GB 5009.210-2023 English PDF

GB 5009.210-2023: National food safety standard - Determination of pantothenic acid in foods
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GB 5009.210-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standards -- Determination of
pantothenic acid in food
ISSUED ON. SEPTEMBER 06, 2023
IMPLEMENTED ON. MARCH 06, 2024
Issued by. National Health Commission of the People's Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
2 Principle... 4
3 Reagents and materials... 4
4 Instruments and equipment... 5
5 Analysis steps... 6
6 Expression of analysis results... 7
7 Precision... 8
8 Other... 8
9 Principle... 8
10 Reagents and materials... 8
11 Instruments and equipment... 11
12 Analysis steps... 11
13 Expression of analysis results... 14
14 Precision... 15
15 Other... 15
16 Principle... 15
17 Reagents and materials... 15
18 Instruments and equipment... 18
19 Preparation and preservation of bacterial strains... 18
20 Analysis steps... 19
21 Expression of analysis results... 23
22 Precision... 26
23 Other... 26
Annex A Chromatogram of pantothenic acid standard solution... 27
Annex B Preparation of culture medium (liquid)... 29
National food safety standards -- Determination of
pantothenic acid in food
1 Scope
This Standard specifies the method for the determination of pantothenic acid in food.
Method One of this Standard is applicable to the determination of pantothenic acid in
infant formula foods (except infant formula foods for special medical purposes), infant
supplementary foods, dairy products, beverages, and nutritional supplements.
Method Two and Method Three of this Standard are applicable to the determination of
pantothenic acid in food.
Method One -- Liquid chromatography
2 Principle
After the specimen is extracted with hot water, it is separated by a C18 reversed-phase
chromatography column, qualitatively determined by retention time, and quantitatively
by the external standard method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure, and the
water is grade one water specified in GB/T 6682.
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 α-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.
3.2.2 Hydrochloric acid (1.0 mol/L). Pipette 8.3 mL of hydrochloric acid into 80 mL of
water. Add water to dilute to 100 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.Use water to dilute to 1000 mL. Use a 0.45 μm filter membrane
to filter.
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.
3.4.2 Pantothenic acid standard intermediate solution (100 μg/mL). Accurately pipette
20.0 mL of the standard stock solution into a 100 mL volumetric flask. Add water to
dilute to volume. Prepare when needed.
3.4.3 Pantothenic acid standard working solution. Accurately pipette 1.0 mL, 2.0 mL,
4.0 mL, 8.0 mL, 16.0 mL and 32.0 mL of pantothenic acid standard intermediate
solution into 100 mL volumetric flasks. Add water and dilute to the mark to obtain
standard working solutions of pantothenic acid, of which the concentrations are 1.0
μg/mL, 2.0 μg/mL, 4.0 μg/mL, 8.0 μg/mL, 16.0 μg/mL and 32.0 μg/mL. Prepare when
needed.
4 Instruments and equipment
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 Analysis steps
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℃~50℃ warm water and conduct
ultrasonic extraction for 20 min. Transfer to a 50 mL volumetric flask. Dilute to volume
with water and mix thoroughly before transferring to a centrifuge tube. Centrifuge at
8000 r/min for 2 min. Pass the supernatant through a 0.45 μm filter membrane. The
filtrate is ready to be measured on the machine.
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. The liquid specimen is about 20 g. Place in a 100 mL
Erlenmeyer flask. Add about 30 mL of 40℃~50℃ warm water. Specimens that do not
contain starch are directly extracted with ultrasound for 20 min. For starch-containing
specimens, add 0.2 g of α-amylase and shake to mix. Cap the bottle. Shake for
enzymatic hydrolysis in a water bath at 55°C ±5°C for 30 min. Take out the specimen
solution. Cool to room temperature.
Adjust the pH to 5.0 ± 0.1 with 0.1 mol/L hydrochloric acid or 1.0 mol/L hydrochloric
acid. Add 5 mL of 0.5 mol/L zinc sulfate solution. Mix thoroughly. Transfer to a 50 mL
volumetric flask. Dilute to volume with water, mix thoroughly, and transfer to a
centrifuge tube. Centrifuge at 8000 r/min for 2 min. Pass the supernatant through a 0.45
μm filter membrane. The filtrate is ready to be measured on the machine.
5.3 Reference chromatographic conditions for liquid phase
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).
10.1.8 Sodium bicarbonate (NaHCO3).
10.1.9 Potassium bicarbonate (KHCO3).
10.1.10 Toluene (C7H8).
10.1.11 α-amylase. enzyme activity ≥1.5 U/mg.
10.1.12 Anion exchange resin. Dowex 1×8 particle size is 38 μm~75 μm.
10.1.13 Alkaline phosphatase. derived from bovine intestinal mucosa, EC3.1.3.1,
enzyme activity ≥10 U/mg.
10.1.14 Pigeon liver acetone extract dry powder. enzyme activity ≥0.1 U/g.
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.
10.2.4 Tris buffer. Weigh 121.0 g of trishydroxymethylaminomethane and dissolve it in
500 mL of water. Use glacial acetic acid to adjust pH to 8.1 ±0.2.Add water to dilute
to 1000 mL. Mix well. Store in refrigerator at 2℃~8℃.
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.
10.2.6 Alkaline phosphatase solution. Weigh 0.2 g of alkaline phosphatase into a mortar.
Add water in small amounts and grind until it is dissolved. Use water to dilute to 10
mL. Prepare when needed. Store in a refrigerator at 2℃~8℃ to pre-cool.
10.2.7 Hydrochloric acid solution (1 mol/L). Pipette 83 mL of hydrochloric acid into
800 mL of water. Add water to dilute to 1000 mL. Mix well.
10.2.8 Potassium bicarbonate solution (0.02 mol/L). Weigh 2 g of potassium
bicarbonate. Add water to dissolve and dilute to 1000 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. Add a small amount of water to the anion
exchange resin. Mix well. Add Tris buffer dropwise to the anion exchange resin. Adjust
pH to 8.0±0.1.Store in a refrigerator at 2℃~8℃. Use within 2 d.
10.2.10 Pigeon liver extract. At the day before preparing this reagent, place the
container in a refrigerator at 2℃~8℃ overnight. Weigh 30 g of pigeon liver acetone
extract dry powder and put it into a pre-cooled mortar. Add 300 mL of 0.02 mol/L
potassium bicarbonate solution in portions under ice bath conditions. Grind to a
homogeneous suspension. Transfer the suspension to a pre-cooled capped centrifuge
tube. Cover tightly, shake thoroughly, and place in a -20°C refrigerator for 10 min. After
taking out, centrifuge at 3000 r/min for 5 min. Transfer the supernatant to a 500 mL
pre-cooled jar. Add partially activated resin (approximately 150 g) to the supernatant.
Place in an ice water bath and shake for 5 min. Pour into a pre-cooled centrifuge tube
and centrifuge at 3000 r/min for 5 min. Transfer the supernatant to another 500 mL pre-
cooled jar. At -20℃, let stand for 10 mins. Then add the remaining activated resin (about
150 g). Place in an ice bath and shake for 5 min. Pour into a centrifuge tube. Centrifuge
at 3000 r/min for 5 min. Collect the supernatant. At -20℃, let stand for 10 min. Aliquot
into pre-cooled stoppered test tubes. Store frozen at -20°C. It can be stored for 1 year.
Defrost in the refrigerator at 2℃~8℃ before use.
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
mL of Tris buffer and 30 mL of water. Shake to mix. Hydrolyze under high pressure
conditions at 121°C for 20 min. Cool to room temperature. Add 0.1 mL of sodium
bicarbonate solution, 0.4 mL of alkaline phosphatase solution, and 0.2 mL of pigeon
liver extract in sequence. After mixing, add 100 μL of toluene and stopper. Conduct
constant temperature oscillation at 37℃ ±1℃ with an amplitude of 100 r/min ± 20
r/min for 8 h~10 h. Transfer to a 100 mL volumetric flask. Add water to the mark. Filter.
Pipette 10.0 mL of the above liquid into a 50 mL centrifuge tube. Add 100 μL of internal
standard stock solution. Add water to 20 mL. Vortex for 10 s. Add 0.4 mL of zinc acetate
solution and potassium ferrocyanide solution, respectively. Add water to 25 mL. Vortex
for 10 s. After letting it stand for 10 to 30 min, centrifuge at 8000 r/min for 2 min. The
supernatant is passed through a 0.22 μm nylon filter and injected for analysis.
12.1.2 Infant formula food, infant supplementary food, formula food for special medical
purposes, dairy products, beverages, nutrient supplements, jelly
Accurately weigh 5 g of solid specimen (accurate to 0.01 g). Add water to 50 g (accurate
to 0.01 g). Add 0.2 g of α-amylase. Shake to mix. Cap the bottle. Shake for 30 min in a
water bath at 55°C ±5°C. Weigh 0.5 g ~ 5.0 g of the above liquid (accurate to 0.1 mg)
and place it in a 50 mL centrifuge tube. After mixing the liquid specimen, directly weigh
0.5 g~5.0 g (accurate to 0.1 mg) and place it in a 50 mL centrifuge tube.
Add 100 μL of internal standard stock solution. Add water to 20 mL. Vortex for 10 s.
Add 0.4 mL of zinc acetate solution and potassium ferrocyanide solution, respectively.
Add water to 25 mL. Vortex for 10 s. After letting it stand for 10 to 30 min, centrifuge
at 8000 r/min for 2 min. The supernatant is passed through a 0.22 μm nylon filter and
injected for analysis.
12.2 Reference conditions for instruments
12.2.1 Reference chromatographic conditions for liquid phase
The liquid phase reference chromatography conditions are as follows.
a) Chromatographic column. C18, 1.8 μm, 100 mm × 2.1 mm, or equivalent.
b) Column temperature. 35℃.
c) Flow rate. 0.3 mL/min.
d) Injection volume. 2 μL.
e) Mobile phase. Phase A is 0.1% formic acid solution; phase B is acetonitrile. The
liquid chromatography gradient elution conditions are shown in Table 1.
Table 1 -- Gradient elution conditions for liquid chromatography
17.1 Reagents
17.1.1 Hydrochloric acid (HCl).
17.1.2 Glacial acetic acid (C2H4O2).
17.1.3 Sodium hydroxide (NaOH).
17.1.4 Sodium chloride (NaCl).
17.1.5 Sodium bicarbonate (NaHCO3).
17.1.6 Potassium bicarbonate (KHCO3).
17.1.7 Sodium acetate trihydrate (C2H3O2Na·3H2O).
17.1.8 Trishydroxymethylaminomethane (C4H11NO3). <...