This standard specifies the HPLC method for the determination of lasalocid sodium in animal feed. This standard is applicable to the determination of lasalocid sodium in compound feed, concentrated feed and additive premix feed. The detection limit of this method is 5 mg/kg. NY/T 724-2003 Determination of lasalocid sodium in feed HPLC method NY/T724-2003 Standard download decompression password: www.bzxz.net

NY/T724—2003
This standard was developed based on the general analytical methods recommended by the National Feed Industry Association (NFIA) of the United States and a large number of domestic and foreign literatures according to the level of technological development in my country. It adopts the high performance liquid chromatography (HPILC)-fluorescence detection method. This standard was proposed by the Ministry of Agriculture of the People's Republic of China and the National Feed Industry Standardization Technical Committee. This standard is under the jurisdiction of the National Feed Industry Standardization Technical Committee. The drafting unit of this standard: National Veterinary Drug Evaluation Center (College of Veterinary Medicine, China Agricultural University). The main drafters of this standard: Shen Jianzhong, Zhang Suxia, Liu Jinfeng, and Li Yuming. Scope
Determination of Lasalocid Sodium in Feed
High Performance Liquid Chromatography
This standard specifies the high performance liquid chromatography method for detecting the content of Lasalocid Sodium in animal feed. NY/T724—2003
This standard is applicable to the determination of the content of Lasalocid Sodium in compound feed, concentrated feed and additive premix feed. The detection limit of this method is 5 mg/kg.
2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all references with dates, all subsequent amendments (excluding errata) or revised versions are not applicable to this standard. However, parties reaching an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For all references without dates, the latest versions apply to this standard. GB/T6682 Specifications and test methods for water used in analytical laboratories GB/T14699.1 Feed sampling method
3 Principle of the method
The sodium lasalocid in the sample is extracted with methanol, and separated and determined by high performance liquid chromatography-fluorescence detection method with phosphate buffer-acetonitrile-methanol as the mobile phase.
4 Reagents and solutions
Unless otherwise specified, the reagents used in this method are analytically pure, and the water is deionized water, which complies with the provisions of GB/T6682 for secondary water. 4.1 Ethylene: chromatographically pure.
4.2 Methanol: chromatographically pure.
4.3 Phosphate buffer (pH-7): Accurately weigh 3.121g sodium dihydrogen phosphate (NaHPO·2HO) and 7.164g disodium hydrogen phosphate (NazHPO·12H2O), dissolve them in water, and dilute to 1000ml to obtain sodium dihydrogen phosphate solution (liquid A) with a concentration of c(NaHPO·2HO)=0.02mol/I and disodium hydrogen phosphate solution (liquid B) with a concentration of c(NazHPO·12H2O)=0.02mol/I. Mix liquid A and liquid B in a ratio of 3.9+6.1 to prepare a phosphate buffer (adjust pH=7). 4.4 Lasalocid sodium standard solution:
4.4.1 Lasalocid sodium standard stock solution: Accurately weigh 0.1000g of lasalocid sodium standard (purity ≥ 98%), place in a 100ml volumetric flask, dissolve with methanol, and make up to volume. The concentration is 1000μg/mL of the stock solution, which is stored in a 4℃ refrigerator. 4.4.2 Lasalocid sodium standard working solution: Accurately take a certain amount of standard stock solution (4.4.1), place in a 10mL volumetric flask, dilute with methanol, and make up to volume. Prepare standard working solutions with concentrations of 2.5μg/ml., 5.0ug/ml, 7.5μg/ml., 10.0ug/ml, 12.5ug/ml..17.5 μg/mL.
5 Instruments and equipment
Commonly used instruments and equipment in the laboratory.
5.1 High performance liquid chromatograph: equipped with a fluorescence detector. 5.2 Centrifuge.
5.3 Oscillator. bzxz.net
NY/T 724—2003
Glass stoppered conical flask (250ml.).
5.5 Microinjector.
5.6 Microporous filter membrane (0.45μm).
6 Sample preparation
According to GB/T14699.1, take a representative sample, reduce it by quartering to about 200g, crush it, pass it through a 1mm mesh sieve, mix it and put it into a ground-mouth bottle for later use.
7 Determination steps
7.1 Sample extraction
Weigh a certain amount of sample (10.0g compound feed, or 5.0g concentrated feed, or 1.0g additive premix feed), place it in a 250mL glass stoppered conical flask, add 40mL methanol, and shake it back and forth for 30min. Let stand for 10 minutes, filter. Then add 30 mL of methanol to the sample and repeat the extraction twice. Combine the three extracts and make up to 100 mL with methanol. Take 10 mL and place it in a centrifuge tube, centrifuge at 3000 r/min for 5 minutes, take the supernatant and filter it with a 0.45 μm microporous organic filter membrane as the sample solution for high performance liquid chromatography analysis. 7.2HPLC chromatographic conditions
a) Chromatographic column: C1g column, 150 mm long, 4.6 mm inner diameter, 5 μm particle size, or equivalent. b)
Column temperature: room temperature.
Mobile phase: phosphate buffer (4.3) + ethyl + methanol (25+35+40). c)
Mobile phase flow rate: 1.0 mL/min.
Excitation wavelength: 310 nm.
Emission wavelength: 420 nm.
Injection volume: 20 μL.
7.3HPLC determination
Take appropriate amount of sample solution (7.1) and standard working solution (4.4.2) of corresponding concentration, make single-point or multi-point calibration, and quantify by the integral value of chromatographic peak area.
8 Calculation and expression of results
8.1 The content of lasalocid sodium in the sample is calculated according to formula (1): X=mxn
Wherein:
X--the content of lasalocid sodium in the sample, in milligrams per kilogram (mg/kg); mi
The mass of lasalocid sodium corresponding to the chromatographic peak of the HPILC sample, in micrograms (ug); m--the mass of the sample, in grams (g);
n--the dilution factor.
8.2 The determination result is expressed as the arithmetic mean of parallel determinations, and is retained to one decimal place. 9 Allowable error
The relative deviation of two parallel determinations shall not exceed 10%. 2
（1）
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