GB 31614.1-2023 English PDF

GB 31614.1-2023: National food safety standard - Determination of sialic acid in foods
Delivery: 9 seconds. Download (& Email) true-PDF + Invoice.
Get Quotation: Click GB 31614.1-2023 (Self-service in 1-minute)
Historical versions (Master-website): GB 31614.1-2023
Preview True-PDF (Reload/Scroll-down if blank)

GB 31614.1-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of Sialic
Acid in Foods
ISSUED ON. SEPTEMBER 6, 2023
IMPLEMENTED ON. MARCH 6, 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
Method 1 - Liquid Chromatography - UV Detection... 4
2 Principle... 4
3 Reagents and Materials... 4
4 Instruments and Equipment... 5
5 Analytical Procedures... 6
6 Expression of Analysis Results... 8
7 Precision... 8
8 Others... 8
Method 2 - Liquid Chromatography - Fluorescence Detection... 9
9 Principle... 9
10 Reagents and Materials... 9
11 Instruments and Equipment... 10
12 Analytical Procedures... 10
13 Expression of Analysis Results... 12
14 Precision... 13
15 Others... 13
Method 3 - Liquid Chromatography - Mass Spectrometry / Mass Spectrometry... 13
16 Principle... 13
17 Reagents and Materials... 13
18 Instruments and Equipment... 15
19 Analytical Procedures... 15
20 Expression of Analysis Results... 18
21 Precision... 18
22 Others... 18
Appendix A Liquid Chromatogram of Sialic Acid Standard Solution... 19
Appendix B Liquid Chromatogram of Sialic Acid Standard Solution... 20
Appendix C Characteristic Ion Chromatogram of Liquid Chromatography - Mass
Spectrometry / Mass Spectrometry of Sialic Acid Standard Solution... 21
National Food Safety Standard - Determination of Sialic
Acid in Foods
1 Scope
This Standard specifies the methods for the determination of sialic acid in foods.
In this Standard, Method 1 “liquid chromatography - UV detection” is applicable to the
determination of bound sialic acid in swiftlets nest and swiftlets nest products; Method 2 “liquid
chromatography - fluorescence detection” and Method 3 “liquid chromatography - mass
spectrometry / mass spectrometry” are applicable to the determination of sialic acid in liquid
milk, milk powder, pastries and beverages.
Method 1 - Liquid Chromatography - UV Detection
2 Principle
The sample is heated and hydrolyzed in hydrochloric acid solution to release bound sialic acid.
The sample test solution is separated by a strong anion exchange chromatography column and
detected by a UV detector or diode array detector. Adopt the external standard method for
quantitative determination.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and
the water is Grade-1 water specified in GB/T 6682.
3.1 Reagents and Materials
3.1.1 Acetonitrile (CH3CN). chromatographically pure.
3.1.2 Phosphoric acid (H3PO4). chromatographically pure.
3.1.3 Concentrated hydrochloric acid (HCl). 12 mol/L.
3.1.4 Dialysis bag. capable of passing through compounds with a relative molecular mass of
less than 7,000.Before use, soak in water for 1 hour.
3.1.5 Microporous filter membrane. 0.45 m, organic phase type.
3.2 Preparation of Reagents
5 Analytical Procedures
5.1 Pre-treatment of Samples
5.1.1 Preparation of specimens
5.1.1.1 Swiftlets nest (cups, strips and chips, etc.) and solid swiftlets nest products
Weigh-take 10 g of swiftlets nest and solid swiftlets nest products and dry them in an oven at
101 C ~ 105 C to a constant mass (the mass difference between the two times shall not exceed
2 mg). After cooling in a dryer, grind them and pass them all through a 100-mesh sieve (with a
mesh size of 0.150 mm), then, put them into a clean container, place it in a dryer and seal for
later use.
5.1.1.2 Liquid swiftlets nest products
After evenly mixing liquid swiftlets nest products, take 200 g, homogenize it at a low speed
first. Then, slowly raise the speed to 10,000 r/min, homogenize it for 3 minutes, let it stand,
until the foam disappears. Put it into a clean container, seal it, keep it in the dark, and store it in
a refrigerator.
5.1.2 Specimen extraction
5.1.2.1 Swiftlets nest (cups, strips and chips, etc.) and solid swiftlets nest products
The determination of total sialic acid content. weigh-take 0.1 g (accurate to 0.0001 g) of sample
into a 25 mL stoppered colorimetric tube, add 10 mL of hydrochloric acid solution, use a glass
stopper to cover it, and conduct vortex mixing. Place it in 80 C water bath to conduct
hydrolysis for 40 min. During the water bath, oscillate it every 5 min, then, take out the
centrifuge tube and cool to room temperature. Transfer the hydrolysate to a 100 mL volumetric
flask, use an appropriate amount of water to wash the centrifuge tube twice and transfer it to a
volumetric flask. Use 0.1% phosphoric acid solution - acetonitrile (4 + 6) to reach a constant
volume to the scale and evenly mix it. Transfer-take 2 mL into a centrifuge tube, at 15,000 r/min,
centrifuge for 3 min. Take the supernatant, pass it through a 0.45 m microporous filter
membrane, and reserve it for determination.
The determination of free sialic acid content. weigh-take 0.1 g (accurate to 0.0001 g) of sample
into a 25 mL stoppered colorimetric tube, add 10 mL of water, and conduct vortex oscillation
for 3 minutes. Transfer the specimen solution to a 100 mL volumetric flask, use an appropriate
amount of water to wash the centrifuge tube twice and transfer it to a volumetric flask. Use 0.1%
phosphoric acid solution - acetonitrile (4 + 6) to reach a constant volume to the scale and evenly
mix it. Transfer-take 2 mL into a centrifuge tube, at 15,000 r/min, centrifuge for 3 min. Take
the supernatant, pass it through a 0.45 m microporous filter membrane, and reserve it for
determination.
5.1.2.2 Liquid swiftlets nest products
The determination of bound sialic acid content. weigh-take 10 g (accurate to 0.01 g) of sample
and place it in a dialysis bag (the specimen volume does not exceed 20% of the capacity of the
dialysis bag). Tie both ends of the dialysis bag, place it in a 1,000 mL beaker and dialyze it
under flowing tap water for 24 hours. After dialysis, transfer the test solution in the dialysis bag
to a 25 mL stoppered colorimetric tube, add a small amount of water to rinse the dialysis bag,
and combine it into the colorimetric tube. Add 104 L of concentrated hydrochloric acid, add
water to reach a constant volume of 25 mL, and evenly mix it, until the mass concentration of
hydrochloric acid in the hydrolysate is 0.05 mol/L. Use a glass stopper to cover it, place it in
80 C water bath to conduct hydrolysis for 40 min, then, take out the colorimetric tube and cool
it to room temperature. Transfer the hydrolysate to a 100 mL volumetric flask, use an
appropriate amount of water to wash the centrifuge tube twice and transfer it to a volumetric
flask. Use 0.1% phosphoric acid solution - acetonitrile (4 + 6) to reach a constant volume to the
scale and evenly mix it. Transfer-take 2 mL into a centrifuge tube, at 15,000 r/min, centrifuge
for 3 min. Take the supernatant, pass it through a 0.45 m microporous filter membrane, and
reserve it for determination.
5.2 Reference Conditions of Liquid Chromatography
The reference conditions of liquid chromatography are as follows.
a) Chromatographic column. SAX strong anion exchange column, 250 mm  4.6 mm
(inner diameter), 5 m; or one with equivalent performance.
b) Column temperature. 30 C.
c) Detection wavelength. 205 nm.
d) Mobile phase. 0.1% phosphoric acid solution - acetonitrile (40 + 60, volume ratio).
e) Flow rate. 1.0 mL/min.
f) Injection volume. 10 L.
5.3 Drawing of Standard Curve
Respectively inject the standard series of working solutions into the liquid chromatograph to
determine the corresponding peak area. Take the mass concentration of sialic acid in the
standard series of working solutions as the x-coordinate, and the peak area as the y-coordinate
to draw a standard curve. For the chromatogram of sialic acid standard solution, see Figure A.1
in Appendix A.
5.4 Determination of Specimen Solution
Inject the specimen solution into the liquid chromatograph to obtain the peak area. In
accordance with the standard curve, obtain the mass concentration of sialic acid in the solution
to be tested. The response values of sialic acid in the standard working solution and the
specimen solution to be tested shall be within the linear response range of the instrument. If the
of 1. 9.
10.2.3 O-phenylenediamine hydrochloride solution (50 mg/mL). weigh-take 2.5 g of o-
phenylenediamine hydrochloride, add 50 mL of 0.05 mol/L hydrochloric acid solution to
dissolve and evenly mix it. Prepare it right before use.
10.2.4 0.2% phosphoric acid solution. draw-take 2 mL of phosphoric acid, dissolve it in water
and dilute to 1,000 mL.
10.3 Reference Material
Sialic acid (N-acetylneuraminic acid, C11H19NO9, CAS No.. 131-48-6). purity  96%, or a
standard substance certified by the state and awarded a reference material certificate.
10.4 Preparation of Standard Solutions
10.4.1 Sialic acid standard stock solution (1,000 mg/L). accurately weigh-take an appropriate
amount of sialic acid reference material (converted in accordance with purity), use water to
dissolve and prepare it into a standard stock solution with a mass concentration of 1,000 mg/L.
Store it in the dark at 4 C. It shall remain valid for 6 months.
10.4.2 Sialic acid standard series of working solutions. respectively draw-take 0.2 mL, 1.0 mL,
2.0 mL, 4.0 mL, 10.0 mL and 20.0 mL of the sialic acid standard stock solution (1,000 mg/L)
into 100 mL volumetric flasks, use acetonitrile - water solution (1 + 9) to reach a constant
volume to the scale, evenly mix them. The mass concentration of the sialic acid standard series
of working solutions is respectively. 2.0 mg/L, 10.0 mg/L, 20.0 mg/L, 40.0 mg/L, 100.0 mg/L
and 200.0 mg/L. Prepare them right before use.
11 Instruments and Equipment
11.1 Liquid chromatograph. equipped with fluorescence detector.
11.2 Constant-temperature water bath.
11.3 Vortex mixer.
11.4 Analytical balance. with a division value of 0.1 mg and 0.01 g, respectively.
11.5 Homogenizer. with a rotation speed not lower than 10,000 r/min.
11.6 High-speed centrifuge.
12 Analytical Procedures
12.1 Pre-treatment of Samples
12.1.1 Preparation of specimens
For liquid specimens, shake them well. For semi-solid specimens and powdered specimens with
uniform matrix, directly use them for the next step of specimen extraction. Other specimens
need to be homogenized or evenly crushed.
12.1.2 Extraction of specimens
Weigh-take 1 g (accurate to 0.01 g) of sample into a 25 mL stoppered colorimetric tube (for
solid samples, add 3 mL of water first and conduct vortex oscillation for 30 s). Along the wall
of the tube, slowly add 10 mL of hydrochloric acid solution. While adding, oscillate it; use a
glass stopper to cover it and conduct vortex mixing. Place it in 80 C water bath to conduct
hydrolysis for 40 min. During the water bath, oscillate it every 5 min. Take out the colorimetric
tube and cool to room temperature. Use acetonitrile - water solution (1 + 9) to reach a constant
volume of 25 mL, evenly mix it and let it stand for 5 min. Take 10 mL of the upper-layer solution
and put it into a centrifuge tube. Add 5 mL of chloroform, conduct vortex mixing, then, at 9,000
r/min, centrifuge for 5 min. Take the supernatant for purification.
12.1.3 Purification of specimens
The solid-phase extraction column is successively activated with 3 mL of methanol and 3 mL
of water, and drained dry under negative pressure. The specimen solution passes through the
solid-phase extraction column at a flow rate of less than 1 mL/min. Collect the effluent and pass
it through a 0.45 m microporous filter membrane for derivatization.
12.1.4 Derivatization
Accurately draw-take 1.0 mL of the specimen solution into a 10 mL stoppered colorimetric tube,
accurately add 1.0 mL of o-phenylenediamine hydrochloride solution, use a glass stopper to
cover it and evenly mix it. Place it in 80 C water bath for reaction for 35 min. Then, take out
the colorimetric tube, cool to room temperature, pass it through a 0.45 m microporous filter
membrane for determination.
Meanwhile, conduct derivatization of the sialic acid standard series of working solutions. After
derivatization, the mass concentration of the standard series of working solutions is respectively.
1.0 mg/L, 5.0 mg/L,10.0 mg/L, 20.0 mg/L, 50.0 mg/L and 100.0 mg/L.
12.2 Reference Conditions of Liquid Chromatography
The reference conditions of liquid chromatography are as follows.
a) Chromatographic column. C18 column, 250 mm  4.6 mm (inner diameter), 5 m; or
one with equivalent performance.
b) Column temperature. 30 C.
c) Fluorescence detector excitation wavelength. 235 nm, emission wavelength. 414 nm.
d) Flow rate. 1.0 mL/min.
17.1.3 Formic acid (HCOOH). chromatographically pure.
17.1.4 Concentrated hydrochloric acid (HCl). 12 mol/L.
17.1.5 Trichloromethane (CHCl3).
17.1.6 Reversed-phase polymer solid-phase extraction column containing polar groups. 60 mg,
3 mL, or equivalent. The polar group-containing reversed-phase polymer solid-phase extraction
column adsorbent is a macro-porous polymer polymerized from two monomers, lipophilic
divinylbenzene and hydrophilic N-vinylpyrrolidone, in a certain proportion.
17.1.7 Microporous filter membrane. 0.22 m, organic phase type.
17.2 Preparation of Reagents
17.2.1 Hydrochloric acid solution (0.05 mol/L). draw-take 4.17 mL of concentrated
hydrochloric acid, dissolve it in water and dilute to 1,000 mL.
17.2.2 Acetonitrile - water solution (1 + 9). evenly mix acetonitrile and water in a volume ratio
of 1. 9.
17.2.3 0.1% formic acid solution. draw-take 1 mL of formic acid, dissolve it in water and dilute
to 1,000 mL.
17.3 Reference Material
Sialic acid (N-acetylneuraminic acid, C11H19NO9, CAS No.. 131-48-6). purity  96%, or a
standard substance certified by the state and awarded a reference material certificate.
17.4 Preparation of Standard Solutions
17.4.1 Sialic acid standard stock solution (1,000 mg/L). accurately weigh-tak...