CN112034063A - Method for measuring concentration of zanthoxylum schinifolium alkali in biological matrix - Google Patents
Method for measuring concentration of zanthoxylum schinifolium alkali in biological matrix Download PDFInfo
- Publication number
- CN112034063A CN112034063A CN202010908722.2A CN202010908722A CN112034063A CN 112034063 A CN112034063 A CN 112034063A CN 202010908722 A CN202010908722 A CN 202010908722A CN 112034063 A CN112034063 A CN 112034063A
- Authority
- CN
- China
- Prior art keywords
- alkali
- sample
- biological matrix
- zanthoxylum schinifolium
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for measuring the concentration of zanthoxylum schinifolium alkali in a biological matrix, which realizes concentration measurement based on a liquid chromatography-mass spectrometry. The method comprises the following steps: preparing a sample to be detected, and pre-treating the biological matrix which is quantitatively absorbed by a protein precipitation method to remove protein which interferes with the determination of the content of the zanthoxylum schinifolium alkali; quantitatively analyzing, transferring the sample to be detected obtained after the pretreatment to a chromatographic column, separating the zanthoxylum schinifolium alkali and the interfering substances in the sample by using the chromatographic column, and finally, entering a mass spectrometer for analysis to obtain a concentration result of nanogram per milliliter. The invention is a brand-new method for determining the concentration of the zanthoxylum schinifolium alkali in a biological matrix, in the process of developing the method, two detection means of comparing ultraviolet detection and mass spectrum detection, two extraction methods of protein precipitation and liquid-liquid extraction are adopted, the protein precipitation is combined with the mass spectrum detection as a final method, the lower limit of the quantitative determination of the zanthoxylum schinifolium alkali in the biological matrix can reach the level of 1.0 ng/ml by using the method, compared with other methods, the method can ensure enough sensitivity, can effectively control the experiment cost, and has simple and clear steps and strong operability.
Description
Technical Field
The invention relates to an analytical method of sinoacutine, in particular to a method for measuring sinoacutine concentration in a biological matrix in a high-precision quantitative manner.
Background
Zanthoxyline, the first 4-quinolinone alkaloid found in Zanthoxylum plants. The research reports that the zanthoxylum schinifolium alkali has the bacteriostatic action and can selectively inhibit the activity of gram-positive bacteria. The unique biological characteristics make the compound have wide application prospect in the aspects of medicine, food, cosmetics, agriculture and the like. Many papers about the study of the zanthoxylum schinifolium alkali are provided, the determination of the content of the zanthoxylum schinifolium alkali is a basic premise of the continuous deep study, and a sensitive and rapid determination method is the guarantee of the analysis accuracy and the result reliability of the study and greatly accelerates the study speed. At present, an analysis method for the content or concentration of the zanthoxylum schinifolium alkali in the biological matrix is not reported, and a high-efficiency and reliable analysis method is developed, so that great help is brought to the follow-up research of the zanthoxylum schinifolium alkali.
Disclosure of Invention
The invention aims to provide a method for measuring the concentration of the zanthoxylum schinifolium alkali in the biological matrix, so as to solve the beneficial problems of acceleration and deep research of the zanthoxylum schinifolium alkali.
The technical scheme for realizing the aim of the invention is that the method for measuring the concentration of the zanthoxylum schinifolium alkali in the biological matrix comprises the following steps: preparing a sample to be detected, and then quantitatively analyzing the concentration of the zanthoxylum schinifolium alkali in the sample to be detected by adopting a liquid chromatography-mass spectrometry method.
Further, the determination method specifically comprises the following steps:
pretreating the biological matrix which is quantitatively absorbed by a protein precipitation method to remove protein which interferes with the determination of the content of the zanthoxylum schinifolium alkali;
and transferring the sample to be detected obtained after the pretreatment to a chromatographic column, separating the zanthoxylum schinifolium alkali and the interfering substances in the sample by using the chromatographic column, and finally, feeding the sample into a mass spectrometer for analysis to obtain a concentration result of nanogram per milliliter.
Further, the pretreatment step is as follows: the biological matrix is transferred to a processing container, acetonitrile with 5 times of sample volume relative to the biological matrix is used for vortex mixing, and centrifugation is carried out, so that supernate serving as a sample to be detected and bottom sediment serving as an interfering substance are obtained.
Further, the rotation speed of the centrifugal treatment is 3200-4000 revolutions per minute, and the centrifugal time is 5-20 minutes.
Further, an internal standard methanol solution with 50% of the sample volume is added into the biological matrix in the pretreatment step.
Further, in the liquid chromatography-mass spectrometry, the first mobile phase is sterile water for injection containing 0.1% formic acid, the second mobile phase is acetonitrile containing 0.1% formic acid, and the two mobile phases are driven by respective pumps to merge into a sample injector in front of a chromatographic column.
Further, the mass spectrometer used in the quantitative analysis was of the type ACQUITY UPLC I-Class + Xevo TQ-S.
Further, the biological matrix includes plasma, whole blood and tissue homogenates.
The invention achieves the following beneficial effects:
(1) the method adopts simple and quick protein precipitation for pretreatment, removes proteins interfering with the determination of the content of the zanthoxylum schinifolium alkali, and utilizes a chromatographic column to separate the zanthoxylum schinifolium alkali and interfering substances in a sample, so that the measurement result is more accurate.
(2) The invention is a brand new method for measuring the concentration of the zanthoxylum schinifolium alkali in the biological matrix, the lower limit of the quantitative measurement of the zanthoxylum schinifolium alkali in the biological matrix can reach the level of 1ng/ml by using the method, the operation is simple and rapid, and the sensitivity is high.
Drawings
FIG. 1 is a mass spectrum of the quantitative off-line of zanthoxylum schinifolium base in the biological matrix measured in example 1.
Figure 2 is a chromatogram of the quantitative bottom line of zanthoxylum schinifolium base in the biological matrix using UPLC assay.
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
The invention relates to a method for measuring the concentration of zanthoxylum schinifolium alkali in a biological matrix, which adopts the technical scheme that the concentration measurement is realized based on a liquid chromatogram-mass spectrometry. The outline steps of the determination scheme mainly comprise two parts, namely, preparing a sample to be determined, and pre-treating the biological matrix quantitatively absorbed by a protein precipitation method to remove protein precipitates interfering with the determination of the content of the zanthoxylum schinifolium alkali; and transferring the sample to be detected obtained after the pretreatment to a chromatographic column, separating the zanthoxylum schinifolium alkali and the interfering substances in the sample by using the chromatographic column, and finally, feeding the sample into a mass spectrometer for analysis to obtain a concentration result of nanogram per milliliter.
Example 1
1. Preparing a sample to be detected, and pretreating the biological matrix quantitatively absorbed by a protein precipitation method: firstly, sucking a proper volume of biological matrix into a processing container, then adding an internal standard (tolbutamide) methanol solution with 50% of sample volume into the processing container, and uniformly mixing by vortex; adding acetonitrile with the volume 5 times of that of the sample of the biological matrix into the processing container, uniformly mixing by vortex again, and then putting the mixture obtained in the previous step into a centrifuge for high-speed centrifugation, wherein the rotation speed of the centrifugation treatment is 3200-4000 revolutions per minute, and the centrifugation time is 5-20 minutes. Of course, according to the amount of the sample used in the routine determination experiment, the centrifugation process preferably has a rotation speed of 4000 rpm, and the centrifugation time is 10 minutes.
2. Quantitative analysis: and transferring the sample to be detected obtained after the pretreatment to a chromatographic column, separating the zanthoxylum schinifolium alkali and the interfering substances in the sample by using the chromatographic column, and finally, feeding the sample into a mass spectrometer for analysis. Wherein the first mobile phase is sterile water for injection containing 0.1% formic acid, the second mobile phase is acetonitrile containing 0.1% formic acid, and the two mobile phases are driven by respective pumps to converge into a chromatographic column sample injector and finally inject into a mass spectrometer. The mass spectrometer used in the quantitative analysis was of the type ACQUITY UPLC I-Class + Xevo TQ-S or higher.
More specifically, liquid phase conditions:
a chromatographic column: ACQUITY
BEH C18 1.7μm 2.1mm×50mm；
Elution gradient:
mass spectral parameters (appropriate variations for different instrument conditions):
the measurement results of example 1 are shown in tables 1 and 2, and the mass spectrum of the quantitative off-line of zanthoxylum schinifolium alkali in the measured biological matrix is shown in fig. 1.
TABLE 1 Standard Curve
TABLE 2 quality control samples
Comparative example 1
The concentrations of zanthoxylum schinifolium alkali in the biological matrix were measured by Ultra Performance Liquid Chromatography (UPLC) method, and the measurement results are shown in tables 3 and 4. The chromatogram of the quantitative off-line of zanthoxylum schinifolium base in the biological matrix measured by UPLC is shown in fig. 2.
TABLE 3 Standard Curve
TABLE 4 quality control samples
By comparing the linear range (1-1000ng/mL) and the UPLC linear range (2.5-7.5 mu g/mL), the invention has wider detection range and wide applicability; by comparing the lower detection limit of the invention (1ng/mL) with the lower detection limit of UPLC (2.5. mu.g/mL), which is 1/2500 for UPLC detection, the invention has a very significant advantage in terms of sensitivity.
In addition, in the exploration process, the differences of different extraction methods in the same detection range are transversely compared, the protein precipitation method and the liquid-liquid extraction method are emphatically compared, the protein precipitation method and the liquid-liquid extraction method have no obvious difference in sensitivity, but the liquid-liquid extraction method uses various volatile reagents in the experimental process, and the method has higher practicability from the perspective of personnel health.
In conclusion, the invention is a brand-new method for measuring the concentration of the zanthoxylum schinifolium alkali in the biological matrix, the lower limit of the quantitative measurement of the zanthoxylum schinifolium alkali in the biological matrix can reach the level of 1ng/ml by using the method, the method adopts simple and quick protein precipitation for pretreatment, the steps are simple and clear, and the operability is strong.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for measuring the concentration of zanthoxylum schinifolium alkali in a biological matrix is characterized by comprising the following steps: preparing a sample to be detected, and then quantitatively analyzing the concentration of the zanthoxylum schinifolium alkali in the sample to be detected by adopting a liquid chromatography-mass spectrometry method.
2. The method for determining the concentration of zanthoxylum schinifolium alkali in the biological matrix as claimed in claim 1, which comprises the following steps:
pretreating the biological matrix which is quantitatively absorbed by a protein precipitation method to remove protein which interferes with the determination of the content of the zanthoxylum schinifolium alkali;
and transferring the sample to be detected obtained after the pretreatment to a chromatographic column, separating the zanthoxylum schinifolium alkali and the interfering substances in the sample by using the chromatographic column, and finally, feeding the sample into a mass spectrometer for analysis to obtain a concentration result of nanogram per milliliter.
3. The method for determining the concentration of zanthoxylum schinifolium alkali in the biological matrix as claimed in claim 2, wherein the pretreatment step comprises: the biological matrix is transferred to a processing container, acetonitrile with 5 times of sample volume relative to the biological matrix is used for vortex mixing, and centrifugation is carried out, so that supernate serving as a sample to be detected and bottom sediment serving as an interfering substance are obtained.
4. The method as claimed in claim 3, wherein the rotation speed of the centrifugation is 3200 and 4000 rpm, and the centrifugation time is 5-20 min.
5. The method for determining the concentration of zanthoxylum schinifolium alkali in the biological matrix as claimed in claim 2, wherein the internal standard methanol solution with 50% of the sample volume is added into the biological matrix in the pretreatment step.
6. The method of claim 1, wherein the first mobile phase of the liquid chromatography-mass spectrometry is sterile water for injection containing 0.1% formic acid, the second mobile phase is acetonitrile containing 0.1% formic acid, and the two mobile phases are driven by separate pumps to feed the sample before the column.
7. The method as claimed in claim 1, wherein the apparatus used for quantitative analysis is ACQUITY UPLC I-Class + Xevo TQ-S Mass Spectrometry.
8. The method of claim 1, wherein the biological substrate comprises plasma, whole blood, and tissue homogenate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010908722.2A CN112034063A (en) | 2020-09-02 | 2020-09-02 | Method for measuring concentration of zanthoxylum schinifolium alkali in biological matrix |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010908722.2A CN112034063A (en) | 2020-09-02 | 2020-09-02 | Method for measuring concentration of zanthoxylum schinifolium alkali in biological matrix |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112034063A true CN112034063A (en) | 2020-12-04 |
Family
ID=73591109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010908722.2A Pending CN112034063A (en) | 2020-09-02 | 2020-09-02 | Method for measuring concentration of zanthoxylum schinifolium alkali in biological matrix |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112034063A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104030937A (en) * | 2014-06-16 | 2014-09-10 | 中华全国供销合作总社南京野生植物综合利用研究所 | Method for quickly preparing high-content zanthoxylum unsaturated amide components |
| CN108129420A (en) * | 2016-12-01 | 2018-06-08 | 云南爱尔康生物技术有限公司 | A kind of method of dimethyl ether fluid extraction Phaeodactylum tricornutum fucoxanthin |
| CN108294060A (en) * | 2018-02-02 | 2018-07-20 | 森知（上海）国际贸易有限公司 | Plant-based bacteriostat and preparation method thereof |
| CN111217864A (en) * | 2020-02-28 | 2020-06-02 | 营山椒宝宝花椒有限责任公司 | Extraction method of green pepper alkaloid |
-
2020
- 2020-09-02 CN CN202010908722.2A patent/CN112034063A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104030937A (en) * | 2014-06-16 | 2014-09-10 | 中华全国供销合作总社南京野生植物综合利用研究所 | Method for quickly preparing high-content zanthoxylum unsaturated amide components |
| CN108129420A (en) * | 2016-12-01 | 2018-06-08 | 云南爱尔康生物技术有限公司 | A kind of method of dimethyl ether fluid extraction Phaeodactylum tricornutum fucoxanthin |
| CN108294060A (en) * | 2018-02-02 | 2018-07-20 | 森知（上海）国际贸易有限公司 | Plant-based bacteriostat and preparation method thereof |
| CN111217864A (en) * | 2020-02-28 | 2020-06-02 | 营山椒宝宝花椒有限责任公司 | Extraction method of green pepper alkaloid |
Non-Patent Citations (3)
| Title |
|---|
| SHENSHEN YANG 等: "Analysis of E. rutaecarpa Alkaloids Constituents In Vitro and In Vivo by UPLC-Q-TOF-MS Combined with Diagnostic Fragment" * |
| 关荣琴;张鸣镝;石雪萍;张卫明;: "高速逆流色谱法分离花椒生物碱" * |
| 王莲萍 等: "基于UFLC-MS/分子模拟计算的吴茱萸醇提取物中胆碱酯酶抑制剂筛选" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111239267B (en) | Method for detecting short-chain fatty acids in serum and lymph tissue based on GC-MS | |
| CN111679028B (en) | High performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics | |
| CN112748198A (en) | Method and device for detecting antifungal drugs in serum by liquid chromatography tandem mass spectrometry technology | |
| CN106645477A (en) | Method for detecting florfenicol amine residue and application | |
| CN107422053B (en) | Method for detecting trichloroacetic acid in cosmetics by liquid chromatography-mass spectrometry | |
| CN112034063A (en) | Method for measuring concentration of zanthoxylum schinifolium alkali in biological matrix | |
| CN109254104A (en) | The detection method of residues of organophosphate pesticides in a kind of grape wine | |
| CN110824081A (en) | Lipidomics analysis method of blood deficiency mouse model | |
| CN106770765B (en) | The detection method and application of a kind of albendazole and its metabolin | |
| CN112051343B (en) | Method for determining antibiotic residues | |
| EP0429082A2 (en) | Method for analysis of catecholamine | |
| CN114669280A (en) | Purification filler for sulfonamide residues and pretreatment method | |
| Kraak et al. | Application of on-column concentration of deproteinized serum to the hplc-determination of anticonvulsants | |
| Kline et al. | Liquid chromatographic determination of valproic acid in human serum | |
| CN108051510B (en) | Method for determining environmental hormone in meat matrix sample | |
| CN117074556A (en) | Method for determining pregabalin and riluzole concentrations | |
| CN106404962B (en) | A kind of method for measuring peanut leaf jasmine acid content | |
| CN110988210A (en) | Method for reducing nonspecific adsorption of fat-soluble vitamin 96-well plate | |
| CN112684018B (en) | Method for detecting contents of bisphenol S and bisphenol F in animal-derived food and application of method | |
| CN103869026B (en) | The preprocess method of mulberry leaf compound preparation and the detection method of 1-DNJ | |
| CN111366672B (en) | Detection method of health wine fingerprint | |
| CN115015407B (en) | Method for determining parahydroxybenzoate isomer in essence | |
| CN117388414A (en) | Method for detecting water-soluble vitamin content in milk powder | |
| Defilippi et al. | An HPLC screening method for the detection of isonicotinic acid hydrazide in cattle milk | |
| CN103323559B (en) | Pan paper solid phase microextraction method and device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |