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Pretreatment and detection method of biogenic amine neurotransmitter, and detection kit

A technology for neurotransmitters and biogenic amines, applied in the field of detection, can solve the problems of complicated steps, interference of mass spectrometry signals, and reduced recovery rate, and achieve the effects of avoiding instability, high sensitivity, and rapid determination

Active Publication Date: 2017-08-15
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of derivatization treatment is the introduction of high concentrations of strong acid or strong alkali salts. These salts will not only interfere with the mass spectrometry signal, but also affect the life of the chromatographic column and even the liquid-mass system. Therefore, it must be removed as much as possible before detection. These salts
[0007] The existing liquid-liquid extraction and off-line solid-phase extraction desalination pre-treatment technologies all need to be re-concentrated after extraction, which makes the pre-treatment steps complicated; moreover, the stability of biogenic amine neurotransmitters and their metabolites Poor stability, concentration and other methods will cause the degradation of biogenic amine neurotransmitters and reduce the recovery rate

Method used

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  • Pretreatment and detection method of biogenic amine neurotransmitter, and detection kit
  • Pretreatment and detection method of biogenic amine neurotransmitter, and detection kit
  • Pretreatment and detection method of biogenic amine neurotransmitter, and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Determination of biogenic amine neurotransmitters and their metabolites (1)

[0099] 1. Reagents and materials used:

[0100] Rat blank brain tissue homogenate: the rat was decapitated, the skull was carefully cut and the brain tissue was taken out. Accurately weigh rat blank brain tissue, add 9 times the volume of 10 mM ascorbic acid aqueous solution, and use a high-speed homogenizer to homogenize to obtain a 10% rat blank brain tissue homogenate.

[0101] Internal standard solution: acetaminophen solution diluted in acetonitrile.

[0102] Neurotransmitter standard solution: prepared by adding 10 mM ascorbic acid aqueous solution to the biogenic amine neurotransmitter standard. Wherein, biogenic amine neurotransmitter standard products were purchased from Sigma Company.

[0103] Sodium carbonate-sodium hydroxide buffered saline, and 1 mg / mL p-dimethylaminobenzenesulfonyl chloride in acetonitrile.

[0104] Wherein, p-dimethylaminobenzenesulfonyl chlorid...

Embodiment 2

[0137] Example 2: Stability study of biogenic amine neurotransmitters and their derivatized products

[0138] 1. Experimental method:

[0139] Investigate the stability of various biogenic amine neurotransmitters and their metabolites in different pH media and other conditions, and the stability of biogenic amine neurotransmitter derivative products.

[0140] Accurately weigh biogenic amine neurotransmitters or their metabolites, and prepare a 1 mg / mL solution with 10 mM ascorbic acid aqueous solution. Dilute with 1% formic acid (pH=1), 10mM ammonium acetate (pH=5) and artificial cerebrospinal fluid (pH=7.4) respectively, so that the concentrations are 10ng / mL, 100ng / mL and 1000ng / mL or 40ng / mL, 400ng / mL and 4000ng / mL. The sample was placed at 37° C., 50 rpm, and heated for 0, 2, 4, 8, 16 and 24 hours respectively to terminate the heating. Take each sample and derivatize biogenic amine neurotransmitters or their metabolites according to the method in Example 1, place the ...

Embodiment 3

[0148] Example 3: Determination of biogenic amine neurotransmitters and their metabolites (2)

[0149] 1. Determination method:

[0150] Referring to the assay method of Example 1.

[0151] The difference is that the derivatization reagent solution uses benzenesulfonyl chloride in acetonitrile solution with a concentration of 1 mg / mL. The ion pairs and mass spectrometry conditions corresponding to the quantitative analysis of biogenic amine neurotransmitters and their metabolites are shown in Table 6.

[0152] Table 6

[0153]

[0154]

[0155] 2. Measurement results:

[0156] 2.1 Draw a standard curve:

[0157] Accurately weigh the solid powders of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, disodium hydrogen phosphate and 4-hydroxyethylpiperazineethanesulfonic acid (HEPEs) into 1L of double-distilled water, shake and dissolve, so that they contain 147mmol / L sodium chloride, 3mmol / L potassium chloride, 1.2mmol / L calcium chloride, 1....

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Abstract

The invention belongs to the technical field of detection, and relates to a pretreatment method for biogenic amine neurotransmitters and / or metabolites thereof, comprising derivatizing samples containing biogenic amine neurotransmitters and / or metabolites thereof, for On-line solid-phase extraction of derivatized products. On this basis, it also relates to a detection method, detection kit and use of biogenic amine neurotransmitters and / or metabolites thereof. The pretreatment method of the present invention can remove impurities in the derivatized product, enrich the derivatized product and transfer it to the subsequent detection step at an appropriate concentration. The detection method of the invention can quickly and accurately measure the contents of biogenic amine neurotransmitters and metabolites in biological samples, and has high detection sensitivity.

Description

technical field [0001] The invention belongs to the technical field of detection, and relates to a pretreatment method, detection method, detection kit and application of biogenic amine neurotransmitters and metabolites. Background technique [0002] Biogenic amine neurotransmitters mainly include dopamine (DA), norepinephrine (NE), epinephrine (E), 5-hydroxytryptamine (5-HT), etc., which are ubiquitous and very important neurons in the human brain. It plays a broad regulatory role in human nervous, cardiovascular, endocrine and other tissue systems, and has an important impact on physiological activities such as mood, emotion, stress behavior, sleep and awakening. [0003] The content changes of biogenic amine neurotransmitters and metabolites are closely related to many diseases, and are an important basis for the diagnosis of pheochromocytoma, Alzheimer's disease, Down's syndrome, depression and Parkinson's disease. At the same time, biogenic amine neurotransmitters can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C309/88
Inventor 李迎梅兴国张有志
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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