A serially coupled stationary phase method for the determination of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine by liquid chromatography ion trap tandem mass spectrometry

Abstract

Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations. Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5 ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46 nmol/mmol creatinine. The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations.

Keywords: 15[N5]2-dG, 15[N5]2′-deoxyguanosine; 15[N5]8-oxodG, 8-oxo-7, 8-dihydro-15[N5]2′-deoxyguanosine; 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxodG, 8-oxo-7, 8-dihydro-2′-deoxyguanosine; EIC, Extracted Ion Chromatogram; ESI, electrospray ionization; IQC, internal quality control; IS, internal standard; Ion trap; LC-MS/MS; LC-MS/MS, liquid chromatography tandem mass spectrometry; LOQ, limit of quantitation; MRM, multiple reaction monitoring; MTH1, Nudix hydrolase mut T homologue 1; NER, nucleotide excision repair system; NIR, nucleotide incision repair system; Oxidative stress; ROS, reactive oxygen species; Reactive oxygen species; SACI, surface-activated ionization; TIC, Total Ion Chromatogram; Urine.

Fig. 1

Extracted ion chromatogram (EIC) and MS/MS scan of 2 ng/ml 8-oxodG and of ¹⁵[N₅]8-oxodG (theoretic concentration 25 ng/ml) added to urine matrix (matrix 3 diluted 1:2). Panel A: EIC of 8-oxodG product ion. Retention time (rt) 21.7 min., 167.9±0.2 m/z (284 m/z parent ion); smoothed (3.04 Gauss, 1 cycle). Panel B: MS/MS mass spectrum of 8-oxodG product ion (167.9 m/z). Panel C: EIC of ¹⁵[N₅]8-oxodG product ion. rt 21.8 min., 172.9±0.2 m/z (289 m/z parent ion) smoothed (3.03 Gauss, 1 cycle). Panel D: MS/MS mass spectrum of ¹⁵[N₅]8-oxodG product ion (172.9 m/z).

Fig. 2

Comparison of 8-oxodG standard in different urine matrixes (1:2 dilution). Results reported as 8-oxodG peak area/IS peak area. Each matrix was employed to build up from four to seven distinct standard curves. Data are shown as mean±standard deviation (SD). Matrix 1 did not include the standard point at 20 ng/ml.

Fig. 3

MS/MS analysis of a urine sample. 8-OxodG/IS peak area ratio=0.74 and correspondent 8-oxodG concentration=2.3 ng/ml (mean of 3 analytical sessions). Upper panel: TIC+ all MSn. 8-OxodG chromatographic peak is indicated by an arrow. Lower panels: panel A, EIC of 8-oxodG product ion. Rt 21.6 min., 167.8±0.2 m/z (284 m/z parent ion); upper box, unsmoothed; lower box, smoothed (3.01 Gauss, 1 cycle) arrow in correspondence of 8-oxodG product ion. Panel B: EIC of ¹⁵[N₅]8-oxodG product ion. Rt 21.7 min., 172.8±0.2 m/z (289 m/z parent ion); upper box, unsmoothed; lower box, smoothed (3.01 Gauss, 1 cycle). an arrow in correspondence of IS product ion.