Development and Validation of a Micellar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC. Alberto de Oliveira, Claudinei A. Silva, Adalberto M. Silva, Marina F.M. Tavares and Massuo J. Kato* ABSTRACT: Introduction – A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective – To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology – The extracts were analysed in an uncoated fused-silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results – A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2. The analytical curve in the range 10.0–50.0 mg/mL (r 2 = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 mg/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0  1.4% of recovery. Conclusions – A novel high-performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright © 2010 John Wiley & Sons, Ltd. Keywords: MEKC; butenolides; Piper malacophyllum Introduction Phytochemical investigations of Piper species have led to the iso- lation of several classes of physiologically active compounds such as alkaloids, amides, pyrones, dihydrochalcones, flavonoids, phe- nylpropanoids, chromenes, lignans and neolignans (Kato and Furlan, 2007; Parmar et al., 1997). Bioactivity-guided fractionation of the dichloromethane extract from leaves of Piper malacophyllum using the bioauto- graphic assay against the fungi Cladosporium cladosporioides and C. sphaerospermum led to the isolation of two bioactive butenolides: 4,6-dimethoxy-5-E-phenylbutenolide (1) and 4,6- dimethoxy-5-Z-phenylbutenolide (2). The higher activity for 1 was associated to E configuration, since the minimum amounts required for the growth inhibition of C. cladosporioides and C. sphaerospermum were 1.0/1.0 mg(1) and 5.0/10.0 mg(2), respec- tively (Lago et al., 2005). A large number of natural and synthetic compounds having butenolides as a core unit have been described (Negishi and Kotora, 1997; Xu et al., 2007). Many of them displayed a wide range of biological activities including cytotoxicity, antibiotic, herbicide and antimicrobial activities (Fang et al., 1991; Kar et al., 2005; Barbosa et al., 2006; Teixeira et al., 2008). Because of the biological and economic importance of such classes of compounds occurring in Piperaceae species, several analytical methods have been developed. Thus, piperamides (Scott et al., 2005; Navickiene et al., 2003) and kavalactones (Bobeldijk et al., 2005; Bilia et al., 2004) have been analysed by HPLC in Piper species. In spite of a number of advantages associ- ated with the variety of mobile and stationary phases, high sen- sitivity and variety of detection modes, HPLC has as major disadvantage. The requirement for clean-up steps for the analysis of crude extracts is demanding. Additionally, the costs of supplies including columns are significant and the use of a large amount of solvents implies high disposal costs. Capillary electrophoresis (CE) has emerged as an alternative and powerful analytical tool for rapid separation of a variety of analytes. This technique overcomes many of the drawbacks of * Correspondence to: M. J. Kato, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo, SP, Brazil. E-mail: majokato@iq.usp.br Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo, SP, Brazil Research Article Received: 28 January 2009; Revised: 24 March 2010; Accepted: 25 January 2010 Published online in Wiley InterScience: 23 March 2010 (www.interscience.wiley.com) DOI 10.1002/pca.1213 428 Phytochem. Anal. 2010, 21, 428–433 Copyright © 2010 John Wiley & Sons, Ltd.