Journal of Chromatography A, 1216 (2009) 4485–4491 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Densitometric determination of (+)-catechin and (-)-epicatechin by 4-dimethylaminocinnamaldehyde reagent Vesna Glavnik, Breda Simonovska, Irena Vovk ∗ National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia article info Article history: Received 23 December 2008 Received in revised form 6 March 2009 Accepted 10 March 2009 Available online 17 March 2009 Keywords: Flavan-3-ols Catechins 4-Dimethylaminocinnamaldehyde DMACA detection reagent Vanillin detection reagent Thin-layer chromatography TLC Densitometry abstract We report the optimization of a sensitive, selective and robust derivatization method using 4-dimethylaminocinnamaldehyde (DMACA) for densitometric determination of (+)-catechin and (-)- epicatechin. The separation of these compounds was achieved by thin-layer chromatography (TLC) on cellulose plates developed with water. With DMACA in HCl, both compounds gave blue bands, while under the same conditions, vanillin produced a fast fading red coloration of bands. Quantitation at 655 nm showed that for both compounds the calibration curve was linear from 2 to 12 ng and polynomial from 2 to 30 ng, and the repeatability of chromatography of 20 ng was 3.5% (RSD, n = 6). The visible limit of detec- tion of both standards was 1 ng, but the densitometric limit of detection was lower (0.2 ng). The optimized DMACA reagent is superior to the more frequently used vanillin reagent and is applicable also for determi- nation of mixtures containing other catechins ((-)-catechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, procyanidin A2, procyanidin B1 and procyanidin B2). © 2009 Elsevier B.V. All rights reserved. 1. Introduction Flavan-3-ols or catechins in addition to oligomeric proantho- cyanidins derived from them, and the related 3-O-gallate esters occur throughout the plant kingdom and are recognized as impor- tant bioactive compounds. In plants, they act as allelopaths [1,2], protectants against excess UV radiation [1], troposferic ozone [3] and even against infections by microorganisms [1,4,5]. Consump- tion of food (chocolate, fruits [6–9], nuts [7], vegetables [6]), beverages (e.g. green tea, cocoa, wine) [6,7,10,11], and spices [e.g. cinnamon (Cinnamomum sp.), juniper (Juniperus sp.)] [7,12,13] con- taining flavan-3-ols and their oligomeric and polymeric derivatives has been shown to be associated with possible prevention and even cure of chronic diseases such as cancer [14], atherosclerosis, cardio- vascular diseases [15], diabetes type II [13]), infections [5,9], and photoprotection of the skin against UV radiation [11]. High-performance liquid chromatography (HPLC) with different detection modes such as UV, fluorescence and MS [6–8,10,13,16–21] is the most frequently used technique for separation, detection and identification of catechins and proanthocyanidins. TLC is also very useful for rapid screening [22–27] and also for quantita- tion [28,29] of catechins. Chromophores enable their detection at 280 nm [6,10,16–21,29], but due to their rather low molar ∗ Corresponding author. Tel.: +386 1 4760 341; fax: +386 1 4760 300. E-mail address: irena.vovk@ki.si (I. Vovk). extinction compared to phenolic acids present in plant extracts derivatization of catechins and proanthocyanidins is desirable [10,17–19]. Catechins and proanthocyanidins react with aromatic aldehy- des in strong acid yielding colored reaction products [30], which can be employed for qualitative and quantitative analysis using colorimetric and spectrophotometric assays [18,31–36], post- chromatographic derivatization HPLC [6,10,17–20], TLC [12,16, 20–26,28–30,35,38–40] and even for histological [3,4,35, 38,39,41–43] investigations. To date, TLC detection of catechins has mainly been performed with the aromatic aldehyde vanillin [21–30], or with p-methoxybenzaldehyde (anisaldehyde) [23,37] or 4-dimethylaminocinnamaldehyde (DMACA) [12,16,35,38–40]. DMACA, originally used in TLC as a detection reagent for indoles [44], was applied to colorimetric assays of these compounds [45,46]. A DMACA colorimetric approach for estimation and TLC screening of catechins using this reagent was first published in 1971 [31]. The application of DMACA was used in histological assays to determine the location of flavanols in different plant tissues [41] and for HPLC post-column derivatization for detection of catechins [17]. Post-chromatographic derivatization with DMACA is used to enhance sensitivity and selectivity in TLC and HPLC analyses of catechins and proanthocyanidins. The same reagent consisting of 1% DMACA in 1.5M H 2 SO 4 dissolved in MeOH [17] has enjoyed general use in HPLC post-column derivatization assays, but differ- ent compositions of DMACA TLC spray reagents have been used for screening of flavanols and their oligomeric derivatives, the proan- 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.03.026