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GB 5009.299-2024: National food safety standards--Determination of lactoferrin in food Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See step-by-step procedure Status: Valid
Similar standardsGB 5009.299-2024: National food safety standards--Determination of lactoferrin in food---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5009.299-2024GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National food safety standard - Determination of lactoferrin in food Issued on. FEBRUARY 08, 2024 Implemented on. AUGUST 08, 2024 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation. Table of Contents1 Scope... 3 2 Principle... 3 3 Reagents and materials... 3 4 Instruments and equipment... 5 5 Analysis procedure... 5 6 Expression of analysis results... 7 7 Precision... 7 8 Others... 7 Annex A Liquid chromatogram of bovine lactoferrin standard solutions... 8 Annex B Heparin affinity column validation method... 9 National food safety standard Determination of lactoferrin in food1 ScopeThis Standard specifies the high-performance liquid chromatography method for the determination of lactoferrin in food. This Standard applies to the determination of bovine lactoferrin content in pasteurized milk, prepared milk, milk-containing beverages, prepared milk powder and milk-based infant formula. This Standard does not apply to the determination of bovine lactoferrin in flavored fermented milk and soy-based infant formula.2 PrincipleThe bovine lactoferrin in the sample is extracted with phosphate buffer, enriched and purified with a heparin affinity column, then separated by a reversed-phase high- performance liquid chromatography column, detected with a UV detector at 280 nm, and quantified by the external standard method.3 Reagents and materialsUnless otherwise stated, the reagents used in this method are of analytical regent, and the water is Grade 1 water specified in GB/T 6682. 3.1.1 Acetonitrile (CH3CN). chromatographically pure. 3.1.5 Disodium hydrogen phosphate (Na2HPO4). 3.2 Preparation of reagents 3.2.1 Phosphate buffer I. Weigh 7.1 g of disodium hydrogen phosphate and 58.4 g of sodium chloride, add 900 mL of water to dissolve, adjust the pH to 8.0 ± 0.2 with phosphoric acid, add water to make the volume to 1000 mL, and freshly prepare each time before use. 3.3 Reference material Bovine lactoferrin reference material (CAS number. 146897-68-9). purity ≥ 95 %, iron content ≤ 35 mg/100 g, or a reference material certified by the country and awarded a reference material certificate. 3.4 Preparation of standard solutions 3.4.1 Bovine lactoferrin standard stock solution (5.0 mg/mL). Weigh 50 mg (accurate to 0.1 mg) of bovine lactoferrin reference material, dissolve it in water, transfer to a 10 mL volumetric flask and dilute to the mark, and mix well. Store at 0℃ ~ 4 ℃, it can be stored for 3 months. 3.5 Materials 3.5.1 Heparin affinity column. 1 mL, column capacity ≥ 2 mg, or one with equivalent performance (see Annex B for verification method). 3.5.2 Filter membrane, aqueous phase, 0.45 μm. 3.5.3 Aqueous syringe filter. polyethersulfone material, 0.45 μm.4 Instruments and equipment4.1 High-performance liquid chromatograph. equipped with a UV detector or equivalent. 4.5 Vortex oscillator. 4.6 Constant temperature water bath. 4.7 Solid phase extraction column device.5 Analysis procedure5.1 Pre-treatment of samples 5.1.1 Preparation of samples Take at least 200 g of a representative sample. If it is a liquid sample, shake well and store it in a container as a sample, store it at 0 ℃ ~ 4 ℃ for later use. If it is a solid sample, mix well and store it in a container as a sample, store it dry at room temperature for later use. 5.1.2 Extraction of samples 5.1.3 Purification of samples Before use, add 5.0 mL of phosphate buffer II to activate the heparin affinity column. Accurately pipette 10.0 mL of the above sample solution to pass through the column, control the flow rate at 1 mL/min ~ 3 mL/min. 5.2 Instrument reference conditions 5.2.1 Chromatographic column. C4, 5 μm, 300Å, 250 mm × 4.6 mm (inner diameter) or equivalent. 5.3 Plotting of standard curve Respectively inject the bovine lactoferrin standard series working solutions into the high-performance liquid chromatograph, determine the corresponding peak areas, and plot a standard curve with the concentrations of bovine lactoferrin in the standard working solutions as the abscissa and the peak areas as the ordinate. 5.4 Determination of sample solution Inject the sample solution into the high-performance liquid chromatograph to obtain the peak area, then obtain the concentration of bovine lactoferrin in the sample according to the standard curve.6 Expression of analysis resultsThe bovine lactoferrin content in the sample is calculated according to formula (1).7 PrecisionThe absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10 % of the arithmetic mean.8 OthersWhen the sampling volume of the liquid sample is 5.00 g and the constant volume is 25 mL, the detection limit of the liquid sample in this method is 0.3 mg/100 g and the quantification limit is 1 mg/100 g; when the sampling volume of the solid sample is 5.00 g and the constant volume is 50 mL, the detection limit is 2 mg/100 g and the quantification limit is 6 mg/100 g.Annex ALiquid chromatogram of bovine lactoferrin standard solutions The liquid chromatogram of bovine lactoferrin standard solutions (50.0 μg/mL) is shown in Figure A.1. Figure A.1 -- Liquid chromatogram of bovine lactoferrin standard solutions (50.0 μg/mL)Annex BHeparin affinity column validation method B.1 Column capacity verification Take 0.40 mL of the 5.0 mg/mL bovine lactoferrin standard stock solution and pass it through the heparin affinity column instead of the sample solution, operate according to 5.1.3, use a liquid chromatograph to measure the bovine lactoferrin content and calculate its recovery rate. Test at least 2 heparin affinity columns from the same batch. B.2 Result judgment If the recovery rate of bovine lactoferrin is ≥ 95 %, it is a usable product. ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al. 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