927 (2001) 39–45 Journal of Chromatography A, www.elsevier.com / locate / chroma Assay of 2,3-dihydroxybenzoic acid and related compounds in plant materials by high-performance liquid chromatography * Retno A. Budi Muljono, Farida L. Darsono, Johannes J.C. Scheffer, Robert Verpoorte Division of Pharmacognosy, Leiden / Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, P .O. Box 9502, 2300 RA Leiden, The Netherlands Received 2 April 2001; received in revised form 28 May 2001; accepted 5 July 2001 Abstract Salicylic acid and its putative biosynthetic precursors were assayed isocratically by RP-HPLC with UV detection at 280 nm. Optimum resolution was provided by an HPLC mobile phase consisting of MeOH–1% aqueous HOAc (40:60, v / v), at pH 4. Furthermore, for the analysis of 2,3-dihydroxybenzoic acid (2,3-DHBA) in Catharanthus roseus cell cultures after elicitation, a mobile phase consisting of acetonitrile–1% aqueous HCOOH containing 0.25% trichloroacetic acid (1:5, v / v), at pH 2, was used. The recovery for the free form of 2,3-DHBA was about 80% after a one-step extraction of the cells. The detection limit of 2,3-DHBA was 3 mg by using saligenin as an internal standard.  2001 Elsevier Science B.V. All rights reserved. Keywords: Plant materials; Catharanthus roseus; Dihydroxybenzoic acid; Benzoic acids; Salicylic acid 1. Introduction (BA) as an intermediate [3,4]. However, little is known about the biosynthetic pathway of a variety of Salicylic acid (SA) is widely distributed in angio- other substituted BA derivatives, such as 2,3-di- sperms and has an important regulatory role in plants. hydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxy- It serves for example as an endogenous inducer of benzoic acid (2,5-DHBA) in plants. In previous thermogenesis and it acts as an endogenous signal studies on the biosynthesis of hydroxybenzoic acids for the induction of systemic acquired resistance in it was shown that those two compounds might plants infected by pathogens [1,2]. Therefore, the originate from SA, as this compound administered to biosynthetic pathway of SA is intensively studied in plants was hydroxylated in the 3- or 5-position different plants. SA is thought to be derived from giving rise to 2,3-DHBA and 2,5-DHBA [5]. phenylalanine via trans-cinnamic acid (CA), through In the course of our studies on the biosynthetic either o-coumaric acid ( o-COA) or benzoic acid pathway of SA and the closely related compound 2,3-DHBA in plants, we used Catharanthus roseus cell suspension cultures as a model. This system has several advantages, since a high production of 2,3- DHBA was observed after elicitation of C. roseus *Corresponding author. Tel.: 131-71-527-4528; fax: 131-71- cell cultures with a cell wall preparation of the 527-4511. E-mail address: verpoort@lacdr.leidenuniv.nl (R. Verpoorte). fungus Pythium aphanidermatum [6]; SA can also be 0021-9673 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0021-9673(01)01098-6