Eucommia ulmoides Oliver is a traditional Chinese medicine and material for functional food with a strong antioxidant activity. To investigate antioxidants in E. ulmoides Oliver flower, an improved on-line high performance liquid chromatography method with the radical cation 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS') as a stable model free radical for the detection of radical scavenging ability was developed. The radical concentration, flow rate, and length of reaction coil were optimized with rutin as a model antioxidant. Under the optimized instrumental set-up, a radical solution of 0.32 mM ABTS(center dot+) is delivered by a pump equipped with a superloop at 0.5 mL/min through a reaction coil of 10 m x 0.25 mm. This method showed a high sensitivity since the system noise was reduced by the superloop. The minimum detecting concentration of the method was in the range of 0.03-0.10 mu M for the negative peaks of rutin, quercetin, ascorbic acid, and alpha-tocopherol. Importantly, the method could be not only used for detection of antioxidants in plant extracts but also combined with mass spectrometry to obtain the structural messages of peaks in high performance liquid chromatography profiles. After analysis by the method, nineteen antioxidants were found in E. ulmoides Oliver flower extract, and the main active compound was identified as chlorogenic acid. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.