This standard specifies the high performance liquid chromatography (HPLC) method for the determination of monensin in animal feed. This standard is applicable to the determination of monensin in compound feed, concentrated feed and additive premix feed. The detection limit of this method is 5 mg/kg. NY/T 725-2003 Determination of monensin in feed High performance liquid chromatography NY/T725-2003 Standard download decompression password: www.bzxz.net

NY/T725—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, and in accordance with the level of technological development in my country. It adopts the high performance liquid chromatography (HPLC)-post-column derivatization method. This standard was proposed by the Ministry of Agriculture of the People's Republic of China and the National Technical Committee for Standardization of Feed Industry. This standard is under the jurisdiction of the National Technical Committee for Standardization of Feed Industry. The drafting unit of this standard: National Veterinary Drug Evaluation Center (College of Veterinary Medicine, China Agricultural University). The main drafters of this standard: Yuming Wang, Jianzhong Shen, Suxia Zhang, Jianmin Yuan. 1 Scope
Determination of monensin in feed
High performance liquid chromatography
This standard specifies the high performance liquid chromatography (HPLC) method for detecting the content of monensin in animal feed. NY/T 725--2003
This standard is applicable to the determination of monensin content 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 suitable for this standard. However, the parties who reach 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
Monensin in the sample is extracted with methanol-water, methanol-glacial acetic acid-water is used as the mobile phase, vanillin is used as the derivatization agent, and the sample is separated and determined by high performance liquid chromatography-post-column derivatization-ultraviolet detection. 4 Reagents and solutions
Unless otherwise specified, the reagents used in this method are analytically pure, and the water is deionized water, which meets the requirements of GB/T6682 for secondary water. 4.1 Sample extract: [Methanol (chromatographic grade) + water] (90+10). 4.2 Vanillin derivative solution: Measure 5 ml of concentrated sulfuric acid and slowly add it to 250 mL of methanol. Place in an ice-water bath and slowly add 20 g of vanillin. Mix well. After degassing, store in a dark place and prepare before use. 4.3 Monensin standard solution:
4.3.1 Monensin standard stock solution: Accurately weigh 0.1000 g of monensin standard (purity ≥ 90%) and place in a 100 mL volumetric flask. Dissolve with methanol and make up to volume. The concentration is 1000 μg/mL of stock solution and store in a refrigerator at 4°C. 4.3.2 Monensin standard working solution: Accurately pipette a certain amount of standard stock solution (4.4.1) into a 10mL volumetric flask, dilute with methanol, and make up to the standard working solution with concentrations of 2.0μg/ml, 4.0μg/ml, 6.0μg/ml., 8.0μg/mL, 10.0μg/ml. 12.0μg/ml.
5 Instruments and equipment
Commonly used laboratory instruments and equipment.
5.1 High performance liquid chromatograph: equipped with post-column derivatization device and UV detector. 5.2 Centrifuge.
5.3 Oscillator.
5.4 Glass stoppered conical flask (250mL). 5.5 Microinjector.
5.6 Microporous filter membrane (0.45μm).
NY/T 725—2003
6 Sample preparation
According to GB/T14699.1, take a representative sample, cut it down to about 200g by quartering, crush it, pass all of it through a 1mm mesh sieve, mix it and put it in 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 sample extract (4.1), and shake it back and forth for 30min. Let it stand for 10min and filter it. Then add 30ml of sample extract (4.1) to each sample, and repeat the extraction twice. Combine the three extracts and make up the volume to 100mL with the sample extract. Take 1.mL and filter it with 0.45um microporous organic filter membrane as the sample solution for high performance liquid chromatography analysis. 7.2HPLC Chromatographic Conditions
Chromatographic column: Cs column, length 150mm, inner diameter 4.6mm, particle size 5μm, or equivalent. a)
Column temperature: room temperature.
Mobile phase: methanol: glacial acetic acid: water (94:3:3). Mobile phase flow rate: 0.7mL/min.
Derivatization liquid flow rate: 0.7mL/min.
Reaction tube: stainless steel, length 10m, inner diameter 0.2mm. Reaction temperature: 95℃.
Detection wavelength: 520nm.
Injection volume: 20uL.
7.3HPLC determination
Take an appropriate amount of sample solution (7.1) and the standard working solution of corresponding concentration (4.3.2), perform single-point or multi-point calibration, and quantify by the integral value of the chromatographic peak area.
8 Calculation and expression of results
8.1 The content of monensin in the sample is calculated according to formula (1): X=mi×n
Wherein:
X—the content of monensin in the sample, in milligrams per kilogram (mg/kg); ml--the mass of monensin corresponding to the HPI.C sample chromatographic peak, in micrograms (μg); m—--the mass of the sample, in grams (g); n
dilution factor.
8.2 The determination result is expressed as the arithmetic mean of parallel determinations, and is retained to one decimal place. The allowable error
The relative deviation of two parallel determinations shall not exceed 10%. 2wwW.bzxz.Net
(1)
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.