AOAC Official Method_aoac23.1.17
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FA0EF27DEE21451D93BC60728FB4B223 |
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0.05 |
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4 |
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日期： |
2024-7-30 |
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23.1.17,AOAC Official Method 995.09,Chlortetracycline, Oxytetracycline,and Tetracycline in Edible Animal Tissues,Liquid Chromatographic Method,First Action 1995,Final Action 1999,(Applicable to determination of oxytetracycline and tetracycline in,bovine muscle tissues, chlortetracycline and oxytetracycline in porcine,muscle tissues, oxytetracycline and tetracycline in bovine kidney,tissues, and chlortetracycline and oxytetracycline in porcine,kidney tissues.),See Table 995.09 for the results of the interlaboratory study supporting,the acceptance of the method.,A. Principle,Tetracyclines are extracted from tissue with pH 4 buffer. Filtered,extract is cleaned up on C18 solid-phase extraction column.,Tetracyclines are separated by liquid chromatography using C8 column,and measured with UV detector at 350 nm. Results are corrected,for recovery for each analyte for each analytical run.,B. Apparatus,(a) Liquid chromatograph (LC).—With solvent delivery system,providing flow rate 1–2 mL/min with low pulsation using specified,LC column and mobile phase; UV detector at 350 nm,0.005 absorbance unit full scale (AUFS); selectable time constant;,manual injector or autosampler; strip chart recorder. Operating conditions:,injection volume 10–60 mL; time constant, 3.0; chart speed,0.5 cm/min; run time, 10 min.,(b) LC columns.—(1) For tetracyclines determination.—250 ′,4.6mmid, 5 mmC8 reverse phase deactivated silica packing (LC column,of the same physical dimensions containing similar 10 mmparticulate,material is suitable); flow rate, 1.2 mL/min for 5 mm LC,column and 2.0 mL/min for 10 mmLC column. (2) For confirmation,of quantitative results.—Another column, 250 ′ 4.6 mm id, 5 or,10 mm C18 reverse phase deactivated silica packing. (C8 column of,the same physical dimensions may be substituted if retention characteristics,are known to be different from those of the standard analytical,column [i.e., made by different manufacturer or with different,degree of deactivation].),(c) Balance.—Weighing to ±0.001 g.,(d) Büchner funnel.—5.5 cm.,(e) Centrifuge.—Holding 50mLpolypropylene tubes; providing,2500 ′ g.,(f) Centrifuge tubes.—50 mL, polypropylene, disposable.,(g) Automatic dispenser.—Graduated to deliver 2–10 mL.,(h) Filter paper.—Glass microfiber, grade GF/B, 5.5 cm.,(i) Erlenmeyer flask.—Sidearm, 125 mL.,(j) Volumetric flasks.—5, 10, 500, and 1000 mL.,(k) Homogenizer.—With cutting blades to disintegrate and,homogenize animal tissue. Recommended volume capacity of,probe, 2–500 mL.,(l) Mechanical shaker.—Flat bed, 2-speed, oscillating horizontally.,(m) pH Meter.—Measuring ±0.05 unit.,(n) Pipet.—Pasteur, disposable, 2 mL.,(o) Filtration cartridge.—To filter tissue supernates; 13 mm,id, 0.45 mm porosity, with Luer-lock.,(p) Solid-phase extraction (SPE) apparatus.—With vacuum,block with 10–12 ports, vacuum gauge, and 75 mL reservoirs.,(q) SPE cartridges.— 6 mL, 500 mg, C18 packing. Column,must meet system suitability requirements, D. Varian Bond Elutò,columns, or equivalent, have been found satisfactory.,(r) Vortex mixer.,(s) Vacuum pump.,C. Reagents,(a) McIlvaine buffer.—pH 4.0 ± 0.05. Place 28.4 g anhydrous,Na2HPO4 (reagent grade) into 1 L volumetric flask and dissolve in,distilledH2O. Dilute to volume withH2Oand mix. Place 21.0 g citric,acid monohydrate into another 1Lvolumetric flask and dilute to volume,with distilled H2O and mix well. Combine 1 L citric acid solution,with 625 mL Na2HPO4 solution in 2 L flask. Adjust pH to,4.0 ± 0.05 by adding dropwise either 0.1M HCl (8.3 mL HCl/L) or,0.1M NaOH (4.0 g/L). Prepare weekly.,(b) McIlvaine buffer–EDTA solution.—Adjust McIlvaine buffer,(a), to contain 0.1M disodium ethylenediamine–tetracetate as follows:,To 1.625 L McIlvaine buffer, add 60.5 g disodium EDTA,dihydrate and mix until dissolved. Prepare weekly.,(c) Methanolic oxalic acid.—Dissolve 1.26 g oxalic acid,dihydrate (reagent grade) in methanol (LC grade) in 1 L volumetric,flask. Dilute to volume with methanol and mix. Prepare daily.,(d) Tetracyclines (TC) analytical standards.—USP reference,standards of chlortetracycline hydrochloride, oxytetracycline hydrochloride,and tetracycline hydrochloride. (1) TC stock standard,solutions.—1000 mg/mL. Weigh 108 ± 0.1 mg each tetracycline,standard into separate weighing dishes (weights corrected for assayed,content) and transfer with methanol into separate 100 mL volumetric,flasks. Dilute to volume with methanol at room temperature,and mix until dissolved. Prepare TC stock standard solution every 3,months and store at –20°C. (2) TC combined stock standard solution.—,100mg/mL. Pipet 10mLeachTCstock standard solution into,one 100 mL volumetric flask, dilu……
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