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.
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) 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).


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.
Hu
Chiung-Wen
CW
Department of Public Health, Chung Shan Medical University, Taichung, Taiwan.
Wang
Chien-Jen
CJ
Chang
Louis W
LW
Chao
Mu-Rong
MR
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D015415:Biomarkers / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D003367:Cotinine / Q000652:urine; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine; D006801:Humans; D007201:Indicator Dilution Techniques; D018384:Oxidative Stress; D012680:Sensitivity and Specificity; D012907:Smoking / Q000652:urine; D021241:Spectrometry, Mass, Electrospray Ionization
D015415:Biomarkers; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine; D003367:Cotinine