CN111879885A - LC-MS (liquid chromatography-mass spectrometry) determination method for doxycycline residues in eggs - Google Patents
LC-MS (liquid chromatography-mass spectrometry) determination method for doxycycline residues in eggs Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention discloses an LC-MS (liquid chromatography-mass spectrometry) determination method for doxycycline residues in eggs, which comprises the steps of sample extraction and purification, preparation of a standard working solution, determination, result calculation and the like, wherein a chromatographic column is C18; gradient elution is carried out on a mobile phase A which is 0.05-0.5% formic acid aqueous solution and a mobile phase B which is acetonitrile solution; the mass-to-charge ratio of doxycycline parent ions is 445.0, the mass-to-charge ratio of daughter ions detected by secondary mass spectrometry is 154 and 428, and the corresponding collision voltages are 37eV and 29eV respectively. The method has the advantages of average recovery rate of 78.51-108.82%, average Relative Standard Deviation (RSD) of 5.96-11.07%, detection limit of 0.5 mu g/kg, quantification limit of 0.5 mu g/kg, simple operation, rapidness, accuracy, high sensitivity, good repeatability and the like.
Description
Technical Field
The invention relates to a method for determining doxycycline residue in eggs, in particular to a method for qualitatively and quantitatively determining doxycycline residue in eggs by adopting liquid chromatography tandem mass spectrometry (LC-MS/MS), belonging to the technical field of veterinary drug residue detection.
Background
The eggs are rich in high-quality protein and vitamins, have high nutritional value and are popular food. China is a big country for egg production and consumption, and according to statistics, the yield of eggs is 3309 ten thousand tons in 2019, wherein eggs account for about 90%, and squat is the first world. Therefore, the quality safety of eggs is always of great concern to consumers.
Veterinary drug residues are important factors influencing the quality and safety of eggs. In the feeding process of laying hens, a plurality of farmers use antibiotics randomly for antibiosis and growth promotion due to the driving of economic benefits or the understanding of scientific medicine knowledge, so that the residual of the antibiotics in eggs exceeds the standard, and food potential safety hazards are left. Doxycycline (doxycycline) is a tetracycline drug used in laying hen breeding, and in recent years, the phenomenon that doxycycline residues in eggs exceed standards sometimes occurs. LvBing et al summarized the egg national food safety supervision spot check results in 2018, and found that 77 unqualified samples were detected in 1310 spot checked eggs, and the total reject rate was 5.88%. The situation of tetracycline antibiotic residues in chicken and eggs in the areas of Hubei in 2016-2018 is analyzed by the Sun Phoenix et al, and the result shows that the detection rate of the tetracycline antibiotic in the 538 sample subjected to spot inspection is 15.61%, wherein the doxycycline is the only item with the highest detection rate and exceeding the standard in the chicken and egg samples. 2016-shaped and 2017 such as Yanghuanchun and the like randomly extract 124 parts of eggs and chicken sold in 8 cities, counties (regions) in Ningxia, and the result shows that the situation of tetracycline antibiotic residue in the eggs and the chicken is not negligible, wherein the standard exceeding rate of the doxycycline in the eggs is 13.5 percent. According to the report, doxycycline is detected from eggs produced by a chicken farm of a certain Xinwan town Liza in an urban and rural integrated demonstration area in 2017, 11, 5 days and in the process of carrying out safe spot inspection activities on the quality of veterinary drugs, feeds and livestock products by the livestock husbandry office in Nanyang city.
The residue detection is an important means for discovering and investigating the overproof residue of the animal-derived food, and is a technical guarantee for scientific law enforcement of law enforcement agencies. Currently, many methods are available for detecting doxycycline residues in eggs. The Wangxudang pill and the like establish a high performance liquid chromatography for simultaneously detecting 4 tetracycline drug residues of oxytetracycline, tetracycline, chlortetracycline and doxycycline in eggs, the detection limit of the method is 10ng/g, the quantification limit is 50ng/g, and the average recovery rate is 67.3-81.4%. Wuningpeng and the like establish a high performance liquid chromatography for measuring the residual quantity of oxytetracycline, tetracycline, chlortetracycline and doxycycline in eggs, the detection limit of the method is 10 mug/kg, the quantification limit is 50 mug/kg, and the average recovery rate is 60-110%. The research progress of chromatographic and mass spectrometric detection technology for tetracycline drug residue in animal derived food has been reviewed by dawna et al. From the above, most of the existing detection of tetracycline residues is focused on animal edible tissues such as pork, chicken, fish, honey and the like; the detected objects mainly focus on oxytetracycline, chlortetracycline and tetracycline; few HPLC-MS methods for detecting doxycycline residues in eggs have high detection limit and low accuracy, and cannot meet the current requirement for detecting doxycycline residues in eggs.
Therefore, the LC-MS/MS detection method which is simple, convenient, rapid, accurate, durable and capable of accurately qualitatively and quantitatively analyzing the doxycycline residual quantity in the eggs has important significance.
Disclosure of Invention
The invention aims to provide an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for doxycycline residues in eggs, and aims to improve the detection limit and accuracy of doxycycline residues in eggs and improve the detection efficiency.
In order to realize the purpose, the technical scheme adopted by the invention is as follows:
an LC-MS (liquid chromatography-mass spectrometry) determination method for doxycycline residues in eggs, which comprises the following steps:
(1) extracting and purifying sample
Taking the homogenized egg sample to be detected, and adding Na2-EDTA-Mclvaine buffer solution, vortex for 1-5min, then add acetonitrile solution, oscillate for 5-10min, ultrasonic at room temperatureExtracting for 10-30min, centrifuging to obtain supernatant, passing the supernatant through a solid phase extraction column, collecting filtrate, drying with 40-50 deg.C water bath nitrogen, diluting to constant volume with mobile phase solution of initial proportion, and filtering with organic microporous membrane to obtain sample solution to be measured;
(2) preparation of standard working solutions
Taking a blank egg sample, extracting and purifying according to the method in the step (1), adding different amounts of doxycycline standard solutions, and preparing into series of standard working solutions with different concentration gradients;
(3) measurement and calculation of results
Performing LC-MS/MS measurement on the standard working solution with each concentration gradient in the step (2), and performing regression analysis on the corresponding concentration of the standard working solution according to the chromatographic peak area of the standard working solution to obtain a standard working curve; injecting the sample solution to be tested in the step (1) into LC-MS/MS for determination under the same conditions, measuring the chromatographic peak area of doxycycline in the sample solution to be tested, substituting the chromatographic peak area into a standard working curve to obtain the content of doxycycline in the sample solution to be tested,
the LC-MS/MS liquid chromatography conditions are as follows: the chromatographic column is C18; the sample amount is 5-20 μ L; the flow rate is 0.1-0.5 mL/min; gradient elution with mobile phase A of 0.05-0.5 vol% formic acid solution and mobile phase B of acetonitrile solution;
the mass spectrum conditions of the LC-MS/MS are as follows: using ESI ion source, positive ion (ES +) mode scan, multiple ion reaction monitoring (MRM) mode measurement, the mass to charge ratio of doxycycline parent ion was 445.0, the mass to charge ratios of daughter ions detected by secondary mass spectrometry were 154 and 428, and the corresponding collision voltages were 37eV and 29eV, respectively.
In the step (3), the procedure of gradient elution is as follows:
| time/min | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 2 | 90 | 10 |
| 3 | 30 | 70 |
| 5 | 30 | 70 |
| 7 | 90 | 10 |
| 8 | 90 | 10 |
The working parameters of the mass spectrum are as follows: desolventizing temperature is 500 ℃; desolventizing agent flow rate, 800L/hr; cone voltage, 27V; cone orifice gas flow rate, 50L/hr; capillary voltage, 3.50 kV; source temperature, 150 ℃; the secondary taper hole extraction voltage is 3.0V; collision cell inlet voltage, 0.5V; collision cell exit voltage, 0.5V.
The concentration gradient of the series of standard working solutions is as follows: 0.5, 1, 2, 5 and 10 mu g/kg.
In the step (1), the temperature of the centrifugation is 4 ℃, and the speed is 10000 r/min.
In the step (1), the pore diameter of the organic microporous filter membrane is 0.22 μm.
The sample injection amount in the step (3) is 10 mu L; the flow rate was 0.2 mL/min.
The chromatographic Column was a Waters acquisition UPLCTM BEH C18Column (2.1X 50mm, 2.5 μm).
Detecting parent ions and ion pairs of the medicine in the filtrate in the step (3), and if the retention time of an ion chromatographic peak of the medicine is consistent with that of a standard working solution; and when the deviation between the relative abundance of two daughter ions of the target compound in the filtrate (sample) and the relative abundance of ions of the blank matrix standard solution with the equivalent concentration is not more than 30%, judging that the veterinary drug exists in the sample; if the two conditions cannot be met simultaneously, the veterinary drug is judged not to be contained.
The invention has the beneficial effects that:
the invention successfully establishes an LC-MS/MS method for qualitatively confirming and quantitatively detecting doxycycline in eggs, the average recovery rate of the method is 78.51-108.82%, the average Relative Standard Deviation (RSD) is 5.96-11.07%, the detection limit is only 0.5 mug/kg, and the quantitative limit is only 0.5 mug/kg.
Drawings
Figure 1 is an LC-MS/MS multiple reaction monitoring chromatogram of a 10.0ng/mL doxycycline standard solution added to a blank egg base.
Figure 2 is a LC-MS/MS multiple reaction monitoring chromatogram of an egg blank sample without doxycycline.
Figure 3 is a standard working curve of doxycycline formulated with an egg blank sample without doxycycline as a base and a standard curve of a standard solution.
Detailed Description
The present invention is illustrated below by way of specific examples, but is not limited to the scope of the present invention.
Instruments and reagents used in the examples
Liquid chromatography-mass spectrometry/mass spectrometer: a power distribution spray ion source; analytical balance: 0.00001g of sensory quantity and 0.001g of sensory quantity; volumetric flask: 100mL, 1000 mL; freezing a high-speed centrifuge (10000 r/min); a constant-temperature water bath kettle; a vortex mixer; a solid phase extraction device; an ultrasonic cleaning machine; a nitrogen blow-drying device; solid phase extraction column: waters prime HLB 200mg/6mL, or equivalent; and (3) filtering the membrane: 0.22 μm.
Reagent: acetonitrile, methanol and formic acid are in chromatographic purity. Na (Na)2EDTA, sodium hydroxide, sodium dihydrogen phosphate, citric acid, oxalic acid, etc. are available from the national pharmaceutical group. Doxycycline (DOTC) standards were purchased from the chinese veterinary medicine institute. All reagents were analytically pure except as otherwise specified, and water was first grade water in accordance with the GB/T6682 specification.
Example 1: detection of doxycycline residual quantity in eggs
(1) Sample pretreatment
Extraction:
taking 1g (accurate to 0.01g) of a homogeneous egg sample, placing the homogeneous egg sample in a 10mL centrifuge tube, and precisely adding Na21.5mL of EDTA-Mclvaine buffer solution, swirling for 1min, precisely adding 4.5mL of acetonitrile solution, fully oscillating for 10min, ultrasonically extracting for 15min at normal temperature, centrifuging for 10min at 10000r/min and 4 ℃ and taking supernatant.
And (3) purification:
selecting a solid phase extraction column without activation, taking 3mL of the extraction filtrate, passing the extraction filtrate through the column, filtering, collecting the filtrate, drying the filtrate in 45 ℃ water bath nitrogen, fixing the volume to 0.5mL by using a mobile phase solution (aqueous formic acid solution/acetonitrile solution is 90/10; v/v) with an initial proportion, filtering by using a 0.22 mu m organic microporous filter membrane, and determining by using a high performance liquid chromatography tandem mass spectrometry.
(2) Preparation of matrix matching curves
Taking 6 parts of blank egg samples, extracting and purifying, adding a proper amount of doxycycline standard solution, preparing into standard working solutions with the concentrations of 0.5, 1, 2, 5 and 10 mu g/kg respectively, and detecting by LC-MS/MS. And drawing a standard curve by taking the measured peak area as a vertical coordinate and the corresponding standard solution concentration as a horizontal coordinate, and solving a regression equation and a correlation coefficient.
(3) Liquid chromatography tandem mass spectrometry (LC-MS/MS) assay
Respectively injecting standard working solutions with different concentration gradients into LC-MS/MS, and performing quantitative analysis on doxycycline content by an external standard method, namely performing regression analysis on corresponding concentrations of the standard working solutions according to chromatographic peak areas of the standard working solutions to obtain a standard curve; injecting the sample extracting solution into LC-MS/MS under the same condition for determination, measuring the chromatographic peak area of doxycycline in the sample solution, substituting the chromatographic peak area into a standard curve to obtain the content of doxycycline in the sample solution, and then calculating according to the mass of the sample represented by the sample solution to obtain the residual amount of doxycycline in the sample.
Wherein the liquid chromatography conditions are
A chromatographic column: waters ACQUITY UPLCTM BEH C18Column (2.1X 50mm, 2.5 μm).
Mobile phase: phase A is 0.1% formic acid water solution; and the phase B is acetonitrile solution.
Flow rate: 0.2 mL/min.
Sample introduction amount: 10 μ L.
Gradient elution: the gradient elution procedure is shown in table 1.
TABLE 1 gradient elution conditions
| Time/min | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 2 | 90 | 10 |
| 3 | 30 | 70 |
| 5 | 30 | 70 |
| 7 | 90 | 10 |
| 8 | 90 | 10 |
Using ESI ion source, positive ion (ES +) mode scan, multiple ion reaction monitoring (MRM) mode measurement, the mass to charge ratio of doxycycline parent ion was 445.0, the mass to charge ratios of daughter ions detected by secondary mass spectrometry were 154 and 428, and the corresponding collision voltages were 37eV and 29eV, respectively. As shown in table 2.
TABLE 2 doxycycline Mass Spectrometry parameters
| Compound (I) | Parent ion (m/z) | Ionic acid (m/z) | Collision voltage (eV) |
| 445 | 154 | 37 | |
| 428 | 29 |
The parameters are set as follows: desolventizing temperature is 500 ℃; desolventizing agent flow rate, 800L/hr; cone voltage, 27V; cone orifice gas flow rate, 50L/hr; capillary voltage, 3.50 kV; source temperature, 150 ℃; the secondary taper hole extraction voltage is 3.0V; collision cell inlet voltage, 0.5V; collision cell exit voltage, 0.5V.
Example 2 assay
1. Qualitative determination
And comparing and determining the retention time of the sample chromatogram with the retention time of the standard substance, and comparing the characteristic ions of the chromatographic peak with the characteristic ions of the chromatographic peak of the standard substance with corresponding concentrations. The relative deviation of the retention time of the sample from the standard is not more than 2.5%; the relative abundance of the characteristic ions of the sample is consistent with that of the standard solution with the equivalent concentration, and the deviation of the relative abundance does not exceed the specification of the table 3, so that the corresponding detected object in the sample can be judged. The retention time deviation is within ± 5%, and the relative abundance of the detected ions should be consistent with the corrected standard solution relative abundance at comparable concentrations. The allowable deviation should meet the requirements of table 3.
TABLE 3 maximum permissible error in relative ion abundance for qualitative confirmation
Units are percentages
| Relative ion abundance | ＞50 | > 20 to 50 | > 10 to 20 | ≤10 |
| Maximum deviation allowed | ±20 | ±25 | ±30 | ±50 |
2. Quantitative determination
Taking a sample solution and a corresponding standard working solution, carrying out single-point or multi-point calibration, quantifying by using a peak area according to an external standard method, wherein the response values of doxycycline in the standard working solution and the sample solution are in a linear range detected by an instrument. The results of the 20 blank tests showed an average response value of 51025 and a sample standard deviation of 8647.83 (as shown in table 4).
TABLE 420 blank test results
| 5.05E+04 | 5.06E+04 | 5.33E+04 | 5.67E+04 | 5.05E+04 |
| 5.25E+04 | 5.36E+04 | 5.00E+04 | 3.02E+04 | 4.77E+04 |
| 6.94E+04 | 4.90E+04 | 5.24E+04 | 5.37E+04 | 5.70E+04 |
| 5.09E+04 | 5.35E+04 | 2.98E+04 | 5.39E+04 | 5.53E+04 |
Note: mean response value 51025, sample standard deviation 8647.83
And (3) diluting the standard stock solution by using a blank matrix solution to obtain a standard matrix working solution with the doxycycline concentration of 0, 0.5, 1, 2, 5 and 10 mu g/kg, then carrying out high performance liquid chromatography-tandem mass spectrometer analysis, quantifying the peak area of each component in a quantitative ion chromatogram, drawing a matrix matching working curve, and obtaining that the matrix matching property of the doxycycline medicament in the eggs is 0.5-10 mu g/L, and the correlation coefficient is more than 0.99. The standard curve is shown in fig. 3: the ratio of the slope of the doxycycline standard curve to the slope of the matrix standard curve is 1.004, the influence of the sample matrix is small, and the recovery rate can be directly calculated through a standard linear curve.
Example 3
1. Sensitivity of the probe
The reliability of the measurement result in the trace analysis depends on the size and fluctuation condition of the blank value to a great extent, let Wt represent the total value of the tested sample, Wb represent the blank value, the lower limit of the sample detection is the content (Wt-Wb) of the tested component is 3 sigma, the lower limit of the quantitative detection is 10 sigma, the lower limit of the actual detection by the method is 0.5 mug/kg, and the lower limit of the quantitative detection is 1 mug/kg.
2. Accuracy and precision
Under the optimal test conditions, the samples of the egg are extracted and measured at different adding concentrations, the accuracy of the method is expressed by the recovery rate, and the precision is expressed by the relative deviation. Specifically, 3 concentration levels of standard doxycycline solution at 1, 2 and 5 μ g/kg were added to eggs without doxycycline and the residual amount was determined according to the above-described treatment procedure. The measured concentrations were compared with the theoretical drug addition concentrations to obtain the drug addition recovery rates, and each addition level was measured in parallel 6 times to obtain the relative standard deviation, the measurement results are shown in table 5. As can be seen from table 3, at 3 spiking levels, the average recovery rate of doxycycline is 79.7% to 100.0%, and the average Relative Standard Deviation (RSD) is 2.16% to 5.41%, indicating that the method of the present invention has high recovery rate and good repeatability.
Table 5 recovery of doxycycline in egg samples
The above examples are only for describing the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by the ordinary engineering in the art without departing from the spirit of the present invention are intended to fall within the protection scope defined by the claims of the present invention.
Claims (8)
1. An LC-MS (liquid chromatography-mass spectrometry) determination method for doxycycline residues in eggs is characterized by comprising the following steps:
(1) extracting and purifying
Taking the homogenized egg sample to be detected, and adding Na2-EDTA-Mclvaine buffer solution, whirling for 1-5min, adding acetonitrile solution, oscillating for 5-10min, performing ultrasonic extraction for 10-30min at normal temperature, centrifuging to obtain supernatant, passing the supernatant through a solid phase extraction column, collecting filtrate, drying the filtrate with nitrogen in water bath at 40-50 ℃, performing constant volume with mobile phase solution of initial proportion, and filtering with an organic microporous filter membrane to obtain a sample solution to be detected;
(2) preparation of standard working solutions
Taking a blank egg sample, extracting and purifying according to the method in the step (1), adding different amounts of doxycycline standard solutions, and preparing into series of standard working solutions with different concentration gradients;
(3) measurement and calculation of results
Performing LC-MS/MS measurement on the standard working solution with each concentration gradient in the step (2), and performing regression analysis on the corresponding concentration of the standard working solution according to the chromatographic peak area of the standard working solution to obtain a standard working curve; injecting the sample solution to be tested in the step (1) into LC-MS/MS for determination under the same conditions, measuring the chromatographic peak area of doxycycline in the sample solution to be tested, substituting the chromatographic peak area into a standard working curve to obtain the content of doxycycline in the sample solution to be tested,
the LC-MS/MS liquid chromatography conditions are as follows: the chromatographic column is C18; the sample amount is 5-20 μ L; the flow rate is 0.1-0.5 mL/min; gradient elution is carried out on a mobile phase A which is 0.05-0.5% formic acid aqueous solution and a mobile phase B which is acetonitrile solution;
the mass spectrum conditions of the LC-MS/MS are as follows: using ESI ion source, positive ion (ES +) mode scan, multiple ion reaction monitoring (MRM) mode measurement, the mass to charge ratio of doxycycline parent ion was 445.0, the mass to charge ratios of daughter ions detected by secondary mass spectrometry were 154 and 428, and the corresponding collision voltages were 37eV and 29eV, respectively.
2. The LC-MS method of determining doxycycline residues in chicken eggs of claim 1, wherein the gradient elution procedure is:
。
3. The LC-MS method for determining doxycycline residues in eggs of claim 1, wherein the operating parameters of the mass spectrum are: desolventizing temperature is 500 ℃; desolventizing agent flow rate, 800L/hr; cone voltage, 27V; cone orifice gas flow rate, 50L/hr; capillary voltage, 3.50 kV; source temperature, 150 ℃; the secondary taper hole extraction voltage is 3.0V; collision cell inlet voltage, 0.5V; collision cell exit voltage, 0.5V.
4. The LC-MS method for determining doxycycline residues in eggs of claim 1, wherein the concentration gradient of the series of standard working solutions is: 0.5, 1, 2, 5 and 10 mu g/kg.
5. The LC-MS assay of doxycycline residues in eggs of claim 1, wherein: the temperature of the centrifugation in the step (1) is 4 ℃, and the speed is 10000 r/min.
6. The LC-MS assay of doxycycline residues in eggs of claim 1, wherein: the aperture of the organic microporous filter membrane in the step (1) is 0.22 μm.
7. The LC-MS assay of doxycycline residues in eggs of claim 1, wherein: the sample injection amount in the step (3) is 10 mu L; the flow rate was 0.2 mL/min.
8. The LC-MS assay of doxycycline residues in eggs of claim 1, wherein: the Column was a Waters acquisition uplcBEH C18Column (2.1X 50mm, 2.5 μm).
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