National Standards of the People's Republic of China
gb 31658.20-2022
National standards for food safety
Determination of residues of amide alcohols and their metabolites in animal foods
Liquid chromatographyTandem mass spectrometry
national food safety standard-
determination of amphenicols and metabolite residues in animal
derived food by liquid chromatography-tandem mass spectrometry method
Scope
This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry determination method for the determination of amide alcohol drugs and their metabolites residues in animal foods.
This document applies to the determination of chloramphenicol, thionomycin, florfenicol and florfenicol amide residues in the muscle, liver, kidney, adipose tissue of pigs, chickens, cattle and sheep, as well as eggs, milk and goat's milk.
Normative references
The content of the following documents constitutes an essential provision of this document by means of normative references in the text. Among them, the reference document with a date is noted, and only the version corresponding to the date applies to this document; For undated references, the most recent version of the document (including all change orders) applies to this document.
GB T 6682 Analytical Laboratory Water Specifications and Test Methods.
Terms and Definitions
There are no terms and definitions that need to be defined in this document.
Principle
The residual chloramphenicol, thionomycin, florfenicol and florfenicol amide in the sample were extracted with 2% ammoniated ethyl acetate solution, n-hexane debinding, ammoniated ethyl acetate reverse extraction, determined by liquid chromatography-tandem mass spectrometry, and quantified by internal standard method.
Reagents and materials
Unless otherwise specified, all reagents are analytically pure, and the water is grade 1 water in accordance with GB T 6682.
5.1 reagent.
5.1.1 Methanol (CH3OH): chromatographically pure.
5. 1.2 Acetonitrile (CH3CN): chromatographically pure.
5. 1.3 Ammonia (NH3·H2O).
5. 1.4 Ethyl acetate (C4H8O2).
5.1.5 Anhydrous sodium sulfate (Na2SO4).
5.1.6 Sodium chloride (NaCl).
5.1.7 ammonium formate (HCOONH4).
5.1.8 n-hexane (C6H14).
5.2. Solution preparation.
5.2.1 2% ammoniated ethyl acetate solution: take 20 ml of ammonia and dilute it with ethyl acetate to 1 000 ml.
5.2.2 4% sodium chloride solution: take 4 g of sodium chloride, dissolve it in water and dilute it to 100 ml.
5.2.3 4% sodium chloride saturated n-hexane: take an appropriate amount of 4% sodium chloride solution, add excess n-hexane, mix, stand and stratify, and take the upper layer of n-hexane.
5.2.4 20% methanol solution: take 20 ml of methanol and dilute it to 100 ml with water.
5.2.5 10 mmol l ammonium formate solution: take ammonium formate 063 g, dissolved in water and diluted to 1 000 ml.
5.3 Standards.
Chloramphenicol, thionomycin, florfenicol and florfenicol amide were all 99%, and chloramphenicol-d5, thiomycin-d3, florfenicol-d3, and florfenicol amide-d3 were all 98%, as detailed in Appendix A.
5.4. Preparation of standard solutions.
5.4.1. Standard stock solution: take an appropriate amount of chloramphenicol, thionomycin, florfenicol and florfenicol amide standards (equivalent to about 10mg of each active ingredient), weigh them accurately, add an appropriate amount of methanol to dissolve and dilute them into a 100ml volumetric flask, and prepare a standard stock solution with a concentration of 100 ml for a concentration of 100 ml. -18 The following will be kept for a period of 12 months.
5.4.2. Internal standard stock solution: take an appropriate amount of chloramphenicol-d5, thionomycin-d3, florfenicol-d3, and florfenicolamide-d3 internal standard (equivalent to about 1 mg of each active ingredient), weigh it accurately, add an appropriate amount of methanol to dissolve and dilute it into a 10 ml volumetric flask, and prepare an internal standard stock solution with a concentration of 100 g ml. -18 The following will be kept for a period of 12 months.
5.4.3. Mix the standard intermediate solution: 01ml, 0 each of the standard stock solutions of methammycin, florfenicol and florfenicolamide5 ml in a 10 ml volumetric flask, diluted to the scale with methanol, prepared a mixed standard intermediate solution with a concentration of 1 g ml of chloramphenicol and a concentration of 5 g ml of thionycin, florfenicol and florfenicolamide. -18 The following will be stored for 3 months.
5.4.4. Mix the intermediate solution of the internal standard: 01 ml, 0 each of methammycin-d3, florfenicol-d3, and florfenicolamide-d3 internal standard stocks5 ml, diluted to the scale with methanol in a 10 ml volumetric flask, prepared with chloramphenicol-d5 at a concentration of 1 g ml, and thionomycin-d3, florfenicol-d3, and florfenicomade-d3 at a concentration of 5 g ml mixed internal standard intermediate. -18 The following will be stored for 3 months.
5.4.5. Mixed internal standard working solution: take the mixed internal standard intermediate solution and dilute it with 20% methanol solution into chloramphenicol-d5 with a concentration of 10 ng ml, and sulfomycin-d3, florfenicol-d3, and florfenicolamide with a concentration of 50 ng ml mixed internal standard working solution, which is now ready for use.
5.5 Materials.
Nylon microporous membrane:m
Instruments and equipment
6.1 Liquid Chromatography-Tandem Mass Spectrometer: Power Distribution Spray Ion Source (ESI).
6.2 Balance: Inductance 00001 g and 001 g。
6.3 Homogenizer.
6.4. Vortex mixer.
6.5. Multi-tube vortex oscillator.
6.6 High-speed refrigerated centrifuge: up to 8 000 r min.
6.7. Nitrogen blowing instrument.
Preparation and preservation of specimens
7.1. Preparation of the sample.
7.1.1Muscle, liver, kidney, and adipose tissue.
Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and homogenize it.
a) Take the homogenized test sample as the test sample;
b) Take a blank sample after homogenization as a blank sample;
c) Take the homogenized blank sample, add the standard working solution of appropriate concentration, and use it as the blank sample.
7.1.2 milk.
Take an appropriate amount of fresh or thawed blank or test milk or goat's milk and mix well.
a) Take the test sample after mixing evenly as the test sample;
b) take a blank sample after mixing uniformly, as a blank sample;
c) Take the homogenized blank sample, add the standard working solution of appropriate concentration, and use it as the blank sample.
7.1.3 eggs.
Take an appropriate amount of fresh or chilled blank or test eggs, remove the shell, and homogenize.
a) Take the homogenized test sample as the test sample;
b) Take a blank sample after homogenization as a blank sample;
c) Take the homogenized blank sample, add the standard working solution of appropriate concentration, and use it as the blank sample.
7.2. Preservation of the sample.
18 Save below.
Assay steps
8.1 Extraction.
Take 2 g of sample (accurate to 0.).05 g), placed in a 50 ml centrifuge tube, added 100 l of mixed internal standard working solution, vortex to mix well, and then added 10 ml of 2% ammoniated ethyl acetate solution (3 g of anhydrous sodium sulfate should be added to milk and goat milk samples), vortex for 30 s, vortex for 10 min, and centrifugation at 8 000 r min for 5 min. Transfer the supernatant to another 50 ml centrifuge tube, add 10 ml of 2% ammoniated ethyl acetate solution to the residue, and repeat the extraction once. The two extracts were combined, dried in 50C nitrogen, and purified.
8.2 Purification.
Take the residue to be purified, add 3ml of 4% sodium chloride solution, vortex to dissolve, then add 5ml of n-hexane saturated with 4% sodium chloride, vortex for 30s, centrifuge at 8 000 r min for 5 min, discard the upper n-hexane layer, and repeat the degreasing with n-hexane saturated with 4% sodium chloride. Add 5 ml of 2% ammoniated ethyl acetate solution, vortex for 5 min, centrifuge at 8 000 r min for 5 min, and take the upper organic phase. Repeat the extraction once with 5 ml of 2% ammoniated ethyl acetate solution, combine the organic phases, blow dry with 50 nitrogen, and add 20% methanol solution 10 ml, vortex for 30 s, over 022 m filter membrane for liquid chromatography tandem mass spectrometry.
8.3. Preparation of standard curves.
Accurately take an appropriate amount of mixed standard intermediate solution and mixed internal standard working solution, dilute with 20% methanol solution, and prepare chloramphenicol at a concentration of 02 μg/l、0.The concentrations of 5 g l, 1 g l, 2 g l, 5 g l, 10 g l, thionomycin, florfenicol and florfenicol were 1 g l and 2, respectively5 g L, 5 g L, 10 g L, 25 g L, 50 g L, chloramphenicol-D5 concentration is 1 g L, thionomycin-D3, florfenicol-D3, florfenicol amide-D3 concentration is 5 g L, ready for liquid chromatography tandem mass spectrometry determination. The standard curve was plotted with the quantitative ion peak area ratio as the ordinate and the concentration as the abscissa, and the regression equation and correlation coefficient were obtained.
8.4 Assay.
8.4.1 Liquid chromatography reference conditions.
a) Column:c18Columns (100 mm2.1 mmm, or equivalent;
b) Column temperature: 30;
c) Injection volume: 5 l;
d) Flow rate: 03 ml/min；
e) Mobile phase: a is 10 mmol l ammonium formate solution; b is acetonitrile; The gradient elution procedure is shown in Table 1.
8.4.2Mass spectrometry reference conditions.
a) Ion source: electrospray ion source;
b) Scanning method: positive ion scanning, negative ion scanning;
c) Detection method: multiple reactive ion monitoring (MRM);
d) Desolvation gas, cone gas and collision gas are all high-purity nitrogen or other suitable gases;
e) Parameters such as spray voltage and collision energy should be optimized to the optimal sensitivity;
f) The reference values of analyte ion source, qualitative ion pair, quantitative ion pair, cone voltage and collision energy are shown in Table 2.
8.4.3 Assay.
8.4.3.1. Qualitative determination.
Under the same test conditions, the relative deviation between the retention time of amide alcohols and their metabolites in the sample solution and the retention time of amide alcohols and their metabolites in the standard working solution was 25% and the relative ion abundance detected should be consistent with the relative ion abundance of a calibration standard solution of comparable concentrations. The allowable deviation shall comply with the requirements of Table 3.
8.4.3.2 Quantitative determination.
The sample solution and the corresponding standard solution were calibrated at a single point or multiple points, and quantified by chromatographic peak area ratio according to the internal standard method. The ratio of amide alcohols and their metabolites to their corresponding internal standard peak areas in the standard solution and sample solution should be within the linear range of the instrument. For the residue beyond the linear range of the instrument, the amount of internal standard working solution added is increased accordingly according to the drug concentration during extraction, so that the concentration of amide alcohols and their metabolites after dilution of the sample solution is within the range of the curve, and the corresponding internal standard concentration is consistent with that of the standard working solution. The characteristic ion mass chromatograms of the standard solutions are shown in Appendix B.
8.5 Blank test.
Take a blank sample, except for the addition of no drug, use the exact same measurement steps for determination.
Calculation and presentation of results
The residues of amide alcohols and their metabolites in the sample are calculated according to the standard curve or formula (1).
Where: x - the value of the residue of amide alcohol drugs and their metabolites in the sample, in micrograms per kilogram (g kg);
CIS - the value of the internal standard concentration of amide alcohols and their metabolites in the sample solution, in micrograms per liter (g l);
CS - the concentration of amide alcohols and their metabolites in a standard solution in micrograms per liter (g L);
c'is - the value of the internal standard concentration of amide alcohols and their metabolites in standard solution, in micrograms per liter (g l);
AI - the peak area of amide alcohols and their metabolites in the sample solution;
AIS——the peak area of the internal standard of amide alcohols and their metabolites in the sample solution;
AS - the peak area of amide alcohols and their metabolites in standard solution;
a'is - the peak area of the internal standard of amide alcohols and their metabolites in the standard solution;
v - the value of the volume of 20% methanol solution of the dissolved residue in milliliters (ml);
m – the numerical value of the mass of the specimen in grams (g).
Method sensitivity, accuracy, and precision
10.1Sensitivity.
The detection limit of chloramphenicol in this method is 01 g kg, the limit of quantification is 02 μg/kg；The limit of detection of thionomycin, florfenicol, and florfenicol amide is 05 g kg, the limit of quantification is 1 g kg.
10.2 Accuracy.
This method chloramphenicol is at 02 g kg 1 g kg addition concentration level, thionomycin at 1 g kg 100 g kg addition concentration level, florfenicol and florfenicol amide at 1 g kg 6 000 g kg addition concentration level ** rate were 70% 120%.
10.3 Precision.
The relative standard deviation of this method is 15% within the assay and 20% the relative standard deviation between assays.
*From: