GB/T 39995-2021.Determination of sterols.
1 Scope
GB/T 39995 specifies the liquid chromatography-mass spectrometry/mass spectrometry method for the determination of sterols.
GB/T 39995 is applicable to the determination of free cholesterol, rapeseed sterol, campesterol, stigmasterol, β-sitosterol, fucosterol, lanosterol, stigmasterol and ergosterol in lard, rapeseed oil, walnut, hawthorn, lettuce, kudzu root, rice, wheat and black green beans. ||
tt||2 Normative references
The contents of the following documents constitute the essential terms of this document through normative references in the text. Among them, for dated references, only the version corresponding to that date applies to this document; for undated references, the latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water used in analytical laboratories
GB/T 15687 Preparation of animal and vegetable oil samples
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Sterols
A class of compounds containing a cyclopentane-polyhydrogen phenanthrene carbon skeleton structure.
4 Principle
The sample is extracted by reflux with anhydrous ethanol, and the extract is filtered through a 0.22μm nylon filter membrane and then subjected to liquid chromatography-mass spectrometry/mass spectrometry for determination. The standard curve internal standard method is used for quantification.
5 Reagents or materials
Unless otherwise specified, the reagents used in this method are of analytical grade. Water is the first-grade water specified in GB/T 6682.
5.1 Methanol: chromatographic grade.
5.2 Anhydrous ethanol: chromatographic grade.
5.3 Sterol standards: Cholestanol, rapeseed sterol, campesterol, stigmasterol, β-sitosterol, fucosterol, lanosterol, stigmasterol, ergosterol, 6-ketocholestanol. For compound information, see Table A.1 in Appendix A. Purity is ≥ 93%. ||tt
||5.4 Internal standard stock solution: Accurately weigh an appropriate amount of 6-ketocholestanol standard (accurate to 0.01 mg), dissolve it in anhydrous ethanol and prepare an internal standard stock solution with a mass concentration of 500 mg/L. Store it at 0 °C~4 °C in the dark for future use. The validity period is 1 month.
5.5 Internal standard working solution: Accurately transfer 2.0 mL of the internal standard stock solution into a 1000 mL volumetric flask, dilute it with anhydrous ethanol and make up to the mark, mix well, and prepare an internal standard working solution with a mass concentration of 1.0 μg/mL. Prepare it for immediate use.
This document specifies the liquid chromatography-mass spectrometry/mass spectrometry determination method for sterols. This document is applicable to the determination of free cholesterol, rapeseed sterol, campesterol, stigmasterol, β-sitosterol, fucosterol, lanosterol, stigmasterol and ergosterol in lard, rapeseed oil, walnut, hawthorn, lettuce, kudzu root, rice, wheat and black green beans.

1CS07.080
CCS A 40
National Standard of the People's Republic of China
GB/T39995—2021
Determination of sterols
Published on April 30, 2021
State Administration for Market Regulation
National Standardization Administration
Implemented on November 1, 2021
People's Republic of China
National Standard
Determination of alcohols
GB/T39305—2021
Published and distributed by China Standards Publishing House
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First edition in April 2021
Book number: 155066 -1-67034
Copyright reserved
Infringements must be investigated
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GB/T 39995—2021
This document is drafted in accordance with the provisions of GB/T1.12020% Guidelines for Standardization Work Part 1: Structure and Drafting Rules of Standardization Documents.
Please note that some contents of this document may involve patents. The issuing agency of this document does not assume the responsibility for identifying patents. This document is proposed and managed by the National Technical Committee for Biochemical Testing Standardization (SAC/TC387). The drafting units of this document are: Institute of Biology, China Institute of Testing Technology, China Institute of Metrology, Sichuan Institute of Technology, Huizhou Food and Drug Inspection Institute, Beijing Institute of Industry and Commerce, Zhejiang Institute of Inspection and Quarantine Science and Technology, Hebei Institute of Food Inspection, Beijing Sambo Technology Co., Ltd.,
The main drafters of this document are: Feng Dejian, Zhang Ya Fen, Duo Yanfang, Wu Wei, Ye Zihong, Huang Xiuli, Wang Xiaoqin, Ma Aixun, Li Huaiping, Yu Xiaoping, Huang Chaoqun, Ye Shanrong, Zhou Lihua, Cui Haifeng, Xu Yipeng, Zhong Ju Jiaping, Li Yi, Chen Li, Guan Juan, Jia Yingmin, Zhang Yan, Hao Shuai-riKaeerKAca-
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1 Scope
Determination of sterols
This document specifies the liquid chromatography-mass spectrometry/mass spectrometry method for the determination of sterols. GB/T 39995—2021
The wooden document is applicable to the determination of free cholanol, rapeseed oil, walnut, hawthorn, lettuce, kudzu root, rice, wheat, black green lard, rapeseed oil, rongyoudiol, bezoarol, 3-sitol, fucoxanthinol, lanolinol, bezoarol, ergosterol in lard ...ergosterol in lard, rapeseed oil, walnut, hawthorn, lettuce, kudzu root, rice, wheat, black green lard, rapeseed oil, rongyoudiol, bezoarol, ergosterol in lard, rapeseed oil, walnut, hawthorn, lettuce, kudzu root, rice, wheat, black green lard 2 Normative references
The contents of the following documents constitute essential clauses of this document through normative references in the text. Among them, for references with a date, only the version corresponding to that date applies to this document; for references without a date, the latest version (including all amendments) applies to this document.
: Specifications and test methods for water for analytical laboratories GB/T 6682
GB/T15687 Preparation of animal and vegetable fat samples 3 Terms and definitions
The following terms and definitions apply to this document. 3.1
Sterols
Compounds containing cyclopentane polyhydrogen non-carbon framework structure. 4 Principle
The sample is extracted with anhydrous ethanol by reflux, and the extract is filtered through a 0.22um nylon filter membrane and then subjected to liquid chromatography-mass spectrometry/mass spectrometry determination. The standard curve internal standard method is used for quantification
5 Reagents or materials
Unless otherwise specified, the reagents used in this method are of analytical grade. Water is (grade 1 water specified in 13/T6682, 5.1 Methanol: chromatographically pure.
5.2 Anhydrous ethanol: chromatographically pure
5.3 Standard alcohols: cholecalciferol, rapeseed alcohol, campesterol, styrol, 3-sitosterol, fucoxanthinol, lanosterol, styrol, 6-ketocholesterol. 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-ketocholesterol standard (accurate to 0.01m), dissolve it in anhydrous ethanol and prepare an internal standard stock solution with a mass concentration of 500mg/1. Store it in the dark at 0~4℃ for use, with a validity period of 1 month. 5.5 Internal standard working solution: Accurately transfer 2.01mL of the internal standard stock solution into a 1000nL volume 5.6 Alcohol mixed standard solution: Accurately weigh appropriate amounts of alcohol standard (accurate to 0.01 mg), dissolve in anhydrous ethanol to prepare 1 liter of alcohol standard stock solution with a mass concentration of 500 mg/L, and store in dark at 0℃~4℃ for future use. The validity period is 1 tablet. 5.7 Alcohol mixed standard solution: Accurately transfer 2.0 ml of each alcohol standard stock solution into a 50 ml stoppered centrifuge tube, blow dry with nitrogen at 10 ℃, and accurately add 10 ml of internal standard working solution. Dissolve and mix well. Prepare mixed standard solutions of 100 mg/L alcohols, ready for immediate use.
5.8 Mixed standard working solution of 1-ethanol: Accurately pipette an appropriate amount of mixed standard solution of 1-ethanol. Dilute with internal standard working solution to prepare a series of mixed standard working solutions 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 alcohols, ready for immediate use.
5.9 0.22 um nylon filter membrane.
6 Instruments and equipment
Liquid chromatography-mass spectrometry/mass spectrometer (IC-MS/MS): equipped with atmospheric pressure chemical ionization source (APCI) 6.2
Analytical balance with a sensitivity of 0.000 1g.0.01g and 0.01mg6.3 Vortex mixer.
Reflux condenser.
Nitrogen blower.
Crusher.
Slurry mixer.
6.8 Centrifuge.
7 Samples
7.1 Lard and rapeseed oil
Prepare according to the provisions of (G13/T15687, 7.2 Walnut, hawthorn, rice, wheat, lettuce, kudzu root, black green beans Take about [kg of representative dry samples of walnut, hawthorn, rice, wheat, black green beans, etc. Use a crusher to crush and mix. The prepared samples are divided into two parts and placed in a clean sample container. Seal and label. Store at 0℃4℃ away from light and measure as soon as possible. Take about 50% representative fresh samples of lettuce, kudzu root, etc.! k, use a homogenizer to make a homogenate, divide the prepared sample into two parts, put them into clean sample containers, seal and label them, and use them immediately. 8 Test steps
8.1 Sample treatment
8.1.1 Lard and rapeseed oil
Weigh 0.4g of the sample (accurate to 0.001g) into a 250mL round-bottom flask, add 70mL of internal standard T solution (5.5). Weigh. After fully mixing, put it into a countercurrent condenser, heat and keep the extract at a slight boil, stop heating after 30min and cool to room temperature, use anhydrous ethanol (5.2) to make up for the weight loss, mix well, and absorb 5ml. Centrifuge the extract at 8000r/min for 5min. Take 2ml of the supernatant and filter it through a 0.22pm nylon filter membrane for testing.
8.1.2 Walnut, hawthorn, rice, wheat, lettuce, kudzu root, black green bean Weigh 1.0g of the sample (accurate to 0.001g) into a 250mL round-bottom flask, add 70mL of the internal standard solution (5.5) and weigh it. Fill it up for 2 minutes and mix it. Put it into a reflux condenser. Heat and keep the extract at a slight boil. After 30 minutes, stop heating and cool it to room temperature. Make up the heat with anhydrous ethanol (5.2) and mix well. Take 5ml. Centrifuge the extract at 8000r/min for 5 minutes. Take 2ml. Pass the clear liquid through a 0.22μm nylon filter membrane for testing.
8.2 Determination
Liquid chromatography reference conditions
Liquid chromatography reference conditions are as follows:
Chromatographic column: C column, 1.8um, 3.0mm×150mm, or chromatographic column with equivalent performance; a
b) Mobile phase: Mobile phase A is water, mobile phase B is methanol. Gradient elution program is shown in Table 1; Flow rate: 0.5ml/min;
Column temperature: 30℃:
Injection volume: 5
Table 1 Mobile phase gradient elution program
Time/min
Note: Different types of instruments can be adjusted appropriately. Mass spectrometry reference conditions
Mass spectrometry reference conditions are as follows:
Mobile phase A fraction/%
a) Ion source: Atmospheric pressure chemical ionization (APCI): Scan mode: Positive ionization mode ;
Detection method: Multiple reaction monitoring (MRM): c
Mobile phase B integral fraction/%
The drying gas, nebulizer gas and collision gas are all high-purity nitrogen: The flow rate of each gas should be adjusted before use to make the mass spectrometer sensitivity meet the detection requirements. For reference conditions, see Appendix B:
Parameters such as spray voltage, source fragmentation voltage, collision gas energy, etc. should be optimized to the maximum sensitivity. For reference conditions, see Appendix 3 Note: Different models of instruments can be adjusted appropriately. 8.2.3 Qualitative determination
When the sample is determined under the same test conditions (see 8.2.1 and 8.2.2), if the retention time of the substance to be measured in the sample is consistent with the corresponding retention time in the standard working solution (the variation range is within 2.5%), the relative abundance of the measured component monitored in the sample spectrum is consistent with the relative abundance of the standard, and the allowable deviation does not exceed the specified range of 2, then it can be determined that the corresponding substance to be measured exists in the sample. Table 2 Maximum allowable deviation of relative ion abundance during qualitative confirmation Relative ion abundance/%
Allowable relative deviation/%
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GB/T39995—2021
8.2.4 Quantitative determination
Under the same experimental conditions (8.2.1, 8.2.2), the sterol series mixed standard working solutions are injected and analyzed in sequence from low to high concentrations. The ratio of the quantitative ion peak area of ​​the analyte and the internal standard is used as the ordinate, and the concentration of the analyte is used as the abscissa to draw the internal standard working curve. The internal standard working curve is used to quantify the sample. The response value of each analyte in the sample solution should be within the linear range of the instrument. If the content exceeds the standard working curve range, it should be diluted to an appropriate concentration before injection and analysis. Under the above conditions, the reference retention time of each sterol substance is shown in Table 1 in the Appendix, and the multiple reaction monitoring diagram is shown in Appendix (8.3 Parallel test
According to the provisions of 8.1 and 8.2, the same sample trace is subjected to two parallel determination test data processing
The calculation of the content of the analyte in the sample is shown in formula (1): -0xV
Wherein:
X—the content of the measured component in the sample. The unit is milligram per kilogram (mg/kg); P——the mass concentration of the measured component solution obtained from the internal standard working curve, the unit is microgram per milliliter (g/ml); the volume of the sample solution, the unit is milliliter (mL); V
m—the mass of the sample, the unit is gram (g). The calculation results are based on two independent measurements obtained under repeatability conditions. The arithmetic mean of the determination results is estimated and the results are rounded to two decimal places. Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 15% of the arithmetic mean. 11 Others
For the quantitative limits of the nine alcohols determined by this method, please refer to Table A.1. rKaeerkAca-
Appendix A
(Informative)
Information on sterol compounds and quantitative limits
Information on sterol compounds and quantitative limits are shown in Table A.1Table A.1
Chinese name
Ergosterol
Brassic acid alcohol
Fucosqualane alcohol
Lanolin alcohol
Peptidestanol
Dodecyl alcohol
Raisin alcohol
Zinc Sitosanol
Stigmastanol
6-Ketocholestanol
English Name
Ergosterol
Brassicasicrol
Fucosteral
L.anoste:rol
3-Cholestanol
Stigmasterol
Carnpesterol
β-Sitosterol
Stigmastanol
6-Ketocholestanol
Compound Information and Quantitative Limit
Relative Molecular Weight
CAS No.
474-67-9
17605-87-3
79-63-0
80-97-7
83-18-7
474-62-41
8:3-46-5
19166-47-8
1175-06-0
Molecular formula
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Lard,
seed oil
GB/T39995—2021
Quantitative limit/(mg/kg)
Walnut, hawthorn, kudzu root, oil friend Rong
Rice, wheat, black green beans
GB/T 39995—2021
The reference mass spectrometry conditions are as follows:
Appendix B
(Informative)
Liquid chromatography-mass spectrometry/mass spectrometry parameter reference conditionsIon source: atmospheric pressure chemical ion source (APCI):Scan mode: positive ionization mode:
Detection method: multiple reaction monitoring (MRM);1Dry gas, nebulizer gas and collision gas are all high-purity nitrogen:Nebulizer gas pressure: 0.14MPa (20psi);Ion spray voltage: 3000V:
Electroplastic needle current: 4μA:
1Dry gas temperature: 350℃;
Nebulizer temperature: 350℃;
+Dry gas flow rate: 5.0L/min;
Quantitative ionization The retention time, monitoring ion pair, collision gas energy and source fragmentation voltage of 10 kinds of alcohols are shown in Table 13.1. The retention time, monitoring ion pair, collision gas energy and source fragmentation voltage of 10 kinds of alcohols are shown in Table B.1
Chinese name
ergofluidol
rapeseedfluidol
fucofluidol
lanosterol
hepatofluidol
stigmasterol
campesinofluidol
3-sitosterol
stigmasterol
polyketocholestanol
retention time/min
quantitative ion pair m/t
379.4/69.0
381.4/69.0
395.5/81.0||t t | Li Guang pairs /
379.4/69.0.379.4/125.0
381.4/147.0;381.4/69.0
395.5/81.0:395.5/147.0
409.4/109.6:409.4/119.0
37 1.4/ 81.1:371.4/109.0
411.3/109.0:111.3/139.0
383.1/161.1;383.4/05.1
397.5/161.0;397.5/135.1
389.5/81.1399.5/149.0
385.3/367.4;385.3/159.2
Collision gas energy/V
In-source fragmentation voltage/V
Note: The listed parameters were completed on Agilr:nt6460 mass spectrometer 1. The listed test instrument models are for reference only. Standard users can use instruments of different manufacturers or models.
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Appendix C
(Informative)
LC-MS/MS multiple reaction monitoring diagrams of 10 sterols LCMS/MS multiple reaction monitoring diagrams of 10 pre-alcohols are shown in Figures C.1 to C.10+MRM(385.3->367.4)std6.d
13.13muin
MRM diagram of 6-ketocholestanol
+MRM(395.5 -> 81.0) STD-fux103
17.87 min
MRM diagram of fucosterol
+MRM(411.3 > 109. 0) STD-s
Figure C.7 MRM diagram of soybean alcohol
+ MRM(379. 4 -> 69. 0) STD-cr x10 5
16.64 min
MRM diagram of ergosterol
+MRM(409. 4 -> 109. 0) STD-1xio 4Www.bzxZ.net
Figure C.5 MRM diagram of lanosterol
+MRM(383. 4 > 161. 1) STD-c
Figure C.8 MRM diagram of campesterol
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GB/T 39995-2021
+MRM(381.4 -> 69. 0) brassicx1o 4
MRM diagram of brassic alcohol
+MRM(371.4 -> 81. 1) STD-ch
MRM diagram of cholestanol
+MRM(397. 5 -> 161. 0) STD-bx104-
Figure C.9MRM diagram of β-glutamyl alcohol
GB/T 39995—2021
GB/T39995-2021
+MRM(399. 5 -> 81. 1) STD 8ti3
MRM diagram of stigmanol
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