Simultaneous direct quantification of urinary 8-oxodGuo and 8-oxoGua using on-line SPE LC-MS/MS method

• Main Authors: Ciao-Han, 謝樵漢
• Other Authors: 胡瓊文
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/35733288974066935507

碩士 === 中山醫學大學 === 公共衛生學系碩士班 === 97 === A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to simultaneously determine 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxodGuo) in urine. 15N5-labeled 8-oxoGua was also successfully synthesized to serve as an internal standard in this method. After spiking internal standard, the urine sample can be directly analyzed within 15 min without further sample purification. With this newly developed method, we further investigated the stability of 8-oxoGua under several common storage conditions. The results showed that the stability of 8-oxoGua was affected by pH, temperature and its initial concentration. It was found that the high concentration of 8-oxoGua (10 μg/mL) was the most stable at 4°C with pH value ≤ 11 (the half-life was estimated to be around 105 days), while it was the less stable at -20°C with pH value of 12 (the half-life was estimated to be 4 days). On the other hand, the low concentration of 8-oxoGua (50 ng/mL) had the best stability at -20°C with pH value < 8 (half-life: 578 days), and the worst stability at -20°C with pH value of 12 (half-life: 4 days). Meanwhile, this “on-line SPE LC-MS/MS” method was also applied to determine the background levels of 8-oxoGua/8-oxodGuo in urine of healthy adults. The urinary levels of 8-oxoGua and 8-oxodGuo were 5.95 ± 5.20 and 1.74 ± 0.84 (nmol/mmol creatinine), respectively. The ratio of 8-oxoGua and 8-oxodGuo levels in urine was around 3.41. This “on-line SPE LC-MS/MS” method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.