GB/T 39995-2021 PDF English
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Determination of sterols
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GB/T 39995-2021: Determination of sterols---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GBT39995-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 07.080
CCS A 40
Determination of sterols
ISSUED ON: APRIL 30, 2021
IMPLEMENTED ON: NOVEMBER 1, 2021
Issued by: State Administration for Market Regulation;
Standardization Administration of the PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Terms and definitions ... 4
4 Principle ... 4
5 Reagents or materials ... 5
6 Apparatus ... 6
7 Samples ... 6
8 Test procedure ... 6
9 Test data processing ... 9
10 Precision ... 9
11 Other ... 9
Appendix A (Informative) Information and limits of quantitation for sterol
compounds ... 10
Appendix B (Informative) Reference conditions for liquid chromatography-mass
spectrometry/mass spectrometry parameters ... 11
Appendix C (Informative) LC-MS/MS multiple reaction monitoring diagrams of
10 sterols ... 13
Determination of sterols
1 Scope
This document specifies a method for the determination of sterols by liquid
chromatography-mass spectrometry/mass spectrometry.
This document applies to the determination of the content OF β-Cholestanol,
brassicasterol, campesterol, stigmasterol, β-Sitosterol, fucosterol, lanosterol,
stigmastanol, and ergosterol in the free state IN lard, rapeseed oil, walnuts, hawthorn,
lettuce, kudzu root, rice, wheat, and black green beans.
2 Normative references
The contents of the following documents, through normative references in this text,
constitute indispensable provisions of this document. Among them, for dated references,
only the edition corresponding to that date applies to this document. For undated
references, the latest edition (including all amendments) applies to this document.
GB/T 6682 Water for analytical laboratory use - Specification and test methods
GB/T 15687 Animal and vegetable fats and oils - Preparation of test sample
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Sterols
A class of compounds containing a cyclopentano-perhydrophenanthrene carbon frame
structure.
4 Principle
Use reflux of absolute ethanol to extract the sample. After the extract passes through a
0.22 μm nylon filter membrane, use liquid chromatography-mass spectrometry/mass
spectrometer for determination; use the standard curve internal standard method for
quantification.
5 Reagents or materials
Unless otherwise stated, the reagents used in this method are analytically pure. The
water shall be Grade 1 water stipulated in GB/T 6682.
5.1 Methanol: Chromatographically pure.
5.2 Absolute ethanol: Chromatographically pure.
5.3 Sterol standard substances: β-Cholestanol, brassicasterol, campesterol, stigmasterol,
β-Sitosterol, fucosterol, lanosterol, stigmastanol, ergosterol, and 6-Ketocholestanol. For
compound information, see Table A.1 in Appendix A. The purity is ≥93%.
5.4 Internal standard stock solution: Accurately weigh an appropriate amount of 6-
Ketocholestanol standard substance (accurate to 0.01 mg); use absolute ethanol to
dissolve it and prepare an internal standard stock solution with a mass concentration of
500 mg/L. Store in the dark at 0 °C~4 °C for later use. The validity period is 1 month.
5.5 Internal standard working solution: Accurately pipette 2.0 mL of internal standard
stock solution into a 1000 mL volumetric flask; add absolute ethanol to dilute and make
up to volume and mix well; prepare an internal standard working solution with a mass
concentration of 1.0 μg/mL. This solution is used right after it is prepared.
5.6 Sterol single standard stock solution: Accurately weigh an appropriate amount of
sterol standard substances (accurate to 0.01 mg), respectively. Use absolute ethanol to
dissolve and prepare into sterol single standard stock solutions with a mass
concentration of 500 mg/L, respectively. Store in the dark at 0 °C~4 °C for later use.
The validity period is 1 month.
5.7 Sterol mixed standard solution: Accurately pipette 2.0 mL of each sterol single
standard stock solution into a 50 mL stoppered centrifuge tube; use nitrogen to blow
dry at 40 °C. Accurately add 10 mL of internal standard working solution to redissolve
and mix well; prepare a sterol mixed standard solution with a mass concentration of
100 mg/L. This solution is used right after it is prepared.
5.8 Sterol mixed standard working solution: Accurately pipette an appropriate amount
of sterol mixed standard solution. Use the internal standard working solution to dilute
AND prepare a series of sterol mixed standard working solutions with mass
concentrations of 20 mg/L, 10 mg/L, 5 mg/L, 2.5 mg/L, 1.0 mg/L, 0.5 mg/L, and 0.25
mg/L, respectively. The solutions are used right after they are prepared.
5.9 0.22 μm nylon filter membrane.
of each sterol; refer to Appendix C for the multiple reaction monitoring diagram.
8.3 Parallel tests
According to the provisions of 8.1 and 8.2, carry out two parallel determinations on the
same test sample.
9 Test data processing
The calculation of the content of the analyte in the sample is shown in formula (1):
Where:
X - The content of the component tested in the test sample, in milligrams per kilogram
(mg/kg);
ρ - The mass concentration of the tested component solution obtained from the internal
standard working curve, in micrograms per milliliter (μg/mL);
V - The constant volume of test sample solution, in milliliters (mL);
m - The mass of test sample, in grams (g).
The calculation result shall be expressed as the arithmetic mean of two independent
determination results obtained under repeatability conditions. The result is rounded to
two decimal places.
10 Precision
The absolute difference between two independent determination results, which are
obtained under repeatability conditions, must not exceed 15% of the arithmetic mean.
11 Other
See Table A.1 for the limits of quantification of the 9 sterols determined by this method.
Appendix B
(Informative)
Reference conditions for liquid chromatography-mass spectrometry/mass
spectrometry parameters
The reference mass spectrometry conditions are as follows:
a) Ion source: Atmospheric-pressure chemical ion source (APCI);
b) Scanning mode: Positive ion mode;
c) Detection method: Multiple reaction monitoring (MRM);
d) Drying gas, atomizing gas, and collision gas are all high-purity nitrogen;
e) Atomizing gas pressure: 0.14 MPa (20 psi);
f) Ion spray voltage: 3000 V;
g) Corona needle current: 4 μA;
h) Drying gas temperature: 350 °C;
i) Atomizer temperature: 350 °C;
j) Drying gas flow rate: 5.0 L/min;
k) Quantitative ion pairs, qualitative ion pairs, collision gas energy, and in-source
fragmentation voltage are shown in Table B.1.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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