Determination of Flavomycin A in Aquatic Products by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

The present invention relates to the technical field of analytical chemistry, and more specifically to a method for detecting flavomycin A in aquatic products using liquid chromatography-tandem mass spectrometry. The method involves extracting, purifying, and concentrating flavomycin A from aquatic products before determination. First, extraction is performed using ammonia-methanol, followed by purification using the QuEChERS method, and concentration followed by determination using a liquid chromatography-tandem mass spectrometer. The method employs an external standard method for quantification and is suitable for determining flavomycin A residues in edible tissues of fish, shrimp, crab, shellfish, and turtles. This method is environmentally friendly, reduces detection costs, and is simple to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of flavomycin A detection, and specifically provides a liquid chromatography-tandem mass spectrometry detection method for flavomycin A in aquatic products. Background Art

[0002] Flavomycin, also known as fulaphos, moenomycin, and bambomycin, is a multicomponent phosphoglycolipid antibiotic produced by anaerobic fermentation of Streptomyces glaucovitae. Flavomycin boasts advantages such as growth promotion, improved feed utilization efficiency, antibacterial and anti-inflammatory properties, disease prevention, and high efficacy at low dosages. It is widely used in livestock, poultry, and aquaculture. In the United States, flavomycin is approved for use in cattle, pigs, and poultry at dosages of 0.5 to 20 mg / kg. It can be used alone or in combination with ionophore antibiotics such as monensin, lasalocid, or salinomycin to improve weight gain and feed efficiency. In 2002, my country's former Ministry of Agriculture officially approved flavomycin premix as a new veterinary drug. However, long-term consumption of animal-derived foods containing flavomycin residues can disrupt the human intestinal flora and affect bone development in animals. Excessive intake can even cause hematopoietic dysfunction. Furthermore, residues in animal products or excretion into the environment through animal feces can contaminate soil, surface water, and other environmental factors, posing a threat to human safety. Therefore, the 2005 EU Agriculture Ministers' Meeting decided to ban the addition of flavonoids to feed starting January 1, 2006. The Ministry of Agriculture and Rural Affairs' Announcement No. 194 stated that starting January 1, 2020, all growth-promoting drug feed additives, except for traditional Chinese medicine, would be withdrawn, including flavonoids premixes.

[0003] Existing research indicates that flavomycin has five active components, all of which possess similar chemical properties and antimicrobial activity. Flavomycin A is the most predominant active component, accounting for 60% to 80% of the total active components. Due to the structural similarities among the five active components and the difficulty in producing single-component standards, flavomycin residues are currently determined indirectly by measuring flavomycin A residues. Currently, the only published standards for flavomycin residues in China are "DB34 / T 1358-2011 Determination of Flavomycin in Feed - High-Performance Liquid Chromatography" and "DB32 / T1279-2008 Determination of Flavomycin A in Feed and Feed Additives - High-Performance Liquid Chromatography." The lack of a standard for the detection of flavomycin residues in aquatic products in my country presents significant challenges in monitoring flavomycin residues in these products.

[0004] Therefore, through research on the detection method of flavomycin A residues in aquatic products, a scientific detection method has been developed to provide technical support for risk assessment and quality and safety supervision of aquatic product breeding processes. At the same time, in order to meet the increasingly prominent detection and supervision needs of drug residues with significant safety risks, it is imperative to develop a detection method for flavomycin A residues in aquatic products. Summary of the Invention

[0005] The present invention is to address the above-mentioned deficiencies and provides a method for detecting flavomycin A in aquatic products by liquid chromatography-tandem mass spectrometry. To achieve this object, the specific scheme adopted by the present invention is as follows:

[0006] The liquid chromatography-tandem mass spectrometry method for detecting flavomycin A in aquatic products provided by the present invention comprises the following steps:

[0007] A. Extraction and purification of flavomycin A

[0008] The edible part of the aquatic product was taken out, homogenized, and then flavomycin A in the sample was extracted using ammonia-methanol. The extract was purified using the QuEChERS method and concentrated by nitrogen blowing. The sample to be tested was obtained after redissolution and filtration.

[0009] B. Detection

[0010] Liquid chromatography-tandem mass spectrometry was used for detection. The specific detection conditions were as follows:

[0011] (1) Liquid chromatography conditions: chromatographic column: T3; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 10 μL; mobile phase A: water containing 10 mmol / L ammonium acetate; mobile phase B: methanol;

[0012] Elution gradient: 0.5 min, 10% B, 90% A; 0.5-4 min, 10%-95% B, 90%-5% A; 4-6.5 min, 95% B, 5% A; 6.5-6.6 min, 95%-10% B, 5%-90% A; 6.6-10 min, 10% B, 90% A,

[0013] (2) Mass spectrometry analysis conditions: electrospray ionization source, negative ion scanning, multiple reaction monitoring; spray voltage: -4500 V; ion transfer capillary temperature: 550°C; collision gas CAD: Medium; curtain gas CUR: 40.0 psi; nebulizer gas GS1: 65.0 psi; auxiliary gas GS2: 60.0 psi.

[0014] Preferably, the aquatic products include fish, shrimp, crab, shellfish and turtle. The present invention uses grass carp, large yellow croaker, shrimp, crab, clam and turtle as examples to detect flavomycin A in them. The results show that the detection limit of the method of the present invention is 1.0 μg / kg and the quantification limit is 2.0 μg / kg, with excellent detection effect.

[0015] The specific operating steps in step A and step B are preferably as follows:

[0016] In step A, the steps for extracting flavomycin A from the sample using ammonia-methanol are as follows: a certain mass of sample is weighed and placed in a plastic centrifuge tube with a volume 25 times the sample weight, and a volume of ammonia-methanol extracting solution 2 to 3 times the sample weight is added. The tube is vortexed for 5 minutes, sonicated for 10 minutes, and centrifuged at 4000 rpm for 10 minutes. The supernatant is transferred to another centrifuge tube, and the sample residue is again added with the aforementioned volume of ammonia-methanol extracting solution. The extraction is repeated once, and the tube is centrifuged at 8000 rpm for 10 minutes. The supernatants are then combined for purification. The ammonia-methanol extracting solution is an ammonia-methanol solution containing 5 to 30% ammonia by volume.

[0017] The extract was concentrated, redissolved, and filtered as follows: C18 adsorbent was added to the supernatant of the extract, vortexed for 30 seconds, and centrifuged at 4000 rpm for 5 minutes; a certain volume of the supernatant was accurately measured in a centrifuge tube, dried to approximately 1 / 10 of the volume with nitrogen at 20-60° C., diluted to 2 times the volume with methanol, centrifuged at 4000 rpm for 5 minutes, and filtered through a 0.22 μm filter membrane to obtain a sample for liquid chromatography-tandem mass spectrometry.

[0018] In step B, the specifications of the T3 chromatographic column used in the chromatographic analysis were 2.1×100 mm, 1.8 μm;

[0019] When mass spectrometry was used for qualitative detection, the parent ion was 789.9 m / z, the daughter ion was 554.1 m / z, the declustering voltage was -100 V, and the collision energy was -45 eV; when mass spectrometry was used for quantitative detection, the parent ion was 789.9 m / z, the daughter ion was 575.6 m / z, the declustering voltage was -100 V, and the collision energy was -36 eV.

[0020] Functions and effects of the invention

[0021] The present invention combines the characteristics of flavomycin A, applies ammoniacal methanol for extraction, and uses the QuEChERS method for purification. Liquid chromatography-tandem mass spectrometry is used to determine the flavomycin A residues in the edible parts of aquatic products, including fish, shrimp, crab, shellfish and turtle. A detection method suitable for flavomycin A residues in aquatic products is established, which fills the gap in the detection of flavomycin A residues in aquatic products in my country and provides a method for the detection and supervision of flavomycin A drug residues in aquatic products.

[0022] Furthermore, the high sensitivity of high-resolution mass spectrometry reduces the separation requirements for quantitative analysis, eliminates interference from other substances in complex matrices, and enhances the accuracy of both qualitative and quantitative results. Pretreatment methods have also undergone significant innovation, with the QuEChERS cleanup method significantly simplifying the purification process, avoiding the introduction of additional matrix interferences associated with complex pretreatment, which can affect the accuracy of results. Furthermore, this method saves significant pretreatment time and utilizes an external standard method for quantification, which is environmentally friendly, reduces testing costs, and is simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the selected ion chromatogram of the flavomycin A standard solution of the present invention (50 μg / L).

[0024] Figure 2 This is the linear regression curve of flavomycin A in grass carp of the present invention.

[0025] Figure 3 This is the linear regression curve of flavomycin A in the large yellow croaker of the present invention.

[0026] Figure 4 This is the linear regression curve of flavomycin A in shrimp of the present invention.

[0027] Figure 5 This is the linear regression curve of flavomycin A in crab of the present invention.

[0028] Figure 6 This is the linear regression curve of flavomycin A in the clam of the present invention.

[0029] Figure 7 This is the linear regression curve of flavomycin A in the soft-shelled turtle of the present invention. DETAILED DESCRIPTION

[0030] The following detailed description of the implementation of the present invention is given in conjunction with the drawings and embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] This embodiment of the present invention uses liquid chromatography-tandem mass spectrometry to detect flavomycin A in aquatic products, including grass carp, large yellow croaker, shrimp, crab, clam, and soft-shell turtle. First, the edible portion of the aquatic product is homogenized and used as a sample. A certain amount of the sample is then extracted with ammonia-methanol, purified using the QuEChERS method, concentrated, reconstituted, and filtered through a membrane before being measured using liquid chromatography-tandem mass spectrometry. Quantification is performed using an external standard method, which is environmentally friendly and simple to operate.

[0036] 1. Experimental Methods

[0037] 1. Prepare standard solution

[0038] 1.1 Preparation of standard stock solution

[0039] Weigh 10 mg (accurate to 0.01 mg) of flavomycin A standard substance, dissolve it in methanol, transfer it to a 100 mL volumetric flask, dilute to the mark with methanol to obtain a 100 μg / mL standard solution, and store it at -4°C in the dark.

[0040] 1.2 Preparation of standard working solution

[0041] Pipette 100 μL of 100 μg / mL flavonoids A standard stock solution into a 10 mL volumetric flask, dilute to the mark with methanol to obtain a 1 μg / mL standard working solution, and store at -4°C in the dark.

[0042] 2. Prepare extraction solution

[0043] 10% ammoniated methanol: Accurately measure 10 mL of ammonia water and then accurately measure 90 mL of methanol, shake thoroughly to mix evenly.

[0044] 3. Sample extraction

[0045] Weigh 2 g (accurate to 0.02 g) of sample and place it in a 50 mL plastic centrifuge tube. Add 5 mL of ammoniacal methanol. Vortex for 5 minutes, sonicate for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Transfer the supernatant to another centrifuge tube, add another 5 mL of ammoniacal methanol to the sample residue, repeat the extraction, centrifuge at 8000 rpm for 10 minutes, and combine the supernatants for purification.

[0046] 4. Sample purification

[0047] Add 200 mg of C18 adsorbent to the supernatant, vortex for 30 seconds, and centrifuge at 4000 rpm for 5 minutes. Accurately measure 5 mL of the supernatant into a 10 mL centrifuge tube, blow dry with nitrogen at 40°C to approximately 0.5 mL, dilute to 1 mL with methanol, centrifuge at 4000 rpm for 5 minutes, and filter through a 0.22 μm filter for analysis by liquid chromatography-tandem mass spectrometry.

[0048] 5. Preparation of matrix-matched standard curve

[0049] Accurately pipette an appropriate amount of standard working solution into each of the six blank sample extracts that have been extracted and purified. Blow dry to approximately 0.5 mL with nitrogen at 40°C, dilute to 1 mL with methanol, and centrifuge at 4000 rpm for 5 minutes to prepare a series of matrix-matched standard solutions with flavomycin A concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL for determination by liquid chromatography-tandem mass spectrometry. See the selected ion chromatogram of the 50 ng / mL flavomycin A standard solution for details. Figure 1 .

[0050] 6. Liquid chromatography-mass spectrometry instrument conditions

[0051] 6.1 Chromatographic analysis conditions

[0052] 1) Chromatographic column: ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm);

[0053] 2) Flow rate: 0.4 mL / min;

[0054] 3) Column temperature: 40°C;

[0055] 4) Injection volume: 10 μL;

[0056] 5) Mobile phase A was water (containing 10 mmol / L ammonium acetate), mobile phase B was methanol, and the gradient elution conditions were shown in Table 1.

[0057] Table 1 Gradient elution conditions

[0058] Time (min) A(％) B(％) 0.0 90 10 0.5 90 10 4.0 5 95 6.5 5 95 6.6 90 10 10.0 90 10

[0059] 6.2 Mass spectrometry analysis conditions

[0060] 1) Ion source: electrospray ionization (ESI);

[0061] 2) Scanning mode: negative ion scanning;

[0062] 3) Detection method: multiple reaction monitoring;

[0063] 4) Spray voltage: -4500V;

[0064] 5) Ion transport capillary temperature: 550°C;

[0065] 6) Collision gas CAD: Medium;

[0066] 7) Curtain air CUR: 40.0psi;

[0067] 8) Atomizing gas GS1: 65.0 psi;

[0068] 9) Auxiliary gas GS2: 60.0 psi;

[0069] 10) The qualitative ion pairs, quantitative ion pairs and collision energies are shown in Table 2.

[0070] Table 2 Qualitative ion pairs, quantifier ions and collision energies

[0071]

[0072] 7. Sample determination

[0073] 7.1 Qualitative determination

[0074] The sample solution is measured under the same instrument conditions as the standard working solution; if the retention time of the chromatographic peak detected in the sample is consistent with the retention time of the chromatographic peak of flavomycin A, the allowable deviation is less than 0.05 min; after deducting the background, the relative abundance of the qualifier ion in the sample is compared with the relative abundance of the corresponding qualifier ion in the mixed standard working solution with similar concentration. If the deviation is within the range specified in Table 3, it can be determined that the corresponding flavomycin A is present in the sample.

[0075] Table 3 Maximum allowable deviation of ion relative abundance

[0076] Relative ion abundance (%) ＞50 ＞20～50 ＞10～20 ≤10 Maximum allowable deviation (%) ±20 ±25 ±30 ±50

[0077] 7.2 Quantitative determination

[0078] Inject equal volumes of the sample solution and a series of matrix-matched standard solutions for measurement. Perform single-point or multi-point calibration. Quantify by characteristic ion mass chromatographic peak area and calculate using the external standard method. The response values ​​of the analyte in both the matrix-matched standard solution and the sample solution should be within the linear range of the instrument.

[0079] 8. Result calculation

[0080] The residual amount of flavonoids A in the sample was calculated using the following formula. The blank value was subtracted from the calculated result, and the result was expressed as the arithmetic mean of two parallel determinations, with three significant figures retained.

[0081]

[0082] Where:

[0083] X——the value of the residual amount of flavonoids A in the sample, in micrograms per kilogram (μg / kg);

[0084] C s ——The value of flavonoids A in the sample solution, in nanograms per milliliter (ng / mL);

[0085] V——The volume of the sample reconstitution solution, in milliliters (mL);

[0086] m——the numerical value of the mass of the test material, in grams (g);

[0087] f——dilution factor.

[0088] 2. Experimental Method Optimization

[0089] (1) Optimization of purification materials

[0090] Due to the characteristics of the aquatic matrix, many proteins and other impurities will be extracted together with flavonoids A, so it is necessary to select a suitable purification method to reduce the interference of impurities on the target peak and the pollution of the instrument, so as to make the detection results more accurate. Therefore, the applicant tried to purify the extract after adding the spike to the blank matrix. The QuEChERS method of HLB solid phase extraction, n-hexane liquid-liquid extraction and C18 and N-propylethylenediamine (PSA) adsorption was selected. The results showed that when using the HLB solid phase extraction column, methanol was difficult to completely elute flavonoids A from the column, resulting in a low recovery rate; when using n-hexane liquid-liquid extraction, there would be a large loss and a low recovery rate; after comparing C18 and PSA, after using PSA, the recovery rate of flavonoids A was about 90%, and the recovery rate of C18 adsorbent was greater than 98%. Therefore, the applicant finally chose to use C18 as the purification material, and its cheapness, speed and excellent retention effect fully met the experimental expectations.

[0091] (2) Optimization of different concentration methods

[0092] After concentration, the extract showed a sensitivity that met the method's requirements. Therefore, the applicant spiked the purified extract and concentrated it using rotary evaporation and nitrogen purge. The results showed that rotary evaporation had a recovery rate of only 61%, while nitrogen purge achieved a recovery rate of over 97%. Therefore, nitrogen purge was selected for the extraction.

[0093] 3. Verification Experiment

[0094] (1) Testing of different aquatic products

[0095] A series of standard working solutions containing 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL of flavomycin A were prepared using blank matrix extracts of grass carp, large yellow croaker, shrimp, crab, clam, and turtle, respectively. Standard curves were prepared by testing the series of standard working solutions under the determined analytical conditions. The results showed that flavomycin A showed good linearity in the content range of 1.0 to 100 ng / mL, with correlation coefficients greater than 0.995. The regression equations and correlation coefficients for each matrix are shown in Table 4, and the linear regression curves are shown in Table 4. Figures 2 to 7 .

[0096] Table 4 Regression equations and correlation coefficients of flavonoids A in different matrices

[0097]

[0098] (2) Recovery and precision

[0099] Spiked recovery experiments were conducted using negative samples of grass carp, large yellow croaker, shrimp, crab, clam, and soft-shell turtle. Three spike levels were used, with six replicates at each level and three replicates for each spike level. The recoveries of flavomycin A ranged from 77.8% to 118% across different samples and spike levels. The intra-assay coefficient of variation (n=6) and inter-assay coefficient of variation (m=3) were less than 15%. Specific spike concentrations, recoveries, and precision are shown in Table 5.

[0100] Table 5 Recovery and precision test results of flavomycin A in different aquatic products

[0101]

[0102]

[0103]

[0104] (3) Limit of quantification and detection limit

[0105] The detection limit and quantification limit were determined when the peak height was approximately 3 and 10 times the baseline noise, respectively, i.e., S / N = 3 and S / N = 10. The detection limit and quantification limit achieved by this assay were 1.0 μg / kg and 2.0 μg / kg, respectively.

[0106] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A liquid chromatography-tandem mass spectrometry method for the detection of flavomycin A in aquatic products, characterized in that: The steps include: A. Extraction and purification of flavomycin A The edible part of the aquatic product is removed, homogenized, and then flavomycin A in the sample is extracted using ammonia-methanol. The extract is purified using the QuEChERS method with a C18 adsorbent and then concentrated by nitrogen blowing. The sample is redissolved and filtered to obtain a test sample; the aquatic product includes fish, shrimp, crab, shellfish, and turtle; B. Detection Liquid chromatography-tandem mass spectrometry was used for detection. The specific detection conditions were as follows: (1) Chromatographic analysis conditions: chromatographic column: T3; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 10 μL; mobile phase A: water containing 10 mmol / L ammonium acetate; mobile phase B: methanol; Elution gradient: 0.5 min, 10% B, 90% A; 0.5-4 min, 10%-95% B, 90%-5% A; 4-6.5 min, 95% B, 5% A; 6.5-6.6 min, 95%-10% B, 5%-90% A; 6.6-10 min, 10% B, 90% A, (2) Mass spectrometry analysis conditions: electrospray ionization source, negative ion scanning, multiple reaction monitoring; spray voltage: -4500 V; ion transfer capillary temperature: 550°C; collision gas CAD: Medium; curtain gas CUR: 40.0 psi; nebulizer gas GS1: 65.0 psi; Auxiliary gas GS2: 60.0psi, When qualitative detection was performed by mass spectrometry, the parent ion was 789.9 m / z, the product ion was 554.1 m / z, the declustering voltage was -100 V, and the collision energy was -45 eV; When quantitative detection was performed by mass spectrometry, the parent ion was 789.9 m / z, the product ion was 575.6 m / z, the declustering voltage was -100 V, and the collision energy was -36 eV.

2. The detection method according to claim 1, wherein: in, In step A, the steps of extracting flavomycin A from the sample using ammonia-methanol are as follows: Weigh a certain mass of sample and place it in a plastic centrifuge tube with a volume 25 times the sample weight. Add ammonia-methanol extract with a volume 2-3 times the sample weight. Vortex and oscillate for 5 minutes, sonicate for 10 minutes, and centrifuge at 4000r / min for 10 minutes. Take the supernatant and transfer it to another centrifuge tube. Add the above volume of ammonia-methanol extract to the sample residue, repeat the extraction once, centrifuge at 8000r / min for 10 minutes, and combine the supernatants for purification.

3. The detection method according to claim 2, wherein: in, The ammoniated methanol extract is an ammoniated methanol solution with an ammonia volume fraction of 5-30%.

4. The detection method according to claim 1, wherein: in, In step A, the extract is concentrated, redissolved, and filtered as follows: Add C18 adsorbent to the supernatant of the extract, vortex for 30 seconds, and centrifuge at 4000 r / min for 5 minutes; accurately measure a certain volume of the supernatant into a centrifuge tube, blow dry with nitrogen at 20-60°C to about 1 / 10 volume, add methanol to dilute to 2 times the volume, centrifuge at 4000 r / min for 5 minutes, and pass through a 0.22 μm filter membrane to obtain a sample for liquid chromatography-tandem mass spectrometry determination.

5. The detection method according to claim 1, wherein: in, The specifications of the T3 chromatographic column are 2.1×100 mm, 1.8 μm.

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