Establishment of fingerprint and determination of multi-components in formula granules of oyster

Abstract

The application discloses a method for establishing a fingerprint spectrum of oyster formula granules, which comprises the following steps: preparing a qualitative control mother liquor; preparing a test sample solution of the oyster formula granules; determining the sample by using pre-column derivatization-high performance liquid chromatography, establishing the fingerprint spectrum of the qualitative control mother liquor and the fingerprint spectrum of the test sample solution under the same chromatographic conditions; and obtaining the fingerprint spectrum of the oyster formula granules by comparing and analyzing the fingerprint spectrum of the qualitative control mother liquor and the fingerprint spectrum of the test sample solution. The application further discloses a method for determining the multi-component content of the oyster formula granules. The application performs relevant research on the types and multi-component content of amino acids contained in the oyster formula granules by using pre-column derivatization-high performance liquid chromatography, establishes the fingerprint spectrum of the oyster formula granules, identifies 14 kinds of amino acids, simultaneously establishes five amino acid content methods, and the established method is good in repeatability and high in accuracy, thereby providing a basis for quality control of the oyster formula granules.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine component detection technology, specifically to a method for establishing fingerprint spectra of oyster formula granules and determining the content of multiple components. Background Technology

[0002] Oyster shells, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), are derived from the shells of the Pacific oyster (*Ostrea gigas* Thunberg), Dalian Bay oyster (*Ostrea talienwhanensis* Crosse), or riparian oyster (*Ostrea rivularis* Gould). As a unique species possessing both animal and mineral medicinal properties, oysters are known for their calming, sedative, and yin-nourishing effects, as well as their ability to soften and disperse masses. Clinically, they are primarily used to treat symptoms such as palpitations, insomnia, dizziness, tinnitus, scrofula, and abdominal masses. Modern research indicates that the main component of oyster shells is CaCO3, along with various inorganic elements such as iron (Fe) and magnesium (Mg), as well as various amino acids including lysine and glycine. Amino acids are the basic building blocks of proteins and are essential substances in biological metabolism. They possess antioxidant, anti-aging, immune-boosting, metabolism-enhancing, and nutritional properties, and many are the material basis for the efficacy of traditional Chinese medicine.

[0003] Traditional Chinese medicine (TCM) formula granules are prepared using water as the extraction solvent and TCM decoction pieces as raw materials. Modern processes and technologies are employed to extract, separate, concentrate, dry, granulate, and package the effective components from the decoction pieces. This process preserves the main active ingredients of the decoction while overcoming the problems of traditional decoctions, such as difficulty in carrying and preparation. Therefore, to further improve the quality control research of oyster formula granules, this invention establishes an HPLC fingerprint of oyster formula granules and a multi-component amino acid determination method from the perspective of organic component amino acids. This aims to promote the overall quality control level of these formula granules and provide a scientific reference for the research of other shellfish mineral medicine formula granules. Summary of the Invention

[0004] To overcome the above-mentioned technical problems, this invention discloses a method for establishing fingerprint spectra of oyster formula granules and determining the content of multiple components.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A method for establishing a fingerprint spectrum of oyster formula granules, the method comprising the following steps:

[0007] Step 1, Preparation of qualitative reference standard stock solution;

[0008] Step 2, Preparation of oyster formula granule test solution;

[0009] Step 3: The fingerprint chromatograms of the qualitative reference standard mother liquor and the test solution were established under the same chromatographic conditions using pre-column derivatization-high performance liquid chromatography.

[0010] Step 4: Obtain the fingerprint spectrum of oyster formula granules by comparing and analyzing the fingerprint spectrum of the qualitative reference mother liquor and the fingerprint spectrum of the test solution.

[0011] The above-mentioned method for establishing the fingerprint spectrum of oyster formula granules, wherein in step 3, the chromatographic conditions are as follows:

[0012] Chromatographic column: Shim-pack Scepter C18-120 column, 4.6×150mm, 3μm;

[0013] Mobile phase: A is an acetonitrile aqueous solution with a volume ratio of 4:1, and B is an acetonitrile-0.1 mol / L sodium acetate solution with a volume ratio of 7:93. The pH of the sodium acetate solution is adjusted to 6.5 with acetic acid.

[0014] Gradient elution program: 0–15 min, 100%–98% B; 15–22 min, 98%–90% B; 22–28 min, 90%–82% B; 28–40 min, 82%–75% B; 40–45 min, 75%–62% B; 45–50 min, 62%–0% B; 50–55 min, 0%–100% B;

[0015] Flow rate: 0.8 mL / min;

[0016] Column temperature: 25℃;

[0017] Detection wavelength: 254nm;

[0018] The injection volume was 5 μL.

[0019] The method for establishing the fingerprint spectrum of the oyster formula granules described above, wherein in step 1, the preparation method of the qualitative reference stock solution is as follows: accurately weigh 3.90 mg of glutamic acid, 5.17 mg of serine, 5.60 mg of glycine, 3.59 mg of histidine, 3.69 mg of threonine, 4.16 mg of arginine, 5.07 mg of alanine, 4.79 mg of proline, 5.10 mg of tyrosine, 6.04 mg of valine, 4.53 mg of leucine, 6.22 mg of isoleucine, 4.57 mg of phenylalanine, and 4.77 mg of lysine and place them in a 25 mL volumetric flask, and add 0.1 mol / L hydrochloric acid solution to each. Dissolve and dilute to the mark, shake well, and prepare the qualitative reference stock solution containing 152.88 μg glutamic acid, 202.664 μg serine, 219.520 μg glycine, 140.728 μg histidine, 198.744 μg alanine, 187.768 μg proline, 144.648 μg threonine, 164.248 μg arginine, 199.920 μg tyrosine, 236.768 μg valine, 177.576 μg leucine, 243.824 μg isoleucine, 190.904 μg phenylalanine, and 186.984 μg lysine per 1 mL.

[0020] The above-mentioned method for establishing the fingerprint spectrum of oyster formula granules, wherein in step 2, the preparation method of the oyster formula granule test solution is as follows: Take an appropriate amount of oyster formula granule sample, grind it finely, weigh about 1.0 g, accurately weigh it, place it in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to fully dissolve it, centrifuge at a speed of 12000 rpm for 15 min, discard the supernatant layer, take the precipitate layer and place it in a 25 mL stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, stopper tightly, hydrolyze at 150℃ for 3 h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper several times with a small amount of water, combine the filtrates, evaporate to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid, transfer it to a 10 mL volumetric flask and dilute to the mark, shake well, and you will get solution A;

[0021] Accurately measure 5 mL of solution A and place it in a 10 mL volumetric flask. Add 1.5 mL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mL of 1 mol / L triethylamine acetonitrile solution. Shake well and let stand at room temperature for 3 hours. Then, add 50% acetonitrile to the mark. Take 5 mL of the solution and add 10 mL of n-hexane. Shake and let stand for 10 minutes. Take the lower layer solution and filter it through a 0.45 μm microporous membrane. Take the filtrate to obtain the oyster formula granule test solution.

[0022] The above-mentioned method for establishing the fingerprint spectrum of oyster formula granules, wherein in step 4, the superimposed spectrum and the reference spectrum are generated by using the Chinese herbal chromatographic fingerprint spectrum similarity evaluation system 2012 version, and the multi-point correction method and Mark peak matching method are used to identify the characteristic peaks of the components to be tested.

[0023] The fingerprint spectrum of the oyster formula granules has 14 common peaks, numbered from left to right: peak 1 L-glutamic acid, peak 2 L-serine, peak 3 glycine, peak 4 L-histidine, peak 5 L-threonine, peak 6 L-arginine, peak 7 alanine, peak 8 proline, peak 9 L-tyrosine, peak 10 L-valine, peak 11 L-isoleucine, peak 12 L-leucine, peak 13 L-phenylalanine, and peak 14 L-lysine.

[0024] A method for determining the content of multiple components in oyster formula granules, the method comprising the following steps:

[0025] Step A: Preparation of quantitative reference standard stock solution;

[0026] Step B, Preparation of mixed standard solutions: Prepare a series of mixed standard solutions with concentration gradients using the quantitative reference stock solution;

[0027] Step C: Pre-column derivatization-high performance liquid chromatography was used for injection and determination. Under the same chromatographic conditions, the peak area was recorded. Linear analysis was performed with the peak area as the ordinate and the concentration of the reference standard as the abscissa to establish a standard curve equation and plot the standard curve of oyster formula granules.

[0028] Step D, preparation of oyster formula granule test solution;

[0029] Step E: Pre-column derivatization-high performance liquid chromatography (HPLC) is used for sample injection and determination. Under the same chromatographic conditions, the peak area of ​​the analyte is recorded, and the content of the analyte is calculated.

[0030] The above-mentioned method for determining the content of multiple components in oyster formula granules, wherein the chromatographic conditions in steps C and E are as follows:

[0031] Chromatographic column: Shim-pack Scepter C18-120 column, 4.6×150mm, 3μm;

[0032] Mobile phase: A is an acetonitrile aqueous solution with a volume ratio of 4:1, and B is an acetonitrile-0.1 mol / L sodium acetate solution with a volume ratio of 7:93. The pH of the sodium acetate solution is adjusted to 6.5 with acetic acid.

[0033] Gradient elution program: 0–15 min, 100%–98% B; 15–22 min, 98%–90% B; 22–28 min, 90%–82% B; 28–40 min, 82%–75% B; 40–45 min, 75%–62% B; 45–50 min, 62%–0% B; 50–55 min, 0%–100% B;

[0034] Flow rate: 0.8 mL / min;

[0035] Column temperature: 25℃;

[0036] Detection wavelength: 254nm;

[0037] The injection volume was 5 μL.

[0038] The above-mentioned method for determining the content of multiple components in oyster formula granules, wherein in step A, the method for preparing the quantitative reference standard stock solution is as follows:

[0039] Accurately weigh 17.70 mg of glycine into a 25 mL volumetric flask. Separately, accurately weigh 6.55 mg of alanine, 6.09 mg of proline, 5.55 mg of tyrosine, and 6.36 mg of lysine into a 50 mL volumetric flask. Dissolve each in 0.1 mol / L hydrochloric acid solution and dilute to the mark. Shake well to prepare the quantitative reference standard stock solutions containing 693.840 μg of glycine, 128.380 μg of alanine, 119.364 μg of proline, 108.780 μg of tyrosine, and 124.656 μg of lysine per mL.

[0040] The above-mentioned method for determining the content of multiple components in oyster formula granules, wherein the linearity test results of each reference standard in step C are as follows:

[0041] glycine y = 66.46x - 46.88 0.9996 6.938～27.754 alanine y = 52.11x - 3.49 0.9997 1.284～12.838 proline y = 46.69x - 1.60 1.0000 1.194～11.936 Tyrosine y = 30.45x - 1.06 0.9999 1.088～8.702 Lysine y = 61.12x - 8.84 0.9997 1.247～6.233

[0042] The above-mentioned method for determining the content of multiple components of oyster formula granules, wherein in step D, the preparation method of the oyster formula granule test solution is as follows: Take an appropriate amount of oyster formula granule sample, grind it finely, weigh about 1.0 g accurately, place it in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to dissolve it completely, centrifuge at 12000 rpm for 15 min, discard the supernatant layer, take the precipitate layer and place it in a 25 mL stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, stopper tightly, hydrolyze at 150℃ for 3 h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper several times with a small amount of water, combine the filtrates, evaporate to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid, transfer it to a 10 mL volumetric flask and dilute to the mark, shake well to obtain solution A;

[0043] Accurately measure 5 mL of solution A and place it in a 10 mL volumetric flask. Add 1.5 mL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mL of 1 mol / L triethylamine acetonitrile solution. Shake well and let stand at room temperature for 3 hours. Then, add 50% acetonitrile to the mark. Take 5 mL of the solution and add 10 mL of n-hexane. Shake and let stand for 10 minutes. Take the lower layer solution and filter it through a 0.45 μm microporous membrane. Take the filtrate to obtain the oyster formula granule test solution.

[0044] The beneficial effects of this invention include:

[0045] (1) This invention uses pre-column derivatization-high performance liquid chromatography to study the types and contents of amino acids and multiple components in oyster formula granules, establishes fingerprint chromatogram of oyster formula granules, identifies a total of 14 amino acids, including 6 essential amino acids and 8 non-essential amino acids, and the characteristic peaks of the 14 amino acids are well characterized in the fingerprint chromatogram of oyster formula granules, which can be used for quality control research.

[0046] (2) This invention also establishes a method for the content of five amino acids: glycine, alanine, proline, tyrosine and lysine. The established method has good repeatability and high accuracy, providing a basis for the quality control of oyster formula granules and also providing a new idea for the quality control of other shell mineral formula granules. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0048] Figure 1 The images show the superimposed fingerprint chromatograms of 10 batches of oyster formula granules and the control fingerprint chromatogram in this invention, wherein S1 to S10 are 10 batches of oyster formula granules and R is the control.

[0049] Figure 2 These are HPLC chromatograms of the mixed reference standard and oyster formulation granules in this invention, wherein A is the HPLC chromatogram of the mixed reference standard and B is the HPLC chromatogram of the oyster formulation granules.

[0050] 1 is L-glutamic acid (Glu); 2 is L-serine (Ser); 3 is glycine (Gly); 4 is L-histidine (His); 5 is L-threonine (Thr); 6 is L-arginine (Arg); 7 is alanine (Ala); 8 is proline (Pro); 9 is L-tyrosine (Tyr); 10 is L-valine (Val); 11 is L-isoleucine (Ile); 12 is L-leucine (Leu); 13 is L-phenylalanine (Phe); 14 is L-lysine (Lys). Detailed Implementation

[0051] The present invention will be further described below through specific embodiments, so as to make the technical solution of the present invention easier to understand and master, rather than to limit the present invention.

[0052] In this embodiment, unless otherwise specified, all reagents are commercially available.

[0053] In this embodiment, unless otherwise specified, all methods used are conventional methods.

[0054] The instruments and reagents used in this embodiment are as follows:

[0055] 1. Materials

[0056] 1.1 Instruments

[0057] XS205DU electronic analytical balance (Mettler-Toledo, Switzerland), 1260Ⅱ HPLC chromatograph (Agilent Technologies, USA); BIORIDGE high-speed centrifuge (Shenzhen Amy Innovation Technology Co., Ltd.), DHG benchtop drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.), Genie G5 ultrapure water system (Shanghai Lefeng Biotechnology Co., Ltd.).

[0058] 1.2 Materials and Reagents

[0059] Amino acid reference standards: L-glutamic acid (Glu, batch number DSTDG007801), L-serine (Ser, batch number DSTDS019001), glycine (Gly, batch number DSTDG006801), L-histidine (His, batch number DSTDZ006901), L-threonine (Thr, batch number DSTDS013501), L-arginine (Arg, batch number DSTDF003902), alanine (Ala, batch number DSTDB011201), proline (Pro, batch number DSTDF003902), L-tyrosine (Tyr) The following amino acids were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.: L-valine (Val, batch number DSTDL03001), L-isoleucine (Ile, batch number DSTDX006101), L-leucine (Leu, batch number DSTDL012401), L-phenylalanine (Phe, batch number DSTDB013601), and L-lysine (Lys, batch number DSTD010801). All had an HPLC purity ≥98%. Acetonitrile was chromatographically pure (Thermo Fisher Scientific). Triethylamine (Guangzhou brand chemical reagent, batch number 20230701) was also used. 19); n-Hexane (Guangzhou brand chemical reagent, batch number 2023070119); anhydrous sodium acetate (Guangzhou brand chemical reagent, batch number 20230302 26); phenyl isothiocyanate (Maclean's reagent, batch number C15329525); water was self-made ultrapure water. All 10 batches of oyster shell formula granules were provided by Guangdong Yifang Pharmaceutical Co., Ltd. Detailed sample information is shown in Table 1.

[0060] Table 1 Information on 10 batches of oyster formula granules

[0061] 1 S1 Omi oyster 2 S2 Omi oyster 3 S3 Omi oyster 4 S4 Omi oyster 5 S5 Omi oyster 6 S6 Omi oyster 7 S7 Omi oyster 8 S8 Omi oyster 9 S9 Omi oyster 10 S10 Omi oyster

[0062] 2. Methods and Results

[0063] 2.1 Chromatographic conditions

[0064] A Shim-pack Scepter C18-120 (4.6×150 mm, 3 μm) column was used. The mobile phase A was acetonitrile:water (4:1), and the mobile phase B was acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93). Gradient elution was used (0–15 min, 100%–98% B; 15–22 min, 98%–90% B; 22–28 min, 90%–82% B; 28–40 min, 82%–75% B; 40–45 min, 75%–62% B; 45–50 min, 62%–0% B; 50–55 min, 0%–100% B). The flow rate was 0.8 mL / min; the column temperature was 25℃; the detection wavelength was 254 nm; and the injection volume was 5 μL.

[0065] 2.2 Preparation of Qualitative Reference Standard Stock Solution

[0066] Accurately weigh 3.90 mg of glutamic acid, 5.17 mg of serine, 5.60 mg of glycine, 3.59 mg of histidine, 3.69 mg of threonine, 4.16 mg of arginine, 5.07 mg of alanine, 4.79 mg of proline, 5.10 mg of tyrosine, 6.04 mg of valine, 4.53 mg of leucine, 6.22 mg of isoleucine, 4.57 mg of phenylalanine, and 4.77 mg of lysine into a 25 mL volumetric flask. Dissolve each ingredient in 0.1 mol / L hydrochloric acid solution and dilute to the mark. Shake well to prepare a solution containing 152.88 mg of glutamic acid per mL. A qualitative reference stock solution containing μg of serine, 202.664 μg of glycine, 219.520 μg of histidine, 140.728 μg of alanine, 198.744 μg of proline, 187.768 μg of threonine, 144.648 μg of arginine, 164.248 μg of tyrosine, 199.920 μg of valine, 236.768 μg of leucine, 177.576 μg of isoleucine, 243.824 μg of phenylalanine, 190.904 μg of lysine, and 186.984 μg of lysine was used for subsequent HPLC chromatographic identification.

[0067] 2.3 Preparation of quantitative reference standard stock solution

[0068] Accurately weigh 17.70 mg of glycine into a 25 mL volumetric flask; separately, accurately weigh 6.55 mg of alanine, 6.09 mg of proline, 5.55 mg of tyrosine, and 6.36 mg of lysine into 50 mL volumetric flasks, dissolve them in 0.1 mol / L hydrochloric acid solution, and dilute to the mark. Shake well to prepare quantitative reference stock solutions containing 693.840 μg of glycine, 128.380 μg of alanine, 119.364 μg of proline, 108.780 μg of tyrosine, and 124.656 μg of lysine per mL, respectively, for later use.

[0069] 2.4 Preparation of Oyster Formula Granules Test Solution

[0070] Take an appropriate amount of sample, grind it finely, accurately weigh about 1.0 g of this product, place it in a 50 mL centrifuge tube, add 10 mL of dilute hydrochloric acid solution to dissolve it completely, centrifuge for 15 min (12000 rpm), discard the supernatant, take the precipitate layer and place it in a 25 mL stoppered hydrolysis tube, accurately add 10 mL of 9 mol / L hydrochloric acid, stopper tightly, hydrolyze at 150℃ for 3 h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper several times with a small amount of water, combine the filtrates, evaporate to dryness, dissolve the residue in 0.1 mol / L hydrochloric acid, transfer it to a 10 mL volumetric flask and dilute to the mark, shake well, and the product is obtained.

[0071] Accurately measure 5 mL of the above solution and place it in a 10 mL volumetric flask. Add 1.5 mL of 0.1 mol / L phenyl isothiocyanate (PITC) acetonitrile solution and 1 mL of 1 mol / L triethylamine acetonitrile solution. Shake well and let stand at room temperature for 3 hours. Then, add 50% acetonitrile to the mark. Take 5 mL of the solution, add 10 mL of n-hexane, shake, and let stand for 10 minutes. Take the lower layer solution and filter it through a 0.45 μm microporous membrane. Collect the filtrate to obtain the oyster shell formula granule test solution.

[0072] 3. Fingerprint Spectrum Study of Oyster Formula Granules

[0073] 3.1 Methodological Investigation of Fingerprint Spectroscopy for Oyster Formula Granules

[0074] 3.1.1 Precision Test

[0075] Take oyster formula granules (S2), prepare the test solution according to the method in section "2.3", and inject it continuously 6 times according to the chromatographic conditions in section "2.1". Using proline as the reference peak, calculate the relative retention time RSD value of each common peak. The RSD value of the relative peak area is 0.01% to 0.20%, and the RSD value of the relative peak area is 0.41% to 1.03%, indicating that the instrument precision is good. See Tables 2 and 3.

[0076] Table 2. Results of Precision Study of Oyster Formula Granule Characteristic Map (Relative Retention Time)

[0077] Peak 1 0.2332 0.2320 0.2320 0.2322 0.2323 0.2320 0.20 Peak 2 0.5342 0.5333 0.5331 0.5331 0.5328 0.5325 0.11 Peak 3 0.5920 0.5913 0.5909 0.5910 0.5907 0.5903 0.09 Peak 4 0.7171 0.7165 0.7167 0.7166 0.7160 0.7158 0.07 Peak 5 0.8818 0.8815 0.8815 0.8815 0.8818 0.8812 0.03 Peak 6 0.9221 0.9222 0.9225 0.9224 0.9224 0.9223 0.02 Peak 7 0.9604 0.9604 0.9603 0.9606 0.9607 0.9605 0.01 Peak 8 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 9 1.6449 1.6473 1.6453 1.6476 1.6502 1.6491 0.13 Peak 10 1.6681 1.6706 1.6685 1.6708 1.6732 1.6723 0.12 Peak 11 1.9824 1.9867 1.9842 1.9867 1.9902 1.9886 0.14 Peak 12 2.0095 2.0138 2.0113 2.0138 2.0176 2.0158 0.15 Peak 13 2.2741 2.2788 2.2759 2.2790 2.2846 2.2814 0.16 Peak 14 2.4519 2.4555 2.4522 2.4562 2.4610 2.4583 0.14

[0078] Table 3. Precision results of oyster formulation particle characteristic spectrum (relative peak area)

[0079] Peak 1 0.5346 0.5339 0.5262 0.5258 0.5260 0.5229 0.91 Peak 2 0.8943 0.8961 0.8900 0.8738 0.8833 0.8821 0.95 Peak 3 3.1743 3.1589 3.1493 3.1565 3.1585 3.1348 0.41 Peak 4 0.1483 0.1472 0.1440 0.1468 0.1467 0.1464 0.98 Peak 5 0.3507 0.3430 0.3450 0.3458 0.3407 0.3419 1.03 Peak 6 0.3799 0.3808 0.3799 0.3790 0.3775 0.3769 0.41 Peak 7 1.2244 1.2205 1.2166 1.2105 1.2074 1.2048 0.63 Peak 8 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 9 0.2887 0.2875 0.2882 0.2890 0.2864 0.2843 0.61 Peak 10 0.8080 0.8016 0.7981 0.7964 0.7944 0.7881 0.84 Peak 11 0.4960 0.4944 0.4921 0.4908 0.4936 0.4890 0.51 Peak 12 1.0607 1.0544 1.0519 1.0518 1.0516 1.0458 0.46 Peak 13 0.4505 0.4518 0.4457 0.4566 0.4446 0.4474 0.99 Peak 14 0.6573 0.6515 0.6485 0.6470 0.6461 0.6425 0.78

[0080] 3.1.2 Stability Test

[0081] Take the oyster formula granules (S2) test solution and inject it at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions in section "2.1". The relative retention time RSD of each common peak of the oyster formula granules is calculated to be 0.06% to 0.18% and the relative peak area RSD is 0.73% to 2.98%, indicating that the test solution has good stability within 24 hours. See Tables 4 and 5.

[0082] Table 4. Results of stability study of oyster formulation granule characteristic spectrum (relative retention time)

[0083] Peak 1 0.2332 0.2320 0.2323 0.2324 0.2324 0.2320 0.18 Peak 2 0.5342 0.5331 0.5328 0.5335 0.5336 0.5322 0.13 Peak 3 0.5920 0.5909 0.5907 0.5914 0.5916 0.5900 0.12 Peak 4 0.7171 0.7167 0.7160 0.7166 0.7171 0.7149 0.12 Peak 5 0.8818 0.8815 0.8818 0.8815 0.8819 0.8805 0.06 Peak 6 0.9221 0.9225 0.9224 0.9224 0.9229 0.9203 0.10 Peak 7 0.9604 0.9603 0.9607 0.9608 0.9607 0.9593 0.06 Peak 8 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 9 1.6449 1.6453 1.6502 1.6490 1.6490 1.6472 0.13 Peak 10 1.6681 1.6685 1.6732 1.6720 1.6718 1.6710 0.12 Peak 11 1.9824 1.9842 1.9902 1.9885 1.9878 1.9882 0.15 Peak 12 2.0095 2.0113 2.0176 2.0158 2.0151 2.0156 0.15 Peak 13 2.2741 2.2759 2.2846 2.2816 2.2801 2.2800 0.17 Peak 14 2.4519 2.4522 2.4610 2.4583 2.4580 2.4546 0.15

[0084] Table 5. Results of stability study of oyster formulation particle characteristic spectrum (relative peak area)

[0085] Peak 1 0.5346 0.5262 0.5260 0.5248 0.5182 0.5089 1.66 Peak 2 0.8943 0.8900 0.8833 0.8567 0.8676 0.8504 2.08 Peak 3 3.1743 3.1493 3.1585 3.1387 3.1225 3.1122 0.73 Peak 4 0.1483 0.1440 0.1467 0.1439 0.1427 0.1394 2.17 Peak 5 0.3507 0.3450 0.3407 0.3385 0.3374 0.3286 2.19 Peak 6 0.3799 0.3799 0.3775 0.3742 0.3715 0.3642 1.61 Peak 7 1.2244 1.2166 1.2074 1.1941 1.1877 1.1643 1.82 Peak 8 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 9 0.2887 0.2882 0.2864 0.2851 0.2836 0.2774 1.45 Peak 10 0.8080 0.7981 0.7944 0.7830 0.7702 0.7434 2.98 Peak 11 0.4960 0.4921 0.4936 0.4866 0.4796 0.4695 2.07 Peak 12 1.0607 1.0519 1.0516 1.0453 1.0344 1.0164 1.52 Peak 13 0.4505 0.4457 0.4446 0.4397 0.4395 0.4386 1.05 Peak 14 0.6573 0.6485 0.6461 0.6402 0.6331 0.6227 1.90

[0086] 3.1.3 Repeatability Test

[0087] Take oyster formula granule sample (S2), prepare 6 test solutions in parallel according to the method in section "2.3", and inject and determine according to the chromatographic conditions in section "2.1". The RSD values ​​of the relative retention times of each common peak are calculated to be 0.05% to 0.18%, and the RSD values ​​of the relative peak areas are 1.02% to 2.83%, indicating that the repeatability of the analytical method for oyster formula granules is good. See Tables 6 and 7.

[0088] Table 6. Results of repeatability study of oyster formulation granule characteristic profiles (relative retention time)

[0089] Peak 1 0.2294 0.2293 0.2295 0.2299 0.2297 0.2295 0.09 Peak 2 0.5337 0.5345 0.5346 0.5350 0.5347 0.5350 0.09 Peak 3 0.5916 0.5923 0.5924 0.5928 0.5925 0.5928 0.08 Peak 4 0.7133 0.7143 0.7151 0.7154 0.7152 0.7155 0.12 Peak 5 0.8814 0.8817 0.8821 0.8823 0.8825 0.8825 0.05 Peak 6 0.9203 0.9210 0.9220 0.9223 0.9224 0.9226 0.10 Peak 7 0.9602 0.9604 0.9607 0.9611 0.9614 0.9614 0.05 Peak 8 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 9 1.6558 1.6524 1.6499 1.6514 1.6546 1.6518 0.13 Peak 10 1.6793 1.6757 1.6728 1.6742 1.6773 1.6744 0.14 Peak 11 1.9934 1.9887 1.9840 1.9854 1.9893 1.9854 0.18 Peak 12 2.0207 2.0157 2.0109 2.0123 2.0164 2.0124 0.18 Peak 13 2.2853 2.2793 2.2741 2.2760 2.2805 2.2756 0.18 Peak 14 2.4707 2.4643 2.4598 2.4626 2.4673 2.4626 0.16

[0090] Table 7. Results of repeatability study of oyster shell formulation particle characteristic spectrum (relative peak area)

[0091] Peak 1 0.5256 0.5083 0.5107 0.5223 0.5209 0.5323 1.75 Peak 2 0.9322 0.9095 0.8840 0.9267 0.8652 0.8980 2.83 Peak 3 3.1603 3.1156 3.1099 3.1909 3.1752 3.1274 1.07 Peak 4 0.1456 0.1396 0.1412 0.1413 0.1421 0.1385 1.73 Peak 5 0.3614 0.3535 0.3451 0.3656 0.3392 0.3492 2.82 Peak 6 0.3763 0.3764 0.3717 0.3855 0.3730 0.3718 1.39 Peak 7 1.1846 1.1780 1.1656 1.2188 1.1676 1.1744 1.66 Peak 8 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 9 0.2678 0.2672 0.2685 0.2803 0.2781 0.2704 2.09 Peak 10 0.7514 0.7438 0.7404 0.7618 0.7397 0.7343 1.32 Peak 11 0.4764 0.4714 0.4691 0.4782 0.4700 0.4652 1.02 Peak 12 1.0339 1.0367 1.0255 1.0576 1.0314 1.0283 1.11 Peak 13 0.3767 0.3666 0.3644 0.3713 0.3651 0.3648 1.33 Peak 14 0.6607 0.6533 0.6528 0.6935 0.6565 0.6607 2.32

[0092] 3.2 Fingerprint pattern establishment and chemical pattern recognition

[0093] 3.2.1 Fingerprint spectrum common peak identification

[0094] Ten batches of oyster formula granule samples were prepared into test solutions according to the method described in section "2.4". The solutions were injected and analyzed under the chromatographic conditions described in section "2.1", and chromatograms were recorded. The AIA data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Overlay chromatograms and control chromatograms were generated using the multi-point correction method and Mark peak matching method. Relevant chromatograms were plotted using Origin 2021 software. (See attached image.) Figure 1Fourteen common peaks were identified in the oyster-based formula granules. Through comparison with a mixed amino acid standard, all 14 amino acid components were identified. (See attached image) Figure 2 The relative retention times and relative peak areas of the 10 batches of oyster formula granules are detailed in Tables 8 and 9.

[0095] Table 8. Relative retention time of 10 batches of oyster granule formulations.

[0096]

[0097] Table 9. Relative peak area of ​​oyster shell granules in 10 batches of formulation.

[0098]

[0099]

[0100] 3.2.2 Similarity Evaluation

[0101] Using the fingerprint spectrum (R) of oyster formula granules as a reference spectrum, the similarity of 10 batches of oyster formula granule samples was calculated, and the results are shown in Table 10. The similarity results of the 10 batches of oyster formula granules ranged from 0.978 to 0.999, indicating that the oyster formula granules have stable quality and strong consistency, and can well reflect the common characteristic components of oyster formula granules, and can be used for the quality control of oyster formula granules.

[0102] Table 10. Similarity evaluation results of fingerprint spectra of 10 batches of oyster formulation granules.

[0103] 1 S1 0.981 2 S2 0.998 3 S3 0.999 4 S4 0.995 5 S5 0.978 6 S6 0.998 7 S7 0.998 8 S8 0.995 9 S9 0.995 10 S10 0.982

[0104] 4. Methodological Investigation of Multi-Indicator Content Determination in Oyster Formula Granules

[0105] 4.1 Drawing the Standard Curve

[0106] Accurately pipette appropriate volumes of glycine, alanine, proline, tyrosine, and lysine reference standard stock solutions into five 25 mL volumetric flasks. Prepare six mixed standard solutions with different concentration gradients according to the test sample treatment method in "2.4". Make up to volume and shake well. Inject 5 μL of each solution sequentially under the chromatographic conditions in "2.1" and record the peak areas. Plot the oyster formula granule standard curve with peak area as the ordinate (y) and reference standard concentration as the abscissa (x). The results are shown in Table 11.

[0107] Table 11. Linearity Study Results of Five Amino Acids in Oyster Formula Granules

[0108] glycine y = 66.46x - 46.88 0.9996 6.938～27.754 alanine y = 52.11x - 3.49 0.9997 1.284～12.838 proline y = 46.69x - 1.60 1.0000 1.194～11.936 Tyrosine y = 30.45x - 1.06 0.9999 1.088～8.702 Lysine y = 61.12x - 8.84 0.9997 1.247～6.233

[0109] 4.2 Precision

[0110] Take oyster formula granules (S2), prepare the test solution according to the method under section "2.4", and repeat the injection 6 times under the chromatographic conditions under section "2.1". Record the peak area and calculate the RSD values ​​of the peak areas of glycine, alanine, proline, tyrosine and lysine in oyster formula granules. The results are 0.38%, 0.69%, 0.14%, 0.60% and 0.81%, respectively, indicating that the instrument precision is good. The relevant results are shown in Table 12.

[0111] Table 12 Results of Precision Testing for Oyster Formula Granule Content Determination

[0112]

[0113] 4.3 Stability

[0114] Oyster formula granules (S2) were taken, and test solutions were prepared according to the method in section "2.4". The chromatographic conditions in section "2.1" were used to inject the samples at 0, 2, 4, 8, 12, and 24 hours, respectively. The RSD values ​​of the peak areas of glycine, alanine, proline, tyrosine, and lysine in the oyster formula granules were calculated to be 0.45%, 1.53%, 0.42%, 1.15%, and 1.59%, respectively. This indicates that the test solution of oyster formula granules has good stability within 24 hours. The results are shown in Table 13.

[0115] Table 13 Results of stability study on oyster shell formulation granule content determination

[0116]

[0117]

[0118] 4.4 Repeatability

[0119] Six samples of oyster formula granules (S2) were accurately weighed, and test solutions were prepared according to the method in section "2.4". The chromatographic conditions in section "2.1" were used for determination. The results showed that the average contents of glycine, alanine, proline, tyrosine, and lysine in the oyster formula granules were 0.407 mg / g, 0.148 mg / g, 0.126 mg / g, 0.089 mg / g, and 0.064 mg / g, respectively, with RSD values ​​of 1.52%, 1.32%, 1.59%, 3.64%, and 0.70%, respectively, indicating good repeatability of the method. The results are shown in Table 14.

[0120] Table 14 Results of Repeatability Study for Oyster Granule Content Determination

[0121]

[0122] 4.5 Recovery rate

[0123] Accurately weigh approximately 0.50 g of oyster formula granules (S2) with known content and place it in a 25 mL hydrolysis tube. Perform six parallel treatments, adding glycine, alanine, proline, tyrosine, and lysine with content close to that of the known oyster formula granules to each sample. Prepare the oyster formula granule test solution according to the method under section "2.4" and determine it according to the chromatographic conditions under section "2.1". The results show that the recoveries of glycine, alanine, proline, tyrosine, and lysine in the oyster formula granules ranged from 91.39% to 98.16%, 93.90% to 101.49%, 95.59% to 101.12%, 95.15% to 104.45%, and 102.18% to 109.67%, respectively, with RSD values ​​of 2.56%, 2.92%, 1.88%, 3.75%, and 2.97%, respectively. The results are shown in Tables 15 to 19.

[0124] Table 15 Results of glycine recovery from oyster shell formulation granules (n=6)

[0125]

[0126] Table 16 Results of Alanine Recovery from Oyster Formula Granules (n=6)

[0127]

[0128] Table 17 Results of proline recovery from oyster shell formulation granules (n=6)

[0129]

[0130] Table 18 Results of tyrosine recovery from oyster shell formulation granules (n=6)

[0131]

[0132] Table 19 Results of Lysine Recovery from Oyster Formula Granules (n=6)

[0133]

[0134] 4.6 Determination of Oyster Formula Granule Content

[0135] Accurately weigh each batch of oyster formula granules, prepare the test solution according to the method in section 2.3, and determine the chromatographic conditions of each batch of oyster formula granules according to section 2.1. The results of the determination of 5 amino acids in 10 batches of oyster formula granules are shown in Table 20.

[0136] Table 20 Results of Oyster Granule Content Determination in 10 Batches of Formula (mg / g)

[0137]

[0138] 5. Conclusion

[0139] This invention utilizes pre-column derivatization-high performance liquid chromatography (HPLC) to study the types and contents of multiple components of amino acids in oyster-based formulation granules. A fingerprint chromatogram of oyster-based formulation granules was successfully established, identifying 14 amino acids, including 6 essential amino acids and 8 non-essential amino acids. The similarity of 10 batches of formulation granules was 1.000, indicating stable quality and strong consistency among different batches. Furthermore, the shared pattern of the fingerprint chromatogram effectively reflects the common characteristic components of oyster-based formulation granules, which can be used for quality control research. In addition, this invention also established methods for the content of five amino acids: glycine, alanine, proline, tyrosine, and lysine. The established methods exhibit good repeatability and high accuracy, providing a basis for the quality control of oyster-based formulation granules and offering new insights for the quality control of other shell mineral-based formulation granules.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solution of the present invention, or modify it into equivalent embodiments, without departing from the scope of the present invention, using the disclosed technical means and content. Therefore, all equivalent changes made based on the shape, structure, and principle of the present invention without departing from the scope of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of oyster formula granules, characterized in that, The establishment method includes the following steps: Step 1, Preparation of qualitative reference standard stock solution; Step 2, Preparation of oyster formula granule test solution; Step 3: The fingerprint chromatograms of the qualitative reference standard mother liquor and the test solution were established under the same chromatographic conditions using pre-column derivatization-high performance liquid chromatography. Step 4: Obtain the fingerprint spectrum of oyster formula granules by comparing and analyzing the fingerprint spectrum of the qualitative reference mother liquor and the fingerprint spectrum of the test solution. In step 3, the chromatographic conditions are as follows: Chromatographic column: Shim-pack Scepter C18-120 column, 4.6×150mm, 3μm; Mobile phase: A is an acetonitrile aqueous solution with a volume ratio of 4:1, and B is an acetonitrile-0.1 mol / L sodium acetate solution with a volume ratio of 7:

93. The pH of the sodium acetate solution is adjusted to 6.5 with acetic acid. Gradient elution program: 0 ~ 15 min, 100% ~ 98% B; 15 ~ 22 min, 98% ~ 90% B; 22 ~ 28 min, 90% ~ 82% B; 28 ~ 40 min, 82% ~ 75% B; 40 ~ 45 min, 75% ~ 62% B; 45 ~ 50 min, 62% ~ 0% B; 50 ~ 55 min, 0% ~ 100% B; Flow rate: 0.8 mL / min; Column temperature: 25℃; Detection wavelength: 254 nm; The injection volume was 5 μL; In step 1, the qualitative reference stock solution is prepared as follows: Accurately weigh 3.90 mg of glutamic acid, 5.17 mg of serine, 5.60 mg of glycine, 3.59 mg of histidine, 3.69 mg of threonine, 4.16 mg of arginine, 5.07 mg of alanine, 4.79 mg of proline, 5.10 mg of tyrosine, 6.04 mg of valine, 4.53 mg of leucine, 6.22 mg of isoleucine, 4.57 mg of phenylalanine, and 4.77 mg of lysine and place them in a 25 mL volumetric flask. Dissolve each in 0.1 mol / L hydrochloric acid solution and dilute to the mark. Shake well. A qualitative reference stock solution was prepared, containing 152.88 μg glutamic acid, 202.664 μg serine, 219.520 μg glycine, 140.728 μg histidine, 198.744 μg alanine, 187.768 μg proline, 144.648 μg threonine, 164.248 μg arginine, 199.920 μg tyrosine, 236.768 μg valine, 177.576 μg leucine, 243.824 μg isoleucine, 190.904 μg phenylalanine, and 186.984 μg lysine per 1 mL. In step 2, the preparation method of the oyster formula granule test solution is as follows: Take an appropriate amount of oyster formula granule sample, grind it finely, weigh about 1.0g accurately, place it in a 50mL centrifuge tube, add 10mL of dilute hydrochloric acid solution to dissolve it completely, centrifuge at 12000 rpm for 15min, discard the supernatant layer, take the precipitate layer and place it in a 25mL stoppered hydrolysis tube, accurately add 10mL of 9mol / L hydrochloric acid, stopper tightly, hydrolyze at 150℃ for 3h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper several times with a small amount of water, combine the filtrates, evaporate to dryness, dissolve the residue in 0.1mol / L hydrochloric acid, transfer it to a 10mL volumetric flask and dilute to the mark, shake well to obtain solution A; Accurately measure 5 mL of solution A and place it in a 10 mL volumetric flask. Add 1.5 mL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mL of 1 mol / L triethylamine acetonitrile solution. Shake well and let stand at room temperature for 3 hours. Then, add 50% acetonitrile to the mark. Take 5 mL of the solution and add 10 mL of n-hexane. Shake and let stand for 10 minutes. Take the lower layer solution and filter it through a 0.45 μm microporous membrane. Take the filtrate to obtain the oyster formula granule test solution.

2. The method for establishing the fingerprint spectrum of oyster formula granules according to claim 1, characterized in that, In step 4, the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine is used in 2012. The multi-point correction method and Mark peak matching method are used to generate superimposed chromatograms and reference chromatograms to identify the characteristic peaks of the components to be tested. The fingerprint spectrum of the oyster formula granules has 14 common peaks, numbered from left to right: peak 1 L-glutamic acid, peak 2 L-serine, peak 3 glycine, peak 4 L-histidine, peak 5 L-threonine, peak 6 L-arginine, peak 7 alanine, peak 8 proline, peak 9 L-tyrosine, peak 10 L-valine, peak 11 L-isoleucine, peak 12 L-leucine, peak 13 L-phenylalanine, and peak 14 L-lysine.

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