High-Throughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format DEJIAN HUANG, ² BOXIN OU,* ,² MAUREEN HAMPSCH-WOODILL, ² JUDITH A. FLANAGAN, ² AND RONALD L. PRIOR ‡ Brunswick Laboratories, 6 Thatcher Lane, Wareham, Massachusetts 02571, and Arkansas Children’s Nutrition Center, Agriculture Research Service, U.S. Department of Agriculture, 120 Marshall Street, Little Rock, Arkansas 72202 The oxygen radical absorbance capacity (ORAC) assay has been widely accepted as a standard tool to measure the antioxidant activity in the nutraceutical, pharmaceutical, and food industries. However, the ORAC assay has been criticized for a lack of accessibility due to the unavailability of the COBAS FARA II analyzer, an instrument discontinued by the manufacturer. In addition, the manual sample preparation is time-consuming and labor-intensive. The objective of this study was to develop a high-throughput instrument platform that can fully automate the ORAC assay procedure. The new instrument platform consists of a robotic eight-channel liquid handling system and a microplate fluorescence reader. By using the high-throughput platform, the efficiency of the assay is improved with at least a 10-fold increase in sample throughput over the current procedure. The mean of intra- and interday CVs was e15%, and the limit of detection and limit of quantitation were 5 and 6.25 µM, respectively. KEYWORDS: ORAC; antioxidant activity; free radical; plate reader; high throughput INTRODUCTION Nutritionists, clinical researchers, and various segments of the food and pharmaceutical industries have an increasing need to know the antioxidant capacity of physiological fluids, foods, beverages, and natural products. This need is derived from the proven evidence of the importance of antioxidants to scavenge the reactive oxygen/nitrogen species (ROS/RNS), which are known to be involved in the pathogenesis of aging and many common diseases (1). Several methods for measuring antioxidant capacity in vitro have been developed and reviewed (2). The very complicated reaction among free radical, substrate, and antioxidant makes it impossible to use a fixed equation to express the kinetic order. Therefore, the accurate measurement of antioxidant capacity requires both inhibition degree and inhibition time to be taken into account. The oxygen radical absorbance capacity (ORAC) is the only method so far that combines both inhibition time and degree of inhibition into a single quantity (3). The early version of the ORAC assay developed by Cao et al. was time-consuming and labor-intensive, particularly for analyses of large numbers of samples (4). Later, the ORAC method was semiautomated by adapting it to a COBAS FARA II analyzer, an instrument discontinued by the manufacturer (5). This semiautomated ORAC can analyze up to nine samples at a single concentration per run. However, the reading of a single concentration usually does not fall within the linear concentration range of standards; thus, repeated runs are needed until a satisfactory result is obtained. Moreover, the long sample preparation times often consume most of a day, whereas the COBAS FARA II “waits” for sample. In addition to the low efficiency of sample throughput, the earlier version of ORAC has several limitations, including interactions between the fluorescent probe and tested antioxidants and incompatibility with lipid soluble antioxidants. Most recently, the ORAC assay was significantly improved by Ou, Huang, and co-workers using fluorescein as the new fluorescent probe (6-7). The improved ORAC was demonstrated to be robust and compatible with lipid soluble antioxidant. However, the new version of ORAC was developed on the COBAS FARA II platform and, as such, is available only in a few laboratories equipped with the COBAS FARA II. This situation not only severely limits access to the assay by other researchers but also causes low productivity. Therefore, development of a high-throughput instrument plat- form that fully automates the ORAC assay from sample preparation to final measurement is necessary. In the present study, we investigated the feasibility of full automation for the ORAC assay using a widely utilized platform consisting of a * Author to whom correspondence should be addressed [e-mail bou@brunswicklabs.com; telephone (508) 291-1830; fax (508) 295-6615]. ² Brunswick Laboratories. ‡ U.S. Department of Agriculture. J. Agric. Food Chem. 2002, 50, 4437-4444 4437 10.1021/jf0201529 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/03/2002