CN111141837A

CN111141837A - Method for detecting residual quantity of cefquinome in vegetables

Info

Publication number: CN111141837A
Application number: CN201811314179.2A
Authority: CN
Inventors: 胡丽芳; 范芳; 徐俊; 戴廷灿; 王冬根
Original assignee: Institute Of Agricultural Products Quality Safety And Standard Jiangxi Academy Of Agricultural Sciences
Current assignee: Institute Of Agricultural Products Quality Safety And Standard Jiangxi Academy Of Agricultural Sciences
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2020-05-12

Abstract

The invention relates to a method for detecting the residual quantity of cefquinome in vegetables by ultra-high performance liquid chromatography-electrospray tandem mass spectrometry, and provides a technical means for monitoring the residual quantity of cefquinome drugs in vegetables.

Description

Method for detecting residual quantity of cefquinome in vegetables

Technical Field

The invention belongs to the field of drug residue detection, and particularly relates to a method for qualitatively and quantitatively detecting cefquinome residue in vegetables.

Background

Cephalosporin antibiotics are mainly used for treating various infections caused by gram bacteria, and have the advantages of wide antibacterial spectrum, high curative effect, low toxicity, less anaphylactic reaction and the like, but excessive antibiotics have toxic and side effects and are mainly reflected in damage to cranial nerves, auditory sensation and kidney. China has already stipulated the limit of cephalosporin drugs in beef, pork and other products.

Cefquinome is a special 4 th generation cephalosporin antibiotic for the first animal, achieves a bactericidal effect by inhibiting the synthesis of cell walls, has broad-spectrum antibacterial property and strong antibacterial activity, and can reach higher concentration in mammary tissues. Cefquinome is registered in China at present and approved for veterinary use, and is mainly used for treating cow mastitis caused by escherichia coli and pig respiratory diseases caused by pasteurella multocida or actinobacillus pleuropneumoniae clinically. At present, domestic detection standards and methods for cefquinome mainly comprise high performance liquid chromatography and ultra high performance liquid chromatography-tandem mass spectrometry, and are mostly concentrated on livestock and poultry products. There is no literature report on cefquinome in vegetables.

The invention establishes the ultra-high performance liquid chromatography-electrospray tandem mass spectrometer determination method for the cefquinome residue in the vegetables, and the method has the advantages of simple and convenient pretreatment, easy operation, good accuracy and high sensitivity.

Ultra Performance Liquid Chromatography (Ultra Performance Liquid Chromatography/UPLC) is a brand new category in separation science, and the UPLC covers brand new technologies such as small particle packing, very low system volume, rapid detection means and the like by means of the theory and principle of HPLC (high Performance Liquid Chromatography), and increases the flux, sensitivity and chromatographic peak capacity of analysis. At present, the ultra high performance liquid chromatograph has begun to be gradually put into liquid phase experiments.

The working principle of triple quadrupole tandem mass spectrometry is as follows:

under the action of electron flow bombardment or strong electric field, the compound molecules are ionized into ions and certain chemical bonds are regularly broken to generate positively charged ions with different masses, and the ions are collected and recorded according to the mass-to-charge ratio (m/z).

Electrospray ionization (ESI):

the sample solution forms a fog-like small droplet with high charge under the action of an electric field, the droplet is reduced due to continuous volatilization of a solvent in the process of moving to the mass analyzer, the surface charge density is increased continuously, when the repulsive force between charges is enough to overcome the surface tension of the droplet, the droplet is split, and the steps are repeated, and finally ions with single charge or multiple charges are obtained.

Triple quadrupole tandem mass spectrometry advantages:

(1) derivatization is not required.

(2) Confirmation quantification was achieved in a single assay.

(3) Low detection limit in complex, very dirty matrices.

(4) More reliable and trustworthy test results.

(5) The mass range is as follows: 5-1200amu

(6) High sensitivity and good quantitative reproducibility

Disclosure of Invention

The invention aims to provide a simple, convenient, rapid, sensitive, accurate and economic method for detecting the residual quantity of cefquinome in vegetables.

The invention comprises the following steps:

according to the invention, the ultra-high performance liquid chromatography-electrospray tandem mass spectrometry is used as a detection means, and a modern ultra-high performance liquid chromatography-electrospray tandem mass spectrometry system is provided with a data acquisition and processing system comprising a chemical workstation, a computer and a software system, so that the residual quantity of the cefquinome in the vegetables can be accurately detected.

The ultra-high performance liquid chromatography-electrospray tandem mass spectrometry comprises two steps: data acquisition and data processing. The mobile phase in the liquid storage device is pumped into the system by a high pressure pump, the sample solution enters the mobile phase through the sample injector and is loaded into a chromatographic column (stationary phase) by the mobile phase, and because each component in the sample solution has different distribution coefficients in two phases, when the two phases move relatively, through the distribution process of repeated adsorption-desorption, each component generates a large difference in the moving speed, is separated into single components and flows out of the column in sequence, when the sample solution passes through the detector, the concentration of the sample is converted into an electric signal and is transmitted to a recorder, and data is printed in a map form.

Preparation of standard solution:

standard stock solutions: weighing appropriate amount of cefquinome standard, preparing into 1.0mg/mL standard stock solutions with methanol, and storing at-18 deg.C in dark place.

Standard intermediate solution: taking 1mL of each standard stock solution, diluting the stock solutions with methanol to obtain standard intermediate solutions with the mass concentration of 10.0mg/L, and storing the intermediate solutions at the temperature of minus 18 ℃ in a dark place.

Standard mixed working solution: taking 1mL of each standard intermediate solution, diluting with methanol to obtain a standard mixed working solution with a mass concentration of 1.0mg/L, and storing at-4 ℃ in a dark place.

This method is qualitative and quantitative based on the selected ions. Taking a blank vegetable sample, adding cefquinome standard solutions with different mass concentrations, and detecting according to optimized experimental conditions from high mass concentration to low mass concentration until the signal-to-noise ratio is equal to 3(S/N is 3) and the signal-to-noise ratio is equal to 10(S/N is 10), determining that the detection Limit (LOD) of cefquinome in the pig hair and chicken hair samples is 1.0 mug/L, and the quantitative Limit (LOQ) is 3.0 mug/L.

Standard working curve

Preparing a series of standard solutions with the mass concentrations of the cefquinome standard solutions of 4.0, 10.0, 15.0, 20.0 and 50.0 mu g/L, and making a standard curve by using the quantitative ion peak area (y) to the concentration (x, mu g/L). The results show that the cefquinome has good linear relation (r) in the range of 4-50.0 mu g/L²> 0.999). The linear regression equation and the related coefficient of cefquinome in vegetables are shown in table 1.

TABLE 1 Cefquinome Linear regression equation and correlation coefficient in vegetables

Accuracy and precision:

taking a blank vegetable sample, adding cefquinome standard solutions of 3.0, 5.0 and 20.0 mu g/kg respectively, then carrying out sample pretreatment and determination according to the method, wherein each addition level is determined for 6 times, the in-day precision is obtained by determining 6 parallel samples under 3 standard addition levels within 1d, and the in-day precision is obtained by determining the samples under 3 standard addition levels continuously for 6d (1 time per day), and the result shows that when the cefquinome addition concentrations are 3.0, 5.0 and 20.0 mu g/kg, the average recovery rate of the method is 79.5-95.1%, the in-day relative standard deviation is 2.67-5.14%, and the in-day relative standard deviation is 4.25-9.37%. The accuracy, precision and relative standard deviation are shown in table 2.

Table 2 blank vegetables with added cefquinome daily and diurnal precisions and relative standard deviations (n ═ 6)

The cefquinome residue in the sample is calculated by mass fraction X, the value is expressed by microgram per kilogram (mu g/kg), and the single point correction is calculated according to the following formulas (1) and (2):

in the formula:

C_i-cefquinome concentration in the sample solution (μ g/L);

C_s-cefquinome concentration in control solution (μ g/L);

A_ipeak area of cefquinome in the sample solution;

A_speak area of cefquinome in control solution;

x represents the residual quantity (mu g/kg) of cefquinome in the sample;

v is the final volume (mL) of the sample solution;

m-sample size (g);

the standard curve calibration is calculated as equation (3):

C_i-cefquinome concentration (. mu.g/L) in the sample checked on the standard curve; (ii) a

V is the final volume (mL) of the sample solution;

m-sample weight (g).

Results are presented as the arithmetic mean of two independent measurements obtained under repetitive conditions, with three significant figures remaining.

Under repetitive conditions, the absolute difference between the two independent test results obtained is not more than 20% of the arithmetic mean of the two measurements.

Optimizing mass spectrum conditions:

respectively preparing 1.0mg/L cefquinome standard solution, directly entering an ion source without a chromatographic column, and carrying out ESI (ion implantation technique)⁺And respectively optimizing the mass spectrum conditions in the modes. Performing first-order mass spectrogram full scanning, determining parent ions of each target compound, optimizing to obtain in-source fragmentation voltage, performing sub-ion scanning on the selected parent ions under the condition of the selected in-source fragmentation voltage, selecting 1 pair of fragment ions with relatively strongest abundance as quantitative ions, and selecting 1 pair or 2 pairs of fragment ions with relatively strongest abundance as qualitative ions, and optimizing the collision energy of the sub-ions respectively. And finally, optimizing parameters such as the pressure of the atomizing gas, the temperature of the drying gas, the flow and the like in an MRM mode. The optimized mass spectrum conditions are shown in table 3.

TABLE 3 Mass Spectrometry optimization conditions for amantadine, rimantadine, moroxydine and D15-amantadine in MRM monitoring mode

Quantitative ions

To further illustrate the features and effects of the present invention, the following description is further provided in conjunction with the accompanying drawings.

Drawings

FIG. 1: TIC diagram of adding 2.0 mu g/kg of cefquinome to vegetable sample

Examples

The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention.

Accurately weighing 2.00g of vegetable sample into a 50mL polypropylene centrifuge tube, adding 15mL of 1% acetic acid acetonitrile solution, carrying out vortex mixing, then carrying out ultrasonic extraction for 3min, centrifuging at 4500r/min for 10min, transferring the supernatant into another 50mL polypropylene centrifuge tube, repeatedly extracting the residue once with 15mL of 1% acetic acid acetonitrile solution, and combining the two extracting solutions. Blowing nitrogen gas to near dry in 40 deg.C water bath, adding 1.00mL water, dissolving, swirling for 30s, filtering with 0.22 μm filter membrane, and measuring.

Setting the conditions of the liquid chromatogram as follows:

mobile phase: a is 0.1 percent of formic acid water, B is acetonitrile,

gradient elution: keeping 8% B at 0-2min, linearly changing 8% B to 70% B at 2-4min, keeping 70% B at 4-8min, linearly changing 70% B to 8% B at 8-8.5min, and keeping 8% B at 8.5-12 min. Flow rate: 0.3mL/min

Sample introduction amount: 5.00 mu L

Mass spectrum conditions: ionization mode: electrospray ionization positive ion mode (ESI +); the detection mode is as follows: multiple Reaction Monitoring (MRM); capillary voltage: 4000V; ion source Temperature (TEM): 325 ℃; temperature of the drying gas: 300 ℃; flow rate of drying gas: 15L/min; atomizing gas pressure: 50 psi; temperature of sheath gas: 400 ℃; the flow rate of the sheath gas: 12L/min.

Of course, those skilled in the art should realize that the above embodiments are illustrative only and not limiting of the present invention, and that changes and modifications to the above described embodiments are intended to fall within the scope of the appended claims, as long as they fall within the true spirit and scope of the present invention.

Claims

1. A method for detecting the residual quantity of cefquinome in vegetables is characterized by comprising the following steps:

(1) accurately weighing 2.00g of vegetable sample into a 50mL polypropylene centrifuge tube, adding 15mL of 1% acetic acid acetonitrile solution, carrying out vortex mixing, then carrying out ultrasonic extraction for 3min, centrifuging at 4500r/min for 10min, transferring the supernatant into another 50mL polypropylene centrifuge tube, repeatedly extracting the residue once with 15mL of 1% acetic acid acetonitrile solution, and combining the two extracting solutions. Blowing nitrogen gas to near dry in 40 deg.C water bath, adding 1.00mL water, dissolving, swirling for 30s, filtering with 0.22 μm filter membrane, and measuring.

(2) And liquid phase conditions:

mobile phase: a is 0.1 percent of formic acid water, B is acetonitrile,

gradient elution: o-2min keeps 8% B,2-4min 8% B linearly changes to 70% B, 4-8min keeps 70% B, 8-8.5min 70% B linearly changes to 8% B, 8.5-12min keeps 8% B.

Flow rate: 0.3mL/min

Sample introduction amount: 5.00. mu.L.