Oyster formula granule fingerprint spectrum establishment and multi-component content determination method

Abstract

The invention discloses a method for establishing a fingerprint spectrum of oyster formula granules. The method comprises the following steps: preparing a qualitative reference substance mother solution; preparing an oyster formula granule test solution; pre-column derivatization-high performance liquid chromatography is adopted for sample injection and determination, and a fingerprint spectrum of a qualitative reference substance mother solution and a fingerprint spectrum of a test solution are established under the same chromatographic condition; and comparing and analyzing the fingerprint spectrum of the qualitative reference substance mother solution and the fingerprint spectrum of the test solution to obtain the fingerprint spectrum of the oyster formula granules. The invention further discloses a method for determining the content of multiple components of the oyster formula granules. According to the present invention, the types and the multi-component content of the amino acids contained in the oyster formula granules are subjected to the related research by using the pre-column derivatization-high performance liquid chromatography, the oyster formula granule fingerprint spectrum is established, the 14 amino acids are identified, the 5 amino acid content method is established, and the established method has characteristics of good repeatability, high accuracy, and good clinical application prospect. And a basis is provided for quality control of the oyster formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine component detection, and in particular to a method for establishing a fingerprint spectrum of oyster formula granules and determining the content of multiple components. Background Art

[0002] The Chinese medicine oyster was first recorded in Shennong's Herbal Classic. It comes from the shells of the oyster family, Ostrea gigas Thunberg, Ostrea talienwhanensis Crosse or Ostrea rivularis Gould. It is a special variety with both animal and mineral properties. It has the effects of calming the mind, nourishing yang and tonifying yin, softening and dispersing nodules. It is mainly used clinically to treat symptoms such as palpitations, insomnia, dizziness and tinnitus, scrofula, phlegm, and lumps. Modern research shows that the main component of oyster is CaCO3. In addition, it also contains a variety of inorganic elements such as iron (Fe) and magnesium (Mg), as well as a variety of amino acids including lysine and glycine. Among them, amino acids are the basic units of protein and are an important type of substance in the biological metabolism process. They have the effects of anti-oxidation, anti-aging, improving immunity, enhancing body metabolism, providing nutrition, etc., and are mostly the material basis for the efficacy of traditional Chinese medicine.

[0003] Chinese medicine formula granules use water as the extraction solvent and Chinese medicine pieces as the raw materials. Modern processes and technologies are used to extract, separate, concentrate, dry, granulate and package the effective ingredients in the pieces. This not only retains the main active ingredients in the decoction, but also makes up for the problems of traditional decoctions being difficult to carry and difficult to decoct. Therefore, in order to further improve the quality control research of oyster formula granules, the present invention establishes the HPLC fingerprint of oyster formula granules and the multi-component amino acid determination method from the perspective of organic component amino acids, in order to promote the improvement of the overall quality control level of the formula granules and provide scientific reference for the research and development of other shell mineral medicine formula granules. Summary of the invention

[0004] In order to overcome the above technical problems, the present invention discloses a method for establishing a fingerprint spectrum of oyster formula particles and determining the content of multiple components.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

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

[0007] Step 1, preparation of qualitative reference substance mother solution;

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

[0009] Step 3, using pre-column derivatization-high performance liquid chromatography for sample injection and determination, and establishing the fingerprint of the qualitative reference substance mother solution and the fingerprint of the test solution under the same chromatographic conditions;

[0010] Step 4, obtaining the fingerprint of the oyster formula granules by comparing and analyzing the fingerprint of the qualitative reference substance mother solution and the fingerprint of the test solution.

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

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

[0013] Mobile phase: 4:1 acetonitrile-water solution as mobile phase A, 7:93 acetonitrile-0.1 mol / L sodium acetate solution as mobile phase B, wherein the pH value of the sodium acetate solution was 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°C;

[0017] Detection wavelength: 254nm;

[0018] The injection volume was 5 μL.

[0019] The method for establishing the fingerprint of the oyster formula granules mentioned above, wherein in step 1, the preparation method of the qualitative reference substance mother solution is: 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, and add 0.1 mol / L hydrochloric acid solution to each solution. The solution was dissolved and diluted to the mark, and shaken to prepare a mother solution of the qualitative reference substance containing 152.88 μg of glutamic acid, 202.664 μg of serine, 219.520 μg of glycine, 140.728 μg of histidine, 198.744 μg of alanine, 187.768 μg of proline, 144.648 μg of threonine, 164.248 μg of arginine, 199.920 μg of tyrosine, 236.768 μg of valine, 177.576 μg of leucine, 243.824 μg of isoleucine, 190.904 μg of phenylalanine, and 186.984 μg of lysine per 1 mL.

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

[0021] Accurately measure 5 mL of the 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 leave at room temperature for 3 h. Then, add 50% acetonitrile to the scale, take 5 mL, add 10 mL of n-hexane, shake, and leave for 10 minutes. Take the lower layer of solution, filter through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the oyster formula granule test solution.

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

[0023] Among them, the oyster formula granule fingerprint has a total of 14 common peaks, numbered from left to right, peak No. 1 L-glutamate, peak No. 2 L-serine, peak No. 3 glycine, peak No. 4 L-histidine, peak No. 5 L-threonine, peak No. 6 L-arginine, peak No. 7 alanine, peak No. 8 proline, peak No. 9 L-tyrosine, peak No. 10 L-valine, peak No. 11 L-isoleucine, peak No. 12 L-leucine, peak No. 13 L-phenylalanine and peak No. 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 a quantitative reference substance mother solution;

[0026] Step B, preparation of mixed standard solution: using the quantitative reference substance mother solution to prepare a series of mixed standard solutions with concentration gradients;

[0027] Step C, using pre-column derivatization-high performance liquid chromatography for sample injection determination, recording the chromatographic peak area under the same chromatographic conditions, performing linear analysis with the peak area as the ordinate and the reference substance concentration as the abscissa, establishing a standard curve equation, and drawing a standard curve for oyster formula granules;

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

[0029] Step E, using pre-column derivatization-high performance liquid chromatography for sample injection and determination, under the same chromatographic conditions, recording the peak area of ​​the component to be measured, and calculating the content of the component to be measured.

[0030] In the above-mentioned method for determining the content of multiple components in oyster formula granules, in step C and step E, the chromatographic conditions are:

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

[0032] Mobile phase: 4:1 acetonitrile-water solution as mobile phase A, 7:93 acetonitrile-0.1 mol / L sodium acetate solution as mobile phase B, wherein the pH value of the sodium acetate solution was 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°C;

[0036] Detection wavelength: 254nm;

[0037] The injection volume was 5 μL.

[0038] In the above-mentioned method for determining the content of multiple components in oyster formula granules, in step A, the preparation method of the quantitative reference substance mother solution is:

[0039] Accurately weigh 17.70 mg of glycine and place it in a 25 mL volumetric flask. Accurately weigh 6.55 mg of alanine, 6.09 mg of proline, 5.55 mg of tyrosine, and 6.36 mg of lysine and place them in a 50 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve and dilute to the scale, shake well, and prepare the quantitative reference substance mother solution 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 1 mL.

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

[0041] Element Regression equation <![CDATA[r 2 ]]> Linear range / (μg / mL) 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 the step D, the preparation method of the oyster formula granule test solution is: take an appropriate amount of oyster formula granule sample, grind it, weigh about 1.0g, accurately weigh it, put it in a 50mL centrifuge tube, add 10mL of dilute hydrochloric acid solution to fully dissolve it, centrifuge it at a speed of 12000 revolutions per minute for 15min, discard the supernatant layer, take the precipitate layer and put it in a 25mL stoppered hydrolysis tube, accurately add 10mL of 9mol / L hydrochloric acid, plug it tightly, hydrolyze it at 150℃ for 3h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrate, evaporate it to dryness, add 0.1mol / L hydrochloric acid to dissolve the residue, transfer it to a 10mL volumetric flask and dilute it to the scale, shake it well, and obtain solution A;

[0043] Accurately measure 5 mL of the 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 leave at room temperature for 3 h. Then, add 50% acetonitrile to the scale, take 5 mL, add 10 mL of n-hexane, shake, and leave for 10 minutes. Take the lower layer of solution, filter through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the oyster formula granule test solution.

[0044] The beneficial effects of the present invention include:

[0045] (1) The present invention uses pre-column derivatization-high performance liquid chromatography to conduct relevant research on the types and multi-component contents of amino acids contained in oyster formula granules, establishes the fingerprint of oyster formula granules, and identifies a total of 14 amino acids, including 6 essential amino acids and 8 non-essential amino acids. The characteristic peaks of the 14 amino acids are well characterized in the fingerprint of oyster formula granules, which can be used for quality control research;

[0046] (2) The present invention simultaneously establishes methods for determining the contents of five kinds of amino acids, namely, glycine, alanine, proline, tyrosine, and lysine. The established methods have good repeatability and high accuracy, which provides a basis for the quality control of oyster formula granules and also provides a new idea for the quality control of other shell mineral formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0048] Figure 1 It is the superposition of fingerprints of 10 batches of oyster formula granules in the present invention and the reference fingerprint, wherein S1 to S10 are 10 batches of oyster formula granules, and R is the reference substance;

[0049] Figure 2 HPLC graphs of the mixed reference substance and oyster formula granules in the present invention, wherein A is the HPLC graph of the mixed reference substance, and B is the HPLC graph of the oyster formula 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 DESCRIPTION

[0051] The present invention is further described below by means of specific embodiments to make the technical solution of the present invention easier to understand and grasp, rather than to limit the present invention.

[0052] In this embodiment, unless otherwise specified, all reagents can be purchased on the market.

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

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

[0055] 1 Materials

[0056] 1.1 Instrument

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

[0058] 1.2 Materials and reagents

[0059] Amino acid reference substances 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 , batch number DSTDL03001), L-valine (Val, batch number DSTDX006101), L-isoleucine (Ile, batch number DSTDY026601), L-leucine (Leu, batch number DSTDL012401), L-phenylalanine (Phe, batch number DSTDB013601), L-lysine (Lys, batch number DSTD010801) were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., with HPLC purity ≥98%; acetonitrile was chromatographically pure (Thermo Fisher Scientific Co., Ltd.); triethylamine (Guangzhou brand chemical reagent, batch number 20230701 19); n-hexane (Guangzhou brand chemical reagent, batch number 2023070119); anhydrous sodium acetate (Guangzhou brand chemical reagent, batch number 20230302 26); phenyl isothiocyanate (McLean reagent, batch number C15329525); water is homemade ultrapure water. All 10 batches of oyster formula granules were provided by Guangdong Yifang Pharmaceutical Co., Ltd. Detailed information of the samples is shown in Table 1.

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

[0061] Serial number batch number Origin 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 (4.6×150mm, 3μm) chromatographic column was used; acetonitrile:water (4:1) was used as mobile phase A, acetonitrile-0.1mol / L sodium acetate solution (the pH value was adjusted to 6.5 with acetic acid) (7:93) was used as mobile phase B, gradient elution (0-15min, 100%-98% B; 15-22min, 98%-90% B; 22-28min, 90%-82% B; 28-40min, 82%-75% B; 40-45min, 75%-62% B; 45-50min, 62%-0% B; 50-55min, 0%-100% B); flow rate: 0.8mL / min; column temperature: 25℃; detection wavelength: 254nm, injection volume: 5μL.

[0065] 2.2 Preparation of qualitative reference substance 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 in 0.1 mol / L hydrochloric acid solution and dilute to the mark, shake well, and make a volume containing 152.88 g of glutamic acid per mL. The mother solution of qualitative reference substance was prepared by mixing 1 μg of 404 μg of amino acids, 1 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, 236.768 μg of valine, 177.576 μg of leucine, 243.824 μg of isoleucine, 190.904 μg of phenylalanine and 186.984 μg of lysine, which was used for identification of subsequent HPLC spectrum.

[0067] 2.3 Preparation of quantitative reference substance stock solution

[0068] Accurately weigh 17.70 mg of glycine and place it in a 25 mL volumetric flask. Accurately weigh 6.55 mg of alanine, 6.09 mg of proline, 5.55 mg of tyrosine, and 6.36 mg of lysine and place them in a 50 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve and dilute to the scale, shake well, and prepare quantitative reference substance mother 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 1 mL for use.

[0069] 2.4 Preparation of oyster formula granule test solution

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

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

[0072] 3 Study on fingerprint of oyster formula granules

[0073] 3.1 Methodological investigation of fingerprint of oyster formula granules

[0074] 3.1.1 Precision test

[0075] Take oyster formula granules (S2), prepare the test solution according to the method under "2.3", and inject 6 times continuously according to the chromatographic conditions under "2.1". Take proline as the reference peak, and calculate the RSD value of the relative retention time of each common peak to be 0.01%~0.20%, and the RSD value of the relative peak area to be 0.41%~1.03%, indicating that the instrument has good precision, see Table 2 and Table 3.

[0076] Table 2 Results of precision study on characteristic spectrum of oyster formula granules (relative retention time)

[0077] Peak Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 RSD(%) 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 investigation results of characteristic spectrum of oyster formula granules (relative peak area)

[0079] Peak Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 RSD(%) 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 test solution of oyster formula granules (S2) and inject samples at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions under "2.1". The relative retention time RSD values ​​of the common peaks of oyster formula granules are calculated to be 0.06% to 0.18%, and the relative peak area RSD values ​​are 0.73% to 2.98%, indicating that the test solution has good stability within 24 hours, see Table 4 and Table 5.

[0082] Table 4 Results of stability study on characteristic spectrum of oyster formula granules (relative retention time)

[0083] Peak 0h 2h 4h 8h 12h 24h RSD(%) 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 on characteristic spectrum of oyster formula granules (relative peak area)

[0085] Peak 0h 2h 4h 8h 12h 24h RSD(%) 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 the oyster formula granule sample (S2), prepare 6 test sample solutions in parallel according to the method under "2.3", and inject and measure according to the chromatographic conditions under "2.1". The RSD value of the relative retention time of each common peak is calculated to be 0.05% to 0.18%, and the RSD value of the relative peak area is 1.02% to 2.83%, indicating that the repeatability of the oyster formula granule analysis method is good, see Table 6 and Table 7.

[0088] Table 6 Results of repeatability study of characteristic spectra of oyster formula particles (relative retention time)

[0089] Serial number Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 RSD(%) 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 characteristic spectrum of oyster formula granules (relative peak area)

[0091] Serial number Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 RSD(%) 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 establishment and chemical pattern recognition

[0093] 3.2.1 Identification of common peaks in fingerprints

[0094] According to the method under "2.4", 10 batches of oyster formula granule samples were prepared into test solutions, and the samples were injected and measured according to the chromatographic conditions under "2.1". The chromatograms were recorded, and the AIA data were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The multi-point correction method and Mark peak matching method were used to generate superimposed spectra and reference spectra, and the Origin2021 software was used to draw relevant spectra. Figure 1. Fourteen common peaks were identified in the oyster formula granules. By comparing with the amino acid mixed reference substance, all 14 amino acid components were identified. Figure 2 The relative retention times and relative peak areas of the 10 batches of oyster formula granules are shown in Tables 8 and 9.

[0095] Table 8 Relative retention time of 10 batches of oyster formula particles

[0096]

[0097] Table 9 Relative peak areas of 10 batches of oyster formula particles

[0098]

[0099]

[0100] 3.2.2 Similarity evaluation

[0101] The similarities of 10 batches of oyster formula granules were calculated using the oyster formula granule control fingerprint (R) as the reference spectrum, and the results are shown in Table 10. The similarity results of the 10 batches of oyster formula granules were 0.978-0.999, indicating that the quality of oyster formula granules was stable and consistent, and it could better reflect the common characteristic components of oyster formula granules and could be used for quality control of oyster formula granules.

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

[0103] Serial number batch number Similarity 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. Investigation of the methodological approach for determining the content of multiple parameters in oyster formula granules

[0105] 4.1 Drawing of standard curve

[0106] Accurately pipette an appropriate amount of the mother solution of glycine, alanine, proline, tyrosine, and lysine reference substances into 5 25 mL volumetric flasks, and prepare 6 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 the solution in sequence according to the chromatographic conditions under "2.1" and record the chromatographic peak area. Use the peak area as the ordinate (y) and the reference substance concentration as the abscissa (x) to draw the standard curve of oyster formula granules. The results are shown in Table 11.

[0107] Table 11 Linearity test results of 5 amino acids in oyster formula granules

[0108] Element Regression equation <![CDATA[ 2 ]]> Linear range / (μg / mL) 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 "2.4", repeat the injection 6 times according to the chromatographic conditions under "2.1", record the peak area, and calculate the RSD values ​​of the peak areas of glycine, alanine, proline, tyrosine, and lysine in the oyster formula granules, which are 0.38%, 0.69%, 0.14%, 0.60%, and 0.81%, respectively, indicating that the instrument has good precision. The relevant results are shown in Table 12.

[0111] Table 12 Results of precision test on the content of oyster formula granules

[0112]

[0113] 4.3 Stability

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

[0115] Table 13 Results of stability study on content determination of oyster formula granules

[0116]

[0117]

[0118] 4.4 Repeatability

[0119] Weigh 6 samples of oyster formula granules (S2) accurately, prepare the test solution according to the method under "2.4", and determine according to the chromatographic conditions under "2.1". The results show that the average contents of glycine, alanine, proline, tyrosine, and lysine in oyster formula granules are 0.407 mg / g, 0.148 mg / g, 0.126 mg / g, 0.089 mg / g, and 0.064 mg / g, respectively, and the RSD values ​​are 1.52%, 1.32%, 1.59%, 3.64%, and 0.70%, respectively, indicating that the method has good repeatability. The results are shown in Table 14.

[0120] Table 14 Results of repeatability test of oyster formula granule content determination

[0121]

[0122] 4.5 Sample recovery rate

[0123] Accurately weigh about 0.50g of oyster formula granules (S2) with known content, place in a 25mL hydrolysis tube, process 6 portions in parallel, add glycine, alanine, proline, tyrosine, and lysine close to the known content of oyster formula granules respectively. Prepare the oyster formula granule test solution according to the method under "2.4", and determine according to the chromatographic conditions under "2.1". The results show that the sample recovery ranges of glycine, alanine, proline, tyrosine, and lysine in oyster formula granules are 91.39% to 98.16%, 93.90% to 101.49%, 95.59% to 101.12%, 95.15% to 104.45%, 102.18% to 109.67%, respectively, and the RSD values ​​are 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 the recovery rate of glycine in oyster formula granules (n=6)

[0125]

[0126] Table 16 Oyster formula granules alanine sample recovery rate investigation results table (n = 6)

[0127]

[0128] Table 17 Results of the recovery rate of proline in oyster formula granules (n=6)

[0129]

[0130] Table 18 Oyster formula granule tyrosine sample recovery rate investigation results (n = 6)

[0131]

[0132] Table 19 Lysine recovery rate of oyster formula granules (n=6)

[0133]

[0134] 4.6 Determination of content of oyster formula granules

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

[0136] Table 20 Results of content determination of 10 batches of oyster formula particles (mg / g)

[0137]

[0138] 5 Conclusion

[0139] The present invention uses pre-column derivatization-high performance liquid chromatography to conduct relevant research on the types and multi-component contents of amino acids contained in oyster formula granules, and successfully establishes a fingerprint of oyster formula granules, identifying a total of 14 amino acids, including 6 essential amino acids and 8 non-essential amino acids; the similarity of 10 batches of formula granules is 1.000, indicating that the quality of samples from different batches is stable and the consistency is strong, and the common pattern of the fingerprint spectrum better reflects the common characteristic components of oyster formula granules, which can be used for quality control research; in addition, the present invention also establishes a method for the content of five amino acids, namely glycine, alanine, proline, tyrosine, and lysine. The established method has good repeatability and high accuracy, which provides a basis for the quality control of oyster formula granules, and also provides a new idea for the quality control of other shell mineral formula granules.

[0140] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any technician familiar with the field can make many possible changes and modifications to the technical solution of the present invention by using the technical means and technical contents disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, all equivalent changes made according to the shape, structure and principle of the present invention without departing from the content of the technical solution of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint of oyster formula particles, characterized in that: The establishment method comprises the following steps: Step 1, preparation of qualitative reference substance mother solution; Step 2, preparation of oyster formula granule test solution; Step 3, using pre-column derivatization-high performance liquid chromatography for sample injection and determination, and establishing the fingerprint of the qualitative reference substance mother solution and the fingerprint of the test solution under the same chromatographic conditions; Step 4, obtaining the fingerprint of the oyster formula granules by comparing and analyzing the fingerprint of the qualitative reference substance mother solution and the fingerprint of the test solution.

2. The method for establishing the fingerprint of oyster formula granules according to claim 1, characterized in that: In step 3, the chromatographic conditions are: Chromatographic column: Shim-pack Scepter C18-120 chromatographic column, 4.6×150mm, 3μm; Mobile phase: 4:1 acetonitrile-water solution as mobile phase A, 7:93 acetonitrile-0.1 mol / L sodium acetate solution as mobile phase B, wherein the pH value of the sodium acetate solution was 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°C; Detection wavelength: 254nm; The injection volume was 5 μL.

3. The method for establishing the fingerprint of oyster formula granules according to claim 2, characterized in that: In step 1, the preparation method of the qualitative reference substance mother 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 into a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve each and dilute to the mark, shake well, and , and a mother solution of the qualitative reference substance was prepared, containing 152.88 μg of glutamic acid, 202.664 μg of serine, 219.520 μg of glycine, 140.728 μg of histidine, 198.744 μg of alanine, 187.768 μg of proline, 144.648 μg of threonine, 164.248 μg of arginine, 199.920 μg of tyrosine, 236.768 μg of valine, 177.576 μg of leucine, 243.824 μg of isoleucine, 190.904 μg of phenylalanine, and 186.984 μg of lysine per 1 mL.

4. The method for establishing the fingerprint of oyster formula granules according to claim 3, characterized in that: In the 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, weigh about 1.0g, accurately weigh it, put it in a 50mL centrifuge tube, add 10mL of dilute hydrochloric acid solution to fully dissolve it, centrifuge it at a speed of 12000 revolutions per minute for 15min, discard the supernatant layer, take the precipitate layer and put it in a 25mL stoppered hydrolysis tube, accurately add 10mL of 9mol / L hydrochloric acid, plug it tightly, hydrolyze it at 150℃ for 3h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrate, evaporate it to dryness, add 0.1mol / L hydrochloric acid to dissolve the residue, transfer it to a 10mL volumetric flask and dilute it to the scale, shake it well, and obtain solution A; Accurately measure 5 mL of the 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 leave at room temperature for 3 h. Then, add 50% acetonitrile to the scale, take 5 mL, add 10 mL of n-hexane, shake, and leave for 10 minutes. Take the lower layer of solution, filter through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the oyster formula granule test solution.

5. The method for establishing the fingerprint of oyster formula granules according to claim 4, characterized in that: In step 4, the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system is used to generate superimposed spectra and reference spectra using the multi-point correction method and the Mark peak matching method to identify the characteristic peaks of the components to be tested; Among them, the oyster formula granule fingerprint has a total of 14 common peaks, numbered from left to right, peak No. 1 L-glutamate, peak No. 2 L-serine, peak No. 3 glycine, peak No. 4 L-histidine, peak No. 5 L-threonine, peak No. 6 L-arginine, peak No. 7 alanine, peak No. 8 proline, peak No. 9 L-tyrosine, peak No. 10 L-valine, peak No. 11 L-isoleucine, peak No. 12 L-leucine, peak No. 13 L-phenylalanine and peak No. 14 L-lysine.

6. A method for determining the content of multiple components in oyster formula granules, characterized in that: The determination method comprises the following steps: Step A, preparation of a quantitative reference substance mother solution; Step B, preparation of mixed standard solution: using the quantitative reference substance mother solution to prepare a series of mixed standard solutions with concentration gradients; Step C, using pre-column derivatization-high performance liquid chromatography for sample injection determination, recording the chromatographic peak area under the same chromatographic conditions, performing linear analysis with the peak area as the ordinate and the reference substance concentration as the abscissa, establishing a standard curve equation, and drawing a standard curve for oyster formula granules; Step D, preparation of oyster formula granule test solution; Step E, using pre-column derivatization-high performance liquid chromatography for sample injection and determination, under the same chromatographic conditions, recording the peak area of ​​the component to be measured, and calculating the content of the component to be measured.

7. The method for determining the content of multiple components in oyster formula granules according to claim 6, characterized in that: In step C and step E, the chromatographic conditions are: Chromatographic column: Shim-pack Scepter C18-120 chromatographic column, 4.6×150mm, 3μm; Mobile phase: 4:1 acetonitrile-water solution as mobile phase A, 7:93 acetonitrile-0.1 mol / L sodium acetate solution as mobile phase B, wherein the pH value of the sodium acetate solution was 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°C; Detection wavelength: 254nm; The injection volume was 5 μL.

8. The method for determining the content of multiple components in oyster formula granules according to claim 7, characterized in that: In step A, the preparation method of the quantitative reference substance mother solution is: Accurately weigh 17.70 mg of glycine and place it in a 25 mL volumetric flask. Accurately weigh 6.55 mg of alanine, 6.09 mg of proline, 5.55 mg of tyrosine, and 6.36 mg of lysine and place them in a 50 mL volumetric flask, add 0.1 mol / L hydrochloric acid solution to dissolve and dilute to the scale, shake well, and prepare the quantitative reference substance mother solution 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 1 mL.

9. The method for determining the content of multiple components in oyster formula granules according to claim 8, characterized in that: In step C, the linearity test results of each reference substance are as follows:

10. The method for determining the content of multiple components in oyster formula granules according to claim 9, characterized in that: In the 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, weigh about 1.0g, accurately weigh it, put it in a 50mL centrifuge tube, add 10mL of dilute hydrochloric acid solution to fully dissolve it, centrifuge it at a speed of 12000 revolutions per minute for 15min, discard the supernatant layer, take the precipitate layer and put it in a 25mL stoppered hydrolysis tube, accurately add 10mL of 9mol / L hydrochloric acid, plug it tightly, hydrolyze it at 150℃ for 3h, take it out, cool it, filter it, and wash the hydrolysis tube and filter paper with a small amount of water several times, combine the filtrate, evaporate it to dryness, add 0.1mol / L hydrochloric acid to dissolve the residue, transfer it to a 10mL volumetric flask and dilute it to the scale, shake it well, and obtain solution A; Accurately measure 5 mL of the 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 leave at room temperature for 3 h. Then, add 50% acetonitrile to the scale, take 5 mL, add 10 mL of n-hexane, shake, and leave for 10 minutes. Take the lower layer of solution, filter through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the oyster formula granule test solution.

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