阅读说明：本技术 一种单面针薄层色谱鉴别方法 (Single-side needle thin-layer chromatography identification method ) 是由 凌勇根 龚云 白璐 王月秀 何娟娟 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种单面针薄层色谱鉴别方法。该方法以木兰花碱和氯化两面针碱为对照品,明确了单面针薄层色谱的特有斑点,可用于鉴别单面针、区分单面针与两面针。该方法重复性好、准确度高、分离好,斑点清晰,且简单可行、快速、成本低,适合用于区分单面针与两面针,以及单面针药材的质量控制和真伪鉴别。而且该方法安全性好、经济环保,安全,效率高,应用前景好。(The invention discloses a single-side needle thin-layer chromatography identification method. The method uses magnoflorine and nitidine chloride as reference substances, defines the unique spot of single-side needle thin layer chromatography, and can be used for identifying single-side needle and distinguishing single-side needle from double-side needle. The method has the advantages of good repeatability, high accuracy, good separation, clear spots, simplicity, feasibility, rapidness and low cost, and is suitable for distinguishing single-face needles from double-face needles, and quality control and authenticity identification of single-face needles. The method has the advantages of good safety, economy, environmental protection, safety, high efficiency and good application prospect.)

1. A single-side needle thin-layer chromatography identification method is characterized by comprising the following steps:

(1) preparing a test solution and ethanol as a solvent;

(2) preparing reference substance solutions, wherein the reference substances are respectively magnoflorine, nitidine chloride and ethanol solvent;

(3) and (3) thin-layer chromatography detection:

respectively dropping the reference solution and the sample solution on the same silica gel thin layer plate, taking saturated developing agent vapor of chloroform, methanol, water and diethylamine as developing agent, taking out, drying, and inspecting;

(4) and (5) inspecting and analyzing results: in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatogram of the control: if there are two red spots at Rf (0.76-0.88) and Rf (0.90-0.98), the test article is a single-face needle, and both the double-face needle and the counterfeit have no such spots.

2. The single-needle thin layer chromatography identification method according to claim 1, wherein the preparation of the test solution in step (1): according to (1.2-2.8 g): (30-50ml) adding the sample into ethanol, performing ultrasonic treatment for 60-90min under sealed condition, filtering, evaporating in water bath, and dissolving the residue with ethanol to obtain sample solution.

3. The single jerusalem artichoke thin-layer chromatography identification method according to claim 1, wherein the concentration of the magnoflorine reference substance in the step (1) is 0.8-1.5mg/mL, and the concentration of the nitidine chloride reference substance is 0.4-0.7 mg/mL.

4. The single-side needle thin-layer chromatography identification method of claim 1, wherein the developing solvent is chloroform, methanol, water, diethylamine (35-45), water (8-12), water (0.5-1.2), water (0.06-0.12).

5. The single-side needle thin layer chromatography identification method of claim 1, wherein in the step (3), the developing agent steam is developed after reaching saturation for 15-30min to be developed to 6-12 cm.

6. The single-face needle thin layer chromatography identification method according to claim 1, characterized in that the spot sample amount in step (3): 5-10 mul of reference solution and 5-10 mul of test solution.

7. The single-face needle thin-layer chromatography identification method of claim 1, wherein the silica gel thin-layer plate in the step (3) is a silica gel GF254 thin-layer plate.

8. The single-needle thin layer chromatography identification method of claim 1, wherein the drying manner in the step (3) is air drying.

9. The single-side needle thin-layer chromatography identification method of claim 1, wherein the color development mode is iodine fumigation until the color development is clear; the inspection methods are 254nm, 366nm before color development and visible light after color development.

10. A single jersey thin layer chromatography identification method according to any one of claims 1 to 9, comprising the steps of:

(1) preparing a test solution:

sieving 2g of test sample powder with a second sieve, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in water bath, and dissolving the residue with 2ml of ethanol to obtain test sample solution;

(2) preparation of control solutions:

taking a magnoflorine reference substance, and adding ethanol to prepare a reference substance solution of 1 mg/mL;

adding ethanol into nitidine chloride reference to obtain 0.5mg/mL reference solution;

(3) and (3) thin-layer chromatography detection:

respectively dropping 5 μ l of reference solution and 5-10 μ l of sample solution on the same silica gel GF254 thin layer plate, taking chloroform, methanol, water, diethylamine (40:10:1:0.1) as developing agent, developing after the developing agent steam reaches saturation for 20min, spreading to 8cm, taking out, air drying, and inspecting at 366 nm;

(4) and (5) inspecting and analyzing results: in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatogram of the control: if there are two red spots at Rf 0.85 and Rf 0.95, the test article is a single-side needle, but there are no such spots on both sides of the needle and the counterfeit.

Technical Field

The invention belongs to the technical field of medicines. More particularly, it relates to a single-side needle thin-layer chromatography identification method.

Background

The Zanthoxylum nitidum (Zanthoxylum bungeanum) is Zanthoxylum bungeanum (Zanthoxylum dissitum Hemsley) and Zanthoxylum spiniferum (Z.echinocarpum Hemsley) evergreen woody lian, and the Zanthoxylum bungeanum is mainly named as Zanthoxylum bungeanum and is recorded in Chinese monograph, Hunan province Chinese medicinal material standard, Guangxi characteristic Chinese medicinal herb resource selective edition and other Chinese medicinal monographs. Folk therapy is commonly used for treating lumbago, toothache, traumatic injury, hernia pain, menorrhagia, etc. The root, stem and leaf can be used as medicine, has high medicinal value, and is mainly used for treating toothache, lumbago, menorrhagia, postpartum irregular menstruation and the like. The single-side needle mainly uses the clam shell pepper as the main raw material medicine of Chinese patent medicines such as gynecological Qianjin capsules, gynecological Qianjin tablets and the like, and has wide application.

The Zanthoxylum nitidum is a traditional Chinese medicine with a long history in China, and has been recorded in the Chinese pharmacopoeia. Radix Zanthoxyli has effects of promoting blood circulation for removing blood stasis, activating qi-flowing, relieving pain, dispelling pathogenic wind, dredging collaterals, removing toxic substance and detumescence, and is mainly used for traumatic injury, stomach ache, toothache, rheumatalgia, venomous snake bite; it can be used for external treatment of burn and scald.

The single-sided needle and the double-sided needle are extremely similar in appearance and difficult to distinguish, and the chemical components of the single-sided needle and the double-sided needle are also similar. The phenomenon of serious confusion and use of single-sided needles and double-sided needles is caused because the single-sided needles lack perfect quality control standards. Therefore, the study of the single-sided needle and the double-sided needle is more and more focused by the researchers. For example, in patent 200710064499.2 of the applicant, in order to better control the quality of the single-sided needle decoction pieces and improve the standard of the single-sided needle decoction pieces, the applicant group continuously studies the standard of the single-sided needle decoction pieces, so as to improve the control index of the single-sided needle decoction pieces and effectively ensure the quality of the single-sided needle decoction pieces.

Disclosure of Invention

The invention aims to provide a thin-layer chromatography detection method for identifying single-sided needles and double-sided needles, and provides a brand new method for distinguishing the single-sided needles from the double-sided needles and for quality control and authenticity identification of single-sided needle medicinal materials. The invention obtains a thin-layer chromatography characteristic spot which can be used for identifying single-sided needles and double-sided needles and controlling the quality and the authenticity of the single-sided needles through a large number of experiments, constructs a thin-layer chromatography detection method for identifying the single-sided needles and the double-sided needles, has good repeatability, high accuracy, simplicity, feasibility, quickness and low cost, and is suitable for distinguishing the single-sided needles and the double-sided needles and controlling the quality and identifying the authenticity of the single-sided needle medicinal materials.

The above purpose of the invention is realized by the following technical scheme:

a single-side needle thin-layer chromatography identification method comprises the following steps:

(1) preparing a test solution and ethanol as a solvent;

(2) preparing reference substance solutions, wherein the reference substances are respectively magnoflorine, nitidine chloride and ethanol solvent;

(3) and (3) thin-layer chromatography detection:

respectively dropping the reference solution and the sample solution on the same silica gel thin layer plate, taking saturated developing agent vapor of chloroform, methanol, water and diethylamine as developing agent, taking out, drying, and inspecting;

(4) and (5) inspecting and analyzing results: in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatogram of the control: two red spots (named as DD-1 and DD-2 respectively) are obvious at Rf (0.76-0.88) and Rf (0.90-0.98), so that the test article is a single-side needle, and the two-side needle and the fake article have no spots.

In the above method, in order to more accurately control the stability and accuracy of the discrimination result, it is preferable that the preparation of the test solution in step (1): according to (1.2-2.8 g): (30-50ml) adding the sample into ethanol, performing ultrasonic treatment for 60-90min under sealed condition, filtering, evaporating in water bath, and dissolving the residue with ethanol to obtain sample solution.

Preferably, the concentration of the magnoflorine reference substance in the step (1) is 0.8-1.5mg/mL, and the concentration of the nitidine chloride reference substance is 0.4-0.7 mg/mL.

More preferably, the concentration of the magnoflorine control is 1mg/mL, and the concentration of the nitidine chloride control is 0.5 mg/mL.

Preferably, the developing solvent is chloroform, methanol, water, diethylamine (35-45), 8-12, 0.5-1.2, 0.06-0.12.

More preferably, the developing solvent is chloroform, methanol, water, diethylamine, 40:10:1: 0.1.

Preferably, in the step (3), the developing agent steam is developed after reaching saturation for 15-30min to be developed to 6-12 cm.

More preferably, the developing agent steam is developed to 8cm after reaching saturation for 20min in the step (3).

Preferably, the dot sample amount in step (3): 5-10 mul of reference solution and 5-10 mul of test solution.

More preferably, the dot sample amount in step (3): 5 mul of the reference solution and 5-10 mul of the test solution.

Preferably, the silica gel thin layer plate in the step (3) is a silica gel GF254 thin layer plate.

Preferably, the drying mode is air drying.

Preferably, the color development mode is that the iodine is smoked to be clear.

Preferably, the inspection is carried out at 254nm, 366nm and visible light after development.

Further preferably, the ethanol used herein is 95% ethanol.

As a most preferred embodiment, the single-side needle thin layer chromatography identification method comprises the following steps:

(1) preparing a test solution:

sieving 2g of test sample powder with a second sieve, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating to dryness in water bath, dissolving the residue with 2ml of ethanol to obtain test sample solution (preferably dissolving with 2ml of ethanol, filtering, and using as test sample solution);

(2) preparation of control solutions:

taking a magnoflorine reference substance, and adding ethanol to prepare a reference substance solution of 1 mg/mL;

adding ethanol into nitidine chloride reference to obtain 0.5mg/mL reference solution;

(3) and (3) thin-layer chromatography detection:

respectively dropping 5 μ l of reference solution and 5-10 μ l of sample solution on the same silica gel GF254 thin layer plate, taking chloroform, methanol, water, diethylamine (40:10:1:0.1) as developing agent, developing after the developing agent steam reaches saturation for 20min, spreading to 8cm, taking out, air drying, and inspecting at 366 nm;

(4) and (5) inspecting and analyzing results: in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatogram of the control: two red spots (named as DD-1 and DD-2 respectively) are obvious near Rf & lt 0.85 and Rf & lt 0.95, so that the test article is a single-face needle, and the two-face needle and the fake article have no spots.

In addition, in order to obtain more accurate detection results, the method needs to be carried out under the condition that the humidity of the environment is not too high, and preferably below 50% RH (preferably 31% RH-45% RH).

The invention has the following beneficial effects:

the invention provides a thin-layer chromatography detection method for identifying single-side needles and double-side needles, which defines the special spots of the thin-layer chromatography of the single-side needles and can be used for identifying the single-side needles and distinguishing the single-side needles from the double-side needles. The method has the advantages of good repeatability, high accuracy, simplicity, feasibility, rapidness and low cost, is suitable for distinguishing the single-side needles from the double-side needles and is suitable for quality control and authenticity identification of the single-side needles, and has good application prospect.

In addition, in the method, ethanol is used as an extraction solvent, so that the safety is better; the GF254 thin-layer plate has better separation degree and is more economic and environment-friendly; chloroform, methanol, water and diethylamine (40:10:1:0.1) are used as developing agents, so that the separation degree is better, and spots are clearer; the color development method is direct 366nm inspection, does not need a color development agent, such as 10% sulfuric acid ethanol heating treatment, can directly develop color, and is economical, safe and high in efficiency.

Drawings

FIG. 1 is a thin layer chromatogram of a single-side needle and a control; 1-blank solvent (ethanol), 2-magnoflorine reference substance solution, 3-chloridized nitidine reference substance solution, 4-10-single-side needle test sample, 11-14-double-side needle test sample and 15-17-single-side needle fake test sample.

FIG. 2 is a graph showing the examination results of the extraction solvent of the sample in example 4; 1-reference substance mixing, 2-Zanthoxylum nitidum 95% ethanol extraction, 3-70% ethanol extraction, 4-methanol extraction, and 5-ethyl acetate extraction.

FIG. 3 is a diagram showing the examination results of the sample extraction method in example 4; 1-reference substance mixing, 2-single-side needle Z2019030073-ultrasonic extraction, 3-reflux extraction and 4-water bath extraction.

FIG. 4 is a graph showing the examination result of the extraction time of the sample in example 4; 1-standard mixing of reference substances, 2-single-side needle Z2019030073-ultrasonic extraction for 30min, 3-ultrasonic extraction for 60min, and 4-ultrasonic extraction for 90 min.

FIG. 5 shows the results of examination of developer-1 in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 6 shows the results of examination of developer-2 in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 7 shows the results of examination of developer-3 in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 8 shows the results of examination of developer-4 in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 9 shows the results of examination of the developer-5 in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 10 shows the results of examination of developer-6 in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 11 shows the results of examining the first color development mode in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 12 shows the results of examining the first color development mode in example 4; 1-reference product mixed label, 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030075, 5-single-side needle Z2019030079, 6-single-side needle fake product Z2019030098 and 7-single-side needle fake product Z2019030099.

FIG. 13 is the result of thin layer chromatography identification after optimization in example 4; 1-reference substance mixed standard of-5 mul, 2-single-side needle Z2019030073-5 mul, 3-single-side needle Z2019030074-5 mul, 4-single-side needle Z2019030077-5 mul, 5-double-side needle Z2019030093-1 mul and 6-single-side needle counterfeit substance Z2019030098-1 mul.

FIG. 14 shows the results of the specificity test of the method (366 nm before the upper color development and visible light after the lower color development); 1-blank solvent (ethanol), 2-magnoflorine reference substance solution, 3-chloridized nitidine reference substance solution, 4-7-single-face needle test articles (with the batch numbers of Z2019030073, Z2019030074, Z2019030075 and Z2019030076 in sequence), 8-10-zanthoxylum spinosum test articles (with the batch numbers of Z2019030100, Z2019030101 and Z2019030102 in sequence), 11-14-double-face needle test articles (with the batch numbers of Z2019030093, Z2019030094, Z2019030095 and Z2019030096 in sequence), and 15-17-single-face needle fake test articles (with the batch numbers of Z2019030097, Z2019030098 and Z2019030099 in sequence).

FIG. 15 shows the results of examination at different temperatures (A-10 ℃, B-20 ℃, C-30 ℃, Nicotiana Summinck chemical industry institute/HSGF₂₅₄Left-366 nm before color development, right-visible light after color development); 1-mixing a reference substance solution (from top to bottom, nitidine chloride and magnoflorine), 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 16 shows the results of examination under different humidities (A-31% RH, B-45% RH, C-78% RH, Nicoti City chemical industry institute/HSGF₂₅₄Left-366 nm before color development, right-visible light after color development); 1-mixing a reference substance solution (from top to bottom, nitidine chloride and magnoflorine), 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077, 5-single-side needle Z2019030078 and 6-single-side needle Z2019030085.

FIG. 17 shows the results of examination of different spotting patterns (A-dots, B-bands, Nicotine chemical industry institute/HSGF)₂₅₄Left-366 nm before color development, right-visible light after color development); 1-mixed reference substance solution (from top to bottom, nitidine chloride and magnoflorine), 2-single-side needle Z2019030073, 3-single-side needle Z2019030074, 4-single-side needle Z2019030077 and 5-single-side needle Z2019030077Z2019030078 and 6-single-side needle Z2019030085.

FIG. 18 shows the results of the solution stability test of the test sample and the control sample (HSGF/HSGF of the institute of chemical industry of the cigarette Taishi city)₂₅₄Left-366 nm before color development, right-visible light after color development); 1-mixing reference substance solution (nitidine chloride and magnoflorine in sequence from top to bottom) and 2-6-single-side needle test samples (0h, 8h, 48h, 72h and 96 h).

FIG. 19 shows the detection of multiple batches of single-side needle samples (HSGF/HSGF research institute of chemical industry in cigarette end)₂₅₄Left-366 nm before color development, right-visible light after color development); 1-mixed reference substance solution (nitidine chloride and magnoflorine in sequence from top to bottom), and 2-17-single-face needle test sample (with batch numbers of Z2019030077, Z2019030078, Z2019030079, Z2019030080, Z2019030081, Z2019030082, Z2019030083, Z2019030084, Z2019030085, Z2019030086, Z2019030087, Z2019030088, Z2019030089, Z2019030090, Z2019030091 and Z2019030092 in sequence).

FIG. 20 is a comparison of thin layer identification of magnoflorine in a single needle of the experiment of comparative example 1; a: developing solvent (dichloromethane: methanol ═ 6: 4); b: developing agent (chloroform: methanol: 6: 4); c: developing solvent (chloroform: methanol: water: ammonia water: 6:4:1: 0.25).

FIG. 21 is a comparison graph of thin layer identification of magnoflorine in the second panel needle of experiment in comparative example 1 (A: 4. mu.L; B: 8. mu.L; C: 10. mu.L).

FIG. 22 is a thin layer identification chart of method 14 two-faced needles (LS 26-LS 39) and 13 lot single-faced needles (S1-S13) of comparative example 1 for magnoflorine.

FIG. 23 is a photograph showing the results of examination of comparative example 2 (254nm before development) (HSGF 254, national institute of chemical industry, Nicoti).

FIG. 24 is a view showing the examination of the method of comparative example 2 (366 nm before coloration, HSGF254, national institute of chemical industry, Nicoti); 1-chelerythrine, 2-magnoflorine, 3-dictamnine, 4-chloridized nitidine, 5-single-side needle Z2019030073, 6-single-side needle fake Z2019030097, 7-single-side needle fake Z2019030098, 8-single-side needle fake Z2019030099, 9-double-side needle Z2019030093, 10-double-side needle Z2019030094, 11-double-side needle Z2019030095, 12-double-side needle Z2019030096, 13-different-base single-side needle Z2019030100, 14-different-base single-side needle Z2019030101 and 15-different-base single-side needle Z9030102.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

The experimental instruments and materials used in the following examples were derived as follows:

TABLE 1 apparatus

TABLE 2 reagents

Serial number	Name of article	Specification of	Purity of	Batch number	Source
						1	Methanol	500ml	Analytical purity	P1441123	General-Reagent
2	95% ethanol	500ml	Analytical purity	P1433419	General-Reagent
						3	Trichloromethane	500ml	Analytical purity	P1318320	General-Reagent
4	Ethanol (Anhydrous)	500ml	Analytical purity	P1437228	General-Reagent
						5	Ethyl acetate	500ml	Analytical purity	P1429095	General-Reagent
6	Aqueous ammonia	500ml	Analytical purity	P1375833	General-Reagent
						7	Methylene dichloride	500ml	Analytical purity	P1454839	General-Reagent
8	Diethylamine	500ml	Analytical purity	P1382819	General-Reagent

TABLE 3 control

TABLE 4 sample sources

TABLE 5 sample batch number

Example 1 Single-needle thin-layer chromatography identification method

The thin-layer chromatography analysis is carried out by taking a single-side needle test sample, a double-side needle test sample and a single-side needle counterfeit test sample as samples and taking a blank ethanol solvent as a reference, and comprises the following steps:

(1) preparing a test solution:

taking 2g of sample powder (sieved by a No. two sieve), placing in a conical flask with a plug, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in water bath, and dissolving residues in 2ml of ethanol to obtain a sample solution;

(2) preparation of control solutions:

taking a magnoflorine reference substance, and adding ethanol to prepare a reference substance solution of 1 mg/mL;

adding ethanol into nitidine chloride reference to obtain 0.5mg/mL reference solution;

the ethanol used was 95% ethanol.

(3) And (3) thin-layer chromatography detection: (refer to Chinese pharmacopoeia 2015 year edition four parts appendix 0502)

Respectively dispensing 5 μ l of reference solution and 8 μ l of sample solution on the same silica gel GF254 thin layer plate, wherein the sample dispensing mode is 8mm strip, chloroform, methanol, water, diethylamine (40:10:1:0.1) are used as developing agent, the developing agent steam is saturated for 20 minutes and then developed to 8cm, and the solution is taken out, dried and inspected under 366 nm;

(4) and (5) inspecting and analyzing results: in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatogram of the control: there were two red spots evident at Rf-0.85 and Rf-0.95 (named DD-1 and DD-2, respectively). As shown in figure 1, the single-side needle fake product and the double-side needle have no spots.

Example 2 Single-needle thin-layer chromatography identification method

A single-side needle thin-layer chromatography identification method comprises the following steps:

(1) preparing a test solution:

taking 1.2g of single-side needle sample powder (sieved by a No. two sieve), placing in a conical flask with a plug, adding 30ml of ethanol, sealing, performing ultrasonic treatment for 90min, filtering, evaporating in a water bath, and dissolving residues in 2ml of ethanol to obtain a sample solution;

(2) preparation of control solutions:

taking a magnoflorine reference substance, and adding ethanol to prepare a reference substance solution of 0.8 mg/mL;

adding ethanol into nitidine chloride reference to obtain 0.4mg/mL reference solution;

(3) and (3) thin-layer chromatography detection: (refer to Chinese pharmacopoeia 2015 year edition four parts appendix 0502)

Respectively dispensing 10 μ l of reference solution and 10 μ l of test solution on the same silica gel GF254 thin layer plate, wherein the dispensing mode is 8mm strip, developing after the developing agent steam reaches saturation for 15min by chloroform, methanol, water and diethylamine 35:8:0.5:0.06, spreading to 6cm, taking out, air drying, and inspecting under 254 nm;

(4) the test chromatogram showed two spots of the same color as the control chromatogram in example 1 at the positions corresponding to the control chromatogram: there were two red spots evident at Rf-0.85 and Rf-0.95 (named DD-1 and DD-2, respectively).

Example 3 Single-needle thin-layer chromatography identification method

A single-side needle thin-layer chromatography identification method comprises the following steps:

(1) preparing a test solution:

taking 2.8g of single-side needle sample powder (sieved by a No. two sieve), placing in a conical flask with a plug, adding 50ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in a water bath, and dissolving residues in 2ml of ethanol to obtain a sample solution;

(2) preparation of control solutions:

taking a magnoflorine reference substance, and adding ethanol to prepare a reference substance solution of 1.5 mg/mL;

adding ethanol into nitidine chloride reference to obtain 0.7mg/mL reference solution;

(3) and (3) thin-layer chromatography detection: (refer to Chinese pharmacopoeia 2015 year edition four parts appendix 0502)

Respectively dispensing 8 μ l of reference solution and 5 μ l of sample solution on the same silica gel GF254 thin layer plate, wherein the dispensing mode is 8mm strip, using chloroform, methanol, water and diethylamine 45:12:1.2:0.12 as developing agent, developing after the developing agent steam reaches saturation for 30min, spreading to 12cm, taking out, air drying, and inspecting under 366 nm;

(4) the test chromatogram showed two spots of the same color as the control chromatogram in example 1 at the positions corresponding to the control chromatogram: there were two red spots evident at Rf-0.85 and Rf-0.95 (named DD-1 and DD-2, respectively).

Example 4 Single-pin thin layer chromatography method optimization

This example presents optimization experiments and data (sample extraction method, developing agent, and color development method) of conditions in a part of single-side needle thin-layer chromatography identification methods.

1. Optimization of test article extraction process

(1) Investigation of sample extraction solvent

Preparation of a test solution: taking 2g of sample powder (sieved by a second sieve), placing in a conical flask with a plug, respectively adding 95% ethanol, 70% ethanol, methanol and 40ml of ethyl acetate, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in water bath, and dissolving the residue with 1ml of ethanol to obtain a sample solution.

Preparation of control solutions: adding ethanol into magnoflorine control solution to obtain control solution containing 1mg per 1 ml. Chlorinating nitidine control, adding ethanol to make control solution containing 0.5mg per 1 ml.

Sample amount of spotting: control (5 μ l); test article (10 μ l)

Sample application mode: strip, 8mm

Developing agent: chloroform methanol water diethylamine (40:10:1:0.1)

Spreading to: 8cm

And (3) drying mode: air drying

The inspection method comprises the following steps: after development, the film is dried in the air and inspected at 254nm and 366 nm.

The results are shown in FIG. 2: the extraction effect of 95% ethanol, 70% ethanol and methanol is relatively consistent, and the extraction component of ethyl acetate is less. In general terms, 95% ethanol was chosen as the extraction solvent.

(2) Examination of sample extraction method

Preparation of a test solution: taking 2g of sample powder (sieved by a second sieve), placing in a conical flask with a plug, adding 40ml of 95% ethanol, sealing, performing reflux treatment for 60min with ultrasound and water bath at 50 ℃, filtering, evaporating in water bath, and dissolving residue with 1ml of ethanol to obtain a sample solution.

Preparation of control solutions: adding ethanol into magnoflorine control solution to obtain control solution containing 1mg per 1 ml. Chlorinating nitidine control, adding ethanol to make control solution containing 0.5mg per 1 ml.

Sample amount of spotting: control (5 μ l); test article (10 μ l)

Sample application mode: strip, 8mm

Developing agent: chloroform methanol water diethylamine (40:10:1:0.1)

Spreading to: 8cm

And (3) drying mode: air drying

The inspection method comprises the following steps: inspection was performed at 254nm and 366 nm.

The results are shown in FIG. 3: the extraction effects of the three extraction modes are relatively consistent. And (4) considering all factors, and selecting ultrasound as an extraction mode.

(3) Investigation of sample extraction time

Preparation of a test solution: taking 2g of sample powder (sieved by a second sieve), placing in a conical flask with a plug, adding 40ml of 95% ethanol, sealing, performing ultrasonic treatment for 30min, 60min and 90min respectively, filtering, evaporating in water bath, and dissolving the residue with 1ml of ethanol to obtain a sample solution.

Preparation of control solutions: adding ethanol into magnoflorine control solution to obtain control solution containing 1mg per 1 ml. Chlorinating nitidine control, adding ethanol to make 1mg of control solution per 1 ml.

Sample amount of spotting: control (5 μ l); test article (10 μ l)

Sample application mode: strip, 8mm

Developing agent: chloroform methanol water diethylamine (40:10:1:0.1)

Spreading to: 8cm

And (3) drying mode: air drying

The inspection method comprises the following steps: inspection was performed at 254nm and 366 nm.

The results are shown in FIG. 4: the extraction effect is consistent with that of 90min at 60min, and the chromatographic band is slightly shallow after 30min of extraction. 60min was selected as the extraction time.

2. Optimization of developing agents

The developing agents are respectively:

developer-1: chloroform methanol water ammonia (6:4:1:0.25)

Developer-2: chloroform acetone methanol formic acid (15:0.5:0.5:1)

Developing agent-3: chloroform methanol (25:1)

Developer-4: chloroform methanol water ammonia (40:10:1:0.2)

Developing agent-5: chloroform methanol water diethylamine (40:10:1:0.2)

Developer-6: chloroform, methanol, water, diethylamine (40:10:1: 0.1).

Preparation of a test solution: taking 2g of sample powder (sieved by a second sieve), placing in a conical flask with a plug, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in a water bath to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution.

Preparation of control solutions: magnoflorine and nitidine chloride reference substances are added with ethanol to prepare reference substance solution containing 1mg per 1 ml.

Sample amount of spotting: control (5 μ l); test article (10 μ l)

Sample application mode: strip, 8mm

Spreading to: 8cm

And (3) drying mode: air drying

The inspection method comprises the following steps: inspection was performed at 254nm and 366 nm.

The results are shown in FIGS. 5-10:

developer-1 (fig. 5): the corresponding positions of the reference substance and the test substance have the same spots which are clearly visible, but the Rf of the nitidine chloride is too high, and the strip is shielded.

Developer-2 (fig. 6): the overall band was clear, but the control shift value was too low.

Developer-3 (fig. 7): the band smeared and the control shift value was too low.

Developer-4 (fig. 8): the band smeared and the control shift value was too low.

Developer-5 (fig. 9): the band smeared and the control shift value was too low.

Developer-6 (fig. 10): the specific shift value of the reference strip is proper and is in the range of 0.2-0.8.

In conclusion, the single-side needle thin layer identification developing agent is determined to be developing agent-6, namely chloroform, methanol, water and diethylamine (40:10:1: 0.1).

3. Investigation of different color development modes

Preparation of a test solution: taking 2g of sample powder (sieved by a second sieve), placing in a conical flask with a plug, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in a water bath to dryness, and dissolving residues in 1ml of ethanol to obtain a sample solution.

Preparation of control solutions: magnoflorine and nitidine chloride reference substances are added with ethanol to prepare reference substance solution containing 1mg per 1 ml.

Sample amount of spotting: control (5 μ l); test article (5 μ l)

Sample application mode: strip, 8mm

Developing agent: chloroform methanol water diethylamine (40:10:1:0.1)

Spreading to: 8cm

And (3) drying mode: air drying

The color development modes are respectively as follows:

the first color development mode: spraying diluted potassium bismuth iodide, standing for 10min, spraying 10% sodium nitrite 50% ethanol, heating to 105 deg.C, and developing.

And a second color development mode: fumigating with iodine until the color is clear.

The results are shown in FIGS. 11-12:

first color development mode (see fig. 11): no obvious optimization is performed after the color development, and the color development method is not selected.

Color development method two (see fig. 12): after the color development, one more identification spot is arranged at the position of the ratio shift value of 0.1 between the single-side needle and the fake product, and the color development method is selected.

In conclusion, through the investigation of the experimental parameters, the optimized thin-layer chromatography method has the following conditions:

preparation of a test solution: taking 2g of sample powder (sieved by a second sieve), placing in a conical flask with a plug, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in a water bath to dryness, and dissolving residues in 2ml of ethanol to obtain a sample solution.

Preparation of control solutions: adding ethanol into magnoflorine reference substance to obtain 1mg/mL reference substance solution. Chlorinating nitidine reference substance, adding ethanol to make into 0.5mg/mL reference substance solution.

Sample amount of spotting: control (5 μ l); test article (5-10 μ l)

Sample application mode: strip, 8mm

Developing agent: chloroform methanol water diethylamine (40:10:1:0.1)

Spreading to: 8cm

And (3) drying mode: air drying

Color development method: fumigating with iodine until color development is clear

The inspection method comprises the following steps: 254nm and 366nm before color development and visible light after color development. The results are shown in FIG. 13.

Example 5 methodological validation of the Single-needle thin layer chromatography method

This example performs a methodological validation of the optimized thin layer chromatography method described above to demonstrate the scientific rationality, reproducibility and feasibility of the detection method.

1. Method overview after optimization (same as example 1): taking appropriate amount of magnoflorine and nitidine chloride reference substances, and adding ethanol to obtain 1ml reference substance solution containing 1mg and 0.5 mg. Performing thin layer chromatography (appendix 0502 of the four parts of the year 2015 in the Chinese pharmacopoeia), and sucking 5 μ l of the above control solution; dropping 5-10 μ l of sample solution on the same silica gel GF₂₅₄Spreading chloroform-methanol-water-diethylamine (40:10:1:0.1) as developing agent on the thin layer plate, after the developing agent steam is saturated for 20min, taking out, air drying, inspecting at 366nm, fumigating in iodine jar until the color is clear, and inspecting under visible light. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.

2. Method specificity

Thin-layer chromatography behaviors of 7 batches of single-face needle medicinal materials (including 3 batches of zanthoxylum bungeanum maxim), 4 batches of double-face needles and 3 batches of single-face needle counterfeits are examined. The results are shown in FIG. 14.

And (4) conclusion: the blank solvent (ethanol) has no interference to the chromatographic color spots. The spots of the same color are detected on the corresponding positions of the test sample and the reference sample. The method can be used as a discrimination point of a single-side needle (comprising different base sources), a double-side needle and a fake product, and has the advantages that fluorescent spots with the same color are displayed at positions corresponding to a reference product in a single-side needle sample chromatogram under the condition of 366nm before color development, and two bright red spots are respectively arranged at positions of Rf (0.85) and Rf (0.95).

3. Adaptation to different deployment temperatures

The development at 10 deg.C, 20 deg.C and 30 deg.C was examined, and the experimental results are shown in FIG. 15.

And (4) conclusion: when the method is inspected under 366nm before and visible light after development at different development temperatures, spots with the same color are displayed on the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference sample, which indicates that the method has good durability at 10-30 ℃.

4. Application of different spreading humidity

The results of examination are shown in FIG. 16, which shows the results of examination under the humidity conditions of 31% RH, 45% RH and 78% RH.

And (4) conclusion: when the method is inspected under 366nm before and under visible light after color development under the humidity of 31% RH and 45% RH, spots with the same color are displayed on the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference sample, which shows that the method has good reproducibility of humidity within 31% RH-45% RH. When the test sample is inspected under 366nm before and under visible light after color development under the humidity of 78% RH, spots with the same color are displayed on the positions, corresponding to the chromatogram of the magnoflorine reference substance, of the chromatogram of the test sample, but the separation degree of the bright green spots of the nitidine chloride reference substance in the test sample is not high, and blue spots are displayed, namely, the spots with the same color are not displayed on the positions, corresponding to the chromatogram of the nitidine chloride reference substance. Therefore, the method is not suitable for high-humidity environment, so that the humidity should be controlled during the experiment.

7. Different sample application modes

The way of dot spotting and stripe spotting were examined separately. The results of the inspection are shown in FIG. 17.

And (4) conclusion: dot spotting and strip spotting are respectively adopted, when the detection is carried out under 366nm before color development and under visible light after color development, spots with the same color are respectively displayed on the positions corresponding to the color spectrum of a reference substance in the color spectrum of a test product, and the thin-layer color spectrum behaviors of the dot spotting and the strip spotting are basically consistent.

8. Stability of

(1) Stability of test article

After the test solution and the reference solution were prepared, they were stored at room temperature for different time points (0h, 8h, 48h, 72h, 96h) and then subjected to spotting, development and color examination, and the results are shown in FIG. 18.

And (4) conclusion: and (4) inspecting at different analysis time points (the strip serial number is 2-6) under 366nm before and visible light after color development. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution. Thus indicating that the test solution is stable within 96 h.

In addition, the control substances used in the development and verification of the experimental method in two months are all taken for the initial preparation, and the same effect is shown in each chromatographic experiment, so that the control substance solution has good stability.

And (4) conclusion: the method uses magnoflorine and nitidine chloride as reference indexes to detect the effective components of the single-face needles, improves and optimizes the original thin-layer identification method, and the optimized thin-layer identification method has strong specificity and good reproducibility. Under 366nm before color development, in the chromatogram of the single-side needle sample, two bright red spots are arranged at positions of Rf (0.85) and Rf (0.95), and can be used as identification points of single-side needles (including different base sources) and double-side needles and counterfeits; after color development, one identification spot is added to the single-side needle at the position of the specific shift value of 0.1 compared with the fake product.

Example 6 thin layer chromatography identification of multiple batches of single-sided needle samples

The method is the same as example 1, and the thin layer chromatography identification of the single-sided needle is carried out under the optimal conditions of the method of the invention. The results are shown in FIG. 19, and in the chromatogram of 16 single-side needle test samples, spots of the same color were observed at positions corresponding to those of the chromatogram of the control sample when examined under visible light at 366nm before and after development.

Comparative example 1

1. Experiment one:

taking 1g of test sample powder, adding 40ml of ethanol, carrying out ultrasonic extraction for 1 hour, filtering, evaporating in water bath, and dissolving the residue with 1ml of ethanol to obtain a test sample solution. Collecting magnoflorine control, adding methanol to obtain solution containing 1mg per 1ml as control solution. Performing thin layer chromatography (0502 of the general provisions of the Chinese pharmacopoeia 2015), sucking the two solutions 8 μ l, respectively dropping on the same silica gel G thin layer plate, simultaneously inspecting the following A, B, C developing agents, developing, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).

Three developing agents:

a: dichloromethane: methanol 6: 4;

b: trichloromethane: methanol 6: 4;

c: trichloromethane: methanol: water: ammonia water is 6:4:1: 0.25.

the results show that: in the test chromatogram, the C-developed system showed the same color of fluorescent spot at the corresponding position of the control chromatogram, and the spot was round but not too clear (see FIG. 20).

2. Experiment two:

on the basis of the first experiment, different sample amounts are compared, and two solutions A of a reference substance and a test substance are respectively taken: 4 mu L of the solution; b: 8 mu L of the solution; c: spreading 10 μ L of the mixture on different thin layer plates, spreading with chloroform-methanol-water-ammonia (6:4:1:0.25) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). The results show that: in the chromatogram of the test sample, A, B, C spots are all at the corresponding positions of the chromatogram of the control sample, and fluorescence spots with the same color are shown, wherein B, C spots are round and clear, and the B spots are only used according to the economic and efficiency principles (see figure 21).

3. In conclusion, the thin-layer identification method for magnoflorine in the zanthoxylum dissitum Hemsl is determined as follows: taking 1g of the product powder, adding 40ml of ethanol, performing ultrasonic extraction for 1 hour, filtering, evaporating in water bath, and dissolving the residue with 1ml of ethanol to obtain a sample solution. Taking appropriate amount of magnoflorine reference substance, adding methanol to make into 1mg solution per 1ml, and using as reference substance solution. Performing thin layer chromatography (according to 0502 of the general rules of the design of Chinese pharmacopoeia 2015), collecting 8 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water-ammonia (6:4:1:0.25) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). And (4) displaying fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution.

4. The above method was used to discriminate between single and double sided needles:

the results of the identification by adopting 13 batches of single-sided needle samples and 14 batches of double-sided needle samples according to the thin-layer identification method show that fluorescent spots with the same color are displayed on the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference sample. This thin layer identification method is not effective in distinguishing between single-sided and double-sided needles (see fig. 22).

Comparative example 2

Preparation of a test solution: taking 1g of sample powder (sieved by a second sieve), placing into a conical flask with a plug, adding 40ml of ethanol, sealing, performing ultrasonic treatment for 60min, filtering, evaporating in a water bath to dryness, dissolving the residue in 1ml of ethanol, and filtering to obtain a sample solution.

Preparation of control solutions: collecting magnoflorine and nitidine chloride reference substances, and adding methanol to obtain reference substance solution containing 1mg per 1 ml.

Sample amount of spotting: control (10 μ l); test article (10 μ l)

Sample application mode: strip, 8mm

Developing agent: chloroform methanol Water Ammonia (6:4:1:0.25) (layering, taking the lower layer)

Spreading to: 8cm

And (3) drying mode: air drying

The inspection method comprises the following steps: inspection was performed at 254nm and 366nm before development.

The results are shown in fig. 23 and 24, the magnoflorine is only visible at the position of 254nm Rf of 0.4 before color development, the chelerythrine, dictamnine and nitidine chloride have excessively high Rf, the concentrations of the nitidine and the zanthoxylum dissitum are overloaded, and no obvious identification point exists.