Discrimination method of processed product of Galli Recurvi Shell and Galli Recurvi Shell

Abstract

The application relates to a method for identifying processed products of Radix Scrophulariae and raw products of Radix Scrophulariae, which comprises the following steps: preparing a test sample solution of a to-be-tested sample, wherein the to-be-tested sample is a processed product of Radix Scrophulariae or a raw product of Radix Scrophulariae; the processed product of Radix Scrophulariae is fried Radix Scrophulariae decoction pieces, fried Radix Scrophulariae standard decoction or fried Radix Scrophulariae formula granules; the raw product of Radix Scrophulariae is Radix Scrophulariae medicinal materials, Radix Scrophulariae standard decoction or Radix Scrophulariae formula granules; and the test sample solution is detected by using high performance liquid chromatography-mass spectrometry. The above method uses a high performance liquid chromatography-mass spectrometer to analyze and detect the to-be-tested sample, has strong specificity and high sensitivity, can efficiently and accurately identify and distinguish the processed product of Radix Scrophulariae or the raw product of Radix Scrophulariae, can provide a strong basis for quality control and evaluation of the processed product of Radix Scrophulariae, and solves the problem that it is difficult to identify the processed product of Radix Scrophulariae and the raw product of Radix Scrophulariae due to the loss of appearance characteristics after processing.

Description

Technical Field

[0001] This application relates to the field of identification techniques for Chinese medicinal materials, and in particular to a method for differentiating processed chicken gizzard lining from raw chicken gizzard lining. Background Technology

[0002] Chicken gizzard lining is the dried inner wall of the gizzard of the domestic chicken (Gallus gallus domesticus Brisson). It has a sweet taste and neutral properties, and enters the spleen, stomach, small intestine, and bladder meridians. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is listed as a superior-grade herb. Its main components are gastric hormones, pepsin, amino acids, and polysaccharides. It has the functions of promoting digestion, strengthening the stomach, astringing essence, stopping seminal emission, and clearing urinary tract stones. Clinically, it is often used to treat symptoms such as indigestion, vomiting, diarrhea, and infantile malnutrition. Chicken gizzard lining can be produced throughout China, and as a traditional Chinese medicine used for both food and medicine, its development and utilization value is very high.

[0003] Currently, chicken gizzard lining is mostly used in clinical medicine as a processed product, with processing methods including stir-frying, sand-scalding, and vinegar-processing. The 2020 edition of the *Chinese Pharmacopoeia* includes 22 compound preparations containing chicken gizzard lining, of which 14 use stir-fried chicken gizzard lining, 7 use raw chicken gizzard lining, and 1 uses vinegar-processed chicken gizzard lining. The processing method for stir-fried chicken gizzard lining is as follows: place clean river sand in a stir-frying container, heat over high heat until it becomes slippery, add the cleaned chicken gizzard lining, stir constantly, and stir-fry until the surface puffs up, turns dark yellowish-brown or charred yellow, and appears granular or with fine bubbles. Remove, sift out the river sand, and let cool. After stir-frying, the properties and composition of chicken gizzard lining change. Although traditional experience can distinguish chicken gizzard lining from stir-fried chicken gizzard lining slices, after being prepared into standard Chinese medicine decoctions, especially the new dosage form of Chinese medicine formula granules needed clinically, which lose the properties of raw medicinal materials, experience cannot effectively distinguish them.

[0004] Currently, most research on processed chicken gizzard lining products focuses on processing techniques, changes in chemical composition before and after processing, pharmacological mechanisms of action, and quality standards. Identification between processed and raw products remains a challenge, and there are no literature or patent reports on this topic. Summary of the Invention

[0005] Therefore, it is necessary to provide a method for distinguishing between processed and raw chicken gizzard products.

[0006] One embodiment of this application provides a method for distinguishing between processed chicken gizzard lining and raw chicken gizzard lining, comprising the following steps:

[0007] Prepare a test solution for the sample to be tested, wherein the sample to be tested is a processed product of chicken gizzard or a raw product of chicken gizzard; the processed product of chicken gizzard is stir-fried chicken gizzard slices, stir-fried chicken gizzard standard decoction, or stir-fried chicken gizzard formula granules; the raw product of chicken gizzard is chicken gizzard medicinal material, chicken gizzard standard decoction, or chicken gizzard formula granules.

[0008] The test solution was detected using high performance liquid chromatography-mass spectrometry.

[0009] In one embodiment, the high-performance liquid chromatography-mass spectrometry (HPLC-MS) conditions of the HPLC system meet one or more of the following conditions:

[0010] (1) The chromatographic column is a C18 column;

[0011] (2) The elution method is gradient elution, wherein mobile phase A is acetonitrile and mobile phase B is a formic acid aqueous solution with a volume fraction of 0.2% to 0.4%;

[0012] (3) The flow rate is 0.2 mL to 0.4 mL per minute;

[0013] (4) The column temperature is 28℃~32℃; and

[0014] (5) The injection volume is 1 μl to 3 μl.

[0015] In one embodiment, the gradient elution process includes:

[0016] From 0 to 5 minutes, the volume percentage of mobile phase A increases from 20% to 30%, and the volume percentage of mobile phase B decreases from 80% to 70%.

[0017] Within 5 to 10 minutes, the volume percentage of mobile phase A increased from 30% to 80%, while the volume percentage of mobile phase B decreased from 70% to 20%.

[0018] In one embodiment, the mass spectrometry conditions of the high-performance liquid chromatography-mass spectrometry system meet one or more of the following conditions:

[0019] (1) Electrospray positive ion mode is adopted;

[0020] (2) Multiple reaction monitoring scan;

[0021] (3) The capillary voltage is 1kV to 3kV;

[0022] (4) The ion source temperature is 300℃~400℃;

[0023] (5) The flow rate of the solvent gas is 800 L / Hr to 1000 L / Hr;

[0024] (6) The air curtain airflow rate is 10L / Hr~30L / Hr;

[0025] (7) Tapered hole voltage 30V~50V; and

[0026] (8) Collision energy 20V~40V.

[0027] In one embodiment, the method for preparing the test solution of the sample to be tested includes the following steps:

[0028] Weigh the sample to be tested, add water, perform ultrasonic extraction, filter, and collect the filtrate.

[0029] In one embodiment, identification is performed based on the characteristic ion pairs in the mass spectrometry detection results and the signal-to-noise ratio of the characteristic ion pairs;

[0030] If the characteristic ion pair includes a first characteristic ion pair and a second characteristic ion pair, and the signal-to-noise ratio of the first characteristic ion pair and the second characteristic ion pair is greater than 3:1, then the sample to be tested is a processed chicken gizzard lining product; otherwise, it is a raw chicken gizzard lining product.

[0031] The first characteristic ion pair has a parent ion mass-to-charge ratio of 706.41 and a daughter ion mass-to-charge ratio of 369.3, while the second characteristic ion pair has a parent ion mass-to-charge ratio of 706.41 and a daughter ion mass-to-charge ratio of 300.2.

[0032] In one embodiment, the identification method further includes the following steps:

[0033] Preparation of a control solution for processed chicken gizzard lining products;

[0034] Thin-layer chromatography was used to analyze the test solution and the reference solution of the processed chicken gizzard.

[0035] In one embodiment, the chromatographic conditions for thin-layer chromatography satisfy one or more of the following conditions:

[0036] (1) The thin-layer chromatography plate is a silica gel G thin-layer plate;

[0037] (2) The developing solvent is a mixed solution of toluene, ethyl acetate and glacial acetic acid;

[0038] (3) Inspection method: under ultraviolet light with a wavelength of 360nm~370nm;

[0039] (4) The sample volume is 3 μl to 15 μl.

[0040] In one embodiment, the volume ratio of toluene, ethyl acetate and glacial acetic acid in the developing solvent is (14-18):(3-7):(0.3-0.7).

[0041] In one embodiment, the method for preparing the chicken gizzard lining preparation control solution includes:

[0042] Weigh out the reference material of chicken gizzard lining, add water, perform ultrasonic extraction, filter, and collect the filtrate.

[0043] In one embodiment, the reference material for the processed chicken gizzard is stir-fried chicken gizzard reference slices.

[0044] The above method utilizes high performance liquid chromatography-mass spectrometry to analyze and detect the sample. It has high specificity and sensitivity, and can efficiently and accurately identify and distinguish between processed chicken gizzard products and raw chicken gizzard products. It can provide a strong basis for the quality control and evaluation of processed chicken gizzard products, and solve the problem of difficulty in identifying processed chicken gizzard products and raw chicken gizzard products due to the loss of appearance characteristics after processing. Attached Figure Description

[0045] Figure 1 TIC chromatograms of mass spectrometry analysis of stir-fried chicken gizzard slices and chicken gizzard medicinal material in Example 1;

[0046] Figure 2 The TIC chromatograms of the standard decoction for stir-fried chicken gizzard lining and the standard decoction for chicken gizzard lining in Example 1 are obtained by mass spectrometry detection.

[0047] Figure 3 TIC chromatograms of the stir-fried chicken gizzard granules and chicken gizzard granules from Example 1, obtained by mass spectrometry detection.

[0048] Figure 4 The following are thin-layer chromatograms of 6 batches of chicken gizzard lining medicinal materials and 6 batches of stir-fried chicken gizzard lining slices from Example 1. Figure 4 The numbers 1-6 represent chicken gizzard lining medicinal materials with batch numbers G1804017, G1804018, G1805097, G1805098, G1805099, and G1805100, respectively. Figure 4 The numbers 7-12 represent the batch numbers GP11804017, GP11804018, GP11805097, GP11805098, GP11805099, and GP11805100, respectively, for stir-fried chicken gizzard slices. Figure 4 The number 13 in the text represents the stir-fried chicken gizzard reference slices with batch number GP11805119;

[0049] Figure 5 The following are thin-layer chromatograms of 6 batches of chicken gizzard lining medicinal materials and 6 batches of stir-fried chicken gizzard lining slices from Example 1. Figure 5 The numbers 1-6 represent chicken gizzard lining medicinal materials with batch numbers G1805119, G1805120, G1805121, G1805122, G1805123, and G1805124, respectively. Figure 5 The numbers 7-12 represent the stir-fried chicken gizzard slices with batch numbers GP11805119, GP11805120, GP11805121, GP11805122, GP11805123, and GP11805124, respectively. Figure 5The number 13 in the text represents the stir-fried chicken gizzard reference slices with batch number GP11805119;

[0050] Figure 6 The following are thin-layer chromatograms of 6 batches of chicken gizzard lining medicinal materials and 6 batches of stir-fried chicken gizzard lining slices from Example 1. Figure 6 The numbers 1-6 represent chicken gizzard lining medicinal materials with batch numbers G1810259, G1810260, G1810261, YG2007007, YG2007008, and YG2007009, respectively. Figure 6 The numbers 7-12 represent the batch numbers GP11810259, GP11810260, GP11810261, GP12007701, GP12007702, and GP12007703, respectively, for the stir-fried chicken gizzard slices. Figure 6 The number 13 in the text represents the stir-fried chicken gizzard reference slices with batch number GP11805119;

[0051] Figure 7 The following are thin-layer chromatograms of six batches of chicken gizzard standard decoction and six batches of stir-fried chicken gizzard standard decoction from Example 1. Figure 7 The numbers 1-6 represent chicken gizzard standard decoctions with batch numbers GT1804017, GT1804018, GT1805097, GT1805098, GT1805099, and GT1805100, respectively. Figure 7 The numbers 7-12 represent the standard decoction of stir-fried chicken gizzard lining with batch numbers GT11804017, GT11804018, GT11805097, GT11805098, GT11805099, and GT11805100, respectively.

[0052] Figure 8 The following are thin-layer chromatograms of six batches of chicken gizzard standard decoction and six batches of stir-fried chicken gizzard standard decoction from Example 1. Figure 8 The numbers 1-6 represent the standard chicken gizzard decoction with batch numbers GT1805119, GT1805120, GT1805121, GT1805122, GT1805123, and GT1805124, respectively. Figure 8 The numbers 7-12 represent the standard decoction of stir-fried chicken gizzard lining with batch numbers GT11805119, GT11805120, GT11805121, GT11805122, GT11805123, and GT11805124, respectively.

[0053] Figure 9 The following are thin-layer chromatograms of six batches of chicken gizzard standard decoction and six batches of stir-fried chicken gizzard standard decoction from Example 1. Figure 9The numbers 1-6 represent chicken gizzard standard decoctions with batch numbers GT1810259, GT1810260, GT1810261, GT2007701, GT2007702, and GT2007703, respectively. Figure 9 The numbers 7-12 represent the standard decoction of stir-fried chicken gizzard lining with batch numbers GT11810259, GT11810260, GT11810261, GT12007701, GT12007702, and GT12007703, respectively.

[0054] Figure 10 The following are thin-layer chromatograms of three batches of chicken gizzard granules and three batches of stir-fried chicken gizzard granules from Example 1. Figure 10 The numbers 1-3 represent chicken gizzard granules with batch numbers CG2008016, CG2008017, and CG2008018, respectively. Figure 10 The numbers 4-6 represent the chicken gizzard granules with batch numbers CG2008020, CG2008021, and CG2008022, respectively. Figure 10 The number 7 in the text represents the reference slices of stir-fried chicken gizzard with batch number GP11805119. Detailed Implementation

[0055] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0056] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0057] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0058] In traditional techniques, the identification of raw chicken gizzard and its processed products, such as stir-fried chicken gizzard, mainly focuses on appearance and has few objective identification indicators. There is even less research on the differences between the two in standard decoctions and formula granules of traditional Chinese medicine. There are the following difficulties in conducting specific identification of stir-fried chicken gizzard slices, standard decoctions of stir-fried chicken gizzard, and formula granules of stir-fried chicken gizzard and raw chicken gizzard medicinal materials, standard decoctions of chicken gizzard, and formula granules of chicken gizzard: (1) The chemical composition of chicken gizzard is complex. The processed product stir-fried chicken gizzard, which is obtained by sand scalding, has a highly similar composition and shows the same efficacy in clinical practice. The mechanism of change of various components during the processing is still unclear. (2) During the preparation of standard decoctions and formula granules, some components are decomposed by heat. The preparation process of standard decoctions and formula granules determines that most of their chemical components are water-soluble, while most of the fat-soluble components cannot be transferred. (3) When formula granules are prepared, excipients need to be added, which interferes with the identification of raw and processed products. The above problems have a significant impact on the identification of raw chicken gizzard and its processed products. Therefore, there is an urgent need to provide an identification method that can overcome the above problems and achieve effective differentiation between the two.

[0059] Based on this, one embodiment of this application provides a method for distinguishing between processed chicken gizzard lining and raw chicken gizzard lining, comprising the following steps:

[0060] Prepare a test solution for the sample to be tested, wherein the sample to be tested is a processed product of chicken gizzard or a raw product of chicken gizzard; the processed product of chicken gizzard is stir-fried chicken gizzard slices, stir-fried chicken gizzard standard decoction or stir-fried chicken gizzard formula granules; the raw product of chicken gizzard is chicken gizzard medicinal material, chicken gizzard standard decoction or chicken gizzard formula granules.

[0061] The test solution was detected using high performance liquid chromatography-mass spectrometry.

[0062] The above method uses high performance liquid chromatography-mass spectrometry to analyze and detect the sample. The method is highly specific and sensitive, and can efficiently and accurately identify and distinguish between processed chicken gizzard products and raw chicken gizzard products. It can provide a strong basis for the quality control and evaluation of processed chicken gizzard products and solve the problem of difficulty in distinguishing between processed and raw chicken gizzard products due to the loss of appearance characteristics after processing.

[0063] In one embodiment, the step of detecting the test solution using high performance liquid chromatography-mass spectrometry is performed in accordance with the high performance liquid chromatography-mass spectrometry method (General Chapter 0512 and General Chapter 0431 of the Chinese Pharmacopoeia 2020 edition).

[0064] In one embodiment, the chromatographic column used for high-performance liquid chromatography (HPLC) elution is a C18 column. Further, the column is packed with octadecylsilane-bonded silica gel. Further, the column can be, for example, an Agilent SB C18 column, wherein the column length can be, for example, 100 nm, the inner diameter can be, for example, 2.1 mm, and the particle size can be, for example, 1.8 μm.

[0065] In one embodiment, the elution method of the high-performance liquid chromatography is gradient elution, wherein mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume fraction of 0.2% to 0.4%. It is understood that the volume fraction of the aqueous formic acid solution in mobile phase B can be, for example, but not limited to, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, etc. Preferably, the volume fraction of the aqueous formic acid solution in mobile phase B is 0.3%.

[0066] Furthermore, the gradient elution procedure includes:

[0067] From 0 to 5 minutes, the volume percentage of mobile phase A increased from 20% to 30%, while the volume percentage of mobile phase B decreased from 80% to 70%.

[0068] Within 5 to 10 minutes, the volume percentage of mobile phase A increased from 30% to 80%, while the volume percentage of mobile phase B decreased from 70% to 20%.

[0069] In one embodiment, the elution flow rate of the high-performance liquid chromatography is 0.2 mL to 0.4 mL per minute. It is understood that the flow rate may be, for example, but not limited to, 0.2 mL, 0.25 mL, 0.3 mL, 0.35 mL, 0.4 mL per minute, etc. Preferably, the flow rate is 0.3 mL per minute.

[0070] In one embodiment, the column temperature of the high-performance liquid chromatography (HPLC) is 28°C to 32°C. It is understood that the column temperature may be, for example, but not limited to, 28°C, 28.5°C, 29°C, 29.5°C, 30°C, 30.5°C, 31°C, 31.5°C, 32°C, etc. Preferably, the column temperature is 30°C.

[0071] In one embodiment, the injection volume of the test solution for high-performance liquid chromatography (HPLC) is 1 μl to 3 μl. It is understood that the injection volume can be, for example, but is not limited to, 1 μl, 2 μl, 3 μl, etc. Preferably, the injection volume is 2 μl.

[0072] In one implementation, mass spectrometry detection employs electrospray positive ion mode (ESI+).

[0073] In one implementation, mass spectrometry detection employs multiple reaction monitoring (MRM) scanning.

[0074] In one embodiment, mass spectrometry detection uses a capillary voltage of 1kV to 3kV. Preferably, the capillary voltage is 1.5kV.

[0075] In one embodiment, mass spectrometry detection uses an ion source temperature of 300°C to 400°C. Preferably, the ion source temperature is 350°C.

[0076] In one embodiment, mass spectrometry detection uses a desolvation gas flow rate of 800 L / Hr to 1000 L / Hr. Preferably, the desolvation gas flow rate is 900 L / Hr.

[0077] In one embodiment, mass spectrometry detection uses a curtain gas flow rate of 10 L / Hr to 30 L / Hr. Preferably, the curtain gas flow rate is 20 L / Hr.

[0078] In one embodiment, mass spectrometry detection uses a cone voltage of 30V to 50V. Preferably, the cone voltage is 40V.

[0079] In one embodiment, mass spectrometry detection uses a collision energy of 20V to 40V. Preferably, the collision energy is 30V.

[0080] In one embodiment, the method for preparing the test solution of the sample to be tested includes the following steps:

[0081] Weigh the sample to be tested, add water, perform ultrasonic extraction, filter, and collect the filtrate.

[0082] Furthermore, during the ultrasonic extraction process, the ultrasonic power is 200W–300W and the ultrasonic frequency is 30kHz–50kHz. Further, during the ultrasonic extraction process, the ultrasonic power is 250W and the ultrasonic frequency is 40kHz.

[0083] In one embodiment, the sample to be tested is stir-fried chicken gizzard slices or chicken gizzard medicinal material, and the preparation method of the test solution includes the following steps:

[0084] Take 0.3g to 0.7g of the sample powder to be tested, add 3ml to 7ml of water, extract by ultrasonication for 25 minutes to 35 minutes, filter, and collect the filtrate.

[0085] Furthermore, the sample to be tested is stir-fried chicken gizzard slices or chicken gizzard medicinal material, and the preparation method of the test solution includes the following steps:

[0086] Take 0.5g of the sample powder to be tested, add 5ml of water, extract by ultrasonication for 30 minutes, filter, and collect the filtrate.

[0087] In one embodiment, the sample to be tested is a standard decoction of stir-fried chicken gizzard or chicken gizzard, and the preparation method of the test solution includes the following steps:

[0088] Take an appropriate amount of the sample to be tested, grind it into a fine powder, take 0.1g to 0.3g, add 3ml to 7ml of water, extract by ultrasonication for 25 minutes to 35 minutes, filter, and take the filtrate.

[0089] Furthermore, the sample to be tested is a standard decoction of stir-fried chicken gizzard lining or a standard decoction of chicken gizzard lining, and the preparation method of the test solution includes the following steps:

[0090] Take an appropriate amount of the sample to be tested, grind it into a fine powder, take 0.2g, add 5ml of water, extract by ultrasonication for 30 minutes, filter, and take the filtrate.

[0091] In one embodiment, the sample to be tested is stir-fried chicken gizzard granules or chicken gizzard granules, and the preparation method of the test solution includes the following steps:

[0092] Take an appropriate amount of the sample to be tested, grind it into a fine powder, take 0.3g to 0.7g, add 3ml to 7ml of water, extract by ultrasonication for 25 minutes to 35 minutes, filter, and take the filtrate.

[0093] Furthermore, the sample to be tested is either stir-fried chicken gizzard lining granules or chicken gizzard lining granules, and the preparation method of the test solution includes the following steps:

[0094] Take an appropriate amount of the sample to be tested, grind it into a fine powder, take 0.5g, add 5ml of water, extract by ultrasonication for 30 minutes, filter, and take the filtrate.

[0095] In one embodiment, identification is performed based on the characteristic ion pairs and their signal-to-noise ratios in the mass spectrometry detection results. If the characteristic ion pairs include a first characteristic ion pair and a second characteristic ion pair, and the signal-to-noise ratio of the first characteristic ion pair and the second characteristic ion pair is greater than 3:1, then the sample to be tested is a processed chicken gizzard lining product; otherwise, it is a raw chicken gizzard lining product. The mass-to-charge ratio of the parent ion of the first characteristic ion pair is 706.41, and the mass-to-charge ratio of the daughter ion is 369.3. The mass-to-charge ratio of the parent ion of the second characteristic ion pair is 706.41, and the mass-to-charge ratio of the daughter ion is 300.2.

[0096] For example, if the test result includes only one of the first and second characteristic ion pairs, the sample to be tested is a raw chicken gizzard product, regardless of whether the signal-to-noise ratio is greater than 3:1.

[0097] For example, if the test results include the first characteristic ion pair and the second characteristic ion pair, but the signal-to-noise ratio of one or both characteristic ion pairs is less than or equal to 3:1, the sample to be tested is also a raw chicken gizzard.

[0098] In one embodiment, the identification method further includes the following steps:

[0099] Preparation of a control solution for processed chicken gizzard lining products;

[0100] Thin-layer chromatography was used to analyze the test solution and the reference solution of chicken gizzard lining.

[0101] Thin-layer chromatography (TLC) was used to further analyze the test solution, distinguishing between raw chicken gizzard lining and its processed form. This further validated the mass spectrometry identification method and improved the accuracy of the detection and identification results.

[0102] In one embodiment, the thin-layer chromatography plate used is a silica gel G thin-layer plate.

[0103] In one embodiment, the developing solvent used in thin-layer chromatography is a mixed solution of toluene, ethyl acetate, and glacial acetic acid. Further, the volume ratio of toluene, ethyl acetate, and glacial acetic acid in the developing solvent is (14–18):(3–7):(0.3–0.7). Further, the volume ratio of toluene, ethyl acetate, and glacial acetic acid in the developing solvent is (15–17):(4–6):(0.4–0.6). Even further, the volume ratio of toluene, ethyl acetate, and glacial acetic acid in the developing solvent is 16:5:0.5.

[0104] In one embodiment, the thin-layer chromatography is performed under ultraviolet light with a wavelength of 360 nm to 370 nm. Further, it is performed under ultraviolet light with a wavelength of 365 nm.

[0105] In one embodiment, the sample spotting volume used in thin-layer chromatography is 3 μl to 15 μl.

[0106] Furthermore, the sample volume of the chicken gizzard preparation control solution was 15 μl.

[0107] Furthermore, the sample to be tested was chicken gizzard lining material or stir-fried chicken gizzard lining slices, and the sample volume was 15 μl.

[0108] Furthermore, the sample to be tested was a standard decoction of chicken gizzard or a standard decoction of stir-fried chicken gizzard, and the sample volume was 3 μl.

[0109] Furthermore, the sample to be tested was chicken gizzard granules or stir-fried chicken gizzard granules, and the sample volume was 10 μl.

[0110] In one embodiment, the thin-layer chromatography uses a spray-on strip spotting method with a strip width of 5 mm to 7 mm. Further, the strip width is 6 mm.

[0111] In one embodiment, the thin-layer chromatography uses a dual-groove developing tank with a developing distance of 6 cm to 10 cm. Further, the developing distance is 8 cm.

[0112] In one embodiment, the temperature is controlled at 20°C to 30°C during thin-layer chromatography analysis. Further, the temperature is controlled at 22°C to 25°C.

[0113] In one embodiment, during thin-layer chromatography analysis, the humidity is controlled at 50%–70%. Further, the humidity is controlled at 60%–70%.

[0114] In one embodiment, the method for preparing the reference solution of chicken gizzard lining product includes:

[0115] Weigh out the reference material of chicken gizzard lining, add water, perform ultrasonic extraction, filter, and collect the filtrate.

[0116] Furthermore, during the ultrasonic extraction process, the ultrasonic power is 200W–300W and the ultrasonic frequency is 30kHz–50kHz. Further, during the ultrasonic extraction process, the ultrasonic power is 250W and the ultrasonic frequency is 40kHz.

[0117] In one embodiment, the preparation method of the reference solution for the processed chicken gizzard includes the following steps:

[0118] Take 0.3g to 0.7g of the reference material of processed chicken gizzard lining, add 3ml to 7ml of water, extract by ultrasonication for 25 minutes to 35 minutes, filter, and collect the filtrate.

[0119] Furthermore, the preparation method of the reference solution for processed chicken gizzard includes the following steps:

[0120] Take 0.5g of the chicken gizzard lining preparation reference, add 5ml of water, extract by ultrasonication for 30 minutes, filter, and collect the filtrate.

[0121] In one embodiment, the reference material for the processed chicken gizzard is stir-fried chicken gizzard slices.

[0122] The above method utilizes high-performance liquid chromatography-mass spectrometry (HPLC-MS) for mass spectrometry identification based on characteristic ion pairs, combined with thin-layer chromatography (TLC) for differentiation. Mass spectrometry offers high sensitivity, while TLC is simple to operate. Both methods are specific for detecting processed chicken gizzard lining and have their own advantages. Combining the two methods allows for mutual verification of results, making the identification method more reliable. This fills the technical gap in differentiating processed chicken gizzard lining products from raw chicken gizzard lining standard decoctions and granules. It can serve as a dedicated identification method for distinguishing processed chicken gizzard lining products from raw products, providing a basis for the quality control and evaluation of processed chicken gizzard lining products and their further processed products.

[0123] The following specific embodiments further illustrate the identification method between processed chicken gizzard and raw chicken gizzard of this application.

[0124] Example 1

[0125] I. Detection using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS)

[0126] 1.1 Mass spectrometry detection of characteristic ion pairs

[0127] Characteristic ion pairs used to distinguish chicken gizzard lining medicinal materials, chicken gizzard lining standard decoction, chicken gizzard lining formula granules, and stir-fried chicken gizzard lining slices, stir-fried chicken gizzard lining standard decoction, and stir-fried chicken gizzard lining formula granules, with the mass-to-charge ratio of the parent ion and the mass-to-charge ratio of the daughter ion being m / z 706.41→369.3 and m / z 706.41→300.2, respectively.

[0128] 1.2 Research Samples

[0129] The samples studied in this embodiment totaled 78 batches, including 18 batches each of stir-fried chicken gizzard slices and standard stir-fried chicken gizzard decoction, 3 batches of stir-fried chicken gizzard formula granules, 18 batches each of chicken gizzard medicinal materials and standard chicken gizzard decoction, and 3 batches of chicken gizzard formula granules. The specific information is shown in Table 1 below.

[0130] Table 1 Sample Information Table

[0131]

[0132]

[0133] 1.3 Preparation of the test solution

[0134] 1.3.1 Preparation method of test sample for stir-fried chicken gizzard slices and chicken gizzard medicinal material: Take 0.5g of the powder of this product, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the test sample solution.

[0135] 1.3.2 Preparation method of test samples for stir-fried chicken gizzard standard decoction and chicken gizzard standard decoction

[0136] Take an appropriate amount of this product, grind it into a fine powder, take 0.2g, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the test solution.

[0137] 1.3.3 Preparation method of test samples of stir-fried chicken gizzard granules and chicken gizzard granules

[0138] Take an appropriate amount of this product, grind it into a fine powder, take 0.5g, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the test solution.

[0139] 1.4 Chromatographic and Mass Spectrometric Conditions

[0140] 1.4.1 Chromatographic conditions: An Agilent SB C18 (100 mm × 2.1 mm, 1.8 μm) column was used; acetonitrile was used as mobile phase A, and 0.3% (v / v) formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 2; the flow rate was 0.3 mL / min, the column temperature was 30 °C, and the injection volume was 2 μl.

[0141] Table 2 Gradient Elution Table

[0142]

[0143] 1.4.2 Mass spectrometry conditions: positive ion mode (ESI+); multiple reaction monitoring (MRM); capillary voltage 1.5 kV; ion source temperature 350 °C; desolvent gas flow rate 900 L / Hr; curtain gas flow rate 20 L / Hr; cone voltage 40 V; cone voltage and collision energy are shown in Table 3 below.

[0144] Table 3. Detection of ion pairs

[0145]

[0146] 1.5 Comparative mass spectrometry identification of raw and processed chicken gizzard lining

[0147] Accurately pipette 2 μL of each of the following test solutions—fried chicken gizzard slices, standard fried chicken gizzard decoction, and granulated fried chicken gizzard, as well as raw chicken gizzard, standard chicken gizzard decoction, and granulated chicken gizzard—into the liquid chromatograph. Elute under the chromatographic conditions described in 1.4.1, and perform mass spectrometry detection under the conditions described in 1.4.2. The results are shown in Table 4. Figures 1-3 .

[0148] Table 4. Test results of stir-fried chicken gizzard slices, standard decoction of stir-fried chicken gizzard, and formula granules of stir-fried chicken gizzard.

[0149]

[0150]

[0151] As shown in Table 4 and Figures 1-3 The results showed that the mass-to-charge ratio (m / z) of stir-fried chicken gizzard slices, standard decoction of stir-fried chicken gizzard, and formula granules of stir-fried chicken gizzard were significantly detected as 706.41→369.3 and m / z as 706.41→300.2, while the corresponding chicken gizzard raw materials, standard decoction of chicken gizzard, and formula granules were not detected.

[0152] II. Thin-layer chromatographic identification of raw and processed chicken gizzard lining

[0153] 2.1 Preparation of the test solution

[0154] 2.1.1 Preparation method of test samples for stir-fried chicken gizzard slices and chicken gizzard medicinal materials

[0155] Take 0.5g of the powder, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and use the filtrate as the test solution.

[0156] 2.1.2 Preparation method of test samples for stir-fried chicken gizzard standard decoction and chicken gizzard standard decoction

[0157] Take an appropriate amount of this product, grind it into a fine powder, take 0.2g, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the test solution.

[0158] 2.1.3 Preparation method of test samples of stir-fried chicken gizzard granules and chicken gizzard granules

[0159] Take an appropriate amount of this product, grind it into a fine powder, take 0.5g, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the test solution.

[0160] 2.2 Preparation of the reference solution for processed chicken gizzard lining

[0161] Take about 0.5g of stir-fried chicken gizzard lining reference slices, add 5ml of water, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the reference solution for the processed chicken gizzard lining product.

[0162] 2.3 Thin-layer chromatography conditions

[0163] Thin-layer plate: Merck silicone G thin-layer plate.

[0164] Developing solvent: toluene, ethyl acetate and glacial acetic acid in a volume ratio of 16:5:0.5.

[0165] Spotting method: spray strip spotting, strip width 6.0mm.

[0166] Unfolding method: A double-groove unfolding cylinder is used, with an unfolding distance of approximately 8cm.

[0167] Temperature: 24.5℃.

[0168] Humidity: 66%.

[0169] Inspection: Examine under ultraviolet light (365nm).

[0170] 2.4 Comparison of thin-layer chromatograms of chicken gizzard lining and processed chicken gizzard lining products

[0171] Take 15 μl each of the test solutions of chicken gizzard lining material, stir-fried chicken gizzard lining slices, and chicken gizzard lining processed product control solution; 3 μl each of the test solutions of chicken gizzard lining standard decoction and stir-fried chicken gizzard lining standard decoction; and 10 μl each of the test solutions of chicken gizzard lining formula granules and stir-fried chicken gizzard lining formula granules. Spot these samples onto the same silica gel G thin-layer plate. Use a mixed solution of toluene, ethyl acetate, and glacial acetic acid (volume ratio 16:5:0.5) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365 nm). The thin-layer chromatography spectrum is shown below. Figures 4 to 10 .

[0172] Depend on Figures 4 to 10 As can be seen, the chromatograms of 18 batches of stir-fried chicken gizzard slices, standard decoctions of stir-fried chicken gizzard, and formula granules of stir-fried chicken gizzard all showed fluorescent spots corresponding to the chromatograms of the control slices of stir-fried chicken gizzard. However, the corresponding chicken gizzard materials, standard decoctions, and formula granules of the 18 batches did not show fluorescent spots at the response positions of the control slices of stir-fried chicken gizzard. Therefore, thin-layer chromatography can be used to further distinguish between raw chicken gizzard and processed chicken gizzard.

[0173] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0174] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for identifying a processed product of Radix Typhonii and a raw product of Radix Typhonii, characterized in that, It comprises the following steps: Preparation of test sample solution of the sample to be tested, which is prepared from the chicken gallbladder or chicken gallbladder raw product; The chicken gallbladder product is fried chicken gallbladder decoction pieces, fried chicken gallbladder standard soup or fried chicken gallbladder formula granules; the chicken gallbladder raw product is chicken gallbladder medicinal material, chicken gallbladder standard soup or chicken gallbladder formula granules; The test sample solution is detected by using ultra-high performance liquid chromatography-mass spectrometry, and the chromatographic column is a C18 chromatographic column; the elution mode is gradient elution, wherein the mobile phase A is acetonitrile, and the mobile phase B is 0.2%~0.4% formic acid aqueous solution; The program of the gradient elution comprises: 0 min~5 min, the volume percentage of the mobile phase A is increased from 20% to 30%, and the volume percentage of the mobile phase B is decreased from 80% to 70%; 5 min~10 min, the volume percentage of the mobile phase A is increased from 30% to 80%, and the volume percentage of the mobile phase B is decreased from 70% to 20%; The mass spectrometry conditions meet the following conditions: (1) using electrospray positive ion mode; (2) multiple reaction monitoring scanning; (3) capillary voltage is 1kV~3kV; (4) ion source temperature is 300℃~400℃; (5) desolvation gas flow is 800L / Hr~1000L / Hr; (6) gas curtain gas flow is 10L / Hr~30L / Hr; (7) cone hole voltage is 30V~50V; (8) collision energy is 20V~40V; The preparation method of the test sample solution of the sample to be tested comprises the following steps: weighing the sample to be tested, adding water, ultrasonic extraction, filtering, and taking the filtrate; According to the characteristic ion pair in the detection result of mass spectrometry and the signal-to-noise ratio of the characteristic ion pair, the sample to be tested is identified; If the characteristic ion pair comprises a first characteristic ion pair and a second characteristic ion pair, and the signal-to-noise ratio of the first characteristic ion pair and the second characteristic ion pair is greater than 3:1, then the sample to be tested is a chicken gallbladder product, otherwise it is a chicken gallbladder raw product; The mass-to-charge ratio of the parent ion of the first characteristic ion pair is 706.41, and the mass-to-charge ratio of the daughter ion is 369.3; the mass-to-charge ratio of the parent ion of the second characteristic ion pair is 706.41, and the mass-to-charge ratio of the daughter ion is 300.

2.

2. The method for identifying the processed product of Radix Typhonii with the raw product of Radix Typhonii according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions of the ultra-high performance liquid chromatography-mass spectrometer meet the following conditions: (1) the flow rate is 0.2mL / min~0.4mL / min; (2) the column temperature is 28℃~32℃; (3) the injection volume is 1μl~3μl.

3. The method for identifying processed product of Radix Typhonii and raw product of Radix Typhonii according to any one of claims 1-2, characterized in that, The identification method further comprises the following steps: Preparation of chicken gallbladder product control solution; The test sample solution and the chicken gallbladder product control solution are analyzed by using thin layer chromatography.

4. The method for identifying processed Radix Scrophulariae and raw Radix Scrophulariae according to claim 3, characterized in that, The chromatographic conditions of thin layer chromatography meet one or more of the following conditions: (1) the thin layer chromatography plate is a silica gel G thin layer plate; (2) the developing agent is a mixed solution of toluene, ethyl acetate and glacial acetic acid; (3) the viewing method is: viewing under a UV light lamp with a wavelength of 360nm~370nm; (4) the sample size is 3μl~15μl.

5. The method for identifying the processed product of Radix Typhonii with the raw product of Radix Typhonii according to claim 4, characterized in that, The volume ratio of toluene, ethyl acetate and glacial acetic acid in the developing agent is (14~18):(3~7):(0.3~0.7).

6. The method for identifying processed product of Radix Typhonii and raw product of Radix Typhonii according to any one of claims 4-5, characterized in that, The preparation method of the prepared Scrophulariae Herba processed product control solution comprises the following steps: The Scrophulariae Herba processed product control is weighed, water is added, ultrasonic extraction is carried out, filtration is carried out, and the filtrate is taken.

7. The method for identifying the processed product of Radix Typhonii with the raw product of Radix Typhonii according to claim 6, characterized in that, The Scrophulariae Herba processed product control is a fried Scrophulariae Herba control decoction piece.

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