A feed additive ingredient detection device and a detection method

Abstract

The present application relates to a kind of feed additive component detection device and detection method;The feed additive component detection device includes the following equipment optimized by parameter: muffle furnace, microwave digestion instrument, high-speed centrifuge, ultrasonic extraction instrument, electronic balance, with online enrichment liquid chromatograph, liquid chromatography-tandem mass spectrometer, atomic absorption spectrometer, near infrared spectrometer, laser particle size analyzer, inductively coupled plasma mass spectrometer, microbiological incubator, colony counter, portable near infrared spectrometer, portable liquid chromatograph, portable enzyme label instrument, sample pretreatment is carried out by above-mentioned equipment, multiple components are synchronously detected, detection precision control, trace component detection, new additive detection and on-site rapid detection, compared with prior art, the present application is accurate and efficient, can multiple components be synchronously detected, while detection precision is significantly improved, can be suitable for new additive high-precision detection.

Description

Technical Field

[0001] This invention belongs to the field of additive detection technology, specifically relating to a device and method for detecting feed additive components. Background Technology

[0002] As a core component of the feed industry, the accuracy of feed additives' composition directly affects livestock and poultry farming efficiency, feed product quality, and animal food safety. With the widespread application of new products such as compound additives, coated additives, nano-additives, and microbial additives, the industry's demand for simultaneous qualitative and quantitative analysis of multiple components, accurate detection of trace substances, and rapid on-site screening continues to increase.

[0003] Current feed additive testing primarily employs traditional pretreatment methods combined with conventional techniques such as chromatography, spectroscopy, and atomic absorption spectrometry. While these methods can perform basic component analysis, the overall technical system suffers from significant shortcomings: cumbersome sample pretreatment procedures and poor parameter matching can easily lead to target analyte loss or severe matrix interference; multi-component detection often relies on single-component analysis, resulting in weak simultaneous detection capabilities and low efficiency; detection limits for low-content antibiotics and trace heavy metals are too high, easily leading to false positives or false negatives; dedicated detection procedures are lacking for novel additives such as coated and nano-sized additives, making it difficult to accurately characterize release rates and particle size distribution; rapid on-site detection methods are scarce, and it is difficult to balance instrument portability with detection accuracy. Existing detection methods and equipment combinations are highly versatile but lack specificity, and their overall detection accuracy, efficiency, and applicability fail to meet the actual needs of strict feed quality and safety control and large-scale industrial development. Therefore, there is an urgent need for a precise, efficient, and low-cost feed additive component detection device and method. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a device and method for detecting feed additive components.

[0005] The technical solution of the present invention is as follows: A method for detecting the components of feed additives, characterized by comprising the following steps: (1) Sample pretreatment: The feed sample was crushed to a particle size ≤0.15mm. 1g, 5g and 10g of premix, compound feed and fish meal / meat meal raw materials were weighed using an electronic balance with an accuracy ≤0.1mg and calibrated with standard weights. Chromium was ashed in a muffle furnace at 450–500℃ for 4–6h. The ash was dissolved in 5%–10% nitric acid by microwave digestion under reflux for 30–60min. Low-content vitamins and antibiotics were enriched online by liquid chromatography with an enrichment factor ≥50 times. Ultrasonic extraction power was 200–400W, extraction time was 15–30min, high-speed centrifugation speed was 6000–10000r / min, and the pretreatment time for a single sample was ≤30min. (2) Simultaneous detection of multiple components: The liquid chromatography-tandem mass spectrometry multiple reaction monitoring mode is adopted, using a 4.6 mm × 250 mm, 5 μm C18 reversed phase column, with methanol-water gradient elution, methanol volume fraction 30%–80%; ion source temperature 300–350℃, collision energy 20–30 eV, target ion peak intensity ≥90%, and simultaneous detection of chromium, lead, arsenic heavy metals, chloramphenicol, reserpine antibiotics, and B vitamins and fat-soluble vitamins in a single injection; (3) Detection accuracy control: Near-infrared spectra are preprocessed with baseline correction and Savitzky-Golay noise reduction, and a calibration model is established according to the feed matrix. The model R²≥0.95; the peak shape matching degree of characteristic ion peaks of liquid chromatography-tandem mass spectrometry is ≥85%, reducing false positives and false negatives; (4) Trace component detection: Atomic absorption graphite furnace drying 100–150℃, ashing 700–900℃, atomization 2500–3000℃, lamp current 8–12mA, hollow cathode lamp wavelength error ≤±0.1nm; liquid chromatography-tandem mass spectrometry ion source temperature 320℃, spray voltage 4000–5000V, collision energy 23–27eV, chloramphenicol and reserpine detection limits ≤10fg; chromium element was detected by spectrophotometry, detection limit ≤0.01μg / g; (5) Detection of novel additives: The release rate of coated additives was detected by column temperature 30–40℃ and elution rate 0.8–1.2 mL / min, with a cumulative release rate RSD ≤ 5%; Nanoparticle additives were detected by 0.1 nm–100 μm laser particle size analyzer combined with inductively coupled plasma mass spectrometry, with a particle size D90 deviation ≤ 3%; Microbial additives were cultured at 35–39℃ for 8–12 h, with a viable count error ≤ 10%; (6) Rapid on-site detection: Use a portable near-infrared spectrometer with a wavelength of 1000–2500 nm. Single sample detection time is ≤5 min. Use a portable liquid chromatograph and enzyme-linked immunosorbent assay (ELISA) with an antigen-antibody ratio of 1:30–1:70. React at 25–37℃ for 15–25 min. The limit of quantitation is ≤0.1 ng / mL.

[0006] Furthermore, in step (1), the muffle furnace ashing temperature is 480℃, the time is 5h, the nitric acid concentration is 8%, the microwave reflux is 30min, the ultrasonic power is 300W, the extraction is 25min, and the centrifugation speed is 8000r / min.

[0007] Furthermore, in step (2), the C18 column is an Agilent ZORBAX SB-C18, methanol is increased from 30% to 80% in a gradient, elution time is 0–25 min, ion source temperature is 320℃, and collision energy is 23–27 eV.

[0008] Furthermore, in step (3), Origin2023 or SIMCA16 is used to build the model, with R²≥0.98 and characteristic ion peak recognition accuracy≥90%.

[0009] Furthermore, in step (4), the graphite furnace is dried at 120°C, ashed at 800°C, and atomized at 2800°C, with a lamp current of 10mA; the detection limit of chloramphenicol is ≤8fg, and the detection limit of chromium is ≤0.005μg / g.

[0010] Furthermore, in step (5), the coating additive detection column temperature is 35℃, the elution rate is 1.0mL / min, the laser particle size analyzer wavelength is 633nm, and the inductively coupled plasma mass spectrometry nebulization pressure is 0.3MPa.

[0011] Furthermore, in step (5), the microbial culture temperature is 37℃ and the time is 12h, with a quantitative error of ≤8% for the number of viable bacteria.

[0012] Furthermore, in step (6), the portable near-infrared spectroscopy wavelength is 1200–2200 nm, the ELISA antigen-antibody ratio is 1:50, the reaction is carried out at 37℃ for 20 min, and the limit of quantitation is ≤0.05 ng / mL.

[0013] A feed additive component detection device according to the above detection method is characterized in that it includes the following equipment with optimized parameters: muffle furnace, microwave digester, high-speed centrifuge, ultrasonic extractor, electronic balance, liquid chromatograph with online enrichment, liquid chromatography-tandem mass spectrometry, atomic absorption spectrometer, near-infrared spectrometer, laser particle size analyzer, inductively coupled plasma mass spectrometer, microbial incubator, colony counter, portable near-infrared spectrometer, portable liquid chromatograph, and portable enzyme-linked immunosorbent assay (ELISA) reader.

[0014] Compared with the prior art, the beneficial effects of the present invention are: Precise and efficient pretreatment: Samples are weighed according to raw material type, and a calibrated electronic balance and muffle furnace-microwave digestion technology are used. Combined with online enrichment and optimized ultrasonic and centrifugation parameters, the pretreatment time for a single sample is ≤30 minutes, reducing the loss of target substances and matrix interference.

[0015] Simultaneous detection of multiple components: Through liquid chromatography-tandem mass spectrometry multiple reaction monitoring mode, multiple components such as heavy metals, antibiotics, and vitamins can be detected simultaneously in a single injection, which greatly improves detection efficiency and meets the needs of large-scale operations.

[0016] Significantly improved detection accuracy: After spectral denoising preprocessing and calibration model optimization (R²≥0.95), combined with mass spectrometry peak shape matching control, the detection limit of trace components reaches the fg level, with chromium ≤0.01μg / g, reducing the probability of false detection.

[0017] Adaptation to novel additive testing: Establish dedicated testing procedures for coated, nanoscale, and microbial additives to accurately characterize release rate, particle size distribution, and viable cell count, thus expanding the scope of application.

[0018] Supports rapid on-site testing: Portable devices enable rapid screening of single samples within ≤5 minutes, with a quantitation limit of ≤0.1ng / mL, balancing portability and accuracy to meet on-site control requirements.

[0019] Strong equipment adaptability: Optimized equipment parameter linkage reduces redundant operations and costs, improves the stability of test results, and meets the needs of high-quality development in the industry. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1: Simultaneous detection of multiple components including heavy metals, antibiotics, and vitamins in compound feed 1. Experimental materials and equipment: Test sample: Compound feed for commercial pigs Standard substances: chromium, lead, and arsenic standard solutions; chloramphenicol and reserpine standards; B vitamins and fat-soluble vitamin standards. Reagents: nitric acid, methanol, ultrapure water, etc. Instruments and equipment: muffle furnace, microwave digester, high-speed centrifuge, ultrasonic extractor, electronic balance, liquid chromatograph with online enrichment function, liquid chromatography-tandem mass spectrometry, atomic absorption spectrometer, near-infrared spectrometer. The electronic balance has an accuracy of no more than 0.1 mg and is calibrated with standard weights; the liquid chromatography-tandem mass spectrometer is an Agilent 1290-6460C model; the atomic absorption spectrometer is a PerkinElmer PinAAcle900T model; and the near-infrared spectrometer is a Bruker MPAII model.

[0022] 2. Sample pretreatment: The compound feed sample was crushed and sieved until the particle size was no greater than 0.15 mm.

[0023] Weigh 5.0000g of the sample using an electronic balance and place it in a crucible.

[0024] Ashing was carried out in a muffle furnace at 450℃ for 4 hours, and then cooled to room temperature after ashing.

[0025] Add 5% nitric acid (by volume) to the ash and dissolve it by heating under reflux for 30 minutes using a microwave digester.

[0026] Take another sample from the same batch, add extraction solvent, and extract for 25 minutes using an ultrasonic extractor at a power of 200W.

[0027] The extract was centrifuged using a high-speed centrifuge at 6000 r / min, and the supernatant was collected.

[0028] Low-content vitamins and antibiotics were enriched online by liquid chromatography with an enrichment factor of not less than 50 times.

[0029] The total pretreatment time for a single sample should not exceed 30 minutes.

[0030] 3. Simultaneous detection of multiple components: Detection was performed using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode. The chromatographic column used was a 4.6 mm × 250 mm, 5 μm particle size C18 reversed-phase column, model Agilent ZORBAX SB-C18.

[0031] The mobile phase was a methanol-water gradient elution system, with the methanol volume fraction increasing linearly from 30% to 80% over a period of 15–25 min.

[0032] The ion source temperature was set to 320℃, and the collision energy was set to 23~27eV.

[0033] A single injection can simultaneously detect heavy metals such as chromium, lead, and arsenic, antibiotics such as chloramphenicol and reserpine, as well as B vitamins and fat-soluble vitamins.

[0034] The intensity of the target ion peak should be controlled to be no less than 90%, and the peak shape matching degree of the characteristic ion peak should be no less than 85%.

[0035] 4. Detection accuracy control: Near-infrared spectral data were subjected to baseline correction and Savitzky-Golay denoising preprocessing in sequence.

[0036] A calibration model was established based on the compound feed matrix, and the model was built using SIMCA16 software. The model's coefficient of determination R² ≥ 0.95.

[0037] The accuracy rate of characteristic ion peak identification by liquid chromatography-tandem mass spectrometry is no less than 90%, reducing false positive and false negative results.

[0038] 5. Trace component detection: The atomic absorption spectrometer uses a graphite furnace mode with a drying temperature of 100℃, an ashing temperature of 700℃, and an atomization temperature of 2500℃.

[0039] The hollow cathode lamp has a current of 10mA and a wavelength error of no more than ±0.1nm.

[0040] The liquid chromatography-tandem mass spectrometry (LC-MS / MS) ion source temperature is 320℃, the spray voltage is 4000~5000V, and the collision energy is 23~27eV.

[0041] The detection limits for chloramphenicol and reserpine are not higher than 8 fg; chromium is detected by spectrophotometry, and the detection limit is not higher than 0.005 μg / g.

[0042] 6. Test Results: In this embodiment, the recovery rates of heavy metals were 90%~96%, antibiotics were 92%~101%, and vitamins were 88%~96%, with relative standard deviations (RSD) all less than 5%, meeting the requirements for accurate detection.

[0043] Example 2: Detection of Coated Additives and Nano-Additives in Premixes 1. Instruments and equipment: Muffle furnace, microwave digester, liquid chromatograph, laser particle size analyzer, inductively coupled plasma mass spectrometer, ultrasonic extractor, electronic balance.

[0044] 2. Sample pretreatment: The feed additive premix is ​​pulverized to a particle size ≤0.15mm, and 1.0000g is weighed using an electronic balance.

[0045] Chromium was ashed in a muffle furnace at 500℃ for 6 hours and then microwave-digested with 10% nitric acid for 60 minutes.

[0046] Coated additive samples were extracted and detected directly using a liquid chromatography system.

[0047] 3. Detection of novel additives: Release rate detection of coated additives: HPLC column temperature 35℃, elution rate 1.0 mL / min, cumulative release rate determined, relative standard deviation RSD ≤ 5%.

[0048] Nanoparticle additive detection: Laser particle size analyzer combined with inductively coupled plasma mass spectrometry was used for detection. The laser wavelength was 633nm, the nebulization pressure of the inductively coupled plasma mass spectrometry was 0.3MPa, and the particle size D90 deviation was ≤3%.

[0049] 4. Experimental Results: The cumulative release rate RSD of the coated additive over 60 min was 3.6%; the particle size D90 deviation of the nano-additive was 2.6%, which met the method requirements.

[0050] Example 3: Detection of Microbial Additives and Rapid On-Site Detection 1. Instruments and equipment: Microbial incubator, colony counter, portable near-infrared spectrometer, portable liquid chromatograph, portable enzyme-linked immunosorbent assay (ELISA) reader.

[0051] 2. Detection of microbial additives: Weigh the microbial additive sample and dilute it serially with sterile physiological saline.

[0052] Incubate at 35°C for 12 hours using a microbial incubator.

[0053] Viable bacteria were counted using a colony counter, with a count error of ≤8%.

[0054] 3. On-site rapid testing: A portable near-infrared spectrometer was used, with a wavelength range of 1000~2500nm, and the detection time for a single sample was ≤5min.

[0055] Enzyme-linked immunosorbent assay (ELISA) was performed using a portable ELISA reader with an antigen-antibody ratio of 1:50 and the reaction was carried out at 25-37°C for 20 minutes.

[0056] The method has a limit of quantitation of ≤0.05 ng / mL, enabling rapid on-site screening.

[0057] 4. Experimental Results: The relative deviation between on-site testing and laboratory values ​​is less than 4%, and the viable bacteria count error is 6.5%, meeting the needs for rapid and accurate on-site testing.

[0058] Example 4: Detection of Microbial Additives and Rapid On-Site Detection 2 1. The instruments and equipment are the same as in Example 3. 2. Detection of microbial additives: Weigh the microbial additive sample and dilute it serially with sterile physiological saline.

[0059] The microbial culture was carried out at 39°C for 8 hours using a microbial incubator.

[0060] Viable bacteria were counted using a colony counter, with a count error of ≤8%.

[0061] 3. On-site rapid testing: A portable near-infrared spectrometer was used, with a wavelength range of 1000~2500nm, and the detection time for a single sample was ≤5min.

[0062] Enzyme-linked immunosorbent assay (ELISA) was performed using a portable ELISA reader with an antigen-antibody ratio of 1:50 and the reaction was carried out at 25-37°C for 20 minutes.

[0063] The method has a limit of quantitation of ≤0.05 ng / mL, enabling rapid on-site screening.

[0064] 4. Experimental Results: The relative deviation between on-site testing and laboratory values ​​is less than 4%, and the viable bacteria count error is 6.3%, meeting the needs for rapid and accurate on-site testing.

[0065] The feed additive component detection device of the present invention includes, but is not limited to, the following equipment units: Muffle furnace, microwave digester, high-speed centrifuge, ultrasonic extractor, electronic balance, liquid chromatograph with online enrichment, liquid chromatography-tandem mass spectrometry, atomic absorption spectrometer, near-infrared spectrometer, laser particle size analyzer, inductively coupled plasma mass spectrometer, microbial incubator, colony counter, portable near-infrared spectrometer, portable liquid chromatograph, portable enzyme-linked immunosorbent assay (ELISA) reader.

[0066] The key parameters of each device must meet the following requirements: The accuracy of the electronic balance is ≤0.1mg; The temperature range of the muffle furnace is 450~500℃; The microwave digester can achieve heating and reflux for 30~60 minutes; High-speed centrifuges operate at speeds of 6000~10000 r / min; The ultrasonic extractor has a power of 200~400W; The liquid chromatography-tandem mass spectrometer supports multiple reaction monitoring mode; The graphite furnace temperature of the atomic absorption spectrometer can reach 2500~3000℃; The laser particle size analyzer has a detection range of 0.1 nm to 100 μm. The temperature control range of the microbial incubator is 35~39℃; Portable near-infrared spectrometer with wavelengths of 1000~2500nm, single sample detection time ≤5min.

[0067] In other embodiments of the present invention, the corresponding equipment parameters and detection steps can be adjusted according to the type of feed additive being detected, such as coated vitamins, microbial additives, etc., and the same detection effect can be achieved. Those skilled in the art can complete the detection operation by referring to this embodiment according to actual detection needs, and the present invention can be realized without creative labor.

Claims

1. A method for detecting the components of feed additives, characterized in that, Includes the following steps: (1) Sample pretreatment: The feed sample was crushed to a particle size ≤0.15mm. 1g, 5g and 10g of premix, compound feed and fish meal / meat meal raw materials were weighed using an electronic balance with an accuracy ≤0.1mg and calibrated with standard weights. Chromium was ashed in a muffle furnace at 450–500℃ for 4–6h. The ash was dissolved in 5%–10% nitric acid by microwave digestion under reflux for 30–60min. Low-content vitamins and antibiotics were enriched online by liquid chromatography with an enrichment factor ≥50 times. Ultrasonic extraction power was 200–400W, extraction time was 15–30min, high-speed centrifugation speed was 6000–10000r / min, and the pretreatment time for a single sample was ≤30min. (2) Simultaneous detection of multiple components: The liquid chromatography-tandem mass spectrometry multiple reaction monitoring mode is adopted, using a 4.6 mm × 250 mm, 5 μm C18 reversed phase column, with methanol-water gradient elution, methanol volume fraction 30%–80%; ion source temperature 300–350℃, collision energy 20–30 eV, target ion peak intensity ≥90%, and simultaneous detection of chromium, lead, arsenic heavy metals, chloramphenicol, reserpine antibiotics, and B vitamins and fat-soluble vitamins in a single injection; (3) Detection accuracy control: Near-infrared spectra are preprocessed with baseline correction and Savitzky-Golay noise reduction, and a calibration model is established according to the feed matrix. The model R²≥0.95; the peak shape matching degree of characteristic ion peaks of liquid chromatography-tandem mass spectrometry is ≥85%, reducing false positives and false negatives; (4) Trace component detection: Atomic absorption graphite furnace drying 100–150℃, ashing 700–900℃, atomization 2500–3000℃, lamp current 8–12mA, hollow cathode lamp wavelength error ≤±0.1nm; liquid chromatography-tandem mass spectrometry ion source temperature 320℃, spray voltage 4000–5000V, collision energy 23–27eV, chloramphenicol and reserpine detection limits ≤10fg; chromium element was detected by spectrophotometry, detection limit ≤0.01μg / g; (5) Detection of novel additives: The release rate of coated additives was detected by column temperature 30–40℃ and elution rate 0.8–1.2 mL / min, with a cumulative release rate RSD ≤ 5%; Nanoparticle additives were detected by 0.1 nm–100 μm laser particle size analyzer combined with inductively coupled plasma mass spectrometry, with a particle size D90 deviation ≤ 3%; Microbial additives were cultured at 35–39℃ for 8–12 h, with a viable count error ≤ 10%; (6) Rapid on-site detection: Use a portable near-infrared spectrometer with a wavelength of 1000–2500 nm. Single sample detection time is ≤5 min. Use a portable liquid chromatograph and enzyme-linked immunosorbent assay (ELISA) with an antigen-antibody ratio of 1:30–1:

70. React at 25–37℃ for 15–25 min. The limit of quantitation is ≤0.1 ng / mL.

2. The method according to claim 1, characterized in that, In step (1), the muffle furnace ashing temperature is 480℃, the time is 5h, the nitric acid concentration is 8%, the microwave reflux is 30min, the ultrasonic power is 300W, the extraction is 25min, and the centrifugation speed is 8000r / min.

3. The method according to claim 1, characterized in that, In step (2), the C18 column is an Agilent ZORBAX SB-C18, methanol is increased from 30% to 80% in a gradient, elution time is 0–25 min, ion source temperature is 320℃, and collision energy is 23–27 eV.

4. The method according to claim 1, characterized in that, In step (3), Origin2023 or SIMCA16 is used to build the model, with R²≥0.98 and characteristic ion peak recognition accuracy≥90%.

5. The method according to claim 1, characterized in that, In step (4), the graphite furnace is dried at 120℃, ashed at 800℃, and atomized at 2800℃, with a lamp current of 10mA; the detection limit of chloramphenicol is ≤8fg, and the detection limit of chromium is ≤0.005μg / g.

6. The method according to claim 1, characterized in that, In step (5), the coating additive detection column temperature was 35℃, the elution rate was 1.0mL / min, the laser particle size analyzer wavelength was 633nm, and the inductively coupled plasma mass spectrometry nebulization pressure was 0.3MPa.

7. The method according to claim 1, characterized in that, In step (5), the microbial culture temperature is 37℃ and the time is 12h, with a quantitative error of ≤8% for the number of viable bacteria.

8. The method according to claim 1, characterized in that, In step (6), the portable near-infrared spectroscopy wavelength is 1200–2200 nm, the ELISA antigen-antibody ratio is 1:50, the reaction is carried out at 37℃ for 20 min, and the limit of quantitation is ≤0.05 ng / mL.

9. A feed additive component detection device for implementing any one of claims 1-8, characterized in that, The following equipment has been optimized for parameters: muffle furnace, microwave digester, high-speed centrifuge, ultrasonic extractor, electronic balance, liquid chromatograph with online enrichment, liquid chromatography-tandem mass spectrometry, atomic absorption spectrometer, near-infrared spectrometer, laser particle size analyzer, inductively coupled plasma mass spectrometer, microbial incubator, colony counter, portable near-infrared spectrometer, portable liquid chromatograph, and portable enzyme-linked immunosorbent assay (ELISA) reader.