Thin-layer identification method for isofraxidin in Yao medicine monsoon

Abstract

The invention discloses a thin-layer identification method for isofraxidin in Yao medicine four monsoon, and belongs to the technical field of medicine analysis. Aiming at the problems of difficulty in separation, high false positive rate, low extraction efficiency and the like in thin-layer identification of isofraxidin in the Yao medicine four monsoon in the prior art, the key points of the technical scheme provided by the invention are as follows: firstly, preparing a four monsoon standard reference medicinal material solution, a to-be-detected product solution and an isofraxidin reference substance solution; wherein the preparation of the first two comprises the steps of ethanol extraction, step-by-step extraction with petroleum ether and dichloromethane and the like, and dissolving a reference substance solution with methanol to a constant volume; respectively taking 4-6 microliters of each solution, and spotting the solutions on a silica gel G thin-layer plate; and finally, developing by taking a mixed solvent prepared from dichloromethane, methanol and formic acid according to a specific volume ratio as a developing solvent, observing fluorescent spots under the ultraviolet wavelength of 300-305nm, and developing by iodine vapor fuming. The method is mainly used for qualitative identification of isofraxidin in the Yao medicine 4 monsoon, and ensures the quality of the medicinal material.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a thin-layer identification method for isoflurane in the Yao medicine Sijifeng. Background Art

[0002] Thin-layer chromatography (TLC) is widely used for the identification of components in traditional Chinese medicines, but the specific identification of isofraquinone in the Yao medicinal herb Sijifeng remains challenging. Sijifeng contains complex components such as polysaccharides, tannins, volatile oils, and coumarin congeners (such as cinnamomum camphora). These impurities are eluted simultaneously with isofraquinone during conventional ethanol extraction. Directly dissolving the sample in a single solvent results in similar mobility between the impurity and the target compound during TLC separation (Rf value difference often less than 0.05), resulting in masking of the isofraquinone spot or tailing (tailing factor exceeding 1.5), making effective separation difficult.

[0003] Existing general-purpose developing systems lack selectivity for isoflurane. For example, in the coumarin-based developing systems specified in the Chinese Pharmacopoeia (e.g., cyclohexane-ethyl acetate), high-polarity developing agents (methanol ratio greater than 1%) cause isoflurane to migrate too quickly (Rf value greater than 0.7), resulting in overlap with polar impurities. Low-polarity developing agents (e.g., pure dichloromethane) cause isoflurane to remain at the origin (Rf value less than 0.2), making it impossible to separate lipid-soluble impurities. Adding formic acid can improve spot morphology, but its traditional dosage (over 0.03%) is prone to edge effects. Furthermore, its high volatility leads to unstable concentrations within the chromatography chamber, with Rf values ​​fluctuating by up to ±0.1.

[0004] A high false-positive rate in identification results is also a prominent issue. Metal ions (such as Fe³⁺ / Mn²⁺) in the silica gel G plate quench isofraquinone fluorescence (quenching rate exceeds 30%), and oxygen molecules in the environment also reduce its fluorescence quantum yield (less than 0.3). To meet detection requirements, it is often necessary to increase the sample volume (greater than 8 μL), but high sample concentrations can exacerbate spot diffusion. Iodine fumigation is generally effective for coumarins, but it cannot distinguish isofraquinone from structural analogs (such as Zanthoxytoxin), further reducing specificity. These factors collectively result in a false-positive rate of more than 15% for traditional methods.

[0005] This problem arises from multiple challenges: the isofraquinone content in fennel is typically less than 0.1% (w / w), and the composition varies significantly between batches (e.g., due to interference from degradation products). Its physicochemical properties are unique, with low solubility in common solvents like methanol (less than 1.5 mg / mL) and pH sensitivity (formic acid concentrations exceeding 0.02% can trigger ring-opening degradation). Furthermore, the TLC procedure itself is significantly affected by variables such as temperature and humidity, sample placement technique, and the saturation level of the chromatography cylinder, making interlaboratory reproducibility difficult to ensure. Existing technologies have yet to effectively address the need for the specific identification of isofraquinone in the complex matrix of fennel. Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] The present invention solves the following technical problems: This study addresses the lack of a dedicated thin-layer chromatography (TLC) method for the identification of isoflurane in Sijifeng. Existing technologies struggle to clearly separate isoflurane spots in complex matrices, resulting in a high false-positive rate in identification results. Solve the spot tailing problem caused by impurity co-migration during gradient development. A single polarity developing solvent cannot balance impurity migration and target separation, affecting the accuracy of the Rf value. This solves the problem of extraction efficiency fluctuations caused by differences in herbal medicine particle size. Uniform pulverization leads to insufficient dissolution of fibrous tissue and excessive gelatinization of parenchyma tissue, reducing the recovery rate of isoflurane. Solve the stubborn emulsification problem caused by plant colloids in dichloromethane extraction. The emulsion layer envelops the target compound, reducing extraction recovery and TLC spot intensity consistency. This solution addresses the problem of solute recrystallization and uneven diffusion caused by rapid solvent evaporation during spotting. The coffee ring effect causes spot distortion, reducing resolution and quantitative accuracy. Solve the frontier effect and resolution degradation caused by the volatilization of trace formic acid in the developing solvent. The imbalance of gas phase polarity causes the Rf value to fluctuate by ±0.05, affecting reproducibility. Solve the problem of uneven background fluorescence on silica gel interfering with spot interpretation. Metal ion quenching and oxygen quenching cause isoflurane fluorescence intensity fluctuations of ±20%. Solve the problem of water-soluble impurities forming a viscoelastic film at the dichloromethane extraction interface. This film hinders droplet coalescence and causes isoflurane tailing (tailing rate >30%). Optimize extraction oscillation parameters to balance emulsification suppression and extraction efficiency. Conventional oscillation modes are prone to microemulsion formation, requiring precise control of shear force and delamination time. In order to achieve these objects and other advantages of the present invention, a thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng is provided, comprising the following steps: S1: Prepare the Sijifeng standard control medicinal material solution, the Sijifeng test medicinal material solution and the isoflurane reference solution; The preparation of the Sijifeng standard control medicinal material solution and the Sijifeng test product medicinal material solution includes: S101: taking dried Sijifeng medicinal material powder, soaking it in 70%-90% ethanol solution with a liquid-to-solid ratio of 1:8-1:12 for 1.5-2.5 hours, heating, condensing and refluxing for 1.5-2.5 hours, filtering and recovering ethanol to obtain an ethanol extract; S102: dissolving the ethanol extract in distilled water, adding an equal volume of petroleum ether, extracting 2-4 times, discarding the petroleum ether layer, and then adding an equal volume of dichloromethane, extracting 2-4 times, and recovering the dichloromethane to obtain a dichloromethane extract; S103: dissolving the dichloromethane extract in a mixed solvent having a volume ratio of dichloromethane to methanol of 1:0.8-1.2 to obtain a Sijifeng standard reference medicinal material solution and a Sijifeng test medicinal material solution; The preparation of the isoflurane reference solution comprises: taking the isoflurane reference, dissolving it in methanol and making up the volume to obtain a solution with a concentration of 0.8-1.2 mg / mL; S2: Take 4-6 μL of the Sijifeng standard control medicinal material solution, Sijifeng test medicinal material solution, isoflurane reference solution, and negative control solution, and spot them on the same silica gel G thin layer plate; S3: Develop the plate using a mixed solvent consisting of dichloromethane, methanol, and formic acid in a volume ratio of 10:0.3-0.5:0.005-0.015. Observe the fluorescent spots on the developed thin layer plate under ultraviolet light at a wavelength of 300-305 nm, and then color it with iodine vapor. Preferably, the expansion operation in step S3 of the present invention is performed according to the following steps: a) placing the spotted thin layer plate in a sealed chromatography chamber, injecting a first developing solvent prepared by mixing dichloromethane, methanol, and formic acid in a volume ratio of 10:0.8-1.2:0.01-0.03, and developing until the solvent front is 35-45 mm from the origin of the thin layer plate; b) Take out the thin layer plate and place it in a ventilated environment with a temperature of 20-25°C and a relative humidity of ≤40% for 1-2 minutes to allow the surface solvent to completely evaporate; c) Place the thin layer plate back in the same chromatography tank and inject a second developing solvent prepared by mixing dichloromethane, methanol, and formic acid in a volume ratio of 10:0.3-0.5:0.005-0.015. Develop the plate until the solvent front is 80-100 mm from the origin of the thin layer plate. Preferably, in step S101 of the present invention, the dried Sijifeng medicinal material powder is prepared in the following manner: a) passing the coarse powder of Sijifeng medicinal material through 20-mesh, 40-mesh, and 60-mesh standard pharmacopoeia sieves in sequence to separate into a coarse particle fraction, a medium particle fraction, and a fine particle fraction, wherein the coarse particle fraction is particles retained on the 20-mesh sieve; the medium particle fraction is particles passing through the 20-mesh sieve but retained on the 40-mesh sieve; and the fine particle fraction is particles passing through the 40-mesh sieve but retained on the 60-mesh sieve; b) mixing the coarse particle fraction with a 70%-90% ethanol solution at a liquid-to-material ratio of 1:6-1:8, soaking for 1.5-2.5 hours, then adding the medium particle fraction, maintaining the total liquid-to-material ratio at 1:8-1:12, and continuing to soak for 0.5 hours, and finally adding the fine particle fraction, maintaining the total liquid-to-material ratio unchanged, and heating the whole to condense and reflux for 2.0-2.5 hours; c) filtering after the reflux is completed, combining the filtrates and recovering ethanol to obtain the ethanol extract. Preferably, in step S102 of the present invention, after the dichloromethane extraction is completed, the dichloromethane layer is subjected to the following treatment: a) placing the combined dichloromethane extracts in a centrifuge tube, incubating at 4-6°C for 10-15 minutes, and then centrifuging at 2500-3500 rpm for 5-8 minutes; b) taking the dichloromethane layer after centrifugation and passing it through a microcolumn filled with 200-400 mesh diatomaceous earth with a column bed height of 10-15 mm and an inner diameter of 5-8 mm at a flow rate of 1-2 mL / min; c) collecting the filtrate, and recovering the dichloromethane under reduced pressure to obtain the dichloromethane extract. Preferably, the spotting operation in step S2 of the present invention is performed according to the following steps: a) adding ethylene glycol dimethyl ether to the Sijifeng standard control medicinal material solution, the Sijifeng test medicinal material solution, and the negative control solution, so that its volume fraction in the solution is 8-12%; b) Use a microinjection needle with an inner diameter of 0.5-0.8 mm. Preheat the needle tip to 30-35°C and maintain constant temperature before injection. c) Take 4-6 μL of each solution and apply it to a silica gel G thin layer plate twice. Apply 2-3 μL the first time, and add the remaining 2-3 μL after the solvent has evaporated completely, 20-30 seconds later. Preferably, before starting the operation in step S3 of the present invention, the chromatography cylinder is pretreated in the following manner: a) Take a silica gel G plate, 20 x 40 mm, immerse it in a 10-15% methanol-water solution for 10 seconds, remove it and air dry it until it is half dry, and then fix it to the top wall of the chromatography tank with the silica gel surface facing the tank space; b) Inject the developing agent into the chromatography tank to a liquid layer thickness of 3-5 mm, close the tank lid, and let it equilibrate at 20-25°C for 30-40 minutes; c) Place the spotted thin layer plate on the support in the cylinder, with the plate surface at an angle of 75-85° to the liquid surface and the bottom of the plate 3-5 mm from the liquid surface. Preferably, the following processing steps are added to the thin layer plate after being developed in step S3 of the present invention before ultraviolet light observation: a) Place the developed and dried TLC plate under a 254 nm UV lamp at an intensity of 5-7 μW / cm² at a distance of 10-15 cm for 2-3 minutes. b) Move the chamber into a sealed transparent chamber and introduce high-purity nitrogen to replace the air at a flow rate of 0.8-1.2 L / min for 5-8 minutes; c) The treated thin layer plate is observed under 300-305 nm UV light while the ambient humidity is controlled at 35-45% RH. Preferably, in step S102 of the present invention, before adding an equal volume of dichloromethane for extraction, the aqueous phase solution is subjected to the following treatments before adding petroleum ether for extraction: a) dissolving the ethanol extract in distilled water, adding saturated sodium sulfate solution, and stirring to a final concentration of 0.3-0.5 mol / L; b) Add 100–200 μm polytetrafluoroethylene powder at a concentration of 10–20 mg / mL of aqueous phase and stir magnetically for 1 minute. Preferably, the method of the present invention comprises adding an equal volume of petroleum ether, manually shaking up and down 30 times, standing for 10 minutes to separate layers, and discarding the petroleum ether layer; adding an equal volume of dichloromethane, manually shaking up and down 40 times, standing for 8-10 minutes to separate layers; collecting the dichloromethane layer, repeating the extraction 2-4 times, and combining the extracts. The present invention has at least the following beneficial effects: By optimizing the extraction solvent system (70-90% ethanol) and the developing solvent ratio (dichloromethane:methanol:formic acid = 10:0.3-0.5:0.005-0.015), baseline separation of isoflurane and coexisting impurities was achieved. UV detection (300-305 nm) combined with iodine fumigation resulted in a stable Rf value of 0.45±0.03. The specificity and reproducibility significantly improved over the standard coumarin TLC method in the Chinese Pharmacopoeia, and the false positive rate was reduced from 15% to below 3%. The two-stage gradient development design enables moderately polar impurities to migrate to the Rf>0.7 region at high methanol concentrations (0.8-1.2%) in the first stage, while isoflurane is focused and separated in the second stage at low methanol concentrations (0.3-0.5%). This reduces the spot tailing factor from 1.8 to 1.1, and improves the resolution (Rs) from 0.8 to 1.5, meeting the requirements for trace component identification in complex matrices. A graded pulverization and sequential feeding strategy ensured that the coarse particles (>20 mesh) were fully infiltrated at a low liquid-to-solid ratio (1:6-1:8), while the fine particles (40-60 mesh) were added later to prevent gelatinization. This significantly reduced the impact of particle size differences on the results, increasing the extraction yield of isoflurane from 82% to 95% and reducing the RSD from 9.2% to 2.5%. Low-temperature centrifugation (4-6°C) combined with adsorption on diatomaceous earth microcolumns (200-400 mesh) effectively removes colloids and saponin emulsifiers. Dichloromethane recovery increases from 78% to 96%, impurity peak areas in the extract decrease by 90%, and TLC background cleanliness improves threefold. Ethylene glycol dimethyl ether (8-12%) slows volatilization, tip temperature control (30-35°C) inhibits crystallization, and step-by-step spotting eliminates the coffee ring effect. The standard deviation of spot diameters is reduced from 0.3 mm to 0.1 mm, and the recovery rate of isoflurane spotting reaches 98.5±0.8%. The silica gel lining (20×40 mm impregnated with 10-15% methanol aqueous solution) adsorbs volatile formic acid. Pre-saturation (30-40 minutes) combined with placement at a 75-85° inclination angle reduces the formic acid concentration fluctuation in the cylinder from ±25% to ±5%, with an Rf value RSD of <1.5%, breaking through the application bottleneck of ultra-low concentration additives. The 254 nm UV-light reduction of the metal quenching center (Fe³⁺→Fe²⁺) was used, while nitrogen protection (O2 <0.001%) blocked oxygen quenching. Humidity control (35-45% RH) stabilized fluorescence emission. The fluorescence quantum yield of isoflurane increased from 0.28 to 0.42, the background signal-to-noise ratio improved eightfold, and the detection limit dropped to 0.05 μg / spot. Sodium sulfate (0.3-0.5 mol / L) compresses the double layer, promoting impurity precipitation, while PTFE powder (100-200 μm) disrupts the interfacial viscoelastic film. This increases the interfacial tension from 45 mN / m to 68 mN / m, shortens the extraction and delamination time by 40%, and reduces the loss of isoflurane encapsulation from 18% to 2%. The precise matching of oscillation times (30 for petroleum ether and 40 for dichloromethane) and rest time (10 minutes for petroleum ether and 8-10 minutes for dichloromethane) optimizes droplet size distribution within the 50-200 μm range. Extraction efficiency RSD is less than 3%, emulsification incidence is reduced from 35% to 5%, and operational reproducibility meets pharmacopoeial standards.

[0008] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The results of thin layer identification when developed with a developing agent; Figure 2 The results of thin-layer identification when developed with another developing agent; Figure 3 This is the result of thin layer identification when developed using another developer. DETAILED DESCRIPTION

[0010] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0011] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0012] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0013] <Example 1> A thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng comprises the following steps: S1: Prepare the Sijifeng standard control medicinal material solution, the Sijifeng test medicinal material solution and the isoflurane reference solution; The preparation of the Sijifeng standard control medicinal material solution and the Sijifeng test product medicinal material solution includes: S101: 1.0 g of dried Sijifeng medicinal material powder was soaked in 10 mL of 90% ethanol solution at a liquid-to-solid mass ratio of 1:10 for 2.0 hours, heated and refluxed for 2 hours, filtered, and ethanol was recovered to obtain an ethanol extract; S102: dissolving the ethanol extract in distilled water, adding an equal volume of petroleum ether, extracting three times, discarding the petroleum ether layer, and then adding an equal volume of dichloromethane, extracting three times, and recovering the dichloromethane to obtain a dichloromethane extract; S103: dissolving the dichloromethane extract in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1 to obtain a Sijifeng standard reference medicinal material solution or a Sijifeng test medicinal material solution; Preparation of the isoflurane reference solution includes: taking 10.0 mg of the isoflurane reference, dissolving it in methanol and making up the volume to 10 mL to obtain a solution with a concentration of 1.0 mg / mL; S2: Take 5 μL each of the Sijifeng standard control medicinal material solution, Sijifeng test medicinal material solution, isoflurane reference solution, and negative control solution, and spot them on the same silica gel G thin layer plate; S3: Develop the plate using a mixed solvent consisting of dichloromethane, methanol, and formic acid in a volume ratio of 10:0.4:0.010. Place the spotted TLC plate in a presaturated chromatography chamber (25°C) and develop until the solvent front is 85 mm from the origin. Remove the plate, evaporate the solvent, and observe the fluorescent spots under ultraviolet light at a wavelength of 302 nm. Color development is then performed using iodine vapor fumigation.

[0014] The expansion results of this embodiment are as follows Figure 1When the developing solvent is petroleum ether: ethyl acetate: formic acid = 5:10:0.01, the result is as follows Figure 2 When the developing solvent is dichloromethane:methanol:formic acid=10:0.1:0.01, the result is as follows Figure 3 As shown. In the TLC chromatograms of the three development systems, the isofraquinone reference, the Sijifeng reference medicinal material, and the Sijifeng test products (Nos. 1, 2, and 3) all showed identical spots at the corresponding locations. The spots from development system 1 exhibited clear fluorescence, moderate Rf values, and good resolution, observed under 302nm fluorescence (FL) and visualized by iodine fumigation. Development systems 2 and 3, while showing clear spots, exhibited poor resolution. In the figure, 1 is the isofraquinone reference; 2 is the Sijifeng standard reference medicinal material; 3 is Sijifeng test product No. 1; 4 is Sijifeng test product No. 2; 5 is Sijifeng test product No. 3; and 6 is the negative control. A negative control solution was prepared by following the same procedures as in S101-S103 using a Sijifeng simulated matrix (e.g., a starch and tannin mixture) that did not contain isofraquinone.

[0015] <Example 2> A thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in <Example 1>, except that the expansion operation in step S3 is performed according to the following steps: a) Place the spotted thin layer plate in a sealed chromatography chamber and inject a first developing solvent prepared by mixing dichloromethane, methanol, and formic acid in a volume ratio of 10:1.0:0.02. Develop the plate until the solvent front is 40 mm from the origin of the thin layer plate. b) Take out the thin layer plate and place it in a ventilated environment at a temperature of 25°C and a relative humidity of ≤40% for 1.5 minutes to allow the surface solvent to evaporate completely; c) Place the TLC plate back in the same chromatography chamber and inject a second developing solvent prepared by mixing dichloromethane, methanol, and formic acid in a volume ratio of 10:0.4:0.01. Develop the plate until the solvent front is 90 mm from the origin of the TLC plate.

[0016] <Example 3> A thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng is based on the method provided in Example 2, except that, in step S101, the dried Sijifeng medicinal material powder is prepared as follows: a) passing the coarse powder of Sijifeng medicinal material through 20-mesh, 40-mesh, and 60-mesh standard pharmacopoeia sieves in sequence to separate into a coarse particle fraction, a medium particle fraction, and a fine particle fraction, wherein the coarse particle fraction is particles retained on the 20-mesh sieve; the medium particle fraction is particles passing through the 20-mesh sieve but retained on the 40-mesh sieve; and the fine particle fraction is particles passing through the 40-mesh sieve but retained on the 60-mesh sieve; b) mixing the coarse particle fraction with an 80% by mass ethanol solution at a liquid-to-solid ratio of 1:7, soaking for 2 hours, then adding the medium particle fraction, maintaining the total liquid-to-solid ratio at 1:10, and continuing to soak for 0.5 hours, and finally adding the fine particle fraction, maintaining the total liquid-to-solid ratio unchanged, and heating the whole to condense and reflux for 2.3 hours; c) filtering after the reflux is completed, combining the filtrates and recovering ethanol to obtain the ethanol extract.

[0017] <Example 4> A thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in <Example 3>, except that, in step S102, after the dichloromethane extraction is completed, the dichloromethane layer is subjected to the following treatment: a) Place the combined dichloromethane extracts in a centrifuge tube, incubate at 5°C for 12 minutes, and then centrifuge at 3000 rpm for 6 minutes; b) The dichloromethane layer after centrifugation was passed through a microcolumn filled with 300-mesh diatomaceous earth (12 mm bed height, 6 mm inner diameter) at a flow rate of 1.5 mL / min. c) collecting the filtrate, and recovering the dichloromethane under reduced pressure to obtain the dichloromethane extract.

[0018] <Example 5> A thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in <Example 4>, except that the spotting operation in step S2 is performed according to the following steps: a) adding ethylene glycol dimethyl ether to the Sijifeng standard control medicinal material solution, the Sijifeng test product medicinal material solution, and the negative control solution so that its volume fraction in the solution is 10%; b) Use a micro-sampling needle with an inner diameter of 0.5 mm. Preheat the needle tip to 33°C and maintain constant temperature before sampling. c) Take 5 μL of each solution and apply it to a silica gel G thin layer plate twice. Apply 2 μL the first time, and add the remaining 3 μL after 25 seconds after the solvent has evaporated completely.

[0019] <Example 6> A thin-layer chromatography identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in <Example 5>, except that, before the operation in step S3, the chromatography cylinder is pretreated in the following manner: a) Take a 20 x 40 mm silica gel G plate, immerse it in a 12% methanol-water solution for 10 seconds, remove it and air dry it until it is half dry, then fix it to the top wall of the chromatography tank with the silica gel surface facing the tank interior; b) Inject the developing agent into the chromatography tank to a liquid layer thickness of 4 mm, close the tank lid, and let it equilibrate at 23°C for 35 minutes; c) Place the spotted thin layer plate on the holder in the cylinder, with the plate surface at an 80° angle to the liquid surface and the bottom of the plate 4 mm from the liquid surface.

[0020] <Example 7> A thin-layer identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in Example 6, except that the following processing steps are added before the developed thin-layer plate is observed under ultraviolet light in step S3: a) After developing and drying the solvent, place the TLC plate under a 254 nm UV lamp at an intensity of 6 μW / cm² at a distance of 12 cm for 2 minutes. b) Move the chamber into a sealed transparent chamber and introduce high-purity nitrogen to replace the air at a flow rate of 1 L / min for 6 minutes; c) The treated thin layer plate was observed under 302 nm UV light while the ambient humidity was controlled at 40% RH.

[0021] <Example 8> A thin-layer chromatography identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in Example 7, except that, in step S102, before adding an equal volume of dichloromethane for extraction, the following treatment is added to the aqueous phase solution before adding petroleum ether for extraction: a) Dissolve the ethanol extract in distilled water, add saturated sodium sulfate solution, and stir to a final concentration of 0.4 mol / L; b) Add 150 μm polytetrafluoroethylene powder at a concentration of 15 mg / mL of aqueous phase and stir magnetically for 1 minute.

[0022] <Example 9> A thin-layer chromatography identification method for isoflurane in the Yao medicine Sijifeng adopts the method provided in <Example 8>, except that an equal volume of petroleum ether is added, the mixture is manually shaken up and down 30 times, the mixture is allowed to stand for 10 minutes to separate layers, and the petroleum ether layer is discarded; an equal volume of dichloromethane is added, the mixture is manually shaken up and down 40 times, the mixture is allowed to stand for 9 minutes to separate layers; the dichloromethane layer is collected, the extraction is repeated 3 times, and the extracts are combined.

[0023] <Comparative Example 1> The method is the same as that of <Example 1>, except that the obtained ethanol extract is directly used for spotting.

[0024] <Comparative Example 2> The method is the same as that of Example 1, except that the dichloromethane extract is dissolved in methanol and then used for spotting.

[0025] The test results of the spot Rf value, tailing factor, separation, fluorescence intensity, false positive rate, recovery rate of isoflurane, and operational reproducibility (RSD) of Examples 1-9 and Comparative Examples 1-2 are shown in Table 1 below.

[0026] Table 1 Spot Rf value tailing factor Separation (Rs) Fluorescence intensity (au) False positive rate Isofraxidin recovery rate Operational reproducibility (RSD) Example 1 0.46±0.02 1.10 1.50 285 ± 12 2.8% 95.2% 1.8% Example 2 0.45±0.01 1.08 1.52 290 ± 10 2.7% 95.5% 1.7% Example 3 0.45±0.01 1.06 1.53 292 ± 9 2.5% 95.8% 1.6% Example 4 0.46±0.01 1.05 1.54 295 ± 8 2.4% 96.0% 1.5% Example 5 0.45±0.01 1.07 1.53 293 ± 8 2.6% 95.7% 1.6% Example 6 0.45±0.01 1.06 1.55 298 ± 7 2.3% 96.2% 1.4% Example 7 0.45±0.01 1.05 1.56 300 ± 6 2.2% 96.3% 1.3% Example 8 0.46±0.01 1.05 1.55 302 ± 6 2.1% 96.5% 1.2% Example 9 0.45±0.01 1.04 1.57 305 ± 5 2.0% 96.8% 1.1% Comparative Example 1 0.22±0.15 1.85 0.75 95 ± 38 22.3% 78.2% 11.2% Comparative Example 2 0.68±0.18 1.78 0.82 110 ± 43 19.8% 80.5% 9.8% Rf value stability: The Rf values ​​of the spots in Examples 1-9 were stable at 0.45±0.03, with minimal fluctuation (RSD <1.5%), while the Rf values ​​of Comparative Example 1 (direct ethanol extract spotting) and Comparative Example 2 (single methanol dissolution) were 0.22±0.15 and 0.68±0.18, respectively, showing significant fluctuations. This may be due to the precise control of the developing agent polarity by the present invention through the design of a gradient developing agent (dichloromethane:methanol:formic acid = 10:0.3-0.5:0.005-0.015) and chromatography cylinder pretreatment (formic acid adsorption on the silica gel lining and pre-saturation equilibrium), thus avoiding the Rf value fluctuation caused by formic acid volatilization in traditional methods and achieving stable and controllable isoflurane migration rate.

[0027] Tailing Factor and Resolution: The tailing factor of the examples was reduced to 1.04-1.10 (close to the ideal value of 1), and the resolution (Rs) reached 1.50-1.57, meeting baseline separation requirements. In contrast, the comparative examples exhibited tailing factors >1.78, resolution <0.82, and severe tailing and overlapping of spots. This is likely due to the combination of a two-stage gradient development (a high methanol ratio in the first stage to remove polar impurities, and a low methanol ratio in the second stage to focus on the target) and fractionated extraction (petroleum ether to remove fat-soluble impurities and dichloromethane to enrich isoflurane) to effectively address the issue of close mobility between impurities and target compounds in complex matrices, resulting in a nearly 200% improvement in separation efficiency compared to conventional methods.

[0028] Improved Fluorescence Intensity: The fluorescence intensity of the examples reached 285-305 a.u., a 2-3-fold increase compared to the comparative examples (95-110 a.u.). This is likely due to UV irradiation reducing the metal quenching center (Fe³⁺→Fe²⁺), nitrogen protection eliminating oxygen quenching (O2 <0.001%), and a controlled humidity environment (35-45% RH) stabilizing fluorescence emission. This increased the fluorescence quantum yield of isoflurane from 0.28 to 0.42, improving the background signal-to-noise ratio by 8-fold and lowering the detection limit to 0.05 μg / spot.

[0029] False-positive rates were significantly reduced: The false-positive rates for the examples were all less than 3% (minimum 2.0%), while those for the comparative examples were as high as 19.8%-22.3%. This is likely due to the enhanced specificity of the iodine fumigation colorimetric assay combined with UV-fluorescence detection. Furthermore, precise control of the formic acid concentration (0.005-0.015%) prevented co-colorization of structural analogs (such as Zanthoxylum bungeanum toxin), thus addressing the false-positive issues associated with traditional methods involving a single colorimetric assay.

[0030] Isofraxidin recovery: The recovery rates in the examples reached 95.2%-96.8%, an increase of over 15% compared to the comparative examples (78.2%-80.5%). This may be due to 1) graded pulverization and sequential feeding: coarse particles (>20 mesh) were first thoroughly infiltrated with a low liquid-to-solid ratio (1:6-1:8), while fine particles (40-60 mesh) were added later to prevent gelatinization. This increased the extraction rate from 82% to 95%, and the RSD decreased from 9.2% to 2.5%. 2) Low-temperature centrifugation combined with diatomaceous earth microcolumn impurity removal: Centrifugation at 4-6°C (2500-3500 rpm) combined with 200-400 mesh diatomaceous earth for colloid adsorption increased the dichloromethane extraction recovery from 78% to 96%, and reduced the impurity peak area by 90%.

[0031] Reproducibility: The RSD for the examples was <2%, while for the comparative examples it was >9%. This was likely due to optimized spotting techniques (delayed volatilization of ethylene glycol dimethyl ether, tip temperature control at 30-35°C, and step-by-step spotting), which eliminated the coffee ring effect and reduced the standard deviation of spot diameter from 0.3 mm to 0.1 mm, achieving a spot recovery of 98.5 ± 0.8%. Furthermore, standardized chromatography cylinder pretreatment (75-85° tilt angle, 30-40 min equilibration) minimized the impact of environmental variables.

[0032] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A thin layer chromatography identification method for isoflurane in the Yao medicine Sijifeng, characterized in that: The following steps are involved: S1: Prepare the Sijifeng standard control medicinal material solution, the Sijifeng test medicinal material solution and the isoflurane reference solution; The preparation of the Sijifeng standard control medicinal material solution and the Sijifeng test product medicinal material solution includes: S101: taking dried Sijifeng medicinal material powder, soaking it in 70%-90% ethanol solution at a liquid-to-solid mass ratio of 1:8-1:12 for 1.5-2.5 hours, heating, condensing and refluxing for 1.5-2.5 hours, filtering and recovering ethanol to obtain an ethanol extract; S102: dissolving the ethanol extract in distilled water, adding an equal volume of petroleum ether, extracting 2-4 times, discarding the petroleum ether layer, and then adding an equal volume of dichloromethane, extracting 2-4 times, and recovering the dichloromethane to obtain a dichloromethane extract; S103: dissolving the dichloromethane extract in a mixed solvent having a volume ratio of dichloromethane to methanol of 1:0.8-1.2 to obtain a Sijifeng standard reference medicinal material solution or a Sijifeng test medicinal material solution; The preparation of the isoflurane reference solution comprises: taking the isoflurane reference, dissolving it in methanol and making up the volume to obtain a solution with a concentration of 0.8-1.2 mg / mL; S2: Take 4-6 μL of the Sijifeng standard control medicinal material solution, Sijifeng test medicinal material solution, isoflurane reference solution, and negative control solution, and spot them on the same silica gel G thin layer plate; S3: Develop the plate using a mixed solvent consisting of dichloromethane, methanol, and formic acid in a volume ratio of 10:0.3-0.5:0.005-0.

015. Observe the fluorescent spots on the developed thin layer plate under ultraviolet light at a wavelength of 300-305 nm, and then color it with iodine vapor.

2. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 1, characterized in that: The expansion operation in step S3 is performed as follows: a) Place the spotted thin layer plate in a sealed chromatography chamber and inject a first developing solvent prepared by mixing dichloromethane, methanol, and formic acid in a volume ratio of 10:0.8-1.2:0.01-0.

03. Develop the plate until the solvent front is 35-45 mm from the origin of the thin layer plate. b) Take out the thin layer plate and place it in a ventilated environment with a temperature of 20-25°C and a relative humidity of ≤40% for 1-2 minutes to allow the surface solvent to completely evaporate; c) Place the thin layer plate back in the same chromatography tank and inject a second developing solvent prepared by mixing dichloromethane, methanol, and formic acid in a volume ratio of 10:0.3-0.5:0.005-0.

015. Develop the plate until the solvent front is 80-100 mm from the origin of the thin layer plate.

3. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 1, characterized in that: In step S101, the dried Four Seasons Wind medicinal material powder is prepared in the following manner: a) passing the coarse powder of Sijifeng medicinal material through 20-mesh, 40-mesh, and 60-mesh standard pharmacopoeia sieves in sequence to separate into a coarse particle fraction, a medium particle fraction, and a fine particle fraction, wherein the coarse particle fraction is particles retained on the 20-mesh sieve; the medium particle fraction is particles passing through the 20-mesh sieve but retained on the 40-mesh sieve; and the fine particle fraction is particles passing through the 40-mesh sieve but retained on the 60-mesh sieve; b) mixing the coarse particle fraction with a 70%-90% ethanol solution at a liquid-to-solid ratio of 1:6-1:8, soaking for 1.5-2.5 hours, then adding the medium particle fraction, maintaining the total liquid-to-solid ratio at 1:8-1:12, and continuing to soak for 0.5 hours, and finally adding the fine particle fraction, maintaining the total liquid-to-solid ratio unchanged, and heating the whole to condense and reflux for 2.0-2.5 hours; c) filtering after the reflux is completed, combining the filtrates and recovering ethanol to obtain the ethanol extract.

4. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 3, characterized in that: In step S102, after the dichloromethane extraction is completed, the dichloromethane layer is subjected to the following treatment: a) placing the combined dichloromethane extracts in a centrifuge tube, incubating at 4-6°C for 10-15 minutes, and then centrifuging at 2500-3500 rpm for 5-8 minutes; b) The dichloromethane layer after centrifugation was passed through a microcolumn filled with 200-400 mesh diatomaceous earth with a bed height of 10-15 mm and an inner diameter of 5-8 mm at a flow rate of 1-2 mL / min; c) collecting the filtrate, and recovering the dichloromethane under reduced pressure to obtain the dichloromethane extract.

5. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 1, characterized in that: The spotting operation in step S2 is performed according to the following steps: a) adding ethylene glycol dimethyl ether to the Sijifeng standard control medicinal material solution, the Sijifeng test medicinal material solution, and the negative control solution, so that its volume fraction in the solution is 8-12%; b) Use a microinjection needle with an inner diameter of 0.5-0.8 mm. Preheat the needle tip to 30-35°C and maintain constant temperature before injection. c) Take 4-6 μL of each solution and apply it to a silica gel G thin layer plate twice. Apply 2-3 μL the first time, and add the remaining 2-3 μL after the solvent has evaporated completely, 20-30 seconds later.

6. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 1, characterized in that: Before the operation in step S3 is carried out, the chromatography cylinder is pretreated in the following manner: a) Take a 20 x 40 mm silica gel G plate, immerse it in a 10-15% methanol-water solution for 10 seconds, remove it and air dry it until it is half dry, then fix it to the top wall of the chromatography tank with the silica gel surface facing the tank interior; b) Inject the developing agent into the chromatography tank to a liquid layer thickness of 3-5 mm, close the tank lid, and let it equilibrate at 20-25°C for 30-40 minutes; c) Place the spotted thin layer plate on the holder in the cylinder, with the plate surface at an angle of 75-85° to the liquid surface and the bottom of the plate 3-5mm from the liquid surface.

7. The thin layer chromatography identification method of isofraxidin in the Yao medicine Sijifeng according to claim 1, characterized in that: Before the ultraviolet light observation of the thin layer plate after being developed in step S3, the following processing steps are added: a) Place the developed and dried TLC plate under a 254 nm UV lamp at an intensity of 5-7 μW / cm² at a distance of 10-15 cm for 2-3 minutes. b) Move the chamber into a sealed transparent chamber and introduce high-purity nitrogen to replace the air at a flow rate of 0.8-1.2 L / min for 5-8 minutes; c) The treated thin layer plate is observed under 300-305 nm UV light while the ambient humidity is controlled at 35-45% RH.

8. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 1, characterized in that: In step S102, before adding an equal volume of dichloromethane for extraction, the aqueous phase solution is subjected to the following treatments and then subjected to petroleum ether for extraction: a) Dissolve the ethanol extract in distilled water, add saturated sodium sulfate solution, and stir to a final concentration of 0.3-0.5 mol / L; b) Add 100–200 μm polytetrafluoroethylene powder at a concentration of 10–20 mg / mL of aqueous phase and stir magnetically for 1 minute.

9. The thin layer chromatography identification method of isoflurane in the Yao medicine Sijifeng according to claim 1, characterized in that: Add an equal volume of petroleum ether, shake up and down manually 30 times, let it stand for 10 minutes to separate the layers, and discard the petroleum ether layer; add an equal volume of dichloromethane, shake up and down manually 40 times, let it stand for 8-10 minutes to separate the layers; collect the dichloromethane layer, repeat the extraction 2-4 times, and combine the extracts.

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