Journal of Chromatography A, 1145 (2007) 155–164 Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs: Characterisation and quantification of antioxidant phenolic acids Maarit Kivilompolo, Tuulia Hy¨ otyl¨ ainen ∗ Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, FIN-00014, Helsinki, Finland Received 7 December 2006; received in revised form 16 January 2007; accepted 19 January 2007 Available online 31 January 2007 Abstract A fast and effective dynamic sonication assisted ethanol extraction method was developed for extracting phenolic acids from basil, oregano, rosemary, sage, spearmint and thyme of the Lamiaceae family. The results were compared with results obtained by conventional solvent extraction techniques. A comprehensive two-dimensional liquid chromatography (LC × LC) system interfaced to electrospray ionisation time-of-flight (TOF) mass spectrometry was then optimised for analysis and quantification of the herb extracts. The optimised LC × LC system employed a combination of C18 and cyano columns. The relative standard deviations for the retention times were better than 0.05% (rosmarinic acid 0.1%) and those for the peak areas 2–14% (2 mg/l, n = 3). Limits of detection were 18–90 ng/ml. The LC × LC–MS method was applied to the quantitative analysis of phenolic acids, and the results were compared with those obtained with conventional LC–MS. © 2007 Elsevier B.V. All rights reserved. Keywords: Comprehensive two-dimensional liquid chromatography; Lamiaceae; Phenolic acids; Sonication assisted extraction; Time-of-flight mass spectrometry 1. Introduction There is great demand in the food industry for compounds that effectively inhibit the oxidation of lipids, which is the major cause of food deterioration. This auto-oxidation process may be delayed or inhibited by antioxidants. Although several synthetic antioxidants are available, their use is severely restricted by law, and there is strong worldwide interest in new and safe natural antioxidants. Antioxidants are important not only as preserva- tion agents but for their possible beneficial effects on human health [1,2]. In numerous studies on antioxidativity, herbs of the Lamiaceae family have been indicated as a potential source of natural antioxidants [3–12]. The herb extracts have also proven useful as stabilisers of edible oils, as reviewed by Yanishlieva and Marinova [13]. The research on antioxidant compounds in the Lamiaceae family has focused on phenolic diterpenes, flavonoids and phenolic acids [14–19]. ∗ Corresponding author. Tel.: +358 9 191 50267; fax: +358 9 191 50253. E-mail address: tuulia.hyotylainen@helsinki.fi (T. Hy¨ otyl¨ ainen). Traditionally, the water soluble antioxidative compounds in plant materials have been determined by solvent extrac- tion and liquid chromatography [6,10,12,20–23]. Recently, also capillary electrophoretic techniques have been used [21,24]. Tra- ditional extraction methods employ organic solvents such as ethanol, methanol, acetone and hexane, which in industrial use would leave residues prohibited in food. Attention is now being directed to extraction techniques that rely on solvents that are not hazardous to human health. Among these techniques are extraction with pressurised hot water [25,26], carbon dioxide (supercritical fluid extraction) [27] and ethanol/water mixtures (e.g. ultrasound-assisted extraction) [28–30]. Herb extracts are complex mixtures containing a variety of compounds in different amounts. Their analysis by conventional chromatographic techniques, such as LC, is challenging. For such complex matrices, the extra separation efficiency given by a powerful multi-dimensional separation technique is of particular value. In a traditional heart-cutting LC–LC–system, only selected fractions from the first column are separated in the second dimension column. In a comprehensive LC × LC system, in turn, all fractions from the first column are con- tinuously sampled and transferred by a switching valve to the 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.01.090