Abstract: The aim of this study was to develop a high-throughput quantitative assay for detecting the total flavonoids in large-scale samples from the broth of Phellinus sp.. The orthogonal experimental design was preformed according to L₉(3⁴) orthogonal table using 96 microplate.Based on the variance analysis of the results from the orthogonal experiments,the optimum experimental chromogenic conditions were obtained as follows:the addition volume of 5% sodium nitrite,10% aluminum nitrate,10% sodium hydroxide were 30 μL,30 μL,100 μL,respectively,and then the sample should be kept for 10 min at room temperature.The maximum absorption wavelength for detecting the total flavonoids from the broth of Phellinus sp.was determined to be 510 nm.The results showed that the developed method was precision and stable.The average recovery was 100.7%.The relative standard deviations of the evaluation test was 0.607%,and the linear range was 0-50 μg/mL (r²=0.9994).The comparative results between different methods indicated that the developed highthroughput assay for the total flavonoids content determination assay of large-scale samples hereby was more accurate,rapid,reliable and using less sample than the traditional method.

Key words: high-throughput assay;multi-volume spectrophotometer system;sodium nitrite-aluminum nitrate colorimetry, Phellinus sp, the total flavonoids