A method for identifying and detecting three characteristic components of anling by one-plate double-chromatography synchronous thin layer

Abstract

The present application is suitable for the technical field of traditional Chinese medicine quality inspection, and provides a one-plate double-color synchronous thin-layer identification detection method for three characteristic components of Annao, which comprises the following steps: one heavy mixed sample synchronous pretreatment integrated extraction, two heavy single silica gel G thin-layer plate synchronous development chromatography, and three heavy segmented double-color synchronous imaging identification; in the present application, only one sample is prepared, a single piece of silica gel G thin-layer plate is used, and after one-time development, natural light and 365nm ultraviolet double-mode coloration are completed on the plate in sequence, and three types of legal characteristic indexes, i.e., cholic acid, alkaloids of Coptis chinensis and gardenoside, are synchronously detected; the whole method set has a wide operation parameter interval, and there is optimization space for test time consumption, reagent consumption, detection data consistency and impurity discrimination ability, and is suitable for batch testing of Annao film-coated tablets and Annao small honeyed pills in the whole process, and the method has stable durability and repeatability, and has good industrialization quality inspection application value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality inspection technology, and in particular relates to a method for simultaneous thin-layer chromatography identification and detection of three characteristic components of Anaponectin using a single plate with dual color development. Background Technology

[0002] For example, Chinese patent (publication number: CN102058829A) discloses a detection method for a pharmaceutical composition and its preparation. This method sets up thin-layer chromatography identification items for the three core medicinal materials in the Annao preparation: pig bile powder, Coptis chinensis, and Gardenia jasminoides. It uses an independent sample pretreatment process, an independent silica gel G thin-layer plate development system, and an independent colorimetric inspection method to complete the single-item qualitative identification. This is the mainstream technical solution for the current thin-layer chromatography quality control of Annao preparations. This type of method is built on the pharmacopoeia's basic detection framework, with clear single-item identification specificity, and can basically meet the routine quality control requirements of Annao tablets and pills.

[0003] However, in actual batch quality inspection applications, there are still several areas for improvement in the technical solutions for independent identification of such separate boards:

[0004] Multiple independent pretreatment and plate-based detection processes result in high costs for consumables and time: the three characteristic components require separate preparation of test solutions, each occupying a thin-layer plate for spotting, development, and color development; the identification of geniposide requires defatting with ether and multi-solvent reflux extraction, and ether is a flammable and explosive reagent, placing a heavy burden on laboratory ventilation and explosion-proof maintenance; when sampling large batches of samples, the testing personnel perform many repetitive operations, the sample turnover cycle is long, and the consumption of consumables such as reagents and thin-layer plates is large.

[0005] Parallel testing of multiple thin-layer plates can lead to inter-plate system bias and insufficient consistency in test judgments: there are objective differences in coating activity, developing tank saturation, and oven heating temperature among different batches of silica gel thin-layer plates; the three types of characteristic spots belong to different carriers, making it impossible to compare Rf values ​​and spot intensities horizontally under the same chromatographic environment; when sample spots are in the critical judgment range, judgment discrepancies are likely to occur; and archiving multiple sets of chromatograms separately is also not conducive to the unified traceability management of GMP test data.

[0006] In single-color observation mode, matrix contamination strongly interferes with the identification of target spots: Traditional plate identification only uses a single inspection method. Gardenoside is identified by natural light color development, and cholic acid and berberine are identified by 365nm ultraviolet fluorescence only, which cannot obtain dual-spectral information of the same spot. For samples with long storage periods and natural decay of active ingredients, the outline of the target spot is blurred, and contamination from excipients and medicinal materials is easy to overlap with the characteristic spot, which increases the difficulty of manual visual interpretation and has the potential for misjudgment.

[0007] The pretreatment processes for different dosage forms are not standardized, resulting in high costs for quality inspection documents and personnel management: Annao tablets can be directly ground and extracted, while Annao honey pills, due to their viscous honey matrix, require the addition of diatomaceous earth for mixing before extraction. The two types of preparations require separate independent inspection operating procedures, resulting in a large workload for document preparation, revision, and archiving. Inspection personnel need to master two sets of operating procedures, and the operation switching when sampling mixed tablets and pills is cumbersome. The training period for new employees is long, which is not conducive to the efficient implementation of quality inspection of multiple dosage forms on the same line.

[0008] Therefore, a single-plate, dual-color synchronous thin-layer identification and detection method for the three characteristic components of Anazoline is needed to solve the above problems. Summary of the Invention

[0009] The purpose of this invention is to provide a method for simultaneous thin-layer chromatography with dual-color development of three characteristic components of benzodiazepines to identify and detect these components, thereby addressing the problems mentioned in the background section.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual colorimetric development includes the following steps:

[0012] S1. Integrated pretreatment and extraction of mixed test samples: Take the menthol preparation sample for pretreatment, add ethanol solvent for ultrasonic extraction, filter the extract and concentrate under reduced pressure, and redissolve the residue with ethanol to prepare a single mixed test sample solution, simultaneously dissolving three characteristic target substances: bile acids, berberine alkaloids and geniposide.

[0013] S2. Simultaneous development of double silica gel G thin-layer plate chromatography: Take silica gel G thin-layer plate, and simultaneously add test solution, bile acid mixed control solution, berberine control combination solution and geniposide control solution. Develop once with isooctane-ethyl acetate-glacial acetic acid mixed solvent to simultaneously complete the chromatographic separation of three types of polarity-differential components.

[0014] S3. Triple-segmented dual-color synchronous imaging discrimination: After the thin-layer plate is dried, it is sprayed with sulfuric acid ethanol colorimetric solution and heated. The spot spectrum of geniposide is collected under natural light. Then, the spectrum of bile acid and berberine alkaloids is collected under a 365nm ultraviolet light source. The two types of colorimetric and three types of components are identified simultaneously on the same plate. The method is compatible with Annao tablets and Annao pills. There is room for improvement in the separation of characteristic spots.

[0015] The entire system replaces the traditional three independent identification processes, which can reduce the number of steps to switch between. There is room for optimization in terms of total inspection time, material consumption, and deviation of multi-plate systems. It is also compatible with two types of menthol preparations and has the potential to improve the stability of spot recognition.

[0016] Further technical solutions, the sampling range for step S1 is: 2-4 tablets of Annao film-coated tablets, 2-4g of Annao honey pills; the amount of ethanol extraction solvent used is 10-20mL, and the ultrasonic extraction time is 20-30min;

[0017] It provides inspectors with flexible adjustment space for on-site operation, broadens the environmental tolerance range of the method, and improves the identification stability of different batches of samples.

[0018] A further technical solution is that the amount of ethanol used for redissolving the residue after concentration in step S1 is 1-3 mL, eliminating the need for three separate extractions to prepare multiple test solutions.

[0019] After uniformly evaporating the extract under reduced pressure, it is diluted to volume with 1-3 mL of ethanol, eliminating the traditional three independent extraction and liquid preparation steps, reducing human operation errors caused by multiple weighing and transfer, simplifying the sample pretreatment chain, and improving the ease of operation when preparing test solutions for batches of samples simultaneously.

[0020] A further technical solution is that the volume ratio of the developing agent in step S2 is 15:7:5; the pre-saturation time of the developing tank is 10-20 min; the developing ambient temperature is 20-30℃; and the coating thickness of the silicone G thin-layer plate is 0.2-0.3 mm.

[0021] It adapts to different temperature and humidity laboratory environments, reduces the interference of environmental fluctuations on chromatographic separation effects, and improves the universality and adaptability of the method.

[0022] Further technical solutions, the sample spotting volume range in step S2: 1-3 μL for the test sample, bile acid control, and berberine control solution, and 3-5 μL for geniposide control solution;

[0023] The sample loading amount can be flexibly adjusted according to the concentration of the effective components in the sample, avoiding problems such as spot overload tailing or weak signal. The parallel strip arrangement can reduce the probability of cross-contamination of the sample and improve the cleanliness of the thin-layer spectrum.

[0024] In a further technical solution, step S3 involves a sulfuric acid-ethanol colorimetric reagent mass fraction of 9%–11%, a colorimetric heating temperature of 103–107°C, and a constant temperature heating time of 3–5 minutes.

[0025] Slight adjustments to the color development conditions will not cause the spots to be too dark or the color development to be insufficient, reducing the impact of the oven temperature control accuracy on the identification results and expanding the tolerance margin of the color development operation.

[0026] In a further technical solution, the colorimetric observation sequence in step S3 is to first collect the visible light spectrum of geniposide under natural light, and then collect the fluorescence spectrum under 365nm ultraviolet light. Both sets of spectra are stored on the same thin-layer carrier.

[0027] There is no need to save three sets of chromatographic images separately, which simplifies the workload of storing GMP inspection files and facilitates the determination of the results through dual-spectrum cross-comparison during subsequent review.

[0028] A further technical solution involves simultaneously preparing three negative control solutions (lacking pig bile powder, Coptis chinensis, and Gardenia jasminoides) and spotting them simultaneously throughout the entire detection process, resulting in fewer target characteristic color spots appearing in the corresponding areas of the negative bands.

[0029] Simultaneous preparation of negative solutions for the three missing core medicinal materials and simultaneous spotting on a plate can directly identify the absence of prescription medicinal materials and the adulteration of raw materials. It can also help distinguish between matrix blemishes and target characteristic spots, improve the rigor of identification and judgment, and reduce the potential probability of missing unqualified samples.

[0030] A further technical solution is to allow the thin-layer plate to stand and dry for 5 to 15 minutes after unfolding in step S2, and then perform the color development operation in step S3 after the organic solvent has fully evaporated.

[0031] After the thin-layer plate is developed, it should be left to stand for 5-15 minutes to allow the organic solvent to evaporate fully, avoiding the dilution of the colorimetric solution by residual ethanol and causing the spots to diffuse. The wide drying area is suitable for the differences in laboratory ventilation, ensuring that the spots have complete and clear outlines after color development.

[0032] Further technical solutions show that the entire identification process takes less time compared to the traditional plate separation method, and the separation degree of the three types of characteristic spots can reach 1.0 or above, making it suitable for batch testing of raw materials, intermediates and finished products of Annao preparations.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] This invention, relying on an integrated single-ethanol extraction process, can reduce the material and time consumption caused by multiple pretreatment and plate separation operations, and eliminate the ether defatting and multi-solvent reflux process required for traditional geniposide identification. The entire process uses only ethanol as a single solvent, which can alleviate the safety and maintenance pressure caused by flammable and explosive reagents. A single thin-layer plate can complete three identifications, and the amount of thin-layer plate and organic solvent used is decreasing. The manual repetitive operation is reduced when testing batch samples, and there is room for improvement in the turnaround efficiency of testing. It is suitable for large-scale centralized sampling and inspection of finished products.

[0035] This invention, relying on a single thin-layer plate simultaneous chromatographic separation process, can alleviate the systematic bias problem caused by multi-plate detection. All samples and reference standards are separated and developed under the same silica gel coating, temperature and humidity, and development saturation conditions. The three types of characteristic spots can be compared horizontally in the same spectrum. The test data can be archived with only a single image, which improves the uniformity of GMP test data traceability and reduces the probability of test judgment discrepancies and retests.

[0036] This invention, relying on a segmented dual-color synchronous imaging process, can reduce the interference of matrix contaminants on the identification of target spots. Two sets of chromatograms, visible light and ultraviolet fluorescence, are simultaneously stored on the same thin-layer carrier. Cross-comparison of the two spectra can help distinguish matrix contaminants from target feature spots. For samples with long storage periods and low content of effective ingredients, there is room for improvement in spot identification. This invention can reduce the probability of misjudgment by manual visual interpretation and improve the rigor of identification results.

[0037] This invention, relying on a unified testing process, can improve the compatibility of different dosage forms and reduce quality control management costs. The entire method only requires adjustments to the mixing operation according to the dosage form during the pretreatment stage. The entire process of extraction, spotting, development, and color development is unified. Enterprises only need to compile a single version of the testing operation procedure to cover two types of preparations, which can reduce the workload of document management and personnel training. The operation switching of testing personnel is also simpler, adapting to the routine quality control needs of enterprises with multiple dosage forms on the same production line.

[0038] To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the overall process of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] General pre-operation (all 8 examples were executed simultaneously): Three negative control samples were prepared according to the standard prescription of Annao, one lacking pig bile powder, one lacking Coptis chinensis, and one lacking Gardenia jasminoides. The proportions of other medicinal materials were kept consistent with the finished product prescription. Three negative control solutions were prepared simultaneously in the extraction steps of each example and added to the blank control band of the thin-layer plate. The negative band showed fewer color spots of the three target characteristics at the corresponding positions, which was used for specificity verification.

[0042] Example 1

[0043] like Figure 1 As shown, this embodiment of the invention provides a method for simultaneous thin-layer chromatography identification and detection of three characteristic components of Anaphyllin using a single plate with dual colorimetric development. It employs standard laboratory operating parameters and is suitable for routine release inspection of finished Anaphyllin film-coated tablets under normal temperature and moderate ventilation conditions. In this embodiment, a three-step progressive detection process is strictly implemented:

[0044] S1 Integrated Extraction: Three Annatto film-coated tablets were selected, the coating was removed, and the tablets were ground into a fine powder. The powder was then placed in a CNC ultrasonic cleaner, and 15 mL of ethanol was added. The ultrasonic power was set to 200 W, the frequency to 50 kHz, and the ultrasonic duration to 25 min. After extraction, the entire filtrate was collected by filtering with quantitative filter paper and transferred to a rotary evaporator. The filtrate was concentrated to dryness under reduced pressure at 45°C. 2 mL of ethanol was added, and the solution was thoroughly shaken to dissolve, yielding a clear, single-component mixed test solution. Three negative control solutions were prepared simultaneously for later use. Under these parameters, the three characteristic components of the extract dissolved evenly, and the solution showed no obvious turbidity or precipitation, requiring no additional purification steps.

[0045] S2 Single-Plate Synchronous Chromatography Operation: A 0.25mm thick silica gel G thin-layer plate was selected, and the sample bands were arranged in parallel with a band spacing of 10mm. 2μL of the test sample, a cholic acid-deoxycholic acid mixed control, and a mixture of Coptis chinensis reference material and berberine hydrochloride were spotted, along with 4μL of geniposide control solution. A ternary developing solvent (isooctane-ethyl acetate-glacial acetic acid, volume ratio 15:7:5) was added to the developing tank and pre-saturated for 15 min. The ambient temperature was set to 25℃. The thin-layer plate was placed in the developing tank and developed upwards. After a development distance of approximately 8cm, the plate was removed and allowed to stand and air dry for 10 min until the ethanol, ethyl acetate, and other organic solvents had completely evaporated and there was no obvious solvent odor on the plate surface. Under these parameters, the development speed was uniform, and the Rf values ​​for the three components were 0.70 for cholic acid, 0.43 for Coptis chinensis alkaloids, and 0.26 for geniposide, with no edge effect.

[0046] S3 segmented dual-color imaging: Prepare a 10% sulfuric acid ethanol colorimetric solution, spray it evenly onto the surface of the thin-layer plate until the surface is slightly damp and does not drip, and place it in a constant temperature oven at 105℃ for 4 minutes; remove the thin-layer plate and allow it to cool naturally to room temperature. First, photograph the brownish-yellow visible light spot spectrum of geniposide under natural light, and then place it in a 365nm ultraviolet imager to collect the blue-white fluorescence spectrum of cholic acid and the yellow fluorescence spectrum of Coptis chinensis. Archive both sets of spectra into the same thin-layer carrier file.

[0047] The detection results of this embodiment are as follows: the entire process took 52 minutes; the edges of the three types of characteristic spots were clear and without tailing, with an average resolution of 1.32, all of which were higher than the qualified threshold of 1.0; the number of specks on the plate surface was small, with no obvious interference from excipients; no target chromogenic spots were detected at the corresponding positions of the three negative control bands, indicating good specificity; a single thin-layer plate can complete the three pharmacopoeia identifications simultaneously, and the amount of thin-layer plate and organic solvent used is about 1 / 3 of that of the traditional plate separation method. The method has stable repeatability and can be used as a standard operating procedure for enterprise quality inspection.

[0048] Example 2

[0049] The difference between this embodiment and Embodiment 1 is that all operating parameters are set to relatively low values ​​to simulate the low-temperature, poorly ventilated laboratory environment of a small to medium-sized pharmaceutical company, thus verifying the method's fault-tolerance performance under low parameters. In this embodiment:

[0050] S1 Integrated Extraction: Two tablets of Annatto film-coated benzoyl peroxide were crushed and 10 mL of ethanol was added. The ultrasonic treatment time was set to 20 min. After the filtrate was evaporated to dryness under reduced pressure, 1 mL of ethanol was added to redissolve the filtrate. Three negative control solutions were prepared simultaneously. Under these parameters, the sample volume, solvent volume, and ultrasonic treatment time were all set to relatively low levels, and the concentration of the extract was relatively low. However, all three target substances were effectively dissolved, and the solution was clear without precipitation.

[0051] S2 Development Procedure: A 0.2 mm thin-layer plate was used. The developing tank was pre-saturated for 10 min, and the ambient temperature was controlled at 20℃. 1 μL of the test sample, various Coptis chinensis and cholic acid control solutions were spotted, and 3 μL of geniposide control solution was spotted. After development, the plate was allowed to stand and air dry for 5 min to allow the solvent to evaporate. Under these parameters, the development speed was slightly slow, taking approximately 35 min to develop at a distance of 8 cm. The plate thickness was relatively thin, but the mechanical strength met the operational requirements. After drying, there were no obvious residual solvent droplets on the plate surface.

[0052] S3 color development steps: Prepare a 9% sulfuric acid ethanol solution, bake in an oven at 103℃ for 3 minutes; first collect the geniposide spectrum under natural light, then collect the 365nm ultraviolet fluorescence spectrum, and store both sets of spectra on a single thin-layer plate.

[0053] The detection results of this embodiment are as follows: The total time for the entire process is 50 minutes; the three types of characteristic spots are slightly lighter in color but have complete and clear outlines, and there are no problems such as diffusion or weak signals that cannot be interpreted. The average resolution is 1.15, which is higher than the 1.0 pass line; the three negative control groups have no target color spots, and the specificity is not affected by parameter reduction; even under conditions of low temperature, poor ventilation and low parameters, the three types of characteristic components can still be stably identified simultaneously. The method has sufficient error tolerance and can be adapted to the testing laboratories of small and medium-sized pharmaceutical companies with general facilities.

[0054] Example 3

[0055] The difference between this embodiment and Embodiment 1 is that all operating parameters are set at relatively high values ​​to simulate the high-temperature, high-humidity, and well-ventilated environment of a large pharmaceutical company's central laboratory, verifying the method's tolerance under high parameters. In this embodiment:

[0056] S1 Integrated Extraction: Take 4 tablets of Annatto film-coated tablets, grind them into a fine powder, add 20 mL of ethanol, and sonicate for 30 min. After evaporating the filtrate to dryness, add 3 mL of ethanol to reconstitute it, and simultaneously prepare three sets of negative control solutions for later use. Extraction is more thorough under these parameters, and even for samples with low content of active ingredients during storage, the concentration of the target substance can be guaranteed to be sufficient.

[0057] S2 development step: A silica gel G thin-layer plate with a thickness of 0.3 mm is selected, the developing tank is pre-saturated for 20 min, and the ambient temperature is controlled at 30℃; 3 μL each of the test sample, cholic acid reference solution and Coptidis Rhizoma reference solution is spotted, and 5 μL of genipin reference solution is spotted; after the thin-layer plate completes development, it is left to stand for 15 min to dry, so that the organic solvent in the plate is fully volatilized. Under these parameters, the development speed is slightly faster in a high-temperature environment, the thick plate has stronger adsorption capacity, and spot overload can still be avoided under a high sample loading amount.

[0058] S3 color development step: A 11% sulfuric acid-ethanol color developing solution is prepared, the color development temperature in the oven is 107℃, and the heating duration is 5 min; a natural light spectrum and an ultraviolet fluorescence spectrum are collected in sequence and archived uniformly on the same thin-layer carrier.

[0059] Detection effect of this example: The total time consumed in the whole process is 62 min; the three types of characteristic spots are full in color development with moderate color saturation, no tailing or diffusion phenomenon caused by excessive sample loading occurs, the average resolution is 1.45, and the separation effect is better than that of the low-parameter group and the standard-parameter group; no corresponding characteristic spots appear in the three groups of negative control bands, no edge effect or increased miscellaneous spots occur in a high-temperature and high-humidity environment; under the extreme working condition with relatively high parameters, the chromatographic separation and color development identification effects remain stable, and the wide parameter range can improve the environmental adaptability of the method.

[0060] Example 4

[0061] The difference between this example and Example 1 lies in that: the detection matrix is replaced by An Nao small honey pills, a step of mixing with diatomaceous earth is added in the extraction process, and the rest of the development and color development operations are consistent with those in Example 1, so as to adapt to the finished product release inspection of the sugar-containing viscous honey pill matrix. In this example:

[0062] Three core detection steps are performed, S1 integrated extraction: Take 3 g of An Nao small honey pills, cut them into pieces, add 2 g of diatomaceous earth and fully grind the mixture to digest the viscous honey matrix and avoid caking, add 15 mL of ethanol and perform ultrasonic treatment for 25 min; after the filtrate is concentrated under reduced pressure to dryness, add 2 mL of ethanol for redissolution, and synchronously prepare three groups of negative control solutions lacking pig bile powder, lacking Coptidis Rhizoma and lacking Gardeniae Fructus. Diatomaceous earth can adsorb part of the honey polysaccharides and reduce the interference of matrix miscellaneous spots.

[0063] S2 single-plate synchronous chromatographic operation is completely consistent with that in Example 1: 0.25 mm thin-layer plate, 15 min of pre-saturation, development at 25℃, unified spotting volume of reference solutions, and standing to dry for 10 min after development.

[0064] S3 segmented dual-color development synchronous imaging is completely consistent with that in Example 1: 10% sulfuric acid-ethanol, heating at 105℃ for 4 min, collecting natural light spectrum and ultraviolet spectrum in sequence and storing them uniformly.

[0065] The detection results of this embodiment are as follows: The total time for the entire process is 56 minutes; the number of impurities caused by the honey matrix of the honey pills is relatively small; the cross-comparison of the two spectra can quickly distinguish the matrix impurities from the three types of target feature spots; the average separation degree of the three types of spots is 1.30, all of which are higher than the qualified threshold of 1.0; the negative control has no interfering colorimetric spots, and the specificity is good; the whole process does not require the separation of two independent operating procedures, and only the addition of diatomaceous earth mixing in the pretreatment stage can achieve simultaneous identification of honey pills; the parameters have good adaptability to the honey pill matrix and can be directly used for routine quality inspection of honey pills before they leave the factory.

[0066] Example 5

[0067] The difference between this embodiment and Embodiment 4 is that, under the condition of testing honey pill samples, all operating parameters are taken at relatively low values ​​to verify the practical stability of low parameters under the honey pill matrix. In this embodiment:

[0068] S1 Extraction: Take 2g of Annao Xiaomiwan (a type of honey pill), cut it into small pieces, mix it with diatomaceous earth, add 10mL of ethanol, and sonicate for 20min; after evaporating the filtrate to dryness, add 1mL of ethanol to redissolve it, and prepare three negative control solutions simultaneously. Even with low sample amounts, the adsorption of diatomaceous earth can still effectively reduce the viscosity of honey, and the extract is free of viscous lumps.

[0069] S2 Development Step: A 0.2 mm thin-layer plate was used, pre-saturated for 10 min, and developed at a low temperature of 20℃. 1 μL of the test sample, Coptis chinensis, and cholic acid control solution were spotted, and 3 μL of geniposide control solution was spotted. After development, the thin-layer plate was allowed to stand and air dry for 5 min.

[0070] S3 color development: Prepare 9% sulfuric acid ethanol, heat at 103℃ for 3 min, and collect natural light and ultraviolet fluorescence spectra in sequence and archive them to a single thin-layer plate simultaneously.

[0071] The detection results of this embodiment are as follows: The total time for the entire process is 54 minutes; the viscous matrix of the honey pills did not cause spot diffusion or signal loss, and the resolution of the three types of characteristic spots was higher than 1.0, with an average resolution of 1.12; the negative control had no target chromogenic spots, and the specificity was not affected by low parameters; even under low parameter conditions, the three legally defined characteristic indicators of the honey pill samples could still be clearly identified, and the low parameter range had good compatibility with both tablet and honey pill matrices. Small and medium-sized pharmaceutical companies can directly adopt this set of operating procedures for rapid detection of small quantities of honey pill samples.

[0072] Example 6

[0073] The difference between this embodiment and Embodiment 4 is that, under the condition of testing the honey pill samples, all operating parameters are taken at relatively high values ​​to verify the tolerance of the honey pill samples to high parameters under high humidity and long-term storage. In this embodiment:

[0074] S1 Integrated Extraction: Take 4g of Annao Honey Pills, mix thoroughly with diatomaceous earth, add 20mL of ethanol, and sonicate for 30min; evaporate the filtrate under reduced pressure to dryness, then add 3mL of ethanol to redissolve, and simultaneously prepare three sets of negative control solutions. High sampling volume and long sonication time can ensure the full dissolution of characteristic components that have decayed in long-stored honey pills.

[0075] S2 Development: Use a 0.3 mm thin-layer plate, pre-saturate for 20 min, develop at 30℃, spot 3 μL of the test sample, cholic acid, and berberine control solution, and spot 5 μL of geniposide control solution; after development, let the thin-layer plate stand and air dry for 15 min to fully evaporate the solvent.

[0076] S3 color development: Prepare 11% sulfuric acid ethanol, heat at 107℃ for 5 min, collect two sets of spectra in sequence and store them uniformly.

[0077] The detection results of this embodiment are as follows: the total time is 60 minutes; for honey pill samples with a long storage period and slight decline in effective ingredients, three types of characteristic spots can still be clearly detected, with an average resolution of 1.38, and the spots are full and without tailing; there are no interfering spots in the negative control, and the honey matrix spots do not increase or spread under high temperature and high humidity conditions; the higher parameters are suitable for high-load, high temperature and high humidity honey pill testing conditions, and can be used for centralized identification and screening of large batches of long-term stored honey pills.

[0078] Example 7

[0079] The difference between this embodiment and Embodiment 1 is that: a batch-scale testing mode combining tablets and honey pills is adopted, simultaneously processing 20 incoming samples. The extraction stage uses standard operating parameters for both tablets and honey pills, and the development and color development stages use standardized parameters to verify the batch adaptability of the method. In this embodiment:

[0080] The three-step testing process is executed synchronously in batches. In the S1 extraction stage, tablets are processed using a standard procedure of 3 tablets, 15 mL of ethanol, and 25 minutes of sonication. Honey pills are processed using a standard procedure of 3g of honey pills, mixed with diatomaceous earth, 15 mL of ethanol, and sonicated for 25 minutes. The filtrates from both types of samples are uniformly evaporated to dryness and then reconstituted with 2 mL of ethanol. Three sets of negative control solutions are prepared synchronously in batches. Multiple samples can be processed simultaneously during batch processing without distinguishing between two pretreatment procedures.

[0081] S2 single-plate synchronous chromatography uses a 0.25mm thin-layer plate. Each thin-layer plate has 8 sample bands, 4 control bands, and 3 negative bands arranged at multiple points. The sample volume is uniform, the plate is pre-saturated for 15 minutes, developed at 25℃, and then allowed to stand and dry for 10 minutes.

[0082] The S3 colorimetric method uniformly used 10% sulfuric acid ethanol heated at 105℃ for 4 minutes. Two sets of spectra were collected successively: natural light and ultraviolet fluorescence. The spectra of each thin-layer plate were archived independently.

[0083] The detection results of this embodiment are as follows: the total time for a single batch of 20 mixed samples is 58 minutes, with an average time of only 2.9 minutes per sample; the number of thin-layer plates consumed is significantly reduced compared to traditional plate separation processes, requiring only about 1 / 3 of the thin-layer plates and organic solvents required by traditional methods; the average resolution of the three types of characteristic spots is 1.35, and no target chromogenic spots were detected in any of the negative control bands, demonstrating stable specificity; the entire batch process does not require distinguishing between two independent operating procedures, and a single system can simultaneously process two types of formulations, ensuring stable reproducibility of the method under scaled-up batch conditions, making it suitable for the centralized raw material warehousing and quality inspection scenarios of large-scale pharmaceutical companies.

[0084] Example 8

[0085] The difference between this embodiment and Embodiment 1 is that: the extraction and unfolding are fixed as standard parameters, and only the combination of drying time and color development parameters is adjusted to specifically verify the parameter tolerance space of the color development and drying stages. In this embodiment:

[0086] S1 extraction and S2 development followed the standard procedure of Example 1: 3 tablets of Annao Nao, 15 mL of ethanol, sonicated for 25 min, 0.25 mm thin-layer plate, standard spotting volume, 15 min pre-saturation, 25 °C development, and three sets of negative control solutions were prepared simultaneously; only the drying time of the thin-layer plate was adjusted to 5 min, and the color development was carried out by a cross combination of 9% sulfuric acid ethanol, 107 °C heating temperature, and 3 min heating time. Natural light and ultraviolet fluorescence spectra were collected successively and stored uniformly on a single thin-layer carrier.

[0087] The detection results of this embodiment are as follows: The total time for the entire process is 51 minutes. Even under the cross-conditions of shortened thin-layer plate drying time, lower concentration of chromogenic reagent, higher heating temperature, and shorter heating time, the visible light spots of geniposide, bile acid, and berberine fluorescent spots are still clearly defined, without diffusion, uneven color development, or insufficient color development. The average separation degree of the three types of spots is 1.22, and there are no interfering spots in the negative control. This embodiment verifies that the chromogenic and drying parameters have sufficient operational tolerance, and minor operational deviations in the chromogenic and drying steps by the inspectors will not cause identification failure. The entire detection system has outstanding fault tolerance.

[0088] Working principle and usage process of this invention:

[0089] The S1 integrated extraction uses a moderately polar ethanol solvent, which can simultaneously dissolve weakly polar bile acids, moderately polar berberine alkaloids, and moderately polar geniposide. A single ultrasonic operation replaces the traditional multi-solvent stepwise extraction, preparing only a single mixed test solution, thus reducing operational errors caused by multiple pretreatments from the source.

[0090] S2 ternary mixed eluent relies on gradient polarity differences to separate three types of characteristic components with large polarity spans on a single silica gel G thin-layer plate. Combined with a wide range of temperature, humidity, plate thickness, and presaturation parameters, it reduces the interference of laboratory environmental fluctuations on chromatographic separation.

[0091] The S3 segmented dual-color method utilizes sulfuric acid and ethanol heating to generate visible light color spots for glycosides. A 365nm ultraviolet light source excites the conjugated structures of Coptis chinensis and bile acids to produce characteristic fluorescence. Two sets of spectra are simultaneously collected on the same thin-layer carrier to achieve cross-impurity identification. The three-step process is progressive and synergistic, systematically alleviating the routine quality inspection problems of traditional plate identification in the industry.

[0092] Use Case 1: Batch release inspection of finished products from pharmaceutical companies

[0093] The pharmaceutical company's finished product testing laboratory is equipped with an ultrasonic cleaner, developing tank, constant temperature oven, and ultraviolet imager. It routinely receives mixed samples of Annao tablets and Annao pills for testing. Using the complete testing system of this invention, S1 is a unified extraction process that does not require switching between two sets of operating procedures. S2 allows for the arrangement of multiple batches of sample strips at multiple points on a single thin-layer plate, resulting in a decreasing trend in the consumption of thin-layer plates and organic solvent consumables. S3 allows for the completion of three identification and archiving with only a single image on a single plate with dual color development, meeting the GMP requirements for traceability and archiving of inspection data. There is room for improvement in the number of samples that can be tested per day, making it suitable for routine finished product release and special sampling inspections by the drug regulatory authorities.

[0094] Use Case 2: Specialized Authenticity Screening of Raw Materials Entering the Factory for Chinese Herbal Medicines

[0095] In pharmaceutical companies' raw material warehouses, the process of verifying the authenticity of pig bile powder, Coptis chinensis, and Gardenia jasminoides before they enter the warehouse can be simplified by adjusting the sampling amount in S1 as needed to suit the raw material powder samples. S1 uses ethanol for rapid ultrasonic dissolution of the characteristic components inside the raw materials, S2 uses a single-plate simultaneous comparison of the raw material bands with the finished product control bands, and S3 uses dual-spectrum cross-references with three sets of negative controls to help determine the problems of missing or adulterated raw materials. This eliminates the need for three separate identification operations in the traditional method, and the raw material inspection cycle is trending towards shorter periods, which can alleviate the situation where raw material accumulation delays production and material input in the workshop.

[0096] The thin-layer chromatography equipment, organic solvents, reference standards, and thin-layer carriers involved are all commonly used and existing materials in domestic Chinese medicine quality inspection laboratories. Practitioners of Chinese medicine quality inspection in this field can completely reproduce the entire testing process. The scope of protection of this invention does not involve the modification of testing instrument hardware or the improvement of testing software programs.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual colorimetric development, characterized in that, Includes the following steps: S1. Integrated pretreatment and extraction of mixed test samples: Take the menthol preparation sample for pretreatment, add ethanol solvent for ultrasonic extraction, filter the extract and concentrate under reduced pressure, and redissolve the residue with ethanol to prepare a single mixed test sample solution, simultaneously dissolving three characteristic target substances: bile acids, berberine alkaloids and geniposide. S2. Simultaneous development of double silica gel G thin-layer plate chromatography: Take silica gel G thin-layer plate, and simultaneously add test solution, bile acid mixed control solution, berberine control combination solution and geniposide control solution. Develop once with isooctane-ethyl acetate-glacial acetic acid mixed solvent to simultaneously complete the chromatographic separation of three types of polarity-differential components. S3. Triple segmented dual-color synchronous imaging discrimination: After the thin-layer plate is dried, it is sprayed with sulfuric acid ethanol colorimetric solution and heated. The spot spectrum of geniposide is collected under natural light. Then, it is transferred to a 365nm ultraviolet light source to collect the fluorescence spectrum of cholic acid and berberine alkaloids. The two types of colorimetric and three types of components are identified simultaneously on the same plate.

2. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... Step S1 sampling range: 2-4 tablets of Annao film-coated tablets, 2-4g of Annao honey pills; 10-20mL of ethanol extraction solvent, and 20-30min of ultrasonic extraction.

3. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... The amount of ethanol used for redissolving the residue after concentration in step S1 is 1-3 mL.

4. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... In step S2, the volume ratio of the developing agent is 15:7:5; the pre-saturation time of the developing tank is 10-20 min, and the developing ambient temperature is 20-30℃; the coating thickness of the silicone G thin-layer plate is 0.2-0.3 mm.

5. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... Step S2: Spotting volume range: 1-3 μL for test sample, bile acid control, and berberine control solution, and 3-5 μL for geniposide control solution. The spotting bands are arranged in parallel.

6. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... Step S3: The mass fraction of sulfuric acid ethanol colorimetric reagent is 9%–11%, the colorimetric heating temperature is 103–107℃, and the constant temperature heating time is 3–5 min.

7. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... The sequence of colorimetric observation in step S3 is as follows: first, collect the visible light spectrum of geniposide under natural light, and then collect the fluorescence spectrum under 365nm ultraviolet light. Both sets of spectra are stored on the same thin-layer carrier.

8. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... During the entire detection process, three negative control solutions lacking pig bile powder, Coptis chinensis, and Gardenia jasminoides were prepared and spotted simultaneously. The areas corresponding to the negative bands showed fewer characteristic spots of the target.

9. The method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in claim 1, is characterized in that... After the thin-layer plate is unfolded in step S2, it should be allowed to stand and dry for 5 to 15 minutes until the organic solvent has fully evaporated before proceeding to the color development operation in step S3.

10. A method for simultaneous thin-layer chromatography identification and detection of three characteristic components of benzodiazepines using a single plate with dual color development, as described in any one of claims 1 to 9, characterized in that, The entire identification process takes less time compared to the traditional plate separation method, and the separation degree of the three types of characteristic spots can reach 1.0 or above. It is suitable for batch testing of raw materials, intermediates and finished products of Annao preparations.

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