GB 5009.210-2023National food safety standard - Determination of pantothenic acid in foods (English PDF)

3.1 Reagents

3.1.1 Hydrochloric acid (HCl).

3.1.2 Phosphoric acid (H3PO4).

3.1.3 Potassium dihydrogen phosphate (KH2PO4).

3.1.4 Zinc sulfate heptahydrate (ZnSO4 . 7H2O).

3.1.5 Acetonitrile (CH3CN). chromatographically pure.

3.1.6 alpha-amylase. enzyme activity >=1.5 U/mg.

3.2 Preparation of reagents

3.2.1 Hydrochloric acid (0.1 mol/L). Pipette 8.3 mL of hydrochloric acid into 800 mL

of water. Add water to dilute to 1000 mL. Mix well.

3.2.3 Zinc sulfate solution (0.5 mol/L). Weigh 14.4 g of zinc sulfate heptahydrate. Add

water to dissolve and dilute to 100 mL.

3.2.4 Potassium dihydrogen phosphate solution (0.02 mol/L). Weigh 2.722 g of

potassium dihydrogen phosphate. Add 500 mL water to dissolve. Use phosphoric acid

to adjust pH to 3.0+/-0.1.

3.3 Standard product

D-calcium pantothenate standard product (C18H32CaN2O10, CAS number. 137-08-6).

purity >=99%, or standard product certified by the country and awarded a reference

material certificate.

3.4 Preparation of standard solution

3.4.1 Pantothenic acid standard stock solution (500 µg/mL). Accurately weigh 136 mg

of D-calcium pantothenate standard (accurate to 0.1 mg). Add water to dissolve and

transfer to a 250 mL volumetric flask. Dilute to volume. Mix well. The standard stock

solution shall be stored in the dark at -18°C and below. It can be kept for 3 months.

4.1 Balance. resolution is 0.1 mg.

4.2 Constant temperature oscillation water bath. oscillation frequency is 100 r/min+/-20 r/min.

4.3 Ultrasonic oscillator.

4.4 pH meter. accuracy is +/-0.01.

4.5 Centrifuge. rotation speed >=8000 r/min.

4.6 0.45 µm filter.

4.7 High performance liquid chromatograph. with UV detector or diode array detector.

5.1 Specimen preparation

The solid specimen is crushed and mixed evenly. Carbonated drinks require ultrasonic

removal of carbon dioxide, and other liquid specimens need to be shaken well.

5.2 Specimen extraction

5.2.1 Beverages, nutrient supplements

Accurately weigh or measure an appropriate amount of specimen to the nearest 0.001

g. Generally, the solid specimen is 0.2 g~2 g. The liquid specimen is 10 g~20 g. Place

in a 50 mL Erlenmeyer flask. Add about 30 mL of 40 degrees C~50 degrees C warm water and conduct

ultrasonic extraction for 20 min.

5.2.2 Infant formula food (except infant formula food for special medical

purposes), infant supplementary food, dairy products

Accurately weigh an appropriate amount of specimen to the nearest 0.001 g. Generally,

the solid specimen is about 5 g.

5.3 Reference chromatographic conditions for liquid phase

The reference chromatographic conditions for the liquid phase are as follows.

5.4 Determination

5.4.1 Determination of standard curve

Carry out chromatographic analysis of the pantothenic acid standard working solution

in sequence (see Figure A.1 in Annex A for the standard chromatogram). Draw a

standard curve with the concentration of the standard working solution as the abscissa

and the peak area as the ordinate.

5.4.2 Determination of specimen solution

The specimen measurement liquid is subjected to chromatographic analysis. Find the

corresponding pantothenic acid concentration from the standard curve based on the

sample peak area.

8.1 For solid specimens, when the sampling volume is 5 g and the volume is adjusted

to 50 mL, the detection limit is 0.025 mg/100 g and the quantitation limit is 0.08 mg/100 g.

8.2 For liquid specimens, when the sampling volume is 20 g and the volume is adjusted

to 50 mL, the detection limit is 0.0063 mg/100g and the quantification limit is 0.02 mg/100g.

10.1 Reagents

10.1.1 Acetonitrile (CH3CN). chromatographically pure.

10.1.2 Formic acid (HCOOH). chromatographically pure.

10.1.3 Hydrochloric acid (HCl).

10.1.4 Glacial acetic acid (C2H4O2).

10.1.5 Zinc acetate [Zn (CH3COO)2].

10.1.6 Potassium ferrocyanide [K4Fe (CN)6].

10.1.7 Trishydroxymethylaminomethane (C4H11NO3).

10.2 Preparation of reagents

10.2.1 Formic acid solution (0.1%). Pipette 1 mL of formic acid. Use water to dilute to 1000 mL. Mix well.

10.2.2 Zinc acetate solution (300 g/L). Weigh 30.0 g of zinc acetate. Use water to

dissolve and dilute to 100 mL.

10.2.3 Potassium ferrocyanide solution (150 g/L). Weigh 15.0 g of potassium

ferrocyanide. Use water to dissolve and dilute to 100 mL.

10.2.5 Sodium bicarbonate solution (0.1 mol/L). Weigh 0.84 g of sodium bicarbonate.

Add water to dissolve and dilute to 100 mL. Mix well.

10.2.9 Resin activation. Weigh 100 g of anion exchange resin into a 2 L Erlenmeyer

flask. Add 1 L of 1 mol/L hydrochloric acid solution and shake thoroughly on an

oscillator for 10 min. Filter through a Buchner funnel lined with filter paper. Transfer

the anion exchange resin back to the Erlenmeyer flask. Then add 1 L of 1 mol/L

hydrochloric acid solution, shake repeatedly for 10 min, and filter. Add 1 L of water to

the anion exchange resin and shake for 10 min. filter. Repeat washing with water 10

times. Transfer to the Erlenmeyer flask.

10.3 Standard product

10.3.1 D-calcium pantothenate standard (C18H32CaN2O10, CAS No.. 137-08-6). purity

>=99%, or a standard certified by the country and awarded a reference material certificate.

10.3.2 13C6, 15N2-Calcium Pantothenate (13C6C12H32Ca15N2O10, CAS No.. 356786-94-

2). Purity >=97%.

10.4 Preparation of standard solution

10.4.1 Pantothenic acid standard stock solution (500 µg/mL). Accurately weigh 136 mg

of D-calcium pantothenate standard (accurate to 0.1 mg). Add water to dissolve and

transfer to a 250 mL volumetric flask. Dilute to volume. Mix well. Standard stock

solutions shall be stored in the dark at -18°C and below.

11.1 Liquid chromatography-tandem mass spectrometer. equipped with electrospray

ionization source (ESI).

11.2 Analytical balance. resolution is 0.1 mg and 0.01 g, respectively.

11.3 pH meter. accuracy is +/-0.01.

11.6 Pressure steam sterilizer. 121 degrees C.

11.8 Vortex mixer.

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