GB 5009.210-2016 English PDF

GB 5009.210-2016: Determination of pantothenic acid in foods
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GB 5009.210-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of pantothenic acid in food
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 01, 2017
Issued by. National Health and Family Planning Commission of the PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Principles ... 4 
3 Reagents and materials ... 4 
4 Instruments and equipment... 8 
5 Preparation and preservation of strain ... 9 
6 Analytical procedures ... 9 
7 Precision ... 15 
8 Other ... 15 
9 Principles ... 15 
10 Reagents and materials ... 15 
11 Instruments and equipment ... 16 
12 Analytical procedures ... 17 
13 Analysis results expression ... 18 
14 Precision ... 19 
15 Other ... 19 
Appendix A Method for preparing culture solution for determination of
pantothenic acid ... 20 
Appendix B Liquid chromatogram of pantothenic acid standard solution ... 23 
National Food Safety Standard -
Determination of pantothenic acid in food
1 Scope
This Standard specifies the method for determination of pantothenic acid and
calcium pantothenate in food.
Method 1 of this Standard is applicable to the determination of pantothenic acid
in food. Method 2 is applicable to the determination of pantothenic acid (calcium)
in nutrient supplement health foods and formula foods.
Method 1 Microbiological method
2 Principles
Pantothenic acid is an essential nutrient for the growth of Lactobacillus
plantarum (ATCC 8014). Under certain control conditions, the Lactobacillus
plantarum solution is inoculated into the culture solution containing the sample
solution. After being cultured for a certain period of time, the light transmittance
(or absorbance value) is determined. According to a standard curve of
pantothenic acid content and light transmittance (or absorbance value), the
content of pantothenic acid in the sample is calculated.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytically
pure; the water is Grade II water specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Glacial acetic acid (C2H4O2).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sodium chloride (NaCl).
3.2.5 Sodium hydroxide solution (1 mol/L). WEIGH 40 g of sodium hydroxide;
ADD water to dissolve and dilute to 1000 mL, and MIX well.
3.2.6 Sodium hydroxide solution (0.1 mol/L). WEIGH 4 g of sodium hydroxide;
ADD water to dissolve and dilute to 1000 mL, and MIX well.
3.2.7 Tris buffer solution. WEIGH 121.0 g of trishydroxymethyl aminomethane
dissolved in 500 mL of water; USE glacial acetic acid to adjust the pH to 8.1±0.1;
ADD water to 1000 mL, and MIX well. STORE in a refrigerator at 2 °C~4 °C. It
can be stored for 2 weeks.
3.2.8 Normal saline. WEIGH 9 g of sodium chloride; ADD water to dissolve and
dilute to 1000 mL, and MIX well. Pre-sterilized before use. Autoclaved at 121 °C
for 10 min and then used.
3.2.9 Ethanol solution (20%). TAKE 200 mL of absolute ethanol and mix with
800 mL of water.
3.2.10 Sodium carbonate solution (0.08 mol/L). WEIGH 8.5 g of sodium
carbonate; ADD water to dissolve and dilute to 1000 mL, and MIX well.
3.2.11 Potassium bicarbonate solution (0.02 mol/L). WEIGH 2 g of potassium
bicarbonate; ADD water to dissolve and dilute to 1000 mL, and MIX well.
3.2.12 Alkaline phosphatase solution. WEIGH 2 g of alkaline phosphatase; ADD
water to dissolve and dilute to 100 mL. Prepared when used. Precooled in a
refrigerator at 2 °C~4 °C.
3.2.13 Pigeon liver extract
3.2.13.1 Activated Dowex 1×8. WEIGH 100 g of Dowex 1×8 into a conical flask;
ADD 1 L of hydrochloric acid solution; PLACE on an oscillator to fully shake for
10 min; USE a Buchner funnel covered with filter paper to filter. TRANSFER
Dowex 1×8 back to the conical flask; then ADD 1 L of hydrochloric acid solution;
and repeatedly shake and filter. Dowex 1×8 is added with 1 L of water and
shaken for 10 min; filtered; and repeatedly USE water to wash 10 times. ADD
the Tris buffer solution to adjust the Dowex 1×8 pH to 8.0±0.1. STORE in a
refrigerator at 2 °C~4° C; USE up within 2 days.
3.2.13.2 Pigeon liver extract. One day before the preparation of this reagent,
the containers used are placed in a refrigerator at 2 °C~4 °C for overnight cold
storage. WEIGH 30 g of liver acetone powder from pigeon into the mortar.
Under ice bath conditions, ADD 300 mL of potassium bicarbonate solution twice,
and grind to homogenate. TRANSFER to a centrifuge tube with stopper, cover
well, shake fully. After freezing at -20 °C for 10 min, CENTRIFUGE at 3000 r/min
for 5 min. TRANSFER the supernatant to a 500 mL jar. ADD 150 g of activated
determination culture medium of equivalent potency from a reagent company;
and before use, prepare it according to the instructions.
3.4 Standard
D-calcium pantothenate (C18H32CaN2O10). purity≥99%.
3.5 Preparation of standard solutions
3.5.1 Standard stock solution of pantothenic acid (40.0 μg/mL). accurately
WEIGH 43.5 mg of D-calcium pantothenate which is pre-dried to constant
weight. ADD water to dissolve; and TRANSFER to a 1000 mL volumetric flask.
ADD 10 mL of acetic acid solution, 100 mL of sodium acetate solution; USE
water to dilute to volume. STORE in a brown bottle; ADD 3~5 drops of toluene.
In a refrigerator at 2 °C~4 °C, it can be stored for 2 years.
3.5.2 Standard intermediate solution of pantothenic acid (1.00 μg/mL).
accurately PIPETTE 25.0 mL of standard stock solution of pantothenic acid in
a 1000 mL volumetric flask. ADD 10 mL of acetic acid solution, 100 mL of
sodium acetate solution; USE water to dilute to volume. ADD 3~5 drops of
toluene. In a refrigerator at 2 °C~4 °C, it can be stored for 1 year.
3.5.3 Standard working solution of pantothenic acid (20 ng/mL). accurately
PIPETTE 2.00 mL of standard intermediate solution of pantothenic acid in a 100
mL volumetric flask. USE water to dilute to volume, and MIX well. Prepared
before use.
4 Instruments and equipment
4.1 Balance. The sensitivity is 0.1 mg.
4.2 Constant temperature incubator. 37 °C±1 °C.
4.3 Pressure steam sterilizer. 121 °C.
4.4 Vortex oscillator.
4.5 Centrifuge. speed≥3000 r/min.
4.6 Inoculating needle and inoculating loop.
4.7 pH meter. accuracy±0.01.
4.8 UV-spectrophotometer.
4.9 Super clean bench.
and sieved (The pore diameter of sieve plate is 0.3 mm~0.5 mm). Meat, eggs,
nuts, etc. shall be made into chyme using a homogenizer. Samples of fruits and
vegetables, semi-solid foods, etc. shall be homogenized and mixed. The liquid
sample, before use, shall be shaken and mixed. In a refrigerator at 4 °C, it can
be stored for 1 week.
6.2 Sample extraction
6.2.1 Direct extraction method
Nutrient supplements, enhancers, or premixes in the form of granules, powders,
tablets, liquids, and formulas or health foods to which calcium pantothenate is
added may be extracted directly.
Accurately WEIGH the appropriate amount of sample (m); 0.2 g~2 g of solid
sample; 5 g~10 g of liquid sample, accurate to 0.001 g. TRANSFER to a 100
mL conical flask; ADD 80 mL of ethanol solution; STOPPER; ultrasonically
OSCILLATE to extract for more than 4 h until the sample is completely dissolved;
and USE water to dilute to 100 mL (V1).
6.2.2 Enzymolysis method
Food samples of general grains, tubers, meat, eggs, milk, fruits and vegetables,
mushrooms and algae, beans, and nuts, etc. shall be extracted by enzymolysis.
6.2.2.1 Hydrolysis. Accurately WEIGH the appropriate amount of sample (m,
containing about 0.02 mg~0.2 mg of pantothenic acid), accurate to 0.001 g. 1
g~5 g of general grains, tubers, meat, eggs, beans, and their products are
weighed. 5 g~10 g of fresh fruits and vegetables, milk, and their products are
weighed. TRANSFER to a 100 mL conical flask; ADD 10 mL of Tris buffer
solution, 40 mL of water; and MIX well by shaking. At 121 °C under high
pressure, HYDROLYZE for 15 min. COOL to room temperature; TRANSFER to
a 100 mL volumetric flask; USE water to dilute to volume (V1), and filter.
6.2.2.2 Enzymolysis. Accurately PIPETTE an appropriate amount of sample
filtrate (1.0 mL~10.0 mL, V2) to the bottom of a 25 mL graduated test tube with
stopper; ADD water to 10 mL; ADD 5 mL of Tris buffer solution. Under ice bath
conditions, pre-cooled 0.1 mL of sodium bicarbonate solution, 0.4 mL of alkaline
phosphatase solution, 0.2 mL of pigeon liver extract, and 0.4 mL of water are
carefully added in sequence. Carefully MIX to avoid adhering the mixture to the
tube wall; ADD 1 drop of toluene; and INCUBATE overnight (8 h or more) in a
37 °C±1 °C incubator. ADD water to 20 mL; USE glacial acetic acid to adjust
the pH to 4.5±0.1; ADD water to dilute to 25.0 mL (V3), and filter. Adjust pH.
PIPETTE an appropriate amount of sample enzymatic hydrolysate (2.0
mL~20.0 mL, V4) into a 25 mL graduated test tube with stopper; ADD water to
20 mL; USE 0.1 mol/L sodium hydroxide solution to adjust the pH to 6.8±0.1;
for 15 min.
6.6 Inoculation and culture
After the determination series tubes are cooled to room temperature, under
aseptic operation conditions, 50 μL of inoculum is added dropwise to each
determination tube. STOPPER the tampons; and PLACE in a 37 °C±1 °C
constant temperature incubator to incubate for 16 h~20 h, until the maximum
turbidity is reached. That is, after another 2 h of incubation, the light
transmittance (or absorbance value) does not change significantly. Another
standard 0 tube (containing 0 ng of pantothenic acid) is prepared and not
inoculated as a 0 control tube.
6.7 Determination
MIX the cultured standard series tubes, sample and enzyme blank series tubes
using a vortex mixer. USE a cuvette with a thickness of 1 cm, at 550 nm, USE
the uninoculated 0 control tube to adjust the light transmittance to 100% (Or the
absorbance value is 0). The light transmittance (or absorbance value) of the
standard series tubes, sample and enzyme blank series tubes is measured in
sequence. If the 0 control tube has obvious bacterial growth; or as compared
with the 0 control tube, the light transmittance of standard 0 tube is below 90%
(or the absorbance value is above 0.2); or the maximum variation of light
transmittance of standard series tubes is < 40% (or the variation of absorbance
value is < 0.4), there may be mixed of miscellaneous bacteria or pantothenic
acid from unidentified sources, and the test shall be repeated.
Note. The suitable spectral range for the determination of pantothenic acid is 540 nm~660
nm.
6.8 Analysis results expression
6.8.1 Standard curve. USE the pantothenic acid content of standard series
tubes as the abscissa. USE the mean value of light transmittance (or
absorbance value) of each standard point as the ordinate. The standard curve
is drawn.
6.8.2 Sample results calculation. The corresponding content (ρx) of pantothenic
acid in sample and enzyme blank series tubes is obtained from the standard
curve. If there are more than 3 in the 4 sample series tubes of each sample
having pantothenic acid content of 10 ng~80 ng; and if the pantothenic acid
concentration (ρ) per milliliter of sample diluent is calculated according to
formula (1), and the relative deviation between the tubes is less than 15%, then
according to formula (2) or formula (3)~formula (5), the results can be
calculated. Otherwise, re-sampling is required for determination.
The calculation result is expressed as the arithmetic mean of the two
independent determination results obtained under repeated conditions. The
result retains three significant figures.
7 Precision
The absolute difference between the two independent determination results
obtained for general foods under repeated conditions shall not exceed 15% of
the arithmetic mean. The absolute difference between the two independent
determination results obtained for nutrient supplements and fortified foods
under repeated conditions shall not exceed 5% of the arithmetic mean.
8 Other
When the sample weighing amount of general food is 5 g, the detection limit is
0.03 mg/100 g; and the limit of quantitation is 0.06 mg/100 g. When the sample
weighing amount of nutrition enhancer health food and formula food is 2 g, the
detection limit is 0.025 mg/100 g; and the limit of quantitation is 0.05 mg/100 g.
Method 2 High performance liquid chromatography
9 Principles
USE the physicochemical properties that pantothenic acid is easily soluble in
water and is stable under weak acidic to neutral conditions (pH 5.0~7.0). The
sample is extracted with hot water under ultrasonic oscillation, separated by C18
reversed phase chromatographic column, and is detected at a wavelength of
200 nm of UV-detector. Qualitative analysis is based on the retention time of
chromatographic peak and the UV spectrogram. USE external standard method
to quantify. CALCULATE the content of pantothenic acid in the sample.
10 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytically
pure; the water is Grade I water specified in GB/T 6682.
10.1 Reagents
10.1.1 Hydrochloric acid (HCl).
10.1.2 Phosphoric acid (H3PO4).
11.2 Constant temperature incubator. 55 °C±5 °C.
11.3 Ultrasonic oscillator.
11.4 pH meter. accuracy±0.01.
11.5 High performance liquid chromatograph. with UV-detector or diode array
detector.
12 Analytical procedures
12.1 Sample preparation
The solid sample shall be crushed and mixed uniformly. Carbonated beverages
require ultrasonic removal of carbon dioxide. Other liquid samples shall be
shaken well.
12.2 Preparation of sample determination solution
12.2.1 Nutrient supplement health food
Accurately WEIGH or MEASURE the appropriate amount of sample (m),
accurate to 0.001 g. 0.2 g~2 g of general solid sample; 10 g~20 g of liquid
sample. PLACE in a 50 mL conical flask; ADD about 30 mL of 40 °C~50 °C
warm water to ultrasonically extract for 20 min; USE water to dilute to volume
(V). TRANSFER to a centrifuge tube to centri...