CN111208230B - Method for detecting American cockroach intestinal flora metabolite - Google Patents
Method for detecting American cockroach intestinal flora metabolite Download PDFInfo
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a method for detecting a periplaneta americana intestinal flora metabolite. The detection method is an ultra-high performance liquid chromatography, can simultaneously determine the content of 6 nitrogen-containing compounds in the periplaneta americana feces by limiting the chromatographic conditions, is efficient, sensitive and simple to operate, and provides a new technology for the research of periplaneta americana intestinal flora metabolites.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for detecting a periplaneta americana intestinal flora metabolite.
Background
Periplaneta Americana (Periplaneta Americana L.) is an insect belonging to the genus Periplaneta of the family Blattaceae, is a traditional Chinese medicine, and is commonly used in traditional Chinese medicine clinical practice for removing cold and heat, eliminating accumulation, invigorating spleen and eliminating malnutrition, etc. Modern researches show that the periplaneta americana has rich active ingredients and good pharmacological action. In recent years, Periplaneta americana is used as a raw material, various new drugs for preventing and treating gastrointestinal diseases, chronic hepatitis B or cardiovascular system diseases and the like are successfully developed, and the new drugs are low in cost, small in toxic and side effects and good in prevention and treatment effect.
The intestinal flora is a microorganism which is combined or adhered with intestinal mucosa in the intestinal tract of animals, can carry out biotransformation on food ingested by a host, and the produced metabolite can influence the health and vitality of the host. Modern medicine focuses on research on animal intestinal flora and metabolites thereof, explores functional characteristics of intestinal microorganisms, analyzes a metabolic regulation mechanism between the intestinal flora and a host, and tries to treat diseases by using the metabolites of the animal intestinal flora.
Periplaneta americana is widely distributed and has strong adaptability to complex environments, which is presumed to be related to active ingredients in metabolites of intestinal flora. Therefore, the periplaneta americana intestinal flora metabolites need to be detected, but no method for detecting the nitrogen-containing metabolites of the intestinal flora exists at present.
Disclosure of Invention
Aiming at the problem that the prior art is lack of a method for detecting the metabolite of the periplaneta americana intestinal flora, the invention provides a method for detecting the metabolite of the periplaneta americana intestinal flora containing nitrogen.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a method for detecting American cockroach intestinal flora metabolites, wherein the metabolites comprise 5-hydroxyindolin-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone, 8-hydroxy-2-quinolinone, oxindole, 8-hydroxy-3, 4-dihydro-2-quinolinone and 8-hydroxy-2-quinolinic acid;
the detection method is ultra-high performance liquid chromatography, and the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
the mobile phase A is 0.08-0.12% (v/v) formic acid aqueous solution, the mobile phase B is acetonitrile, and the gradient elution procedure is as follows: 0-5 min, and 98-93% of mobile phase A; 5-9 min, and 93% -90% of a mobile phase A; 9-19 min, and 90% -78% of a mobile phase A; 19-20 min, and 78% -98% of a mobile phase A;
the flow rate is 0.3-0.35 mL/min;
the column temperature is 40-60 ℃;
the detection wavelength is 252-256 nm.
The ultra-high performance liquid chromatography provided by the detection method is high in efficiency, sensitive and simple to operate, and can simultaneously detect the content of the 6 nitrogen-containing compounds in the periplaneta americana intestinal flora metabolite, so that a foundation can be laid for the periplaneta americana intestinal flora metabolite research. The 6 nitrogen-containing compounds 5-oxindole-2-one (A), 6-hydroxy-3, 4-dihydro-1H-2-quinolinone (B), 8-hydroxy-2-quinolinone (C), oxindole (D), 8-hydroxy-3, 4-dihydro-2-quinolinone (E) and 8-hydroxy-2-quinolinic acid (F) have the following structural formulas:
in the gradient elution procedure, the proportion range of the mobile phase A means that the mobile phase A linearly changes within a limited proportion range within a set time, for example, "0-5 min, 98% -93% of the mobile phase A" means that the mobile phase A changes from 98% to 93% in a gradient within 0-5 min, and the same is applied below.
Preferably, the mobile phase a is 0.10% (v/v) aqueous formic acid.
Preferably, the flow rate is 0.3 mL/min.
Preferably, the column temperature is 50 ℃.
Preferably, the detection wavelength is 254 nm.
Preferably, the detection method comprises the following operations:
preparing a mixed reference substance solution of the metabolite by taking a methanol aqueous solution as a solvent; detecting the mixed reference solution by using ultra-high performance liquid chromatography under the chromatographic condition, and establishing a concentration-peak area linear regression equation of each metabolite according to the obtained peak area;
preparing the periplaneta americana feces into a test solution by taking a methanol water solution as a solvent; and under the chromatographic condition, detecting the test solution by using the ultra-high performance liquid chromatography, substituting the obtained peak area into the concentration-peak area linear regression equation, and calculating the content of the metabolite in the test solution.
Preferably, the volume percentage concentration of methanol in the methanol aqueous solution for preparing the mixed control solution is 30-70% (v/v). Preferably, the methanol aqueous solution contains 0-1% (v/v) formic acid, so that the repeatability of the detection method is better, and the RSD of the sample is smaller when parallel samples are processed.
Preferably, the preparation method of the test solution comprises the following steps: the periplaneta americana excrement is crushed, placed in 50-70% (v/v) methanol water solution which is 20-100 times of the weight of the periplaneta americana excrement, subjected to ultrasonic treatment for 30-50 min, filtered, and diluted to 2-3 times of filtrate by water. The higher the methanol concentration, the higher the extraction efficiency, but the more pronounced the solvent effect. Selecting 50-70% (v/v) methanol aqueous solution with 20-100 times weight as extraction solvent, and diluting with water to obtain good chromatographic peak shape and no solvent effect. The filtration is carried out by adopting a conventional 0.45 mu m filter membrane. Preferably, the methanol aqueous solution contains 0-1% (v/v) formic acid. Preferably, the water contains 0-1% (v/v) formic acid.
Preferably, the power of the ultrasound is 540W and the frequency is 35 kHz.
Preferably, the preparation method further comprises centrifuging at 14000rpm for 10min before the ultrasonic treatment, and taking the supernatant.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a UPLC chromatogram of a test solution in example 1 of the present invention;
FIG. 2 is a UPLC chromatogram of a mixed control solution of example 1 of the present invention;
in the figure:
1 is 5-hydroxyindolin-2-one;
2 is 6-hydroxy-3, 4-dihydro-1H-2-quinolinone;
3 is 8-hydroxy-2-quinolinone;
4 is oxindole;
5 is 8-hydroxy-3, 4-dihydro-2-quinolinone;
6 is 8-hydroxy-2-quinolinic acid.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following instruments and reagents were used in the examples and the test examples as follows:
acquisty UPLC ultra high liquid chromatograph (Waters corporation, USA) equipped with Waters PDA e lambda detector; ACQUITY
HST 3 column (2.1X 100mm, 1.8 μm, Waters Corp.); Milli-Q ultrapure water system (Millipore, USA); an ME204 ten thousandth electronic balance, an MS105DU ten thousandth electronic balance (Mettler toledo, switzerland); DL720B ultrasonic cleaner (Shanghai Xin apparatus Co., Ltd.); centrifuge 5424R desk high speed Centrifuge (Eppendorf, Germany).
5-hydroxyindolin-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone, 8-hydroxy-2-quinolinone, oxindole, 8-hydroxy-3, 4-dihydro-2-quinolinone and 8-hydroxy-2-quinolinic acid reference substances (the purity is more than or equal to 95 percent, the reference substances are prepared by laboratories); methanol, acetonitrile (chromatographically pure, Sigma company, usa); formic acid (chromatographically pure, Shanghai Allatin Biotech, Inc.); the experimental water was ultrapure water.
The periplaneta americana feces sample is taken from the GAP culture base of the stone mountain periplaneta americana of Yunnan Teng pharmaceutical products GmbH, the feeding conditions are strictly executed according to the GAP standard, and the main component of the feed is bean flour. Collecting the periplaneta americana feces in the adult stage and storing in a refrigerator at-20 ℃.
Example 1
The embodiment of the invention provides a method for detecting nitrogen-containing metabolites of periplaneta americana intestinal flora, which comprises the following steps:
1. preparation of control solutions
Weighing appropriate amounts of 5-hydroxyindoline-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone and 8-hydroxy-2-quinolinone respectively, placing in a volumetric flask, and taking 70% (v/v) methanol water solution (containing 0.5% (v/v) formic acid) as a solvent to respectively prepare reference stock solutions of 5-hydroxyindoline-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone and 8-hydroxy-2-quinolinone, wherein the concentrations are 1.061mg/mL, 0.515mg/mL and 0.548mg/mL respectively;
appropriate amounts of oxindole, 8-hydroxy-3, 4-dihydro-2-quinolinone and 8-hydroxy-2-quinolinic acid are respectively weighed and placed in a volumetric flask, and control stock solutions of the oxindole, the 8-hydroxy-3, 4-dihydro-2-quinolinone and the 8-hydroxy-2-quinolinic acid are respectively prepared by taking 30% (v/v) methanol aqueous solution (containing 0.5% (v/v) formic acid) as a solvent, wherein the concentrations are respectively 0.513mg/mL, 0.529mg/mL and 1.072 mg/mL.
Respectively taking a proper amount of each control stock solution, placing the solutions into a 5mL volumetric flask, metering the volume by 30% (v/v) methanol aqueous solution (containing 0.5% (v/v) formic acid), and shaking the solutions evenly to prepare a mixed control solution containing 42.4 mu g/mL of 5-hydroxyindolin-2-one, 10.3 mu g/mL of 6-hydroxy-3, 4-dihydro-1H-2-quinolinone, 49.3 mu g/mL of 8-hydroxy-2-quinolinone, 15.4 mu g/mL of oxindole, 37.0 mu g/mL of 8-hydroxy-3, 4-dihydro-2-quinolinone and 85.8 mu g/mL of 8-hydroxy-2-quinolinic acid.
2. Preparation of test solution
Taking a proper amount of periplaneta americana excrement, crushing the periplaneta americana excrement by a traditional Chinese medicine crusher, sieving the periplaneta americana excrement by a 50-mesh sieve, uniformly mixing, taking 0.5g of periplaneta americana excrement powder, precisely weighing, placing the periplaneta americana excrement powder into a 25mL volumetric flask, carrying out ultrasonic treatment (power is 540W and frequency is 35kHz) for 30min, placing the periplaneta americana excrement powder to room temperature, fixing the volume to a scale by using 70% (v/v) methanol aqueous solution (containing 0.5% (v/v) formic acid), uniformly mixing, centrifuging the mixture at 14000rpm for 10min, taking supernate, filtering the supernate by using a 0.45-micron filter membrane, and diluting the filtrate by using equal-volume 0.5% (v/v) formic acid water to obtain a test solution.
3. Detecting the mixed reference substance solution and the test substance solution by using ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: ACQUITY
HSS T3(2.1×100mm，1.8μm)；
Mobile phase a was 0.10% (v/v) aqueous formic acid, mobile phase B was acetonitrile, and the gradient elution procedure was: 0-5 min, and 98-93% of mobile phase A; 5-9 min, and 93% -90% of a mobile phase A; 9-19 min, and 90% -78% of a mobile phase A; 19-20 min, and 78% -98% of a mobile phase A;
the sample volume is 2 mu L;
the flow rate is 0.3 mL/min;
the column temperature is 50 ℃;
the detection wavelength is 254 nm.
And establishing a concentration-peak area linear regression equation of each metabolite according to the peak area obtained by mixing the reference solution, substituting the peak area obtained by the test solution into the concentration-peak area linear regression equation, and calculating the content of the metabolite in the test solution.
Example 2
The embodiment of the invention provides a method for detecting nitrogen-containing metabolites of periplaneta americana intestinal flora, which comprises the following steps:
1. preparation of control solutions
Weighing appropriate amounts of 5-hydroxyindoline-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone and 8-hydroxy-2-quinolinone respectively, placing in a volumetric flask, and taking 70% (v/v) methanol water solution (containing 1% (v/v) formic acid) as a solvent to prepare reference stock solutions of 5-hydroxyindoline-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone and 8-hydroxy-2-quinolinone respectively with the concentrations of 1.061mg/mL, 0.515mg/mL and 0.548 mg/mL;
respectively weighing appropriate amounts of oxindole, 8-hydroxy-3, 4-dihydro-2-quinolinone and 8-hydroxy-2-quinolinic acid, placing the weighed materials in a volumetric flask, and respectively preparing reference stock solutions of the oxindole, the 8-hydroxy-3, 4-dihydro-2-quinolinone and the 8-hydroxy-2-quinolinic acid by taking 30% (v/v) methanol aqueous solution (containing 1% (v/v) formic acid) as a solvent, wherein the concentrations are respectively 0.513mg/mL, 0.529mg/mL and 1.072 mg/mL.
Respectively taking a proper amount of each control stock solution, placing the solutions into a 5mL volumetric flask, fixing the volume by 30% (v/v) methanol aqueous solution (containing 1% (v/v) formic acid), shaking up to prepare a mixed control solution containing 42.4 mu g/mL of 5-hydroxyindolin-2-one, 10.3 mu g/mL of 6-hydroxy-3, 4-dihydro-1H-2-quinolinone, 49.3 mu g/mL of 8-hydroxy-2-quinolinone, 15.4 mu g/mL of oxindole, 37.0 mu g/mL of 8-hydroxy-3, 4-dihydro-2-quinolinone and 85.8 mu g/mL of 8-hydroxy-2-quinolinic acid.
2. Preparation of test solution
Taking a proper amount of periplaneta americana excrement, crushing the periplaneta americana excrement by a traditional Chinese medicine crusher, sieving the periplaneta americana excrement by a 50-mesh sieve, uniformly mixing, taking 0.5g of periplaneta americana excrement powder, precisely weighing, placing the periplaneta americana excrement powder into a 25mL volumetric flask, carrying out ultrasonic treatment (power is 540W and frequency is 35kHz) for 30min, placing the periplaneta americana excrement powder to room temperature, fixing the volume to a scale by using a 50% (v/v) methanol aqueous solution (containing 1% (v/v) formic acid), uniformly mixing, centrifuging the mixture at 14000rpm for 10min, taking supernate, filtering the supernate by using a 0.45-micron filter membrane, and diluting the filtrate by using 1% (v/v) formic acid water with the same volume to obtain a test solution.
3. Detecting the mixed reference substance solution and the test substance solution by using ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: ACQUITY
HSS T3(2.1×100mm，1.8μm)；
The mobile phase A is 0.08% (v/v) formic acid water solution, the mobile phase B is acetonitrile, and the gradient elution procedure is as follows: 0-5 min, and 98-93% of mobile phase A; 5-9 min, and 93% -90% of a mobile phase A; 9-19 min, and 90% -78% of a mobile phase A; 19-20 min, and 78% -98% of a mobile phase A;
the sample volume is 2 mu L;
the flow rate is 0.3 mL/min;
the column temperature is 40 ℃;
the detection wavelength is 252 nm.
And establishing a concentration-peak area linear regression equation of each metabolite according to the peak area obtained by mixing the reference solution, substituting the peak area obtained by the test solution into the concentration-peak area linear regression equation, and calculating the content of the metabolite in the test solution.
Example 3
The embodiment of the invention provides a method for detecting nitrogen-containing metabolites of periplaneta americana intestinal flora, which comprises the following steps:
1. preparation of control solutions
Respectively weighing appropriate amounts of 5-hydroxyindoline-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone and 8-hydroxy-2-quinolinone, placing in a volumetric flask, and respectively preparing reference stock solutions of 5-hydroxyindoline-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone and 8-hydroxy-2-quinolinone by taking 70% (v/v) methanol aqueous solution as a solvent, wherein the concentrations are respectively 1.061mg/mL, 0.515mg/mL and 0.548 mg/mL;
respectively weighing appropriate amounts of oxindole, 8-hydroxy-3, 4-dihydro-2-quinolinone and 8-hydroxy-2-quinolinic acid, placing the weighed materials in a volumetric flask, and respectively preparing reference stock solutions of the oxindole, the 8-hydroxy-3, 4-dihydro-2-quinolinone and the 8-hydroxy-2-quinolinic acid by taking 30% (v/v) methanol aqueous solution as a solvent, wherein the concentrations are respectively 0.513mg/mL, 0.529mg/mL and 1.072 mg/mL.
Respectively taking a proper amount of each control stock solution, placing the solutions into a 5mL volumetric flask, fixing the volume by 30% (v/v) methanol aqueous solution, and shaking the solutions evenly to prepare a mixed control solution containing 42.4 mu g/mL of 5-hydroxyindolin-2-one, 10.3 mu g/mL of 6-hydroxy-3, 4-dihydro-1H-2-quinolinone, 49.3 mu g/mL of 8-hydroxy-2-quinolinone, 15.4 mu g/mL of oxindole, 37.0 mu g/mL of 8-hydroxy-3, 4-dihydro-2-quinolinone and 85.8 mu g/mL of 8-hydroxy-2-quinolinic acid.
2. Preparation of test solution
Taking a proper amount of periplaneta americana excrement, crushing the periplaneta americana excrement by a traditional Chinese medicine crusher, sieving the periplaneta americana excrement by a 50-mesh sieve, uniformly mixing, taking 0.5g of periplaneta americana excrement powder, precisely weighing, placing the periplaneta americana excrement powder into a 25mL volumetric flask, carrying out ultrasonic treatment (power of 540W and frequency of 35kHz) for 30min, placing the periplaneta americana excrement powder to room temperature, fixing the volume to a scale by using 70% (v/v) methanol aqueous solution, uniformly mixing, centrifuging the mixture at 14000rpm for 10min, taking supernatant, filtering the mixture by using a 0.45-micron filter membrane, and diluting the filtrate by using 2-fold volume water to obtain a test solution.
3. Detecting the mixed reference substance solution and the test substance solution by using ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: ACQUITY
HSS T3(2.1×100mm，1.8μm)；
Mobile phase a was 0.12% (v/v) aqueous formic acid, mobile phase B was acetonitrile, and the gradient elution procedure was: 0-5 min, and 98-93% of mobile phase A; 5-9 min, and 93% -90% of a mobile phase A; 9-19 min, and 90% -78% of a mobile phase A; 19-20 min, and 78% -98% of a mobile phase A;
the sample volume is 2 mu L;
the flow rate is 0.35 mL/min;
the column temperature is 60 ℃;
the detection wavelength was 256 nm.
And establishing a concentration-peak area linear regression equation of each metabolite according to the peak area obtained by mixing the reference solution, substituting the peak area obtained by the test solution into the concentration-peak area linear regression equation, and calculating the content of the metabolite in the test solution.
Examination example
The test example provides a methodology research of the method for detecting the nitrogen-containing metabolites of the periplaneta americana intestinal flora:
1. survey of precision within and during the day
A test solution was prepared in a single portion per day according to the method for preparing a test solution in example 1, and the sample injection was repeated 6 times according to the chromatographic conditions in example 1 for three consecutive days to calculate the contents of each compound and the RSD value, and the results are shown in Table 1.
TABLE 1 precision test results
2.4.2 repeatability test
6 parts of the sample solution was prepared by the method for preparing the sample solution in example 1, and the content of each compound and the RSD value were measured by injecting the sample under the chromatographic conditions in example 1, and the reproducibility of the method was examined, and the results are shown in Table 2.
TABLE 2 results of the repeatability tests
| Compound (I) | Repeatability (RSD,%) |
| 5-hydroxyindolin-2-ones | 1.1 |
| 6-hydroxy-3, 4-dihydro-1H-2-quinolinones | 0.7 |
| 8-hydroxy-2-quinolinones | 0.8 |
| Oxindoles | 0.6 |
| 8-hydroxy-3, 4-dihydro-2-quinolinones | 0.5 |
| 8-hydroxy-2-quinolinic acid | 2.6 |
2.4.3 stability test
1 part of the sample solution was prepared according to the method for preparing the sample solution in example 1, and the sample was injected for 0, 2, 4, 6, 8, 10, and 12 hours under the chromatographic conditions in example 1, and the content of each compound was measured, and the RSD value was calculated to examine the stability, and the results are shown in Table 3.
TABLE 3 stability test results
| Compound (I) | Stability (RSD,%) |
| 5-hydroxyindolin-2-ones | 1.5 |
| 6-hydroxy-3, 4-dihydro-1H-2-quinolinones | 0.8 |
| 8-hydroxy-2-quinolinones | 0.3 |
| Oxindoles | 1.1 |
| 8-hydroxy-3, 4-dihydro-2-quinolinones | 0.4 |
| 8-hydroxy-2-quinolinic acid | 0.8 |
2.4.4 sample recovery test
Taking 0.25g of periplaneta americana fecal powder, precisely weighing, accurately adding a mixed reference substance solution with the same mass as each compound in the sample, preparing 6 parts of a test solution according to the preparation method of the test solution in example 1, injecting sample according to the chromatographic conditions in example 1, measuring the content of each compound, and calculating the sample injection recovery rate and the RSD value, wherein the results are shown in Table 4.
TABLE 4 sample recovery test results
2.4.5 Linear relationship
The mixed control solution obtained in example 1 was designated as "mixed standard 1", and was diluted stepwise with 30% methanol (containing 0.5% (v/v) formic acid) to prepare a series of mixed standard solutions, and the peak area of each compound was measured under the chromatographic conditions in example 1. The concentration of the compound control was plotted as x (μ g/mL) abscissa and y (peak area) ordinate, a standard curve was plotted, a linear regression equation was calculated, and the detection limit (LOD, S/N. apprxeq.3) and the quantitation limit (LOQ, S/N. apprxeq.10) were determined, and the results are shown in Table 5.
TABLE 5 results of linear relationship examination
The results showed that 6 compounds were in good linear relationship (R) in the respective concentration ranges2Not less than 0.999), the concentration range of the detection limit is 0.054 to 0.134 mug/mL, and the concentration range of the quantification limit is 0.179 to 0.447 mug/mL.
The above results show that the method meets the requirements of related methodology and can be used for content determination of samples. The chromatogram of the mixed control solution is shown in FIG. 1.
2.5 detection of nitrogen-containing metabolites of periplaneta americana intestinal flora
The sample solution of 10 batches of periplaneta americana stool samples was prepared according to the method for preparing the sample solution in example 1, the peak areas of 6 nitrogen-containing compounds were measured according to the chromatographic conditions in example 1, and the obtained peak areas were substituted into the above concentration-peak area linear regression equation to calculate the content of the above metabolites in the sample solution. The results are shown in Table 6, and the chromatograms are shown in FIG. 2.
TABLE 610 batch Periplaneta americana intestinal flora metabolite assay results (n 2, mg/g)
In the above samples, the content of 5-hydroxyindolin-2-one is 0.230-0.259 mg/g, and the content of 6-hydroxy-3, 4-dihydro-1H-2-quinolinone is 0.114-0.143 mg/g; the content of 8-hydroxy-2-quinolinone is 0.550-0.707 mg/g; the content of oxindole is 0.071-0.079 mg/g; the content of 8-hydroxy-3, 4-dihydro-2-quinolinone is 0.373-0.471 mg/g; the content of the 8-hydroxy-2-quinolinic acid is 1.267-1.714 mg/g.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The method for detecting the periplaneta americana intestinal flora metabolite is characterized in that the metabolite comprises 5-hydroxyindolin-2-one, 6-hydroxy-3, 4-dihydro-1H-2-quinolinone, 8-hydroxy-2-quinolinone, oxindole, 8-hydroxy-3, 4-dihydro-2-quinolinone and 8-hydroxy-2-quinolinic acid; the detection method is ultra-high performance liquid chromatography, and the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
the mobile phase A is 0.08-0.12% (v/v) formic acid aqueous solution, the mobile phase B is acetonitrile, and the gradient elution procedure is as follows: 0-5 min, and 98-93% of mobile phase A; 5-9 min, and 93% -90% of a mobile phase A; 9-19 min, and 90% -78% of a mobile phase A; 19-20 min, and 78% -98% of a mobile phase A;
the flow rate is 0.3-0.35 mL/min;
the column temperature is 40-60 ℃;
the detection wavelength is 252-256 nm.
2. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 1, wherein the mobile phase a is 0.10% (v/v) formic acid aqueous solution.
3. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 1, wherein the flow rate is 0.3 mL/min; and/or
The column temperature is 50 ℃; and/or
The detection wavelength is 254 nm.
4. The method for detecting the periplaneta americana intestinal flora metabolite according to any one of claims 1 to 3, wherein the method comprises the following operations:
preparing a mixed reference substance solution of the metabolite by taking a methanol aqueous solution as a solvent; detecting the mixed reference solution by using ultra-high performance liquid chromatography under the chromatographic condition, and establishing a concentration-peak area linear regression equation of each metabolite according to the obtained peak area;
preparing the periplaneta americana feces into a test solution by taking a methanol water solution as a solvent; and under the chromatographic condition, detecting the test solution by using the ultra-high performance liquid chromatography, substituting the obtained peak area into the concentration-peak area linear regression equation, and calculating the content of the metabolite in the test solution.
5. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 4, wherein the volume percentage concentration of methanol in the methanol aqueous solution for preparing the mixed control solution is 30-70%.
6. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 5, wherein the methanol aqueous solution contains 0-1% (v/v) formic acid.
7. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 4, wherein the preparation method of the test solution comprises the following steps: the periplaneta americana excrement is crushed, placed in 50-70% (v/v) methanol water solution which is 20-100 times of the weight of the periplaneta americana excrement, subjected to ultrasonic treatment for 30-50 min, filtered, and diluted to 2-3 times of filtrate by water.
8. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 7, wherein the methanol aqueous solution contains 0-1% (v/v) formic acid; and/or
The water contains 0-1% (v/v) formic acid.
9. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 7, wherein the power of the ultrasound is 540W and the frequency is 35 kHz.
10. The method for detecting the periplaneta americana intestinal flora metabolite according to claim 7, wherein the preparation method further comprises the step of centrifuging for 10min at 14000rpm before the ultrasonic treatment, and taking the supernatant.
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| CN107367557A (en) * | 2017-07-05 | 2017-11-21 | 四川好医生攀西药业有限责任公司 | A kind of UPLC assay methods of blattaria protocatechuic acid content |
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Non-Patent Citations (2)
| Title |
|---|
| Intestinal Microbial Flora of the American Cockroach, Periplaneta americana L.;J. W. BRACKE等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19791130;第38卷(第5期);第945-955页 * |
| 美洲大蠊提取物CⅡ-3 UPLC指纹图谱研究;周姣姣等;《大理大学学报》;20190228;第4卷(第2期);第27-32页 * |
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