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GB 5009.154-2016

Chinese Standard: 'GB 5009.154-2016'
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Detail Information of GB 5009.154-2016; GB5009.154-2016
Description (Translated English): Determination of vitamin B6 in foods
Sector / Industry: National Standard
Classification of Chinese Standard: X09
Date of Issue: 2016-12-23
Date of Implementation: 2017-06-23
Older Standard (superseded by this standard): GB 5413.13-2010; GB/T 5009.154-2003
Regulation (derived from): National Health and Family Planning Commission Notice No.17 of 2016

GB 5009.154-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Vitamin B6 in Foods
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People’s Republic of China;
State Food and Drug Administration of the People’s Republic
of China.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
Method I High-performance Liquid Chromatography ... 4 
2 Principle ... 4 
3 Reagents and Materials ... 4 
4 Instruments and Equipment ... 6 
5 Analytical Procedures ... 6 
6 Expression of Analysis Results ... 8 
7 Precision ... 9 
8 Others ... 9 
Method II Microbial Method ... 9 
9 Principle ... 9 
10 Reagents and Materials ... 9 
11 Instruments and Equipment ... 11 
12 Analytical Procedures ... 11 
13 Expression of Analysis Results ... 13 
14 Precision ... 14 
15 Others ... 14 
Appendix A Method of Concentration Correction of Vitamin B6 in Each
Component of Standard Solution ... 15 
Appendix B Liquid Chromatogram of Vitamin B6 ... 17 
Appendix C Medium Component and Preparation Method ... 18 
National Food Safety Standard -
Determination of Vitamin B6 in Foods
1 Scope
This Standard specifies methods of determining vitamin B6 in foods.
In this Standard, Method I is high-performance liquid chromatography, which is
applicable to the determination of foods that contain vitamin B6; Method II is microbial
method, which is applicable to the determination of vitamin B6 in all kinds of food.
Method I -- High-performance Liquid Chromatography
2 Principle
After pre-processing of the sample, for example, extraction, the sample is separated
with C18 chromatographic column and detected with high-performance liquid
chromatography - fluorescence detector. The content of vitamin B6 (pyridoxine,
pyridoxal and pyridoxamine) is quantitatively determined with the external standard
method.
3 Reagents and Materials
Unless otherwise indicated, the reagents adopted under this method are of analytical
purity. The water is first-grade water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Sodium octane sulfonate (C8H17NaO3S).
3.1.2 Glacial acetic acid (C2H4O2).
3.1.3 Triethylamine (C6H15N). chromatographic purity.
3.1.4 Methanol (CH4O). chromatographic purity.
3.1.5 Hydrochloric acid (HCl).
3.1.6 Sodium hydroxide (NaOH).
3.1.7 Amylase. enzyme activity ≥1.5 U/mg.
3.2 Preparation of Reagents
then, mix it up. Place it evenly for 5 min~10 min, then, cool it down to the room
temperature.
b) Liquid sample. weigh-take approximately 20 g (accurate to 0.01 g) of thoroughly
mixed liquid sample; place it in 150 mL conical flask. Place it evenly for 5 min~10 min.
5.1.3 Preparation of test solution
Use hydrochloric acid solution to adjust the above-mentioned sample solution to pH
1.7±0.1; place evenly for around 1 min. Use sodium hydroxide solution to adjust the
sample solution to pH 4.5±0.1. Place the above-mentioned conical flask into ultrasonic
oscillator; start ultrasonic oscillation for around 10 min. Transfer the sample solution
into 50 mL volumetric flask; use water to rinse the conical flask. Combine the lotion in
50 mL volumetric flask; use water to dilute to the constant volume of 50 mL. Take
another 50 mL conical flask, place a funnel and filter paper on top of it; pour the
constant-volume sample solution into the conical flask, then, naturally filter it. Use 0.45
μm microporous membrane to filter the filtrate, then, use a tube to collect it; transfer 1
mL of filtrate to inlet bottle as the test solution.
Note. avoid strong light exposure during the operation.
5.2 Instrument Reference Conditions
Please see instrument reference conditions below.
a) Chromatographic column. C18 column, length. 150 mm, internal diameter. 4.6
mm, particle size of column filling. 5 μm, or any equivalent;
b) Mobile phase. methanol 50 mL, sodium octane sulfonate 2.0 g, triethylamine 2.5
mL; use water to dissolve to the constant volume of 1,000 mL, then, use glacial
acetic acid to adjust to pH 3.0±0.1; use 0.45 μm microporous membrane to filter
it;
c) Flow rate. 1 mL/min;
d) Column temperature. 30 °C;
e) Detection wavelength. excitation wavelength. 293 nm, emission wavelength. 395
nm;
f) Inlet volume. 10 μL.
5.3 Draw a Standard Curve Line
Respectively inject vitamin B6 mixed standard series working solution into high-
performance liquid chromatograph, then, measure the peak area of each component;
take the concentration of corresponding standard working solution as x-coordinate,
take the peak area as y-coordinate to draw a standard curve line.
10.1.6 Sodium chloride (NaCl).
10.1.7 Bromo cresol green (C21H14Br4O5S).
10.2 Preparation of Reagents
10.2.1 Hydrochloric acid solution (0.01 mol/L). take 0.9 mL of hydrochloric acid; use
water to dilute to the constant volume of 1,000 mL.
10.2.2 Sulfuric acid solution (0.22 mol/L). add 700 mL of water and 12.32 mL of sulfuric
acid to 2,000 mL beaker; use water to dilute to 1,000 mL.
10.2.3 Sulfuric acid solution (0.5 mol/L). add 700 mL of water and 28 mL of sulfuric
acid to 2,000 mL beaker; use water to dilute to 1,000 mL.
10.2.4 Sodium hydroxide solution (10 mol/L). weigh-take 40 g of sodium hydroxide,
then, add 40 mL of water to dissolve it; cool it down and use water to dilute to the
constant volume of 100 mL.
10.2.5 Sodium hydroxide solution (0.1 mol/L). transfer-take 1 mL of 10 mol/L sodium
hydroxide solution, then, add water to dilute to the constant volume of 100 mL.
10.2.6 Saline (9 g/L). weigh-take 9 g of sodium chloride, use water to dissolve it to the
constant volume of 1,000 mL; start autoclaved sterilization under 121 °C for 15 min;
cool it down and reserve for later usage.
10.2.7 Bromo cresol green solution (0.4 g/L). accurately weigh-take 0.1 g of bromo
cresol green and place it in a mortar; add 1.4 mL of 0.1 mol/L sodium hydroxide solution
to grind it; add a little water and continue to grind it, till it completely dissolves; use
water to dilute to 250 mL.
10.3 Medium (refer to Appendix C for medium component and
preparation method)
10.3.1 Pyridoxine Y medium.
10.3.2 Pyridoxine Y agar medium.
10.3.3 Malt extract powder agar medium.
10.3.4 YM broth medium.
10.3.5 YM broth agar medium.
10.4 Standards
Pyridoxine hydrochloride (C8H12ClNO3, CAS No.. 58-56-0). purity≥99%, or standard
substance with a national-level certificate and a certificate of standard substance.
12.1.2 Monthly-reserved strain preparation. inoculate strain rejuvenation medium on
the bevel of YM broth agar medium (subculture medium) through streak inoculation;
start culture under 30 °C for 20 h~24 h; store in the refrigerator under 2 °C~8 °C. This
strain is the first generation of monthly-reserved strain. Inoculate the previous
generation of monthly-reserved strain on the bevel of YM broth agar medium
(subculture medium) through streak inoculation every month in the following period;
start culture under 30 °C for 20 h~24 h; store in the refrigerator under 2 °C~8 °C. This
monthly-reserved strain can remain valid for 1 month.
12.1.3 Weekly-reserved strain preparation. inoculate monthly-reserved strain on the
bevel of YM broth agar medium (subculture medium) every week; start culture under
30 °C for 20 h~24 h; store in the refrigerator under 2 °C~8 °C. It can remain valid for 7
days. Strain that’s reserved for over several weeks cannot be immediately adopted for
the preparation of inoculum. Before usage, it must be transferred once a day for 2
days~3 days in a row, otherwise, the growth cannot be guaranteed.
12.1.4 Inoculant suspension preparation. one day before the determination of vitamin
B6, transfer weekly-reserved strain in 10 mL of YM broth medium (seed culture
medium). 2 tubes can be prepared simultaneously. Start oscillating culture under 30 °C
for 20 h~24 h, then, obtain seed culture medium for determination. The number of total
generations from monthly-reserved strain to seed culture medium shall be ≤5. Start
centrifugation of the seed culture medium under 3,000 r/min for 10 min, dump the
supernatant; use 10 mL of saline to rinse it, start centrifugation and dump the
supernatant, then, use saline to repeatedly rinse it for 2 times. Add 10 mL of sterilized
saline, then, place centrifuge tube on vortex mixer to thoroughly mix it; turn the strain
into suspension, then, pour the suspension into a sterilized syringe; use it immediately.
12.2 Sample Processing
12.2.1 Weigh-take 0.5 g~10 g (accurate to 0.01 g, the content of vitamin B6 shall be
≤10 ng), place it in 100 mL conical flask, then, add 72 mL of 0.22 mol/L sulfuric acid
solution. Place it in autoclave under 121 °C and start hydrolysis for 5 h; take it out and
cool it down. Adopt 10.0 mol/L sodium hydroxide solution and 0.5 mol/L sulfuric acid
solution to adjust to pH 4.5; use bromo cresol green as the indicator (indicator turns
from yellow to yellow-green). Transfer the reagent in the conical flask into 100 mL
volumetric flask; use distilled water to dilute to the constant volume of 100 mL; use
filter paper to filter it, store the filtrate in the refrigerator for later usage (the period of
validity shall be ≤36 h).
Note. keep away from light during sample processing.
12.2.2 Preparation of standard curve line. respectively add 0.00 mL, 0.02 mL, 0.04 mL,
0.08 mL, 0.12 mL and 0.16 mL of pyridoxine working solution to 3 groups of tube; add
pyridoxine Y medium to 5.00 mL; mix it up, put on cotton plug.
12.2.3 Preparation of sample tube. respectively add 0.05 mL, 0.10 mL and 0.20 mL of
Appendix C
Medium Component and Preparation Method
C.1 Pyridoxine Y Medium
Solution (per liter) contains. glucose 40.0 g, L-asparagine 4.0 g, ammonium sulfate 4.0
g, potassium dihydrogen phosphate3.0 g, magnesium sulfate 1.0 g, calcium oxide 0.49
g, DL-methionine 40.0 mg, DL-tryptophan 40.0 mg, DL-isoleucine 40.0 mg, DL-proline
40.0 mg, histidine hydrochloride 20.0 mg, riboflavin 20.0 mg, biotin 8.0 mg, inositol 5.0
mg, ferrous sulfate 500.0 μg, thiamine hydrochloride 400.0 μg, calcium pantothenate
400.0 μg, choline acid 400.0 μg, boric acid 200.0 μg, potassium iodide 200.0 μg,
ammonium molybdate 40.0 μg, manganese sulfate 80.0 μg, copper sulfate 90.0 μg,
zinc sulfate 80.0 μg, distilled water 1,000 mL.
Weigh-take 5.3 g of the above-mentioned pyridoxine Y medium, then, dissolve it in 100
mL of distilled water; adjust to pH 4.1±0.05; start sterilization under 121 °C for 15 min;
reserve for later usage.
C.2 Pyridoxine Y Agar Medium
Weigh-take 5.3 g of the above-mentioned pyridoxine Y medium, then, dissolve it in 100
mL of distilled water; adjust to pH 4.1±0.05. Add 1.2 g of agar, heat it up and boil it to
melt the agar. Thoroughly mix it up and divide it into tubes (10 mL in each tube). Start
sterilization under 121 ......
Related standard:   GB 5009.149-2016  GB 5009.150-2016
   
 
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