Talanta 79 (2009) 695–699 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Multivariate optimization of hydrodistillation-headspace solvent microextraction of thymol and carvacrol from Thymus transcaspicus Vahid Kiyanpour a , Ali Reza Fakhari a,b,∗ , Reza Alizadeh b , Behvar Asghari a , Mehdi Jalali-Heravi c a Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran b Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, G. C., Evin, Tehran, Iran c Department of Chemistry, Sharif University of Technology, Azadi Avenue, P.O. Box 11155-9516, Tehran, Iran article info Article history: Received 26 November 2008 Received in revised form 25 April 2009 Accepted 27 April 2009 Available online 9 May 2009 Keywords: Thymol Carvacrol Hydrodistillation Headspace solvent microextraction Response surface methodology Thymus transcaspicus abstract In this paper multivariate response surface methodology (RSM) has been used for the optimization of hydrodistillation-headspace solvent microextraction (HD-HSME) of thymol and carvacrol in Thy- mus transcaspicus. Quantitative determination of compounds of interest was performed simultaneously using gas chromatography coupled with flame ionization detector (GC-FID). Parameters affecting the extraction efficiency were assessed and the optimized values were 5min, 2 L and 3 min for the extrac- tion time, micro-drop volume and cooling time after extraction, respectively. The amounts of analyte extracted increased with plant weight. The calibration curves were linear in the ranges of 6.25–81.25 and 1.25–87.50 mg L -1 for thymol and carvacrol, respectively. Limit of detection (LOD) for thymol and car- vacrol was 1.87 and 0.23 mg L -1 , respectively. Within-day and between-day precisions for both analytes were calculated in three different concentrations and recoveries obtained were in the range of 89–101% and 95–116% for thymol and carvacrol, respectively. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Essential oils are naturally occurring compounds extracted from vegetal materials and due to their various properties such as antimicrobial, antioxidant and antifungal have been used in pharmaceutical and food industries [1]. The thyme essential oil is one of the most important oils used in traditional medicine to treat cold, flu and cough [2]. New properties of thyme oil such as strong acetylcholinesterase (Alzheimer disease agent) inhibitory effect have been reported [3]. Based on our previ- ous research, the above mentioned properties of thyme oil are related to its high phenolic content (thymol, carvacrol, linalool, etc.) [4]. A wide variety of extraction method has been used for the isolation of the essential oil from plant materials. Common tech- niques for the isolation and extraction of the essential oils include hydrodistillation and steam distillation, supercritical-fluid extrac- tion (SFE), solid-phase microextraction (SPME), headspace solvent microextraction (HSME) and hydrodistillation-headspace solvent microextraction (HD-HSME). HD-HSME is a rapid, simple and cost effective method for extracting volatile compounds. There are vari- ∗ Corresponding author at: Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, G. C., Evin, Tehran, Iran. Tel.: +98 21 22431683; fax: +68 21 22431661. E-mail address: a-zavareh@sbu.ac.ir (A.R. Fakhari). ous parameters such as extraction time, micro-drop volume, sample mass, etc., affecting the extraction efficiency of this method [5–11]. Different chromatographic methods have been used for the determination of thymol and carvacrol in various matrixes. The method of quantitative thin-layer chromatography (TLC) combined with densitometry has been used to determine the thymol and carvacrol content of Oleum thymi essential oil [12]. Vinas and co- workers have determined phenol, thymol and carvacrol in honey using high performance liquid chromatography with fluorimetric detection [13]. These compounds have also been determined using gas chromatographs equipped with flame ionization [14,15] and mass spectrometric detection [2,16]. The aim of the present work was optimizing a HD-HSME method for the extraction of thymol and carvacrol in Thymus transcaspicus followed by their quantitative determination using a simple and reliable GC-FID analysis procedure. 2. Experimental 2.1. Reagents and materials Aerial parts of T. transcaspicus were collected in June 2007 from Aladagh Mountains, Bojnourd, Iran. The plant materials were air dried in the absence of light at ambient temperature and stored at 4 ◦ C. 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.04.068