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Application of GE-200 liquid chromatograph in the determination of apramycin in feed
Application of GE-200 liquid chromatograph in the determination of apramycin in feed
Ministry of Agriculture No. 1486 Announcement-3-2010
This standard specifies high performance liquid chromatography for the determination of apramycin in feed, which is suitable for compound feed. Determination of apramycin in concentrated feed and additive premixed feeds. The detection limits and quantitation limits were 3 mg/kg and 10 mg/kg, respectively.
Method principle
The apramycin in the feed is extracted with hydrochloric acid solution, purified by cationic solid phase extraction column, derivatized with terephthalic acid, and then detected by high performance liquid chromatography and fluorescence detector, and quantified by external standard method.
Kit material
Unless otherwise specified, the reagents used in this standard are of analytical grade, and the water is deionized water, which meets the requirements of GB/T 6682 secondary water.
Anhydrous methanol.
Ammonia, 25%.
Concentrated hydrochloric acid.
Hydrochloric acid solution (c (HCI) = 0.10 mol/L): 8.4 ml of concentrated hydrochloric acid was accurately weighed and diluted to 1000 ml with water.
Ammonia water methanol solution: Measure 20 ml of methanol in a 100 Ml volumetric flask, add 5.00 ml of ammonia water, and then dilute to the mark with methanol.
Sodium hydroxide solution (c (NaOH) = 6 mol/L): 240 g of sodium hydroxide was dissolved in 1000 ml of water.
Borate buffer solution: Accurately weigh 24.73g of boric acid in a 1000ml beaker, dissolve it with about 900ml of water, then adjust the pH to 9.5 with sodium hydroxide solution, and dilute to the mark with water.
Apramycin standard solution
Apramycin white cum storage: accurately weigh the apramycin standard (content ≥ 99%) 0.0250g, as for the 25ml volumetric flask, dissolve in water, dilute to the mark, shake, the concentration is 1mg / Ml. Stored at 4 ° C, valid for 1 month.
Apramycin standard intermediate solution: accurately absorb 10.00 ml of standard stock solution, place it in a 100 ml volumetric flask, dilute with water, and make up the volume, the corresponding concentration is 100 ug/ml.
The standard work of apramycin also: take 2.50ml, 1.25ml, 0.500ml, 0.250ml and 0.125ml of apramycin standard intermediate solution in a 25.0ml volumetric flask, dilute to the mark with borate buffer, shake up The concentrations were 10.0 ug/ml, 5.00 ug/ml, 2.00 ug/ml, 1.00 ug/ml, and 0.500 ug/ml, respectively.
Mobile phase A: Weigh 0.77 g of ammonium acetate in a 1000 ml volumetric flask, add about 800 ml of water, and after it is dissolved, add 40 ml of acetic acid, dilute to the mark with water, and shake well.
Mobile phase B: acetonitrile, chromatographically pure.
A hybrid solid phase extraction cartridge having a reverse retention mechanism of reversed phase and cation exchange.
equipment
Analytical balance: The sensitivity is 0.0001 g.
Magnetic stirrer.
Centrifuge: can reach 3000r/min-4000r/min.
Solid phase extraction unit.
Constant temperature water bath.
Nitrogen blow dryer.
GE-200 High Performance Liquid Chromatograph, Clarify Chromatography Workstation, (Wters 474 Fluorescence Detector).
Sample preparation
According to GB/T 14699.1, a representative sample of 1000g was taken, reduced to about 200g by the quarter method, pulverized, and all passed through a sieve with a pore size of 0.45 mm, and then stored in a grinding bottle for use.
Analysis step
Weigh the appropriate amount of sample (5g of compound feed, 2g-3g of concentrated feed, 1g of additive premixed feed, accurate to 0.0001g), place in a 50ml centrifuge tube, add 40ml of hydrochloric acid solution, cover the lid, stir on a magnetic stirrer The extract was extracted for 25 min and centrifuged at 3000 r/min-4000 r/min for 10 min in a centrifuge. The supernatant was transferred to a 100 ml volumetric flask, and extracted twice with 35 ml of 25 ml hydrochloric acid solution, and the supernatant was collected, diluted with a hydrochloric acid solution to a mark, shaken, filtered with a medium-speed quantitative filter paper, and the filtrate was reserved. Concentrated feed and additives Premixed feed The above filtrates need to be diluted 5 times and 10 times, respectively.
The solid phase extraction cartridge was activated with 1.0 ml of methanol and 1.0 ml of water, respectively, and then 1.00 ml of the sample extract was accurately loaded onto the solid phase extraction cartridge, and the column was passed at a flow rate of 1.0 ml/min without the dog, and then 1.0 was respectively used. The ml color solution and 1.0 ml of methanol were rinsed once, eluted with a solution of ammonia in methanol to a 10 ml test tube, then placed in a water bath at 60 ° C, dried with nitrogen, dissolved in 1.00 ml of borate buffer, and then placed on the machine. Determination.
Column: Vertex C18, column length 300 mm, inner diameter 3.9 mm, particle size 5 um analytical column.
Column temperature: room temperature.
Mobile phase: mobile phase A - mobile phase B = 1 + 1 (v / v), flow rate of 1.0 ml / min, elution time of 15 min.
Detector: excitation wavelength 230nm, emission wavelength 389nm
Injection volume: 100ul.
Accurately transfer 400 ul of the sample solution to the injection bottle of the high performance liquid chromatograph, add 200 ul of the derivatized reagent to mix, control the derivatization time for 5.0 min, and immediately inject the sample for separation and determination.
Accurately transfer the standard work of apramycin to 400 ul, derivatize and measure, and draw a working curve on the peak area with the concentration of the standard working solution.
The content of apramycin in the sample is expressed in mass fraction (mg/kg), X=n*V*p/m
X---Ampmycin content in the sample, in milligrams per kilogram (mg/kg)
V---The volume of the final sample solution is prepared in milliliters (ml)
m---sample quality in grams (g)
P---Ampmycin concentration in the sample solution obtained from the working curve, in micrograms per milliliter (ug/ml)
n---dilution factor
The results of the measurements are expressed as the arithmetic mean of the parallel measurements, with three significant figures retained.
Allowable difference: The relative deviation of 2 parallel measurements performed by the same operator in the same laboratory is not less than 15%.
This standard complies with GB/T 1.1-2009
This standard was proposed by the Animal Husbandry Department of the Ministry of Agriculture of the Republic of China.
Drafting unit: China Agricultural University, Feed Industry Center of the Ministry of Agriculture
Drafters: Zhang Liying, Lin Dongxia, Wang Zongyi, Sing Biying, Yang Wenjun, He Pingli.