Thin-layer identification method for identifying adulteration of dragon's blood in dragon's blood

Abstract

The invention discloses a thin-layer identification method for identifying adulteration of dragon's blood in dragon's blood, which is characterized in that loureirin B is used as a reference substance, dichloromethane-toluene-ethyl acetate-formic acid (19: 2: 6: 0.5) is used as a developing solvent, and spots with the same color cannot be displayed at the corresponding position of a reference substance chromatography in a test substance chromatography. The method has the advantages of simple and accurate operation, good specificity, good separation degree, clear and round spots, and easy result determination. By adopting the method disclosed by the invention, whether the dragon's blood is adulterated with the dragon's blood or not can be quickly and accurately identified, a reliable guarantee is provided for medication safety of the dragon's blood, a basis is provided for quality control of the dragon's blood, the requirements of daily inspection and supervision can be met, and a technical support is provided for a medicine supervision department.

Description

[0001] The invention relates to the technical field of identification of traditional Chinese medicines, and in particular to a thin layer identification method for identifying dragon's blood adulterated with dragon's blood. Background Art

[0002] Dragon's blood is made from the resin exuded from the fruit of Daemonoropsdraco Bl., a palm plant. Dragon's blood is produced in Indonesia, Malaysia, India and other places. According to literature reports and market surveys, the annual import volume of dragon's blood is relatively stable, but the price is increasing year by year. According to research, the amount of dragon's blood actually used by Chinese pharmaceutical companies each year is greater than the import volume, and the companies do not always provide import inspection reports for the dragon's blood they use, so the quality of some dragon's blood cannot be controlled and inspected. After consulting literature, market surveys and laboratory verification, it was found that some dragon's blood was adulterated with dragon's blood.

[0003] Dragon's Blood is a resin extracted from the fat-containing wood of the Liliaceae plant Dracaena cochinchinensis (Lour.) SCChen. Dragon's Blood and Dragon's Blood are both resins, similar in appearance, similar in color, and similar in efficacy. Moreover, Dragon's Blood is expensive, up to about 2,000 yuan per kilogram, while Dragon's Blood only costs about 200 yuan per kilogram. Therefore, some unscrupulous merchants mix some Dragon's Blood with Dragon's Blood because of its high price and small amount of Dragon's Blood to confuse it in order to seek profits. In recent years, fake Dragon's Blood products often appear on the market, and Dragon's Blood is used as Dragon's Blood or Dragon's Blood is mixed with Dragon's Blood in Dragon's Blood. The function and treatment of Dragon's Blood are similar to Dragon's Blood, and its appearance and properties are also similar. It is difficult to distinguish traditional experience. If Dragon's Blood is mixed with Dragon's Blood for use, it is difficult to distinguish only from the appearance and properties. Therefore, in order to ensure the drug safety of Dragon's Blood and meet the needs of daily inspection and supervision, it is urgent to develop a method for quickly identifying whether Dragon's Blood is adulterated in Dragon's Blood. Summary of the invention

[0004] The purpose of the present invention is to provide a thin layer identification method for identifying adulterated dragon's blood in dragon's blood in view of the deficiencies in the prior art. The method is simple to operate and has good specificity. The method can be used to quickly and accurately identify whether dragon's blood is adulterated with dragon's blood, thereby providing reliable protection for the safety of dragon's blood use and providing technical support for drug regulatory authorities.

[0005] The technical solution adopted by the present invention is as follows: A thin layer identification method for identifying adulterated dragon's blood in dragon's blood comprises the following steps: (1) Take an appropriate amount of this product, grind it into powder, take about 0.1g, put it in a conical flask, add 5ml of ethanol, ultrasonically treat it for 5 minutes, cool it, filter it, and use the filtrate as the test solution; (2) Take an appropriate amount of dragon's blood B reference substance and add ethanol to make a solution containing 1 mg per 1 ml as the reference substance solution; (3) According to the thin layer chromatography method (General Chapter 0502 of the 2015 edition of the Chinese Pharmacopoeia), 5-10 μl of the test sample solution and 5 μl of the reference sample solution were respectively spotted on the same silica gel G thin layer plate, and the plate was developed using dichloromethane-toluene-ethyl acetate-formic acid in a volume ratio of 19:2:6:0.5. The plate was taken out, dried, and sprayed with a mixed solution of 5% vanillin sulfuric acid and 10% ethanolic sulfuric acid, and heated to 105°C until the spots were clearly colored. In the chromatogram of the test sample, no spots of the same color should appear at the corresponding positions in the chromatogram of the reference sample.

[0006] Furthermore, in the above-mentioned thin layer identification method for identifying adulterated dragon's blood in dragon's blood, in step (3), in the mixed solution of 5% vanillin sulfuric acid-10% sulfuric acid ethanol, the volume ratio of 5% vanillin sulfuric acid:10% sulfuric acid ethanol is 1:4, and it is freshly prepared before use.

[0007] Dragon's blood element B is a specific component in dragon's blood, with stable properties and mature research. The thin-layer identification method is fast and simple, and has been verified by methodology to have no negative interference. Therefore, the present invention selects dragon's blood element B as an indicator to establish a thin-layer identification method for adulterating dragon's blood in dragon's blood.

[0008] The beneficial effects of the present invention are: The present invention provides a thin-layer identification method for identifying adulterated dragon's blood in dragon's blood, which is simple and accurate to operate, has good specificity and separation, obvious color development, fast development rate, clear and round spots, easy to judge results, good durability and strong applicability. The method of the present invention can quickly and accurately identify whether there is adulterated dragon's blood in dragon's blood, detect whether dragon's blood contains dragon's blood components, provide reliable guarantee for the drug safety of dragon's blood, provide basis for dragon's blood quality control, meet the needs of daily inspection and supervision, and provide technical support for drug regulatory departments. The method of the present invention can be used as a supplementary inspection method for checking dragon's blood components in dragon's blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The selection of indicator components was examined in the chromatogram, in which: 1. Dragon's blood A reference substance, 2. Dragon's blood B reference substance, 3. Dragon's blood reference medicinal material, 4. Dragon's blood sample (LXJ-21), 5. Dragon's blood sample (LXJ-31), spot A is rose red, spot B is orange, thin layer plate: Merck plate, developing agent: dichloromethane-toluene-ethyl acetate-formic acid (19:6:2:0.5), color development and inspection: spray with 5% vanillin sulfuric acid-10% sulfuric acid ethanol (1:4) mixed solution (freshly prepared before use), heat at 105°C until the spots are clearly colored, and inspect under sunlight.

[0010] Figure 2Thin layer chromatograms of different development systems, in the figure, 1. Dragon's Blood reference medicinal material, 2. Dragon's Blood reference medicinal material, 3. Dragon's Blood B reference substance.

[0011] Figure 3 Investigation of the extraction method. In the figure, 1. Ultrasonic extraction, 2. Heating reflux extraction, 3. Dragon's blood B reference substance, spot A is orange, thin layer plate: Qingdao Ocean Chemical Plant Branch.

[0012] Figure 4 Investigation of extraction solvents. In the figure, 1. ethyl acetate extraction, 2. methanol extraction, 3. ethanol extraction, 4. dragon's blood B reference substance, spot A is orange, thin layer plate: Qingdao Ocean Chemical Plant Branch.

[0013] Figure 5 Investigation of extraction time. In the figure, 1. Dragon's blood protein B reference substance, 2. Ultrasonic extraction for 5 minutes, 3. Ultrasonic extraction for 10 minutes. Spot A is orange. Thin layer plate: Qingdao Ocean Chemical Plant Branch.

[0014] Figure 6 Investigation of sampling quantity. In the figure, 1. Dragon's blood element B reference substance, 2. Sampling quantity 0.2g to 5ml, 3. Sampling quantity 0.1g to 1ml, spot A is orange, thin layer plate: Qingdao Ocean Chemical Plant Branch.

[0015] Figure 7 Specificity test chromatogram, in the figure, 1, Dragon's Blood reference medicinal material, 2, Dragon's Blood B reference substance, 3, Dragon's Blood reference medicinal material, spot A is orange-yellow, T: 30℃, RH: 60%.

[0016] Figure 8 Thin layer chromatograms of different sample amounts of 5% adulterated dragon's blood samples, in the figure, 1, dragon's blood B reference solution, 2~11, 5% adulterated dragon's blood samples 1μl~10μl, 12, 5% adulterated dragon's blood sample 15μl, spot A is orange, T: 30℃, RH: 60%.

[0017] Fig. 9 Thin layer chromatograms of different thin layer plates. In the figure, 1. Negative (Dragon's Blood control medicinal material), 2. Dragon's Blood B reference substance, 3. Dragon's Blood control medicinal material, 4. Dragon's Blood sample (XJ-1), 5. Dragon's Blood sample (XJ-15), 6. Dragon's Blood sample (XJ-20). Note: Dragon's Blood XJ-1 and XJ-15 are samples that were detected to be adulterated with Dragon's Blood.

[0018] Fig.10 Thin layer chromatograms of different temperatures, in which: 1. Dragon's blood B reference substance, 2. Dragon's blood reference medicinal material, 3. Dragon's blood sample (XJ-1), 4. Dragon's blood sample (XJ-15), 5. Dragon's blood sample (XJ-20), thin layer plate: Yantai Institute of Chemical Industry batch number: 20170419.

[0019] Fig.11 Thin layer chromatograms of different humidity tests, in the figure, 1. Dragon's blood B reference substance, 2. Dragon's blood reference medicinal material, 3. Dragon's blood sample (XJ-1), 4. Dragon's blood sample (XJ-15), 5. Dragon's blood sample (XJ-20), Thin layer plate: Qingdao Ocean Chemical Plant Branch Batch No.: 20211102.

[0020] Fig.12 Detection limit investigation chromatogram, in the figure, 1, dragon's blood B reference solution, 2, spiked sample (containing dragon's blood B 0.1027 mg), 3, spiked sample (containing dragon's blood B 0.2054 mg), 4, spiked sample (containing dragon's blood B 0.3081 mg), 5, spiked sample (containing dragon's blood B 0.4108 mg), 6, spiked sample (containing dragon's blood B 0.5135 mg), spot A is orange-yellow, T: 30℃, RH: 60%, thin layer plate: Qingdao Ocean Chemical Plant Branch.

[0021] Fig.13 Chromatogram of the investigation of reference substance concentration, in the figure, 1, Loureirin B 0.1mg / ml reference substance solution, 2, Loureirin B 0.2mg / ml reference substance solution, 3, Loureirin B 0.5mg / ml reference substance solution, 4, Loureirin B 0.7mg / ml reference substance solution, 5, Loureirin B 1.0mg / ml reference substance solution, spot A is orange-yellow, T: 30℃, RH: 60%.

[0022] Fig.14 Chromatogram of adulteration investigation, in the figure, 1, dragon's blood protein B reference substance, 2, 1% adulterated sample, 3, 2% adulterated sample, 4, 3% adulterated sample, 5, 4% adulterated sample, 6, 5% adulterated sample, 7, 10% adulterated sample, 8, 20% adulterated sample, 9, 50% adulterated sample, 10, 100% adulterated sample, spot A is orange-yellow, T: 30℃, RH: 60%, thin layer plate: Qingdao Ocean Chemical Plant Branch.

[0023] Fig.15 Identification chromatograms of Dragon's Blood test samples 1-10, in the figure, 1, dragon's blood reference substance 5μl, 2-11, dragon's blood samples (1-10) 5μl, thin layer plate: Qingdao Ocean Chemical Plant Branch, color development: spray with 5% vanillin sulfuric acid-10% sulfuric acid ethanol mixed solution (1:4), heat at 105℃ until the spots are clearly colored.

[0024] Fig.16Identification chromatogram of Dragon's Blood test samples 11-20, in the figure, 1, 5μl of dragon's blood reference substance B, 2-11, 5μl of Dragon's Blood samples (11-20), thin layer plate: Qingdao Ocean Chemical Plant Branch, color development: spray with 5% vanillin sulfuric acid-10% sulfuric acid ethanol mixed solution (1:4), heat at 105℃ until the spots are clearly colored. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments and drawings. Example 1

[0026] A thin layer identification method for identifying adulterated dragon's blood in dragon's blood comprises the following steps: (1) Take an appropriate amount of this product, grind it into powder, take about 0.1g, put it in a conical flask, add 5ml of ethanol, ultrasonically treat it for 5 minutes, cool it, filter it, and use the filtrate as the test solution;

[0027] (2) Take an appropriate amount of dragon's blood B reference substance and add ethanol to make a solution containing 1 mg per 1 ml as the reference substance solution;

[0028] (3) According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 5-10 μl of the test solution and 5 μl of the control solution were respectively spotted on the same silica gel G thin layer plate, and developed with dichloromethane-toluene-ethyl acetate-formic acid (19:2:6:0.5) as the developing solvent. After development, the plate was taken out, dried, and sprayed with a mixed solution of 5% vanillin sulfuric acid-10% sulfuric acid ethanol (1:4) (freshly prepared before use), and heated to 105°C until the spots were clearly colored. In the chromatogram of the test sample, no spots of the same color should be visible at the corresponding positions of the chromatogram of the reference sample. Example 2

[0029] 1Instruments, drugs and reagents Dragon's blood B reference substance (111558-202108, content of 99.7%), dragon's blood reference medicinal material (121252-201303), and dragon's blood reference medicinal material (120906-201611) were all provided by the China Food and Drug Inspection Institute. Dragon's blood was sampled and purchased from the market, as shown in Table 1 for details. Dragon's blood was purchased from the market and pharmaceutical companies, as shown in Table 2 for details.

[0030]

[0031]

[0032]

[0033] 2 Research on inspection methods 2.1 Investigation of extraction methods and development systems 2.1.1 Selection of indicator components Dragon's blood contains dragon's blood A and dragon's blood B, both of which are its characteristic components. The collected dragon's blood was studied by thin layer chromatography. The results showed that the spots of dragon's blood B were clearer and rounder than those of dragon's blood A. Figure 1 Therefore, dragon's blood element B was selected as the indicator component of the inspection method.

[0034] Preparation of reference substance: Take appropriate amount of dragon's blood A and B respectively, add methanol to prepare 1 mg / ml reference substance solution.

[0035] Preparation of Dragon's Blood reference medicinal material: Take 0.1 g of Dragon's Blood reference medicinal material, add 10 ml of methanol, ultrasonically treat for 10 minutes, filter, and concentrate the filtrate to 1 ml.

[0036] Preparation of the test sample: Take 0.1 g of dragon's blood sample, add 10 ml of methanol, ultrasonically treat for 10 minutes, filter, and concentrate the filtrate to 1 ml.

[0037] 2.1.2 Expand system selection Preparation of Loureirin B reference substance: Take an appropriate amount of Loureirin B and add ethanol to prepare a 1 mg / ml reference substance solution.

[0038] Preparation of Dragon's Blood reference medicinal material: Take 0.1 g of Dragon's Blood reference medicinal material, add 10 ml of methanol, ultrasonically treat for 10 minutes, filter, and concentrate the filtrate to 1 ml.

[0039] Preparation of the Dragon's Blood reference medicinal material: Take 0.1 g of the Dragon's Blood reference medicinal material, add 10 ml of methanol, ultrasonically treat for 10 minutes, filter, and concentrate the filtrate to 1 ml.

[0040] Different development and inspection systems were used to investigate the dragon's blood B reference substance, dragon's blood control medicinal material and dragon's blood control medicinal material. The results showed that when development and inspection system 3 was used, i.e., silica gel G thin layer plate, the developing solvent was dichloromethane-toluene-ethyl acetate-formic acid (19:6:2:0.5), sprayed with 5% vanillin sulfuric acid-10% sulfuric acid ethanol (1:4) mixed solution (freshly prepared before use), and heated at 105°C until the spots were clearly colored, the dragon's blood B spots could be observed more clearly with good separation, while dragon's blood B was not detected in the dragon's blood control medicinal material. Therefore, development and inspection system 3 was selected for the next step of research. Figure 2 The thin layer plates from Qingdao Ocean Chemical Plant were used in the inspection.

[0041] Development and inspection system 1: petroleum ether-ethyl acetate-toluene-methanol (12:8:2:1) silica gel GF 254 Thin layer plate, inspected at 254 nm.

[0042] Development and inspection system 2: dichloromethane-toluene-ethyl acetate-formic acid (20:2:6:0.3) silica gel G thin layer plate, sprayed with 10% sulfuric acid ethanol.

[0043] Development and inspection system 3: Dichloromethane-toluene-ethyl acetate-formic acid (19:6:2:0.5) silica gel G thin layer plate, spray with 5% vanillin sulfuric acid-10% sulfuric acid ethanol (1:4) mixed solution (freshly prepared before use), heat at 105℃ until the spots are clearly colored, and inspect under sunlight.

[0044] 2.1.3 Investigation of extraction methods The ultrasound and reflux methods were used to investigate the proposed deployment system. The results showed that there was no significant difference between ultrasound and reflux. Figure 3 Therefore, ultrasonic method was adopted for further research.

[0045] Ultrasonic extraction: Take 0.1 g of dragon's blood reference medicinal material, add 20 ml of methanol, ultrasonicate for 10 minutes, filter, and concentrate the solution to 1 ml.

[0046] Reflux extraction: Take 0.1 g of dragon's blood reference medicinal material, add 20 ml of methanol, heat and reflux for 30 minutes, filter, and concentrate the filtrate to 1 ml.

[0047] 2.1.4 Investigation of extraction solvent Methanol, ethanol and ethyl acetate were used to examine the dragon's blood control medicinal material. The results showed that the spots extracted by ethyl acetate were slightly blurred, while there was no obvious difference between methanol and ethanol. Figure 4 Therefore, non-toxic and non-irritating ethanol is used as the extraction solvent.

[0048] Extraction method: Take 0.1g of dragon's blood control medicinal material, add 20ml of methanol, ethanol and ethyl acetate respectively, ultrasonicate for 10 minutes, filter and concentrate the filtrate to 1ml.

[0049] 2.1.5 Investigation of extraction time According to the proposed method, the ultrasound was performed for 5 minutes and 10 minutes respectively. There was no significant difference in the results, so 5 minutes of ultrasound time was selected. Figure 5 .

[0050] 2.1.6 Investigation of sampling volume Preparation method of the test sample: Take 0.2 g of dragon's blood control medicinal material, add 5 ml of ethanol, ultrasonicate for 5 minutes, and filter to obtain the sample.

[0051] Considering the representativeness of the sampling, the sampling amount was set at 0.2g. In order to simplify and speed up the test method, 5ml of ethanol was added, ultrasonicated and filtered. The results showed that the method was the same as "take 0.1g, add 10ml of ethanol, ultrasonicate for 5 minutes, filter, and concentrate to 1ml". There was no significant difference. Figure 6 .

[0052] 2.2 Methodological validation 2.2.1 Exclusivity 5 μl of dragon's blood B reference solution, dragon's blood draconite reference medicinal material solution, and dragon's blood draconite reference medicinal material solution were respectively spotted on the same silica gel G thin layer plate and tested according to the proposed method.

[0053] Results: The solution of the reference medicinal material of dragon's blood had no interference with the test; in the chromatograms of the reference medicinal material of dragon's blood and the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the reference material of dragon's blood B. The results showed that dragon's blood did not contain dragon's blood B, and the method had good specificity. Figure 7 .

[0054] 2.2.2 Investigation of sample quantity 1μl, 2μl, 3μl, 4μl, 5μl, 6μl, 7μl, 8μl, 9μl, 10μl, and 15μl of the 5% adulterated sample solution of dragon's blood were respectively spotted on the same silica gel G plate and tested according to the proposed method. The results showed that when the spotting volume was 5μl, the spots of dragon's blood B could be observed more clearly, so the spotting volume of 5μl was selected for methodological research (see Figure 8 ).

[0055] 2.3 Durability 2.3.1 Comparison of different thin layer plates Domestic prefabricated silica gel G plates (Qingdao Ocean Chemical Plant, Yantai Chemical Industry Research Institute) and imported prefabricated silica gel G plates (Merck, Germany) were tested according to the proposed method. The results showed that the spots of domestic prefabricated silica gel G plates and imported prefabricated silica gel G plates can be effectively separated, but the imported plates are better, see Fig. 9 In order to improve the reproducibility of the method, domestic plates were used for the investigation of other conditions.

[0056] 2.3.2 Comparison of different temperatures The thin layer plate after spotting was developed, colored and inspected at 4℃ and 30℃ (room temperature). The results showed that there was no significant effect on the inspection when the test was conducted within the range of 4~30℃ (room temperature). Fig.10 .

[0057] 2.3.3 Comparison of different humidity levels Take the thin layer plate after the strip sample is spotted, develop it in the chromatography cylinder adjusted with sulfuric acid at 32% and 88% relative humidity, develop the color, and inspect it. The results show that the test in the relative humidity range of 32% to 88% has no obvious effect on the inspection. Fig.11 .

[0058] The results of the above-mentioned methodology verification test showed that changes in temperature and humidity had little effect on the unfolding effect, so it is believed that the durability of this method is good.

[0059] 2.3.4 Investigation of method detection limit and amount of adulteration 2.3.4.1 Investigation of detection limit Preparation of the stock solution of Loureirin B reference substance: Take 10.30 mg of Loureirin B reference substance (content is 99.7%), place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, the concentration is: 1.027 mg / ml.

[0060] Preparation of detection limit spiked sample solution: Take 5 portions of dragon's blood (XJ-20) sample, 0.2g each, and accurately add 0.5ml, 0.4ml, 0.3ml, 0.2ml, and 0.1ml of the reference stock solution of 1.027mg / ml, respectively, and mix well. According to the preparation method of the test solution under 2.1, the test solutions containing 0.5135mg, 0.4108mg, 0.3081mg, 0.2054mg, and 0.1027mg of lord's blood B are prepared, respectively. Check according to the above method. When the test sample contains 0.2054mg of lord's blood B, spots of lord's blood B can be observed, as shown in Figure 2. Fig.12 Therefore, the detection limit of the method for dragon's blood B is: 0.2054÷test sample sampling amount=0.2054÷0.2=1.027mg / g.

[0061] 2.4.4.2 Investigation of reference substance concentration Take appropriate amount of dragon's blood element B reference substance, add ethanol to make solutions containing 0.1, 0.2, 0.5, 0.7, and 1.0 mg per 1 ml respectively, as reference substance solutions.

[0062] Take 5 μl of each of the above-mentioned reference solution, spot them on the same silica gel G thin layer plate, and test according to the proposed method.

[0063] Results: When the concentration of the reference substance was 0.2 mg / ml, the spot of Loureirin B could be observed, but it was rather blurry. In order to determine the result more accurately, the concentration of the reference substance was set to 0.5 mg / ml so that the color and concentration of the observed reference substance spot were close to those of Loureirin B in the sample. Fig.13 .

[0064] 2.4.4.3 Investigation of the amount of admixture Preparation of adulterated samples: Take 0.198g, 0.196g, 0.194g, 0.192g, 0.190g, 0.180g, 0.160g, 0.10g and 0.0g of Dragon's Blood (XJ-20) sample and add 0.002g, 0.004g, 0.006g, 0.008g, 0.010g, 0.020g, 0.040g, 0.10g and 0.20g of Dragon's Blood sample (L-20) respectively to prepare adulterated samples with 1%, 2%, 3%, 4%, 5%, 10%, 20%, 50% and 100% dragon's blood added.

[0065] Preparation of adulterated sample solutions: Take the above-mentioned adulterated samples with adulteration of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 50%, and 100%, respectively, and prepare them according to the test sample preparation method to obtain a series of adulterated solutions.

[0066] 5 μl of each of the dragon's blood B reference solution, dragon's blood jelly reference medicinal material solution, and a series of adulterated solutions were taken and spotted on the same silica gel G thin layer plate, and tested according to the proposed method.

[0067] Results: When the doping amount is 5%, a spot of the same color can be seen in the chromatogram of the test product at the corresponding position of the chromatogram of the reference product of dragon's blood B. Fig.14 .

[0068] According to the 2020 edition of the "Pharmacopoeia of the People's Republic of China" Part IV 0212 General Rules for the Inspection of Medicinal Materials and Decoctions, impurities should generally not exceed 3%. In order to exclude unintentional adulteration and counterfeiting, and in response to the situation of subjective large-scale adulteration, combined with the results of the adulteration investigation, the limit of adulteration of dragon's blood in dragon's blood is proposed to be 5%.

[0069] 2.4.5 Identification of test products A total of 20 batches of dragon's blood samples were taken for research (see Table 1 for sample information). According to the proposed method, 6 batches (samples 1, 2, 3, 4, 9, and 15) were found to contain dragon's blood B. Fig.15 , Fig.16 .

Claims

1. A thin layer identification method for identifying adulterated dragon's blood in dragon's blood, characterized in that: The following steps are involved: (1) Take an appropriate amount of this product, grind it into powder, take 0.1g, put it in a conical flask, add 5ml of ethanol, ultrasonically treat it for 5 minutes, cool it, filter it, and use the filtrate as the test solution; (2) Take an appropriate amount of dragon's blood B reference substance and add ethanol to make a solution containing 1 mg per 1 ml as the reference substance solution; (3) According to the thin layer chromatography test in General Chapter 0502 of the 2015 edition of the Chinese Pharmacopoeia, 5-10 μl of the test sample solution and 5 μl of the reference sample solution were respectively spotted on the same silica gel G thin layer plate, and the plate was developed with dichloromethane-toluene-ethyl acetate-formic acid in a volume ratio of 19:2:6:0.

5. The plate was taken out, dried, and sprayed with a mixed solution of 5% vanillin sulfuric acid and 10% sulfuric acid ethanol. The plate was heated to 105°C until the spots were clearly colored. In the chromatogram of the test sample, no spots of the same color should appear at the corresponding positions in the chromatogram of the reference sample.

2. The thin layer identification method for identifying adulterated dragon's blood in dragon's blood according to claim 1 is characterized in that: In step (3), in the mixed solution of 5% vanillin sulfuric acid and 10% ethanol sulfuric acid, the volume ratio of 5% vanillin sulfuric acid to 10% ethanol sulfuric acid is 1:4, and the mixture is freshly prepared before use.

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