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GB/T 40179-2021: Determination of organic acids in plant - Liquid chromatography-tandem mass spectrometry
GB/T 40179-2021
Determination of organic acids in plant-Liquid chromatography-tandem mass spectrometry
ICS 07.080
CCSA40
National Standards of People's Republic of China
Determination of organic acids in plants
Liquid chromatography-mass spectrometry/mass spectrometry
Released on 2021-05-21
2021-12-01 implementation
State Administration of Market Supervision and Administration
Issued by the National Standardization Management Committee
Preface
This document is in accordance with the provisions of GB/T 1.1-2020 "Guidelines for Standardization Work Part 1.Structure and Drafting Rules of Standardization Documents"
Drafting.
Please note that some of the contents of this document may involve patents. The issuing agency of this document is not responsible for identifying patents.
This document was proposed and managed by the National Standardization Technical Committee for Biochemical Testing (SAC/TC387).
Drafting organizations of this document. Institute of Biology, China Academy of Testing Technology, Sichuan University, China Jiliang University, Beijing Technology and Business University, Hebei
Provincial Food Inspection Research Institute.
The main drafters of this document. Li Huaiping, Feng Dejian, Song Hang, Wu Wei, Xu Yang, Zou Yan, Ye Shanrong, Zhou Lihua, Ye Zihong, Ma Aijin, Zhang Yan.
Determination of organic acids in plants
Liquid chromatography-mass spectrometry/mass spectrometry
1 Scope
This document specifies the method for the determination of organic acids in plants by liquid chromatography-mass spectrometry/mass spectrometry.
This document is applicable to fumaric acid, trans-aconitic acid, citrus, apple, strawberry, cucumber, tomato, honeysuckle, codonopsis, hawthorn, and wolfberry.
Adipic acid, gallic acid, vanillic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, salicylic acid, 2,5-dihydroxyl
Determination of benzoic acid and 3,4-dihydroxybenzoic acid.
2 Normative references
The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations
Only the version corresponding to that date is applicable to this document; for undated reference documents, the latest version (including all amendments) is applicable to
This document.
GB/T 6682 Analytical laboratory water specifications and test methods
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Organicacids
An acidic organic compound in plant materials.
4 Principle
After the sample is heated and refluxed with 50% methanol solution, it is separated by reversed-phase chromatographic column and determined and confirmed by liquid chromatography-mass spectrometry/mass spectrometry.
Standard working curve method for quantification.
5 Reagents or materials
Unless otherwise stated, the reagents used in this method are all chromatographically pure. The water is the first grade water specified in GB/T 6682.
5.1 Methanol.
5.2 Formic acid.
5.3 0.1% formic acid solution. pipette 1.0mL formic acid in a 1L volumetric flask, and dilute to the mark with water.
5.4 50% methanol solution. Pipette 500.0mL of methanol into a 1L volumetric flask, and dilute to the mark with water.
5.5 Organic acid standards. fumaric acid, trans-aconitic acid, adipic acid, gallic acid, vanillic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid
Acid, ferulic acid, p-hydroxybenzoic acid, salicylic acid, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid and succinic acid-2,2,3,3-d4, compounds
For details, see Table A.1 in Appendix A, and the purity is ≥98%.
5.6 Single standard stock solution. accurately weigh the appropriate amount of each organic acid standard (accurate to 0.01mg), including fumaric acid and trans-aconitic acid
Dissolve with gallic acid in water, adipic acid, vanillic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, salicylic acid
Acid, 2,5-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid are dissolved in methanol, mixed, and formulated into organic acids with a concentration of 1000.0mg/L
Single standard stock solution, sealed and stored at 0℃~4℃, the validity period is 3 months.
5.7 Mixed standard stock solution. accurately draw an appropriate amount of each organic acid single standard stock solution into the same 10mL volumetric flask, and use 50%
Dilute the alcohol solution and dilute to the mark, mix well, prepare a mixed standard stock solution with a concentration of 10.0mg/L, and seal it at 0℃~4℃
Keep it for 3 months.
5.8 Internal standard stock solution. accurately weigh out an appropriate amount of succinic acid-2,2,3,3-d4 standard (accurate to 0.01mg), dissolve it with 50% methanol solution, and mix it.
Evenly, formulated into an internal standard stock solution with a concentration of 100mg/L, sealed and stored at 0℃~4℃, the validity period is 3 months.
5.9 Internal standard solution. accurately pipette 1.0 mL of internal standard stock solution into a 100 mL volumetric flask, dilute with 50% methanol solution and dilute to an hour.
Mix well and prepare an internal standard solution with a concentration of 1.0 mg/L, which is now ready for use.
5.10 Mixed standard working solution. accurately draw an appropriate amount of mixed standard stock solution into a 10mL volumetric flask, and then accurately add it separately
2.5mL internal standard solution, diluted with 50% methanol solution to make the concentration are 0.10mg/L, 0.25mg/L, 0.50mg/L, 0.75,
1.0mg/L, 1.25mg/L series of mixed standard working solutions, ready to use.
5.11 0.22μm hydrophilic nylon microporous filter membrane.
6 Equipment
6.1 Liquid chromatography-mass spectrometry/mass spectrometer. distributed spray ion source (ESI).
6.2 Electronic balance. Sensitivity is 0.0001g and 0.01mg.
6.3 High-speed centrifuge. the speed is not less than 10000r/min.
6.4 High-speed homogenizer.
6.5 High-speed crusher.
6.6 Condensation reflux device.
7 Analysis steps
7.1 Sample preparation
Take about 1kg of representative dried samples such as honeysuckle, dangshen, hawthorn, wolfberry, etc., pulverize with a grinder, mix well, and divide the prepared samples equally
Divide into two parts, put into a clean sample container, seal and label, store at 0℃~4℃ in the dark, and measure within one week.
Take about 1kg of representative fresh samples such as citrus, apple, strawberry, cucumber, tomato, etc., use a homogenizer to make a homogenate, and prepare the sample
Divide them into two parts, put them in a clean sample container, seal and mark them, and make them for immediate use.
7.2 Sample handling
Weigh about 1.5g dry sample or 10g fresh sample (accurate to 0.001g) into a 100mL round bottom flask, add 625μL of internal standard stock solution and
30mL of 50% methanol solution, heated to reflux for 2.5h, filtered while hot, the filtrate was transferred to a 50mL volumetric flask, and 15mL of 50% methanol solution was divided into three
Wash the round bottom flask and sample residue twice, combine the washing liquid into a volumetric flask, dilute to the mark with 50% methanol solution, mix well, and under 10000r/min
Centrifuge for 5min, take 2.0mL supernatant in a 10mL volumetric flask, dilute to the mark with 50% methanol solution, mix well, pass 0.22μm nylon
Filter membrane for liquid chromatography-mass spectrometry/mass spectrometry determination.
7.3 Instrument reference conditions
7.3.1 Reference conditions for liquid chromatography
a) Chromatographic column. HSST3 column, 2.1mm×150mm, particle size 1.8μm, or a column of equivalent performance;
b) Mobile phase. mobile phase A is 0.1% formic acid solution, mobile phase B is methanol, and the gradient elution procedure is shown in Table 1;
c) Flow rate. 0.2mL/min;
d) Column temperature. 30℃;
e) Injection volume. 1μL.
Table 1 Mobile phase gradient elution program
7.3.2 Reference conditions for mass spectrometry
a) Ion source. Electrospray ion source (ESI);
b) Scanning mode. negative ion mode;
c) Detection method. multiple reaction monitoring (MRM);
d) Dry gas, atomizing gas, and collision gas are all high-purity nitrogen; adjust the flow of each gas before use to make the sensitivity of the mass spectrometer meet the detection requirements
See Appendix B for reference conditions;
e) Parameters such as spray voltage, fragmentation voltage in the source, and collision gas energy should be optimized to the best sensitivity. Refer to Appendix B for reference conditions.
7.4 Liquid chromatography-mass spectrometry/mass spectrometry
7.4.1 Qualitative determination
When the sample is measured under the same experimental conditions, if the retention time of the substance to be tested in the sample is
The retention time is consistent (within ±2.5%), and the relative abundance and concentration of the component to be tested in the sample spectrum are equivalent to the standard
If the relative abundance of the sample is consistent, and the allowable deviation does not exceed the range specified in Table 2, it can be judged that the corresponding analyte exists in the sample.
Table 2 Maximum allowable deviation of relative ion abundance during qualitative confirmation
7.4.2 Quantitative determination
Under the same experimental conditions, the organic acid series of mixed standard working solutions were sequentially injected and analyzed from low to high concentration, and the analyte and the internal standard
The ratio of the quantitative ion peak area of the substance is the ordinate, and the concentration of the analyte is the abscissa.
Draw a curve to quantify the sample, so that the response value of each analyte in the sample solution is within the linear range determined by the instrument.
The upper limit of the standard working curve range should be re-measured after appropriately reducing the sampling volume. Under the above conditions, the reference retention time of each organic acid
See Table B.1 in Appendix B, and see Appendix C for multiple reaction monitoring diagrams.
7.5 Parallel test
Perform two parallel measurements on the same sample according to the provisions of 7.2 to 7.4.
8 Test data processing
The calculation of the content of the analyte in the sample is shown in formula (1).
X=ρ×
(1)
Where.
X ---The content of the component to be tested in the sample, in milligrams per kilogram (mg/kg);
ρ --- The mass concentration of the tested component solution obtained from the internal standard standard working curve, in micrograms per milliliter (μg/mL);
V --- the total volume of the sample solution, in milliliter (mL);
m --- The mass of the sample, in grams (g).
The calculation result is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, and the result is to two decimal places.
9 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.
10 other
Refer to Table A.1 in Appendix A for the limits of quantification of 14 organic acids determined by this method.