(1) Reagents and materials Unless otherwise specified, the reagents used are of analytical grade
â‘ Acetonitrile
â‘¡Methanol: pure UV spectrum
â‘¢Triethylamine
â‘£ Phosphoric acid (85%)
⑤Sodium chloride
â‘¥Phosphate buffer
⑦ Mixed extraction solution: phosphate buffer-acetonitrile (1+).
⑧ Potassium borate buffer solution: Dissolve 2.5z boric acid in 80mi water and adjust the pH to 10 with 50g / 100ml potassium hydroxide solution. 5. Make up to 100ml with water.
⑨ Phthalicaldehyde reagent: Dissolve 100mg of phthalaldehyde in 1ml of methanol, add 200ul of 2-erythritol and 10ml of potassium borate buffer, store in a refrigerator with a brown bottle (with lid), effective within 1 week.
â‘© Alkaline buffer solution: Dissolve 76e potassium tetraborate in 400mi water, adjust the pH to 11.0 with 50g / 100ml potassium hydroxide solution, and bring the volume to 500mi with water.
⑾ Eluent: alkaline buffer-methanol (1 10 4).
â‘¿Cation exchange resin; D152.
â’€ Neomycin sulfate standard product: biochemical reagent
â’ Neomycin sulfate standard solution: Weigh an appropriate amount of neomycin sulfate standard (accurate to 0, lmg), and make up the concentration with water to o. The standard stock solution of 100rug / ml, then make up the standard working solution of appropriate concentration with water according to need.
â’‚ Neomycin derivative standard solution: Inject appropriate amount of standard working solution into the cation exchange column to conduct derivatization reaction to prepare neomycin derivative standard solution,
(2) Instruments and equipment
â‘ Liquid chromatograph: equipped with fluorescence detector
â‘¡High-speed tissue masher
â‘¢ Oscillator
â‘£Centrifuge
⑤Filter funnel; filter holes are 80-120um, 40-80 / um
â‘¥Cation exchange column: 120mmX 5mm (inner diameter) glass column. The cation exchange resin was made into suspension with alkaline buffer, equilibrated for 2h, loaded into a glass column, the height was about 6cra, rinsed with 5ml eluent, the flow rate was 0.8ml / min, and then washed with water to the effluent Neutral, spare.
(3) Measurement procedure
①Extract and weigh about 10g (accurate to 0.1g) in a 100ml Erlenmeyer flask, add 60ral mixed extract and 2g sodium chloride, shake for 30min, and pass through a filter funnel with a filter hole of 80-120 ~ m Filter with suction and wash the residue with 3x10ml of mixed extract. Combine the filtrate and wash solution in a 250rnl beaker. Heat in boiling water for 30min, remove, centrifuge at 3000r / min for 20min, place in 4 ° C refrigerator for 20min, and then filter through a filter funnel with a filter hole of 40-80bm to collect the filtrate.
â‘¡ Derivatization Inject the filtrate into the cation exchange column, wash the column with 10 ml of water, and discard the effluent. Inject 1ml of phthalaldehyde reagent into the column and react for 5rain. Then add 2ml of eluent to elute the derivative on the column. Discard the iml eluent in the front, collect the lml eluate in the back, immediately place it in an 18 ~ 0 refrigerator, and perform liquid chromatography measurement after 15 minutes.
â‘¢Determination
a. Chromatographic conditions
Column: ODSHypersi
Column temperature: 35 ~ C:
Mobile phase: 77.5% methanol aqueous solution contains 0.09mol / L triethylamine and o. 45moI / L phosphoric acid
Flow rate: 1.0ml / Illin
Excitation wavelength: 345nm
Emission wavelength: 445nm
Injection volume: 10ul
b. Determination According to the content of neomycin in the sample solution, select a standard solution of neomycin derivatives with similar peak areas. The response values ​​of neomycin derivatives in standard solution and sample solution should be within the linear range of instrument detection. For standard solution and sample solution, the same volume is inserted into the sample for determination. Under the above chromatographic conditions, the retention time of neomycin derivatives is about 6 min.
â‘£Blank test Except that no sample is added, follow the above measurement steps.
(4) Detection limit and recovery rate
The determination limit of this method is 50ug / kg.
The recovery rate at the concentration level of 0.05-0.50mg / kg is 90.4% -96.3%
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