GB 5009.158-2016_English: PDF (GB5009.158-2016)
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National Food Safety Standard -- Determination of Vitamin K1 in Foods
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GB 5009.158-2016
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| GB/T 5009.158-2003 | English | 279 |
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Determination of vitamin K1 in vegetables
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GB/T 5009.158-2003
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Preview PDF: GB 5009.158-2016
| Standard ID | GB 5009.158-2016 (GB5009.158-2016) | | Description (Translated English) | National Food Safety Standard -- Determination of Vitamin K1 in Foods | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 15,163 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB 5413.10-2010; GB/T 5009.158-2003 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 |
GB 5009.158-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Vitamin K1 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 Phase Chromatography - Fluorescence
Detection ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instruments and Equipment ... 7
5 Analytical Procedures ... 7
6 Expression of Analysis Results ... 10
7 Precision ... 10
8 Others ... 11
Method II Liquid Phase Chromatography - Tandem Mass Spectrometry ... 11
9 Principle ... 11
10 Reagents and Materials... 11
11 Instruments and Equipment ... 13
12 Analytical Procedures ... 14
13 Expression of Analysis Results ... 18
14 Precision ... 18
15 Others ... 18
Appendix A Methods of Correcting Vitamin K1 Standard Concentration ... 19
Appendix B High-performance Liquid Phase Chromatogram ... 20
Appendix C Mass Spectrometry Reference Figure ... 21
Foreword
This Standard is drafted as a replacement of GB/T 5009.158-2003 “Determination of
Vitamin K1 in Vegetables” and GB 5413.10-2010 “National Food Safety Standard -
Determination of Vitamin K1 in Foods for Infants and Young Children, Milk and Milk
Products”.
In comparison with GB/T 5009.158-2003, there are several main modifications as
follows.
-- The name of this Standard is modified into “National Food Safety Standard -
Determination of Vitamin K1 in Foods”;
-- The method of high-performance liquid phase chromatography - fluorescence
detection is added;
-- The method of liquid phase chromatography - tandem mass spectrometry is
added;
-- The method of high-performance liquid phase chromatography - UV detection is
deleted.
National Food Safety Standard -
Determination of Vitamin K1 in Foods
1 Scope
This Standard specifies the method of determining vitamin K1 in foods.
In this Standard, Method I is high-performance liquid phase chromatography -
fluorescence detection, Method II is liquid phase chromatography - tandem mass
spectrometry, which are both applicable to the determination of vitamin K1 in all kinds
of formula food, vegetable oil, fruit and vegetable.
Method I -- High-performance Liquid Phase
Chromatography - Fluorescence Detection
2 Principle
Through lipase and amylase enzymolysis of samples like foods for infants and young
children, dairy products and vegetable oil, adopt N-hexane to extract vitamin K1 from
the sample. Adopt C18 liquid phase chromatographic column to separate vitamin K1
from other impurities. Adopt zinc column for reduction, adopt fluorescence detector for
detection; quantify with the external standard method.
Adopt isopropanol and N-hexane to extract vitamin K1 from low-fat plant samples like
fruit and vegetable; adopt neutral alumina column for purification, remove interfering
substances like chlorophyll. Adopt C18 liquid phase chromatographic column to
separate vitamin K1 from other impurities. Adopt zinc column for reduction, adopt
fluorescence detector for detection; quantify 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 Anhydrous ethanol (CH3CH2OH).
sample grinder to grind flaky and granular samples into powder, store it in a sample
bag for later usage; shake liquid samples like liquid milk and vegetable oil, directly
take the ample; take edible part from fruit and vegetable, use water to rinse it, then,
use gauze to remove water on the surface; homogenize with the homogenizer, store
in a sample bottle for later usage. Determine the sample as soon as possible once the
sample is prepared.
5.2 Processing of Samples
Note. during the processing, avoid direct UV light irradiation, keep away from light.
5.2.1 Foods for infants and young children, dairy products and vegetable oil
5.2.1.1 Enzymolysis
Accurately weigh-take 1 g ~5 g (accurate to 0.01 g, the content of vitamin K1 shall be
≥0.05 μg) of homogenized sample, place it in 50 mL centrifuge tube. Add 5 mL of
warm water to dissolve it (directly absorb 5 mL of liquid sample; no water shall be
added to dilute vegetable oil), add 5 mL of phosphate buffer solution (pH8.0), mix it up.
Add 0.2 g of lipase and 0.2 g of amylase (no amylase shall be added if the sample
doesn’t contain starch); put on the lid, start vortex for 2 min~3 min. Mix it up, place it in
37°C±2°C constant-temperature water bath oscillator and start oscillation for over 2 h,
so that it can go through thorough enzymolysis.
5.2.1.2 Extraction
After enzymolysis, take out the sample, respectively add 10 mL of ethanol and 1 g of
potassium carbonate, then, mix it up; add 10 mL of N-hexane and 10 mL of water,
start vortex or oscillation extraction for 10 min, start centrifugation at 6,000 r/min for 5
min; or transfer enzymolysis solution to 150 mL separating funnel for extraction, start
static stratification (if emulsification emerges, more N-hexane or water can be properly
added to eliminate emulsification); transfer the supernatant to 100 mL rotary
evaporation bottle, add 10 mL of N-hexane to the underlayer fluid; repeat the above
operation once, then, combine the supernatant into the above-mentioned rotary
evaporation bottle.
5.2.1.3 Concentration
Start rotary evaporation of the above-mentioned N-hexane extract, till it reaches
dryness (if there’s any residual solution, slightly blow with nitrogen till it reaches
dryness); use methanol to transfer and dilute to the constant volume in 5 mL
volumetric flask, mix it up. Use 0.22 μm membrane to filter it, reserve the filtrate for
inlet.
Add no sample, comply with the same method of operation and start a blank test.
5.2.2 Fruit and vegetable
8 Others
Foods for infants and young children, dairy products and vegetable oil. when the
amount of sampling is 1 g and the constant volume is 5 mL, the detection limit is 1.5
μg/100 g and the quantitation limit is 5 μg/100 g; fruit and vegetable. when the amount
of sampling is 5 g, the volume of dispensed extract is 5 mL and the constant volume is
5 mL, the detection limit is 1.5 μg/100 g and the quantitation limit is 5 μg/100 g.
Method II -- Liquid Phase Chromatography - Tandem
Mass Spectrometry
9 Principle
Through lipase and amylase enzymolysis of samples like foods for infants and young
children, dairy products and vegetable oil, adopt N-hexane to extract vitamin K1 from
the sample. Adopt C18 liquid phase chromatographic column to separate vitamin K1
from other impurities. Adopt tandem mass spectrometry for detection; quantify with
the isotope-labeled internal standard method.
Adopt isopropanol and N-hexane to extract vitamin K1 from low-fat plant samples like
fruit and vegetable; adopt neutral alumina column for purification, remove interfering
substances like chlorophyll. Adopt C18 liquid phase chromatographic column to
separate vitamin K1 from other impurities. Adopt tandem mass spectrometry for
detection; quantify with the isotope-labeled internal standard method.
10 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.
10.1 Reagents
10.1.1 Anhydrous ethanol (CH3CH2OH).
10.1.2 Potassium carbonate (K2CO3).
10.1.3 Potassium hydroxide (KOH).
10.1.4 Formic acid (HCOOH). chromatographic purity.
10.1.5 Ammonium formate (HCOONH4). chromatographic purity.
10.1.6 Isopropanol [(CH3)2CHOH].
standard intermediate fluid of vitamin K1 and place it in 100 mL volumetric flask; add
methanol to the constant volume, mix it up.
10.4.4 Isotope-labeled internal standard stock solution of vitamin K1-D7 (100 μg/mL).
accurately weigh-take 1 mg (accurate to 0.01 mg) of isotope-labeled internal standard
of vitamin K1-D7; use methanol to dissolve and dilute to the constant volume of 10 mL.
10.4.5 Isotope-labeled internal standard working fluid of vitamin K1-D7 (1 μg/mL).
absorb 1.00 mL of isotope-labeled internal standard stock solution of vitamin K1-D7,
then, place it in 100 mL volumetric flask; add methanol to the constant volume, mix it
up.
10.4.6 Standard series of working fluid. respectively and accurately absorb 0.10 mL,
0.20 mL, 0.50 mL, 1.00 mL, 2.00 mL and 4.00 mL of standard working fluid of vitamin
K1, place it in 10 mL volumetric flask; respectively add 0.50 mL of isotope-labeled
internal standard working fluid; add methanol to dilute to the constant volume. The
concentration of the standard series of working fluid of vitamin K1 is 10 ng/mL, 20
ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL and 400 ng/mL respectively.
10.5 Materials
10.5.1 Neutral alumina. particle size 50 μm~150μm.
10.5.2 Neutral alumina column. 2 g/6 mL, 10% of water is contained in the filling.
Commodity column can be purchased directly, or it can be replaced by filling.
Note. the method of filling neutral alumina column.
a) Filling processing. take 200 g of neutral alumina and place it in 500 mL wide-mouth bottle;
bake for 2 h in a drying oven under 150 °C; put on the lid, then, cool it down in the drying
oven, till it reaches the room temperature. Slowly add 20 mL of pure water, shake it
while adding the water; put on the lid, place it in the oven for 3 min~5 min under 80 °C;
take it out, violently shake it, till alumina in the bottle can flow freely without showing any
agglomeration; place it in the drying oven and cool it down for 30 min, reserve for later
usage.
b) Filling of chromatographic column. take 6 mL syringe-shaped column sleeve, add sieve
plate; weigh-take 2.00 of the above-mentioned filling that’s deactivated; add another
sieve plate, then, compress it with filling instrument.
10.5.3 Microporous filter head. equipped with 0.22 μm microporous membrane.
11 Instruments and Equipment
11.1 Liquid phase chromatography - tandem mass spectrometer. equipped with
electrospray ionization (ESI).
starch); put on the lid, start vortex for 2 min~3 min. Mix it up, place it in 37°C±2°C
constant-temperature water bath oscillator and start oscillation for over 2 h, so that it
can go through thorough enzymolysis.
12.2.1.2 Extraction
After enzymolysis, take out the sample, respectively add 10 mL of ethanol and 1 g of
potassium carbonate, then, mix it up; add 10 mL of N-hexane, start vortex extraction
for 10 min, start centrifugation at 6,000 r/min for 3 min. Transfer the supernatant to
another 50 mL centrifuge tube, add 10 mL of N-hexane to the underlayer fluid. Start
vortex for 5 min and centrifugation at 6,000 r/min for 3 min. Combine the supernatant,
add N-hexane solution to dilute to the constant volume of 25 mL, reserve for
purification.
12.2.1.3 Purification
Add 20 mL of water to the above-mentioned extract, shake for 0.5 min. After static
stratification, respectively take 5 mL of the supernatant and place it in 10 mL glass
tube. Use nitrogen to blow it, till it reaches dryness. Add 1 mL of methanol to dissolve
it. Use 0.22 μm membrane to filter it, reserve the filtrate for inlet.
Add no sample, comply with the same method of operation and start a blank test.
12.2.2 Fruit and vegetable
12.2.2.1 Extraction
Accurately weigh-take 1 g~5 g (accurate to 0.01 g, the content of vitamin K1 shall be
≥0.02 μg) of homogenized sample, place it in 50 mL centrifuge tube. Add 0.25 mL of
isotope-labeled internal standard working fluid (1 μg/mL); add 5 mL of isopropanol.
Start vortex for 1 min and ultrasonic for 5 min. Add 10 mL of N-hexane, start vortex or
oscillation extraction for 3 min, start centrifugation at 6,000 r/min for 5 min.
Remove-take the supernatant in 25 mL brown volumetric flask. Add 10 mL of
N-hexane to the underlayer fluid; repeat extraction once, then, combine the
supernatant into the above-mentioned volumetric flask. Use N-hexane to dilute to the
constant volume; use pipette to accurately dispense 5 mL of the supernatant to 10 mL
tube. Use nitrogen to slightly blow it, till it reaches dryness. Add 1 mL of N-hexane to
dissolve it, reserve for purification.
12.2.2.2 Purification
Use a small amount of N-hexane to transfer 1 mL of the above-mentioned extract to
neutral alumina column that’s previously activated with 5 mL of N-hexane. Wait till the
extract is almost drained, use 5 mL of N-hexane to rinse it; use 6 mL of N-hexane -
ethyl acetate mixed solution to elute to 10 mL tube. Use nitrogen to blow it, till it
reaches dryness; add 1 mL of methanol. Adopt 0.22 μm membrane to filter it; reserve
the filtrate for analysis and determination.
Add no sample, comply with the same method of operation and start a blank test.
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