Inspection of gentamicin residues in meat and meat products

â‘  Gentamicin sulfate standard product: a standard product of known purity, such as 8 is equivalent to o. 631ug gentamicin.

â‘¡Phosphate buffer solution: pH 8.0 + 0.1. Weigh 13.3e potassium dihydrogen phosphate and 6.2g potassium hydroxide, dissolve in water and dilute to 1000ml with water. Autoclave at 121 ~ C for 15min.

③ Gentamicin standard stock solution: Weigh accurately an appropriate amount of gentamicin sulfate standard crystals, and prepare a standard stock solution with a concentration of 1000vg / mi with phosphate buffer. Stored in a refrigerator at 4 ℃, can be used for 1 month.

â‘£Gentamicin standard working solution: Draw a certain amount of gentamicin standard stock solution, dilute it with phosphate buffer to an intermediate standard solution with gentamicin concentration of 20ug / m1, and then use phosphoric acid Salt buffer diluted to gentamicin with a concentration of 0.05 ug / m1, 0.10 ug / m1, 0.20 ug / m1, 0.40 ug / m1, 0.80 ug / m1, 1.60 ug / m1 Standard working fluid. The above solution should be prepared on the same day.

⑤ Test strain: Staphylococcus epidermidis (Staphylococcus epidermidis), bacteria number CMCC 12228, provided by the China National Institute for the Control of Pharmaceutical and Biological Products.

⑥Culture I Put tryptone 10.0g, beef extract 5.0g, sodium chloride 2.5g and agar 14.0-16. 0g into 1000ml distilled water and heat to dissolve, adjust the pH value to 6.5-6 ．6. Packed in test tubes or Kirschner bottles, autoclaved at 121 ~ C for 15 minutes. After sterilization, they can be made into inclined surfaces.

⑦ Medium II Tryptone 10.0g, beef extract 5.0g and sodium chloride 2.5g are placed in 1000ml distilled water and heated to dissolve, adjust the pH value to 6.5 ~ 6.6, aliquot in test tube or gram In the bottle, autoclave at 121 ~ C for 15min.

⑧medium Ⅲ Peptone 6.0g, yeast extract 4.0g, beef extract 1.5g glucose 1.0g and agar 15.0g were heated and dissolved in 1000ml distilled water, the pH value was adjusted to 6.5, at 121 ~ C Sterilize for 15min. Before use, adjust the pH to 8 with 1mol / L sodium hydroxide solution. o.

⑨Normal saline: Weigh 8.58 sodium chloride, dissolve it in 1000ml water, aliquot it, and autoclave at 121 ~ C for 15min

â‘©Expansion agent: chloroform-methanol-concentrated ammonia (1 + 1 + 1).

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(2) Instruments and equipment

â‘ Petri dish: an inner diameter of 90mm, a height of 20mm, a flat or smooth glass or plastic dish with a terracotta cover.

â‘¡Oxford Cup: stainless steel cylinder

â‘¢ Kirschner bottle: 800ral.

â‘£ Vernier caliper: measuring range. One 200mm, accuracy o. 02mm or bacteriostatic circle measuring instrument

⑤ Homogenizer: The rotation speed is not less than 10000r / iTlln.

â‘¥Centrifuge: The speed is not less than 4000r / rain.

⑦ Constant temperature water bath: 0 ~ l00 ~ C, accuracy ± 1 ℃.

⑧ Constant temperature incubator: (36 ~ 1.0) ℃, the shelf must be kept level.

⑨ Autoclave.

â‘© Semi-logarithmic graph paper.

â‘£ Rotary evaporator.

â‘© Chromatography cylinder: 20cmXl5cm ~ 30cm.

â‘©Thin-layer silicone G board: 5em ~ tocm or 10cmX20cm

⑩Micro syringe: 10t ~ L or 20tH ·.

(3) Measurement procedure

① Preparation of sample solution Weigh 10g of sample (accurate to 0.1g) in a homogenization cup, add 10ml of phosphate buffer solution, homogenize for 3min (8000r / min), place at room temperature for 60min, move into a centrifuge tube and centrifuge for 30min ( 4000r / rain), take the supernatant in a large test tube, and then add 10ml of phosphate buffer to mix with the residue, perform a second centrifuge, combine the two supernatants in an eggplant-shaped bottle, use lmol / L hydrogen Adjust the pH to 8 with sodium oxide solution. o ± o. 1. Make up to 20ml with buffer solution as the sample solution to be tested.

②Preparation of bacterial suspension Inoculate the test strain in the test tube of medium II, and incubate at (36 ± 1) ° C for (24 ± 1) h. Transfer 1ml of the bacterial solution to the Kirschner flask containing the appropriate amount of medium I, so that the bacterial solution evenly covers the surface of the agar and incubate at (36 ± 1) ° C for (24i1) h. Then wash the fungus with an appropriate amount of saline and set aside. Place in 4 ~ C refrigerator (not more than 1 month).

â‘¢Preparation of the verification plate Before the determination, a pre-test is required to obtain the optimal amount of bacterial suspension. Use o. 10ug / ml gentamicin standard working solution was tested on plates with different amounts of bacterial suspension, after cultivation, it can make o. The 10ug / ml standard working fluid has a diameter greater than or equal to 10mm. The clear and complete bacteriostatic zone is the best dosage.

Melt the medium III, cool to about 50 ~ C, add the best amount of bacterial suspension, mix it thoroughly, take 10ml, and pour it into the sterilized Petri dish, keep it level, wait for it to solidify and make the test plate. The plates used must be prepared on the same day.

④ Preparation of standard curve Use gentamicin standard working solution to draw the standard curve, with o. 2 "8 / ml concentration standard working solution is the reference concentration, and o, 05 / ~ g / ml concentration standard working solution is used as a negative control. For each standard working solution concentration, 3 test plates are taken as a group. In each Place 6 Oxford cups on the flat plate to make the Oxford cups have a 60 * angular spacing on a round surface with a radius of 2.8cm, 3 cups are filled with the standard working solution of the reference concentration, and the other 3 cups are filled with other concentrations A standard working solution. The reference concentration solution and the standard working solution should be placed apart. A total of 15 verification plates of 5 concentration standard working solutions are used to draw the standard curve. In this way, o. 2bg / L reference concentration standard working solution will be obtained. The value of 45 bacteriostatic circle diameters, while other standard working fluids get 9 bacteriostatic circle diameter values ​​respectively.

Cover the dish, place it at (36 ± 1) ° C and incubate for (174) h, then turn over the plate and remove the Oxford cup. Accurately measure the diameter of the bacteriostatic circle at each concentration (accurate to 0.1mm), and obtain the respective average value. Then the average value of the diameters of 45 bacteriostatic circles with the reference concentration of 0.2ug / L was calculated. Use the difference between the average value of the diameter of the inhibition zone of the reference concentration and the average value of the diameter of the inhibition zone of the reference concentration of each group of plates to correct the average value of the reading of the inhibition zone of the standard working solution of each concentration.

Find the L point and H point using the corrected values ​​as follows. On semi-logarithmic graph paper, take the diameter of the bacteriostatic circle (nm) as the ordinate (arithmetic grade) and the concentration of gentamicin as the abscissa (logarithmic grade), and mark the L point and the H point. A straight line through point L and point H is the standard curve.

⑤Determination

Each sample uses 3 test plates, and 6 sterilized Oxford cups are placed on each test plate. Fill three intervals of Oxford cups on each test plate with the standard reference concentration solution of gentamicin, and the other three Oxford cups with the sample solution to be tested. Cover the terracotta cover and incubate at (36 ± 1) ° C for (17i1) h. Turn over the plate and remove the Oxford Cup. If there is a zone of inhibition, accurately measure its diameter.

(4) Sensitivity and recovery rate

The lower limit of the determination of this method is o. 10mg / kg

The recovery range at the concentration level of 0.10 ～ 0.40rug / ke is 92.5% ～ 98.0%

(6) Confirmation test Confirmation test was carried out by thin layer chromatography.

â‘ Activate the ordinary silica gel G thin layer board (5cm ~ 10cm) at 105 ~ C for 2h, and then use it after cooling.

â‘¡Sampling liquid 20.0mi (equivalent to 10.08 sample), adjust the pH value to 8.0 with sodium hydroxide solution (1mol / L), and concentrate to dryness under reduced pressure at 65-70 ~ C. Use 1.0ml phosphate buffer to dissolve the residue and use the solution as the sample solution to be tested.

â‘¢Aspirate 2.0ul of standard working solution of gentamicin and the sample solution to be tested at a concentration of 2ttg / ml with a micro syringe, respectively, and point on the above thin-layer board (spacing 2cm). The thin-layer plate was transferred to an expansion tank containing the developing agent and unfolded, and the solvent was extended to the front of the thin-layer plate (about 20 min) and then taken out. After the solvent evaporates, place the thin-layer plate in another expansion tank filled with saturated iodine vapor and observe the color development. According to the color and position of the spot of the liquid to be tested on the thin layer plate, the brother value is measured, and the spot of the standard solution is compared to determine whether the bacteriostatic substance in the sample solution is gentamicin.