High performance liquid chromatography for simultaneously detecting four indole substances in rat plasma

A high-performance liquid chromatography and indole technology, which can be used in measurement devices, instruments, scientific instruments, etc., can solve the problem of not being widely popularized, and achieve the effects of small sample consumption, good reproducibility and high sensitivity

Pending Publication Date: 2022-02-18
重庆医科大学国际体外诊断研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the common detection method of indoles in rat plasma is ultra-high performance liquid chromatography-mass spectrometry, but this method is not yet widely used

Method used

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  • High performance liquid chromatography for simultaneously detecting four indole substances in rat plasma
  • High performance liquid chromatography for simultaneously detecting four indole substances in rat plasma
  • High performance liquid chromatography for simultaneously detecting four indole substances in rat plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Plasma sample pretreatment

[0036] Take 300 μL of EDTA anticoagulated plasma, add 10 μL of internal standard solution (methylnaphthol, 20.00 mg / L), add ether / ethyl acetate (3 / 5, v / v) mixed extractant 1100 μL, vortex and mix, Shake at 150 rpm for 10 min. Centrifuge at 13,300g for 15min, transfer the supernatant to another centrifuge tube, concentrate and dry by vacuum centrifugation. 200 μL of mobile phase was reconstituted, centrifuged at 12,200g for 10 min, and 20 μL of the supernatant was injected for analysis.

[0037] 2. Liquid chromatography conditions

[0038] Mobile phase: mobile phase A is 10mmol / LNaH 2 PO 4 solution;

[0039] The mobile phase B is methanol, and the isocratic elution method is adopted, and the proportion of mobile phase A is 40%.

[0040] Chromatographic column: Shim-Pack VP-ODS (150mm×4.6mm, 4.6μm).

[0041] Flow rate: 0.8mL / min.

[0042] Column temperature: 30°C.

[0043] Injection volume: 50 μL.

[0044] Fluorescence detector: P...

Embodiment 2

[0049] This example is to investigate the linearity and detection limit of the method for determining 3-INDS, IAA, IPA and IND in plasma. Prepare calibration samples by adding 30 μL of serial mixed standard working solution to 270 μL of mixed plasma respectively. After the sample is pretreated, the measurement is repeated three times. The concentration of the analyte added to the mixed plasma is the abscissa (x), and the ratio of the peak area of ​​the analyte to the internal standard minus the ratio of the peak area of ​​the mixed plasma analyte to the internal standard is the vertical axis. Coordinate (y), draw a calibration curve, calculate the detection limit with three times the signal-to-noise ratio (S / N=3), each analyte R 2 ≥0.9980, see Table 2 for the results.

[0050] Table 2 Standard curve and detection limit of plasma indoles

[0051]

Embodiment 3

[0053] This example is to investigate the precision of the method of the present invention for determining 3-INDS, IAA, IPA and IND in rat plasma. Real samples were used to investigate the precision, and the mixed plasma with high, medium and low concentrations of indole substances was selected. After sample pretreatment, the measurement was repeated 5 times a day, and the measurement was continued for 5 days. The ratio of naphthol was evaluated for precision, and the results are shown in Table 3. The relative standard deviations are all equal to or less than 2.5%, indicating that the present invention has good precision.

[0054] Table 3 Precision (n=5)

[0055]

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Abstract

The invention discloses a high performance liquid chromatography method for simultaneously detecting four indole substances in rat plasma, which belongs to the technical field of detection, and comprises the following steps: (1) preparing a standard substance solution; (2) carrying out liquid-liquid extraction treatment on the sample; and (3) carrying out qualitative and quantitative determination by adopting a high performance liquid chromatography. According to the method, indoxyl sulfate, indoleacetic acid, indolepropionic acid and indole in the rat plasma are fully extracted and separated, four indole substances with large content difference in the rat plasma can be quantitatively detected at the same time by adopting program detection wavelength, and the method has the advantages of high sensitivity, good reproducibility, short analysis time, small sample dosage and the like.

Description

technical field [0001] The invention belongs to the technical field of detection, and in particular relates to a high performance liquid chromatography method for simultaneously detecting four indole substances including indoxyl sulfate, indole acetic acid, indole propionic acid and indole in rat blood plasma. Background technique [0002] As an important "organ" of the body, the intestinal flora plays a vital role in the health of the host. It not only helps the host obtain nutrients and energy from food, but also produces many metabolites to participate in the metabolism of the host. Under normal circumstances, the intestinal flora maintains a dynamic balance with the host and the external environment. Once the intestinal flora is disturbed and the balance is out of balance, it will cause the loss of various functions of the host, such as the loss of barrier function, inflammation, and immune function. thereby inducing disease. [0003] Indole and its derivatives are meta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/74
CPCG01N30/06G01N30/74
Inventor 丁敏潘胜男张晓清马莉刘颖菊燕宇彤李彦茹
Owner 重庆医科大学国际体外诊断研究院
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