multiple optimizations, the relatively better condition 7 is obtained as follows. The specific conditions are as follows.

[0099] Condition 7: Waters 2695 instrument was used, with acetonitrile-water (80:20) as mobile phase A and 50 mmol / L sodium acetate solution (pH adjusted to 5.8 with acetic acid)-acetonitrile (93:7) as mobile phase B; A T3 (4.6 × 250 mm, 5 μm) column was used; gradient elution was performed according to program 10 in Table 6; the flow rate was 1 ml / min; the column temperature was 40 °C; the detection wavelength was 254 nm; and the injection volume was 10 μl. Results are attached. Figure 3 (g). The results show that condition 7 is significantly better than condition 2 in terms of separation effect, but the chromatographic peak may exhibit co-elution at a retention time of about 11 minutes, and this cannot be improved by adjusting the mobile phase ratio.

[0100] The aforementioned scheme, after multiple optimizations, did not yield satisfactory results, so we tried replacing it with a UPLC instrument and a column with a smaller particle size.

[0101] Condition 8: Use Waters H-Class instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; ACQUITY The HSS T3 (2.1×150mm, 1.8μm) column was used; gradient elution was performed according to procedure 8 in Table 6; the flow rate was 0.25 ml / min; the column temperature was 35℃; the detection wavelength was 254 nm; and the injection volume was 2 μl. Results are attached. Figure 3 (h). The results show that under these chromatographic conditions, the separation of each chromatographic peak is good. This method will be used as the initial condition for subsequent system optimization.

[0102] (2) Determination of detection wavelength

[0103] Take the standard decoction (lyophilized powder) test solution and inject it into the liquid chromatograph under the chromatographic conditions described in condition 8 above. Perform a full-wavelength scan and compare the chromatograms at five different absorption wavelengths (220 nm, 240 nm, 254 nm, 270 nm, and 300 nm) based on the chromatographic information. See the attached figures for details. Figure 4 As shown in (a)-(e), the number of chromatographic peaks detected, the absorbance corresponding to the retention time, and the stability of the baseline were used as evaluation criteria.

[0104] The results show that the UV absorption of the chromatographic peak is significantly reduced at the detection wavelength of 270-300nm. Compared with 220nm, 240nm and 254nm, the effective peak absorption at 254nm is more balanced. Taking all factors into consideration, 254nm is selected as the detection wavelength for the characteristic spectrum of oyster standard decoction.

[0105] In summary, the preliminary chromatographic conditions for the optimized characteristic chromatogram of oyster standard decoction (lyophilized powder) in this embodiment are as follows: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; ACQUITY The HSS T3 (2.1×150mm, 1.8μm) column was used; gradient elution was performed according to procedure 8 in Table 6 above; the flow rate was 0.25ml per minute; the column temperature was 35℃; the detection wavelength was 254nm; and the injection volume was 2μl.

[0106] (3) Investigation of different flow velocities

[0107] Based on the preliminary chromatographic conditions determined above, the effects of different flow rates (0.20 ml / min, 0.25 ml / min, and 0.30 ml / min) on the separation effect of the characteristic chromatograms of oyster standard decoction were investigated. The results are shown in the appendix. Figure 5 (a)-(c) and Table 7 below.

[0108] Table 7 Applicability parameters of the system for different flow velocities

[0109]

[0110]

[0111] The results show that as the flow rate increases, each chromatographic peak gradually shifts forward. However, when the flow rate increases to 0.3 ml / min, peak 12 (chromatographic peak 25.782) co-elutes with the derivatization reagent peak. When the flow rate is 0.25 ml / min, the separation of each peak is good. Therefore, a flow rate of 0.25 ml / min was selected for subsequent condition investigation.

[0112] (4) Investigation at different column temperatures

[0113] Based on the preliminary chromatographic conditions determined above, the effect of different column temperatures (30℃, 35℃, and 40℃) on the separation effect of the characteristic chromatograms of oyster standard decoction was investigated. The results at 30℃, 35℃, and 40℃ are shown in the appendix. Figure 6 (a)-(c) and Table 8 below.

[0114] Table 8. Applicability parameters of the system for different column temperatures.

[0115]

[0116]

[0117] The results show that different column temperatures have no significant effect on each chromatographic peak. Taking all factors into consideration, a column temperature of 35℃ was selected for screening and investigation of subsequent conditions.

[0118] (5) Investigation of different mobile phase additives

[0119] Based on the preliminary chromatographic conditions determined above, the effects of different mobile phase additives (formic acid, phosphoric acid, and glacial acetic acid) on the separation of characteristic chromatograms of oyster standard decoction were investigated. The results of the investigations in the acetonitrile-0.1% formic acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.1% glacial acetic acid systems are shown in the appendix. Figure 7 As shown in (a)-(c) and Table 9 below.

[0120] Table 9. Parameters for evaluating the system applicability of different mobile phase additives.

[0121]

[0122]

[0123] The results show that the separation between peaks 5 and 6 decreased when 0.1% formic acid was added to the mobile phase; peak merging occurred when 0.1% glacial acetic acid was added to the mobile phase; and the chromatographic information was rich and the separation was good when 0.1% phosphoric acid was added to the mobile phase. Therefore, phosphoric acid was selected as an additive for subsequent condition screening.

[0124] (6) Investigation of different phosphoric acid concentrations in the mobile phase

[0125] Based on the preliminary chromatographic conditions determined above, the effect of different phosphoric acid concentrations (0.05%, 0.10%, and 0.2%) in the mobile phase on the separation effect of characteristic chromatograms of oyster standard decoction was investigated. The results of the investigation in acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.2% phosphoric acid systems are shown in the appendix. Figure 8 As shown in (a)-(c) and Table 10 below.

[0126] Table 10. Applicability parameters of the system for different phosphoric acid concentrations.

[0127]

[0128]

[0129] The results show that 0.05% phosphoric acid concentration affects the separation of peaks 1 and 2, while 0.1% and 0.2% phosphoric acid concentrations have no significant effect on each chromatographic peak. In order to minimize the acidity of the mobile phase, a phosphoric acid concentration of 0.1% was selected for subsequent condition screening.

[0130] (7) Investigation of different chromatographic columns

[0131] Based on the above chromatographic conditions, the test solution of oyster standard decoction (lyophilized powder) was subjected to two different chromatographic methods. T3 1.6μm, 2.1×150mm; ACQUITY The sample was injected and analyzed using an HSS T3 column (1.8 μm, 2.1 × 150 mm). The chromatograms are shown in the attached figures. Figure 9 (a)-(b) and Table 11 below.

[0132] Table 11 System suitability parameters for chromatographic peaks obtained from different chromatographic columns

[0133]

[0134] The results show that ACQUITY The peak shape and resolution of the chromatograms obtained from the HSS T3 column are superior to those obtained from other chromatograms. The T3 column indicates that this method has some column selectivity; therefore, ACQUITY was chosen. The HSS T3 column was used for subsequent method studies.

[0135] In summary, this embodiment optimizes the chromatographic conditions and system suitability test: using ACQUITY The chromatographic column was HSS T3 (2.1 mm × 150 mm, 1.8 μm); the mobile phase was acetonitrile (A) and 0.1% phosphoric acid solution (B); gradient elution was performed according to the specifications in Table 12; the flow rate was 0.25 mL per minute; the column temperature was 35 °C; and the detection wavelength was 254 nm.

[0136] Table 12 Optimized gradient elution procedure

[0137] Time (min) Mobile phase A (%) Mobile phase B (%) 0～7 17 83 7～10 17→22 83→78 10～15 22 78 15～16 22→35 78→65 16～20 35 65 20～30 35→36 65→64

[0138] Example 4

[0139] This embodiment examines the preparation method of the test solution under the aforementioned determined chromatographic conditions.

[0140] During the initial preparation of the test solution, it was observed that the bubbles generated were quite vigorous and difficult to control when 6 mol / L hydrochloric acid solution was added. Therefore, the method was changed to first slowly adding 10 ml of dilute hydrochloric acid to remove calcium carbonate from the sample, then adding a certain amount of hydrochloric acid, and then hydrolyzing. The specific steps are as follows: Take 0.5 g of the powder, place it in a hydrolysis tube, slowly add 10 ml of dilute hydrochloric acid until no more bubbles are generated, then add 5 ml of hydrochloric acid, hydrolyze at 150℃ for 1 hour, remove, cool, transfer to an evaporating dish, wash 3 times with water (2-3 ml each time), combine the washings with the evaporating dish, evaporate to dryness, add an appropriate amount of 0.1 mol / L hydrochloric acid solution to dissolve the residue, transfer to a 20 ml volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid solution, shake well, and the product is ready.

[0141] Accurately measure 10 ml of the above test solution (centrifuge and collect the supernatant if necessary), place it in a 50 ml centrifuge tube, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution, shake well, and let stand at room temperature for 1 hour. Add 15 ml of n-hexane, shake, let stand for 10 minutes, collect the lower layer solution, filter, and collect the filtrate to obtain the test solution.

[0142] (1) Investigation on the amount of hydrochloric acid added

[0143] This embodiment investigates the hydrolysis effect of different amounts of hydrochloric acid (i.e., the second acid solution) on oyster standard decoction (lyophilized powder), and the results are shown in Table 13 below.

[0144] Table 13 Chromatogram parameters of oyster standard decoction (lyophilized powder) with different amounts of hydrochloric acid added.

[0145]

[0146]

[0147] The results showed that the addition of hydrochloric acid in amounts of 1-5 ml could completely hydrolyze the amino acids in the oyster standard decoction (lyophilized powder). To ensure complete hydrolysis, 3 ml of hydrochloric acid was selected as the amount added for further investigation.

[0148] (2) Hydrolysis time investigation

[0149] Based on the determined amount of hydrochloric acid (i.e., the second acid solution) added above, the effects of different hydrolysis times (0.5 hours, 1 hour, and 1.5 hours) on the hydrolysis effect of oyster standard decoction (lyophilized powder) were investigated. The results are shown in Table 14 below.

[0150] Table 14 Chromatogram parameters of oyster standard decoction (lyophilized powder) at different hydrolysis times

[0151]

[0152]

[0153] The results showed that the hydrolysis time had no significant effect on the information content of the chromatographic peaks and the system suitability parameters. In order to ensure complete hydrolysis, 1 h was selected as the hydrolysis time for subsequent investigation.

[0154] (3) Sampling size investigation

[0155] Based on the determined amount of hydrochloric acid (i.e., the second acid solution) and hydrolysis time, the effects of sampling amounts of 0.25g, 0.5g, and 1g on the extraction efficiency of oyster standard decoction (lyophilized powder) were further investigated. The results are shown in Table 15 below.

[0156] Table 15 Chromatogram parameters of oyster standard decoction (lyophilized powder) at different sampling amounts

[0157]

[0158]

[0159] The results showed that the peak area of ​​the chromatographic peaks increased by a roughly proportional ratio with the increase in sample amount, indicating that the amino acid components in the oyster standard decoction (lyophilized powder) could be completely hydrolyzed within the sample amount range of 0.25-1.0 g. For ease of operation, 0.5 g was selected for subsequent condition investigation.

[0160] In summary, the optimized test solution preparation method of this embodiment is as follows: Take about 0.5g of the powder, accurately weigh it, place it in a hydrolysis tube, slowly add 10ml of dilute hydrochloric acid, then add 3ml of hydrochloric acid, hydrolyze at 150℃ for 1 hour, cool, transfer to an evaporating dish, wash with water 3 times (2-3ml each time), add the washings to the evaporating dish, evaporate to dryness, add an appropriate amount of 0.1mol / L hydrochloric acid solution to dissolve the residue, transfer to a 20ml volumetric flask, dilute to the mark with 0.1mol / L hydrochloric acid solution, shake well, and the test solution is obtained.

[0161] Example 5

[0162] This embodiment investigates the derivatization method of the sample solution under the aforementioned determined chromatographic conditions.

[0163] (1) Investigation on the amount of phenyl isothiocyanate (PITC) added

[0164] Based on the established method for preparing the test solution, the effects of adding 1.5 ml, 2 ml, 2.5 ml, and 3 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution on the characteristic chromatograms of oyster standard decoction (lyophilized powder) were further investigated. The information content of the chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results are shown in Table 16 below.

[0165] Table 16 Chromatogram parameters of oyster standard decoction (lyophilized powder) with different PITC addition amounts

[0166]

[0167]

[0168] The results showed that, except for the smaller chromatographic peak when 1.5 ml of PITC was added, the other added volumes were basically the same. Therefore, 2.5 ml was selected as the amount of PITC added for further investigation.

[0169] (2) Investigation on the amount of triethylamine added

[0170] Based on the determined test solution preparation method and PITC addition amount, the effect of triethylamine addition amounts (2 ml, 2.5 ml, and 3 ml) on the characteristic chromatogram of oyster standard decoction (lyophilized powder) was further investigated. The information content of chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results are shown in Table 17 below.

[0171] Table 17 Chromatogram parameters of oyster standard decoction (lyophilized powder) with different amounts of triethylamine added.

[0172]

[0173]

[0174] The results showed that the amount of triethylamine added between 2.0 and 3.0 ml had no significant effect on the information content of the chromatographic peaks and the suitability of the system. 2.5 ml was selected as the amount of triethylamine added for further investigation.

[0175] (3) Derivative time examination

[0176] Based on the determined test solution preparation method, PITC addition amount, and triethylamine addition amount, the effects of reaction time (0.5 h, 1 h, and 1.5 h) on the characteristic chromatograms of oyster standard decoction (lyophilized powder) were further investigated. The information content of chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results are shown in Table 18 below.

[0177] Table 18 Chromatogram parameters of oyster standard decoction (lyophilized powder) at different derivatization times

[0178]

[0179]

[0180] The results showed that the reaction time between 0.5 and 1.5 h had no significant impact on the information content of the chromatographic peaks and the applicability of the system. In order to ensure complete reaction, 1 h was selected as the reaction time for subsequent investigation.

[0181] (4) Investigation of different injection volumes

[0182] Based on the established test solution preparation method and derivatization conditions, the separation effect of different injection volumes (1 μl, 2 μl, 3 μl, and 4 μl) was further investigated. The information content of the chromatographic peaks, system suitability, and separation effect were used as the main evaluation indicators. The results are shown in Table 19 below.

[0183] Table 19. Parameters of Chromatographic Peak System Suitability under Different Injection Volumes

[0184]

[0185]

[0186] The results showed that as the injection volume increased, the peak area of ​​the chromatographic peak in the sample increased accordingly. The peak shape of 2 μl was better, the peak height and peak width were relatively moderate, and the resolution of the chromatographic peak was relatively good. Therefore, the injection volume was selected as 2 μl.

[0187] In summary, the optimized provisional derivatization method for oyster standard decoction (lyophilized powder) in this embodiment is as follows: Accurately measure 10 ml of the above-mentioned test sample solution (centrifuge if necessary and take the supernatant), place it in a 50 ml centrifuge tube, add 2.5 ml of acetonitrile solution of 0.1 mol / L phenyl isothiocyanate and 2.5 ml of acetonitrile solution of 1 mol / L triethylamine, shake well, let stand at room temperature for 1 hour, shake well again, add 15 ml of n-hexane, shake, let stand for 10 minutes, take the lower layer solution, filter, and take the filtrate to obtain the final product.

[0188] Accurately pipette 2 μl each of the derivatized reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatograms after 35 minutes.

[0189] Example 6

[0190] This embodiment is based on the aforementioned determined chromatographic conditions to conduct methodological validation and investigation.

[0191] (1) Precision test

[0192] Take the same sample solution of oyster standard decoction (lyophilized powder), inject it six times consecutively under chromatographic conditions, and calculate the relative retention time and relative peak area of ​​each characteristic peak. Detailed results are attached. Figure 10 And see Tables 20-21 below.

[0193] Table 20 Results of Instrument Precision and Relative Retention Time Tests

[0194]

[0195]

[0196] Table 21 Results of Instrument Precision Relative Peak Area Test

[0197]

[0198] The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the instrument has good precision.

[0199] (2) Method repeatability test

[0200] Six test solutions were prepared repeatedly using the same batch of oyster standard decoction (lyophilized powder) according to the test solution preparation method. The solutions were then analyzed under chromatographic conditions, and the relative retention times and relative peak areas of each characteristic peak were calculated. Detailed results are attached. Figure 11 And Tables 22-23 below.

[0201] Table 22 Results of Repeatability Relative Retention Time Test

[0202]

[0203]

[0204] Table 23 Results of Repeatability Relative Peak Area Test

[0205]

[0206] The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the method has good repeatability.

[0207] (3) Intermediate precision (different operators)

[0208] Following the method for preparing the test sample solution, three personnel (A, B, and C) each prepared two test sample solutions. These solutions were then analyzed on the same instrument under the same chromatographic conditions. The relative retention times and relative peak areas of each characteristic peak were calculated. Detailed results are shown in the appendix. Figure 12 And see Tables 24-25 below.

[0209] Table 24 Results of Intermediate Precision Relative Retention Time Test

[0210]

[0211]

[0212] Table 25 Results of Intermediate Precision Relative Peak Area Test

[0213]

[0214] The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the method has good intermediate accuracy among different personnel.

[0215] (4) Specificity examination

[0216] The test solution and negative control granule solution were prepared according to the solution preparation method. UPLC analysis was performed under the chromatographic conditions described in the section on "Characteristic Chromatography" to investigate whether the negative control sample of the oyster (Ostrea gigas) decoction would cause interference. The negative control chromatogram is recorded in the appendix. Figure 13 .

[0217] It can be seen that the blank control of the test sample does not interfere with the characteristic spectrum and can be used as a detection method for the characteristic spectrum of standard decoction of oyster (Ostrea gigas) slices.

[0218] (5) Stability test

[0219] Take the same sample solution, prepare it according to the chromatographic conditions under the

Characteristic Chromatography

[0220] Table 26 Results of the relative retention time test for stability

[0221]

[0222] Table 27 Results of Stability Relative Peak Area Test

[0223]

[0224]

[0225] The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 2%, and the RSD values ​​of the relative peak areas were all less than 10%, indicating that the test solution was stable within 48 hours and met the determination requirements.

[0226] (6) Durability - Examination of different instruments

[0227] Two researchers prepared six test solutions each at different times according to the test solution preparation method, and analyzed them using different devices. The relative retention time and relative peak area of ​​each characteristic peak were calculated. Detailed results are shown in the appendix. Figure 15 And see Tables 28-29 below.

[0228] Table 28 Results of Intermediate Precision Relative Retention Time Tests for Different Instruments

[0229]

[0230]

[0231] Table 29 Results of Intermediate Precision Relative Peak Area Tests for Different Instruments

[0232]

[0233]

[0234] The results show that the RSD values ​​of the relative retention times of each characteristic peak are all less than 5%, indicating good intermediate precision among different instruments.

[0235] In summary, based on the above methodological investigation results, the established method for the characteristic chromatogram of oyster standard decoction (lyophilized powder) has certain robustness to column temperature and acid concentration, but poor robustness to flow rate and different column types. Considering the above results, it is recommended that the specified range for relative retention time be controlled within ±10%. Peaks 3 (glycine), 6 (proline), 7 (alanine), and 8 (lysine) are indicator components for content determination; it is recommended that they be identified using reference peaks, without specifying relative retention times, and that the relative retention times and relative peak areas of other characteristic peaks be calculated.

[0236] Example 7: Characteristic chromatographic data of standard decoction of oyster (Ostrea gigas) slices

[0237] Fourteen batches of standard decoction (lyophilized powder) of oyster (Ostrea gigas) were tested using the optimized [characteristic chromatogram] method described above. The results are shown in Tables 30-31 below.

[0238] Table 30. Relative retention time determination results of characteristic spectra of 14 batches of oyster (Ostrea gigas) decoction (lyophilized powder)

[0239]

[0240]

[0241] Table 31. Results of relative peak area determination of characteristic spectra of 14 batches of oyster (Ostrea gigas) decoction (lyophilized powder)

[0242]

[0243]

[0244] The results showed that the characteristic chromatograms of 14 batches of oyster (Ostrea gigas) decoction standard powder (lyophilized) contained 9 characteristic peaks, which should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 6, 7, and 8 should correspond to the retention times of the corresponding reference standard peaks. The peak corresponding to the glycine reference peak is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times of each characteristic peak were all within ±10% of the specified value.

[0245] Example 8: Prescribed values ​​of the characteristic spectrum

[0246] Based on the aforementioned research results, it can be determined that the chromatogram of the test sample should show 9 characteristic peaks, and these peaks should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 6, 7, and 8 should correspond to the retention times of the corresponding reference standard peaks. The peak corresponding to the glycine reference standard peak is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.62 (peak 1), 0.90 (peak 2), 1.19 (peak 4), 1.26 (peak 5), and 3.78 (peak 9).

[0247] The relative retention times and relative peak areas of the obtained control characteristic chromatograms, as well as the results of the control medicinal material characteristic chromatograms, are shown in Tables 32-35 below. The characteristic chromatograms of oyster (Ostrea gigas) control medicinal material, 14 batches of oyster (Ostrea gigas) decoction standard powder, and oyster (Ostrea gigas) formula granules are attached. Figure 16In (a)-(c), peak 2: serine; peak 3: glycine; peak 4: glutamic acid; peak 5: aspartic acid; peak 6: proline; peak 7: alanine; peak 8: lysine; peak 9: phenylalanine.

[0248] The fitting method for the comparison spectrum is Mark peak fitting.

[0249] Table 32. Comparison chart of standard decoctions made from oyster (Ostrea gigas) slices: relative retention time.

[0250] serial number Peak 1 Peak 2 Peak 3(S) Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Retention time 4.779 6.938 7.673 9.122 9.695 12.667 13.291 27.744 29.025 Relative retention time 0.62 0.90 1.00 1.19 1.26 1.65 1.73 3.62 3.78

[0251] Table 33. Comparison of relative peak areas in standard decoctions of oyster (Ostrea gigas) slices.

[0252]

[0253]

[0254] Table 34. Relative retention time of oyster (Ostrea gigas) as a reference medicinal material.

[0255] serial number Peak 1 Peak 2 Peak 3(S) Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Retention time 4.722 6.794 7.528 8.899 9.443 12.496 13.099 27.264 28.509 Relative retention time 0.63 0.90 1.00 1.18 1.25 1.66 1.74 3.62 3.79

[0256] Table 35. Relative peak areas of characteristic spectra of oyster (Ostrea gigas) and other medicinal materials.

[0257] serial number Peak 1 Peak 2 Peak 3(S) Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak area 153331 185486 685320 61072 186524 97006 96327 69877 36896 relative peak area 0.224 0.271 1.000 0.089 0.272 0.142 0.141 0.102 0.054

[0258] Example 9: Differentiation of Contrast Products

[0259] Currently, commonly used shellfish medicinal materials in clinical practice include abalone shell, mother-of-pearl, clam shell, and oyster shell.

[0260] The clinical effects of oyster shell include: calming the mind and soothing the nerves, nourishing Yin and calming Yang, and softening and dispersing masses. It is used for palpitations, insomnia, dizziness, tinnitus, scrofula, phlegm nodules, and abdominal masses. Calcined oyster shell has astringent and consolidating properties, and also neutralizes acid and relieves pain. It is used for spontaneous sweating, night sweats, seminal emission, metrorrhagia, leukorrhea, stomach pain, and acid reflux.

[0261] The clinical effects of oyster shell are: resolving phlegm and removing blood stasis, softening and dispersing masses, neutralizing acid and relieving pain. It is used for stubborn phlegm that is difficult to expectorate, goiter, scrofula, abdominal masses, stomach pain and acid reflux.

[0262] Clam shells have the following effects: clearing heat and resolving phlegm, softening and dispersing nodules, neutralizing acid and relieving pain; externally, they can be used to astringe and heal sores. They are used for cough with phlegm and fire, chest and rib pain, blood in sputum, scrofula, goiter, stomach pain and acid regurgitation; externally, they can be used to treat eczema and burns.

[0263] Mother-of-pearl has the following effects: calming the liver and suppressing yang, soothing the mind and relieving fright, and improving eyesight and clearing away corneal opacity. It is used for headaches, dizziness, palpitations, insomnia, red eyes with corneal opacity, and blurred vision.

[0264] It can be seen that oysters, compared with other shellfish medicines, have a special function of sedation and tranquilization, and should not be used interchangeably in clinical practice. Furthermore, there are few oyster preparations that retain the original medicinal properties and can be easily distinguished; therefore, differentiating oysters from other shellfish medicines using characteristic spectra is particularly important.

[0265] The aforementioned shellfish medicinal materials were collected separately and analyzed using the optimized characteristic mapping method for Pacific oysters. Characteristic maps of Pacific oysters and oyster shells, Pacific oysters and clam shells, Pacific oysters and abalone shells, and Pacific oysters and mother-of-pearl are attached. Figure 17 (a)-(d) and Tables 36-37 below. The results show that the Pacific oyster is somewhat different from other common shellfish medicinal materials.

[0266] Table 36. Characteristic Graphs of Oceanus longifolia-related adulterants and their relative retention times.

[0267]

[0268]

[0269] Table 37 Relative peak areas of characteristic spectra of adulterants related to Pacific oyster.

[0270]

[0271]

[0272] Based on the characteristic spectrum results of the Pacific oyster and the cork oyster, there is a difference between the two in the ratio of peak 2 to peak 3. The relative peak area of ​​peak 2 to peak 3 in cork oyster is 0.198-0.239, while that in Pacific oyster is 0.033-0.172. It is stipulated that the relative peak area of ​​peak 2 to peak 3 should not be greater than 0.19, thus distinguishing Pacific oyster from cork oyster.

[0273] Based on the characteristic spectrum results of the Pacific oyster and clam shell, there is a distinction between the two in the ratio of peak 4 to peak 5. The relative peak area of ​​peak 4 to peak 5 in clam shell is 0.448-0.557, while that in Pacific oyster is 0.727-1.001. It is stipulated that the relative peak area of ​​peak 4 to peak 5 should not be less than 0.65, thus distinguishing Pacific oyster from clam shell.

[0274] Based on the characteristic spectrum results of the Pacific oyster, abalone shell, and mother-of-pearl, there is a distinction between the two in the ratio of peak 6 to peak 7. The relative peak area of ​​peak 6 to peak 7 in abalone shell and mother-of-pearl is 0.136-0.440, while that in Pacific oyster is 0.682-4.053. It is stipulated that the relative peak area of ​​peak 6 to peak 7 should not be less than 0.61, thus distinguishing Pacific oyster from abalone shell and mother-of-pearl.

[0275] As can be seen, by establishing a characteristic spectrum of the Pacific oyster and its preparations, the present invention achieves better separation effect, improves analytical efficiency, and can clearly distinguish the Pacific oyster from common shellfish drugs, thus ensuring accurate drug administration.

[0276] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of oyster and its pharmaceutical preparations, characterized in that, This includes the step of performing ultra-high performance liquid chromatography detection on test solutions of oysters and / or oyster pharmaceutical preparations; The preparation method of the test solution includes the following steps: S1 step: Take the test sample and accurately add it to the first acid solution and mix. Then add the second acid solution to hydrolyze the sample. Collect the hydrolysate, wash it, and evaporate it to dryness. Add the residue to the third acid solution to re-dissolve it. Step S2: Add the solution obtained in Step S1 to an acetonitrile solution containing phenyl isothiocyanate and an acetonitrile solution containing triethylamine, mix, then add n-hexane and mix again. Filter the lower layer solution to obtain the final product. The second acid solution includes concentrated hydrochloric acid with a concentration of 35-37%; The method further includes the steps of preparing a reference solution and constructing a characteristic chromatogram of the reference solution based on the ultra-high performance liquid chromatography. The reference standards include glycine, proline, alanine, and lysine hydrochloride; The preparation method of the reference solution includes: accurately adding the reference standard to the third acid solution and mixing, then adding an acetonitrile solution containing phenyl isothiocyanate and an acetonitrile solution containing triethylamine and mixing, then adding n-hexane and mixing, and finally filtering the lower layer solution to obtain the solution. Chromatographic conditions included: using an ACQUITY UPLC® HSS T3 column, acetonitrile as mobile phase A, 0.08-0.12% phosphoric acid solution as mobile phase B, and a detection wavelength of 254±2 nm, with gradient elution performed according to the following program: From 0 to 7 minutes, the ratio of A to B was 17% to 83%. 7-10 min, A:B ratio changed from 17% to 22%; A:B ratio changed from 83% to 78%. 10-15 min, A:B ratio is 22%:78%; 15-16 min, A:B ratio changed from 22% to 35%; 78%:B ratio changed from 65%. 16-20 min, A:B ratio 35%:65%; Over 20-30 minutes, the ratio of A to B changed from 35% to 36% and from 65% to 64%.

2. The method for constructing characteristic maps of oysters and their pharmaceutical preparations according to claim 1, characterized in that, The ultra-high performance liquid chromatography (UHPLC) detection steps also include the following chromatographic conditions: flow rate of 0.25-0.30 ml / min and column temperature of 30-40℃.

3. The method for constructing characteristic spectra of oysters and their pharmaceutical preparations according to claim 1 or 2, characterized in that, The method also includes the step of preparing a reference solution of the control medicinal material, specifically including: taking oyster reference medicinal material and accurately adding it to the first acid solution for mixing, and continuing to add the second acid solution for hydrolysis, collecting the hydrolysate, washing it and evaporating it to dryness, and adding the residue to the third acid solution for redissolution, thus obtaining the solution.

4. The method for constructing characteristic maps of oysters and their pharmaceutical preparations according to claim 3, characterized in that, The method further includes a step of derivatizing the reference drug solution, comprising: taking the reference drug solution and adding it to an acetonitrile solution containing phenyl isothiocyanate and an acetonitrile solution containing triethylamine, mixing them together, then adding hexane and mixing again, and filtering the lower layer solution to obtain the final product.

5. The method for constructing characteristic spectra of oysters and their pharmaceutical preparations according to claim 1, 2, or 4, characterized in that: The first acid solution comprises dilute hydrochloric acid with a concentration of 9.5-10.5%; and / or, The third acid solution comprises a hydrochloric acid solution with a concentration of 0.08-0.12 mol / L; and / or, The temperature of the hydrolysis step is 120-180℃; and / or, The concentration of the acetonitrile solution containing phenyl isothiocyanate is 0.08-0.12 mol / L; and / or, The concentration of the triethylamine-containing acetonitrile solution is 0.08-0.12 mol / L.

6. The method for constructing characteristic spectra of oysters and their pharmaceutical preparations according to claim 1 or 2, characterized in that, The oyster pharmaceutical preparations include oyster formula granules, oyster slices, or oyster standard decoctions.

7. The method for constructing characteristic spectra of oysters and their pharmaceutical preparations according to claim 1 or 2, characterized in that, The characteristic spectrum has 9 characteristic peaks. The peak corresponding to the glycine reference standard peak is designated as the S peak. The relative retention times of the other characteristic peaks relative to the S peak are within ±10% of the specified values. The specified values ​​of each characteristic peak are: peak 1: 0.62, peak 2: 0.90, peak 4: 1.19, peak 5: 1.26, and peak 9: 3.

78.

8. The application of the method for constructing characteristic spectra of oysters and their pharmaceutical preparations according to any one of claims 1-7 in the field of quality testing of oysters and their pharmaceutical preparations.

9. A method for quality testing of oysters and their pharmaceutical preparations, characterized in that, The method includes the steps of constructing the feature map and the reference feature map according to any one of claims 1-7, and the step of comparing the feature map with the reference feature map.

Images