Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2′-Deoxyguanosine by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry with On-Line Solid-Phase Extraction

Abstract

Quantification of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in urine or blood is used to assess and monitor oxidative stress in patients. We describe the use of on-line solid-phase extraction (SPE) and isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for automated measurement of urinary 8-oxodGuo. Automated purification of urine was accomplished with a switching valve and an Inertsil ODS-3 column. After the addition of 15N5-labeled 8-oxodGuo as an internal standard, urine samples were analyzed within 10 min without sample purification. This method was applied to measure urinary 8-oxodGuo in a group of healthy persons (32 regular smokers and 35 nonsmokers). Urinary cotinine was also assayed by an isotope-dilution LC-MS/MS method. The lower limit of detection was 5.7 ng/L on column (2.0 fmol). Inter- and intraday imprecision (CV) was < 5.0%. Mean recovery of 8-oxodGuo in urine was 99%-102%. Mean (SD) urinary concentrations of 8-oxodGuo in smokers [7.26 (3.14) microg/g creatinine] were significantly higher than those in nonsmokers [4.69 (1.70) microg/g creatinine; P < 0.005]. Urinary concentrations of 8-oxodGuo were significantly correlated with concentrations of cotinine in smokers (P < 0.05). This on-line SPE LC-MS/MS method is sufficiently sensitive, precise, and rapid to provide high-throughput direct analysis of urinary 8-oxodGuo without compromising quality and validation criteria. This method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.