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Analysis method of 8-hydroxy deoxyguanosine, 8-hydroxy guanosine and 8-iso-prostaglandin F2alpha in urine of human body

A technology of hydroxydeoxyguanosine and hydroxyguanosine is applied in the field of evaluation of oxidative stress indicators in human urine, which can solve the problems of not very sensitive detection, unsuitable separation, quantitative interference, etc., and achieve simple and convenient sample analysis and detection. Low limit, high sensitivity effect

Active Publication Date: 2015-03-04
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

When HPLC-MS / MS is analyzed, the sample can be pre-treated and purified by direct dilution or solid-phase extraction, but due to the high polarity of these compounds, they are basically not retained on commonly used reversed-phase chromatographic columns, resulting in these compounds and Other interferences are not suitable for separation, causing quantitative interference, and the electrospray ion source (ESI source) is not very sensitive to the detection of polar compounds separated by reversed-phase columns, and the sensitivity of actual sample compounds may be lower than the detection limit; 8-isomer Prostaglandin F2α can be analyzed by ELISA, GC-MS and HPLC / MS / MS

Method used

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  • Analysis method of 8-hydroxy deoxyguanosine, 8-hydroxy guanosine and 8-iso-prostaglandin F2alpha in urine of human body
  • Analysis method of 8-hydroxy deoxyguanosine, 8-hydroxy guanosine and 8-iso-prostaglandin F2alpha in urine of human body
  • Analysis method of 8-hydroxy deoxyguanosine, 8-hydroxy guanosine and 8-iso-prostaglandin F2alpha in urine of human body

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example 1

[0019] 8-Hydroxydeoxyguanosine, 8-Hydroxydeoxyguanosine, and 8-Isomerized Prostaglandin F2α were prepared into a mixed series of standard working solutions with different concentrations, and the concentration gradient of 8-Hydroxydeoxyguanosine was 0.5, 1, 2, 4, 10, 25ng / ml, the concentration gradient of 8-hydroxyguanosine is 1, 2, 4, 8, 20, 50ng / ml, the concentration gradient of 8-isomeric prostaglandin F2α is 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 ng / ml. The internal standards of the two targets of 8-hydroxydeoxyguanosine and 8-hydroxyguanosine are [ C 13 N 15 ]-8-hydroxyguanosine, the internal standard of 8-isomeric prostaglandin is D4-8-isomeric prostaglandin F2α, and the final concentration of both internal standards is 10ng / ml 。 Determination was carried out by HPLC-MS / MS. Perform linear regression analysis on the ratio of the chromatographic peak area of ​​the target substance to the peak area of ​​the internal standard to its concentration ratio, and obtain the standard cur...

example 2

[0023] For the same urine sample, the sample was purified according to the steps (1) and (2) of the summary of the invention above, and HPLC-MS / MS was used to perform 6 parallel intra-day and inter-day measurements. The results showed that the intra-day and inter-day relative standard deviations of each compound were between 1.7-5.3%, indicating that the method had good reproducibility and stability (results are shown in Table 3 and Table 4).

[0024] Table 3 Intra-day repeatability determination results of the method (ng / ml)

[0025]

[0026] Table 4 Day-to-day repeatability determination results of the method (ng / ml)

[0027]

example 3

[0029]Take the same urine sample, add standard solutions of 8-hydroxydeoxyguanosine, 8-hydroxyguanosine, and 8-isomeric prostaglandin F2α, and prepare three levels of spiked samples at high, medium, and low levels, according to the content of the previous invention Steps (1) (2) of the sample were purified and analyzed by HPLC-MS / MS, and the recovery rate was calculated from the spiked amount and the measured amount, and 3 parallels were made for each concentration level (results are shown in Table 5 ). The results showed that the recoveries of the three levels ranged from 87.2%–108%.

[0030] Table 5 method recoveries

[0031]

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Abstract

The invention relates to an analysis method of 8-hydroxy deoxyguanosine, 8-hydroxy guanosine and 8-iso-prostaglandin F2alpha in urine of a human body. The analysis method has the characteristics that the method can detect a change in in vivo typical oxidative stress biological marker caused by active oxygen relatively conveniently, rapidly and quantitatively, and can be used for evaluating in vivo oxidative damage and restoring degrees and studying a pathological process of a disease. The method employs AgilentBondEluC18 solid phase extraction column, three compounds of two different mass spectrometric detection modes, i.e., 8-hydroxy deoxyguanosine, 8-hydroxy guanosine (cation mode detection) and 8-iso-prostaglandin F2alpha (anion mode detection), in the urine can be purified simultaneously, and are subjected to liquid chromatography tandem mass spectrometry detection by the same liquid chromatography column and the same mobile phase. The method has simple, convenient and quick pretreatment, and is high in sensitivity, good in repeatability and high in recovery rate.

Description

technical field [0001] The invention relates to a new method for pretreatment and analysis of 8-hydroxydeoxyguanosine, 8-hydroxyguanosine and 8-isomeric prostaglandin F2α in human urine, and it is used for oxidative stress indicators in human urine evaluation of. Specifically, Agilent Bond EluC18 solid-phase extraction column was used to simultaneously purify 8-hydroxydeoxyguanosine, 8-hydroxyguanosine and 8-isomeric prostaglandin F2α in urine, and then HPLC-MS / MS technology was used to determine 8- Analytical methods for the content of hydroxydeoxyguanosine, 8-hydroxyguanosine and 8-isomeric prostaglandin F2α. Background technique [0002] Reactive oxygen species (ROS) are chemically active oxygen free radicals produced in the process of cellular aerobic metabolism and substances that can be converted into free radicals. ROS in organisms is an indispensable substance for the body, and its production and elimination are in a state of dynamic balance. At low and medium con...

Claims

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Application Information

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IPC IPC(8): G01N30/88
Inventor 赵阁余晶晶王昇王冰谢复炜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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