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GB 5009.300-2025: National food safety standard - Determination of L-carnitine in the foods 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 5009.300-2025: National food safety standard - Determination of L-carnitine in the foods---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/GB5009.300-2025GB NATIONAL STANDARD OF THE PEOPLE'S REPUBLIC OF CHINA National food safety standard - Determination of L-carnitine in foods Issued on: MARCH 16, 2025 Implemented on: SEPTEMBER 16, 2025 Issued by. National Health Commission of the People's Republic of China; State Administration for Market Regulation. Table of ContentsForeword... 3 1 Scope... 4 Method I. Spectrophotometric method... 4 2 Principle... 4 3 Reagents and materials... 4 4 Instruments and equipment... 6 5 Analysis steps... 6 6 Presentation of analysis results... 7 7 Precision... 8 8 Others... 8 Method II. Pre-column derivatization-high performance liquid chromatography... 8 9 Principle... 8 10 Reagents and materials... 8 11 Instruments and equipment... 10 12 Analysis steps... 11 13 Presentation of analysis results... 13 14 Precision... 13 15 Others... 14 Method III. Liquid chromatography-tandem mass spectrometry... 14 16 Principle... 14 17 Reagents and materials... 14 18 Instruments and equipment... 16 19 Analysis steps... 16 20 Presentation of analysis results... 18 21 Precision... 20 22 Others... 20 Appendix A L-carnitine chromatogram... 21 National food safety standard - Determination of L-carnitine in foods1 ScopeThis standard specifies the determination method of L-carnitine in foods. Method I, the spectrophotometric method, is applicable to the determination of L- carnitine in infant foods (excluding partially hydrolyzed milk protein formula, deeply hydrolyzed milk protein formula and amino acid formula in infant formula foods for special medical purposes) and dairy products. Method II, the pre-column derivatization-high performance liquid chromatography, is suitable for the determination of L-carnitine in foods (excluding infant formula foods for special medical purposes and formula foods for special medical purposes). Method III, the liquid chromatography-tandem mass spectrometry, is suitable for the determination of L-carnitine in infant formula foods and formula foods for special medical purposes. Method I. Spectrophotometric method2 PrincipleThe test specimen is extracted with water and filtered after protein precipitation with perchloric acid. The bound L-carnitine in the filtrate is released by alkaline saponification and reacts with acetyl coenzyme A under the catalysis of acetylcarnitine transferase to generate acetylcarnitine and free coenzyme A. Free coenzyme A reacts with 5,5'-dithiobis(2-nitrobenzoic acid) to generate a yellow substance, the color depth of which is proportional to the content of free coenzyme A. Since free coenzyme A and L-carnitine are in an equimolar reaction relationship, the content of L-carnitine in the test specimen can be indirectly calculated.3 Reagents and materialsUnless otherwise specified, all reagents used in this method are analytically pure, and the water is grade 3 water as specified in GB/T 6682. 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 Disodium ethylenediaminetetraacetate (C10H14N2Na2 • 2H2O). 3.1.7 Acetyl-CoA. With a purity of ≥85%, and stored at 2 ℃~8 ℃. 3.1.8 Acetylcarnitine transferase (CAT). Stored at 2 ℃~8 ℃. 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 and dissolve it in water. After cooling, dilute to 100 mL. 3.2.4 Potassium hydroxide solution (4 mol/L). Weigh 22.4 g of potassium hydroxide and dissolve it in water. After cooling, dilute to 100 mL. 3.2.5 Color development 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 disodium ethylenediaminetetraacetate respectively, and 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 at 4 °C, with a shelf life of 3 months. 3.2.6 Color development working solution. Take 5 mL of color development stock solution and dilute it to 25 mL with water. 3.2.7 Acetyl-CoA solution. Weigh 20 mg of acetyl CoA and dissolve it in 2 mL of water. Prepare it fresh just before use. 3.2.8 Acetylcarnitine transferase (CAT) solution. Pipette 100 μL of acetylcarnitine transferase suspension, add water to 2 mL, and mix well. Prepare it fresh just before use. 3.3 Standards L-carnitine standard (C7H15NO3, CAS No.. 541-15-1). With a purity of ≥98%, or a reference material certified by the nation and awarded with a reference material certificate. Dry in an electrothermal air blast drying oven at 102 ℃±2 ℃ for 2 hours before use. 3.4 Preparation of standard solution 3.4.1 L-carnitine standard stock solution (1 mg/mL). Accurately weigh 10 mg (accurate to 0.01 mg) of L-carnitine and place it in a 10 mL beaker. Add 5 mL of water to dissolve it. Transfer all of it to a 10 mL volumetric flask, dilute to the mark with water, and mix well. Transfer the solution to a brown container and store it at 4 °C away from light. The shelf life is 3 months. 3.4.2 L-carnitine standard intermediate solution (80 μg/mL). Pipette 2.00 mL of L- carnitine standard stock solution into a 25 mL volumetric flask, dilute to the mark with water, and mix well. Transfer the solution to a brown container and store at 4 °C away from light. The shelf life is 1 month. 3.4.3 L-carnitine series standard working solution. 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 into 25 mL volumetric flasks, dilute to the mark with water, and mix well. The mass concentrations of this series of standard working solutions 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 them fresh just before use.4 Instruments and equipment4.1 Analytical balance. with a sensitivity of 0.001 g and 0.01 mg, respectively. 4.2 pH meter. 4.3 Constant temperature water bath device. 4.4 Spectrophotometer. 4.5 Electrothermal air blast drying oven.5 Analysis steps5.1 Test specimen processing 5.1.1 Solid test specimen. Accurately weigh 5.0 g (accurate to 0.001 g) of the mixed test specimen into a beaker, dissolve it with 30 mL of 40 ℃ warm water, and transfer it to a 100 mL volumetric flask. Add 10 mL of perchloric acid solution (13%), mix well, c -- mass concentration of L-carnitine in the sample solution obtained from the standard curve, in micrograms per milliliter (μg/mL); 50 -- the metered volume of the sample solution, in milliliters (mL); m -- the sampling mass of the test specimen, in grams (g); V -- the volume of filtrate, in milliliters (mL); 10 -- conversion factor; 100 -- the metered volume of the sample solution, in milliliters (mL). The calculation result is rounded to 2 decimal places.7 PrecisionThe absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.8 OthersThe detection limit of the solid test specimen method is 0.6 mg/100 g, and the quantification limit is 2 mg/100 g. The detection limit of the liquid test specimen method is 0.12 mg/100 g, and the quantification limit is 0.4 mg/100 g. Method II. Pre-column derivatization-high performance liquid chromatography9 PrincipleThe L-carnitine in the test specimen is extracted with dilute hydrochloric acid, saponified with alkali, and precipitated with acetonitrile for protein, and then purified with a cation exchange solid phase extraction column. The L-carnitine in the purified solution is derivatized with L-propionamide-β-naphthylamine under the catalysis of butyl chloroformate and triethylamine, and the derivatives are determined by high- performance liquid chromatography and quantified by the external standard method.10 Reagents and materialsUnless otherwise specified, all reagents used in this method are of analytical grade, and dissolve it in water, and dilute to 1000 mL. 10.2.5 4% ammonia methanol solution. Pipette 4 mL of ammonia and dilute to 100 mL with methanol. 10.2.6 Derivatization solution. Weigh 0.45 g of L-propionamide-β-naphthylamine, dissolve it in anhydrous ethanol, and dilute to 100 mL. Store at -18 °C. The shelf life is 2 weeks. 10.2.7 Catalyst I. Weigh 0.5 g of triethylamine and dilute to 100 mL with dichloromethane. 10.2.8 Catalyst II. Weigh 0.5 g of butyl chloroformate and dilute to 100 mL with dichloromethane. 10.2.9 Sodium bicarbonate solution (50 mmol/L). Weigh 0.42 g of sodium bicarbonate, dissolve it in water, and dilute to 100 mL. 10.2.10 Phosphate buffer solution. Weigh 3.4 g of dipotassium hydrogen phosphate trihydrate and 0.43 g of sodium octane sulfonate, dissolve them in 900 mL of water, adjust the pH to 2.5~2.8 with phosphoric acid, and dilute to 1000 mL with water. 10.3 Standards Same as 3.3. 10.4 Preparation of standard solution 10.4.1 L-carnitine standard stock solution (1 mg/mL). same as 3.4.1. 10.4.2 L-carnitine standard intermediate solution (10 μg/mL). Pipette 0.25 mL of L- carnitine standard stock solution into a 25 mL volumetric flask, dilute to the mark with methanol, and mix. Prepare it fresh just before use. 10.4.3 L-carnitine series standard working solutions. Pipette 0.20 mL, 0.50 mL, 1.00 mL, 2.00 mL, 5.00 mL, and 10.0 mL of L-carnitine standard intermediate solution into 10 mL volumetric flasks, dilute to the mark with methanol, and mix well. The mass concentrations of this series of standard working solutions are 0.200 μg/mL, 0.500 μg/mL, 1.00 μg/mL, 2.00 μg/mL, 5.00 μg/mL, and 10.0 μg/mL, respectively. Prepare them fresh just before use.11 Instruments and equipment11.1 High-performance liquid chromatograph. equipped with a UV detector or diode array detector. 11.2 Analytical balance. with a sensitivity of 0.001 g and 0.01 mg respectively. 11.3 Vortex mixer. 11.4 Centrifuge, with a speed of ≥4000 r/min. 11.5 Nitrogen concentrator. 11.6 Ultrasonic cleaning apparatus. 11.7 Constant temperature water bath. 11.8 pH meter. 11.9 Electrothermal air blast drying oven.12 Analysis steps12.1 Test specimen preparation Liquid test specimens shall be shaken evenly; semi-solid test specimens and powdered test specimens with uniform matrix can be used directly for extraction; other test specimens need to be homogenized or crushed evenly. 12.2 Test specimen extraction 12.2.1 Solid test specimen. Weigh 5.0 g (accurate to 0.001 g) of the test specimen and place it in a 150 mL conical flask, add 20 mL of hydrochloric acid solution (0.1 mol/L), vortex mix for 1 min, and ultrasonicate for 5 min to dissolve the sample; add 5 mL of potassium hydroxide solution (1 mol/L), vortex mix, saponify in a 60 ℃ water bath for 30 min, and cool to room temperature; add 5 mL of hydrochloric acid solution (1 mol/L), vortex mix for 1 min, transfer to a 50 mL volumetric flask, dilute to the mark with hydrochloric acid solution (0.1 mol/L), and shake well. Pipette 1.00 mL of the extract, add 9.00 mL of acetonitrile, vortex mix for 1 min, centrifuge at 4000 r/min for 5 min, and the obtained supernatant is to be purified. NOTE. For the test specimen with L-carnitine content greater than 1000 mg/kg, the test specimen shall first be dissolved in water and diluted to a L-carnitine content less than or equal to 1000 mg/kg, and then extracted according to the extraction method in 12.2.1. 12.2.2 Liquid test sample. Weigh 25.0 g (accurate to 0.001 g) of the test specimen into a 150 mL conical flask, add 5 mL of potassium hydroxide solution (1 mol/L), vortex mix, saponify in a 60 ℃ water bath for 30 min, and cool to room temperature; add 5 mL of hydrochloric acid solution (1 mol/L), vortex mix for 1 min, transfer to a 50 mL volumetric flask, dilute to the mark with hydrochloric acid solution (0.1 mol/L), and shake well. Pipette 1.00 mL of the extract, add 9.00 mL of acetonitrile, vortex mix for repeatability conditions shall not exceed 10% of the arithmetic mean.15 OthersThe detection limit of the solid test specimen method is 0.6 mg/100 g, and the quantification limit is 2 mg/100 g. The detection limit of the liquid test specimen method is 0.12 mg/100 g, and the quantification limit is 0.4 mg/100 g. Method III. Liquid chromatography-tandem mass spectrometry16 PrincipleThe test specimen is dissolved or diluted with water, extracted by microwave, and then determined by liquid chromatography-tandem mass spectrometry and quantified by the isotope internal standard method.17 Reagents and materialsUnless otherwise specified, all reagents used in this method are of analytical grade, and the water is first-grade water as specified in GB/T 6682. 17.1 Reagents and materials 17.1.1 Nitric acid (HNO3). analytical pure or higher. 17.1.2 Acetonitrile (C2H3N). chromatographically pure. 17.1.3 Ammonium formate (NH4COOH). chromatographically pure. 17.1.4 Formic acid (HCOOH). chromatographically pure. 17.1.5 Microporous filter membrane. 0.22 μm, water system. 17.2 Reagent preparation 17.2.1 Mobile phase A [5 mmol/L ammonium formate acetonitrile-water solution (containing 0.2% formic acid)]. Weigh 0.63 g of ammonium formate, dissolve it in 1 L of water, then add 1 L of acetonitrile and 4 mL of formic acid, mix well, and obtain the mobile phase. 17.2.2 Mobile phase B [30 mmol/L ammonium formate acetonitrile-water solution (containing 0.2% formic acid)]. Weigh 3.78 g of ammonium formate, dissolve it in 1 L of water, then add 1 L of acetonitrile and 4 mL of formic acid, mix well, and obtain mobile phase B. 17.3 Standards L-carnitine standard (C7H15NO3, CAS No.. 541-15-1). with a purity of ≥98%, or a reference material certified by the nation and awarded with a reference material certificate. Dry at 102 ℃±2 ℃ for 2 hours before use. L-carnitine isotope internal standard. L-carnitine-d3 (C7H12D3NO3, CAS No.. 350818- 62-1), with a purity of ≥95%, or a reference material certified by the nation and awarded with a reference material certificate. 17.4 Preparation of standard solutions 17.4.1 L-carnitine standard stock solution (20 mg/mL). Accurately weigh 200 mg (accurate to 0.1 mg) of L-carnitine and place it in a 10 mL beaker. Add 5 mL of water to dissolve it. Transfer all of it to a 10 mL volumetric flask, dilute to the mark with water, and mix well. Transfer the solution to a brown container and store it at 4 °C away from light. The shelf life is 6 months. 17.4.2 L-carnitine standard working solution. Pipette 0.005 mL, 0.01 mL, 0.25 mL, 1.00 mL, 2.00 mL, and 2.50 mL of L-carnitine standard stock solution (20 mg/mL) into 10 mL volumetric flasks, dilute to the mark with water, and mix well. The mass concentrations of this series of standard working solutions are 10.0 μg/mL, 20.0 μg/mL, 500 μg/mL, 2000 μg/mL, 4000 μg/mL, and 5000 μg/mL, respectively. L-carnitine standard working solutions (10.0 μg/mL and 20.0 μg/mL) must be prepared fresh just before use; L-carnitine standard working solutions (500 μg/mL, 2000 μg/mL, 4000 μg/mL, and 5000 μg/mL) must be stored at 4 °C away from light, with a shelf life of 3 months. 17.4.3 Isotope internal standard solution (200 μg/mL). Accurately weigh 10 mg (accurate to 0.1 mg) of L-carnitine-d3 and place it in a 10 mL beaker. Add 5 mL of water to dissolve it. Transfer all of it to a 50 mL volumetric flask, dilute to the mark with water, and mix well. Transfer the solution to a brown container and store it at 4 °C away from light. The shelf life is 3 months. 17.4.4 L-carnitine series standard working solutions. Pipette 50.0 μL of the above L- carnitine standard working solutions of each concentration into microwave digestion tanks respectively, add 50.0 μL of isotope internal standard solution, and operate in the same way as in 19.2.The mass concentrations of this series of standard working solutions are 10.0 ng/mL, 20.0 ng/mL, 500 ng/mL, 2000 ng/mL, 4000 ng/mL, and 5000 ng/mL, respectively.18 Instruments and equipment18.1 Liquid chromatography-tandem triple quadrupole mass spectrometer. equipped with an electrospray ionization (ESI). 18.2 Analytical balance. with a sensitivity of 0.001 g and 0.1 mg respectively. 18.3 Vortex mixer. 18.4 Ultrasonic cleaning apparatus. 18.5 Microwave digestion apparatus. equipped with a polytetrafluoroethylene digestion inner tank. 18.6 Electrothermal air blast drying oven.19 Analysis steps19.1 Test specimen preparation Liquid test specimens shall be shaken evenly; semi-solid test specimens and powdered test specimens with uniform matrix can be used directly for extraction; other test specimens need to be homogenized or crushed evenly. 19.2 Test specimen extraction Solid test specimen. Weigh 5.0 g (accurate to 0.001 g) of test specimen into a 50 mL centrifuge tube, add 40.0 g of water, vortex mix for 1 min, ultrasonicate for 5 min to dissolve the test specimen, cool to room temperature, shake well, and obtain the solid test specimen solution, then weigh it. Weigh 1.0 g (accurate to 0.001 g) of solid test specimen solution into a microwave digestion tank, add 50.0 μL of isotope internal standard solution, 5 mL of water and 2.5 mL of nitric acid, vortex mix well, tighten the tank cover, and place it in a microwave digestion instrument for extraction (rise the temperature to 120 ℃ in 10 min and maintain for 40 min, with the maximum power of 1000 W). Take it out after cooling, slowly open the tank cover to exhaust, dilute with water to 25 mL, mix well, filter with a 0.22 μm microporous filter membrane, and set aside. Take 0.50 mL of the filtrate, add 0.50 mL of acetonitrile, mix well, and use a liquid chromatography-tandem mass spectrometer for determination. Liquid test specimen. Weigh 1.0 g (accurate to 0.001 g) of test specimen into the microwave digestion inner tank, add 50 μL of isotope internal standard solution, 5 mL of water and 2.5 mL of nitric acid, vortex mix, tighten the tank cover, and place it in a microwave digestion instrument for extraction (rise the temperature to 120 ℃ in 10 min and maintain for 40 min, with the maximum power of 1000 W). Take it out after cooling, ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al. 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