Received on November 15, 2005
Accepted on April 18, 2006

Automation and Analytical Techniques

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

¹ Departments of Public Health, Chung Shan Medical University, Taichung, Taiwan
² Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung, Taiwan
³ Departments of Occupational Safety and Health, Chung Shan Medical University, Taichung, Taiwan, and Occupational Safety and Health, Chung Shan Medical University, Taichung, Taiwan

^* To whom correspondence should be addressed. E-mail: mrchao{at}csmu.edu.tw.

Background: 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.

Methods: 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.

Results: 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) µg/g creatinine] were significantly higher than those in nonsmokers [4.69 (1.70) µg/g creatinine; P <0.005]. Urinary concentrations of 8-oxodGuo were significantly correlated with concentrations of cotinine in smokers (P <0.05).

Conclusions: 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.