This standard specifies the method for determining the residual amount of tetracycline antibiotics in honey by the microbial tube plate method. This standard is applicable to the determination of the residual amount of tetracycline antibiotics in natural or processed honey. GB/T 5009.95-2003 Determination of the residual amount of tetracycline antibiotics in honey GB/T5009.95-2003 Standard download decompression password: www.bzxz.net

ICS 67.040
National Standard of the People's Republic of China
GB/T5009.95—2003
! 151
Determination of tetracycline antibiotics residues in honey
Determination of tetracycline antibiotics residues in honey2003-08-11Promulgated
Ministry of Health of the People's Republic of China
Standardization Administration of China
2004-01-01Implementation
G3/T5009.952033
This standard replaces GB/T13110—1 Method for determination of content or dosage. Compared with GB/13:10-1991, the main revisions of this standard are as follows: the Chinese name of the standard has been revised, the non-Chinese name has been changed to GB/2CV.4-252, the structure of the original standard has been revised, and this standard is issued by the People's Republic of China and is under the jurisdiction of the Ministry of Health. The original standard was first issued in 1991. This revision specifies the method for determining the residual tetracyclic antibiotics in honey by the biological plate method. The technical standard is suitable for the determination of the cumulative residues of tetracyclic antibiotics in natural or screened honey. 2 Management
GB/T5009.95—2003
The sample was extracted with Me1-uin buffer and then purified by SFEF-PAKC. The tetracycline antibiotics were used as tetracycline and the mycobacterium was separated and identified by thin layer chromatography biodetection method; Bacillus sp. was used as test powder and the microbial identification method was used.
3 Reagents
3.1Mcllva:ne filtration (pF14): Take sodium phosphate (NuHPO12EI, ()) 27.65, citrate (CHaH2O) 12.0R. Disodium tetraethyl ether 37.2k. Dissolve in water and make up to 1000L, 3.20.1mul/1. Phosphate filtration (pII4.E): Weigh potassium dihydrogen phosphate 13.e dilute with water to 100(ml11 sterilize 2 times 4% Store in refrigerator
3.3 Disodium ethylenediaminetetraacetic acid water solution (50g11.3.4 Wa: UrsSF.P-PAKCa format (made in China PT-CL) first pass through 10mL of neutral alcohol, then connect with 10n:L distilled water, then pass through 1cmL.50g:L disodium ethylenediaminetetraacetic acid, 3.5 Antibiotics: Chinese ginseng, ten toxins, Huiguanhao standard products (provided by the Institute of Drug and Biological Products of the Ministry of Health) 3.6 Standard drop solution
3.6.1 Preparation of antibiotic standard stock solution: weigh four cyclohexane and seven cyclohexane products (converted according to potency), dissolve in 1.01 nmol/mL 4% ethanol and store at 1000 mg/ml. Store in a refrigerator at 4°C [can be used for 7 days]. 3.6.2 Preparation of antibiotic standard solution: prepare the standard solution by adding 0.mol/mL phosphate buffer solution 1 month before the preparation of the standard solution. The standard concentrations of the standard solution of cyclohexane and cyclohexane are 0.16, 0.21, 0.26, and 0.32. , 0.41: n.5m/ml. The concentration of the gold standard curve is 0.33.3.0.1.0.051.0.064.0.080.0.133ug/ml. The liquid base is C.c53mg/mL. The standard for qualitative test is cyclohexane, and the soil needs to be 2g/m.
3.7 Development vehicle: n-butyl-water (1+15), 4 receivers
4.1 Water-limited constant temperature pen.
4.2 4.3 Relative saline solution. 4.4 High pressure sterilizer 4.5 Rotary vacuum evaporator 4.6 Two-speed evaporator 2333/min: 4.7 Half full volume 0.1mA. 4. Multi-layer new cylinder, inner length 2 (cm, width 1m 4.9 Rectangular culture medium: 1.cm × 8cx23cm 977 CB/I5009.95-2003 2, 10 layers of new paper rmXem, natural paper 4. 11 Micro-or syringe; 1U rL, 50 JL: 4.12 Electric hair dryer,
4.13 Vernier caliper.
4.14 Adjustment: Measure 1m. The unit is 1mL. 4.15 Radiation: Capacity is 21m:
4.1G Flat: Inner diameter is 9mm, height 16mm~17mm. Bottom is smooth and submerged, with porcelain belt, 4.17 Small tube without pot (referred to as small tube): Inner diameter 6.0mm±0.1mm, diameter 7.8am±1.1m. Height 11r-n, t mm.
5 Culture medium
5.1 Chrysanthemum seed culture medium:
Egg yolk paste
Sodium amide
Sodium hydroxide
Water
14@--:5R:
Mix the above ingredients in water, stir and heat to decompose. Heat at 113'C for 3min. The final pH value is 7.2~-7.4. 5.2 Test medium:
Egg yolk paste
Beef juice
Sodium hydroxide
Water
14@--:5R:
Mix the above ingredients in hot water, stir and heat until fluid, 110% alcohol, and the total pH is 55. 1.6 Sample treatment
6.1 Quantitative test sample preparation, take 10.1% of the mixed fibroblast sample, add 3nL of H4:1% viscoelastic solution, mix thoroughly, dissolve in a syringe, filter several times, filter with the final pre-treated PA, use 10mL water to wash, elute with 10mL of distilled water, set the deionized water to 3mL of pH 4.5, and prepare for negative detectionbzxz.net
6.2 Qualitative test sample preparation, weigh the honey sample, treat according to 6.1, elute with distilled water, evaporate in a 41% vacuum oven, dissolve with 0.1mL of distilled water, and prepare for qualitative test. ? 7.1 Preparation of test plate: Select CTSR: R. MCIES, No. 6331, provided by the Institute for the Control of Pharmaceutical and Biological Products of the Ministry of Industry and Health. 7.2 Preparation of bacterial blanket: Transfer the seedlings into a flask with a medium of 37°C, culture at 37°C, make the number of cells in the micropipette reach 70%, add 10mL of sterile water, centrifuge for 20min, add 10L of sterile water to the precipitate, mix, and then heat the precipitate in a constant temperature water bath for 30min. Take it out of the water bath, place it in a warm water bath at room temperature, and then store it in ice after cooling. The total amount of sterile water solution can be taken out after 10 months.
7.3 Determination of the dosage of spores: add the same amount of the liquid to the test culture medium, and operate according to this method. 0.2 more than 678
GB/T5009.95-2003
National Environmental Protection Administration concentration can produce more than 15rm1L of complete seedlings, and select the most suitable one. For every 11 mT. of test culture medium, add 0.2 aL~0.5 mE of dilute liquid. 7.4 Preparation of test plates: Spread 200% of the culture medium in advance as the bottom layer for the test. Add the appropriate fresh liquid (determined in 7.3) to the test medium and heat it to 55℃60℃. After filtering, add 5m2 of the bacteria to the above plate. After the plate is rotten, make the bacterial layer evenly distributed on the bottom surface. Place it horizontally and solidify it. Place a small section on the surface of each plate with a radius of 20. The test plate should be prepared quickly. 8 Quantitative test
8.1 Solution of standard curve: 3 calibration plates are used as the vertical axis, and 6 groups of standard curves are required. In each group, a standard concentration of 37° is required in each of the 3 small tubes. The standard concentration is obtained by the average value of the standard within the group and the reference solution. The vertical axis is the corresponding value as the vertical coordinate, and the corresponding value is the vertical coordinate. The standard curve is used as the vertical coordinate.2 Test: Use at least 3 half plates for each test, fill the 3 small spaces on each half plate with C.28m (or 0.25/L or 3.0/ml, depending on the type of bacteria contained) and fill the other 3 small spaces with the test sample liquid: "87=1 times the diameter of the test sample" and calculate the average value of the test sample liquid and the reference value. Under the qualitative test, take 7rX22m of the chromatographic filter group and spray it with 0.1mm/3.5% phosphorus salt. Dry in air for 10 days and prepare the test document. The bottom edge of the filter paper is 2.m long and filled with the qualitative test liquid. Break the filter paper in the filter paper containing the test liquid (3.7), unfold it in the above method, and take out the filter paper when it is 15cm in front of the test liquid. Stick it on a rectangular culture medium containing mL of test liquid in air for 2 minutes. After that, remove the separate paper and incubate it in the air for 2 minutes to ensure that the tetracyclines contained in the test column are removed. Calculation of results: 10
According to the correlation between the filtration rate and the inhibition rate of the test sample, the antibiotic concentration was found from the antibiotic standard curve (/ml.) When two tetracyclines coexist in the sample, except tetracycline, the results are expressed in gold, and other situations are expressed in soil. The concentration of tetracycline family antibiotics in the sample is calculated as follows: x=sxz1000
nXI CC
Formula,
X-…·Tetracycline antibiotic residues in the sample. Single pull is mg/kg.—Test sample solution 4, unit is microgram per unit (gm; Test solution volume, unit is milliliter (ml.): m—Test sample mass, unit is g.
11 Goose density
The cross-correlation of the results obtained under normal conditions of the state drinking water standard shall not exceed the average value (treatment, Er
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