GB 5009.300-2025National food safety standard - Determination of L-carnitine in foods (English PDF)

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 °C~8 °C.

3.1.8 Acetylcarnitine transferase (CAT). Stored at 2 °C~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 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 °C±2 °C 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,

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.

4.1 Analytical balance. with a sensitivity of

0.001 g and

0.01 mg, respectively.

4.3 Constant temperature water bath device.

4.4 Spectrophotometer.

4.5 Electrothermal air blast drying oven.

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 °C 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.

10.2.6 Derivatization solution. Weigh

0.45 g of L-propionamide-beta-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,

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.

11.2 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.

11.9 Electrothermal air blast drying oven.

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 °C 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 °C 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.

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 °C±2 °C 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,

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

20.0 ng/mL, 500 ng/mL, 2000 ng/mL, 4000 ng/mL, and 5000 ng/mL, respectively.

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.

19.2 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 °C 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