Detail of GB 5009.300-2025

Introduction of GB 5009.300-2025

GB 5009.300-2025 National food safety standard - Determination of L-carnitine in foods 1 Scope This standard specifies the methods for determination of L-carnitine in foods. Method I “Spectrophotometry” is applicable to the determination of L-carnitine in foods for infants and young children (excluding partially hydrolyzed milk protein formula, deeply hydrolyzed milk protein formula and amino acid formula in formula and formulas for special medical purposes intended for infants) and dairy products. Method II "Precolumn derivatization-High performance liquid chromatography” is applicable to the determination of L-carnitine in foods (excluding formula and formulas for special medical purposes intended for infants and foods for special medical purpose). Method III "Liquid chromatography-Tandem mass spectrometry” is applicable to the determination of L-carnitine in formula foods for infants and young children and foods for special medical purpose. Method I Spectrophotometry 2 Principle The specimen is extracted with water, and filtered after the protein is precipitated with perchloric acid. The bound L-carnitine in the filtrate is is released through alkaline saponification and reacts with AcetylCoA under the catalysis of carnitine-acetyltransferase to produce acetyl carnitine and free CoA. Free CoA reacts with 5, 5'-disulfobis (2-nitrobenzoic acid) to produce yellow substance whose color intensity is proportional to the content of free CoA. The content of L-carnitine in the specimen may be indirectly determined due to the equimolar reaction between free CoA and L-carnitine. 3 Reagents and materials Unless otherwise specified, only analytically pure reagents and Grade 3 water specified in GB/T 6682 are used for the purpose of this method. 3.1 Reagents 3.1.1 Perchloric acid (HClO4). 3.1.2 Sodium hydroxide (NaOH). 3.1.3 Potassium hydroxide (KOH). 3.1.4 5, 5'-dithiobis (2-nitrobenzoic acid) (C14H8N2O8S2). 3.1.5 N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (C8H18N2O4S). 3.1.6 Ethylenediamine tetraacetic acid disodium salt (C10H14N2Na2·2H2O). 3.1.7 AcetylCoA: Purity ≥ 85%, stored at 2–8℃. 3.1.8 Carnitine-acetyltransferase (CAT): Stored at 2–8°C. 3.2 Reagent preparation 3.2.1 Perchloric acid solution (13%): Pipette 13 mL of perchloric acid, and dilute to 100 mL with water. 3.2.2 Perchloric acid solution (3%): Pipette 3 mL of perchloric acid, and dilute to 100 mL with water. 3.2.3 Sodium hydroxide solution (10 mol/L): Weigh 40 g of sodium hydroxide, dissolve it in water, and dilute the solution to 100 mL after it gets cool. 3.2.4 Potassium hydroxide solution (4 mol/L): Weigh 22.4 g of potassium hydroxide, dissolve it in water, and dilute to 100 mL after it gets cool. 3.2.5 Color-substrate stock solution: Weigh 50 mg of 5, 5'-dithiobis (2-nitrobenzoic acid), 5.96 g of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, and 185 mg of ethylenediamine tetraacetic acid disodium salt respectively. Dissolve them in 30 mL of water, adjust the pH to 7.4–7.6 with 10 mol/L sodium hydroxide solution, and then dilute to 50 mL with water. Store the solution at 4℃, with a shelf life of 3 months. 3.2.6 Color-substrate working solution: Pipette 5 mL of color-substrate stock solution and dilute to 25 mL with water. 3.2.7 AcetylCoA solution: Weigh 20 mg of AcetylCoA and dissolve it in 2 mL of water. Prepare it immediately before use. 3.2.8 Carnitine-acetyltransferase (CAT) solution: Pipette 100 μL of carnitine-acetyltransferase suspension, add water to 2 mL, and mix well. Prepare it immediately before use. 3.3 Standard L-carnitine standard (C7H15NO3, CAS No.: 541-15-1): With purity ≥ 98%, or the reference material certified by the state and granted with the reference material certificate. Dry the standard in an electrothermal blowing dry box at 102±2℃ for 2 h before use. 3.4 Preparation of standard solutions 3.4.1 L-carnitine standard stock solution (1 mg/L): Accurately weigh 10 mg (accurate to 0.01 mg) of L-carnitine and place it in a 10-mL beaker. Dissolve it with 5 mL of water, transfer it to a 10-mL volumetric flask, dilute to the scale with water, and mix well. Transfer the solution to a brown container and store it at 4℃ away from light, with a shelf life of 3 months. 3.4.2 L-carnitine standard intermediate solution (80 μg/ mL): Pipette 2.00 mL of L-carnitine standard stock solution and place it into a 25-mL volumetric flask, dilute to the scale with water, and mix well. Transfer the solution to a brown container and store it at 4℃ away from light, with a shelf life of 1 month. 3.4.3 L-carnitine standard working solution series: Pipette 0.125 mL, 0.50 mL, 1.00 mL, 2.00 mL, 3.00 mL, and 5.00 mL of L-carnitine standard intermediate solution respectively and place them in 25-mL volumetric flasks, dilute to the scale with water, and mix well. The mass concentrations of the standard working solution series are 0.4 μg/mL, 1.6 μg/mL, 3.2 μg/mL, 6.4 μg/mL, 9.6 μg/mL and 16 μg/mL respectively. Prepare it immediately before use. 4 Instruments and apparatus

Contents of GB 5009.300-2025

Contents Foreword 1 Scope Method I Spectrophotometry 2 Principle 3 Reagents and materials 4 Instruments and apparatus 6 Expression of analysis results 7 Precision 8 Others Method II Precolumn derivatization-High performance liquid chromatography 9 Principle 10 Reagents and materials 11 Instruments and apparatus 12 Analytical procedures 13 Expression of analysis results 14 Precision 15 Others Method III Liquid chromatography-Tandem mass spectrometry 16 Principle 17 Reagents and materials 18 Instruments and apparatus 19 Analytical procedures 20 Expression of analysis results 21 Precision 22 Others Annex A L-carnitine chromatogram