Influence of Sample Preparation on Assay of Phenolic Acids from Eggplant DEVANAND L. LUTHRIA* AND SUDARSAN MUKHOPADHYAY Food Composition Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705-3000 Sample preparation is often overlooked and is frequently considered as “a means to an end”. This systematic study with a phenolic-enriched substrate, eggplant (Solanum melongena L.), was undertaken to evaluate the substantial variations in the extraction techniques, solvents, and parameters as described in the published literature. Direct comparison of over 10 extraction procedures or conditions was performed to show the importance and influence of sample preparation on the assay of phenolic compounds. Chlorogenic acid (CA) was the most abundant phenolic acid accounting for >75% of the total phenolic acids content extracted from the eggplant sample. Optimum extraction of CA and total phenolics (TP) from Black Bell cultivar of eggplant were obtained when extractions were performed with a mixture of MeOH/H 2 O at a ratio of 80:20% v/v using a pressurized liquid extractor (PLE) at 100 °C. The amount of CA and TP extracted from eggplant by the previously reported procedures using a wrist shaker, rotary shaker, stirring, sonication, or reflux with different extraction solvents (acetone or varying composition of MeOH/H 2 O solvent mixtures) varied significantly between 5 and 95% as compared to PLE. The predominant phenolic acids in the free phenolic acid fraction of Black Beauty cultivar of eggplant were CA isomers. However, caffeic acid isomers were the major phenolic acids extracted from the base-hydrolyzed fraction. The total amount of caffeic acid extracted from the Italian Neon cultivar was more that twice that of four other eggplant cultivars (Orient Express, Calliope Zebra Stripe, Orient Charm Neon, and Black Beauty). KEYWORDS: Eggplant; Solanum melongena L.; sample preparation; extraction procedures; phenolic acids; Folin-Ciocalteu; HPLC; solvent composition; hydrolysis INTRODUCTION Phenolic acids (PA) are aromatic secondary metabolites biosynthesized by plants and are ubiquitous throughout the plant kingdom (1). The chemical nature of plant phenolics may vary from a simple monomeric unit to highly polymerized structures of varying proportions of monomeric aglycon units (2). In recent years, phenolic acids have received considerable attention due to their health beneficial effect for protection against certain form of cancers and cardiovascular diseases (3, 4). Eggplant (Solanum melongena L.) is ranked among the top 10 vegetables in terms of antioxidant capacity (5). Eggplant is a common vegetable consumed throughout the world. It is a member of the potato family, and it is known worldwide, in various synonyms as aubergine, brinjal, melanzana, garden egg, and patlican. The first report on the extraction and identification of chlorogenic acid and browning in eggplant was published by Kozukue et al. (6). The influence of storage conditions (tem- perature and time) was studied by both Kozukue et al. and Esteban et al. (6, 7). Research related to the beneficial effects of the consumption of eggplant phenolics in animal subjects was carried out by Sudhesh et al. (8). The antioxidant activity of eggplant by different assays was reported by Huang et al. (9). Recently, Stommel and Whitaker carried out a detailed study on the identification and determination of PA in different cultivars of eggplants (10, 11). The procedures used by various research groups for the extraction of phenolics from eggplant varied significantly and are summarized in Table 1. These data clearly illustrate that sample preparation is often overlooked and is frequently considered as “a means to an end”. In recent years, there have been tremendous advancements in chromatographic and spec- troscopic instrumentations for the separation, detection, and identification of phenolic compounds from natural sources. However, sample preparation has received limited attention. Optimum sample preparation is critical for any analyses and is of great importance for phenolic compounds due to the oxidative and thermal labile nature of this class of compounds. In addition, >8000 naturally occurring phenolic compounds with diverse structural configurations have been isolated from natural sources (1). Phenolic acids may exist in multiple forms as free, esterified, glycosylated, or polymerized and may coexist as complexes with * Address correspondence to this author at Room 202 B, Building 161, BARC-E, Food Composition Laboratory, Beltsville Human Nutrition Research Center, USDA, 10300 Baltimore Ave., Beltsville, MD 20705- 3000 [telephone (301) 504-8071; fax (301) 504-8314; e-mail luthriad@ ba.ars.usda.gov]. J. Agric. Food Chem. 2006, 54, 41-47 41 10.1021/jf0522457 This article not subject to U.S. Copyright. Published 2006 by the American Chemical Society Published on Web 12/08/2005