Reversed phase liquid chromatography method for determining content of cyromazine in food

Abstract

The invention relates to a reversed-phase liquid chromatography method for determining the content of cyromazine in food. The reversed-phase liquid chromatography method comprises the following steps: selecting a chromatographic column which takes monodisperse silica gel as a basis and is bonded with a biphenyl functional group; water / an aqueous solution containing acid or buffer salt and an organic phase are used as mobile phases; and carrying out liquid chromatographic analysis under the conditions that the flow rate is 0.5-1.5 mL / min, the detection wavelength is 214-400 nm, the column temperature of a chromatographic column is 25-40 DEG C and the sample size is 5-50 microliters. According to the present invention, the effective separation of the cyromazine from the interference of different matrixes can be achieved only by using the 1% organic solvent during the analysis, the ion pair reagent is not required in the analysis method, the accurate determination of the cyromazine in different food matrixes can be achieved in the reversed-phase system, the determination time is short, and the detection sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a reverse phase liquid chromatography method for determining the content of cyromazine in food. Background Art

[0002] Cyromazine (also known as cyromazine) is a selective insect growth regulator that can induce morphological deformation of Diptera larvae and pupae, resulting in incomplete emergence of adult insects. In 2002, my country's Ministry of Agriculture approved cyromazine as a new Class III veterinary drug. Although cyromazine has low toxicity, studies have shown that the main metabolite of cyromazine in animals is carcinogenic melamine.

[0003] For the detection of cyclopropanezine, the most widely used methods are liquid chromatography-ultraviolet method and liquid chromatography-mass spectrometry method. There are two common systems in the chromatographic separation part. One is the HILIC system. Commonly used are amino liquid chromatography columns, HILIC liquid chromatography columns and silica gel liquid chromatography columns. Among them, amino chromatography columns are the most commonly used chromatography columns for detecting cyclopropanezine. However, when using amino chromatography columns to determine cyclopropanezine, the aminopropyl group of the chromatography column bonded phase is easy to fall off, the chromatography column life is short, the maintenance and storage of the chromatography column are difficult, the chromatography column equilibration time (requires 60 minutes) is long, and it is not suitable for batch detection; when analyzing cyclopropanezine with HILIC chromatography columns, there are also problems such as the proportion of organic reagents (usually acetonitrile) in the mobile phase exceeds 95%, and the time to equilibrate the chromatography column is long. In general, the use of HILIC system consumes a lot of organic reagents, the chromatography column equilibration time is long, and it is not suitable for large-scale sample analysis. The other is the reverse phase system, which commonly uses C18 liquid chromatography columns with different bonding methods. Because cyromazine has a strong polarity, it has a short retention time in the C18 column and a fast peak. It is difficult to separate cyromazine from polar impurities. This is an inevitable problem when using a C18 column to determine cyromazine. If it is mass spectrometry detection, it can still be separated, but under the liquid chromatography-ultraviolet method commonly used in the national standard, the retention of cyromazine is too weak to effectively separate from the matrix interference. The solution to this problem is to add ion pair reagents to the mobile phase, which can achieve better retention of cyromazine. Ion pair reagents solve the problem of weak retention of cyromazine on the C18 column, but at the same time they will also cause irreversible damage to the column, shortening the service life of the column. The test cost is relatively high, and the ion pair reagents are sensitive to pH values, requiring high precision when preparing the mobile phase. Summary of the invention

[0004] In order to overcome the above technical defects, the present invention provides a reverse phase liquid chromatography method for determining the content of cyromazine in food. A biphenyl analytical column bonded with monodisperse silica gel is used to perform reverse phase liquid chromatography analysis on cyromazine in food. Only 1% of organic solvent is used in the analysis method to achieve effective separation of cyromazine and different matrix interferences.

[0005] To achieve the purpose of the invention, the present invention adopts the following technical scheme: a reversed phase liquid chromatography method for determining the content of cyromazine in food, using monodisperse silica gel as a chromatographic column with a base ball bonded functional group, water / aqueous solution containing acid or buffer salt and an organic phase as mobile phases, at a flow rate of 0.5-1.5mL / min, a detection wavelength of 214-400nm, a chromatographic column temperature of 25-40°C, and an injection volume of 5-50μL.

[0006] Preferably, the chromatographic column is C18, C8, or Biphenyl, and has a column length of 50-300 mm, a diameter of 1-10 mm, and a filler particle size of 1-10 μm.

[0007] Preferably, the cyromazine in food is analyzed by reverse phase liquid chromatography using a chromatographic column Biphenyl.

[0008] Preferably, the chromatographic column model is Biphenyl 5 μm, 4.6×250 mm.

[0009] Preferably, the mobile phase of the reverse phase liquid chromatography analysis is: water / aqueous solution containing acid or buffer salt and an organic phase.

[0010] Preferably, the mobile phase of the reverse phase liquid chromatography analysis is: 99 / 1v / v 0.1% phosphoric acid water / acetonitrile, flow rate: 1.0 mL / min.

[0011] Among them, the method of the present invention is applicable to all types of food in "NY / T1725-2009 High Performance Liquid Chromatography for Determination of Cyromazine Residues in Vegetables" and "GB31658.12-2021 National Food Safety Standard High Performance Liquid Chromatography for Determination of Cyromazine Residues in Animal Foods".

[0012] The present invention uses an analytical column of a reversed-phase silica gel-bonded biphenyl stationary phase to measure cyromazine. The mobile phase used for the measurement contains very little organic reagent (only 1% acetonitrile), thus avoiding the use of a large amount of organic reagent and being environmentally friendly.

[0013] The present invention uses an analytical column with a reversed-phase silica gel bonded biphenyl stationary phase, and the column equilibration time is less than 20 minutes, which is greatly shortened compared with the equilibration time of 60 minutes in the prior art. The measurement time is short, the detection efficiency is improved, and the detection sensitivity is high.

[0014] The beneficial effects of the present invention are as follows: 1) when the reversed phase liquid chromatography method of the present invention is used to determine cyromazine, the column equilibration time is effectively shortened, thereby improving the experimental efficiency; 2) very little organic reagent is used in the mobile phase, and the mobile phase is environmentally friendly; 3) no ion pair reagent needs to be added to the mobile phase, thereby extending the service life of the chromatographic column; 4) the separation between impurities and target substances is high, the baseline noise is small, and qualitative and quantitative analysis is more accurate; 5) the chromatographic analysis is not affected by the solvent effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison chart of cyromazine standard, mutton sample, and mutton sample spiked;

[0016] Figure 2 This is a comparison chart of cyromazine standard, chicken sample, and spiked chicken sample;

[0017] Figure 3 This is a comparison chart of cyromazine standard, cowpea sample, and cowpea sample spiked with cyromazine. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0019] Example 1 A method for determining cyromazine in food by reverse phase liquid chromatography, the steps are as follows:

[0020] 1. Sample extraction

[0021] The sample extraction method complies with the provisions of "NY / T1725-2009 High Performance Liquid Chromatography for Determination of Cyromazine Residues in Vegetables" and "GB31658.12-2021 National Food Safety Standard High Performance Liquid Chromatography for Determination of Cyromazine Residues in Animal Foods".

[0022] Animal food: Take 5g of sample (accurate to ±0.02g), put it in a 50mL centrifuge tube, add 15mL of extract (take 15mL of 1% trichloroacetic acid solution, dilute to 100mL with acetonitrile), homogenize at high speed to make it evenly dispersed, centrifuge at 5000rpm for 5min, take the supernatant in a 100mL separatory funnel, add 10mL of extract to the residue, repeat the extraction once, combine the supernatant, add 30mL of n-hexane, shake for 2min, and let stand to separate. Collect the lower layer liquid in a 100mL chicken heart bottle, evaporate it in a 50℃ water bath to 1mL (the vacuum degree needs to be controlled to prevent boiling), transfer it to a 10mL graduated centrifuge tube, rinse the chicken heart bottle with the extract twice, 2mL each time, combine the extract, centrifuge at 10000rpm for 5min, take the supernatant, and set aside.

[0023] Vegetable food: Weigh 20g of sample (accurate to 0.01g) into a 50mL centrifuge tube, add 30mL of extract (absorb 200mL of methanol, use 0.1M hydrochloric acid aqueous solution to make up to 1L volumetric flask), high-speed homogenization for 2min, centrifuge at 5000r / min for 15min, take the supernatant, add 30mL of extract to the residue and centrifuge tube, high-speed homogenization for 2min, centrifuge at 5000r / min for 15min, take the supernatant, add 20mL of extract to the residue and centrifuge tube, high-speed homogenization for 2min, centrifuge at 5000r / min for 15min, take the supernatant, combine the three supernatants, make up to 100mL volumetric flask with the extract, mix well and set aside.

[0024] 2. SPE purification

[0025] SelectCoreMCX60mg / 3mL solid phase extraction column

[0026] (1) Activation: Activate with 3 mL of methanol and 3 mL of water in sequence;

[0027] (2) Sampling: Take all the supernatant of animal samples or vegetable samples and pass them through the column, controlling the flow rate to 1 mL / min;

[0028] (3) Washing: Wash with 3 mL of methanol, 3 mL of 0.1 mol / L hydrochloric acid solution, 3 mL of water, and 3 mL of methanol in sequence, and drain;

[0029] (4) Elution: Elute with 5 mL of 5% ammonia methanol solution, collect the eluate, blow dry with nitrogen at 50°C, reconstitute with 1.0 mL of mobile phase, vortex for 30 seconds, filter with a filter membrane, and use for HPLC determination.

[0030] 3. Reverse phase liquid chromatography detection method

[0031] Column: ChromCoreBiphenyl 5μm, 4.6×250mm

[0032] Mobile phase: 99 / 1v / v 0.1% phosphoric acid water / acetonitrile

[0033] Flow rate: 1.0mL / min

[0034] Column temperature: 30°C

[0035] Injection volume: 20 μL

[0036] Detection wavelength: 214nm

[0037] 4. Chromatogram

[0038] The reverse phase liquid chromatography method of the present invention was used to detect mutton, chicken and cowpea samples, and the chromatograms were obtained as follows: Figure 1 , Figure 2 and Figure 3 ,in, Figure 1 This is a comparison chart of cyromazine standard, mutton sample, and mutton sample spiked; Figure 2 This is a comparison chart of cyromazine standard, chicken sample, and spiked chicken sample; Figure 3 It is a comparison chart of cyromazine standard, cowpea sample, and cowpea sample spiked. It can be seen that the reversed phase liquid chromatography method of the present invention can well detect cyromazine in food, better realize the effective separation of cyromazine and different matrix interferences, the separation between impurities and targets is high, the baseline noise is small, and the qualitative and quantitative analysis is more accurate.

[0039] In the embodiment of the present invention, an analytical column with a reversed-phase silica gel bonded biphenyl stationary phase is used, and the column equilibration time is less than 20 minutes, which is greatly shortened compared with the equilibration time of 60 minutes in the prior art.

[0040] The invention uses a biphenyl analytical column bonded with monodisperse silica gel as the basis to perform reversed phase liquid chromatography analysis on cyclopropane in food. Only 1% of organic solvent is used in the analytical method to achieve effective separation of cyclopropane from interferences of different matrices. No ion pair reagent is required in the analytical method. Accurate determination of cyclopropane in different food matrices can be achieved in a reversed phase system. The determination time is short and the detection sensitivity is high.

[0041] The above-described embodiments are only some preferred solutions of the present invention, but they are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A reverse phase liquid chromatography method for determining the content of cyromazine in food, characterized in that: The method is carried out using a chromatographic column with monodisperse silica as the base ball bonded with functional groups, water / aqueous solution containing acid or buffer salt and organic phase as the mobile phase, a flow rate of 0.5-1.5 mL / min, a detection wavelength of 214-400 nm, a chromatographic column temperature of 25-40° C., and an injection volume of 5-50 μL.

2. The reverse phase liquid chromatography method for determining the content of cyromazine in food according to claim 1, characterized in that: The chromatographic column is C18, C8, Biphenyl, the length of the chromatographic column is 50-300 mm, the diameter is 1-10 mm, and the filler particle size is 1-10 μm.

3. The reverse phase liquid chromatography method for determining the content of cyromazine in food according to claim 1, characterized in that: The chromatographic column is Biphenyl.

4. The reverse phase liquid chromatography method for determining the content of cyromazine in food according to claim 1, characterized in that: The mobile phase of the reversed phase liquid chromatography analysis is: water / aqueous solution containing acid or buffer salt and an organic phase.

5. The reverse phase liquid chromatography method for determining the content of cyromazine in food according to claim 1, characterized in that: The mobile phase of the reversed phase liquid chromatography analysis is: 99 / 1 v / v 0.1% phosphoric acid water / acetonitrile.

6. The reverse phase liquid chromatography method for determining the content of cyromazine in food according to claim 1, characterized in that: The foods mentioned are all food types in "NY / T1725-2009" and "GB31658.12-2021".

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