Thin-layer Identification Method for Shujincao and Shenjincao Formula Granules

Abstract

The present invention provides a thin-layer identification method for the formula granules of Lycopodiastrum casuarinoides and Lycopodium clavatum, comprising: A) taking a sample to be tested, and obtaining a test solution after pretreatment; B) taking the reference medicinal material of Lycopodiastrum casuarinoides, and obtaining a reference medicinal material solution after pretreatment; C) performing thin-layer chromatography detection on the test solution and the reference medicinal material solution, and the thin-layer plate is a silica gel G thin-layer plate; the developing agent is cyclohexane-ethyl acetate; D) visualizing under an ultraviolet lamp. In the chromatogram of the test sample, if there is a blue fluorescent spot at an Rf value of 0.31 to 0.464, it is the formula granules of Lycopodiastrum casuarinoides; if there is no blue fluorescent spot at an Rf value of 0.31 to 0.464, it is the formula granules of Lycopodium clavatum. The thin-layer identification chromatographic method for the formula granules of Lycopodiastrum casuarinoides and Lycopodium clavatum established by the present invention uses the presence or absence of fluorescent spots within a specified retention factor value range in the thin-layer chromatogram as the identification point to quickly and effectively identify the formula granules of Lycopodiastrum casuarinoides and Lycopodium clavatum.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and particularly to a thin-layer identification method for the formula granules of Lycopodiastrum casuarinoides and Lycopodium japonicum. Background Art

[0002] Lycopodiastrum casuarinoides is the dried aerial part of the plant Lycopodiastrum casuarinoides (Spring) Holub of the family Lycopodiaceae. It has the effects of dispelling wind and dampness, and promoting blood circulation and relaxing tendons. It is used for rheumatic numbness, traumatic injury, bone and muscle pain, irregular menstruation, etc.

[0003] Lycopodium japonicum is the dried whole herb of the plant Lycopodium japonicum Thunb of the family Lycopodiaceae. It has the effects of dispelling wind and dampness, and relaxing tendons and activating collaterals. It is used for joint soreness, and difficult flexion and extension, etc.

[0004] Clinically, Lycopodiastrum casuarinoides is mainly used for rheumatic diseases, while Lycopodium japonicum is mainly used for muscle and bone injuries, etc. The two cannot be used interchangeably. However, due to the imperfect thin-layer identification method for the formula granules of Lycopodiastrum casuarinoides in the current standard, there is no effective means to distinguish it from the easily confused Lycopodium japonicum formula granules.

[0005] Therefore, there is a need to establish a thin-layer method that can effectively identify the formula granules of Lycopodiastrum casuarinoides and distinguish them from the easily confused Lycopodium japonicum formula granules. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a thin-layer identification method for the formula granules of Lycopodiastrum casuarinoides and Lycopodium japonicum.

[0007] In this article, the terms "include", "comprise" and "have" can be used interchangeably, aiming to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that using "include", "comprise" and "have" to describe in this article also provides the solution of "consisting of...".

[0008] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0009] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or its similar expression refers to any combination of these items, including any combination of single item (s) or multiple items (s).

[0010] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the following processes do not imply the order of execution. Some or all of the steps may be executed in parallel or sequentially. The execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0011] The present invention provides a thin-layer identification method for Lycopodiastrum casuarinoides and Lycopodium clavatum formula granules, comprising:

[0012] A) Take the sample to be tested, and after pretreatment, obtain the test solution;

[0013] B) Take the reference medicinal material of Lycopodiastrum casuarinoides, and after pretreatment, obtain the reference medicinal material solution;

[0014] C) Perform thin-layer chromatography detection on the test solution and the reference medicinal material solution. The thin-layer plate is a silica gel G thin-layer plate; the developing agent is cyclohexane-ethyl acetate;

[0015] D) Examine under an ultraviolet lamp. In the chromatogram of the test sample, if there is a blue fluorescent spot at an Rf value of 0.31 - 0.464, it is Lycopodiastrum casuarinoides formula granules; if there is no blue fluorescent spot at an Rf value of 0.321 - 0.464, it is Lycopodium clavatum formula granules.

[0016] The pretreatment in step A) of the present invention is specifically: take the sample to be tested, dissolve it with petroleum ether, heat under reflux for extraction, discard the petroleum ether solution, decoct with water, filter, ultrasonically treat with methanol, filter, and dissolve the residue with methanol to obtain the test solution.

[0017] Specifically, the mass-volume ratio of the sample to be tested, water, and methanol is: 1 g: 100 mL: 40 mL.

[0018] In some embodiments, the time for heating under reflux extraction is 2 - 3 h; the decocting time is 1.5 - 2 h; the ultrasonic treatment time is 30 - 40 min; the boiling point of petroleum ether is 60 - 90 °C.

[0019] In some embodiments, the time for heating under reflux extraction is 3 h; the decocting time is 2 h; the ultrasonic treatment time is 30 min; the boiling point of petroleum ether is 60 - 90 °C.

[0020] Take the reference medicinal material of Lycopodiastrum casuarinoides, and after pretreatment, obtain the reference medicinal material solution. Among them, the pretreatment is specifically: take the reference medicinal material of Lycopodiastrum casuarinoides, dissolve it with petroleum ether, heat under reflux for extraction, discard the petroleum ether solution, decoct with water, filter, ultrasonically treat with methanol, filter, and dissolve the residue with methanol to obtain the reference medicinal material solution.

[0021] Specifically, the mass-volume ratio of the reference crude drug, petroleum ether, water, and methanol is: 1 g: mL: 100 mL: 40 mL

[0022] In some embodiments, the time for heating under reflux extraction is 1 - 2 h; the time for decocting is 1.5 - 2 h; the time for ultrasonic treatment is 30 - 40 min; the boiling point of the petroleum ether is 60 - 90 °C.

[0023] In some embodiments, the time for heating under reflux extraction is 2 h; the time for decocting is 2 h; the time for ultrasonic treatment is 30 min; the boiling point of the petroleum ether is 60 - 90 °C.

[0024] Perform thin-layer chromatography detection on the test solution and the reference crude drug solution.

[0025] Preferably: Pipette the test solution and the reference crude drug solution, and spot them on the same silica gel G thin-layer plate respectively. Develop with the developing agent, take out, dry in the air, and examine under an ultraviolet lamp.

[0026] The ultraviolet light wavelength in the present invention is 365 nm.

[0027] The thin-layer plate in the present invention is a silica gel G thin-layer plate. It can be selected from the cuttable thin-layer chromatography plates of Tianjin Silida Technology Co., Ltd., Merck, and the prefabricated silica gel G plates of the Qingdao Marine Chemical Factory Branch.

[0028] The results show that the retention factor (Rf) values of the blue fluorescent spots at 3.193 - 5.130 cm from the origin on the three thin-layer plates are 0.424, 0.310, and 0.354 respectively, all meeting the identification requirements. It is used to identify the Shujincao formula granules.

[0029] Examine under an ultraviolet lamp. If there is a blue fluorescent spot in the test sample chromatogram at an Rf value of 0.31 - 0.464, it is the Shujincao formula granules; if there is no blue fluorescent spot at an Rf value of 0.31 - 0.464, it is the Shenjincao formula granules.

[0030] Wherein the Rf value is 0.31 - 0.464; specifically, it can be 0.421, 0.437, 0.424, 0.310, 0.354, 0.417, 0.428, 0.481, 0.460, 0.468, 0.459, and 0.409.

[0031] The sample application volume of the test solution in the present invention is 1 - 5 μL; the sample application volume of the reference crude drug solution is 1 - 3 μL.

[0032] In some preferred embodiments of the present invention, the mass ratio of cyclohexane - ethyl acetate is 5:4; the boiling point of the petroleum ether is 60 - 90 °C.

[0033] The inspection temperature of the present invention is 4°C to 35°C. The method of the present invention has good adaptability to different temperatures, and at the retention factor values of 0.417, 0.428, and 0.481, the Shujincao formula granules show blue fluorescent spots, while the Shenjincao formula granules have no spots.

[0034] The inspection humidity of the present invention is 32% rh to 75% rh. The method of the present invention has good adaptability to different humidities, and at the retention factor values of 0.584, 0.613, and 0.599, the Shujincao formula granules show blue fluorescent spots, while the Shenjincao formula granules have no spots.

[0035] The test sample of the present invention is Shujincao and / or Shenjincao formula granules.

[0036] The present invention provides a thin-layer identification method for Shujincao and Shenjincao formula granules, including: A) taking the test sample, and obtaining a test solution after pretreatment; B) taking the reference medicinal material of Shujincao, and obtaining a reference medicinal material solution after pretreatment; C) performing thin-layer chromatography detection on the test solution and the reference medicinal material solution, and the thin-layer plate is a silica gel G thin-layer plate; the developing agent is cyclohexane - ethyl acetate; D) observing under an ultraviolet lamp. In the test chromatogram, if there are blue fluorescent spots at the Rf value of 0.31 to 0.464, it is Shujincao formula granules; if there are no blue fluorescent spots at the Rf value of 0.31 to 0.464, it is Shenjincao formula granules. The thin-layer identification chromatographic method for Shujincao and Shenjincao formula granules established by the present invention uses the presence or absence of fluorescent spots within a specified retention factor value range in the thin-layer chromatogram as the identification point to quickly and effectively identify Shujincao and Shenjincao formula granules. Description of the Drawings

[0037] Figure 1 For the investigation of the sample application amount;

[0038] Figure 2 For the investigation of specificity;

[0039] Figure 3 For the investigation of different thin-layer plates - Tianjin Silida.

[0040] Figure 4 Investigation of different thin-layer plates - Merck;

[0041] Figure 5 For the investigation of different thin-layer plates - Qingdao Ocean;

[0042] Figure 6 For different temperatures - 4°C;

[0043] Figure 7 For different temperatures - 25°C;

[0044] Figure 8 For different temperatures - 35°C;

[0045] Figure 9 For different humidities - 32%;

[0046] Figure 10 For different humidities - 51%;

[0047] Figure 11 For different humidities - 75%;

[0048] Figure 12 For the verification results of multiple batches;

[0049] Figure 13 Result diagram of Comparative Example 1 of the present invention;

[0050] Figure 14 Result diagram of the method of Comparative Example 2;

[0051] Figure 15 It is the result diagram of the method of Comparative Example 2. Detailed implementation manners

[0052] In order to further illustrate the present invention, the following describes in detail a thin-layer identification method for a formula granule of Lycopodiastrum casuarinoides and Lycopodium clavatum provided by the present invention in combination with embodiments.

[0053] Water bath, Soxhlet extractor, heating plate, thin-layer imaging system: CAMAG TLC Visualizer, silica gel G thin-layer plate (Qingdao Ocean Chemical Factory, batch number: 20180527, Tianjin Silitda Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0054] Petroleum ether (60 - 90°C), methanol, cyclohexane, ethyl acetate are all of analytical grade, and water is ultrapure water (self-made in the laboratory).

[0055] Control medicinal material of Lycopodiastrum casuarinoides (National Institutes for Food and Drug Control, batch number: 380003 - 202104), formula granule of Lycopodiastrum casuarinoides (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2302078, 2302079, 2302080); formula granule of Lycopodium clavatum (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2002001; 2002002; 2002003).

[0056] Example 1

[0057] Preparation of test solution

[0058] Take 1 g of this product, grind it finely, add an appropriate amount of petroleum ether (60 - 90 °C), place it in a Soxhlet extractor, heat under reflux for 3 hours, discard the petroleum ether solution, evaporate the solvent from the medicinal residues, add 100 ml of water, decoct for 2 hours, filter, evaporate the filtrate to dryness, add 40 ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), take 1 μl of the test solution and 1 μl of the reference medicinal material solution, respectively spot them on the same silica gel G thin-layer plate, use cyclohexane - ethyl acetate (5:4) as the developing agent, develop, take out, dry in air, and examine under ultraviolet light (365 nm). In the chromatogram of the test solution, at the corresponding position to the chromatogram of the reference medicinal material, fluorescent spots of the same color appear.

[0059] Preparation of reference substance and reference medicinal material solution

[0060] Take 2 g of the medicinal material powder, add an appropriate amount of petroleum ether (60 - 90 °C), place it in a Soxhlet extractor, heat under reflux for 2 hours, discard the petroleum ether solution, evaporate the solvent from the medicinal residues, add 100 ml of water, decoct for 2 hours, filter, evaporate the filtrate to dryness, add 40 ml of methanol to the residue, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to obtain the reference medicinal material solution.

[0061] Methodology investigation of Example 2

[0062] 2.1 Investigation of spotting volume

[0063] Under the above - established experimental conditions, take 1 μl, 3 μl, 5 μl, 10 μl, 15 μl, and 20 μl of the reference medicinal material solution of Lycopodiastrum casuarinoides and the test solution of Lycopodiastrum casuarinoides formula granules respectively, spot them on the same silica gel G thin-layer plate. The thin-layer chromatographic fluorescent spots are clearly developed. It is determined that the spotting volume of the reference medicinal material solution is 1 - 3 μl, and the spotting volume of the test solution is 1 - 5 μl. Spots of the same color appear at the position with a retention factor (Rf) of 0.421, which can be used as the identification point for Lycopodiastrum casuarinoides formula granules.

[0064] 2.2 Investigation of specificity

[0065] Prepare the reference medicinal material solution of Lycopodiastrum casuarinoides, the Lycopodiastrum casuarinoides formula granule solution, and the negative solution respectively according to the above test solution preparation method, conduct thin-layer identification experiments, and develop according to the established chromatographic conditions. The results are shown in Figure 2 , as can be seen from the figure, the negative sample has no interference on the test solution of Lycopodiastrum casuarinoides formula granules, and this method has good specificity. It can be used to identify Lycopodiastrum casuarinoides formula granules.

[0066] 2.3 Investigation of durability

[0067] 2.3.1 Comparison of different thin-layer plates

[0068] Select the cuttable thin-layer chromatography plates of Tianjin Silida Technology Co., Ltd., Merck, and the prefabricated silica gel G plates of Qingdao Ocean Chemical Factory Branch, and conduct tests according to the proposed test methods. See Figures 3 - 5 The results show that the Rf values of the blue fluorescent spots at 3.193 - 5.130 cm from the origin on the three thin-layer plates are 0.424, 0.310, and 0.354 respectively, all meeting the identification requirements. It is used to identify the Shujincao formula granules.

[0069] The results are as Figure 3 , Figure 3 For the investigation of different thin-layer plates - Tianjin Silida. Among them: 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0070] Figure 4 For the investigation of different thin-layer plates - Merck; among them: 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0071] Figure 5 For the investigation of different thin-layer plates - Qingdao Ocean; among them 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0072] 2.3.2 Comparison at different temperatures

[0073] Take the spotted thin-layer plates and develop them respectively in the temperature environments of low temperature 4°C, normal temperature 25°C, and high temperature 35°C. It can be seen from Figures 6 - 8 that this method has good adaptability to different temperatures, and the Rf values of the blue spots are 0.417, 0.428, and 0.481 respectively. It is used to identify the Shujincao formula granules.

[0074] Figure 6 For different temperatures - 4°C; among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0075] Figure 7 For different temperatures - 25°C, among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0076] Figure 8 For different temperatures - 35°C, among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0077] 2.3.3 Comparison at different humidities

[0078] Take the spotted thin-layer plates and develop them respectively in the humidity environments of 32% rh, 51% rh, and 75% rh. See Figures 9 - 11As can be seen from the figure, the method has good adaptability to different temperatures, and the Rf values of the blue spots are 0.584, 0.613, and 0.599 respectively, which are used to identify the Shujincao formula granules.

[0079] Figure 9 For different humidities -32%; among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0080] Figure 10 For different humidities -51%; among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0081] Figure 11 For different humidities -75%; among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0082] 2.3.4 Verification

[0083] The thin-layer chromatography identification of 3 batches of Shujincao formula granule spots was verified. The Rf value of the blue fluorescent spot at 5.03 cm from the origin was 0.409. The experimental results are shown in Figure 12 .

[0084] Figure 12 For the verification results of multiple batches; among them, 1 is the solution of the control medicinal material of Shujincao; 2 - 4 are the solutions of the Shujincao formula granules.

[0085] 2.4 Distinguishing between Shujincao formula granules and Shenjincao formula granules

[0086] The solutions of Shujincao formula granules and Shenjincao formula granules were prepared respectively according to the above-mentioned test sample preparation method, and a thin-layer chromatography identification experiment was carried out. The Rf value of the blue fluorescent spot of Shujincao formula granules at 5.2 cm from the origin was 0.591, while there was no spot for Shenjincao formula granules at this position. The results are shown in Figure 13 , among which, 1, 2 are Shujincao formula granules; 3, 4 are Shenjincao formula granules; as can be seen from the figure, this method can be used to distinguish Shujincao formula granules and Shenjincao formula granules.

[0087] 2.5 Determination of the Rf value of the identification points of Shujincao formula granules and Shenjincao formula granules

[0088] The Rf value data of each item in the thin-layer chromatography methodology investigation were summarized, and the results are shown in Table 1. According to the summary results, it was determined that the Rf value of the identification spot of Shujincao formula granules should be within the range of 10%, and the specified value is 0.482.

[0089] Table 1 Summary of Rf values in methodology investigation

[0090]

[0091] Control Example 1

[0092] Take 2 g of this product, grind it finely, add 40 mL of water and decoct for 3 hours, filter, evaporate the filtrate to dryness, add 20 mL of methanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution. Separately, take 1 g of the control crude drug of Lysimachia christinae Hance and 2 g of the negative control without Lysimachia christinae Hance, and prepare the control crude drug solution and the negative control solution in the same method. Pipette 5 μL of each of the above solutions and spot them on the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate (8:5) as the developing solvent, take out, and air dry. Examine under ultraviolet light (365 nm). No red spots appeared at the corresponding positions in the test solution chromatogram compared with the control crude drug chromatogram, and there was no interference from the negative control. The results are shown in Figure 14 . Figure 14 The result diagram of Control Example 1 of the present invention. Among them: 1-2 are the control crude drug solution of Lysimachia christinae Hance; 3-4 are the formula granules solution of Lysimachia christinae Hance; 5 is the negative control solution. The results show that by using the above-mentioned thin-layer identification method, the spots of the control crude drug of Lysimachia christinae Hance are inconsistent with those of the formula granules finished product, and the two cannot be distinguished from each other.

[0093] Control Example 2

[0094] Take 2 g of this product, grind it finely, add 5 mL of ammonia water and stir evenly, then add 20 mL of chloroform, heat under reflux for 2 hours, filter, place the filtrate in a separating funnel, add 20 mL of 2% sulfuric acid solution and shake, take the acid aqueous layer, adjust the pH to 8-9 with sodium hydroxide solution, place it in a separating funnel, extract with chloroform, 10 mL each time for a total of two times, concentrate the chloroform extract to 2 mL to obtain the test solution. Separately, take 2 g of the control crude drug of Lysimachia christinae Hance and 2 g of the negative control without Lysimachia christinae Hance, and prepare the control crude drug solution and the negative control solution in the same method. Pipette 10 μL of each of the above solutions and spot them on the same silica gel G thin-layer plate. Develop with chloroform-methanol-acetone-ammonia water (40:5:5:2) as the developing solvent, take out, and air dry. Color with an equal mixture of iodine-potassium iodide and bismuth potassium iodide. Examine under daylight. Yellow-brown spots appeared at the corresponding positions in the test solution chromatogram compared with the control crude drug chromatogram, and there was no interference from the negative control. The results are shown in Figure 15 .

[0095] Figure 15 It is the result diagram of the method of Control Example 2. 1 Negative control solution; 2 Control crude drug solution of Lysimachia christinae Hance; 3-5 are the formula granules solution of Lysimachia christinae Hance.

[0096] The results show that by using the above-mentioned thin-layer identification method, the developed spots of the formula granules of Lysimachia christinae Hance are too few and not beautiful enough, so the two cannot be distinguished.

[0097] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A thin-layer identification method for Herba Lycopodiellae and Herba Lycopodii formula granules, comprising: A) Take the test sample, and after pretreatment, obtain the test solution; The specific pretreatment is as follows: Take the test sample, dissolve it in petroleum ether, heat under reflux for extraction, discard the petroleum ether solution, decoct with water, filter, ultrasonically treat with methanol, filter again, dissolve the residue in methanol to obtain the test solution; B) Take the reference medicinal material of Herba Lycopodiellae, and after pretreatment, obtain the reference medicinal material solution; The specific pretreatment is as follows: Take the reference medicinal material of Herba Lycopodiellae, dissolve it in petroleum ether, heat under reflux for extraction, discard the petroleum ether solution, decoct with water, filter, ultrasonically treat with methanol, filter again, dissolve the residue in methanol to obtain the reference medicinal material solution; C) Perform thin-layer chromatography detection on the test solution and the reference medicinal material solution. The thin-layer plate is a silica gel G thin-layer plate; The developing agent is cyclohexane-ethyl acetate; The mass ratio of cyclohexane to ethyl acetate is 5:4; D) Examine under an ultraviolet lamp. In the chromatogram of the test sample, if there are blue fluorescent spots at an Rf value of 0.31 - 0.464, it is Herba Lycopodiellae formula granules; If there are no blue fluorescent spots at an Rf value of 0.31 - 0.464, it is Herba Lycopodii formula granules.

2. The method according to claim 1, wherein In the pretreatment of the test sample, the time for heating under reflux extraction is 2 - 3 h; The decocting time is 1.5 - 2 h; The time for ultrasonic treatment is 30 - 40 min; The boiling point of the petroleum ether is 60 - 90 °C; The mass-volume ratio of the test sample, water, and methanol is: 1 g : 100 mL : 40 mL; In the pretreatment of the reference medicinal material of Herba Lycopodiellae, the time for heating under reflux extraction is 1 - 2 h; The decocting time is 1.5 - 2 h; The time for ultrasonic treatment is 30 - 40 min; The boiling point of the petroleum ether is 60 - 90 °C.

3. The method according to claim 1, wherein The spotting volume of the test solution is 1 - 5 μL; The spotting volume of the reference medicinal material solution is 1 - 3 μL.

4. The method according to claim 1, characterized in that The wavelength of the ultraviolet lamp in step D) is 365 nm.

5. The method according to claim 1, characterized in that The temperature for examination in step D) is 4 °C - 35 °C.

6. The method according to claim 5, wherein The humidity for examination in step D) is 32%rh - 75%rh.

7. The method according to claim 1, wherein The test sample in step A) is Herba Lycopodiellae and / or Herba Lycopodii formula granules.

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