| Summary: | 碩士 === 輔英科技大學 === 醫事技術系碩士班 === 98 === Free radicals produced from metabolism of organisms induces oxidative stress such as protein oxidation, DNA damage and lipid peroxidation. Oxidative stress induces not only cell aging and death but also indirect human aging and deterioration of resistance. Therefore, developing a rapid and sensitive analysis of peroxidation is an important event. In this study, we reported a novel method for rapid and sensitive determination of malondialdehyde (MDA) in serum and urine by ultra performance liquid chromatography (UPLC). MDA was reacted with thiobarbituric acid (TBA) to form MDA–TBA2, a red-colored adduct with maximum absorbance at 532 nm. The effects of UPLC parameters (pH of mobile phase, non-polar ratio of mobile phase, flow rate and injection volume) on peak area and retention time were investigated for the optimized operation conditions that could enhance the sensitivity and the rapidity. The linearity, matrixes interference of serum and urine, limit of detection, recovery, precision and the test of real samples were validated in the experimental section. Our results indicated the absorbance at mobile phase pH6.8, KH2PO4: Methanol 60:40(v/v), 5 μL of injection volume by UPLC was the same as at 20 μL of injection volume by HPLC. When the flow rate was 0.4 mL/min, the retention time was 2.5 min. Comparing to HPLC, the high separation capacity and half retention time of UPLC were found. The linear range of standard solution was from 0.61 to 38.88 μmol/L and the detection limit of MDA was 43 fg. The intra-assay or inter-assay precisions of MDA in serum and urine were all below 10%. The recoveries of MDA in serum and urine were 94-110% and 81-110%, respectively. Furthermore, the stable temperatures of MDA were 4℃ and -20℃. The results showed that there were no matrixes interferences for the determination of MDA in serum and urine. Therefore, the calibration curves for the analysis of serum and urine could be established by the aqueous standard solution. The proposed method provided sensitive and rapid measurement of MDA in serum and urine by UPLC can be easily applied to clinical laboratory.
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