GB/T 46279-2025 Determination of amprolium hydrochloride，ethopabate and sulfaquinoxaline in feeds English, Anglais, Englisch, Inglés, えいご
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ICS 65.120 CCS B46
NATIONAL STANDARD OF THE PEOPLE'S REPUBLIC OF CHINA
GB/T 46279-2025
Replacing GB/T 8381.11-2005
Determination of Amprolium Hydrochloride, Ethopabate and Sulfaquinoxaline in Feeds
Issued on 2025-08-29 Implemented on 2026-03-01
Issued by State Administration for Market Regulation
Standardization Administration of China
Contents
1 Scope
2 Normative References
3 Terms and Definitions
4 High Performance Liquid Chromatography (HPLC) Method
5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method
Appendix A (Informative) HPLC Chromatogram of Mixed Standard Solution of Amprolium Hydrochloride, Ethopabate and Sulfaquinoxaline
1 Scope
This document specifies high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of amprolium hydrochloride, ethopabate and sulfaquinoxaline in feeds.
It is applicable to compound feeds, concentrated feeds, supplementary feeds, additive premixes and mixed feed additives.
Detection limits (HPLC method):
2 Normative References
The following documents are indispensable for the application of this standard. For dated references, only the edition cited applies. For undated references, the latest edition (including any amendments) applies.
GB/T 6682: Water for analytical laboratory use - Specifications and test methods
GB/T 20195: Animal feeding stuffs - Preparation of test samples
3 Terms and Definitions
No terms and definitions are defined in this document.
4 High Performance Liquid Chromatography (HPLC) Method
4.1 Principle
The target analytes in samples are extracted with methanol solution, defatted with n-hexane, purified through hydrophilic-lipophilic balance (HLB) solid-phase extraction columns, separated by HPLC, and quantified using tandem UV-fluorescence detectors with external standard method.
4.2 Reagents and Materials
Unless otherwise specified, use analytical-grade reagents.
4.2.1 Water: Grade 1 (GB/T 6682)
4.2.2 Methanol: HPLC grade
4.2.3 n-Hexane
4.2.4 Extraction solution: Methanol-water mixture (4:1, v/v)
4.2.6 Wash solution: 10 mL methanol diluted to 100 mL with water
4.2.7 Sodium heptanesulfonate solution (0.005 mol/L): Dissolve 1 g sodium heptanesulfonate in 800 mL water, add 6 mL triethylamine and 24 mL glacial acetic acid, dilute to 1,000 mL
Standard Solutions:
4.2.9 Stock solutions (1,000 μg/mL):
Amprolium hydrochloride (CAS: 137-88-2, ≥98.0%)
Ethopabate (CAS: 59-06-3, ≥98.0%)
Sulfaquinoxaline (CAS: 59-40-5, ≥98.0%)
Prepared in methanol, stored at <-18°C (validity: 9 months for amprolium/ethopabate; 6 months for sulfaquinoxaline)
4.2.10 Intermediate solutions (100 μg/mL): Diluted from stock solutions, stored at <-18°C (validity: 3 months)
4.2.11 Mixed calibration standards (prepared fresh):
4.2.12 SPE columns: HLB (60 mg/3 mL) or equivalent
4.2.13 Microporous membrane: 0.45 μm (organic phase)
4.3 Apparatus
4.3.1 HPLC system with UV/DAD and fluorescence detectors (connected in series)
4.3.2 Analytical balance (0.1 mg/0.01 mg precision)
4.3.3 Vortex mixer
4.3.4 Ultrasonic cleaner
4.3.5 Centrifuge (≥5,000 rpm)
4.3.6 SPE device
4.3.7 Nitrogen evaporator
4.4 Sample Preparation
Prepare samples according to GB/T 20195: ≥200 g, ground to pass through 0.425 mm sieve, homogenized, stored in glass bottles.
4.5 Procedure
4.5.1 Extraction
Weigh 2 g (accurate to 0.1 mg) into 50 mL centrifuge tube
Add 25 mL extraction solution, vortex, ultrasonicate 30 min (vortex every 10 min)
Centrifuge at 5,000 rpm for 5 min, collect supernatant
Repeat extraction, combine supernatants (V)
4.5.2 Purification
4.5.2.1 Defatting
Transfer 8 mL supernatant, add 5 mL n-hexane, vortex 1 min, centrifuge
Collect 5 mL lower phase (V1; for premixes: collect 1 mL)
Evaporate to near-dryness under N₂ at 40°C
Reconstitute in 3 mL phosphate buffer, centrifuge
4.5.2.2 Solid-Phase Extraction
