Equipment and method for detecting illegal antibiotics in plant extract

Abstract

The invention relates to the technical field of analysis and detection, and discloses a device and method for detecting illegal antibiotics in plant extracts.The device comprises a sample preparation module used for weighing, homogenizing, extracting and centrifugally separating a plant extract sample; the sample purification module is used for removing impurities in the extracting solution and concentrating a target object; the detection module is used for separating and quantitatively analyzing target antibiotics; the data processing module is used for collecting and analyzing detection data; and the control and safety module is used for operation control and safety protection. According to the equipment, high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS / MS) technologies are combined, the problems that a target object is interfered by a matrix and the sensitivity is insufficient in a traditional detection method are solved through efficient purification of the solid-phase extraction module, the extraction and purification efficiency of antibiotics is optimized through sample preparation and purification steps in the method, trace illegal antibiotics can be detected, and the method is suitable for large-scale popularization and application. Meanwhile, high-sensitivity and high-accuracy detection is realized.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a detection device and method for illegal antibiotics in plant extracts. Background Art

[0002] Plant extracts are important raw materials for medicines, health products, food additives and cosmetics. Their natural origin and wide application make them occupy an important position in the market. However, it is not uncommon for some manufacturers to illegally add antibiotics to improve product performance, increase antibacterial effects or extend shelf life. These illegal antibiotics not only affect the pure natural properties of plant extracts, but may also cause consumers to take low doses of antibiotics for a long time, thereby causing antibiotic resistance, allergic reactions and other health risks.

[0003] At present, the detection of illegal antibiotics in plant extracts mainly adopts high performance liquid chromatography (HPLC) or gas chromatography (GC) combined with mass spectrometry (MS). However, these detection methods usually have the following problems: plant extracts contain a large amount of complex matrices such as pigments, sugars, lipids, etc., which can easily interfere with the separation and detection of target antibiotics, resulting in reduced sensitivity and accuracy. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a device and method for detecting illegal antibiotics in plant extracts, which solves the problem that plant extracts contain a large amount of complex matrices such as pigments, sugars, lipids, etc., which easily interfere with the separation and detection of target antibiotics, resulting in a decrease in sensitivity and accuracy during detection.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: a detection device for illegal antibiotics in plant extracts, comprising:

[0006] A sample preparation module for weighing, homogenizing, extracting and centrifuging plant extract samples;

[0007] Sample purification module, used to remove impurities in the extract and concentrate the target substance;

[0008] A detection module for separation and quantitative analysis of target antibiotics;

[0009] A data processing module is used to collect and analyze the detection data;

[0010] Control and safety modules are used for operation control and safety protection.

[0011] Preferably, the sample preparation module comprises:

[0012] Precision electronic balance, using electromagnetic force balance sensor, weighing accuracy is 0.01mg, weighing pan is made of corrosion-resistant stainless steel;

[0013] High speed homogenizer:

[0014] High-speed motor with a speed range of 3000-30,000rpm;

[0015] Homogenizing blade head, made of titanium alloy or 316L stainless steel, resistant to chemical corrosion;

[0016] Built-in temperature control device monitors the temperature during homogenization through a thermal sensor and controls the temperature within 25°C through a cooling fan;

[0017] Ultrasonic Extractor:

[0018] Ultrasonic wave generator with a frequency of 20-40kHz;

[0019] The sample tank is made of chemical corrosion resistant materials and has a tank capacity of 2-5L;

[0020] Constant temperature oscillator:

[0021] The temperature control range is 20℃ to 80℃, and the temperature control accuracy is ±1℃;

[0022] Oscillation speed range is 50-300rpm;

[0023] High-speed centrifuge:

[0024] Driven by a brushless DC motor, the speed range is 5000-15,000rpm;

[0025] The sample rotor supports 10-50mL sample tubes.

[0026] Preferably, the sample purification module comprises:

[0027] Solid phase extraction device:

[0028] Solid phase extraction column, the filling material is C18 bonded silica gel or polymer resin, and the column shell is made of polypropylene or stainless steel;

[0029] Elution device, the flow control range of which is 1-5mL / min;

[0030] Vacuum Concentrator:

[0031] Vacuum pump, minimum pressure up to 10Pa, oil-sealed or oil-free design;

[0032] The condenser tube prevents the loss of solvent from evaporation, and the temperature control range of the sample heating plate is 30°C to 60°C. Preferably, the detection module includes:

[0033] High Performance Liquid Chromatography HPLC:

[0034] Mobile phase delivery system, pump flow rate range 0.1-2.0mL / min;

[0035] Chromatographic column, stationary phase is C18 reverse phase column, particle size is 3-5μm, length is 100-150mm; column oven, temperature control range is 20℃ to 60℃;

[0036] Tandem mass spectrometer MS / MS:

[0037] Electrospray ionization source ESI, used to generate parent ions;

[0038] Triple quadrupole mass analyzer with a mass range of 10-2000Da and a resolution of 0.1Da;

[0039] A collision cell having an adjustable collision energy range of 10-50 eV for optimizing parent and daughter ion fragmentation. Preferably, the data processing module comprises:

[0040] Data Acquisition System:

[0041] An analog signal converter converts the analog signal of the detector into a digital signal;

[0042] A data storage unit with a storage capacity of at least 1 TB for storing detection data;

[0043] Data analysis software:

[0044] Standard curve calibration function, used to generate concentration curve of target antibiotic;

[0045] Quantitative analysis function is used to calculate the concentration of target antibiotics in samples.

[0046] Preferably, the control and safety module includes:

[0047] Touch control panel:

[0048] Parameter input interface, used to input equipment operating parameters, including speed, temperature and pressure;

[0049] Real-time display interface, used to display equipment operation status and test results;

[0050] Safety protection device:

[0051] Overload protection system monitors the motor load through the current sensor and triggers automatic power-off protection when overloaded;

[0052] The temperature control protection device uses a thermistor to detect the device temperature in real time and automatically shuts down when the temperature exceeds the set range.

[0053] Preferably, the method for detecting illegal antibiotics in plant extracts comprises the following steps:

[0054] S1. Sample preparation, processing plant extract samples by weighing, homogenizing, extracting and centrifuging to obtain supernatant;

[0055] S2, sample purification, removing impurities through a solid phase extraction device and concentrating the target substance to dryness;

[0056] S3, detection and analysis, using high performance liquid chromatography and tandem mass spectrometry to separate, identify and quantify the target antibiotics;

[0057] S4. Data processing, generating test results through data acquisition and analysis software.

[0058] Preferably, the S1 sample preparation step comprises:

[0059] S101. Weighing samples:

[0060] Use a precision electronic balance to weigh the plant extract samples with a weighing accuracy of 0.01-0.1 mg and a sample mass range of 1-5 g;

[0061] S102, adding extraction solvent:

[0062] The sample was added to a mixed solvent of acetonitrile and water at a volume ratio of 60:40 to 90:10, and 0.05% to 0.2% of acid was added to the solvent;

[0063] S103, homogenization treatment:

[0064] The samples were homogenized using a high-speed homogenizer with a speed range of 8000-12,000 rpm and a processing time of 20-60 seconds;

[0065] S104, ultrasonic extraction:

[0066] The homogenized sample is placed in an ultrasonic extractor with an ultrasonic frequency of 20-40 kHz, an extraction temperature of 30-50°C, and an extraction time of 10-40 minutes;

[0067] S105, centrifugal separation:

[0068] Centrifuge at 8,000-12,000 rpm for 5-15 minutes in a high-speed centrifuge and collect the supernatant.

[0069] Preferably, the S2 sample purification step comprises:

[0070] S201, loading samples:

[0071] The supernatant collected in step S105 is loaded onto a C18 solid phase extraction column, wherein the filling material of the extraction column is bonded silica gel with a particle size range of 30-60 μm;

[0072] S202, activated extraction column:

[0073] Use 2-5 mL of methanol to activate the extraction column and balance it with 2-5 mL of deionized water;

[0074] S203, sample loading and impurity washing:

[0075] The supernatant was passed through the extraction column at a flow rate of 0.5-2 mL / min and washed with 2-5 mL of 0.05%-0.2% acidified aqueous solution to remove polar impurities;

[0076] S204, target elution:

[0077] Elute the target antibiotic with 2-5 mL of organic solvent and collect the eluate into a concentration container;

[0078] S205, Concentration and Reconstitution:

[0079] Use a vacuum concentrator to concentrate the eluent to dryness, control the vacuum pressure at 5-20Pa, and control the temperature at 30-50°C. After concentration to dryness, add 0.5-2mL of mobile phase to reconstitute.

[0080] Preferably, the S3 detection and analysis step includes:

[0081] S301, HPLC separation:

[0082] The chromatographic column is a C18 reverse phase column with a column length ranging from 100 to 150 mm, an inner diameter ranging from 3.0 to 4.6 mm, and a particle size of 3 to 5 μm;

[0083] The mobile phase was acetonitrile-water gradient elution, containing 0.05%-0.2% acid;

[0084] The flow rate range was 0.8-1.5 mL / min, and the column temperature was controlled at 30-50 °C;

[0085] Gradient Program:

[0086] 0-2 minutes: acetonitrile 30%-40%, aqueous phase 60%-70%;

[0087] 2-10 minutes: linear gradient to acetonitrile 70%-90%, aqueous phase 10%-30%;

[0088] 10-12 minutes: acetonitrile maintained at 70%-90%, aqueous phase 10%-30%;

[0089] S302, tandem mass spectrometry:

[0090] The ionization method was electrospray ionization (ESI);

[0091] The mass ranges of parent and product ions were m / z 700–900 and m / z 150–700;

[0092] The collision energy range is 20-50 eV;

[0093] The scanning mode was selected reaction monitoring SRM;

[0094] The S4 data processing step includes:

[0095] S401, data collection:

[0096] The liquid chromatography and mass spectrometry signals were recorded using a data acquisition system with an acquisition frequency of 1-10 Hz;

[0097] S402, peak area calculation:

[0098] The peak area of ​​the mass spectrometer output signal was integrated by the analysis software, and the peak area range was 1000-10,000 units;

[0099] S403, concentration calculation:

[0100] The standard curve is generated based on the concentration of the standard solution, and the linear fitting coefficient R 2 ≥0.99;

[0101] S404, result output:

[0102] Generate a test report including target concentration, standard curve, spike recovery and detection limit.

[0103] The present invention provides a device and method for detecting illegal antibiotics in plant extracts. It has the following beneficial effects:

[0104] 1. The device of the present invention combines high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS / MS) technology, and solves the problem of target interference and insufficient sensitivity in traditional detection methods through efficient purification of solid phase extraction module. The sample preparation and purification steps in the method optimize the extraction and purification efficiency of antibiotics, and can detect trace amounts of illegal antibiotics, while having high sensitivity and high accuracy detection.

[0105] 2. The equipment of the present invention is equipped with a touch control panel, automatic parameter setting and alarm system. Through functions such as automatic flow rate control, gradient elution and vacuum concentration, human intervention is reduced and sample processing efficiency is improved. The method simplifies the sample pretreatment steps and improves work efficiency through the combination of a homogenizer, an ultrasonic extractor and centrifugal separation.

[0106] 3. The detection method of the present invention reduces the dependence on high-risk chemical reagents by optimizing the sample processing process and solvent use conditions, while reducing the use of organic solvents and waste emissions. Its scientific experimental step design ensures the operational safety of sample extraction, purification and detection, and reduces the risk of sample degradation and environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 The overall structural block diagram of the detection device for illegal antibiotics in plant extracts of the present invention;

[0108] Figure 2 It is a structural block diagram of the sample preparation module of the present invention;

[0109] Figure 3 It is a structural block diagram of the sample purification module of the present invention;

[0110] Figure 4 It is a structural block diagram of the detection module of the present invention;

[0111] Figure 5 It is a structural block diagram of the data processing module of the present invention;

[0112] Figure 6 It is a structural block diagram of the control and safety module of the present invention;

[0113] Figure 7 The present invention is a flow chart of the method for detecting illegal antibiotics in plant extracts. DETAILED DESCRIPTION

[0114] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0115] Please see attached Figure 1 -Attached Figure 6 , an embodiment of the present invention provides a detection device for illegal antibiotics in plant extracts, comprising:

[0116] A sample preparation module for weighing, homogenizing, extracting and centrifuging plant extract samples;

[0117] Sample purification module, used to remove impurities in the extract and concentrate the target substance;

[0118] A detection module for separation and quantitative analysis of target antibiotics;

[0119] A data processing module is used to collect and analyze the detection data;

[0120] Control and safety modules are used for operation control and safety protection.

[0121] Specifically, the present invention achieves high-sensitivity and high-accuracy detection of illegal antibiotics in plant extracts by combining high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS / MS) technology, as well as optimized sample preparation and purification processes. The C18 solid phase extraction column in the device can efficiently remove interferences such as pigments and lipids in the sample matrix, improve the purity of the target, HPLC has excellent separation performance, and MS / MS can perform accurate quantitative and qualitative analysis of low-concentration illegal antibiotics. At the same time, the device is equipped with a touch control panel and automatic parameter setting function, combined with automatic flow rate control, vacuum concentration and alarm system, which greatly reduces human intervention and improves sample processing efficiency.

[0122] The sample preparation module includes:

[0123] Precision electronic balance, using electromagnetic force balance sensor, weighing accuracy is 0.01mg, weighing pan is made of corrosion-resistant stainless steel;

[0124] High speed homogenizer:

[0125] High-speed motor with a speed range of 3000–30,000rpm;

[0126] Homogenizing blade head, made of titanium alloy or 316L stainless steel, resistant to chemical corrosion;

[0127] Built-in temperature control device monitors the temperature during homogenization through a thermal sensor and controls the temperature within 25°C through a cooling fan;

[0128] Ultrasonic Extractor:

[0129] Ultrasonic wave generator with a frequency of 20-40kHz;

[0130] The sample tank is made of chemical corrosion resistant materials and has a tank capacity of 2-5L;

[0131] Constant temperature oscillator:

[0132] The temperature control range is 20℃ to 80℃, and the temperature control accuracy is ±1℃;

[0133] Oscillation speed range is 50-300rpm;

[0134] High-speed centrifuge:

[0135] Driven by a brushless DC motor, the speed range is 5000-15,000rpm;

[0136] The sample rotor supports 10-50mL sample tubes.

[0137] Specifically, the implementation method is:

[0138] Precision electronic balance:

[0139] Structure: The electronic balance adopts a high-precision electromagnetic force balance sensor with a weighing accuracy range of 0.01-0.1mg and a maximum weighing range of 200g.

[0140] Operation: Place the plant extract sample on the weighing pan of the balance and use an external shield to prevent ambient airflow from interfering with the weighing result. The weighing result is displayed in real time on the digital panel.

[0141] High speed homogenizer:

[0142] Construction: The homogenizer includes a high-speed motor (speed range is 3000-30,000rpm), a titanium alloy homogenizing blade and a cooling fan. The homogenizing blade is detachable and adaptable to sample containers of different capacities (10-100mL).

[0143] Operation: Place the sample and solvent in a homogenizing container, adjust the speed to 10,000 rpm, and run for 30 seconds; during the homogenization process, the temperature is controlled within 25°C to ensure that the active ingredients of the sample are not affected by high temperature.

[0144] Ultrasonic Extractor:

[0145] Structure: The extractor includes an ultrasonic generator (frequency range is 20-40kHz), a corrosion-resistant sample tank (capacity is 2-5L) and a cooling water circulation system.

[0146] Operation: Transfer the homogenized sample to the sample tank, set the ultrasonic frequency to 30kHz, the extraction temperature to 40℃, and run for 30 minutes; the cooling system automatically adjusts the circulating water flow rate to maintain a constant temperature.

[0147] High-speed centrifuge:

[0148] Construction: The centrifuge is driven by a brushless DC motor with a speed range of 5000-15,000rpm and is equipped with an automatic balancing device to reduce vibration; the sample rotor supports centrifuge tubes with a capacity of 10-50mL.

[0149] Operation: Place the extract in a centrifuge tube, set the speed to 10,000 rpm, and run for 10 minutes; collect the supernatant into a collection tube using an automatic pouring device.

[0150] Effect:

[0151] Through homogenization and ultrasonic extraction, the extraction efficiency of the target antibiotics is effectively improved, and the extraction recovery rate can reach more than 90%;

[0152] The centrifugation process removes solid impurities, which greatly reduces the risk of clogging in subsequent purification steps;

[0153] The temperature control system ensures the chemical stability of antibiotics in the sample and avoids decomposition or degradation caused by high temperature.

[0154] The sample cleanup module includes:

[0155] Solid phase extraction device:

[0156] Solid phase extraction column, the filling material is C18 bonded silica gel or polymer resin, and the column shell is made of polypropylene or stainless steel;

[0157] Elution device, the flow control range of which is 1-5mL / min;

[0158] Vacuum Concentrator:

[0159] Vacuum pump, minimum pressure up to 10Pa, oil-sealed or oil-free design;

[0160] Condenser to prevent solvent evaporation loss, sample heating plate temperature control range is 30 ℃ to 60 ℃.

[0161] Specifically, the implementation method is:

[0162] Solid phase extraction device:

[0163] Structure: The device consists of a solid phase extraction column, a sample loading unit and an automatic flow rate control valve; the extraction column is filled with C18 bonded silica gel with a particle size of 40-60μm, which is suitable for the purification of plant extract samples.

[0164] operate:

[0165] Pre-column treatment: Activate and equilibrate the extraction column with 3 mL of methanol and 3 mL of deionized water, respectively;

[0166] Sample loading: The supernatant was loaded into the extraction column at a rate of 1 mL / min through the loading unit;

[0167] Impurity removal: Wash with 3 mL of 0.1% formic acid water to remove polar impurities;

[0168] Elution of target: Elute the target antibiotic with 3 mL of acetonitrile and collect the eluate into a concentration bottle.

[0169] Vacuum Concentrator:

[0170] Construction: The concentrator is equipped with a digital temperature control board (temperature control range is 30-50℃), a low-pressure vacuum pump (vacuum pressure range is 5-20Pa) and a condenser to recover volatile solvents.

[0171] operate:

[0172] Place the eluent on the concentrator hot plate;

[0173] Set the vacuum pressure to 10Pa and the temperature to 40℃;

[0174] Run until the liquid is completely concentrated to dryness, and add 1 mL of mobile phase to reconstitute.

[0175] Effect:

[0176] The solid phase extraction column removes non-target substances (such as lipids and pigments) through highly selective adsorption, improving the purity of the target antibiotics, with a purification efficiency of more than 95%;

[0177] Vacuum concentration significantly shortens sample processing time (about 20 minutes) while protecting the chemical stability of the target and providing high-concentration samples for subsequent testing.

[0178] The detection module includes:

[0179] High Performance Liquid Chromatography HPLC:

[0180] Mobile phase delivery system, pump flow rate range 0.1-2.0mL / min;

[0181] Chromatographic column, the stationary phase is C18 reverse phase column, the particle size is 3-5μm, and the length is 100-150mm;

[0182] Column oven, temperature control range is 20℃ to 60℃;

[0183] Tandem mass spectrometer MS / MS:

[0184] Electrospray ionization source ESI, used to generate parent ions;

[0185] Triple quadrupole mass analyzer with a mass range of 10-2000Da and a resolution of 0.1Da;

[0186] Collision cell with adjustable collision energy range of 10-50 eV for optimized parent and daughter ion fragmentation.

[0187] Specifically, the implementation method is:

[0188] High Performance Liquid Chromatography (HPLC):

[0189] Structure: The equipment consists of a high-pressure infusion pump, a C18 reverse phase column (particle size 3-5 μm, column length 100-150 mm) and a UV detector; the mobile phase is acetonitrile-water gradient elution (containing 0.1% formic acid).

[0190] operate:

[0191] Inject the reconstituted sample into the autosampler;

[0192] Set the flow rate to 1 mL / min and the column temperature to 40 °C;

[0193] Run according to the gradient program and record the characteristic peak of the target antibiotic (such as the retention time of erythromycin is 7-9 minutes).

[0194] Tandem mass spectrometry (MS / MS):

[0195] Configuration: Includes electrospray ion source (ESI), triple quadrupole mass analyzer and data acquisition terminal; scanning mode is selected reaction monitoring (SRM).

[0196] operate:

[0197] Set the parent ion and daughter ion pair (e.g., the parent ion m / z of erythromycin is 734.5, and the daughter ion m / z is 158.0);

[0198] The collision energy was adjusted to 30 eV and the scan time was 0–12 min;

[0199] The data terminal records the response value of the target object for quantitative analysis.

[0200] Effect:

[0201] Liquid chromatography achieved efficient separation of target antibiotics, with peak resolution greater than 1.5 between different antibiotics;

[0202] The mass spectrometer provides high-sensitivity detection with a detection limit of up to 0.1 ng / mL, ensuring reliable detection of low-concentration illegal antibiotics.

[0203] The data processing module includes:

[0204] Data Acquisition System:

[0205] An analog signal converter converts the analog signal of the detector into a digital signal;

[0206] A data storage unit with a storage capacity of at least 1 TB for storing detection data;

[0207] Data analysis software:

[0208] Standard curve calibration function, used to generate concentration curve of target antibiotic;

[0209] Quantitative analysis function is used to calculate the concentration of target antibiotics in samples.

[0210] Specifically, the implementation method is:

[0211] Data Acquisition System:

[0212] Structure: includes signal converter, storage module and real-time display unit; storage capacity is at least 1TB for saving detection records.

[0213] Operation: Collect and convert the output signals of liquid chromatography and mass spectrometry into digital data.

[0214] Data analysis software:

[0215] Structure: includes concentration fitting module, report generation module and recovery rate calculation module.

[0216] operate:

[0217] Generate a concentration curve based on the standard solution (linear correlation coefficient R 2 ≥0.99);

[0218] The concentration and spike recovery of the target antibiotics were calculated using software (ranging from 60% to 120%);

[0219] Output detailed report including target concentration, standard curve and detection limit.

[0220] Effect:

[0221] Automatic data processing avoids human calculation errors and improves analysis efficiency;

[0222] The output test report is intuitive and clear, providing highly reliable data support for the determination of illegal antibiotics.

[0223] The control and safety modules include:

[0224] Touch control panel:

[0225] Parameter input interface, used to input equipment operating parameters, including speed, temperature and pressure;

[0226] Real-time display interface, used to display equipment operation status and test results;

[0227] Safety protection device:

[0228] Overload protection system monitors the motor load through the current sensor and triggers automatic power-off protection when overloaded;

[0229] The temperature control protection device uses a thermistor to detect the device temperature in real time and automatically shuts down when the temperature exceeds the set range.

[0230] Specifically, the implementation method is:

[0231] Touch control panel:

[0232] Construction: The touch panel consists of a high-sensitivity capacitive touch screen and an embedded processor. The screen size is 5-10 inches and the resolution is not less than 720p.

[0233] Function: Input equipment operating parameters through the operation interface, including the speed of the high-speed homogenizer, the temperature and time of ultrasonic extraction, the speed of the centrifuge and the gradient flow rate, etc. It supports sub-module adjustment and provides parameter preset function for repeated experimental operations.

[0234] Real-time display system:

[0235] Structure: The real-time display unit is integrated into the touch panel, and the displayed content includes the current operating status (such as equipment temperature, pressure, time, etc.), warning information and completion time countdown.

[0236] Function: The detection data is transmitted from the sensor to the display screen, which can monitor the key operating parameters of the equipment (such as temperature stability ±1°C, vacuum pressure fluctuation range ±5Pa), helping users to make real-time adjustments.

[0237] Effect:

[0238] The parameter control system realizes intuitive operation through the touch interface and can accurately adjust the operating parameters of each module to improve the automation level of the equipment;

[0239] The real-time monitoring function can quickly respond to equipment abnormalities (such as high temperature or low vacuum), reduce human errors and improve experimental reliability.

[0240] Overload protection function:

[0241] Structure: The overload protection module consists of a current sensor and a power-off switch. The current sensor monitors the current load of the equipment in real time. When the load exceeds the rated power (for example, the motor power is overloaded when the high-speed homogenizer is running), the power-off switch is triggered to protect the equipment.

[0242] Function: After automatic power failure, the touch interface displays an overload alarm and stops running related modules.

[0243] Temperature control alarm function:

[0244] Structure: The temperature of the equipment during operation (such as ultrasonic extractors and vacuum concentrators) is monitored through thermistors. When the temperature exceeds the set range (such as 25℃-50℃), the system will sound an audible and visual alarm and suspend the equipment operation. The user can check the cause of overheating on the interface.

[0245] Function: The temperature control alarm function prevents sample degradation or equipment damage due to overheating.

[0246] Effect:

[0247] The overload protection function prolongs the service life of the core components of the equipment (such as motors) and reduces equipment maintenance costs;

[0248] The temperature control alarm and vacuum monitoring functions can effectively avoid experimental failures caused by abnormal equipment operation and improve the stability and reliability of detection.

[0249] Please see attached Figure 7 , a method for detecting illegal antibiotics in plant extracts, comprising the following steps:

[0250] S1. Sample preparation, processing plant extract samples by weighing, homogenizing, extracting and centrifuging to obtain supernatant;

[0251] S2, sample purification, removing impurities through a solid phase extraction device and concentrating the target substance to dryness;

[0252] S3, detection and analysis, using high performance liquid chromatography and tandem mass spectrometry to separate, identify and quantify the target antibiotics;

[0253] S4. Data processing, generating test results through data acquisition and analysis software.

[0254] Specifically, in terms of sample preparation, the combination of high-speed homogenization, ultrasonic extraction and centrifugal separation achieves efficient extraction of target antibiotics while avoiding target degradation, with an extraction efficiency of more than 90% and reduced impurity interference; in terms of sample purification, solid phase extraction technology is used to selectively remove interferences such as pigments, lipids and sugars, with an impurity removal rate of more than 95%, while reducing the amount of solvent by 30%-50%, reducing the detection cost and waste liquid discharge, and being environmentally friendly and economical; in terms of detection and analysis, high-performance liquid chromatography and tandem mass spectrometry are used, with extremely high sensitivity and accuracy, a minimum detection limit of 0.1ng / mL, fast separation speed and high resolution, and accurate qualitative and quantitative analysis of illegal antibiotics; in terms of data processing, automated data acquisition and analysis software is used to achieve rapid generation of standard curve fitting, concentration calculation and result reports, reduce human errors, output results that are intuitive and standardized, and support full process traceability. These advantages together constitute a set of efficient, safe, environmentally friendly and sensitive detection solutions, which significantly improve the efficiency and reliability of illegal antibiotic detection in plant extracts.

[0255] S1 sample preparation steps include:

[0256] S101. Weighing samples:

[0257] Use a precision electronic balance to weigh the plant extract samples with a weighing accuracy of 0.01-0.1 mg and a sample mass range of 1-5 g;

[0258] S102, adding extraction solvent:

[0259] The sample was added to a mixed solvent of acetonitrile and water at a volume ratio of 60:40 to 90:10, and 0.05% to 0.2% of acid was added to the solvent;

[0260] S103, homogenization treatment:

[0261] The samples were homogenized using a high-speed homogenizer with a speed range of 8000-12,000 rpm and a processing time of 20-60 seconds;

[0262] S104, ultrasonic extraction:

[0263] The homogenized sample is placed in an ultrasonic extractor with an ultrasonic frequency of 20-40 kHz, an extraction temperature of 30-50°C, and an extraction time of 10-40 minutes;

[0264] S105, centrifugal separation:

[0265] Centrifuge at 8,000-12,000 rpm for 5-15 minutes in a high-speed centrifuge and collect the supernatant.

[0266] Specifically, S101 sample weighing:

[0267] The sample is a plant extract, which can be in the form of solid powder (such as dry powder extracted from plant roots, stems, and leaves) or viscous liquid (such as concentrated liquid), and is sampled after uniform mixing.

[0268] Use an electronic balance to weigh 1-5g of sample. During the operation, keep the relative humidity of the weighing environment below 60% to avoid weighing deviation caused by sample hygroscopicity.

[0269] S102 Extraction Solvent Addition:

[0270] The extraction solvent is a mixture of acetonitrile and water with a volume ratio of 60:40 to 90:10; for some easily degradable antibiotics, 0.05%-0.2% formic acid can be added to the solvent as an acidifier to improve the solubility and stability of the target.

[0271] S103 sample homogenization:

[0272] After mixing the sample and solvent, use a high-speed homogenizer to homogenize them. The homogenization time ranges from 20 to 60 seconds. The specific time is adjusted according to the sample particle size and the homogenization effect. For liquid samples, the homogenization time can be appropriately reduced.

[0273] S104 Ultrasonic Extraction:

[0274] After the sample is homogenized, it is placed in an ultrasonic extractor and subjected to ultrasonic action at a frequency of 20-40 kHz for 10-40 minutes. For some plant extracts containing volatile components, a sealing film may be covered during the ultrasonic extraction process to prevent solvent loss.

[0275] S105 centrifugal separation:

[0276] The extracted sample is placed in a high-speed centrifuge and centrifuged at 8000-12,000 rpm for 5-15 minutes; the centrifuge tube is made of organic solvent-resistant material (such as polypropylene or glass) with a capacity of 10-50 mL. After centrifugation, the supernatant is taken for subsequent purification.

[0277] The S2 sample cleanup steps include:

[0278] S201, loading samples:

[0279] The supernatant collected in step S105 is loaded onto a C18 solid phase extraction column, wherein the filling material of the extraction column is bonded silica gel with a particle size range of 30-60 μm;

[0280] S202, activated extraction column:

[0281] Use 2-5 mL of methanol to activate the extraction column and balance it with 2-5 mL of deionized water;

[0282] S203, sample loading and impurity washing:

[0283] The supernatant was passed through the extraction column at a flow rate of 0.5-2 mL / min and washed with 2-5 mL of 0.05%-0.2% acidified aqueous solution to remove polar impurities;

[0284] S204, target elution:

[0285] Elute the target antibiotic with 2-5 mL of organic solvent and collect the eluate into a concentration container;

[0286] S205, Concentration and Reconstitution:

[0287] Use a vacuum concentrator to concentrate the eluent to dryness, control the vacuum pressure at 5-20Pa, and control the temperature at 30-50°C. After concentration to dryness, add 0.5-2mL of mobile phase to reconstitute.

[0288] Specifically, S201 loads the sample:

[0289] The filling material of the solid phase extraction column is C18 bonded silica gel with a particle size range of 30-60μm, which is suitable for the separation of antibiotic targets.

[0290] Activation and equilibration steps: First, use methanol (2-5 mL) to activate the column, and then use deionized water (2-5 mL) to equilibrate to ensure that the extraction column reaches the best adsorption state.

[0291] S202 activated extraction column:

[0292] The sample supernatant is loaded through the solid phase extraction column at a flow rate of 0.5-2mL / min, and the flow rate is precisely adjusted by an automatic control valve to prevent the target from leaking due to excessive flow rate.

[0293] S203 sample loading and impurity washing:

[0294] Use 2-5 mL of 0.05%-0.2% formic acid aqueous solution to wash and remove non-target polar impurities to prevent the impurities from affecting the subsequent detection sensitivity.

[0295] S204 target elution:

[0296] Use acetonitrile (or acetonitrile-methanol mixed solvent) to elute the target antibiotic with 2-5 mL, and collect the eluate into a concentration bottle.

[0297] S205 Concentration and Reconstitution:

[0298] The eluent is concentrated to dryness in a vacuum concentrator, the vacuum pressure is controlled at 5-20 Pa, and the temperature control range is 30-50° C. The residue after concentration is reconstituted with 0.5-2 mL of mobile phase, ensuring that the reconstitution is uniform and filtered for detection.

[0299] The S3 detection and analysis steps include:

[0300] S301, HPLC separation:

[0301] The chromatographic column is a C18 reverse phase column with a column length ranging from 100 to 150 mm, an inner diameter ranging from 3.0 to 4.6 mm, and a particle size of 3 to 5 μm;

[0302] The mobile phase was acetonitrile-water gradient elution, containing 0.05%-0.2% acid;

[0303] The flow rate range was 0.8-1.5 mL / min, and the column temperature was controlled at 30-50 °C;

[0304] Gradient Program:

[0305] 0-2 minutes: acetonitrile 30%-40%, aqueous phase 60%-70%;

[0306] 2-10 minutes: linear gradient to acetonitrile 70%-90%, aqueous phase 10%-30%;

[0307] 10-12 minutes: acetonitrile maintained at 70%-90%, aqueous phase 10%-30%;

[0308] S302, tandem mass spectrometry:

[0309] The ionization method was electrospray ionization (ESI);

[0310] The mass ranges of parent and product ions were m / z 700–900 and m / z 150–700;

[0311] The collision energy range is 20-50 eV;

[0312] The scanning mode was selected reaction monitoring SRM;

[0313] S4 data processing steps include:

[0314] S401, Data Collection:

[0315] The liquid chromatography and mass spectrometry signals were recorded using a data acquisition system with an acquisition frequency of 1-10 Hz;

[0316] S402, peak area calculation:

[0317] The peak area of ​​the mass spectrometer output signal was integrated by the analysis software, and the peak area range was 1000-10,000 units;

[0318] S403, concentration calculation:

[0319] The standard curve is generated based on the concentration of the standard solution, and the linear fitting coefficient R 2 ≥0.99;

[0320] S404, result output:

[0321] Generate a test report including target concentration, standard curve, spike recovery and detection limit.

[0322] Specifically, S301 high performance liquid chromatography separation:

[0323] The sample was injected into a liquid chromatograph using a C18 reverse phase column (column length range 100-150 mm, particle size 3-5 μm), and the mobile phase was acetonitrile-water gradient elution containing 0.05%-0.2% acidifying agent (such as formic acid or acetic acid).

[0324] The gradient elution program included:

[0325] The initial mobile phase ratio is 30%-40% acetonitrile and 60%-70% water;

[0326] Within 5-10 minutes, adjust linearly to 70%-90% acetonitrile and 10%-30% water phase;

[0327] Elution was allowed to proceed steadily for 2-5 minutes to achieve complete separation.

[0328] S302 tandem mass spectrometry quantification:

[0329] A tandem mass spectrometer was used, the ionization mode was electrospray ionization (ESI), and the parent ion and product ion mass ranges were m / z 700-900 (parent ion) and m / z 150-700 (daughter ion), respectively;

[0330] The collision energy was adjusted in the range of 20–50 eV, the ion fragmentation mode was optimized according to the chemical properties of the target antibiotics, and the scanning mode was selected reaction monitoring (SRM).

[0331] S401 Data Collection:

[0332] The output signals of liquid chromatography and mass spectrometry are recorded by the acquisition system with a sampling frequency of 1-10 Hz; the characteristic peak signal of each target is calibrated by the response value and retention time.

[0333] S402 concentration calculation:

[0334] The peak area of ​​the target was integrated using the analysis software, and the concentration value was obtained by standard curve fitting. The concentration range of the standard solution was 0.1-50 ng / mL, and the linear regression coefficient R 2 ≥0.99.

[0335] S403 data output:

[0336] Generate a test report including target antibiotic concentration, standard curve, recovery (60%-120%) and detection limit (≤0.1-1 mg / kg).

[0337] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A device for detecting illegal antibiotics in plant extracts, characterized in that: include: A sample preparation module for weighing, homogenizing, extracting and centrifuging plant extract samples; Sample purification module, used to remove impurities in the extract and concentrate the target substance; A detection module for separation and quantitative analysis of target antibiotics; A data processing module is used to collect and analyze the detection data; Control and safety modules are used for operation control and safety protection.

2. The detection device for illegal antibiotics in plant extracts according to claim 1, characterized in that: The sample preparation module comprises: Precision electronic balance, using electromagnetic force balance sensor, weighing accuracy is 0.01mg, weighing pan is made of corrosion-resistant stainless steel; High speed homogenizer: High-speed motor with a speed range of 3000-30,000rpm; Homogenizing blade, made of titanium alloy or 316L stainless steel, resistant to chemical corrosion; Built-in temperature control device monitors the temperature during homogenization through a thermal sensor and controls the temperature within 25°C through a cooling fan; Ultrasonic Extractor: Ultrasonic wave generator with a frequency of 20-40kHz; The sample tank is made of chemical-resistant materials and has a tank capacity of 2-5L; Constant temperature oscillator: The temperature control range is 20℃ to 80℃, and the temperature control accuracy is ±1℃; Oscillation speed range is 50-300rpm; High-speed centrifuge: Driven by a brushless DC motor, the speed range is 5000-15,000rpm; The sample rotor supports 10-50mL sample tubes.

3. The detection device for illegal antibiotics in plant extracts according to claim 1, characterized in that: The sample purification module comprises: Solid phase extraction device: Solid phase extraction column, the filling material is C18 bonded silica gel or polymer resin, and the column shell is made of polypropylene or stainless steel; Elution device, the flow control range of which is 1-5mL / min; Vacuum Concentrator: Vacuum pump, minimum pressure up to 10Pa, oil-sealed or oil-free design; Condenser to prevent solvent evaporation loss, sample heating plate temperature control range is 30 ℃ to 60 ℃.

4. The detection device for illegal antibiotics in plant extracts according to claim 1, characterized in that: The detection module comprises: High Performance Liquid Chromatography HPLC: Mobile phase delivery system, pump flow rate range 0.1-2.0mL / min; Chromatographic column, the stationary phase is C18 reverse phase column, the particle size is 3-5μm, and the length is 100-150mm; Column oven, temperature control range is 20℃ to 60℃; Tandem mass spectrometer MS / MS: Electrospray ionization source ESI, used to generate parent ions; Triple quadrupole mass analyzer with a mass range of 10-2000Da and a resolution of 0.1Da; Collision cell with adjustable collision energy range of 10-50 eV for optimized parent and daughter ion fragmentation.

5. The detection device for illegal antibiotics in plant extracts according to claim 1, characterized in that: The data processing module comprises: Data Acquisition System: An analog signal converter converts the analog signal of the detector into a digital signal; A data storage unit with a storage capacity of at least 1 TB for storing detection data; Data analysis software: Standard curve calibration function, used to generate concentration curve of target antibiotic; Quantitative analysis function is used to calculate the concentration of target antibiotics in samples.

6. The detection device for illegal antibiotics in plant extracts according to claim 1, characterized in that: The control and safety module includes: Touch control panel: Parameter input interface, used to input equipment operating parameters, including speed, temperature and pressure; Real-time display interface, used to display equipment operation status and test results; Safety protection device: Overload protection system monitors the motor load through the current sensor and triggers automatic power-off protection when overloaded; The temperature control protection device uses a thermistor to detect the device temperature in real time and automatically shuts down when the temperature exceeds the set range.

7. A method for detecting illegal antibiotics in plant extracts, characterized in that: The detection device for illegal antibiotics in plant extracts according to any one of claims 1 to 6 comprises the following steps: S1. Sample preparation, processing plant extract samples by weighing, homogenizing, extracting and centrifuging to obtain supernatant; S2, sample purification, removing impurities through a solid phase extraction device and concentrating the target substance to dryness; S3, detection and analysis, using high performance liquid chromatography and tandem mass spectrometry to separate, identify and quantify the target antibiotics; S4. Data processing, generating test results through data acquisition and analysis software.

8. The method for detecting illegal antibiotics in plant extracts according to claim 7, characterized in that: The S1 sample preparation steps include: S101. Weighing samples: Use a precision electronic balance to weigh the plant extract samples with a weighing accuracy of 0.01-0.1 mg and a sample mass range of 1-5 g; S102, adding extraction solvent: The sample was added to a mixed solvent of acetonitrile and water at a volume ratio of 60:40 to 90:10, and 0.05% to 0.2% of acid was added to the solvent; S103, homogenization treatment: The samples were homogenized using a high-speed homogenizer with a speed range of 8000-12,000 rpm and a processing time of 20-60 seconds; S104, ultrasonic extraction: The homogenized sample is placed in an ultrasonic extractor with an ultrasonic frequency of 20-40 kHz, an extraction temperature of 30-50°C, and an extraction time of 10-40 minutes; S105, centrifugal separation: Centrifuge at 8,000-12,000 rpm for 5-15 minutes in a high-speed centrifuge and collect the supernatant.

9. The method for detecting illegal antibiotics in plant extracts according to claim 7, characterized in that: The S2 sample cleanup step includes: S201, loading samples: The supernatant collected in step S105 is loaded onto a C18 solid phase extraction column, wherein the filling material of the extraction column is bonded silica gel with a particle size range of 30-60 μm; S202, activated extraction column: Use 2-5 mL of methanol to activate the extraction column and balance it with 2-5 mL of deionized water; S203, sample loading and impurity washing: The supernatant was passed through the extraction column at a flow rate of 0.5-2 mL / min and washed with 2-5 mL of 0.05%-0.2% acidified aqueous solution to remove polar impurities; S204, target elution: Elute the target antibiotic with 2-5 mL of organic solvent and collect the eluate into a concentration container; S205, Concentration and Reconstitution: Use a vacuum concentrator to concentrate the eluent to dryness, control the vacuum pressure at 5-20Pa, and control the temperature at 30-50°C. After concentration to dryness, add 0.5-2mL of mobile phase to reconstitute.

10. The method for detecting illegal antibiotics in plant extracts according to claim 7, characterized in that: The S3 detection and analysis step includes: S301, HPLC separation: The chromatographic column is a C18 reverse phase column with a column length ranging from 100 to 150 mm, an inner diameter ranging from 3.0 to 4.6 mm, and a particle size of 3 to 5 μm; The mobile phase was acetonitrile-water gradient elution, containing 0.05%-0.2% acid; The flow rate range was 0.8-1.5 mL / min, and the column temperature was controlled at 30-50 °C; Gradient Program: 0-2 minutes: acetonitrile 30%-40%, aqueous phase 60%-70%; 2-10 minutes: linear gradient to acetonitrile 70%-90%, aqueous phase 10%-30%; 10-12 minutes: acetonitrile maintained at 70%-90%, aqueous phase 10%-30%; S302, tandem mass spectrometry: The ionization method was electrospray ionization (ESI); The mass ranges of parent and product ions were m / z 700–900 and m / z 150–700; The collision energy range is 20-50 eV; The scanning mode was selected reaction monitoring SRM; The S4 data processing step includes: S401, Data Collection: The liquid chromatography and mass spectrometry signals were recorded using a data acquisition system with an acquisition frequency of 1-10 Hz; S402, peak area calculation: The peak area of ​​the mass spectrometer output signal was integrated by the analysis software, and the peak area range was 1000-10,000 units; S403, concentration calculation: The standard curve is generated based on the concentration of the standard solution, and the linear fitting coefficient R 2 ≥0.99; S404, result output: Generate a test report including target concentration, standard curve, spike recovery and detection limit.

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