CN113624888B - Detection method of indoleacetic acid and indolopropionic acid in serum and feces - Google Patents
Detection method of indoleacetic acid and indolopropionic acid in serum and feces Download PDFInfo
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Abstract
The invention relates to the field of metabolite detection, in particular to a detection method of indoleacetic acid and indolopropionic acid in serum and feces. Before liquid chromatography mass spectrometry detection, methanol-water-formic acid mixed solvent is adopted for extraction, and a solid phase extraction column is combined for purification and enrichment, so that the pretreatment process can obviously reduce matrix interference, and qualitative and quantitative detection of indoleacetic acid and indolopropionic acid in serum and excrement can be realized without derivatization treatment. The pretreatment mode adopted by the invention is green and environment-friendly, no toxic or corrosive reagent is introduced, the pretreatment process is simple, the cost is low, and the pretreatment method has important significance for researching the action mechanism of the indoleacetic acid and the indolepropionic acid for protecting intestinal barriers and regulating host immunity.
Description
Technical Field
The invention relates to the field of metabolite detection, in particular to a detection method of indoleacetic acid and indolopropionic acid in serum and feces.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Indole derivatives such as indoleacetic acid, indolepronic acid, indolecarboxaldehyde, indoleacetic acid and indoleacetic acid are small molecule compounds produced by the catabolism of tryptophan by intestinal microorganisms, and are endogenous ligands of aromatic hydrocarbon receptors. The aromatic hydrocarbon receptor is an important ligand-activated transcription factor, is widely expressed in various immune or non-immune cells, participates in important biological processes such as signal transduction under the stimulation of endogenous and environmental signals, and plays an important role in the regulation of organism immune balance. Indole derivatives, in particular indoleacetic acid and indolepronic acid, have been reported to protect the intestinal barrier and modulate the host immune response via the aromatic receptor signaling pathway.
At present, the analysis of the indoleacetic acid and the indolepropionic acid mainly comprises liquid chromatography and liquid chromatography-mass spectrometry, and the sensitivity of the liquid chromatography is difficult to meet the trace analysis requirement, so the liquid chromatography-mass spectrometry is popular. Because biological samples such as serum and feces have complex matrixes and more interfering substances, so far, no liquid chromatography mass spectrometry detection method for directly carrying out qualitative and quantitative analysis on indoleacetic acid and indolopropionic acid in serum and feces is available.
In the prior art, a mode of combining derivatization treatment with liquid chromatography mass spectrometry detection is generally adopted, derivatization reaction is adopted between a derivatization reagent and indoleacetic acid or indolopropionic acid to generate corresponding derivatives, the detection sensitivity is improved, the detection difficulty is reduced, but most of derivatization reagents are toxic and irritant substances, the price is high, and certain difficulty exists in complete removal in the later stage. Therefore, a method for directly detecting the indoleacetic acid and the indolepropionic acid in the complex biological sample is established, and a scientific quantitative means can be provided for the research of the indolepropionic acid and the indolepropionic acid in the field of immunology.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a detection method of indoleacetic acid and indolopropionic acid in serum and excrement, which is characterized in that before liquid chromatography mass spectrometry detection, methanol-water-formic acid mixed solvent is directly adopted for extraction, and purification and enrichment are carried out by combining with a solid phase extraction column.
The technical scheme adopted by the invention is as follows:
the invention provides a detection method of indoleacetic acid and indolopropionic acid in serum and feces, which comprises pretreatment and liquid chromatography mass spectrometry detection;
the pretreatment step comprises the following steps:
step one: preparing a mixed solvent of methanol, water and formic acid according to a proportion as an extracting solution, and extracting a fecal or serum sample to obtain an extracting solution;
step two: the extract was purified and enriched on an activated Oasis HLB (3 cc/60 mg) solid phase extraction column and the eluate was collected for measurement.
One or more embodiments of the present invention have at least the following beneficial effects:
(1) According to the invention, by reasonably setting the pretreatment method, derivatization treatment is not needed, the methanol-water-formic acid mixed solvent is directly adopted for extraction, and the solid phase extraction column is combined for purification and enrichment, so that the indoleacetic acid and the indolopropionic acid can be extracted from serum and excrement to the greatest extent, meanwhile, interference caused by other substances in the serum and the excrement is effectively avoided, a good basis is provided for subsequent liquid chromatography mass spectrometry detection, and further qualitative and quantitative detection of the indoleacetic acid and the indolopropionic acid in the serum and the excrement is realized.
(2) The method starts from the pretreatment stage of the detection process to improve the scheme, the pretreatment mode adopted is green and environment-friendly, toxic and corrosive reagents are not introduced, the pretreatment process is simple, the cost is low, and the method has important significance in the field of deep research on intestinal permeability and host immune function based on indoleacetic acid and indolepropionic acid.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a collection channel of quantitative and qualitative ions of indolopropionic acid-d 2, indolopropionic acid, indoleacetic acid-d 5, and indoleacetic acid;
FIG. 2 shows the amounts of indoleacetic acid and indoleacetic acid in the serum of mice in the control group and in the sodium salt solution fed group of indoleacetic acid in example 1;
FIG. 3 shows the amounts of indoleacetic acid and indolopropionic acid in the mouse feces of the control group, indoleacetic acid feed group and indolopropionic acid feed group of example 2.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As described in the background art, in the prior art, the derivatization treatment is generally combined with the liquid chromatography mass spectrometry detection to detect the indoleacetic acid and the indolopropionic acid in serum and excrement, and derivatization reaction is carried out on the derivatization reagent, the indoleacetic acid and the indolopropionic acid to generate corresponding derivatives, so that the detection sensitivity is improved, the detection difficulty is reduced, but most derivatization reagents are toxic and irritant substances, the price is high, and the later complete removal is difficult.
In order to solve the technical problems, the invention provides a detection method of indoleacetic acid and indolopropionic acid in serum and feces, which comprises pretreatment and liquid chromatography mass spectrometry detection;
the pretreatment step comprises the following steps:
step one: methanol is prepared according to the proportion: water: the formic acid mixed solvent is used as an extracting solution, and the feces or serum sample is extracted to obtain an extracting solution;
step two: the extract was purified and enriched on an activated Oasis HLB (3 cc/60 mg) solid phase extraction column and the eluate was collected for measurement.
Among them, pretreatment is particularly important because of the complex matrix of serum and stool samples and the high amount of interfering substances. In the pretreatment process, the detection of the indoleacetic acid and the indolepropionic acid is realized mainly by considering the following two aspects: firstly, the indoleacetic acid and the indolepronic acid in the sample are extracted by a reasonable extractant, and methanol is adopted for the first time in the field: water: the mixed formic acid solvent is used for extracting the indoleacetic acid and the indolepronic acid in blood or excrement, so that the extraction effect is better and the interference of substances can be removed more sensitively compared with the conventional methanol-water extracting solution or the mixed methanol-chloroform-water extracting solution; secondly, the impurities in the extract can be further removed by adopting a solid phase extraction small column for purification and enrichment, and the matrix interference in the data acquisition process is reduced.
Further, methanol: water: the volume ratio of formic acid is (15-16): (4-5): (1-2); preferably 15:4:1.
furthermore, when the fecal sample is extracted, grinding and homogenizing are needed under the low-temperature airtight condition, and the loss of the target in the pretreatment process can be reduced under the low-temperature environment; preferably, grinding is carried out at 3-5 ℃;
in one or more embodiments of the present invention, the specific procedure of the first procedure is: adding mixed internal standard solution of indoleacetic acid-d 5 and indolepropionic acid-d 2 into the fecal or serum sample, adding the extracting solution for ultrasonic extraction, centrifuging to obtain supernatant, adding the same amount of extracting solution, repeating the steps, and combining the supernatants to obtain the extracting solution. The method adopts secondary solvent extraction and combines ultrasonic solvent extraction to fully improve the extraction effect, adopts an internal standard method to quantify, and can correct and eliminate the influence on an analysis result caused by fluctuation of operation conditions so as to improve the accuracy of the analysis result.
In the extraction process, the addition amounts of the extracting solutions corresponding to different extraction objects are different, and when the feces are extracted, the addition ratio of the feces to the extracting solution is 0.05g:1mL; when serum is extracted, the volume ratio of the serum to the extracting solution is 1:9;
further, the extraction process is carried out at 3-5 ℃ under an ultrasonic environment, and the extraction time is 15-20min;
centrifuging at 10000rpm for 10min;
in one or more embodiments of the invention, 500-700 μl of extract is removed from step two and purified and enriched by passing through an activated Oasis HLB (3 cc/60 mg) solid phase extraction column;
in one or more embodiments of the present invention, in the liquid chromatography mass spectrometry detection, the liquid phase detection conditions are:
chromatographic column:BEH C18, mobile phase a is 0.1% formic acid in water, B is acetonitrile;
the elution procedure was: 0 to 0.5min,10 percent of B;0.5 to 2min,10 to 95 percent of B; 2-3 min,95% B;3 to 3.5min,95 to 10 percent of B;3.5 to 5 minutes, 10 percent of B;
further, the specification of the chromatographic column: 2.1X105 mm,1.7 μm; the sample injection amount is 1-3 mu L; the flow rate is 0.1-0.3mL/min;
the mass spectrum detection conditions are as follows: electrospray ion source, positive ion scanning (ESI) + ) Multi-reaction monitoring (MRM) mode, capillary voltage: 2.5kV; ion source temperature: 150 ℃;
further, desolventizing gas temperature: 250 ℃; taper hole air flow: 150L/hf; desolventizing gas flow: 600L/hf; indoloacetic acid qualitative ion pair m/z 176.1/103, cone voltage 25V, collision energy 20eV, quantitative ion pair m/z 176.1/130, cone voltage 25V, collision energy 20eV; indoleacetic acid-d 5 qualitative ion pair m/z181.1/106, cone voltage 20V, collision energy 8eV, quantitative ion pair m/z 181.1/134, cone voltage 20V, collision energy 30eV; indolopropionic acid qualitative ion pair m/z190/171.9, cone voltage 30V, collision energy 19eV, quantitative ion pair m/z 190/130, cone voltage 30V, collision energy 10eV; indolopropionic acid-d 2 qualitative ion pair m/z 192/55.9, cone voltage 12V, collision energy 12eV, quantitative ion pair m/z192/130, cone voltage 12V, collision energy 16eV.
Further, after liquid chromatography mass spectrometry detection, a standard working curve is drawn, the internal standard method is used for quantification, and the concentration of the indoleacetic acid are calculated according to the ratio of the external standard to the corresponding internal standard.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
In the present invention, the materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
The invention adopts a Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph and is provided with a WatersXevo TQ-XS mass spectrometer for data acquisition. The chromatograms of the indoleacetic acid and the indolopropionic acid are shown in figure 1, and the figure shows that the retention time of the indoleacetic acid is 3.27min, the retention time of the indolopropionic acid is 3.41min, the peaks are sharp and smooth, the whole baseline is stable, no solvent peak or other impurity peak exists, and the daily analysis and test requirements are completely met.
Example 1:
determination of indoleacetic acid and indolopropionic acid in mouse serum
1. Ultrasonic solvent extraction: 100. Mu.L of mouse serum is accurately removed by a pipette into a 2mL EP tube, an appropriate amount of internal standard (indoleacetic acid-d 5 and indolopropionic acid-d 2) is added, 0.45mL of extract (methanol: water: formic acid=15:4:1) is added, the mixture is subjected to sealed ultrasonic extraction for 15min, centrifugation is carried out at 10000rpm and 4 ℃ for 10min, after the supernatant is taken, 0.45mL of extract (methanol: water: formic acid=15:4:1) is added, the extraction is repeated once, and the extracts are combined.
2. Enrichment and purification of a solid phase extraction column: first, 4mL of methanol and 4mL of water are used for activating an Oasis HLB (3 cc/60 mg) solid phase extraction column, then 600 mu L of extract is accurately removed for loading, 4mL of water is added for leaching, 3mL of eluent (0.1% methanol formate) is used for eluting after the leaching is finished, and the eluent is collected.
3. Ultra-high performance liquid chromatography mass spectrometry detection: and (5) filtering the collected eluent with a 0.2 mu m filter membrane, and detecting the eluent on a machine.
Chromatographic conditions: chromatographic column:BEH C18, specification: 2.1X105 mm,1.7 μm; the sample injection amount is 2 mu L; the flow rate is 0.2mL/min; mobile phase a was 0.1% aqueous formic acid and B was acetonitrile, elution procedure was as follows: 0 to 0.5min,10 percent of B;0.5 to 2.0min,10 to 95 percent of B; 2-3 min,95% B;3 to 3.5min,95 to 10 percent of B;3.5 to 5 minutes, 10 percent of B; the mass spectrum detection conditions are as follows: electronic sprayMist ion source, positive ion scan (esi+), multiple Reaction Monitoring (MRM) mode, capillary voltage: 2.5kV; ion source temperature: 150 ℃; desolventizing gas temperature: 250 ℃; taper hole air flow: 150L/hf; desolventizing gas flow: 600L/hf; indoloacetic acid qualitative ion pair m/z 176.1/103, cone voltage 25V, collision energy 20eV, quantitative ion pair m/z 176.1/130, cone voltage 25V, collision energy 20eV; indoleacetic acid-d 5 qualitative ion pair m/z181.1/106, cone voltage 20V, collision energy 8eV, quantitative ion pair m/z 181.1/134, cone voltage 20V, collision energy 30eV; indolopropionic acid qualitative ion pair m/z190/171.9, cone voltage 30V, collision energy 19eV, quantitative ion pair m/z 190/130, cone voltage 30V, collision energy 10eV; indolopropionic acid-d 2 qualitative ion pair m/z 192/55.9, cone voltage 12V, collision energy 12eV, quantitative ion pair m/z192/130, cone voltage 12V, collision energy 16eV.
4. Drawing a standard curve: standard solutions of 1000. Mu.g/L of indoleacetic acid and indolepronic acid, and internal standard mixtures of 1000. Mu.g/L of indoleacetic acid-d 5 and indolepronic acid-d 2 were prepared with 0.1% methanol formate, and standard curves of 0.5. Mu.g/L, 1. Mu.g/L, 5. Mu.g/L, 10. Mu.g/L, 20. Mu.g/L, 50. Mu.g/L, 100. Mu.g/L, 200. Mu.g/L and 500. Mu.g/L were prepared, respectively, and the concentration of each of indoleacetic acid-d 5 and indolepronic acid-d 2 was 50. Mu.g/L, and were quantified according to the ratio of the standard to the internal standard.
5. Method recovery rate measurement: three gradient concentrations of 50. Mu.g/L, 500. Mu.g/L and 1000. Mu.g/L were added, 5 parallel samples were set for each addition level, treated and tested according to steps 1-3, quantified by means of standard curves drawn by internal standard methods, with recovery rates between 80% and 110% and RSD <10% (see Table 1).
6. Practical application: the method of the invention is used for detecting the contents of indoleacetic acid and indoleacetic acid in serum samples of 9 mice in a control group (common drinking water group) and a sodium indoleacetic acid salt solution feeding group (see figure 2). The instrument detection Limits (LOD) of both indoleacetic acid and indolopropionic acid were 0.04 μg/L (S/n=3), indicating that the method provided in this example can realize qualitative and quantitative detection of indoleacetic acid and indolopropionic acid in serum.
Example 2
Determination of indoleacetic acid and indolopropionic acid in mouse faeces
1. Homogenizing grinding and ultrasonic solvent extraction: accurately weighing 0.05g of a mouse faecal sample in a 2mL EP tube, adding an appropriate amount of internal standard (indoleacetic acid-d 5 and indolopropionic acid-d 2), adding 0.5mL of an extract (methanol: water: formic acid=15:4:1), adding grinding beads, grinding at 4 ℃, performing ultrasonic extraction for 15min, centrifuging at 4 ℃ and 10000rpm for 10min, taking supernatant, adding 0.5mL of an extract (methanol: water: formic acid=15:4:1), repeatedly extracting once, and combining the extracts.
2. Enrichment and purification of a solid phase extraction column: first, 4mL of methanol and 4mL of water are used for activating an Oasis HLB (3 cc/60 mg) solid phase extraction column, then 600 mu L of extract is accurately removed for loading, 4mL of water is added for leaching, 3mL of eluent (0.1% methanol formate) is used for eluting after the leaching is finished, and the eluent is collected.
3. Ultra-high performance liquid chromatography mass spectrometry detection: and (5) filtering the collected eluent with a 0.2 mu m filter membrane, and detecting the eluent on a machine.
Chromatographic conditions: chromatographic column:BEH C18, specification: 2.1X105 mm,1.7 μm; the sample injection amount is 2 mu L; the flow rate is 0.2mL/min; mobile phase a was 0.1% aqueous formic acid and B was acetonitrile, elution procedure was as follows: 0 to 0.5min,10 percent of B;0.5 to 2.0min,10 to 95 percent of B; 2-3 min,95% B;3 to 3.5min,95 to 10 percent of B;3.5 to 5 minutes, 10 percent of B; the mass spectrum detection conditions are as follows: electrospray ion source, positive ion scan (esi+), multiple Reaction Monitoring (MRM) mode, capillary voltage: 2.5kV; ion source temperature: 150 ℃; desolventizing gas temperature: 250 ℃; taper hole air flow: 150L/hf; desolventizing gas flow: 600L/hf; indoloacetic acid qualitative ion pair m/z 176.1/103, cone voltage 25V, collision energy 20eV, quantitative ion pair m/z 176.1/130, cone voltage 25V, collision energy 20eV; indoleacetic acid-d 5 qualitative ion pair m/z181.1/106, cone voltage 20V, collision energy 8eV, quantitative ion pair m/z 181.1/134, cone voltage 20V, collision energy 30eV; indolopropionic acid qualitative ion pair m/z190/171.9, taper hole voltage 30V, collision energy 19eV,quantitative ion pair m/z 190/130, cone voltage 30V, collision energy 10eV; indolopropionic acid-d 2 qualitative ion pair m/z 192/55.9, cone voltage 12V, collision energy 12eV, quantitative ion pair m/z192/130, cone voltage 12V, collision energy 16eV.
4. Drawing a standard curve: standard solutions of 1000. Mu.g/L of indoleacetic acid and indolepronic acid, and internal standard mixtures of 1000. Mu.g/L of indoleacetic acid-d 5 and indolepronic acid-d 2 were prepared with 0.1% methanol formate, and standard curves of 0.5. Mu.g/L, 1. Mu.g/L, 5. Mu.g/L, 10. Mu.g/L, 20. Mu.g/L, 50. Mu.g/L, 100. Mu.g/L, 200. Mu.g/L and 500. Mu.g/L were prepared, respectively, and the concentration of each of indoleacetic acid-d 5 and indolepronic acid-d 2 was 50. Mu.g/L, and were quantified according to the ratio of the standard to the internal standard.
5. Method recovery rate measurement: two gradient concentrations of 500 mug/L and 1000 mug/L were added, 5 parallel samples were set for each addition level, treated and tested according to steps 1-3, quantified by means of standard curves drawn by internal standard methods, recovery rates between 80% and 120% and RSD <10% (see table 1).
6. Practical application: the control group (normal feed without adding indoleacetic acid and indolopropionic acid), the indoleacetic acid feed group and the indolopropionic acid feed group were tested by the method of this example, the contents of indoleacetic acid and indolopropionic acid in the feces of mice fed with different diets in total 18 samples (the result is shown in fig. 3), and the instrument detection Limits (LOD) of indoleacetic acid and indolopropionic acid are all 0.04 μg/L (S/n=3), which indicates that the method provided by this example can realize qualitative and quantitative detection of indoleacetic acid and indolopropionic acid in the feces.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
TABLE 1 recovery and RSD values for different addition levels in mouse serum and feces
Claims (4)
1. The detection method of indoleacetic acid and indolopropionic acid in serum and feces comprises pretreatment and liquid chromatography mass spectrometry detection, and is characterized in that: the pretreatment steps comprise:
step one: methanol is prepared according to the proportion: water: the formic acid mixed solvent is used as an extracting solution, and the feces or serum sample is extracted to obtain an extracting solution;
step two: purifying and enriching the extract by using an activated Oasis HLB solid phase extraction column, and collecting and measuring the eluent;
the specific process of the first procedure is as follows: adding mixed internal standard solution of indoleacetic acid-d 5 and indolepropionic acid-d 2 into the fecal or serum sample, adding the extracting solution for ultrasonic extraction, centrifuging to obtain supernatant, adding the same amount of extracting solution, repeating the steps, and combining the supernatants to obtain the extracting solution;
methanol: water: the volume ratio of formic acid is (15-16): (4-5): (1-2);
when the fecal sample is extracted, grinding and homogenizing are carried out under the low-temperature airtight condition; grinding at 3-5deg.C;
when the feces is extracted, the addition ratio of the feces to the extract is 0.05g:1mL; when serum is extracted, the volume ratio of serum to extract is 1:9, a step of performing the process;
in the liquid chromatography mass spectrometry detection, the liquid phase detection conditions are as follows:
chromatographic column: ACQUITYBEH C18, mobile phase a is 0.1% formic acid in water, B is acetonitrile;
the elution procedure was: 0 to 0.5min,10 percent of B;0.5 to 2.0min,10 to 95 percent of B; 2-3 min,95% B;3 to 3.5min,95 to 10 percent of B;3.5 to 5 minutes, 10 percent of B;
the specification of the chromatographic column is 2.1X105 mm,1.7 μm; the sample injection amount is 1-3 mu L; the flow rate is 0.1-0.3mL/min;
the mass spectrum detection conditions are as follows: electrospray ion source, positive ion scanning ESI + Multi-reaction monitoring MRM mode, capillary voltage: 2.5kV; ion source temperature: 150 ℃;
desolventizing gas temperature: 250 ℃; taper hole air flow: 150L/hf; desolventizing gas flow: 600L/hf; indoloacetic acid qualitative ion pair m/z 176.1/103, cone voltage 25V, collision energy 20eV, quantitative ion pair m/z 176.1/130, cone voltage 25V, collision energy 20eV; indoleacetic acid-d 5 qualitative ion pair m/z181.1/106, cone voltage 20V, collision energy 8eV, quantitative ion pair m/z 181.1/134, cone voltage 20V, collision energy 30eV; indolopropionic acid qualitative ion pair m/z190/171.9, cone voltage 30V, collision energy 19eV, quantitative ion pair m/z 190/130, cone voltage 30V, collision energy 10eV; indolopropionic acid-d 2 qualitative ion pair m/z 192/55.9, cone voltage 12V, collision energy 12eV, quantitative ion pair m/z192/130, cone voltage 12V, collision energy 16eV.
2. The method of detection according to claim 1, wherein: methanol: water: the volume ratio of formic acid is 15:4:1.
3. the method of detection according to claim 1, wherein: the extraction process is carried out in an ultrasonic environment, and the extraction time is 15-20min.
4. The method of detection according to claim 1, wherein: in the second step, 500-700 mu L of extract is taken and purified and enriched by an activated Oasis HLB solid phase extraction column.
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