Teixeira A J, Ferreira M R, van Dijk W J, van de Werken G, de Jong A P
Laboratory of Organic-Analytical Chemistry, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Anal Biochem. 1995 Apr 10;226(2):307-19. doi: 10.1006/abio.1995.1230.
A previously developed gas chromatographic/mass spectrometric method was applied to the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat liver DNA and in rat urine. For DNA samples, the method included: (i) fortification of samples with [15N]DNA (internal standard), (ii) enzymatic hydrolysis of DNA to deoxynucleosides, (iii) degradation of native nucleosides by treatment with trifluoroacetic acid and hydrazine, (iv) purification by C18 solid-phase extraction (SPE), (v) derivatization (acetylation followed by pentafluorobenzylation, Ac-PFB), and (vi) GC/MS analysis of the derivatives. For urine, the following methodology was used: (i) fortification of the samples with 8-18OHdG, (ii) prepurification by C18/OH SPE, (iii) derivatization, (iv) high-performance liquid chromatography purification of the Ac-PFB derivatives, and (v) GC/MS analysis. The precision of the method was demonstrated by carrying out replicate analysis of several urine and DNA samples: within-run and between-run variability was less than 5 and 8%, respectively. The analytical approaches were sufficiently sensitive to quantitate the urinary excretion of 8-OHdG (490 +/- 70 pmol/kg/24 h; sample size, 600 microliters urine) and to measure the level of 8-OHdG in liver DNA (20 8-OHdG/10(6) deoxynucleosides; sample size, 30 micrograms DNA) of rats not deliberately exposed to oxidative stress. Major advantages over previous methods are increased precision due to the use of proper isotopically labeled internal standards, and increased sensitivity due to the optimization of cleanup procedures. The simultaneous analysis of standards of three different oxidized nucleosides, namely 8-OHdG, thymidine glycol, and 5-hydroxy-methyl-2'-deoxyuridine, is shown.
一种先前开发的气相色谱/质谱法被用于测定大鼠肝脏DNA和大鼠尿液中的8-羟基-2'-脱氧鸟苷（8-OHdG）。对于DNA样本，该方法包括：（i）用[15N]DNA（内标）对样本进行加标；（ii）将DNA酶解为脱氧核苷；（iii）用三氟乙酸和肼处理降解天然核苷；（iv）通过C18固相萃取（SPE）进行纯化；（v）衍生化（乙酰化后进行五氟苄基化，Ac-PFB）；以及（vi）对衍生物进行气相色谱/质谱分析。对于尿液，使用了以下方法：（i）用8-18OHdG对样本进行加标；（ii）通过C18/OH SPE进行预纯化；（iii）衍生化；（iv）对Ac-PFB衍生物进行高效液相色谱纯化；以及（v）气相色谱/质谱分析。通过对多个尿液和DNA样本进行重复分析证明了该方法的精密度：批内和批间变异分别小于5%和8%。这些分析方法足够灵敏，能够定量8-OHdG的尿排泄量（490±70 pmol/kg/24小时；样本量，600微升尿液），并测量未特意暴露于氧化应激的大鼠肝脏DNA中8-OHdG的水平（20个8-OHdG/10(6)个脱氧核苷；样本量，30微克DNA）。与先前方法相比的主要优点是，由于使用了适当的同位素标记内标，精密度提高，并且由于净化程序的优化，灵敏度提高。展示了对三种不同氧化核苷标准品，即8-OHdG、胸苷二醇和5-羟基甲基-2'-脱氧尿苷的同时分析。
