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Determination of methionine hydroxy analogue in feeds - High-performance liquid chromatography
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Feed additive -- Liquid methionine hydroxy analogue
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Basic data
| Standard ID | GB/T 19371-2025 (GB/T19371-2025) |
| Description (Translated English) | Determination of methionine hydroxy analogue in feeds - High-performance liquid chromatography |
| Sector / Industry | National Standard (Recommended) |
| Classification of Chinese Standard | B46 |
| Classification of International Standard | 65.120 |
| Word Count Estimation | 10,139 |
| Date of Issue | 2025-08-29 |
| Date of Implementation | 2026-03-01 |
| Older Standard (superseded by this standard) | GB/T 19371.2-2007 |
| Issuing agency(ies) | State Administration for Market Regulation; Standardization Administration of China |
GB/T 19371-2025: Determination of methionine hydroxy analogue in feeds - High-performance liquid chromatography
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ICS 65.120
CCSB46
National Standards of the People's Republic of China
Replaces GB/T 19371.2-2007
Determination of methionine hydroxy analogues in feed
High performance liquid chromatography
Published on 2025-08-29
Implemented on 2026-03-01
State Administration for Market Regulation
The State Administration for Standardization issued a statement.
Foreword
This document complies with the provisions of GB/T 1.1-2020 "Standardization Work Guidelines Part 1.Structure and Drafting Rules of Standardization Documents".
Drafting.
This document supersedes GB/T 19371.2-2007 "Determination of Methionine Hydroxyl Analogs in Feed - High Performance Liquid Chromatography".
Compared with GB/T 19371.2-2007, apart from structural adjustments and editorial changes, the main technical changes are as follows.
a) The scope of application has been expanded to include concentrate supplements and mixed feed additives (see Chapter 1);
b) The mobile phase was changed to formic acid acetonitrile solution (see 5.4, 4.5 in the.2007 edition);
c) The requirements and related regulations for standard products have been revised (see 5.8, 4.6 of the.2007 edition);
d) Requirements for standard series solutions have been added (see 5.9);
e) Sampling was removed, and sample preparation was changed (see Chapter 7, Chapter 6 in the.2007 edition);
f) The formula for calculating calcium hydroxymethionine has been removed (see Chapter 8 in the.2007 edition);
g) The precision requirements have been changed (see Chapter 10, Chapter 9 of the.2007 edition).
Please note that some content in this document may involve patents. The issuing organization of this document assumes no responsibility for identifying patents.
This document was proposed and is under the jurisdiction of the National Technical Committee on Standardization of Feed Industry (SAC/TC76).
This document was drafted by. Institute of Agricultural Quality Standards and Testing Technology, Chinese Academy of Agricultural Sciences; Tongwei Agricultural Development Co., Ltd.; and Shaanxi Province.
Qin Yun Agricultural Products Inspection and Testing Co., Ltd., and Shandong Xinhecheng Amino Acid Co., Ltd.
The main drafters of this document are. Suo Decheng, Fan Xia, Zhang Fengping, Feng Yuchao, Liu Wuyan, Lu Jiawen, Wang Jianbo, Xiao Zhiming, Zhang Lu, and Wu Hongkai.
He Xiwen, Chen Xiaoru, Zhao Juan, and Wang Junming.
The release history of this document and the document it replaces is as follows.
---First published in.2004 as GB/T 19371.2-2003, and revised for the first time in.2007;
---This is the second revision.
Determination of methionine hydroxy analogues in feed
High performance liquid chromatography
1 Scope
This document describes a high-performance liquid chromatography method for the determination of methionine hydroxy analogues in feed.
This document applies to methionine in compound feed, concentrated feed, concentrate supplements, additive premixes, and mixed feed additives.
Determination of hydroxy analogs and/or calcium salts of methionine hydroxy analogs (calculated as methionine hydroxy analogs).
The quantitative limits for compound feed, concentrated feed, and concentrate supplements in this document are 0.03%. The quantitative limits for additive premixed feed and mixed feed additives are also specified.
The limit of quantification for the additive is 0.1%.
2 Normative references
The contents of the following documents, through normative references within the text, constitute essential provisions of this document. Dated citations are not included.
For references to documents, only the version corresponding to that date applies to this document; for undated references, the latest version (including all amendments) applies.
This document.
GB/T 6682 Specifications and test methods for water used in analytical laboratories
GB/T 20195 Preparation of animal feed samples
3 Terms and Definitions
This document does not contain any terms or definitions that need to be defined.
4 Principles
Methionine hydroxy analogues in the sample were extracted with acetonitrile solution, hydrolyzed with potassium hydroxide solution, determined by high performance liquid chromatography, and quantified by external standard method.
5 Reagents or materials
Unless otherwise specified, use only analytical grade reagents.
5.1 Water. GB/T 6682, Grade I water.
5.2 Acetonitrile. chromatographic grade.
5.3 Formic acid. chromatographic grade.
5.4 Formic acid and acetonitrile solution. Take 0.5 mL of formic acid and 50 mL of acetonitrile, add 950 mL of water, mix well, and degas by sonication.
5.5 10% acetonitrile solution. Measure 100 mL of acetonitrile and 900 mL of water, and mix well.
5.6 50% phosphoric acid solution. Measure 100 mL of phosphoric acid and 100 mL of water, and mix well.
5.7 50% potassium hydroxide solution. Weigh 50g of potassium hydroxide, add 100mL of water to dissolve, and mix well.
5.8 1 mg/mL Standard Stock Solution (calculated as methionine hydroxy analog). Weigh an appropriate amount (accurate to 0.0001 g) of methionine hydroxy analog.
Calcium sulfate (CAS No.. 4857-44-7, purity greater than 98%) standard was dissolved in a 100 mL volumetric flask with 10% acetonitrile solution (5.5).
Mix thoroughly; or weigh an appropriate amount (accurate to 0.0001 g) of methionine hydroxy analogue (CAS No.. 583-91-5, purity greater than 88%) and mix well.
Dissolve and dilute to volume with 10% acetonitrile solution (5.5) in a 100 mL volumetric flask, and mix well. Store below -18℃; shelf life is 6 months. Alternatively, use a standard...
Quasi-materials.
Note. If necessary, use the correction standard stock solution for determining the content of methionine hydroxy analogues according to GB 7300.103.
5.9 Standard Series Solutions. Accurately transfer appropriate amounts of the standard stock solution (5.8), dilute and bring to volume with 10% acetonitrile solution (5.5), mix well, and prepare...
A series of standard solutions with mass concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, and 80 μg/mL were prepared. (Prepared for immediate use)
Prepared on the spot.
5.10 Microporous filter membrane. 0.45μm, organic system.
6 Instruments and Equipment
6.1 High-performance liquid chromatograph. equipped with an ultraviolet detector or a diode array detector.
6.2 Analytical balance. with an accuracy of 0.01g and 0.0001g.
6.3 Centrifuge. speed not less than 8000 r/min.
6.4 Vortex Oscillator.
6.5 Ultrasonic cleaner.
6.6 Reciprocating oscillator.
7 samples
Prepare samples according to GB/T 20195, at least.200g, crush them so that they all pass through a test sieve with a 0.425mm aperture, mix thoroughly, and load into...
In a sealed container.
8.Test Procedure
8.1 Extraction
8.1.1 Compound feed, concentrated feed, and concentrate supplement
Perform two parallel tests. Weigh 5g of the sample (accurate to 0.0001g) and place it in a 150mL Erlenmeyer flask with a stopper. Accurately add 50mL of [unspecified liquid].
Mix 10% acetonitrile solution (5.5), vortex for 30 seconds, shake vigorously on a shaker for 30 minutes, and centrifuge at 8000 rpm for 5 minutes. Collect the supernatant.
spare.
8.1.2 Additives, premixed feeds, and mixed feed additives
Perform two parallel tests. Weigh 2g of the sample (accurate to 0.0001g) and place it in a 150mL Erlenmeyer flask with a stopper. Accurately add 100mL of [unspecified liquid].
Mix 10% acetonitrile solution (5.5), vortex for 30 seconds, shake vigorously on a shaker for 30 minutes, and centrifuge at 8000 rpm for 5 minutes. Collect the supernatant.
spare.
8.2 Hydrolysis
Accurately transfer 5 mL of the stock solution (8.1) and the standard series solution (5.9) into separate 10 mL stoppered test tubes, and accurately add 0.1 mL of each solution.
Add 50% potassium hydroxide solution (5.7), shake for at least 10 seconds, then accurately add 0.2 mL of 50% phosphoric acid solution (5.6), shake for at least 10 seconds.
Centrifuge at 8000 r/min for 5 min, filter the supernatant through a microporous membrane (5.10), and then analyze.
8.3 Reference conditions for liquid chromatography
The reference conditions for liquid chromatography are as follows.
a) Chromatographic column. C18, column length 250 mm, inner diameter 4.6 mm, particle size 5.0 μm, or equivalent;
b) Mobile phase. Formic acid acetonitrile solution (5.4);
c) Flow rate. 1.0 mL/min;
d) Detection wavelength. 210nm;
e) Column temperature. 30℃;
f) Injection volume. 20 μL.
8.4 Measurement
8.4.1 Determination of Standard Series Solutions and Sample Solutions
Under optimal instrument conditions, standard working solutions (8.2) and sample solutions (8.2) were tested using the instrument. Methionine hydroxyl groups
The liquid chromatogram of the similar standard solution is shown in Appendix A.
8.4.2 Qualitative
Qualitative analysis is based on retention time; the retention time of the methionine hydroxy analog in the sample solution should be comparable to that in the standard series solutions (with equivalent mass concentration).
The retention times of methionine hydroxy analogues were consistent, with relative deviations within ±2.5%.
8.4.3 Quantitative
A standard curve was plotted with the mass concentration of methionine hydroxy analogues in the standard series solutions as the x-axis and the peak area as the y-axis.
The correlation coefficient of the standard curve should be no less than 0.99.The mass concentration of the analyte in the sample solution should be within the linear range of the standard curve. If it exceeds this range...
If the sample solution (8.1) is not suitable for quantification, it should be diluted and hydrolyzed with 10% acetonitrile solution (5.5) and then re-measured. For single-point calibration quantification, the analyte in the sample solution...
The mass concentration of the solution should not differ from that of the standard solution by more than 30%.
9.Experimental Data Processing
The content of methionine hydroxy analogues in the sample is expressed as a mass fraction w, with the value expressed as a percentage (%). Multi-point calibration is calculated according to formula (1); single-point calibration...
Calculate according to formula (2).
w=ρ×
V×f×106
m × 100
(1)
In the formula.
ρ --- The mass concentration of methionine hydroxy analogue in the sample solution obtained from the standard curve, in micrograms per milliliter.
(μg/mL);
V --- Total volume of the sample extraction solution, in milliliters (mL);
f --- The dilution factor of the sample solution after exceeding the linear range;
m --- Sample mass, in grams (g).
w=
A×ρs×V×f×106
As×m ×
100 (2)
In the formula.
A --- Peak area of the analyte, a methionine hydroxy analogue;
ρs --- the mass concentration of methionine hydroxy analogue in the standard solution, expressed in micrograms per milliliter (μg/mL);
V --- Total volume of the sample extraction solution, in milliliters (mL);
f --- the dilution factor of the sample solution;
...
