| Summary: | 碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 98 === 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 determine malondialdehyde (MDA) in urine following derivatization with 2,4-dinitrophenylhydrazine (DNPH). After derivatization, the urine samples can be directly analyzed within 11 min without further purification. The method was then applied to investigate the optimal reaction conditions for MDA derivatization using DNPH. The results showed that for a completely MDA derivatization, the additions of DNPH to MDA calibration solutions or urinary samples were extremely different. The optimal addition ratio of DNPH to urinary MDA is 60 times higher than that of MDA standard solution. This finding indicated that there were considerable interfering substances present in urine matrix that could also react with DNPH.
Furthermore, this “on-line SPE LC-MS/MS” method was applied to determine the urinary levels of MDA in 109 healthy adults, including 63 non-smokers and 46 smokers. The mean urinary MDA levels were 1.6 ± 0.9 and 1.8 ± 1.8 μmol/g creatinine for non-smokers and smokers,respectively. There was no significant difference in urinary MDA levels between the two groups (p > 0.05). Overall, this newly developed “on-line SPE LC-MS/MS” method could be applicable in both research and clinical practice for assessing oxidative stress.
In addition to this, the urine samples were also added with a stable isotope d2-MDA prior to derivatization and analyzed by on-line SPE LC-MS/MS. The results showed that the urinary MDA values obtained from both methods (with or without stable isotope) were highly correlated (Pearson correlation coefficient r = 0.9957, p < 0.001, n = 34).
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