CN111896636A - Liquid chromatography tandem mass spectrometry detection method for emodin and aloe-emodin in mouse urine - Google Patents
Liquid chromatography tandem mass spectrometry detection method for emodin and aloe-emodin in mouse urine Download PDFInfo
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- CN111896636A CN111896636A CN202010504470.7A CN202010504470A CN111896636A CN 111896636 A CN111896636 A CN 111896636A CN 202010504470 A CN202010504470 A CN 202010504470A CN 111896636 A CN111896636 A CN 111896636A
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Abstract
The invention discloses a liquid chromatography tandem mass spectrometry detection method of emodin and aloe-emodin in mouse urine, which comprises the following steps: s1, unfreezing the collected urine, and recovering to room temperature; s2, adding deionized water into the urine obtained in the first step for mixing; s3, adding an internal standard into the mixed solution; s4, filtering the mixed solution; s5, taking the filtered mixed solution for determination by a liquid chromatography-mass spectrometer, wherein the liquid chromatography mobile phase is a water-acetonitrile system, the liquid chromatography column is a C18 reversed phase liquid chromatography column, and the flow rate of the liquid chromatography column is not more than 0.5 mL/min.
Description
Technical Field
The invention relates to a detection method of urine metabolites in the technical field of bioengineering, in particular to a liquid chromatography tandem mass spectrometry detection method of emodin and aloe-emodin in mouse urine.
Background
The radix et rhizoma Rhei medicinal material is derived from dried root and rhizome of plant of radix et rhizoma Rhei (Rheum) of Polygonaceae (Polgynoaceae). Has antibacterial, antitumor, hyperlipidemia resisting, blood pressure lowering, stomach invigorating, gallbladder function promoting, liver protecting, heart tonifying, antiinflammatory, antiaging, and immunity regulating effects. Rhubarb is one of four most commonly used Chinese medicines in China, and more than 800 Chinese medicine preparations in 8000 Chinese medicine preparations all over the country contain rhubarb, so that the rhubarb is an important Chinese medicine for treating various diseases and has wide application in clinic. The pharmacological effective components of radix et rhizoma Rhei mainly comprise anthraquinone compounds including rhein, emodin, chrysophanol, aloe-emodin, and physcion.
The determination of emodin and aloe-emodin in urine is of great significance for understanding normal physiological process, disease mechanism, target of drug therapy and evaluation of drug effect. The common detection method for emodin and aloe-emodin in urine is liquid chromatography and liquid chromatography tandem mass spectrometry. The liquid chromatogram tandem mass spectrometry has the advantages of sensitivity, rapidness, accurate quantification, small interference and the like. The method is widely applied to the aspects of pesticide residue, medicine, metabolic analysis and the like. The existing detection method for emodin and aloe-emodin in urine is commonly used by liquid chromatography, the detection sensitivity is not high enough, and the steps of volatilizing a dry solvent after urine sample extraction, concentrating and then sampling are complicated. The method directly samples and analyzes the diluted urine sample, is simple, convenient and quick, has high sensitivity, and is more suitable for the determination of emodin and aloe-emodin in the urine sample.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a liquid chromatography tandem mass spectrometry detection method of emodin and aloe-emodin in rat urine, which is used for detecting emodin and aloe-emodin in rat urine, provides effective information and basis for understanding normal physiological processes, occurrence and development mechanisms of diseases and target spots of drug treatment, and has the advantages of high sensitivity, simple and convenient operation, high stability and reliable result.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a liquid chromatography tandem mass spectrometry detection method of emodin and aloe-emodin in rat urine, which comprises the following steps:
s1, unfreezing the collected urine, and recovering to room temperature;
s2, adding deionized water into the urine obtained in the first step for mixing;
s3, adding an internal standard into the mixed solution;
s4, filtering the mixed solution;
and S5, taking the filtered mixed solution for determination by a liquid chromatography-mass spectrometer, wherein the liquid chromatography mobile phase is a water-acetonitrile system, the liquid chromatography column is a C18 reversed phase liquid chromatography column, and the flow rate of the liquid chromatography column is not more than 0.5 mL/min.
Preferably, in the step S2, the urine is taken in an amount of 200 μ L, and deionized water is added in an amount of 800 μ L.
Preferably, in the step S3, the internal standard is rutin.
Preferably, in the step S5, the mobile phase is: the mobile phase A is 0.1% formic acid water solution, the mobile phase B is acetonitrile, and the elution mode is gradient elution.
Preferably, the gradient elution is performed according to the following conditions:
| time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (mL/min) | Maximum pressure (bar) |
| 0 | 100 | 0 | 0.3 | 600 |
| 30 | 70 | 30 | 0.3 | 600 |
| 50 | 30 | 70 | 0.3 | 600 |
| 53 | 0 | 100 | 0.3 | 600 |
| 54 | 100 | 0 | 0.3 | 600 |
Preferably, in the step S5, the liquid chromatography column is an Agilent Poroshell120 SB-C18 chromatography column.
Preferably, in the step S5, the flow rate of the liquid chromatography column is 0.2-0.4 mL/min
In summary, compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts a liquid chromatography tandem mass spectrometry detection method, selects the combination of ultra-high performance liquid and tandem mass spectrometry, and combines specific chromatographic conditions, thereby greatly improving the detection sensitivity of emodin and other substances in the mouse urine.
(2) The method is characterized in that the method is used for screening in a plurality of mobile phases including acetonitrile-0.1% phosphoric acid, methanol-acetic acid, methanol-phosphoric acid, methanol-formic acid and the like, and finally selecting 0.1% formic acid water solution and acetonitrile as the mobile phases for gradient elution, so that the emodin and aloe-emodin have better separation degree and good system adaptability, can reduce column pressure, and is beneficial to improving the durability of a chromatographic column.
(3) The method for pre-treating the mouse urine is simple, and the detection method is high in sensitivity, simple and convenient to operate, high in stability and reliable in result.
Drawings
FIG. 1 is a diagram of liquid chromatography mass spectrum MRM of a mixed standard solution of emodin and aloe-emodin obtained in example 1 of the present invention.
FIG. 2 is a liquid chromatography mass spectrum MRM of emodin and aloe-emodin in the feed solution obtained in example 1 of the present invention.
FIG. 3 is a liquid chromatography mass spectrum MRM of emodin and aloe-emodin in mouse urine after gavage of 3# material liquid obtained in example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.
Example 1
Pretreatment method
Taking the urine of the mouse after the 3# feed liquid is perfused to unfreeze at room temperature, adding 200 mu L of unfrozen sample into 800 mu L of deionized water, 100 mu L of mixed internal standard solution, filtering by a 0.22 mu m organic phase filter membrane, and then carrying out HPLC-MS/MS analysis.
Drawing of standard curve
1) Preparation of Standard stock solutions
10mg of emodin and 17.8mg of aloe-emodin are accurately weighed and respectively placed in a 100mL brown volumetric flask, diluted to the scale by methanol, and a standard stock solution with the standard stock solution concentration is stored at minus 18 ℃ in the dark, and the validity period is 3 months, which is shown in Table 1.
Table 1 feed liquid ingredient standard stock solutions
| Standard stock solution | Concentration (μ g/mL) |
| Emodin | 100 |
| Aloe-emodin | 178 |
2) Preparation of mixed standard solution
Transferring emodin standard stock solution and aloe-emodin standard stock solution into volumetric flasks of 1.0mL to 100mL respectively, and diluting with methanol to scale. The solution is a standard mixed solution, stored at-18 ℃ in the dark, and has the validity period of 3 months, which is shown in Table 2.
TABLE 2 Standard Mixed solution of feed liquid Components
| Standard mixed solution | Concentration (μ g/mL) |
| Emodin | 1.00 |
| Aloe-emodin | 1.78 |
3) Internal standard stock solution
Accurately weighing 17.8mg of rutin, placing the rutin in a 100mL brown volumetric flask, diluting the rutin to the scale with methanol, and preparing an internal standard stock solution. The stock solution was stored at-18 ℃ in the dark for a period of 3 months, as shown in Table 3.
TABLE 3 internal standard stock solutions
| Standard stock solution | Concentration (μ g/mL) |
| Rutin (Cycleic acid) | 178 |
4) Internal standard solution
Transferring 1.0mL to 100mL volumetric flasks of rutin internal standard stock solution, diluting to scale with methanol, and preparing into mixed internal standard solution. The solution was stored at-18 ℃ in the dark for a period of 3 months, see table 4.
TABLE 4 Mixed internal standard solutions
| Standard stock solution | Concentration (μ g/mL) |
| Rutin (Cycleic acid) | 1.78 |
5) Standard working solution
Transferring 0.1mL, 0.2mL, 0.5mL, 1.0mL, 2.0mL and 5.0mL of mixed standard solution respectively, adding 1mL of internal standard solution, placing in a 10mL volumetric flask, diluting to a constant volume with methanol to a scale, preparing a standard working solution, and storing the solution at-18 ℃ in the dark for 2 weeks.
Elution method
The elution method is gradient elution, wherein a mobile phase A is 0.1% formic acid aqueous solution, and a mobile phase B is acetonitrile; gradient elution conditions were as follows:
TABLE 5 gradient elution conditions
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (mL/min) | Maximum pressure (bar) |
| 0 | 100 | 0 | 0.3 | 600 |
| 30 | 70 | 30 | 0.3 | 600 |
| 50 | 30 | 70 | 0.3 | 600 |
| 53 | 0 | 100 | 0.3 | 600 |
| 54 | 100 | 0 | 0.3 | 600 |
Chromatographic mass spectrometry conditions
Liquid chromatography column used: an Agilent Poroshell120 SB-C18 column (3.0X 100mm i.d., 2.7 μm, Agilent Corp.); sample introduction amount: 10 mu L of the solution; flow rate: 0.30 mL/min;
table 6 shows electrospray ionization source (ESI) set conditions. The scanning mode is as follows: scanning negative ions; the detection mode is as follows: multiple reaction monitoring mode (MRM); the molecular weight, parent ion, daughter ion, residence time, Collision Energy (CE) and peak time for each compound are shown in table 7. Gas Temp: 180 ℃; nebulizer: 20 psi; shear gas flow: 10.0L/min; shear gas Temp: at 300 ℃.
TABLE 6 ion Source conditions
TABLE 7 MRM parameters of analytes and internal standards
Evaluation of detection method
The urine sample of the rat treated by the pretreatment method is detected by the working curve, the elution method and the chromatographic conditions of the mass spectrum, and the detection method is evaluated.
1) Standard curve
Under the chromatographic conditions described above, each ion can be efficiently separated using the gradient elution method described above. As shown in table 8, the correlation was good.
TABLE 8 Standard Curve equation
| Compound (I) | Equation of standard curve | R2 |
| Aloe-emodin | y＝0.16x+0.4173 | 0.9984 |
| Emodin | y＝0.127x+0.9238 | 0.9727 |
2) Precision test
Within the same day, 6 parallel experiments were performed on the urine samples of the rats treated with the pretreatment method described above, and the measured contents of the various analytes were analyzed to obtain the standard deviation of the relative standard within the day. The results are shown in table 9, the in-day precision of the analytes is aloe-emodin: 7.8%, emodin: 15.1 percent.
Precision of the method (relative standard deviation in days)
| Aloe-emodin (mug/mL) | Emodin (mug/mL) | |
| 1 | 13.203 | 1.003 |
| 2 | 12.603 | 0.883 |
| 3 | 11.336 | 0.850 |
| 4 | 11.056 | 0.770 |
| 5 | 11.324 | 0.796 |
| Mean value of | 11.90 | 0.860 |
| RSD (precision) | 7.92％ | 10.60％ |
3) Detection and quantitation limits of methods
Emodin: 4.64ng/mL, 2.51ng/mL aloe-emodin, wherein the signal-to-noise ratio (S/N) > 3 is determined as the detection limit of an instrument method; emodin: 15.45ng/mL, 8.36ng/mL aloe-emodin, signal-to-noise ratio (S/N) > 10, determined as the limit of quantitation of instrumental methods; the detection limit and the quantification limit are sufficient to meet the requirements.
The results show that the detection method for simultaneously determining emodin and aloe-emodin in mouse urine by using LC-MS/MS established by the method adopts an internal standard method to extract and dilute a sample for determination, and has strong specificity, high sensitivity and low detection limit. Through methodology investigation, the method is completely suitable for quantitative detection of emodin and aloe-emodin in a mouse urine sample.
Experimental example 2 detection of sample
The analysis and detection are carried out on the component analysis of the rat urine liquid after the 24h of intragastric administration of 6 (blank control), 6 feed liquids of 3# and 6 feed liquids of 4# by adopting the established analysis method. Analyzing and detecting to obtain two kinds of material liquid metabolic components: aloe-emodin, emodin. The metabolic components exhibit the same laws: control <3# feed <4# feed and the results are shown in table 10.
TABLE 10 emodin and Aloe-emodin content in urine of gavage rat
The invention selects 0.1 percent formic acid water solution and acetonitrile as mobile phases, selects ultra-high performance liquid phase and tandem mass spectrometry to be combined, and has gradient elution, the separation degree of emodin and aloe-emodin meets the requirement, and the system has good adaptability.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
Claims (7)
1. A liquid chromatography tandem mass spectrometry detection method of emodin and aloe-emodin in mouse urine is characterized by comprising the following steps:
s1, unfreezing the collected urine, and recovering to room temperature;
s2, adding deionized water into the urine obtained in the first step for mixing;
s3, adding an internal standard into the mixed solution;
s4, filtering the mixed solution;
and S5, taking the filtered mixed solution for determination by a liquid chromatography-mass spectrometer, wherein the liquid chromatography mobile phase is a water-acetonitrile system, the liquid chromatography column is a C18 reversed phase liquid chromatography column, and the flow rate of the liquid chromatography column is not more than 0.5 mL/min.
2. The method for detecting emodin and aloe-emodin in murine urine according to claim 1, wherein in the step S2, the urine is taken at 200 μ L, and deionized water is added at 800 μ L.
3. The method for detecting emodin and aloe-emodin in murine urine according to claim 1, wherein in the step of S3, the internal standard is rutin.
4. The method of claim 1, wherein the liquid chromatography-tandem mass spectrometry is carried out on emodin and aloe-emodin in murine urine,
characterized in that, in the step S5, the mobile phase is: the mobile phase A is 0.1% formic acid water solution, the mobile phase B is acetonitrile, and the elution mode is gradient elution.
5. The method for detecting emodin and aloe-emodin in murine urine according to claim 4, wherein said gradient elution is performed according to the following conditions:
6. The method of claim 1, wherein the liquid chromatography-tandem mass spectrometry is carried out on emodin and aloe-emodin in murine urine,
wherein in the step S5, the liquid chromatography column is an Agilent Poroshell120 SB-C18 chromatography column.
7. The method of claim 1, wherein the liquid chromatography-tandem mass spectrometry is carried out on emodin and aloe-emodin in murine urine,
characterized in that in the step S5, the flow rate of the liquid chromatographic column is 0.2-0.4 mL/min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022110569A1 (en) * | 2020-11-27 | 2022-06-02 | 海南医学院 | Lc-ms/ms measurement method for aloesin in rat blood plasma |
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| US5652265A (en) * | 1995-03-29 | 1997-07-29 | Wisconsin Alumni Research Foundation | Production of rhein and rhein derivatives |
| CN102854268A (en) * | 2012-10-08 | 2013-01-02 | 成都中医药大学 | Detection method of authentic rheum officinale seeds |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5652265A (en) * | 1995-03-29 | 1997-07-29 | Wisconsin Alumni Research Foundation | Production of rhein and rhein derivatives |
| CN102854268A (en) * | 2012-10-08 | 2013-01-02 | 成都中医药大学 | Detection method of authentic rheum officinale seeds |
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| PEIJINLI 等: "Pharmacokinetics and pharmacodynamics of rhubarb anthraquinones extract in normal and disease rats", 《BIOMEDICINE & PHARMACOTHERAPY》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022110569A1 (en) * | 2020-11-27 | 2022-06-02 | 海南医学院 | Lc-ms/ms measurement method for aloesin in rat blood plasma |
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