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Determination method of 3-alkylated adenine DNA adducts in urine

A technology for DNA adducts and alkylated adenine, which is used in measuring devices, instruments, scientific instruments, etc.

Active Publication Date: 2014-05-21
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method requires a complex and time-consuming pretreatment process of chemical derivatization of the sample, and the sensitivity of the GC-MS method is far from that of the liquid chromatography-tandem mass spectrometry method
At present, the high-throughput analysis method for extraction, purification and detection of three alkylated adducts (3-methyladenine, 3-ethyladenine, 3-hydroxyethyladenine) in urine matrix has not been reported yet

Method used

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  • Determination method of 3-alkylated adenine DNA adducts in urine
  • Determination method of 3-alkylated adenine DNA adducts in urine
  • Determination method of 3-alkylated adenine DNA adducts in urine

Examples

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example 1

[0054] 1. Instruments and reagents:

[0055] Agilent 1200 liquid chromatography (Agilent, USA), equipped with G1367D autosampler, G 1312B binary mixing pump and G1316B column thermostat; API 5500 triple quadrupole tandem mass spectrometer (Applied Biosystems, USA), equipped with Electrospray ion source (ESI) and Analyst 1.5 software data processing system.

[0056] Ammonia was analytically pure; ammonium formate, ethyl acetate, and methanol were all chromatographically pure (TEDIA, USA); Oasis MCX solid-phase extraction column (6 mL, Waters, USA).

[0057] 3-methyladenine, 3-ethyladenine, and 3-hydroxyethyladenine were purchased from Canada TRC Company, deuterated-3-methyladenine, deuterated-3-ethyladenine internal standard was purchased from American CIL laboratory.

[0058] 2. Sample handling:

[0059] The urine was thawed at room temperature and mixed evenly. Take 4 mL of urine, add 4 mL of ultrapure water, add 100 μL of internal standard (100 ng / mL), and adjust the tem...

example 2

[0061] Example 2: As described in Example 1, the method established in this study was used to detect the content of 3-alkylated adenine in the urine of 20 samples. The content of 3-alkylated adenine in the urine is shown in Table 6.

[0062] Table 6 Content of 3-alkylated adenine in urine

[0063] serial number 3-MeA content / 24h (μg) 3-EtA content / 24h (μg) 3-HOEtA content / 24h (μg) serial number 3-MeA content / 24h (μg) 3-EtA content / 24h (μg) 3-HOEtA content / 24h (μg) 1 5.72 108.4 228.8 11 25.64 955.4 120.7 2 13.16 417.4 364.2 12 16.48 142.9 295.9 3 21.75 313.0 345.6 13 25.88 781.6 106.2 4 10.12 237.3 211.5 14 28.45 939.3 332.9 5 6.73 546.7 726.1 15 26.87 399.5 323.2 6 27.73 277.3 144.7 16 28.45 636.0 153.3 7 63.14 216.6 696.8 17 24.58 494.8 154.0 8 12.87 245.3 281.7 18 69.58 572.5 747.0 9 77.35 246.7 172.6 19 29.38 220.7 185.7 10 45.06 301.8 282.3 20 1...

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Abstract

The invention discloses a determination method of 3-alkylated adenine DNA adducts in urine, namely the determination method of 3-methyl adenine (3-MeA), 3-ethyl adenine (3-EtA) and 3-hydroxyethyl adenine (3-HOEtA). The determination method is characterized in that the test process comprises the following steps: unfreezing urine within 24 hours at room temperature, uniformly mixing and centrifugally filtering, purifying and enriching through an OasisMCX solid phase extraction small column, mixing to be uniform and then introducing into an LC-MS / MS system to analyze so as to accurately detect the content level of 3-MeA, 3-EtA and 3-HOEtA in the urine. The invention relates to the determination method of the 3-alkylated adenine DNA adducts in fresh urine, a deuterated standard product is used as an interior standard quantitive analyte so as to reduce the error caused in the pre-treatment process of the sample, and a tandem mass spectrometry well improves the selectivity, accuracy and sensitivity of the method. The chromatographic separation process is better improved through the selection and optimization of a chromatographic column and an elution condition, the chromatographic analysis time is shortened, and the consumption of an inorganic solvent is reduced.

Description

[0001] [0002] Technical field: [0003] The invention belongs to the technical field of physical and chemical testing of urine samples, and mainly relates to 3-alkylated adenine (3-methyladenine 3-MeA; 3-ethyladenine 3-EtA; 3-hydroxyethyl adenine) in urine The technical field of determination of purine 3-HOEtA) DNA adducts, specifically a liquid chromatography-electrospray ionization-tandem mass spectrometer (LC-ESI-MS / MS) for the determination of 3-alkylated adenine in urine (3-methyladenine 3-MeA; 3-ethyladenine 3-EtA; 3-hydroxyethyladenine 3-HOEtA) method. [0004] [0005] Background technique: [0006] 3-Alkylated adenine is the product of the combination of free radicals generated by the metabolism of exogenous compounds in vivo and adenine. Because the background value of endogenous 3-alkylated adenine in urine is low and can more accurately reflect the characteristics of the prototype compound, it is widely used as a biological monitoring indicator for the biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/02
Inventor 田永峰杨进侯宏卫胡清源王安刘勇陈欢刘彤韩书磊吴帅宾
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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