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A method using HPLC-DAD coupled with second-order calibration was developed to simultaneously determine metronidazole and tinidazole in plasma samples in this paper.The second-order calibration method based on APTLD(alternating penalty trilinear decomposition)algorithm was proposed to analyze the three-way HPLC-DAD data from both standard and prediction samples,which makes it possible that calibration can be performed even in the presence of unknown interferences with a simple and green chromatographic condition and short analysis time.The results showed that good recoveries were obtained although the chromatographic and spectral profiles of the analytes of interest as well as background were partially overlapped with each other in plasma samples.