Quantitative determination method for effective components in traditional Chinese medicine capsule

Abstract

The invention relates to the technical field of material testing and analysis, and discloses a quantitative determination method for effective components in a traditional Chinese medicine capsule, which comprises the following steps: mixing choline chloride and ethylene glycol to prepare a deep eutectic solvent system; the traditional Chinese medicine capsule is placed in the eutectic solvent system, a composite frequency ultrasonic field is applied, a sound pressure amplitude step is generated by alternately switching a first working frequency and a second working frequency, and the eutectic solvent system is driven to complete solvent exchange in the medicine powder micropores; according to the method, the diffusion equilibrium of a microscopic interface is broken through the physical suction effect generated by sound pressure step, a solvent saturation dead zone in the compact particles is eliminated, and on the basis of integration of extraction and purification, the extraction and purification efficiency is greatly improved. And the accuracy and the stability of quantitative response signals are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for quantitatively determining effective components in traditional Chinese medicine capsules, and belongs to the technical field of material testing and analysis. BACKGROUND

[0002] Currently, the quantitative determination of effective components in traditional Chinese medicine capsules usually adopts organic solvents combined with ultrasonic assisted extraction, uses a liquid chromatograph to record a response signal, and relies on solvation to realize the migration of target components to a liquid phase, which is a general technical path in this field. A traditional Chinese medicine capsule is composed of medicine powder and a capsule shell, and the capsule shell contains gelatin protein and pigments. In the determination process, the solubility selectivity of conventional organic solvents for target components and matrix components is low. When the ultrasonic power or the energy field distribution is adjusted to improve the recovery rate, the macromolecular components in the capsule shell are physically swollen and simultaneously enter the solvent system, which increases the viscosity of the extraction liquid and produces a matrix inhibition effect in the subsequent chromatographic determination, thereby shortening the operation cycle of the chromatographic column.

[0003] Although solid-phase extraction or multi-stage dilution methods can handle the above-mentioned interference, they involve multiple physical transfer steps, produce detection errors, and the conventional technical improvements focus on the backend purification, without addressing the physical contradictions in the extraction stage. The reason is that the existing solvent system cannot distinguish between the solubilization requirements of target components and the repulsion requirements of matrix molecules based on the hydrogen bond energy level difference, which leads to the formation of solvent saturation dead zones in the internal pores of the medicine powder particles, limiting the migration of components across the interface. The industry explores specific extraction media limited to static formula adjustment, and the dynamic feedback control of physical fields is insufficient. For example, the Chinese invention patent with the publication number CN109171014A discloses a menthol eutectic solvent and a capsule for cigarettes. This technology focuses on component reorganization and does not touch the control of physical interface breakthrough in complex matrix extraction. In the face of aged or high-density medicine powder particles, it lacks real-time monitoring and closed-loop adjustment of ultrasonic cavitation energy level, transducer acoustic impedance, and cavitation noise sub-band energy, cannot judge the shell rupture instant and the internal saturation point of the medicine powder, and has a blind area in the determination of the extraction endpoint, making it difficult to overcome the diffusion lag of high-viscosity solvents in micro-pores. The high-concentration solvent does not consider the strong solvent effect caused by the high-concentration solvent during the sampling stage, resulting in distortion of the chromatographic peak shape.

[0004] Therefore, how to use the hydrogen bond network of the eutectic solvent to construct an extraction environment with selectivity, and cooperate with the electric parameter feedback and polarity gradient adjustment of the physical field to eliminate matrix interference, becomes a technical problem to be solved by the present application. SUMMARY

[0005] To solve the problems raised in the background art, the technical solution of the present application is as follows: a method for quantitatively determining effective components in traditional Chinese medicine capsules, comprising the following steps:

[0006] Step 101: Mix choline chloride and ethylene glycol, controlling the molar ratio of choline chloride to ethylene glycol to be 1:2.02 to 1:2.08, and heat at 75°C. Up to 76 Stir at a constant temperature until a homogeneous liquid is formed, and add 4% to 5% deionized water based on the total mass of choline chloride and ethylene glycol to obtain a eutectic solvent system.

[0007] Step 102: Place the herbal capsules to be tested in a eutectic solvent system, and control the extraction temperature within the range of 40°C. Up to 45 Initiate composite frequency ultrasonic processing: perform continuous cavitation at a first frequency of 38kHz to 40kHz; during continuous cavitation, periodically switch the ultrasonic frequency to a second frequency of 20kHz to 22kHz and maintain it for 2s to 4s, and use the sound pressure amplitude step generated by the frequency switching to perform solvent exchange on the powder inside the Chinese medicine capsule to be tested.

[0008] Step 103: Maintain the extraction temperature so that the gelatin component in the Chinese medicine capsule to be tested after being treated with composite frequency ultrasound in step 102 remains in an undissolved solid precipitate state in the eutectic solvent system.

[0009] Step 104: Centrifuge the extracted mixture to obtain the supernatant. Inject the supernatant into an ultra-high performance liquid chromatograph (UHPLC) and separate the active ingredients by gradient elution to obtain a quantitative response signal. The gradient elution uses an organic phase and an aqueous phase containing an acidic regulator as the mobile phase.

[0010] Preferably, step 102 further includes an extraction endpoint control step: by real-time monitoring of the current phase feedback signal of the ultrasonic transducer, the acoustic impedance characteristics of the eutectic solvent system are obtained; when the acoustic impedance characteristics change abruptly, it is determined to be the shell rupture point of the Chinese medicine capsule to be tested, and the composite frequency ultrasonic processing is switched from the detection mode to the extraction mode.

[0011] Preferably, step 102 further includes a particle infiltration monitoring step: real-time acquisition of the acoustic signal generated by ultrasonic cavitation and extraction of the subband energy integral value in the 150kHz to 300kHz frequency band, based on the rate of change of the subband energy integral value over time. Determine the solvation saturation state and rate of change of the drug powder particles. The following relationship must be satisfied: ,in, The sub-band energy integral value for the 150kHz to 300kHz frequency band. The duration after step 102 is initiated; when the rate of change When the energy of the powder particles approaches zero and the integral value of the sub-band energy remains stable within the preset range for more than 60 seconds, it is determined that the powder particles have reached the fully wetted state.

[0012] Preferably, when the rate of change When the value is less than the preset threshold, the ultrasonic drive frequency is adjusted to perform frequency sweep processing, thereby breaking the acoustic shielding between the powder particles by changing the sound field distribution.

[0013] Preferably, in step 101, the mass fraction of deionized water added is 4.8%.

[0014] Preferably, in step 102, the extraction temperature is controlled at 42.5°C. ; and during continuous cavitation, the ultrasonic frequency is switched to a second frequency every 55 to 65 seconds and maintained for 2 to 4 seconds.

[0015] Preferably, in step 104, the supernatant is mixed with a zone compression reagent before being injected into the ultra-high performance liquid chromatograph. The zone compression reagent is a low-boiling-point ether compound. The zone compression reagent is used to adjust the polarity difference between the supernatant and the mobile phase, so that the active ingredient can be focused in a zone at the head of the chromatographic column.

[0016] Preferably, the injection step adopts a sandwich injection mode, in which the supernatant is placed between two zonal compression reagents in the injection flow path, and the zonal compression reagents are used to perform transient polar dilution of the eutectic solvent system in the supernatant to eliminate the chromatographic peak prolongation caused by the solvent effect.

[0017] Preferably, in step 104, the gradient elution uses acetonitrile and a 0.1% (w / w) aqueous solution of phosphoric acid as the mobile phase, and the active ingredient is separated from the solvent peak within 3.5 min.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. In traditional Chinese medicine capsules, the regular hydrogen bond network formed by hydrogen bond acceptors and donors in the eutectic solvent is utilized to achieve specific anchoring of target molecules by leveraging the matching energy levels of hydrogen bonds between the acceptors and donors and the functional groups of the target active ingredient. Meanwhile, gelatin protein remains physically precipitated because it cannot integrate into this high-density hydrogen bond network, preventing matrix components from entering the liquid phase system. This simplifies the pretreatment process and eliminates background signal interference. By monitoring the acoustic impedance feedback signal of the ultrasonic transducer during the extraction process, the interface breakthrough point where the capsule shell transitions from a rigid, closed state to a flexible, permeable state is captured. The ultrasonic output mode is adjusted according to this interface breakthrough point, ensuring that the extraction energy field precisely acts on the solute exchange stage after the shell structure collapses. This avoids matrix dissolution and component degradation caused by fixed energy excitation, improving the reproducibility of measurements between samples with different storage durations.

[0020] 2. By utilizing the instantaneous switching between continuous cavitation mode and pressure pulse mode of ultrasonic working frequency, a step fluctuation in sound pressure amplitude is generated within the solvent system. This physically draws the micropores inside the powder particles, forcibly emptying the saturated solvent within the micropores and drawing in fresh extraction medium. This disrupts the diffusion balance of high-viscosity solvent at the micro-interface, ensuring complete release of the effective components in the core region of the high-density powder. Based on the adaptive wetting mechanism of cavitation noise subband energy feedback, the solvation saturation inside the powder particles is characterized in real time. By capturing the energy attenuation trajectory in the 150kHz to 300kHz frequency band, the particle wetting rate is inverted, and frequency scanning is triggered to eliminate the acoustic shielding effect between particles. This enables the extraction process to have adaptive compensation capabilities for fluctuations in the properties of complex powders, reducing systematic errors in the recovery rate.

[0021] 3. The sandwich segmented structure constructed in the injection path utilizes the conditioning liquid to perform transient polarity adjustment on the eutectic solvent, forming a viscosity barrier at the column head, causing the analyte to precipitate from the solvent clusters and pin to the front of the stationary phase, thereby achieving forced compression of the physical band and eliminating the chromatographic peak forwarding and splitting caused by high concentration of matrix solvent, thus optimizing the symmetry of the quantitative response signal while maintaining high detection sensitivity. Attached Figure Description

[0022] Fig. 1 This is a flowchart of the quantitative determination process of eutectic solvent and composite frequency ultrasound according to the present invention.

[0023] Fig. 2 This is a timing diagram showing the collaborative interaction of the various execution units in the quantitative measurement system of this invention. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. These embodiments are intended to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0025] This invention provides a method for the quantitative determination of active ingredients in traditional Chinese medicine capsules. It utilizes a eutectic solvent system with a specific hydrogen bond energy level distribution, combined with a composite frequency ultrasonic field to trigger hydrogen bond replacement and interfacial mass exchange, and employs polarity gradient adjustment during sample introduction to achieve zonal focusing of the target component. This enables quantitative analysis of components in gelatin and excipient matrices. The method encompasses solvent preparation, physical field-induced extraction, in-situ matrix shielding, and chromatographic signal acquisition stages. The stability of the determination results is ensured through coupling of physicochemical parameters at each stage. In preparing the eutectic solvent system, hydrogen bond acceptor choline chloride and hydrogen bond donor ethylene glycol are mixed, with their molar ratio controlled at a specific ratio. to If the molar ratio is controlled to be The determination of the molar ratio range depends on the hydrogen bond network density and kinetic viscosity of the equilibrium system. When the molar ratio is lower than... At this time, the intermolecular forces between the hydrogen bond acceptor and the hydrogen bond donor are in an unsaturated state, causing the system to... The viscosity below mPa·s, suppressing ultrasonic cavitation efficiency and reducing the recovery rate of effective components to [value missing]. The following; and when the molar ratio is higher At this time, excessive ethylene glycol molecules cause local collapse of the hydrogen bond network, increasing the solubility of the gelatin component in the tested herbal capsules in the system, resulting in decreased transparency of the extract. The mixture was then placed in a thermostatic magnetic stirrer... to Continue stirring at the specified temperature until a homogeneous, transparent liquid is formed. Add a mass fraction of [missing information] to the liquid. to Deionized water, such as adding a mass fraction of Deionized water, through the intervention of water molecules, adjusts the hydrogen bond network density, allowing the solvent to... At the extraction temperature, the solvent exhibits a preset flowability and solute exchange rate. This eutectic solvent utilizes the hydrogen bond energy level matching characteristics between itself and the functional groups of the target active ingredient to anchor the target molecule, while matrix components such as gelatin protein remain in a physically precipitated state because they cannot integrate into this hydrogen bond network.

[0026] In the physical field-induced extraction process, the herbal capsules to be tested were placed in a eutectic solvent system, and the extraction temperature was controlled at [temperature value missing]. The ultrasonic generator is then activated. The ultrasonic processing initially operates in probe mode, monitoring the current phase feedback signal of the ultrasonic transducer in real time to obtain the acoustic impedance characteristic value of the eutectic solvent system. The system then acquires the voltage signal through a digital signal processing unit. With current signal Real-time phase angle between fundamental components And introduce phase angle deviation rate As a discriminant, the calculation formula is as follows: ;in, This represents the average phase angle reference value during the steady-state operation phase of the detection mode. When the system detects the phase angle deviation rate... When five consecutive sampling points exceed the threshold of 15%, the shell of the Chinese medicine capsule under test is identified as a rupture point, which triggers the switching of ultrasonic processing from the detection mode to the extraction mode. This multi-point continuous determination logic aims to effectively filter out linear impedance drift signals caused by slow fluctuations in extraction temperature, sedimentation of powder particles, or environmental noise through time-domain feature extraction. The system also uses a digital processing unit to capture the real-time voltage signal across the ultrasonic transducer. With real-time current signal The voltage signal is obtained by performing a fast Fourier transform. With current signal The phase difference between fundamental components, i.e., the real-time phase angle. Determine the phase angle deviation rate of the shell rupture point The following relationship must be satisfied: ,in, Indicates the phase angle deviation rate. Indicates the real-time phase angle. This represents the average phase angle reference value during the steady-state operation phase of the detection mode, when the phase angle deviation rate in the current phase feedback signal... When a mutation occurs, it is identified as a rupture point in the shell of the tested herbal capsule. The ultrasonic treatment is then switched from the detection mode to the extraction mode, and the power is adjusted to... W, this step adjusts the ultrasonic output through physical parameter feedback to avoid excessive matrix dissolution caused by fixed energy excitation; the logic for determining the rupture point of the capsule shell of the Chinese medicine to be tested in step 102 includes: driving the ultrasonic transducer to perform a 20kHz to 50kHz frequency sweep process under the detection mode, and collecting the voltage signal at both ends of the transducer. With current signal The real-time phase angle is obtained by calculating the phase difference between the two fundamental components through a digital signal processing unit. The average phase angle was continuously sampled and calculated within 10 seconds before the program started, and used as the average phase angle reference value during the steady-state operation phase of the detection mode. Real-time phase angle Deviation from benchmark value When the percentage of five consecutive sampling points exceeds 15%, it is determined to be a shell rupture point and triggers ultrasonic processing to switch from detection mode to extraction mode.

[0027] The powder extraction stage employs a composite frequency ultrasonic mode that alternates between the first and second frequencies. kHz to A first frequency of kHz performs continuous cavitation, disrupting the intermolecular forces between the active ingredient and the drug powder matrix. During the continuous cavitation process, at each interval... s to s, such as every s, switch the frequency to kHz to The second frequency of kHz and maintained s to The extraction process utilizes a step-like acoustic pressure amplitude generated by frequency switching to physically draw the medicinal powder micropores, driving the exchange between the saturated solvent and fresh extraction medium within the micropores. This ensures complete release of the active ingredients from the core region of the high-density medicinal powder. During extraction, an adaptive wetting mechanism based on cavitation noise subband energy feedback is used to characterize the solvation saturation state of the medicinal powder particles in real time. The acoustic frequency signal generated by ultrasonic cavitation is acquired and extracted in real time. kHz to Subband energy integral value in kHz band Based on the rate of change of the subband energy integral value over time Determine the wetting state and the rate of change. The following relationship must be satisfied: ,in, For the rate of change, for kHz to Subband energy integral value in the kHz band. The duration after ultrasonic treatment is initiated, when the rate of change... The time it takes for the subband energy integral value to approach zero and stabilize within the preset range exceeds [a certain value]. At time s, it is determined that the powder particles have reached a fully wetted state. If the rate of change... If the value is less than a preset threshold, the ultrasonic drive frequency is adjusted to perform frequency sweep processing to break the acoustic shielding between the powder particles; the calibration process for determining the full wetting state of the powder particles in step 102 includes: in a pure eutectic solvent system without the addition of Chinese medicine capsules, ultrasonic processing at a first frequency of 38kHz to 40kHz is initiated, cavitation signals are collected by an acoustic sensor with a sampling frequency of 500kHz, and the initial value of the subband energy integral in the frequency band of 150kHz to 300kHz is obtained by analyzing the 4096-point fast Fourier transform. ,Will The preset range is set to 90% to 95%, and the judgment threshold is set to 0.05. During the extraction process, when the sub-band energy integral value... Entering this interval and over time rate of change When the absolute value is below the judgment threshold for 60 consecutive seconds, it is determined that the internal pores of the powder particles have completed solvent exchange and reached a fully wetted state.

[0028] The extracted mixture is maintained at In a constant-temperature environment, the gelatin protein component in the ultrasonically broken capsule shells remains insoluble in a physical precipitation state in a eutectic solvent system due to the energy level difference of the hydrogen bond network. The mixture is centrifuged to obtain the supernatant. During the injection stage, a zone focusing method is used. The supernatant is mixed with a zone compression reagent (a low-boiling-point ether compound) before injection into the ultra-high performance liquid chromatograph. The injection step uses a sandwich injection mode, placing the supernatant between two zones of the zone compression reagent in the injection path. The zone compression reagent performs transient polar dilution of the eutectic solvent in the supernatant, causing the active ingredient to precipitate from the solvent clusters and concentrate at the front of the stationary phase, achieving forced compression of the physical zones. In step 104, the zone compression reagent is selected from methyl tert-butyl ether or isopropyl ether. When the lower polarity parameter is below 3.0 and the kinetic viscosity is below 0.5 mPa·s, it forms a polarity step when combined with a eutectic solvent system with a kinetic viscosity greater than 20 mPa·s. During sandwich injection, the volume ratio of supernatant to zonal compression reagent is controlled at 1:0.3 to 1:0.4. The supernatant is placed between the two zonal compression reagents in the injection path. The transient polarity dilution of the eutectic solvent by the zonal compression reagents allows the active ingredient to produce a zonal focusing effect at the front of the stationary phase at the column head, eliminating the peak protrusion caused by solvent viscosity and polarity differences. Chromatographic detection uses gradient elution with acetonitrile and [missing information - likely a specific concentration or value]. A phosphoric acid aqueous solution was used as the mobile phase to allow the active ingredient to... Within min, the peak is separated from the solvent peak and a quantitative response signal is obtained.

[0029] Example 1: When the system faces a traditional Chinese medicine capsule sample with high physical resistance due to deep oxidative cross-linking of gelatin molecules in the capsule shell; the sample is placed in a container with a molar ratio of choline chloride to ethylene glycol of [missing information]. And contains a mass fraction of A eutectic solvent system using deionized water is employed; its well-organized hydrogen bond network serves as an anchoring medium for phenolic hydroxyl groups in the analyte, enabling specific desorption of the active ingredient. During the extraction initiation phase, the ultrasonic generator is controlled to operate in sweep frequency mode, and the current phase feedback signal of the ultrasonic transducer is acquired in real time. When a nonlinear abrupt change in the phase angle deviation rate is detected, it is determined to be the interface breakthrough point where the capsule shell transitions from rigid sealing to flexible penetration; subsequently, the process is switched to... W extracts power and initiates composite frequency ultrasonic processing, that is, in kHz to During the continuous cavitation process at the first frequency of kHz, each interval s instantly switch to kHz to The second frequency of kHz and maintained s, through the physical suction effect generated by the step of sound pressure amplitude, breaks the diffusion balance in the micropores of the drug powder, driving the saturated solvent and the fresh extraction medium to complete the solvent exchange at the micro interface.

[0030] Real-time parsing and extraction process kHz to kHz band subband energy integral value And by calculating the rate of change of the integral value over time To determine the solvation saturation state of the drug powder particles, the rate of change is used. Satisfying the relation ,in, For the rate of change, for kHz to Subband energy integral value in the kHz band. Duration; rate of change The time it takes for the subband energy integral value to approach zero and stabilize within the preset range exceeds [a certain value]. After s, the effective components were determined to have achieved molecular-level full-wetting extraction; the obtained extract, after being mixed with a low-boiling-point ether-based zone-compression reagent, was injected into the ultra-high performance liquid chromatograph using a sandwich injection mode. The transient adjustment effect of the polar gradient formed a focused zone at the column head. The separation of the active ingredient and solvent peaks was completed within min, and the quantitative response signal was recorded.

[0031] Example 2: In an experimental scenario to verify the extraction rate of effective components from aged samples of traditional Chinese medicine capsules with highly oxidative cross-linked capsule shells; the physical experimental platform for performing the experiment included a frequency conversion ultrasonic generator and an ultra-high performance liquid chromatograph equipped with a diode array detector; the ultrasonic generator has... kHz to kHz frequency conversion output capability and The power adjustment resolution of W; the temperature control accuracy of the ultra-high performance liquid chromatograph is... And the sampling frequency is not lower than Hz; To simulate mechanical background interference in an industrial setting, a signal-to-noise ratio of Hz is actively superimposed onto the signal acquisition link. dB of Gaussian white noise was used to simulate the signal attenuation environment; experimental data were acquired based on actual measurements from a physical experimental platform. The performance of the coupling between the composite frequency ultrasonic field parameters and the low eutectic solvent energy level was verified by comparing the recovery rates of the effective components under different aging times; the extraction power of the composite frequency ultrasonic treatment was set to [value missing]. W, its engineering basis lies in coordinating the desorption kinetic energy of the effective component with the physical impedance of the gelatin shell, when the power is at Below W, the intermolecular forces within the micropores of the drug powder are difficult to overcome, leading to a stagnation in recovery rate growth. However, when the power exceeds W... W induces thermal degradation of gelatin segments and increases the viscosity resistance of the extract; the mass fraction of deionized water is fixed at 1%. This parameter is determined based on the functional relationship between the fluidity requirement of the hydrogen bond network and the density of the hydrogen bond network. When the moisture content is lower than... At that time, the system's kinetic viscosity limited the ultrasonic cavitation efficiency, while the water content exceeded [a certain threshold]. This weakens the anchoring strength between the hydrogen bond acceptor and the donor, leading to a decrease in extraction selectivity.

[0032] During the experiment, the aging time was as follows: Months Months Months Months and Capsule samples collected over one month were allocated to the experimental group and control group B; control group B used a choline chloride to ethylene glycol molar ratio of [missing value]. Eutectic solvent and perform kHz constant frequency ultrasound extraction; the experimental group followed the alternating frequency switching procedure of the specific implementation method; utilizing Calculate the sub-band energy integral value Over time rate of change ,in, For the rate of change, for kHz to Subband energy integral value within the kHz band, The duration of ultrasonic treatment after it is initiated is shown in Table 1.

[0033] Table 1: Comparison of measured data for samples with different aging times under experimental and control group B modes.

[0034]

[0035] Referring to Table 1, with the increase in physical impedance due to the increase in capsule shell aging time, the recovery rate of the effective ingredient in control group B decreased from... Descending to The recovery rate of the active ingredient in the experimental group consistently remained at a linear deterioration trend across all gradient samples. In summary, the physical suction effect generated by the step amplitude of sound pressure effectively eliminates the solvent saturation dead zone inside high-density particles, enabling the extraction process to have adaptive compensation capability for fluctuations in the properties of complex powders, and achieving simultaneous improvement in extraction efficiency and the stability of quantitative response signal.

[0036] Example 3: This example combines Figs. 1-2 This document describes a method for the quantitative determination of the active ingredients in a traditional Chinese medicine capsule, such as... Fig. 1As shown, the process begins in step 101, where choline chloride and ethylene glycol are mixed in a molar ratio of 1:2.02 to 1:2.08. The mixture is stirred at a constant temperature of 75°C to 76°C to form a homogeneous liquid. Then, 4% to 5% (w / w) of deionized water is added to prepare a eutectic solvent system. Next, step 102 is performed, where the herbal capsule to be tested is placed in the eutectic solvent system. A composite frequency ultrasonic treatment is initiated at 40°C to 45°C, with continuous cavitation performed at a first frequency of 38kHz to 40kHz, periodically switching to 20kHz. The second frequency of z to 22kHz is maintained for 2s to 4s, and solvent exchange is performed using a step of sound pressure amplitude. Then, step 103 is performed to maintain the extraction temperature so that the gelatin component in the Chinese medicine capsule to be tested after ultrasonic treatment of the composite frequency remains in an undissolved solid precipitation state in the eutectic solvent system. Finally, step 104 is performed to centrifuge the extraction mixture to obtain the supernatant, which is injected into an ultra-high performance liquid chromatograph. An organic phase and an aqueous phase containing an acidic regulator are used as the mobile phase. The effective components are separated by gradient elution and the quantitative response signal is obtained.

[0037] like Fig. 2 As shown, the determination process involves the interaction of five main units: operator, solvent system, ultrasonic system, centrifuge, and chromatograph. The operator mixes choline chloride and ethylene glycol in the solvent system, stirs it at a constant temperature until it becomes a homogeneous liquid, and adds deionized water. After the eutectic solvent system is prepared, the herbal capsule to be tested is placed in it. The ultrasonic system starts the detection mode, and after detecting the shell rupture point, it switches to the extraction mode and performs composite frequency ultrasonic treatment. After extraction, the operator transfers the extraction mixture to the centrifuge for centrifugation. Finally, the operator obtains the supernatant and injects it into the chromatograph. The chromatograph performs sandwich injection and zonal focusing, and outputs a quantitative response signal after gradient elution separation.

[0038] Example 4: For cases where the shell wall thickness deviation exceeds... And after In a scenario where the effective components of traditional Chinese medicine capsules aged for several months are extracted through physical field induction; the extraction is achieved by mixing choline chloride (a hydrogen bond acceptor) and ethylene glycol (a hydrogen bond donor) in a controlled molar ratio. With temperature control accuracy of In a thermostatic magnetic stirrer Stir until a homogeneous, transparent liquid is formed, wherein the stirring temperature is set based on the activation equilibrium point of the association energy barrier between hydrogen bond acceptor and hydrogen bond donor molecules; add a mass fraction of [missing information] to the liquid. Deionized water was used to adjust the hydrogen bond network density; the sample was placed in the resulting eutectic solvent system and the ultrasonic transducer was controlled to enter the detection mode.

[0039] The detection mode is transmitted through an ultrasonic transducer. kHz to Frequency sweep within the kHz range and acquisition of acoustic impedance characteristic values ​​in pure solvent state. As a calibration benchmark; real-time monitoring of the ultrasonic transducer's current phase feedback signal to analyze the real-time phase angle. The logical rule for determining the shell rupture point is: when the phase angle deviation rate... continuous The number of sampling points exceeded the threshold. At that time, the ultrasonic processing is triggered to switch from the detection mode to the extraction mode, where To detect the steady-state phase angle reference value recorded during the modal initiation phase; the system then automatically switches the power from W was raised to W and activate composite frequency ultrasonic processing, through During the kHz continuous cavitation process, each interval The duration of one insertion of s is s A kHz pressure pulse generates a step in sound pressure amplitude and exerts a physical suction effect on the micropores of the powder; the system analyzes in real time. kHz to Subband energy integral value in kHz band With duration rate of change ,in The deterministic procedure for determining whether the powder particles have reached a fully wetted state is set as follows: when the rate of change... The absolute value continues s is lower than and Stabilized at the initial integral value of the acoustic characteristic power spectrum to When the sample is within the specified range, the physical wetting process is considered complete, and ultrasonic excitation is terminated. The resulting extract, after being mixed with low-boiling-point ether compounds used as zone-compression reagents, is injected into the ultra-high performance liquid chromatograph using a sandwich injection mode. The transient adjustment effect of the polar gradient creates physical zone compression at the column head to eliminate the solvent effect, and the recovery rate of the effective components in the sample stabilizes within a certain range. And the relative standard deviation within the batch is .

[0040] Example 5: In a scenario where acoustic characteristics are calibrated for a newly replaced ultrasonic transducer; the ultrasonic generator performs a frequency sweep in a pure eutectic solvent system without the addition of traditional Chinese medicine capsules, with a sweep frequency range of [missing information]. kHz to kHz, by continuously acquiring 10 sets of phase angle data and calculating their arithmetic mean as the initial steady-state phase angle reference value. In the simulation experiment, active introduction was made into the extraction system. Temperature fluctuations and simulated powder settling interference were observed. Actual test results showed that the phase angle deviation rate caused by these interferences was [missing information]. All are less than 5% and do not exhibit instantaneous jump characteristics; however, when the capsule shell undergoes physical rupture, the phase angle... This produces a significant nonlinear mutation, making Within 0.05 seconds (5 consecutive sampling periods), the preset threshold of 15% was rapidly exceeded; the acoustic impedance characteristic values ​​at each frequency sweep point were recorded, and a background field power spectrum distribution model was established to control the extracted power to be... When the sound pressure amplitude fluctuation of the transducer is within the rated output value, W... Within the range.

[0041] In scenarios where raw material moisture content is calibrated to address variations in environmental humidity; The mixing molar ratio under constant temperature conditions is Choline chloride and ethylene glycol were mixed and stirred at a constant speed. After the system reaches a transparent and homogeneous state, the dynamic viscosity of the liquid is measured using a rotational viscometer at rpm. If dynamic viscosity Deviation from preset benchmark value mPa·s, then based on viscosity deviation Replenish moisture, among which the measured value Each increase The mass fraction of deionized water is mPa·s. exist Increasing on the basis And the adjusted total moisture content is limited to to Within the range; the calibrated solvent rheological parameters support the hydrogen bond network in The high-density diffusion channels required for phenolic hydroxyl substitution are maintained, ensuring that the consistency deviation of the quantitative results of the active ingredient between batches under different environmental humidity is kept within acceptable limits. Within.

[0042] Example 6: In a scenario where systematic calibration of the cavitation noise subband energy integral parameter is performed during composite frequency ultrasonic processing to eliminate errors in determining the wetting of drug powder through micropores; the sampling frequency is controlled to be... A kHz acoustic sensor acquires cavitation signals from the extract and sets the number of points for a Fast Fourier Transform. for ,in Indicates the number of transformation points; based on the sliding window overlap rate. The time-domain segmentation logic obtains the spectral resolution, and the spectral resolution is used for analysis. kHz to Subband energy integral value in kHz band ,in This represents the sub-band energy integral value; the spectral resolution setting is based on capturing the non-stationary signal characteristics at the instant of the aged gelatin shell rupture, by selecting the number of points when the sampling frequency is fixed. Balancing the constraints between time resolution and frequency resolution supports the determination of the rate of change in powder particles reaching full wetting. The physical precision, of which Satisfying the relation ,in Indicates the rate of change and Indicates duration.

[0043] In scenarios where chromatographic injection polarity dilution ratios are optimized to eliminate solvent peak interference in high-concentration eutectic solvent systems, low-boiling-point ether compounds are selected as zone-compressing reagents, and an injection volume ratio gradient sequence is established. The volume ratio range of the supernatant to the zone-compressing reagent is set as follows: to The physicochemical basis for selecting the zone compression reagent lies in the interfacial polarity difference between the polarity grade of low-boiling-point ether compounds and the eutectic solvent system, which generates transient zone focusing on the stationary phase surface at the column head; when the volume ratio is adjusted to At that time, the physical band width of the active ingredient molecules at the column head is compressed to the initial state due to the step adjustment of the polarity level. Within.

[0044] Example 7: To further verify the driving effect of the sound pressure amplitude step in step 102 on solvent exchange at the microscopic interface of high-viscosity eutectic solvent, this example constructs a visualization verification platform based on a microfluidic chip. A simulated drug powder micropore array is prepared using transparent siloxane material and pre-filled with a saturated extract containing tracer fluorescent microspheres. In a constant-temperature eutectic solvent system at 42.5℃, the control group is subjected to constant-frequency ultrasound at 40kHz. Due to the kinetic viscosity of the eutectic solvent being greater than 20mPa·s at this temperature, the fluorescence signal intensity inside the micropores does not significantly decrease within 300s, proving that solvation alone is insufficient to overcome the diffusion dead zone within the dense micropores. The experimental group strictly follows the composite frequency switching procedure in step 102 of this invention, that is, during continuous cavitation at the first frequency of 38kHz to 40kHz, the frequency is periodically switched to the second frequency of 20kHz to 22kHz every 60s and maintained for 3s.

[0045] Experimental observations show that at the instantaneous switch of ultrasonic frequency from the first frequency to the second frequency, due to the nonlinear abrupt change in acoustic impedance, significant microjets and physical suction phenomena are induced at the simulated microchannel opening, resulting in a measured instantaneous sound pressure difference. achieve As the frequency step cycle repeats, the intensity of the tracer fluorescence in the pores exhibits a significant step-like decay characteristic. Within 4 step cycles, the solvent exchange rate inside the micropores exceeds 92%. This quantitative test data directly confirms that the physical suction force generated by the sound pressure amplitude step is sufficient to break the diffusion equilibrium of the micro-interface. From the physical mechanism level, it ensures the complete release of the effective components in the core area of ​​the high-density drug powder and solves the diffusion lag problem of high-viscosity solvent systems in the extraction of complex matrices.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for quantitative determination of active ingredients in traditional Chinese medicine capsules, characterized in that, Includes the following steps: Step 101: Mix choline chloride and ethylene glycol, controlling the molar ratio of choline chloride to ethylene glycol to be 1:2.02 to 1:2.08, and heat at 75°C. Up to 76 Stir at a constant temperature until a homogeneous liquid is formed, and add 4% to 5% deionized water based on the total mass of choline chloride and ethylene glycol to obtain a eutectic solvent system. Step 102: Place the herbal capsules to be tested in a eutectic solvent system, and control the extraction temperature within the range of 40°C. Up to 45 Initiate composite frequency ultrasonic processing: perform continuous cavitation at a first frequency of 38kHz to 40kHz; during continuous cavitation, periodically switch the ultrasonic frequency to a second frequency of 20kHz to 22kHz and maintain it for 2s to 4s, and use the sound pressure amplitude step generated by the frequency switching to perform solvent exchange on the powder inside the Chinese medicine capsule to be tested. Step 103: Maintain the extraction temperature so that the gelatin component in the Chinese medicine capsule to be tested after being treated with composite frequency ultrasound in step 102 remains in an undissolved solid precipitate state in the eutectic solvent system. Step 104: Centrifuge the extracted mixture to obtain the supernatant. Inject the supernatant into an ultra-high performance liquid chromatograph (UHPLC) and separate the active ingredients by gradient elution to obtain a quantitative response signal. The gradient elution uses an organic phase and an aqueous phase containing an acidic regulator as the mobile phase.

2. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, In step 102, the power of the composite frequency ultrasonic treatment is 385W; the cavitation field generated by the first frequency is used to break the intermolecular forces between the active ingredient and the inner wall of the micropores of the drug powder, and the physical suction effect generated by the step of the sound pressure amplitude is used to expel the saturated extraction medium inside the micropores and draw in the eutectic solvent system.

3. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, Step 102 also includes an extraction endpoint control step: by real-time monitoring of the current phase feedback signal of the ultrasonic transducer, the acoustic impedance characteristics of the eutectic solvent system are obtained; when the acoustic impedance characteristics change abruptly, it is determined to be the shell rupture point of the Chinese medicine capsule to be tested, and the composite frequency ultrasonic processing is switched from the detection mode to the extraction mode.

4. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, Step 102 also includes a particle infiltration monitoring step: real-time acquisition of the acoustic signal generated by ultrasonic cavitation and extraction of the subband energy integral value in the 150kHz to 300kHz frequency band, based on the rate of change of the subband energy integral value over time. Determine the solvation saturation state and rate of change of the drug powder particles. The following relationship must be satisfied: ,in, The sub-band energy integral value for the 150kHz to 300kHz frequency band. The duration after step 102 is initiated; when the rate of change When the energy of the powder particles approaches zero and the integral value of the sub-band energy remains stable within the preset range for more than 60 seconds, it is determined that the powder particles have reached the fully wetted state.

5. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 4, characterized in that, When the rate of change When the value is less than the preset threshold, the ultrasonic drive frequency is adjusted to perform frequency sweep processing, thereby breaking the acoustic shielding between the powder particles by changing the sound field distribution.

6. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, In step 101, the mass fraction of deionized water added is 4.8%.

7. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, In step 102, the extraction temperature is controlled at 42.5°C. ; During continuous cavitation, the ultrasonic frequency is switched to a second frequency every 55 to 65 seconds and maintained for 2 to 4 seconds.

8. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, In step 104, the supernatant is mixed with a zone compression reagent before being injected into the ultra-high performance liquid chromatograph. The zone compression reagent is a low-boiling-point ether compound. The zone compression reagent is used to adjust the polarity difference between the supernatant and the mobile phase, so that the active ingredient can be focused in a zone at the head of the chromatographic column.

9. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 8, characterized in that, The injection step adopts a sandwich injection mode, in which the supernatant is placed between two zonal compression reagents in the injection flow path, and the zonal compression reagents are used to perform transient polar dilution of the eutectic solvent system in the supernatant.

10. The method for quantitative determination of active ingredients in a traditional Chinese medicine capsule according to claim 1, characterized in that, In step 104, gradient elution uses acetonitrile and a 0.1% (w / w) aqueous solution of phosphoric acid as the mobile phase, and the active ingredient is separated from the solvent peak within 3.5 min.

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