Characteristic chromatogram detection method of ginseng donkey-hide gelatin
High-performance liquid chromatography (HPLC) was used to detect multiple medicinal components in ginseng, deer antler and donkey-hide gelatin, which solved the problem of lack of specificity in existing quality standards, realized the controllability and safety of product quality, and enhanced market competitiveness.
Patent Information
- Application Number
- CN202511129342.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-13
- Publication Date
- 2025-11-14
AI Technical Summary
The existing quality standards for ginseng, deer antler and donkey-hide gelatin lack specific methods for identifying authenticity and for determining the content of active ingredients, resulting in large fluctuations in product quality and difficulty in assessing process stability, which poses risks to market supervision and corporate reputation.
High-performance liquid chromatography (HPLC) was used to detect the characteristic chromatograms of various medicinal components in ginseng, deer antler and donkey-hide gelatin by preparing reference and test solutions and combining specific chromatographic conditions and elution procedures. The content of components such as glycyrrhizin, glycyrrhizic acid ammonium and verbascoside was determined.
It enables the overall characterization and evaluation of the quality of ginseng, deer antler and donkey-hide gelatin, ensuring that the product quality is controllable, safe and effective, and is simple, fast, reproducible and low cost.
Smart Images
Figure CN120948652A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for detecting the characteristic chromatograms of ginseng, deer antler and donkey-hide gelatin. Background Technology
[0002] Ginseng and Deer Antler Collagen is composed of 23 herbs, including donkey skin, angelica, chuanxiong, prepared rehmannia root, white peony root, atractylodes macrocephala, poria cocos, prepared pinellia tuber, cinnamon, safflower, costus root, ginseng, deer antler, amomum villosum, polygonatum odoratum, angelica dahurica, tangerine peel, cyperus rotundus, rehmannia root, ophiopogon japonicus, codonopsis pilosula, licorice root, and peony bark. It has the effect of nourishing blood and replenishing essence, and is used for dizziness due to blood deficiency, fatigue, and irregular menstruation.
[0003] Currently, the existing quality standards for ginseng, deer antler and donkey-hide gelatin only include basic inspection items such as appearance, moisture, ash, heavy metals and microbial limits. They lack specific means of distinguishing genuine products from counterfeit products, making it impossible to effectively differentiate between genuine and counterfeit products or monitor the consistency of the main medicinal materials used. They also lack the determination of the content of active ingredients, resulting in large quality fluctuations between product batches and difficulty in assessing process stability, which poses risks to product quality, market supervision and corporate reputation.
[0004] To enhance the scientific rigor, controllability, and market competitiveness of ginseng, deer antler, and donkey-hide gelatin quality standards, it is urgently necessary to construct their exclusive characteristic chromatograms to achieve highly specific identification of authenticity and batch-to-batch monitoring of chemical composition consistency. Simultaneously, it is crucial to establish exclusive, sensitive, and accurate content determination methods for effective components. By setting reasonable content limits, quantitative control of effective substances can be achieved, ensuring stable, safe, and effective product efficacy, ultimately forming a more comprehensive, scientific, and controllable quality standard system. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin.
[0006] The specific technical solution adopted in this invention is as follows: A method for detecting the characteristic chromatograms of ginseng, deer antler, and donkey-hide gelatin includes the following steps: (1) Preparation of the reference solution: Preparation of reference solutions: Glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costus lactone reference standards were dissolved in methanol to prepare reference solutions. Preparation of reference herbal solutions: Take the reference herbs Angelica sinensis, Ligusticum chuanxiong, Rehmannia glutinosa, Paeonia lactiflora, Aucklandia lappa, Panax ginseng, deer antler, Citrus reticulata peel, Cyperus rotundus, Rehmannia glutinosa, Codonopsis pilosula, Glycyrrhiza uralensis, and Paeonia suffruticosa, add 50 ml of water to each, heat under reflux, cool, filter, measure the filtrate, place it in a stoppered conical flask, add organic solvent to extract, filter, measure 50 ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the solution.
[0007] (2) Preparation of test solution: Take ginseng, deer antler and donkey-hide gelatin, add organic solvent to extract, and prepare test solution.
[0008] (3) High-performance liquid chromatography (HPLC) determination: Accurately pipette 10-30 μl of the reference solution and the test solution into the HPLC instrument and determine the characteristic chromatogram with characteristic peaks. The chromatographic conditions are as follows: Column: Packed with octadecylsilane-bonded silica gel; Flow rate: 0.8–1.2 ml per minute; Column temperature: 25~35℃; Detection wavelength: 203–254 nm; The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 2000. Mobile phase: 0.1-0.5% (v / v) formic acid acetonitrile as mobile phase A, and a mixed aqueous solution of 0.1-0.5% (v / v) formic acid and 5-10 mM ammonium formate as mobile phase B; The elution procedure is as follows: From 0 to 30 min, mobile phase A increased from 3 to 8% (v / v) to 12 to 18% (v / v); Over 30–50 minutes, mobile phase A increased from 12–18% (v / v) to 28–32% (v / v); Over 50–70 minutes, mobile phase A increased from 28–32% (v / v) to 58–62% (v / v).
[0009] In a preferred embodiment, in step (1), the amount of the test sample used is 5 to 15 g.
[0010] In a preferred embodiment, in step (2), the concentration of the reference solution of glycyrrhizin, glycyrrhizic acid ammonium, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunol is as follows: per 1 ml, glycyrrhizin 10-20 μg, glycyrrhizic acid ammonium 0.1-0.2 mg, verbascoside 10-20 μg, paeoniflorin 40-60 μg, ferulic acid 30-50 μg, catalpol 30-50 μg, ginsenoside Rg1 0.1-0.2 mg, hesperidin 0.2-0.4 mg, paeonol 0.1-0.2 mg, and costunolol 0.1-0.2 mg.
[0011] In a preferred embodiment, in steps (1) and (2), the organic solvent is acetonitrile-ethyl acetate (8:2, v / v) to acetonitrile-ethyl acetate (6:4, v / v).
[0012] In a preferred embodiment, in steps (1) and (2), the amount of organic solvent used is 100-200 ml.
[0013] In a preferred embodiment, in steps (1) and (2), the extraction method is either shaking or ultrasound.
[0014] In a preferred embodiment, the extraction time in steps (1) and (2) is 30 to 60 minutes.
[0015] In a preferred embodiment, the chromatographic conditions in step (3) are as follows: Column temperature: 30℃; Flow rate: 1.0 ml / min; Detection wavelength: 230nm; The theoretical plate number, calculated based on glycyrrhizin, is not less than 2000; Mobile phase: 0.1% (v / v) formic acid acetonitrile as mobile phase A, and a mixed aqueous solution of 0.1% (v / v) formic acid and 5 mM ammonium formate as mobile phase B.
[0016] The gradient elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 15% (v / v); Over 30–50 minutes, mobile phase A increased from 15% (v / v) to 30% (v / v); Over 50–70 minutes, mobile phase A increased from 30% (v / v) to 60% (v / v).
[0017] The beneficial effects achieved by one or more embodiments of the present invention are as follows: This method can effectively separate various chromatographic peaks in ginseng, deer antler, and donkey-hide gelatin, obtaining comprehensive characteristic spectra. It can identify 13 medicinal materials, including Angelica sinensis, Ligusticum chuanxiong, Rehmannia glutinosa, Paeonia lactiflora, Aucklandia lappa, ginseng, deer antler, Citrus reticulata peel, Cyperus rotundus, Rehmannia glutinosa, Codonopsis pilosula, Glycyrrhiza uralensis, and Paeonia suffruticosa. It can also simultaneously determine the content of major components in ginseng, deer antler, and donkey-hide gelatin, such as glycyrrhizin, glycyrrhizic acid ammonium, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costus lactone. Therefore, this method can achieve comprehensive characterization and evaluation of the quality of ginseng, deer antler, and donkey-hide gelatin, ensuring controllable, safe, and effective product quality. Furthermore, this method is simple, rapid, reproducible, information-rich, and low-cost, providing reliable technical support for the effective control of ginseng, deer antler, and donkey-hide gelatin quality. Attached Figure Description
[0018] Figure 1 The characteristic spectrum of Experiment Example 3; Figure 2 The characteristic spectrum of Experiment Example 1; Figure 3This is the characteristic spectrum of Experiment Example 2. Detailed Implementation
[0019] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0020] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention.
[0021] Experimental Example 1 Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A and 0.1% (v / v) formic acid aqueous solution was used as mobile phase B for elution; the flow rate was 1.0 ml per minute; the column temperature was 30℃; the detection wavelength was 254 nm; the theoretical plate number calculated based on the glycyrrhizin peak should not be less than 2000.
[0022] The specific elution procedure is as follows: From 0 to 20 minutes, mobile phase A increased from 5% (v / v) to 35% (v / v); Over 20–35 minutes, mobile phase A increased from 35% (v / v) to 55% (v / v); Over 35–40 minutes, mobile phase A increased from 55% (v / v) to 70% (v / v); Over 40–55 minutes, mobile phase A increased from 70% (v / v) to 95% (v / v).
[0023] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0024] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, measure 1ml of the filtrate, place it in a stoppered conical flask, add 100ml of 70% acetonitrile, sonicate for 30 minutes, filter, measure 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the final solution.
[0025] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 5g, place it in a stoppered conical flask, accurately add 100ml of 70% acetonitrile, seal tightly, weigh, sonicate for 30 minutes, cool, weigh again, replenish the lost weight with 70% acetonitrile, shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and collect the filtrate to obtain the product.
[0026] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0027] Experimental results: The number of chromatographic peaks in the test sample was relatively small; the chromatographic peaks of the reference sample had poor resolution and poor peak shape.
[0028] Experiment Example 2 Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; elution was performed using 0.1% (v / v) formic acid acetonitrile as mobile phase A and a mixed aqueous solution of 0.1% (v / v) formic acid and 5 mM ammonium formate as mobile phase B; the flow rate was 1.0 ml per minute; the column temperature was 30℃; the detection wavelength was 203 nm; the theoretical plate number calculated based on the glycyrrhizin peak should not be less than 2000.
[0029] The specific elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 20% (v / v); Over 30–50 minutes, mobile phase A increased from 20% (v / v) to 30% (v / v); Over 50–70 minutes, mobile phase A increased from 30% (v / v) to 60% (v / v); For 70–75 min, mobile phase A is maintained at 60% (v / v).
[0030] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0031] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, measure 1ml of the filtrate, place it in a stoppered conical flask, add 100ml of acetonitrile-ethyl acetate (7:3, v / v), sonicate for 30 minutes, filter, measure 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the solution.
[0032] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 10g, accurately weigh it, place it in a stoppered conical flask, accurately add 100ml of acetonitrile-ethyl acetate (7:3, v / v), stopper tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, make up the lost weight with acetonitrile-ethyl acetate (7:3, v / v), shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0033] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0034] Experimental results: The reference standard chromatographic peaks could be separated from adjacent peaks, and the peak shapes were improved; however, the number of chromatographic peaks of the test sample was small, and the separation of the remaining peaks was poor.
[0035] Experimental Example 3 Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; elution was performed using 0.1% (v / v) formic acid acetonitrile as mobile phase A and a mixed aqueous solution of 0.1% (v / v) formic acid and 5 mM ammonium formate as mobile phase B; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 230 nm; the theoretical plate number, calculated based on the glycyrrhizin peak, should not be less than 2000; the specific elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 15% (v / v); Over 30–50 minutes, mobile phase A increased from 15% (v / v) to 30% (v / v); Over 50–70 minutes, mobile phase A increased from 30% (v / v) to 60% (v / v).
[0036] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0037] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, measure 1ml of the filtrate, place it in a stoppered conical flask, add 100ml of acetonitrile-ethyl acetate (7:3, v / v), sonicate for 30 minutes, filter, measure 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the solution.
[0038] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 10g, accurately weigh it, place it in a stoppered conical flask, accurately add 100ml of acetonitrile-ethyl acetate (7:3, v / v), stopper tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, make up the lost weight with acetonitrile-ethyl acetate (7:3, v / v), shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0039] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0040] Experimental results: The reference standard chromatographic peak was well separated from adjacent peaks and the peak shape was symmetrical; all other chromatographic peaks were well separated; the number of chromatographic peaks in the test sample was moderate.
[0041] exist Figure 1In the HPLC characteristic chromatogram shown, ginseng, deer antler and donkey-hide gelatin have 19 characteristic peaks at a wavelength of 230 nm. Peaks 1 and 19 are characteristic components of costus root; peaks 2, 6, 10, 17 and 18 are characteristic components of licorice; peaks 3 and 11 are characteristic components of raw and processed rehmannia root; peak 4 is a characteristic component of cyperus rhizome; peaks 5 and 7 are characteristic components of tangerine peel; peaks 8 and 13 are characteristic components of codonopsis root; and peak 9 is a characteristic component of white peony root. The components are as follows: peak 12 is a characteristic component of deer antler, peak 14 is a characteristic component of angelica and chuanxiong, peak 15 is a characteristic component of ginseng, and peak 16 is a characteristic component of peony bark; peak 3 is catalpol, peak 7 is hesperidin, peak 9 is paeoniflorin, peak 10 is glycyrrhizin, peak 11 is verbascoside, peak 14 is ferulic acid, peak 15 is ginsenoside Rg1, peak 16 is paeonol, peak 18 is ammonium glycyrrhizate, and peak 19 is costus lactone. Peak 10, glycyrrhizin, was used as a reference peak. The relative retention times of the 19 peaks were: 0.132, 0.154, 0.187, 0.213, 0.265, 0.521, 0.694, 0.825, 0.858, 1.000, 1.024, 1.057, 1.079, 1.120, 1.194, 1.215, 1.282, 1.484, and 1.525.
[0042] Experiment Example 4 1. Precision Experiment Take one batch of ginseng and donkey-hide gelatin, prepare one sample of test sample according to the test sample solution preparation method in Experiment Example 3, and inject the sample five times consecutively according to the chromatographic conditions in Experiment Example 3. The results are shown in the table below.
[0043] 2. Repeatability experiment Take one batch of ginseng and donkey-hide gelatin, and prepare 6 test samples according to the test sample solution preparation method in Experiment Example 3. Inject each sample once according to the chromatographic conditions in Experiment Example 3. The results are shown in the table below.
[0044] 3. Stability test Take one batch of ginseng and donkey-hide gelatin, prepare one sample of test sample according to the test sample solution preparation method in Experiment Example 3, and inject the sample at 0h, 4h, 8h, 12h and 24h respectively according to the chromatographic conditions in Experiment Example 3. The results are shown in the table below.
[0045] Example 1 This embodiment illustrates a method for detecting the characteristic spectrum of ginseng, deer antler and donkey-hide gelatin.
[0046] Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; elution was performed using 0.1% (v / v) formic acid acetonitrile as mobile phase A and a mixed aqueous solution of 0.5% (v / v) formic acid and 10 mM ammonium formate as mobile phase B; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 230 nm; the theoretical plate number, calculated based on the glycyrrhizin peak, should not be less than 2000; the specific elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 15% (v / v); Over 30–50 minutes, mobile phase A increased from 15% (v / v) to 30% (v / v); Over 50–70 minutes, mobile phase A increased from 30% (v / v) to 60% (v / v).
[0047] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0048] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, measure 1ml of the filtrate, place it in a stoppered conical flask, add 100ml of acetonitrile-ethyl acetate (7:3, v / v), sonicate for 30 minutes, filter, measure 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the solution.
[0049] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 10g, accurately weigh it, place it in a stoppered conical flask, accurately add 100ml of acetonitrile-ethyl acetate (7:3, v / v), stopper tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, make up the lost weight with acetonitrile-ethyl acetate (7:3, v / v), shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0050] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0051] Experimental results: The reference standard chromatographic peak was well separated from adjacent peaks and the peak shape was symmetrical; all other chromatographic peaks were well separated; the number of chromatographic peaks in the test sample was moderate.
[0052] Example 2 This embodiment illustrates a method for detecting the characteristic spectrum of ginseng, deer antler and donkey-hide gelatin.
[0053] Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; elution was performed using 0.2% (v / v) formic acid acetonitrile as mobile phase A and a mixed aqueous solution of 0.1% (v / v) formic acid and 5 mM ammonium formate as mobile phase B; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 230 nm; the theoretical plate number, calculated based on the glycyrrhizin peak, should not be less than 2000; the specific elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 18% (v / v); Over 30–50 minutes, mobile phase A increased from 18% (v / v) to 30% (v / v); Over 50–70 minutes, mobile phase A increased from 30% (v / v) to 62% (v / v).
[0054] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0055] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, and take 1ml of the filtrate. Place it in a stoppered conical flask, add 100ml of acetonitrile-ethyl acetate (8:2, v / v), sonicate for 30 minutes, filter, take 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the solution.
[0056] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 10g, place it in a stoppered conical flask, accurately add 100ml of acetonitrile-ethyl acetate (8:2, v / v), seal tightly, weigh, sonicate for 30 minutes, cool, weigh again, make up the lost weight with acetonitrile-ethyl acetate (8:2, v / v), shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0057] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0058] Experimental results: The reference standard chromatographic peak was well separated from adjacent peaks and the peak shape was symmetrical; all other chromatographic peaks were well separated; the number of chromatographic peaks in the test sample was moderate.
[0059] Example 3 This embodiment illustrates a method for detecting the characteristic spectrum of ginseng, deer antler and donkey-hide gelatin.
[0060] Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; elution was performed using 0.2% (v / v) formic acid acetonitrile as mobile phase A and a mixed aqueous solution of 0.1% (v / v) formic acid and 5 mM ammonium formate as mobile phase B; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 230 nm; the theoretical plate number, calculated based on the glycyrrhizin peak, should not be less than 2000; the specific elution procedure is as follows: From 0 to 30 min, mobile phase A increased from 5% (v / v) to 12% (v / v); Over 30–50 minutes, mobile phase A increased from 12% (v / v) to 28% (v / v); Over 50–70 minutes, mobile phase A increased from 28% (v / v) to 60% (v / v).
[0061] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0062] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, and take 1ml of the filtrate. Place it in a stoppered conical flask, add 100ml of acetonitrile-ethyl acetate (6:4, v / v), sonicate for 30 minutes, filter, take 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the final solution.
[0063] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 10g, place it in a stoppered conical flask, accurately add 100ml of acetonitrile-ethyl acetate (6:4, v / v), seal tightly, weigh, sonicate for 30 minutes, cool, weigh again, make up the lost weight with acetonitrile-ethyl acetate (6:4, v / v), shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0064] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0065] Experimental results: The reference standard chromatographic peak was well separated from adjacent peaks and the peak shape was symmetrical; all other chromatographic peaks were well separated; the number of chromatographic peaks in the test sample was moderate.
[0066] Example 4 This embodiment illustrates a method for detecting the characteristic spectrum of ginseng, deer antler and donkey-hide gelatin.
[0067] Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; elution was performed using 0.1% (v / v) formic acid acetonitrile as mobile phase A and a mixed aqueous solution of 0.2% (v / v) formic acid and 5 mM ammonium formate as mobile phase B; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 230 nm; the theoretical plate number, calculated based on the glycyrrhizin peak, should not be less than 2000; the specific elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 15% (v / v); Over 30–50 minutes, mobile phase A increased from 15% (v / v) to 28% (v / v); Over 50–70 minutes, mobile phase A increased from 28% (v / v) to 62% (v / v).
[0068] Preparation of the reference solution: Preparation of reference solutions: Accurately weigh the reference standards of glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costunolide, and add methanol to prepare solutions containing 20 μg glycyrrhizin, 0.2 mg ammonium glycyrrhizate, 20 μg verbascoside, 60 μg paeoniflorin, 50 μg ferulic acid, 50 μg catalpol, 0.2 mg ginsenoside Rg1, 0.4 mg hesperidin, 0.2 mg paeonol, and 0.2 mg costunolide per ml.
[0069] Preparation of reference herbal solutions: Take 6g of Angelica sinensis, 4g of Ligusticum chuanxiong, 1g of Rehmannia glutinosa, 2g of Paeonia lactiflora, 1g of Aucklandia lappa, 0.5g of Panax ginseng, 0.5g of deer antler, 2g of Citrus reticulata peel, 5g of Cyperus rotundus, 2g of Rehmannia glutinosa, 3g of Codonopsis pilosula, 12g of Glycyrrhiza uralensis, and 2g of Paeonia suffruticosa. Add 50ml of water to each, heat under reflux for 1 hour, cool, filter, measure 1ml of the filtrate, place it in a stoppered conical flask, add 100ml of acetonitrile-ethyl acetate (7:3, v / v), sonicate for 30 minutes, filter, measure 50ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in methanol and transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the solution.
[0070] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 10g, accurately weigh it, place it in a stoppered conical flask, accurately add 100ml of acetonitrile-ethyl acetate (7:3, v / v), stopper tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, make up the lost weight with acetonitrile-ethyl acetate (7:3, v / v), shake well, filter, accurately measure 50ml of the filtrate, evaporate to dryness, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0071] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0072] Experimental results: The reference standard chromatographic peak was well separated from adjacent peaks and the peak shape was symmetrical; all other chromatographic peaks were well separated; the number of chromatographic peaks in the test sample was moderate.
[0073] The contents (μg / g) of some components in a batch of ginseng and donkey-hide gelatin (batch number: 2204028) were determined by Examples 1-4, and the results are shown in the table below.
[0074] The content of some components in three batches of ginseng, deer antler and donkey-hide gelatin (batch numbers: 2205013, 2204037, 2204032) was determined by Example 1, and the results are shown in the table below.
[0075] Experimental results show that this method can determine the content of major components in ginseng, deer antler and donkey-hide gelatin, including glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol and costus lactone.
Claims
1. A method for detecting the characteristic chromatograms of ginseng, deer antler, and donkey-hide gelatin, characterized in that, Includes the following steps: (1) Preparation of the reference solution: Preparation of reference solutions: Glycyrrhizin, ammonium glycyrrhizate, verbascoside, paeoniflorin, ferulic acid, catalpol, ginsenoside Rg1, hesperidin, paeonol, and costus lactone reference standards were dissolved in methanol to prepare reference solutions. (2) Preparation of test solution: Take ginseng, deer antler and donkey-hide gelatin, add organic solvent to extract, and prepare test solution; (3) High performance liquid chromatography determination: The reference solution and the test solution are precisely pipetted into the high performance liquid chromatograph and measured to obtain characteristic chromatograms with characteristic peaks.
2. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 1, characterized in that, The conditions for high performance liquid chromatography are: Column: Packed with octadecylsilane-bonded silica gel; Flow rate: 0.8–1.2 ml per minute; Column temperature: 25~35℃; Detection wavelength: 203–254 nm; The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 2000. Mobile phase: 0.1-0.5% (v / v) formic acid acetonitrile as mobile phase A, and a mixed aqueous solution of 0.1-0.5% (v / v) formic acid and 5-10 mM ammonium formate as mobile phase B; The elution procedure is as follows: From 0 to 30 min, mobile phase A increased from 3 to 8% (v / v) to 12 to 18% (v / v); Over 30–50 minutes, mobile phase A increased from 12–18% (v / v) to 28–32% (v / v); Over 50–70 minutes, mobile phase A increased from 28–32% (v / v) to 58–62% (v / v).
3. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 2, characterized in that, The conditions for high performance liquid chromatography are: In step (3), the chromatographic conditions are as follows: Column temperature: 30℃; Flow rate: 1.0 ml / min; Detection wavelength: 230nm; The theoretical plate number, calculated based on glycyrrhizin, is not less than 2000; Mobile phase: 0.1% (v / v) formic acid acetonitrile as mobile phase A, and a mixed aqueous solution of 0.1% (v / v) formic acid and 5 mM ammonium formate as mobile phase B; The gradient elution procedure is as follows: From 0 to 30 minutes, mobile phase A increased from 5% (v / v) to 15% (v / v); Over 30–50 minutes, mobile phase A increased from 15% (v / v) to 30% (v / v); Over 50–70 minutes, mobile phase A increased from 30% (v / v) to 60% (v / v).
4. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 1, characterized in that, In step (1), the amount of the test sample used is 5 to 15 g.
5. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 1, characterized in that, In step (2), the concentration of the reference solution is as follows: per 1 ml, glycyrrhizin 10-20 μg, glycyrrhizic acid ammonium 0.1-0.2 mg, verbascoside 10-20 μg, paeoniflorin 40-60 μg, ferulic acid 30-50 μg, catalpol 30-50 μg, ginsenoside Rg1 0.1-0.2 mg, hesperidin 0.2-0.4 mg, paeonol 0.1-0.2 mg, and costone lactone 0.1-0.2 mg.
6. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 1, characterized in that, In steps (1) and (2), the organic solvent is acetonitrile-ethyl acetate in a volume ratio of 8:2 to 6:
4.
7. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 6, characterized in that, In steps (1) and (2), the organic solvent is acetonitrile-ethyl acetate in a volume ratio of 7:
3.
8. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 7, characterized in that, In steps (1) and (2), the amount of organic solvent used is 100-200 ml.
9. The method for detecting the characteristic spectrum of ginseng, deer antler, and donkey-hide gelatin according to claim 1, characterized in that, In steps (1) and (2), the extraction time is 30 to 60 minutes.