Standard number: GB/T 38571-2020
Standard name: Determination of alkaloids as plant secondary metabolites—Liquid chromatography-mass spectrometry
English name: Determination of alkaloids as plant secondary metabolites—Liquid chromatography-mass spectrometry
Standard format: PDF
Release time: 2020-03-31
Implementation time: 2020-03-31
Standard size: 455K
Standard introduction: 1 Scope
This standard specifies the method for detecting alkaloids in plant secondary metabolites using liquid chromatography tandem mass spectrometry.
This standard applies to the determination of (-)-scopolamine, strychnine, aconitine, mesaconitine, mesaconitine, colchicine, atropine, strychnine, philocarpine, pilocarpine and podophyllotoxin
.
2 Normative references
The following documents are indispensable for the application of this document. For dated references, only the dated version applies to this document. For undated references, the latest version (including all amendments) applies to this document.
GB/T6682 Specifications and test methods for water for analytical laboratories
This standard specifies the method for detecting alkaloids of plant secondary metabolites using liquid chromatography-tandem mass spectrometry. This standard is applicable to the determination of (-)-scopolamine, strychnine, aconitine, hypaconitine, metaconitine, colchicine, atropine, strychnine, camptothecin, pilocarpine and podophyllotoxin.

ICS07.080
iiikAacJouakAa
National Standard of the People's Republic of China
GB/T38571-2020
Detection of alkaloids as plant secondary metabolites--Liquid chromatography-mass spectrometry
Determination of alkaloids as plant secondary metabolites--Liquid chromatography-mass spectrometry2020-03-31Issued
State Administration for Market Regulation
National Standardization Administration
2020-03-31Implementation
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. iiiKAa~cJouaKAa
GB/T38571—2020
The drafting units of this standard are: Biological Research Institute of China Testing Technology Research Institute, Hebei Food Inspection Institute, Hebei Medical University of China National Institute of Standardization, and China University of Metrology. The main drafters of this standard are: Zhang Yan, Fan Sufang, Ma Aijin, Feng Dejian, Ma Chunling, Wendi, Cong Bin, Wang Juan, Chang Pin, and Ye Zihong. 1
1Scope
Detection of alkaloids in plant secondary metabolites
Liquid chromatography-mass spectrometry
iiiKAa~cJouakAa
GB/T38571—2020
This standard specifies the method for detecting alkaloids in plant secondary metabolites using liquid chromatography-tandem mass spectrometry. This standard applies to the determination of (-)-toshibapine, strychnine, aconitine, mesaconitine, mesaconitine, colchicine, atropine, styraxine, camptothecin, pilocarpine, and podophyllotoxin. 2 Normative references
The following documents are essential for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water for analytical laboratories 3 Principle
The sample is extracted by ultrasonic extraction and centrifugation, and then measured by liquid chromatography-tandem mass spectrometry, and quantified by external standard method. Reagents or materials
Unless otherwise specified, all reagents used are analytically pure. Water is the first-grade water specified in GB/T6682. 10% (volume fraction) acetonitrile solution
10mL acetonitrile + 90mL deionized water, mix. 4.2 Formic acid
Chromatographically pure.
4.3 0.1% (volume fraction) formic acid aqueous solution Accurately transfer 1.0 mL of formic acid into a 1L volumetric flask, dilute to volume with water, and mix. 4.4 1% (volume fraction) formic acid-acetonitrile solution 1 mL formic acid + 99 mL acetonitrile, mix.
5 100 mmol/L ammonium acetate solution
Weigh 3.854 g of ammonium acetate-dissolve with deionized water and dilute to 500 mL4.6
Ammonium acetate-ammonia buffer solution·pH9
Take 100 mL of 100 mmol/L ammonium acetate solution and adjust the pH to 9.0±0.5 with ammonia. 1
GB/T38571—2020
Alkaloids
Purity is not less than 98%.
1mg/mL alkaloid stock solution
iiiKAa~cJouakAa
Accurately weigh 0.100g of the corresponding alkaloid and place it in a 100mL volumetric flask, dilute to volume with methanol to prepare a stock solution with a concentration of 1mg/mL, and store it in the dark at 4. The validity period is 2 months. 910mg/L mixed standard stock solution
Accurately pipette 1.0mL of (-)-donghuaying, strychnine, aconitine, mesaconitine, colchicine, atropine, thiazolinone, camptothecin, pilocarpine, and podophyllotoxin standard stock solution into a 100mL volumetric flask. Make up to volume with methanol and mix well. Prepare a mixed standard stock solution of (-)-donghuaying, strychnine, aconitine, mesaconitine, colchicine, atropine, thiazolinone, camptothecin, pilocarpine, and podophyllotoxin with a concentration of 10mg/L. Store at 4℃ away from light. It can be used for one week. 4.10 microporous filter membrane
0.22Am, organic phase.
5 Instruments and equipment
5.1 Liquid chromatography-tandem mass spectrometer (LC-MS/MS): equipped with electrospray ion source (ESI) and quadrupole mass analyzer. 2 Electronic balance, sensitivity of 0.0001g and 0.001g. 5.2
5.3 Centrifuge: maximum speed not less than 9500r/min. 5.4 Ultrasonic cleaning instrument.
5 Nitrogen blowing instrument.
Vortex mixer.
5.7 Homogenizer.
High-centrifuge tube: 10mL.50mL.
6 Sample preparation
1 Plant tissue, animal tissue
Solid samples such as plant tissue and animal tissue should be minced and homogenized. 6.2
Liquid samples such as blood and urine should be fully mixed before testing. 7 Test steps
7.1 Sample extraction
Plant tissue, animal tissue
Weigh 1g of plant tissue sample (accurate to 0.001g) into a 50mL centrifuge tube. Add 5mL of deionized water and vortex for 2min. Add 10mL of acetonitrile and vortex for 1min. Perform ultrasonic assisted extraction for 10min. Add 4g of anhydrous magnesium sulfate and shake rapidly for 1min. 9500r/min2
iiiKAa~cJouakAa
GB/T38571—2020
Centrifuge for 5min. Take 5.0mL of supernatant and blow out with hydrogen in a 45℃ water bath. Reduce to dryness, reconstitute with 1.0mL 10% acetonitrile solution. Pass through a microporous filter membrane and wait for determination.
7.1.2 Body fluids
7.1.2.1 Blood
Take 0.5mL of blood sample and put it into a 10mL centrifuge tube. Add 1mL ammonium acetate-ammonia buffer solution, vortex mix for 1min, add 2.0mL methanol, vortex mix for 2min, perform ultrasonic assisted extraction for 10min, centrifuge at 9500r/min for 5min, take the supernatant: add 2.0mL methanol to the residual flow and repeat the extraction once. Combine the supernatants, concentrate to dryness by nitrogen blowing in a 45℃ water bath, reconstitute with 1.0mL 10% acetonitrile solution, pass through a microporous filter membrane and wait for determination.
Take 1.0mL of urine sample in a 10mL centrifuge tube, accurately add 4.0mL of 1% formic acid-acetonitrile solution, vortex and mix for 1min. Add 1.5g of anhydrous magnesium sulfate to the centrifuge tube, shake rapidly for 1min. High speed centrifuge at 9500r/min for 5min. Take 2.0mL of supernatant and concentrate to dryness in a 45℃ water bath with ammonia, re-dissolve with 1.0mL of 10% acetonitrile solution, pass through a microporous filter membrane, and wait for determination. 7.2 Instrument reference conditions
7.2.1 Liquid chromatography reference conditions are as follows:
Chromatographic column: Cls column, 100mm×2.1mm inner diameter). 2.5um, or equivalent. a)t
6b) Mobile phase: Mobile phase A-0.1% formic acid aqueous solution: Mobile phase B-acetonitrile. Gradient elution, see Appendix A for gradient elution procedure.
Flow rate: 0.30mL/min
Column temperature: 30C
Injection volume: 2#L
Mass spectrometry reference conditions are as follows:
Ionization mode: electrospray ionization positive ion mode: b
Spray voltage: 3.5kV:
Ion transmission temperature: 350℃
High ion source temperature: 320C:
Warp gas: 4.6L/min
Auxiliary gas: 8.8L/min
Purge gas: 1.5L/min
h) Scanning mode: Multiple reaction monitoring (MRM) mode. For the mass spectrometry parameters of 11 alkaloids, please refer to Appendix B. 7.3 Preparation of mixed standard working solution
Take the corresponding blank sample and process it according to step 6.2. Use the obtained blank sample extract to dilute the mixed standard stock solution step by step to obtain a series of mixed standard working solutions of 5Ag/L10g/L.20pg/L, 50#g/L, and 100#g/L of dongshobiline, fenbutine, aconitine, mesaconitine, aconitine, colchicine, atropine, dapoxetine, camptothecin, pilocarpine, and podophyllotoxin. The concentrations are tested from low to high, and the quantitative ion peak area is plotted against the mass concentration to make a standard curve regression equation. 7.4 Determination steps
Inject the sample solution into the liquid chromatography-tandem mass spectrometer to obtain the mass spectrometry response values ​​of 11 alkaloids. According to the standard curve, the concentrations of 11 alkaloids in the test solution are obtained. The response values ​​of each component in the sample solution should be within the linear range of the standard curve. If it exceeds the linear range, it should be diluted and then injected for determination.
7.5 Qualitative determination
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Measure the sample solution and the standard solution under the same conditions. If the retention time of the chromatographic peak detected in the sample solution is consistent with the retention time of a certain component peak in the standard solution (the range of variation is within ±2.5%), and the mass-to-charge ratio of the two pairs of selected daughter ions is consistent, when the relative abundance of the qualifier ion in the sample solution is compared with the relative abundance of the qualifier ion in the standard working solution with equivalent concentration, the relative deviation does not exceed the range specified in Table 1, then it can be judged that the component exists in the sample. For the total high ion flow diagram of 11 alkaloid reference materials, see Appendix C Table 1 Maximum allowable deviation of relative ion abundance in qualitative determination Relative ion abundance
Allowed relative deviation
Blank test
>20%50%
Except for no sample, the determination conditions and steps of 7.1 to 7.5 shall be followed. 7.7 Limit of detection and limit of quantification of the method
For the limit of detection and limit of quantification of the method of this standard, see Appendix D. 8 Experimental data processing
Plant tissue, animal tissue
The content of plant tissue and animal tissue components is calculated according to formula (1): X,-PXVXK
Wherein:
X, the content of the component to be tested in the sample, in micrograms per kilogram (pg/kg): 10%20%
-The mass concentration of a certain component in the test solution obtained from the standard curve, in micrograms per liter (g/L): P
VConstant volume, in milliliters (mL): KExplanation multiple:
-The mass of the sample, in grams (g).
The calculation result shall retain three significant figures.
8.2 Body fluids
The content of body fluid components is calculated according to formula (2):
Wherein:
X,=P:×Ve客×K
V武轩
X:-The content of the component to be tested in the sample, in micrograms per liter (g/L)10%
-The mass concentration of a certain component in the test solution obtained from the standard curve, in micrograms per liter (g/L):Vfixed density fixed volume, in milliliters (mL):KDilution multiple:
V-Sample volume, in milliliters (mL). The calculation result shall retain three significant figures.
Precision
iiikAa~cJouakAa
GB/T38571—2020
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5
GB/T38571—2020
Gradient elution program is shown in Table A.1.
Time/min
Appendix A
(Informative Appendix)
Gradient elution program
Gradient elution program table
Mobile phase A/%
iiiKAa~cJouaKAa
Mobile phase B/%
Mass spectrometry parameters of 11 alkaloids are shown in Table B1.
Appendix B
(Informative Appendix)
Mass spectrometry parameters of 11 alkaloids
Quantitative ions.
Substance name
(-)-Orthogonine
Strychnine
Aconitine
Euraconitine
Mesaconitine
Colchicine
Atropine
Strychnine
Camptothecin
Pilocarpine
Polyphyllotoxin
Mass spectrometry parameter table of 11 alkaloids
Parent ion
Lens voltage
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GB/T38571—2020
Daughter ion
Silicon collision energy
GB/T38571—2020
Appendix C
(Informative Appendix)
Extracted ion mass spectra of 11 alkaloid standard substances11 alkaloid standard substances The mass spectrum of the extracted quotient of the quasi-substance is shown in Figure C.1. 1000000
3000000
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1000000
1000000
500000
400000
300000
200000
100000
30 0000 | |tt | 008
No. 100000
40 000
Pilocarpine
Atropine
(-)-Donglanzhu
Zhu Dening
Xigepo
Fanbenjiine
iiikAa~cJouakAa
18192021m
21314151617
Mushuinacirine
Polyphyllotoxin
Mesonaconziaceae
New Aconitum
Aconitum medulla
569101215141516118192021
Retention time/min
Figure C.1Extracted ion mass spectra of 11 alkaloid standard substances. See Table D.1 for method detection limit and quantification limit.
Analyte
Pilocarpine
Atropine
(-)-Orientine
Camptothecin
Strychine
Neornithine
Colchicine
Colonitine
Aconitine
Strychnine
Polydophyllotoxin
Detection limit
Quantification limit
Appendix D
(Normative Appendix)
Formula Detection limit, quantification limit
Method detection limit, quantification limit
Detection limit
Quantification limit
Animal tissue
Detection limit
Quantification limit
Chinese medicinal materials
Detection limit
iiikAa~cJouakAa
GB/T38571—2020
Other plant tissues
Quantification limit
Detection limit
Quantification limit-PXVXK
Where:
X, the content of the component to be tested in the sample, in micrograms per kilogram (pg/kg): 10%20%
-The mass concentration of a component in the test solution obtained from the standard curve, in micrograms per liter (g/L): P
V is a certain volume, in milliliters (mL): K is the dispersion multiple:
-The mass of the sample, in grams (g).
The calculation result is retained to three significant figures.
8.2 Body fluids
The content of body fluid components is calculated according to formula (2):
Wherein:
X,=P:×Ve客×K
V武轩
X:-The content of the component to be tested in the sample, in micrograms per liter (g/L)10%
-The mass concentration of a certain component in the test solution obtained from the standard curve, in micrograms per liter (g/L):Vfixed density fixed volume, in milliliters (mL):KDilution multiple:
V-Sample volume, in milliliters (mL). The calculation result shall retain three significant figures.
Precision
iiikAa~cJouakAa
GB/T38571—2020
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5
GB/T38571—2020
Gradient elution program is shown in Table A.1.
Time/min
Appendix A
(Informative Appendix)
Gradient elution program
Gradient elution program table
Mobile phase A/%
iiiKAa~cJouaKAa
Mobile phase B/%
Mass spectrometry parameters of 11 alkaloids are shown in Table B1.
Appendix B
(Informative Appendix)
Mass spectrometry parameters of 11 alkaloids
Quantitative ions.
Substance name
(-)-Orthogonine
Strychnine
Aconitine
Euraconitine
Mesaconitine
Colchicine
Atropine
Strychnine
Camptothecin
Pilocarpine
Polyphyllotoxin
Mass spectrometry parameter table of 11 alkaloids
Parent ion
Lens voltage
iiikAa~cJouakAa
GB/T38571—2020
Daughter ion
Silicon collision energy
GB/T38571—2020
Appendix C
(Informative Appendix)
Extracted ion mass spectra of 11 alkaloid standard substances11 alkaloid standard substances The mass spectrum of the extracted quotient of the quasi-substance is shown in Figure C.1. 1000000
3000000
2000000
1000000
1000000
500000
400000
300000
200000
100000
30 0000 | |tt | 008
No. 100000
40 000
Pilocarpine
Atropine
(-)-Donglanzhu
Zhu Dening
Xigepo
Fanbenjiine
iiikAa~cJouakAa
18192021m
21314151617
Mushuinaijing
Polyphyllotoxin
Mesonaconziaceae
New Aconitum
Aconitum medulla
569101215141516118192021
Retention time/min
Figure C.1Extracted ion mass spectra of 11 alkaloid standard substances. See Table D.1 for method detection limit and quantification limit.
Analyte
Pilocarpine
Atropine
(-)-Orientine
Camptothecin
Strychine
Neornithine
Colchicine
Colonitine
Aconitine
Strychnine
Polydophyllotoxin
Detection limit
Quantification limit
Appendix D
(Normative Appendix)
Formula Detection limit, quantification limit
Method detection limit, quantification limit
Detection limit
Quantification limit
Animal tissue
Detection limit
Quantification limit
Chinese medicinal materials
Detection limit
iiikAa~cJouakAa
GB/T38571—2020
Other plant tissues
Quantification limit
Detection limit
Quantification limit-PXVXK
Where:
X, the content of the component to be tested in the sample, in micrograms per kilogram (pg/kg): 10%20%
-The mass concentration of a component in the test solution obtained from the standard curve, in micrograms per liter (g/L): P
V is a certain volume, in milliliters (mL): K is the dispersion multiple:
-The mass of the sample, in grams (g).
The calculation result is retained to three significant figures.
8.2 Body fluids
The content of body fluid components is calculated according to formula (2):
Wherein:
X,=P:×Ve客×K
V武轩
X:-The content of the component to be tested in the sample, in micrograms per liter (g/L)10%
-The mass concentration of a certain component in the test solution obtained from the standard curve, in micrograms per liter (g/L):Vfixed density fixed volume, in milliliters (mL):KDilution multiple:
V-Sample volume, in milliliters (mL). The calculation result shall retain three significant figures.
Precision
iiikAa~cJouakAa
GB/T38571—2020
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5
GB/T38571—2020
Gradient elution program is shown in Table A.1.
Time/min
Appendix A
(Informative Appendix)
Gradient elution program
Gradient elution program table
Mobile phase A/%
iiiKAa~cJouaKAa
Mobile phase B/%
Mass spectrometry parameters of 11 alkaloids are shown in Table B1.
Appendix B
(Informative Appendix)
Mass spectrometry parameters of 11 alkaloids
Quantitative ions.
Substance name
(-)-Orthogonine
Strychnine
Aconitine
Euraconitine
Mesaconitine
Colchicine
Atropine
Strychnine
Camptothecin
Pilocarpine
Polyphyllotoxin
Mass spectrometry parameter table of 11 alkaloids
Parent ion
Lens voltage
iiikAa~cJouakAa
GB/T38571—2020
Daughter ion
Silicon collision energy
GB/T38571—2020
Appendix C
(Informative Appendix)
Extracted ion mass spectra of 11 alkaloid standard substances11 alkaloid standard substances The mass spectrum of the extracted quotient of the quasi-substance is shown in Figure C.1. 1000000
3000000
2000000
1000000
1000000
500000
400000
300000
200000
100000
30 0000 | |tt | 008
No. 100000
40 000
Pilocarpine
Atropine
(-)-Donglanzhu
Zhu Dening
Xigepo
Fanbenjiine
iiikAa~cJouakAa
18192021m
21314151617
Mushuinacirine
Polyphyllotoxin
Mesonaconziaceae
New Aconitum
Aconitum medulla
569101215141516118192021
Retention time/min
Figure C.1Extracted ion mass spectra of 11 alkaloid standard substances. See Table D.1 for method detection limit and quantification limit.
Analyte
Pilocarpine
Atropine
(-)-Orientine
Camptothecin
Strychine
Neornithine
Colchicine
Colonitine
Aconitine
Strychnine
Polydophyllotoxin
Detection limit
Quantification limit
Appendix D
(Normative Appendix)
Formula Detection limit, quantification limit
Method detection limit, quantification limit
Detection limit
Quantification limit
Animal tissue
Detection limit
Quantification limit
Chinese medicinal materials
Detection limit
iiikAa~cJouakAa
GB/T38571—2020
Other plant tissues
Quantification limit
Detection limit
Quantification limit1. 1000000www.bzxz.net
3000000
2000000
1000000
1000000
500000
400000
300000
200000
100000
300000||tt ||200000
100000
200000
150000
100000
20000 yuan
200000
150000||tt| |100000
150008
No. 100000
40 000
pilocarpine
atropine
(-)-Donglanzhu
Zhu Dening
Xigepo
Banbenji alkaloid
iiikAa~cJouakAa
18192021m
21314151617
Merchantine
podophyllotoxin
hypoconitum
New Aconitum
Aconitum
569101215141516118192021
Retention time/min
Figure C. Extracted ion mass spectra of 111 alkaloid standard substances Method detection limit, The limits of quantification are shown in Table D.1.
Analyte
Pilocarpine
Atropine
(-)-Orientine
Camptothecin
Strychnine| |tt||neoconitine
colchicine
hypoconitine
aconitine
strychnine
podophyllotoxin| |tt||Detection limit
Quantification limit
Appendix D
(Normative Appendix)
Detection limit, quantification limit
Detection limit
Quantification limit
Animal tissue
Detection limit| |tt||Limit of quantification
Chinese medicinal materials
Limit of detection
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GB/T38571—2020
Other plant tissues||tt ||Limit of Quantitation
Detection Limit
Limit of Quantitation1. 1000000
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1000000
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400000
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100000
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100000
200000
150000
100000
20000 yuan
200000
150000||tt| |100000
150008
No. 100000
40 000
pilocarpine
atropine
(-)-Donglanzhu
Zhu Dening
Xigepo
Banbenji alkaloid
iiikAa~cJouakAa
18192021m
21314151617
Merchine
Polyphyllotoxin
Aconitum
New Aconitum
Aconitum
569101215141516118192021
Retention time/min
Figure C. Extracted ion mass spectra of 111 alkaloid standard substances Method detection limit, The limits of quantification are shown in Table D.1.
Analyte
Pilocarpine
Atropine
(-)-Orientine
Camptothecin
Strychnine| |tt||neoconitine
colchicine
hypoconitine
aconitine
strychnine
podophyllotoxin| |tt||Detection limit
Quantification limit
Appendix D
(Normative Appendix)
Detection limit, quantification limit
Detection limit
Quantification limit
Animal tissue
Detection limit| |tt||Limit of quantification
Chinese medicinal materials
Limit of detection
iiikAa~cJouakAa
GB/T38571—2020
Other plant tissues||tt ||Limit of Quantitation
Detection Limit
Limit of Quantitation
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