CN114216989A - Kit for detecting plasma indole substances by high performance liquid chromatography and application thereof - Google Patents

Abstract

The invention belongs to the technical field of detection, and particularly relates to a kit for detecting 5 kinds of indole substances in blood plasma by high performance liquid chromatography, wherein the 5 kinds of indole substances are respectively as follows: indoxyl sulfate (3-INDS), Indole-3-acetic acid (IAA), Indole-3-propionic acid (IPA), Indole (INDOL, IND), and 3-Methylindole (3-Methylindole, 3-MI). The kit comprises the following reagents: mobile phase, standard substance stock solution, internal standard solution and extraction liquid. The kit can simultaneously detect 5 indole substances in blood plasma, has high detection sensitivity and simple pretreatment process, and is a blood plasma indole substance detection kit with simple reagent, low price and convenient use.

Description

Kit for detecting plasma indole substances by high performance liquid chromatography and application thereof

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a kit for detecting indole substances in blood plasma by using a high performance liquid chromatography and application thereof.

Background

The intestinal flora is the microbial flora inhabiting in human intestinal tracts and plays an important role in the health of a host. Under normal conditions, the intestinal flora keeps dynamic balance with the host and the external environment, and once the intestinal flora is disordered and the balance is unbalanced, the multiple functions of the host, such as barrier function, inflammation, immune function loss and the like, can be caused, so that diseases are induced.

The intestinal flora is mainly closely interacted with a host through small molecular metabolites, and the occurrence and development of diseases are influenced. Indole and its derivatives are metabolites of tryptophan under the action of intestinal flora, and can interact with arene receptors to influence host immunity, metabolism, behavior, etc. Researches show that the indole substances are closely related to bacterial infection, intestinal inflammation, cancer, diabetes, nervous system diseases and the like. Therefore, the detection of indole substances in blood plasma has important significance for disease screening and pathological mechanism research.

At present, a common detection method for plasma indole substances is an ultra-high performance liquid chromatography-mass spectrometry method, but the method cannot be widely popularized. The relatively more popular high performance liquid chromatography only simultaneously detects two indole substances of indole and trimethylindole, the detection range is limited, and the detection efficiency is low. Therefore, the development of a simple method for simultaneously detecting multiple indole substances in blood plasma by high performance liquid chromatography has important value for clinical research.

Disclosure of Invention

The invention aims to provide a kit for simultaneously determining 5 indole substances in blood plasma by using a high performance liquid chromatography and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a kit for detecting 5 Indole substances in blood plasma by high performance liquid chromatography, wherein the 5 Indole substances are Indoxyl sulfate (3-INDS), indoleacetic acid (IAA), indolepropionic acid (IPA), Indole (INDOLE, IND) and 3-Methylindole (3-Methylindole, 3-MI);

the kit comprises the following reagents:

(1) mobile phase:

mobile phase A: 10mmol/LNaH₂PO₄A solution;

mobile phase B: methanol;

(2) standard stock solutions:

a methanol solution containing IAA, IPA, IND, 3-MI and a methanol aqueous solution containing 3-INDS;

(3) internal standard solution: methanolic methyl naphthol solution;

(4) extracting liquid: diethyl ether/ethyl acetate.

Wherein the standard stock solution is a methanol solution containing 100.00 mu mol/L IAA, 60.00 mu mol/L IPA, 4.00 mu mol/L IND, 4.00 mu mol/L3-MI and a methanol aqueous solution containing 4.00 mmol/L3-INDS standard.

Wherein the internal standard solution is 10.00mg/L methyl naphthol methanol solution.

Wherein the volume ratio of the diethyl ether to the ethyl acetate in the extractant is 2: 1.

The preparation method of the kit comprises the following steps:

(1) mobile phase:

mobile phase A: 10mmol/LNaH₂PO₄A solution;

mobile phase B: methanol;

(2) standard stock solutions: 100.00. mu. mol/L IAA, 60.00. mu. mol/L IPA, 4.00. mu. mol/L IND and 4.00. mu. mol/L3-MI standard stock solutions were prepared with methanol, and 4.00 mmol/L3-INDS standard stock solution was prepared with 50% methanol.

(3) Internal standard solution: preparing 10.00mg/L methyl naphthol by using methanol;

(4) extracting liquid: the diethyl ether and ethyl acetate were mixed at a volume of 2:1 and vortexed.

The kit is applied to simultaneously detecting 5 indole substances in blood plasma, and the application method of the kit in simultaneously detecting 5 indole substances in blood plasma by using high performance liquid chromatography is also within the protection scope of the invention.

The method for simultaneously detecting 5 indole substances in blood plasma by high performance liquid chromatography comprises the following steps:

(1) sample pretreatment: taking 300 mu L of EDTA anticoagulated plasma, adding 10 mu L of internal standard solution, adding 810 mu L of diethyl ether/ethyl acetate mixed extractant, uniformly mixing by vortex, and shaking at the speed of 150rpm for 15min at 25 ℃; centrifuging at 13,300g for 10min, collecting supernatant, vacuum centrifuging, concentrating, drying, and re-dissolving in 90 μ L mobile phase;

(2) chromatographic conditions are as follows:

a chromatographic column: Shim-PackVP-ODS (150 mm. times.4.6 mm, 4.6 μm);

mobile phase A: 10mmol/LNaH₂PO₄A solution; mobile phase B: methanol; isocratic elution, wherein the proportion of the mobile phase A is 40%;

flow rate: 0.8 mL/min;

column temperature: 35 ℃;

a fluorescence detector: the excitation wavelength is 280nm, and the emission wavelength is 355 nm;

sample introduction amount: 50 μ L.

Has the advantages that: the kit is applied to high performance liquid chromatography for detecting indole substances in blood plasma, and can simultaneously detect 5 indole substances in blood plasma; the plasma indole substance detection kit has the advantages of simple sample pretreatment process, low cost and high sensitivity, and is simple in reagent, low in price and convenient to use.

Drawings

FIG. 1 is a high performance liquid chromatogram of a plasma sample.

Detailed Description

In order to further understand the present invention, the present invention will be further described with reference to the following examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as further limiting the present invention.

The following examples used the following instruments and material sources:

(1) the instrument comprises the following steps: agilent model 1100 high performance liquid chromatograph (Agilent corporation); a Millipore ultra-pure water filtration device and a Millipore vacuum pump (Millipore, USA); a Fresco 17 low temperature bench top centrifuge (Thermo corporation); ZWY-100H constant temperature culture shaker (Shanghai Zhicheng analytical instruments manufacturing Co., Ltd.); VORTEX-QILNBEIER VORTEX shaker (Scientific Instrument Co.); savant DNA120 vacuum centrifugal concentrator (Thermo Co.); electronic analytical balance (Shanghai Jingtian electronics Co., Ltd.).

(2) Reagent consumables: methanol (chromatographic grade, Tedia corporation); acetonitrile (chromatographic grade, Tedia corporation); Shim-PackVP-ODS column (Shimadzu Corp.).

Example 1

And (3) preparing a plasma indole substance detection kit.

The components in the detection kit are shown in Table 1

TABLE 1 kit Components

The preparation method of the kit specifically comprises the following steps:

(1) mobile phase:

mobile phase A: 10mmol/LNaH₂PO₄A solution;

mobile phase B: methanol;

(2) standard stock solutions: 100.00. mu. mol/L IAA, 60.00. mu. mol/L IPA, 4.00. mu. mol/L IND and 4.00. mu. mol/L3-MI standard stock solutions were prepared with methanol, and 4.00 mmol/L3-INDS standard stock solution was prepared with 50% methanol.

(3) Internal standard solution: preparing 10.00mg/L methyl naphthol by using methanol;

(4) extracting liquid: the diethyl ether and ethyl acetate were mixed at a volume of 2:1 and vortexed.

Example 2

Use of the kit for detecting the plasma indole substances.

1. Plasma sample pretreatment

mu.L of EDTA anticoagulant plasma is taken, 10 mu.L of internal standard solution (methyl naphthol, 10.00mg/L) is added, then, 810 mu.L of diethyl ether/ethyl acetate mixed extractant is added into a sample, and after uniform mixing, the sample is shaken for 15min at the speed of 150rpm at 25 ℃. After centrifugation at 13,300g for 10min, the supernatant was transferred to another centrifuge tube, concentrated to dryness by vacuum centrifugation and redissolved in 90. mu.L of mobile phase. The reconstituted solution was centrifuged at 13,300g for 10min, and 50. mu.L of the supernatant was sampled for analysis.

2. Conditions of liquid chromatography

Mobile phase: the mobile phase A is 10mmol/LNaH₂PO₄Solution, mobile phase B is AAlcohol, adopting isocratic elution method, and the proportion of the mobile phase A is 40%.

A chromatographic column: Shim-PackVP-ODS (150 mm. times.4.6 mm, 4.6 μm).

Flow rate: 0.8 mL/min.

Column temperature: 35 ℃ is carried out.

A fluorescence detector: the excitation wavelength was 280nm and the emission wavelength was 355 nm.

Sample introduction amount: 50 μ L.

Example 3

This example examines the linearity and detection limits of the kits for determining 3-INDS, IAA, IPA, IND and 3-MI in plasma. A30 μ L series of mixed standard working solutions was added to 270 μ L of blank plasma to prepare calibration samples. The sample is subjected to pretreatment and then repeatedly measured for three times, the concentration of the plasma added with the substance to be measured is taken as an abscissa (x), the ratio of the peak area of the plasma substance to be measured to the peak area of the internal standard minus the ratio of the peak area of the blank plasma substance to be measured to the peak area of the internal standard is taken as an ordinate (y), a calibration curve is drawn, the detection limit is calculated by the triple signal-to-noise ratio (S/N is 3), and each analyte R²>0.9990, and the results are shown in Table 2.

TABLE 2 Standard Curve and detection Limit for plasma indoles

Example 4

This example is a study of the precision of the kits of the invention for determining 3-INDS, IAA, IPA, IND and 3-MI in plasma. And (3) adopting a real sample to carry out precision investigation, selecting mixed plasma of the indole substances with high, medium and low concentrations, carrying out sample pretreatment, repeatedly measuring for 5 times in a day, continuously measuring for 5 days, calculating the ratio of the substance to be measured to the alpha naphthol, and carrying out precision evaluation, wherein the result is shown in table 3. The coefficient of variation is equal to or less than 5.2%, which shows that the invention has good precision.

TABLE 3 precision (n ═ 5)

Example 5

This example is a study of the kits of the present invention for determining the recovery of 3-INDS, IAA, IPA, IND and 3-MI in plasma samples. The plasma matrix is added with mixed standard indole substances with high, medium and low concentrations respectively, and after sample pretreatment, each concentration is measured in parallel for 3 times to carry out a recovery rate test. The relative recovery rate (measured concentration of indole in plasma matrix after adding standard-measured concentration of indole in plasma matrix before adding standard)/concentration of indole standard substance added × 100%, the results are shown in table 4. The average recovery rate is between 90.1% and 109.3%, which shows that the method has good accuracy.

TABLE 4 recovery test

Although the invention has been described in detail above with reference to specific embodiments and illustrative embodiments, it will be apparent that modifications and improvements can be made to the invention without departing from the scope of the invention. As will be apparent to those skilled in the art. Accordingly, it is intended that all such modifications and variations be included within the scope of the invention as claimed and not departing from the spirit thereof.

Claims (7)

1. The kit for detecting the indole substances in the blood plasma by the high performance liquid chromatography is characterized in that: the kit comprises the following reagents:

(1) mobile phase:

mobile phase A: 10mmol/LNaH₂PO₄A solution;

mobile phase B: methanol;

(2) standard stock solutions:

a methanol solution containing Indole Acetic Acid (IAA), Indole Propionic Acid (IPA), Indole (IND ) and 3-Methylindole (3-Methylindole, 3-MI), and a methanol aqueous solution containing Indoxyl sulfate (3-INDS);

(3) internal standard solution: methanolic methyl naphthol solution;

(4) extracting liquid: diethyl ether/ethyl acetate.

2. The kit for detecting the indole substances in the blood plasma by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the stock solution of the standard substance was a methanol aqueous solution containing 100.00. mu. mol/L IAA, 60.00. mu. mol/L IPA, 4.00. mu. mol/L IND, 4.00. mu. mol/L3-MI methanol solution, and 4.00 mmol/L3-INDS standard substance.

3. The kit for detecting the indole substances in the blood plasma by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the internal standard solution is 10.00mg/L methyl naphthol methanol solution.

4. The kit for detecting the indole substances in the blood plasma by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the volume ratio of the ethyl ether to the ethyl acetate in the extractant is 2: 1.

5. The method for preparing a kit according to any one of claims 1 to 4, wherein:

(1) mobile phase:

mobile phase A: 10mmol/LNaH₂PO₄A solution;

mobile phase B: methanol;

(2) standard stock solutions: 100.00. mu. mol/L IAA, 60.00. mu. mol/L IPA, 4.00. mu. mol/L IND and 4.00. mu. mol/L3-MI standard stock solutions were prepared with methanol, and 4.00 mmol/L3-INDS standard stock solution was prepared with 50% methanol.

(3) Internal standard solution: preparing 10.00mg/L methyl naphthol by using methanol;

(4) extracting liquid: the diethyl ether and ethyl acetate were mixed at a volume of 2:1 and vortexed.

6. Use of a kit according to any one of claims 1 to 4, wherein: and (3) detecting 5 indole substances in the pretreated blood plasma by adopting a high performance liquid chromatography technology.

7. Use of a kit according to claim 6, wherein:

(1) sample pretreatment: taking 300 mu L of EDTA anticoagulated plasma, adding 10 mu L of internal standard solution, adding 810 mu L of diethyl ether/ethyl acetate mixed extractant, uniformly mixing by vortex, and shaking at the speed of 150rpm for 15min at 25 ℃; centrifuging at 13,300g for 10min, collecting supernatant, vacuum centrifuging, concentrating, drying, and re-dissolving in 90 μ L mobile phase;

(2) chromatographic conditions are as follows:

a chromatographic column: Shim-PackVP-ODS (150 mm. times.4.6 mm, 4.6 μm);

mobile phase A: 10mmol/LNaH₂PO₄A solution; mobile phase B: methanol; isocratic elution, wherein the proportion of the mobile phase A is 40%;

flow rate: 0.8 mL/min;

column temperature: 35 ℃;

a fluorescence detector: the excitation wavelength is 280nm, and the emission wavelength is 355 nm;

sample introduction amount: 50 μ L.

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