CN112858503A

CN112858503A - Method for measuring residual quantity of 10 aminoglycoside antibiotics in eggs

Info

Publication number: CN112858503A
Application number: CN202110021917.XA
Authority: CN
Inventors: 魏莉莉; 薛霞; 刘艳明; 卢兰香; 武传香; 丁一
Original assignee: Shandong Institute for Food and Drug Control
Current assignee: Shandong Institute for Food and Drug Control
Priority date: 2021-01-08
Filing date: 2021-01-08
Publication date: 2021-05-28

Abstract

The invention relates to a method for measuring the residual quantity of 10 aminoglycoside antibiotics in eggs, belonging to the technical field of trace veterinary drug residue detection. The determination method comprises the following specific steps: sample pretreatment, preparing a standard stock solution, a mixed standard intermediate solution and a matrix matching working solution, determining by using the matrix matching standard working solution through a hydrophilic interaction chromatography-triple quadrupole mass spectrometry method, drawing a standard curve, detecting the solution to be detected according to the same instrument parameters to obtain a mass chromatogram of 10 aminoglycosides in a sample, and then carrying out qualitative and quantitative analysis. The invention avoids the pollution of ions to the reagent on the chromatogram and the mass spectrum: the detection method provided by the invention avoids the pollution of ions such as heptafluorobutyric acid and sodium heptanesulfonate on the chromatographic column and the mass spectrum detector; the pretreatment operation is simple, the purification effect is good, the sensitivity is higher, the detection limit and the quantification limit are greatly reduced, and the limit requirement of national standards on aminoglycosides in eggs is completely met.

Description

Method for measuring residual quantity of 10 aminoglycoside antibiotics in eggs

Technical Field

The invention relates to a method for measuring the residual quantity of 10 aminoglycoside antibiotics in eggs, belonging to the technical field of trace veterinary drug residue detection.

Background

The aminoglycoside is a broad-spectrum antibiotic, has obvious antibacterial effect on gram-positive bacteria and gram-negative bacteria, can effectively inhibit the growth and reproduction of bacteria, and has wide application in animal husbandry and breeding industry. The aminoglycoside can be used for treating animal diseases, and can be used as feed additive for promoting animal growth. Research shows that the aminoglycoside medicine has certain ototoxicity, nephrotoxicity and neuromuscular blocking effect.

In order to guarantee food safety, the maximum residual limit of spectinomycin, neomycin and hygromycin in eggs is respectively 2000 mug/kg and 500 mug/kg and cannot be detected in GB 31650 plus 2019 'maximum residual limit of veterinary drugs in national standard food for food safety', and apramycin and kanamycin are forbidden in the egg producing period. At present, most of substrates for detecting aminoglycosides in documents are honey, dairy products, meat products, aquatic products and the like, and few reports are made on detection of aminoglycoside drugs in eggs. The rapid and accurate determination method for the residual quantity of the aminoglycoside drugs in the eggs is established, and has important significance for maintaining the human diet health.

Aminoglycoside antibiotics are strong polar basic compounds, and are more challenging to detect compared with other veterinary drugs. The currently reported detection methods of aminoglycoside antibiotics mainly comprise an enzyme-linked immunosorbent assay, a gas chromatography, a liquid chromatography and a liquid chromatography-tandem mass spectrometry. The enzyme-linked immunosorbent assay is relatively simple in operation, but cannot be accurately characterized, and is often used as a screening method. The gas chromatography requires derivatization and the steps are complicated; the liquid chromatography needs a general detector, is easily interfered by matrix and has high detection limit. The liquid chromatography-tandem mass spectrometry has the advantages of strong matrix interference resistance, high sensitivity and the like, and is most widely applied to the detection of aminoglycoside drugs. GB/T21323 and 2007 'determination of residual quantity of aminoglycoside drugs in animal tissues high performance liquid chromatography-mass spectrometry/mass spectrometry' stipulates reversed phase ion pair chromatography-tandem mass spectrometry of residual quantity of 10 aminoglycoside drugs in animal viscera, muscle and aquatic products, but the used ion pair reagent of heptafluorobutyric acid pollutes a mass spectrometry detector to cause ion inhibition. Therefore, further improvements and developments are needed in the existing detection technology.

Disclosure of Invention

Aiming at the problems of ion pair chromatography-mass spectrometry detection, pollution of a mass spectrometry detector and the vacancy of a pretreatment technology of aminoglycoside medicines in eggs in the prior art, the invention provides a method for measuring the residual quantity of 10 aminoglycoside medicines in eggs, and the rapid, simple and high-sensitivity quantitative detection is realized by establishing a novel pretreatment method and a hydrophilic interaction chromatography-tandem mass spectrometry detection instrument method.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the invention provides a method for measuring the residual quantity of 10 aminoglycoside drugs in eggs, which comprises the following steps:

(1) sample pretreatment: weighing a sample, adding the sample into an extracting solution, mixing in a vortex manner, carrying out ultrasonic extraction, taking out, centrifuging, transferring supernatant, adding the extracting solution into residues, repeatedly extracting for one time, combining the two supernatants, adjusting the pH value, fixing the volume, shaking up, filtering, obtaining filtrate for later use, and purifying to obtain a solution to be detected;

(2) standard stock solutions and mixed standard intermediate solutions: accurately weighing appropriate amount of 10 aminoglycoside antibiotics standard substances, dissolving with water, fixing volume to scale, preparing into standard stock solution with concentration of 1 mg/mL, and storing at 4 deg.C in dark place; the standard stock solution was diluted stepwise with water to make mixed standard intermediate solutions of 10. mu.g/mL, 1. mu.g/mL and 0.1. mu.g/mL.

(3) Matrix matching standard working solution: transferring the standard intermediate solution, and diluting with egg treating solution without the substance to be detected into matrix matching standard working solutions with concentrations of 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL respectively;

(4) analyzing and detecting a sample: the method comprises the steps of determining a matrix matching standard working solution by using a hydrophilic interaction chromatography-triple quadrupole mass spectrometry method, drawing a standard curve, detecting a solution to be detected according to the same instrument parameters to obtain a mass chromatogram of 10 aminoglycosides in a sample, carrying out qualitative analysis on a target object by using retention time and ion ratio, and calculating the concentration of the object to be detected by using the peak area and the linear regression equation of the mass chromatogram.

The specific method for sample pretreatment provided by the invention comprises the following steps: weighing 2.5 g of sample in a 50 mL centrifuge tube, adding 10 mL of extracting solution, uniformly mixing by vortex, carrying out ultrasonic treatment for 15 min, centrifuging for 5 min at 8000 r/min, transferring supernatant into another 50 mL centrifuge tube, repeatedly extracting residue once by using 10 mL of extracting solution, combining the two supernatants, adjusting pH to 6.5 by using 10 mol/L sodium hydroxide solution, and adding water to constant volume to 25 mL. Filtering, collecting filtrate, passing 20 mL of the filtrate through an activated PRIME HLB solid-phase extraction column, discarding the filtrate, rinsing twice with 3mL of ultrapure water, rinsing once with 3mL of 5% methanol water, draining, accurately adding 2.0 mL of eluent for elution, collecting the eluent, performing vortex mixing, and passing through an organic filter membrane of 0.22 mu m to obtain the solution to be detected.

The above extractive solution is 10 mmol/L ammonium acetate solution containing 0.4 mmol/L EDTA and 50 g/L trichloroacetic acid.

Further, the PRIME HLB solid phase extraction column was activated with 3mL of methanol followed by 3mL of water.

Further, the eluent is composed of formic acid, acetonitrile and water according to the volume ratio of 10:5: 85.

The 10 aminoglycoside antibiotics detected by the invention are streptomycin, dihydrostreptomycin, hygromycin B, kanamycin, amikacin, tobramycin, apramycin, spectinomycin, neomycin and gentamycin.

Further, in the step (4), in the hydrophilic interaction chromatography-triple quadrupole mass spectrometry, the conditions of the hydrophilic interaction chromatography are as follows: a chromatographic column: SIELC Obelisc R, column length 150 mm, column inner diameter 2.1mm, filler particle size 5 μm; mobile phase A: acetonitrile; mobile phase B: 1% formic acid (containing 1 mmol/L ammonium formate) solution; sample introduction volume: 5 mu L of the solution; column temperature: 40 ℃; and (3) an elution mode: gradient elution.

The conditions of the mass spectrometry are as follows: an ion source: electrospray ion source (ESI source); the detection mode is as follows: multiple Reaction Monitoring (MRM); the scanning mode is as follows: scanning in a positive ion mode; flow rate of atomizing gas: 3L/min; heating air flow: 10L/min; interface temperature: 300^oC; temperature of DL tube: 250^oC; temperature of the heating block: 400^oC; flow rate of drying gas: 10L/min.

Further, in the step (4), the drawing of the standard curve specifically includes: and detecting the prepared standard working solutions with the concentrations of 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL respectively by using a high performance liquid chromatography-tandem mass spectrometer to obtain a mass chromatogram of the standard solution, and drawing a standard curve by taking the peak area as a vertical coordinate and the concentration of the standard solution as a horizontal coordinate to obtain a linear regression equation.

The invention has the beneficial effects that:

(1) the pollution of ions to the reagent on the chromatogram and the mass spectrum is avoided: the detection method provided by the invention avoids the pollution of ions such as heptafluorobutyric acid and sodium heptanesulfonate on the chromatographic column and the mass spectrum detector.

(2) The pretreatment operation is simple, the purification effect is good: the method comprises the steps of purifying the egg sample by using a PRIME HLB solid-phase extraction column for the first time, cooperating with a specific pretreatment process, and having simple operation and high accuracy.

(3) The sensitivity is higher: the invention adopts a hydrophilic interaction chromatographic column to separate and analyze 10 aminoglycoside drugs in eggs, and takes a high-concentration organic phase as a mobile phase, thereby being beneficial to improving the ionization efficiency of mass spectrometry and further improving the sensitivity of mass spectrometry. The national standard detection limit range of the aminoglycoside drug in the existing animal derived food is 20-100 mug/kg; the detection limit is 2-5 mug/kg, the quantitative limit is 5-10 mug/kg, and the limit requirement of national standards on the quantity of aminoglycoside in eggs is completely met.

Drawings

FIG. 1 is an MRM chromatogram of a blank egg sample.

FIG. 2 is a MRM graph of a blank egg spiked sample at the limit of quantitation (LOQ) level.

FIG. 3 is a diagram showing the separation of 10 aminoglycosides on the column in example 1.

FIG. 4 is a graph showing the separation of 10 aminoglycoside compounds on a BEH HILIC column.

FIG. 5 is a diagram showing the separation of 10 aminoglycoside compounds on a BEH Amide column.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the following explains and explains the present invention with reference to specific embodiments.

Examples

1. Apparatus and materials

Shimadzu LC/MS-8050 ultra high performance liquid chromatography-tandem mass spectrometer (equipped with electrospray ionization source); 3-18K table high speed centrifuge; BT 125D electronic balance; a vortex mixer model MS 3; SB-800DTD ultrasonic cleaning instrument; Milli-Q ultrapure water systems.

Streptomycin, dihydrostreptomycin, hygromycin B, kanamycin, amikacin, tobramycin, apramycin, spectinomycin, neomycin and gentamycin standards; methanol; acetonitrile; formic acid; ammonium formate; trichloroacetic acid; disodium ethylene diamine tetraacetate; hydrochloric acid; sodium hydroxide; ammonia water; ammonium acetate; prime HLB solid phase extraction column (200 mg, 6 mL); obelisc R column (150X 2.1mm, 5 μm).

2. Pretreatment

2.1 Standard stock solution and Mixed Standard intermediate solution

Accurately weighing streptomycin, dihydrostreptomycin, hygromycin B, kanamycin, amikacin, tobramycin, apramycin, spectinomycin, neomycin and gentamycin standards which are equivalent to 10 mg (accurate to 0.01 mg) in a conversion manner, respectively placing the standards in a 10 mL volumetric flask, dissolving the standards with water, fixing the volume to a scale, preparing a standard stock solution with the concentration of 1 mg/mL, and storing the standard stock solution at 4 ℃ in a dark place.

The standard stock solution was diluted stepwise with water to make mixed standard intermediate solutions of 10. mu.g/mL, 1. mu.g/mL and 0.1. mu.g/mL.

2.2 matrix matching working solutions

In order to eliminate the influence of matrix effect, when an ultra-high performance liquid chromatography-tandem mass spectrometer is adopted for detection, a matrix matching curve is usually adopted for quantification, namely, a blank sample treatment solution is adopted to dilute a standard intermediate solution, so that a matrix matching working curve is obtained. And (4) transferring a proper amount of standard intermediate solution, and diluting the standard intermediate solution into matrix matching standard working solutions with the concentrations of 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL respectively by using the egg treatment solution without the substance to be detected.

2.3 sample pretreatment

Extraction: accurately weighing 2.5 g of sample (accurate to 0.01 g) into a 50 mL centrifuge tube, adding 10 mL10 mmol/L ammonium acetate buffer solution (containing 0.4 mmol/L EDTA and 50 g/L trichloroacetic acid), mixing by vortex, performing ultrasonic treatment for 15 min, centrifuging for 5 min at 8000 r/min, transferring the supernatant into another 50 mL centrifuge tube, extracting the residue once again with 10 mL extracting solution, combining the supernatants, adjusting pH to 6.5 with 10 mol/L sodium hydroxide solution, and adding water to reach a constant volume of 25 mL. Filtering and collecting filtrate.

Purifying: the Prime HLB solid phase extraction column was activated with 3mL of methanol followed by 3mL of water. Accurately transferring 20 mL of supernatant, passing through a solid phase extraction column at a flow rate of not more than 1 mL/min, and discarding the filtrate; leaching twice with 3mL of ultrapure water, leaching once with 3mL of methanol-water (5: 95, v/v), discarding the leaching solution, and draining the small column; eluting with 2 mL formic acid-acetonitrile-water (10: 5:85, v/v/v) solution, collecting eluate, draining off solid phase extraction column, mixing, filtering with microporous membrane, and analyzing and determining with HPLC-tandem mass spectrometer.

3. Liquid chromatography and mass spectrometry conditions

3.1 chromatographic conditions: a chromatographic column: SIELC Obelisc R, column length 150 mm, column inner diameter 2.1mm, filler particle size 5 μm; mobile phase A: acetonitrile; mobile phase B: 1% formic acid (containing 1 mmol/L ammonium formate) solution; sample introduction volume: 5 mu L of the solution; column temperature: 40 ℃; and (3) an elution mode: gradient elution, the elution conditions are shown in table 1.

TABLE 1 gradient elution conditions

3.2 Mass Spectrometry conditions: an ion source: electrospray ion source (ESI source); the detection mode is as follows: multiple Reaction Monitoring (MRM); the scanning mode is as follows: scanning in a positive ion mode; flow rate of atomizing gas: 3L/min; heating air flow: 10L/min; interface temperature: 300^oC; temperature of DL tube: 250^oC; temperature of the heating block: 400^oC; flow rate of drying gas: 10L/min, other mass spectral parameters are shown in Table 2.

TABLE 2 Mass Spectrometry parameters

4. Analysis of results

4.1 Linear Range and Standard Curve of the method

And (3) detecting the matrix matching standard working solution (the concentration is respectively 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL) prepared in the step (2.2) by using an ultra-high performance liquid chromatography-tandem mass spectrometer to obtain a mass chromatogram of the standard solution. The peak area was taken as the ordinate and the concentration of the standard solution as the abscissa, and a standard curve was plotted to obtain a linear regression equation, as shown in table 3. As can be seen from Table 3, the peak areas and the concentrations of the 10 aminoglycosides are in good linear relation within the range of 5-200 ng/mL, and the linear correlation coefficients are all larger than 0.99.

TABLE 3 Standard Curve for aminoglycosides

4.2 Process recovery and precision

And (3) respectively adding standard solutions with low, medium and high concentration levels into the blank egg sample, performing 6 parallel experiments for each level, and detecting according to the steps 2 and 3 to obtain the quality chromatograms of the sample and the labeled sample. Wherein, FIG. 1 is the quality chromatogram of the blank egg, and FIG. 2 is the MRM chart of the blank egg labeled sample with the limit level of quantification. The quantification was performed according to step 4 to obtain the recovery and precision of 10 aminoglycosides, and the specific results are shown in Table 4. As can be seen from Table 4, the standard recovery rate of 10 aminoglycoside drugs in the egg sample is 68.1-111.3%, the RSD is 1.2-12.3%, and the accuracy and precision of the method meet the requirement of micro-analysis.

TABLE 4 recovery with addition of standards and precision experiments

4.3 detection and quantitation limits of the method

Adding 1 mug/kg, 2 mug/kg, 5 mug/kg and 10 mug/kg of standard substances into the blank egg sample respectively to obtainS/NNot less than 3, determining the detection limit of the method, so as toS/NAnd (4) determining the quantitative limit of the method, wherein the quantitative limit is more than or equal to 10. The detection limit of the neomycin is 5 mug/kg, the quantitative limit of the neomycin is 10 mug/kg, the detection limits of the rest 9 aminoglycoside compounds are 2 mug/kg, and the quantitative limit of the neomycin is 5 mug/kg.

Comparative example 1

The procedure was as in example 1 except that the extract was a 10 mmol/L ammonium acetate solution containing 50 g/L of trichloroacetic acid. The matrix effect after extraction and purification of 10 aminoglycosides in the comparative example and example was compared. The matrix effect calculation formula is as follows: (slope of substrate matching standard curve/slope of reagent standard curve-1). times.100. After the extract in the comparative example is added, the matrix effects of streptomycin, dihydrostreptomycin and hygromycin B are respectively-79%, -73% and-40%; the matrix effects of streptomycin, dihydrostreptomycin and hygromycin B after the extract liquid in the embodiment is added are respectively-0.3%, -12.5% and-12.1%, which shows that the addition of EDTA can reduce the matrix effects of streptomycin, dihydrostreptomycin and hygromycin B.

Comparative example 2

The procedure was as in example 1 except that the extract was acetonitrile. After the addition of the extract of comparative example, no detection was observed for 10 aminoglycoside compounds.

Comparative example 3

The procedure was as in example 1 except that the column used was BEH HILIC (100 mm. times.2.1 mm, 1.7 μm, Waters). The separation of the 10 aminoglycoside compounds on the column is shown in FIG. 3 in the examples and in the comparative examples in FIG. 4. The aminoglycoside compound has poor peak shape on the chromatographic column in the comparative example, and only spectinomycin, streptomycin, dihydrostreptomycin and hygromycin B have obvious response.

Comparative example 4

The procedure was as in example 1 except that the column used was BEH Amide (100 mm. times.2.1 mm, 1.7 μm, Waters corporation). The separation of the 10 aminoglycoside compounds on the column in the comparative example is shown in FIG. 5. The 10 aminoglycoside compounds all have response, but the separation effect is poor, and the matrix effect is large in the detection of actual samples.

Claims

1. A method for measuring the residual quantity of 10 aminoglycoside drugs in eggs is characterized by comprising the following steps:

2. The measurement method according to claim 1, wherein in the step (1), the specific method of the sample pretreatment is: weighing 2.5 g of sample in a 50 mL centrifuge tube, adding 10 mL of extracting solution, uniformly mixing by vortex, carrying out ultrasonic treatment for 15 min, centrifuging for 5 min at 8000 r/min, transferring supernatant into another 50 mL centrifuge tube, repeatedly extracting residue once by using 10 mL of extracting solution, combining the two supernatants, adjusting pH to 6.5 by using 10 mol/L sodium hydroxide solution, and adding water to constant volume to 25 mL. Filtering, collecting filtrate, passing 20 mL of the filtrate through an activated PRIME HLB solid-phase extraction column, discarding the filtrate, rinsing twice with 3mL of ultrapure water, rinsing once with 3mL of 5% methanol water, draining, accurately adding 2.0 mL of eluent for elution, collecting the eluent, performing vortex mixing, and passing through an organic filter membrane of 0.22 mu m to obtain the solution to be detected.

3. The method according to claim 2, wherein the extract is a 10 mmol/L ammonium acetate solution containing 0.4 mmol/L EDTA and 50 g/L trichloroacetic acid.

4. The assay of claim 2, wherein the PRIME HLB solid phase extraction column is activated with 3mL of methanol followed by 3mL of water.

5. The method according to claim 2, wherein the eluent is composed of formic acid, acetonitrile and water at a volume ratio of 10:5: 85.

6. The method according to claim 1, wherein in step (2), the 10 aminoglycoside antibiotics are streptomycin, dihydrostreptomycin, hygromycin B, kanamycin, amikacin, tobramycin, apramycin, spectinomycin, neomycin, gentamycin.

7. The assay method according to any one of claims 1 to 6, wherein in step (4), in the hydrophilization chromatography-triple quadrupole mass spectrometry, the conditions of the hydrophilization chromatography are: a chromatographic column: SIELC Obelisc R, column length 150 mm, column inner diameter 2.1mm, filler particle size 5 μm; mobile phase A: acetonitrile; mobile phase B: 1% formic acid (containing 1 mmol/L ammonium formate) solution; sample introduction volume: 5 mu L of the solution; column temperature: 40 ℃; and (3) an elution mode: gradient elution.

8. The method of claim 7, wherein in step (4), the conditions of mass spectrometry are: an ion source: electrospray ion source (ESI source); the detection mode is as follows: multiple Reaction Monitoring (MRM); the scanning mode is as follows: scanning in a positive ion mode; flow rate of atomizing gas: 3L/min; heating air flow: 10L/min; interface temperature: 300^oC; temperature of DL tube: 250^oC; temperature of the heating block: 400^oC; flow rate of drying gas: 10L/min.

9. The assay according to any one of claims 1 to 8, wherein in step (4), the standard curve is drawn by: and detecting the prepared standard working solutions with the concentrations of 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL respectively by using a high performance liquid chromatography-tandem mass spectrometer to obtain a mass chromatogram of the standard solution, and drawing a standard curve by taking the peak area as a vertical coordinate and the concentration of the standard solution as a horizontal coordinate to obtain a linear regression equation.