CN113063862A - Method for simultaneously detecting 4 aristolochic acid substances in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry - Google Patents

Method for simultaneously detecting 4 aristolochic acid substances in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry Download PDF

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CN113063862A
CN113063862A CN202110278873.9A CN202110278873A CN113063862A CN 113063862 A CN113063862 A CN 113063862A CN 202110278873 A CN202110278873 A CN 202110278873A CN 113063862 A CN113063862 A CN 113063862A
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aristolochic acid
houttuynia cordata
mass spectrometry
liquid chromatography
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万益群
余璇
郭岚
刘翻
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Nanchang University
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Abstract

The invention discloses an ultrahigh performance liquid chromatography-mass spectrometry analysis method for simply and rapidly detecting 4 aristolochic acid substances (aristolochic acid I, aristolochic acid II, aristolochialactam I and aristolochialactam AII) in houttuynia cordata at the same time, and belongs to the technical field of analytical chemistry. The houttuynia cordata is a traditional substance used as both food and medicine, and has the effects of clearing heat and removing toxicity, eliminating carbuncle and expelling pus, and inducing diuresis and treating stranguria as a herbal medicine; as a dish, it has become a "big way potherb" on the popular dining table. However, the content level of aristolochic acid substances in the houttuynia cordata is also a common concern of the society, and whether the content level is at risk or not is also the concern. The invention provides a novel analysis method capable of simply, quickly and sensitively detecting 4 aristolochic acid substances in houttuynia cordata, the instrument detection limit is 0.0001 mg/L-0.01 mg/L, and the method has important significance for safety evaluation and quality monitoring of houttuynia cordata.

Description

Method for simultaneously detecting 4 aristolochic acid substances in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry
Technical Field
The invention relates to a method for simultaneously detecting four aristolochic acid substances (aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII) in houttuynia cordata based on an ultra-performance liquid chromatography-mass spectrometry, belonging to the technical field of analysis.
Background
Houttuynia cordata is a traditional substance for both food and medicine. On one hand, the houttuynia cordata has the effects of clearing heat and removing toxicity, eliminating carbuncle and expelling pus, inducing diuresis and treating stranguria, treating lung carbuncle and vomiting pus, phlegm heat and asthma and cough, dysentery, carbuncle and swelling and sore toxicity, and simultaneously has remarkable sterilization and antivirus effects, and plays an important role as a sterilization or antivirus medicament in certain epidemic diseases outbreak in China. On the other hand, wild or domestic herba houttuyniae has become a 'wild vegetable' on the popular dining table in various parts of China.
Since 1993, a series of aristolochic acid events occur all over the world, aristolochic acid substances are pushed to the wind gap wave tip of public opinion, and the life safety problem caused by the aristolochic acid substances rapidly becomes the focus of attention of people. In 2002, aristolochic acid was classified as a class I carcinogen by the world health organization. Because the houttuynia cordata contains aristolochic acid substances, the establishment of a rapid, simple and sensitive analysis technology for the aristolochic acid substances in the houttuynia cordata has important significance for safety assessment and quality monitoring of the houttuynia cordata.
At present, a determination method of aristolochic acid substances in houttuynia cordata is not specified in 'Chinese pharmacopoeia' of 2020 edition, and the determination methods of the aristolochic acid substances in documents mainly include a thin layer chromatography, an ultraviolet spectrophotometry, a capillary electrophoresis method, a liquid chromatography ultraviolet detection method and a liquid chromatography mass spectrometry, but the determination of the aristolochic acid substances in the houttuynia cordata is rarely reported.
Disclosure of Invention
The invention aims to provide a rapid, simple and sensitive ultra-high performance liquid chromatography-mass spectrometry method for simultaneously detecting 4 aristolochic acids (aristolochic acid I, aristolochic acid II, aristolochialactam I and aristolochialactam AII) in houttuynia cordata.
The technical scheme of the invention is as follows: an analysis method for simultaneously detecting 4 aristolochic acid substances (aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII) in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry. Ultrasonic extracting 4 aristolochic acids (aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII) in herba Houttuyniae with methanol, diluting the extractive solution with water until the methanol content is 80%, purifying with HC-C18 material, concentrating the supernatant by nitrogen blowing, and measuring with ultra performance liquid chromatography-mass spectrometry.
The invention mainly comprises the following steps: ultra performance liquid chromatography-mass spectrometry conditions; simultaneously detecting aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII; pretreating a sample; and (4) detecting the sample.
An analysis method for simultaneously detecting four aristolochic acid substances (aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII) in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry is characterized in that: mainly comprises the following steps:
(1) ultra-high performance liquid chromatography-mass spectrometry conditions
The instrument used in the present invention is UPLC ultra high performance liquid chromatography (Shimadzu Japan) -Triple TOFTM5600+Quadrupole time-of-flight mass spectrometer (AB SCIEX, USA). Chromatographic conditions are as follows: a chromatographic column: a Shim-pack GIST C18 column (2.1 mm. times.75 mm. times.2.0. mu.m); column temperature: 35 ℃; sample introduction amount: 5 μ L. Mobile phase: phase A was water (containing 0.1% formic acid) and phase B was acetonitrile (containing 0.1% formic acid). Gradient elution procedure: 0min, 40% B; 7 min, 50% B; 7.1-9 min, 90% B; 9.1 min, 40% B; keeping for 3 min. Flow rate: 0.3 mL/min. Mass spectrum conditions: ion source, ESI source; a negative ion mode; air curtain gas (CUR gas) 35 psi; atomizing GAS (GAS 1) 50 psi; assist GAS (GAS 2) 50 psi; the ion source temperature is 550 ℃; primary mass spectrum: scanning range, 50-350 Da; the data acquisition time is 100 ms; declustering voltage (DP) 80V; collision Energy (CE) 10V; second-order mass spectrometry: scanning range: 200- & lt350 & gt.
(2) Detection of aristolochic acid I, aristolochic acid II, aristololactam I and aristololactam AII
Preparing a series of 4 aristolochic acid substance mixed standard solutions with mass concentration, analyzing under the condition of the chromatography-mass spectrometry, and performing linear regression on the mass concentration (x) by using peak areas (y) of each component to obtain a linear equation. Aristolochic acid I (0.04-2 mg/L), aristolochic acid II (0.04-2 mg/L), aristololactam I (0.04-2 mg/L) and aristololactam AII (0.004-2 mg/L) have good linear relation (R)2>0.9987). The detection Limit (LOD) and the quantification Limit (LOQ) of the four target analytes are in the range of 0.0001-0.01 mg/L and 0.0003-0.03 mg/L, respectively.
(3) Pretreatment of samples
Pulverizing dried herba Houttuyniae sample, sieving with 80 mesh sieve, weighing 0.2 g (accurate to 0.001 g) into 50 mL graduated centrifuge tube, adding 15.0 mL methanol, vortex for 2 min, mixing, ultrasonic extracting for 20min, centrifuging at 3000 rpm for 5 min, collecting supernatant, and diluting with water to obtain 80% methanol. Collecting 2.0 mL of above extractive solution, adding 280 mg HC-C18 adsorbent, vortex oscillating for 20min, centrifuging at 13800 rpm for 10 min, blow-drying supernatant with nitrogen gas, dissolving residue with 200 μ L methanol, and filtering with 0.22 μm filter membrane to obtain supernatant.
(4) Detection of samples
Weighing 0.2 g (accurate to 0.001 g) of houttuynia cordata sample, respectively adding the aristolochic acid substance mixed standard solution with high, medium and low concentration levels, processing the sample according to the sample pretreatment method, performing UPLC-Q/TOF-MS analysis, and calculating the sample standard recovery rate.
The invention has the beneficial effects that: the invention designs an ultra-high performance liquid chromatography-mass spectrometry method for simultaneously detecting 4 aristolochic acid substances in houttuynia cordata, which can simply, conveniently, quickly and sensitively detect four aristolochic acid substances in houttuynia cordata, provides convenience for future production and supervision, and can meet the domestic requirements for production and supervision of the four aristolochic acid substances.
Drawings
FIG. 1 is a chromatogram of extracted ions of aristolochic acid I, aristolochic acid II, aristololactam I and aristololactam AII; 1: aristolochia lactam AII (0.008 mg/L); 2: aristolochia lactam I (1 mg/L); 3: aristolochic acid II (5 mg/L); 4: aristolochic acid I (5 mg/L).
Detailed Description
Example 1:
using UPLC ultra-high performance liquid chromatography (Shimadzu Japan) -Triple TOFTM 5600+Quadrupole time-of-flight mass spectrometer (AB SCIEX, USA). Chromatographic conditions are as follows: a chromatographic column: a Shim-pack GIST C18 column (2.1 mm. times.75 mm. times.2.0. mu.m); column temperature: 35 ℃; sample introduction amount: 5 μ L. Mobile phase: phase A is water (containing 0.1% formic acid), phase B is acetonitrile (containing 0.1% formic acid); the gradient elution procedure is shown in table 1; flow rate: 0.3 mL/min. Mass spectrum conditions: an ion source: an ESI source; a negative ion mode; air curtain gas (CUR gas) 35 psi; atomizing GAS (GAS 1) 50 psi; assist GAS (GAS 2) 50 psi; the ion source temperature is 550 ℃; primary mass spectrum: scanning range, 50-350 Da; the data acquisition time is 100 ms; declustering voltage (DP) 80V; collision Energy (CE) 10V. Second-order mass spectrometry: scanning range: 200-350 Da; the collection mode is as follows: an ion scanning mode is selected. Mass spectrometric detection parameters for 4 target compounds: the parent ions, the daughter ions, the spray voltage (ISVF), the declustering voltage (DP) and the Collision Energy (CE) are shown in Table 2.
Figure 179099DEST_PATH_IMAGE001
Figure 359413DEST_PATH_IMAGE002
Example 2:
preparing a series of 4 aristolochic acid substance mixed standard solutions with mass concentration, analyzing under the condition of the chromatography-mass spectrometry, and performing linear regression on the analyte concentration (x) by using the peak area (y), wherein the linear range, the linear equation, the correlation coefficient, the detection limit and the quantitative limit are shown in a table 3.
Figure 785847DEST_PATH_IMAGE003
Example 3:
sample pretreatment: weighing herba Houttuyniae fine powder 0.2 g (accurate to 0.001 g) into 50 mL polypropylene centrifuge tube by Ohanus electronic balance, adding 15.0 mL methanol, violently vortex the mixture at 3000 rpm for 2 min by MS3 vortex mixer, ultrasonically extracting for 20min, and centrifuging at 3000 rpm for 5 min by TDL-5C low-speed desk type large-capacity centrifuge. Diluting the supernatant with water until the methanol content is 80%, placing 2.0 mL of the above extractive solution in 5 mL centrifuge tube, adding 280 mg HC-C18 material, vortex oscillating for adsorption for 20min, and centrifuging the mixture with TG20.5 high speed centrifuge at 13800 rpm for 10 min. Blowing the supernatant with nitrogen, dissolving the residue with 200 μ L methanol, and filtering with 0.22 μm filter membrane to obtain the filtrate.
Example 4:
weighing 0.2 g (accurate to 0.001 g) of houttuynia cordata powder sample, respectively adding three aristolochic acid substance mixed standard solutions with different concentration levels, processing the sample according to the sample pretreatment method, performing UPLC-Q/TOF-MS analysis, and calculating the sample standard addition recovery rate and precision, wherein the results are shown in Table 4.
Figure 54017DEST_PATH_IMAGE004
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. It should be understood by those skilled in the art that various changes and substitutions may be made in accordance with the technical solution and the inventive concept of the present invention, and the same properties or uses should be considered as the protection scope of the present invention.

Claims (2)

1. An analysis method for simultaneously detecting 4 aristolochic acid substances (aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII) in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry is characterized in that: mainly comprises the following steps:
(1) ultra performance liquid chromatography-mass spectrometry conditions;
(2) simultaneously detecting aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII;
(3) pretreating a sample;
(4) and (4) detecting the sample.
2. An analysis method for simultaneously detecting 4 aristolochic acid substances (aristolochic acid I, aristolochic acid II, aristolochia lactam I and aristolochia lactam AII) in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry is characterized in that: mainly comprises the following steps:
(1) ultra-high performance liquid chromatography-mass spectrometry conditions
The instrument used in the present invention is UPLC ultra high performance liquid chromatography (Shimadzu Japan) -Triple TOFTM 5600+Quadrupole time-of-flight mass spectrometer (AB SCIEX, usa); chromatographic conditions are as follows: a chromatographic column: a Shim-pack GIST C18 column (2.1 mm. times.75 mm. times.2.0. mu.m); column temperature: 35 ℃; sample introduction amount: 5 mu L of the solution; mobile phase: phase A is water (containing 0.1% formic acid), phase B is acetonitrile (containing 0.1% formic acid); gradient elution procedure: 0min, 40% B; 7 min, 50% B; 7.1-9 min, 90% B; 9.1 min, 40% B; keeping for 3 min; flow rate: 0.3 mL/min; mass spectrum conditions: ion source, ESI source; a negative ion mode; air curtain gas (CUR gas) 35 psi; atomizing GAS (GAS 1) 50 psi; assist GAS (GAS 2) 50 psi; the ion source temperature is 550 ℃; primary mass spectrum: scanning range, 50-350 Da; the data acquisition time is 100 ms; declustering voltage (DP) 80V; collision Energy (CE) 10V; second-order mass spectrometry: scanning range: 200-350 Da;
(2) simultaneous detection of aristolochic acid I, aristolochic acid II, aristololactam I and aristololactam AII
Preparing a series of 4 aristolochic acid substance mixed standard solutions with mass concentration, analyzing under the condition of the chromatography-mass spectrometry, and performing linear regression on the mass concentration (x) by using peak areas (y) of each component to obtain a linear equation; aristolochic acid I (0.04-2)mg/L), aristolochic acid II (0.04-2 mg/L), aristololactam I (0.04-2 mg/L) and aristololactam AII (0.004-2 mg/L) have good linear relation (R2>0.9987) the detection Limit (LOD) and the quantification Limit (LOQ) of 4 target analytes are respectively in the range of 0.0001-0.01 mg/L and 0.0003-0.03 mg/L;
(3) pretreatment of samples
Pulverizing dried herba Houttuyniae sample, sieving with 80 mesh sieve, weighing 0.2 g (accurate to 0.001 g) into 50 mL graduated centrifuge tube, adding 15.0 mL methanol, vortex for 2 min, mixing, ultrasonic extracting for 20min, centrifuging at 3000 rpm for 5 min, collecting supernatant, and diluting with water to obtain 80% methanol; taking 2.0 mL of the above extractive solution, adding 280 mg HC-C18 adsorbent, performing vortex oscillation adsorption for 20min, centrifuging at 13800 rpm for 10 min, blow-drying the supernatant with nitrogen, dissolving the residue with 200 μ L methanol, and filtering with 0.22 μm filter membrane to obtain the upper computer solution;
(4) detection of samples
Weighing 0.2 g (accurate to 0.001 g) of houttuynia cordata sample, respectively adding the aristolochic acid substance mixed standard solution with high, medium and low concentration levels, processing the sample according to the sample pretreatment method, performing UPLC-Q/TOF-MS analysis, and calculating the sample standard recovery rate.
CN202110278873.9A 2021-03-16 2021-03-16 Method for simultaneously detecting 4 aristolochic acid substances in houttuynia cordata based on ultra-high performance liquid chromatography-mass spectrometry Pending CN113063862A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117471003A (en) * 2023-12-28 2024-01-30 中国中医科学院中药研究所 Separation and quantitative detection method for aristolochic acid component in houttuynia cordata and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117471003A (en) * 2023-12-28 2024-01-30 中国中医科学院中药研究所 Separation and quantitative detection method for aristolochic acid component in houttuynia cordata and application thereof

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