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Method for determining polyamines in urine

A determination method and urine technology, applied in the field of medicine, can solve the problems of low body content and incomplete establishment of a polyamine detection method, and achieve the effects of good repeatability, reduction of side reactions, and easy operation.

Inactive Publication Date: 2012-06-13
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since polyamines are small molecular compounds with the characteristics of simple structure and low content in the body, rapid, sensitive, accurate and economical determination methods need to be established for clinical detection. Establish

Method used

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  • Method for determining polyamines in urine
  • Method for determining polyamines in urine
  • Method for determining polyamines in urine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Urine sample pretreatment

[0037] a. Take 500 μL of urine sample and add 5% HClO 4 250 μL, vortex to mix, and centrifuge.

[0038] b. Take the supernatant after treatment in step a, add 2 mol mL -1 NaOH 800 μL, 250 μL benzoyl chloride mass concentration of 4% mixed solution of benzoyl chloride and acetonitrile, vortex to dissolve and mix well, 30 o C. Ultrasonic reaction for 30 min, adding 1 mL of saturated sodium chloride solution to terminate the reaction, extracting the derivatized polyamine with 4 mL of ether, drying under air flow, reconstitution with 100 μL of methanol, centrifuging at 15000 r.m.p for 3 min, and taking 20 μL The supernatant was analyzed by LC-MS.

[0039] (2) Liquid phase separation: the chromatographic column is Kromasil C 18 Column (250 mm×4.6 mm, 5 μm); mobile phase A: methanol, B: water; gradient elution: 0-14 min: 55 % A→74 % A. Flow rate is 1.0 mL ? min -1 , the column temperature is 35 o c.

[0040] (3) MS measurement

[0...

Embodiment 2

[0046] (1) Urine sample pretreatment

[0047] a. Take 500 μL of urine sample and add 9% HClO 4 250 μL, vortex, and centrifuge.

[0048] b. Take the supernatant after treatment in step a, add 0.5 mol mL -1 NaOH 800 μL, 250 μL benzoyl chloride mass concentration of 4% mixed solution of benzoyl chloride and acetonitrile, vortex to dissolve and mix well, 30 o C. Ultrasonic reaction for 30 min, adding 1 mL of saturated sodium chloride solution to terminate the reaction, extracting the derivatized polyamine with 4 mL of ether, drying under air flow, reconstitution with 100 μL of methanol, centrifuging at 15000 r.m.p for 3 min, and taking 20 μL The supernatant was analyzed by LC-MS.

[0049] (2) Liquid phase separation: the chromatographic column is Kromasil C 18 Column (250 mm×4.6 mm, 5 μm); mobile phase A: methanol, B: water; gradient elution: 0-14 min: 55 % A→74 % A. Flow rate is 1.0 mL ? min -1 , the column temperature is 35 o C, inject 20 μL.

[0050] (3) MS measurem...

Embodiment 3

[0056] (1) Urine sample pretreatment

[0057] a. Take 500 μL of urine sample and add 5% HClO 4 250 μL, vortex to mix, and centrifuge.

[0058] b. Take the supernatant after treatment in step a, add 2 mol mL -1 NaOH 800 μL, 250 μL benzoyl chloride mass concentration of 1% mixed solution of benzoyl chloride and acetonitrile, vortex to dissolve and mix well, 30 o C. Ultrasonic reaction for 30 min, adding 1 mL of saturated sodium chloride solution to terminate the reaction, extracting the derivatized polyamine with 4 mL of ether, drying under air flow, reconstitution with 100 μL of methanol, centrifuging at 15000 r.m.p for 3 min, and taking 20 μL The supernatant was analyzed by LC-MS.

[0059] (2) Liquid phase separation: the chromatographic column is Kromasil C 18 Column (250 mm×4.6 mm, 5 μm); mobile phase A: methanol, B: water; gradient elution: 0-14 min: 55 % A→74 % A. Flow rate is 1.0 mL ? min -1 , the column temperature is 35 o C, inject 20 μL.

[0060] (3) MS mea...

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Abstract

The invention relates to a method for determining polyamines in biological urine sample. Polyamine is an endogenous substance with important physiological effect and plays a significant role in cell proliferation and synthesis of protein and nucleic acid, etc.; therefore, generation of polyamine is closely related to cancer, and polyamine can be applied to diagnosis and treatment of cancer as a tag. Due to difficulty in detecting properties of polyamine by a routine method, the invention provides a sensitive and stable method for determining polyamines in urine of human and provides basis for clinic diagnosis of cancer and research and development of anti-cancer drug.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for determining polyamine substances in urine, more precisely, a method for efficiently and sensitively quantifying polyamine substances in urine. Background technique [0002] Polyamines are a class of small molecule aliphatic amine compounds with two or more primary and secondary amino groups, usually referring to 1,3-propanediamine, putrescine, cadaverine, spermidine, spermine and Its acylated products, etc. Polyamines exist widely in organisms, participate in the regulation of gene expression and translation, cell proliferation and other life activities, and are closely related to the occurrence and development of cancer. [0003] At present, the quantitative determination of endogenous polyamines in vivo mainly includes liquid chromatography-fluorescence derivatization method, liquid chromatography-ultraviolet derivatization method, immunoassay and liq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 毕开顺李清刘然贾英尹然
Owner SHENYANG PHARMA UNIVERSITY