1 Instruments and Conditions
LC-10T High Performance Liquid Chromatograph (HPLC); SPD-10T UV Detector (detection wavelength: 240 nm), column temperature: 40℃; VI2010 Chromatography Workstation; VI-11 Manual Injection Valve (made in Japan); Vertex C18 Liquid Chromatography Column; Column Oven; Centrifuge (rotational speed: ≥8000 rpm); Electronic Balance; Nitrogen Evaporator; Solid-Phase Extraction (SPE) Device.
(1) Chromatographic column: Vertex C18 (4.6×250 mm); Buffer solution: 10 mM citric acid, 10 mM sodium heptanesulfonate; Mobile phase: Buffer solution: acetonitrile = 85:15; Flow rate: 1.0 mL/min.
(2) Chromatographic column: Vertex C8 (4.6×250 mm); Mobile phase: Buffer solution: acetonitrile = 85:15; Buffer solution: 10 mM citric acid, 10 mM sodium octanesulfonate (adjust pH to 3.0); Flow rate: 1.0 mL/min.
Ion-exchange solid-phase extraction column: Agela Clearnert™ PCX
2 Reagents and Samples
Pet food samples; Methanol, acetonitrile; Ammonia water, lead acetate, trichloroacetic acid (TCA); Melamine standard, citric acid, sodium octanesulfonate. Among them, methanol is of chromatographic grade, and other reagents are of analytical grade.
3 Experimental Methods
3.1 Sample Pretreatment Method
(1) Preparation of Standard Sample
Weigh 50 mg of melamine standard, dissolve it in 20% methanol, and dilute to a constant volume of 50 mL to obtain a standard solution with a concentration of 1000 ppm. When in use, dilute it to the required concentration with the extraction solution (0.1% trichloroacetic acid).
(2) Extraction
Weigh 5 g of the feed sample, add 50 mL of 0.1% trichloroacetic acid extraction solution, mix thoroughly, add 2 mL of 2% lead acetate solution, and perform ultrasonic treatment for 20 minutes.
Then transfer a portion of the solution to a 10 mL centrifuge tube, centrifuge at 8000 rpm/min for 10 minutes, and take 3 mL of the supernatant to pass through a mixed-mode cation exchange cartridge (PCX cartridge).
(3) Purification (PCX Cartridge, 60 mg/3 mL)
- a) Activation and equilibration: 3 mL methanol, 3 mL water
- b) Sample loading: Add 3 mL of the extraction solution
- c) Rinsing: 3 mL water; 3 mL methanol; Discard the rinsing solution and dry the cartridge by suction.
- d) Elution: Elute with 5 mL of 5% ammoniated methanol (v/v). (Preparation of 5% ammoniated methanol: 5 mL ammonia water + 95 mL methanol).
- e) Concentration: Dry with nitrogen at 50℃, dilute to a constant volume of 2 mL with 20% methanol/water, and conduct HPLC analysis or GC/MS analysis after derivation.
3.2 HPLC Detection Method
Melamine is a strongly polar compound, which has poor retention on traditional reversed-phase C18 columns. Ion-pair reagent chromatography is required to achieve good retention and separation. According to the melamine detection method issued by the U.S. Food and Drug Administration (FDA) and the melamine detection method published by the Ministry of Agriculture of the People’s Republic of China, using Vertex series hydrophilic chromatographic columns can achieve good separation effects. The analytical chromatogram is as follows:
3.2.1 Melamine HPLC-UV Detection Method
(a) Chromatographic column: Vertex C8 4.6×250 mm; Standard: FDA method; Mobile phase: Buffer solution: acetonitrile = 85:15; Buffer solution: 10 mM citric acid, 10 mM sodium octanesulfonate (adjust pH to 3.0); Flow rate: 1.0 mL/min; Column temperature: 40℃; Wavelength: 240 nm
(b) Chromatographic column: Vertex C18 4.6×250 mm; Standard: Standard method issued by the Ministry of Agriculture of the People’s Republic of China; Buffer solution: 10 mM citric acid, 10 mM sodium heptanesulfonate; Mobile phase: Buffer solution: acetonitrile = 85:15; Flow rate: 1.0 mL/min; Column temperature: 40℃; Wavelength: 240 nm