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1 Scope of application
Suitable for 18 kinds of sulfonamides in royal jelly ( sulfadiazine , sulfathiazole , sulfapyridine, sulfamethazine, sulfamethoxine , sulfamethizole, sulfamethazine, sulfamethoxazole , sulfamethoxazole) Pyrimidine, Sulfachloropyridazine, Sulfamethoxazole, Sulfamethoxazole, Sulfamethoxazole, Sulfamethoxazole, Sulfachloropyrazine, Sulfapyrazole, Sulfamethoxazole Quinoxaline) residual liquid chromatography
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Zhonglian mass spectrometry
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2 Principle of the method
The residues of sulfonamides in royal jelly were extracted with deionized water, and the protein was precipitated with trichloroacetic acid .
After centrifugation, the supernatant was purified by Oasis MCX ion exchange column or equivalent solid phase extraction column, and ammonia-methanol solution (1+19, v /v) After elution and drying, the residue is dissolved with acetonitrile-0.
01mo/L ammonium acetate solution, and after passing through a 0.
2um filter membrane, the sample solution is measured by liquid chromatography-tandem mass spectrometer and quantified by internal standard method
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3 Reagents and materials
Methanol, acetonitrile: chromatographic purity: ammonium acetate, trichloroacetic acid, formic acid, ammonia water: analytically pure; 0.
01mol/L ammonium acetate solution; weigh 0.
77g ammonium acetate and dissolve in 1000mL water; 2% formic acid solution: draw 2mL formic acid and dilute with water To 100mL: 50% trichloroacetic acid solution: Weigh 20g of trichloroacetic acid dissolved in 20mL of water ammonia-methanol solution: (1+19, v/v), suck 5mL ammonia and 95mL methanol and mix well; Acetonitrile -0.
01mo/L Ammonium acetate solution (3+22, v/v): Measure 12mL acetonitrile and 88mL ammonium acetate solution and mix well
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18 kinds of sulfa standard materials: purity>99%
0.
1 mg/mL standard stock solution: accurately weigh an appropriate amount of each sulfonamide standard substance, and use methanol to prepare a 0.
1 mg/mL standard stock solution.
The solution is stored at 4°C
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5.
0ug/mL sulfonamide mixed standard working solution: Pipette 0.
5mL of each standard stock solution into a 10mL volumetric flask, and dilute with methanol to a 5.
0ug/mL mixed standard working solution
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The solution is stored at 4°C
Sulfa internal standard substances: sulfamethoxazole-D4, sulfadiazine-D4, sulfathiazole-D4
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Purity ≥99%
0.
1mg/mL sulfamethoxazole-D4, sulfadiazine-D4, sulfaoxazole-D4 internal standard stock solution: Weigh appropriate amount of sulfamethoxazole-D4, sulfadiazine-D4, sulfathiazole-D4 standards Substances were made up to 0.
1mg/mL with methanol
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The solution is stored at 4°C
Mixed internal standard working solution: Pipette 0.
5 mL each of the sulfamethoxazole-D4, sulfadiazine-D4, and sulfadioxazole-D4 stock solutions into a 10 mL volumetric flask, dilute to the mark with methanol, and keep the solution at 4°C Save
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Matrix standard working solution: draw different volumes of standard working solution and 20μL mixed internal standard working solution, and use blank sample extract to make 5.
0ng/mL, 10.
0ng/mL, 20.
0ng/mL, 50.
0ng/mL, 100.
0ng/mL Different concentrations of matrix standard working solutions
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Prepared on the same day
Oasis MCX column or equivalent: 150mg, 6mL
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Treat with 5mL methanol and 10mL water in sequence before use
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4 Apparatus and equipment
Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source: analytical balance: sensitivity 0.
1mg and 0.
01g; polypropylene test tube with stopper: 50mL; oscillator, liquid mixer; solid phase extraction device: glass storage Liquid container: 50mL; nitrogen concentrator; concentration tube: 10mL
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5 Sample pretreatment
(1) Each sample system
(2) Extraction
Weigh 2g sample, accurate to 0.
01g
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Place it in a 50mL polypropylene test tube with a stopper, add 20uL of internal standard working solution, then add 20mL of water, mix quickly on a liquid mixer for 1 min, then place on a shaker to shake and extract for 10 min, add 0.
(3) Purification
For frozen laboratory samples, stir them evenly after thawing
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Separate 0.
5 kg as a sample
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The prepared sample is placed in a sample bottle, sealed, and marked
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The samples were frozen and stored at -18°C
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Connect the glass reservoir plugged with glass wool to the Oasis MCX column, pour the supernatant into the glass reservoir, adjust the flow rate to less than 3mL/min, and wait for the sample solution to flow out completely, use 5mL formic acid solution and 5mL in turn Wash the column with methanol and discard all the effluent
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Finally, it was eluted with 5 mL ammonia-methanol solution, and the eluate was collected in a 10 mL concentrating tube
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Blow dry at 50°C with a nitrogen concentrator
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Accurately add 1.
0mL acetonitrile-0.
01mo/L ammonium acetate solution to dissolve the residue.
After the sample solution is passed through a 0.
2um filter membrane, it is used for liquid chromatography-tandem mass spectrometer determination
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According to the above extraction and purification steps, a blank sample extract solution for preparing a series of matrix standard working solutions is prepared
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