1. Determination of volatile components and phenolic acids in Satureja montana by GC/MS Józef Rzepa 1, Mieczysław Sajewicz 1, Tomasz Baj 2, Patrycja Gorczyca 1, Magdalena Włodarek 1, Kazimierz Głowniak 2, Monika Waksmundzka-Hajnos 3, and Teresa Kowalska 1 1 Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland 2 Department of Pharmacognosy with Medicinal Plant Unit, Faculty of Pharmacy, Medical University of Lublin, 1 Chodźki Street, 20-093 Lublin, Poland 3 Department of Inorganic Chemistry, Faculty of Pharmacy, Collegium Pharmaceuticum, Medical University of Lublin, 4a Chodźki Street, 20-093 Lublin, Poland INTRODUCTION Our work presents the results of chromatographic analysis of the volatile fraction and the phenolic acids fraction contained in different samples of the culinary plant winter savory (Satureja montana). This herb is highly appreciated and widely utilized as a food additive and it ha got a long history of use in traditional medicine as tonics, carminatives, astringents, and expectorants, and for the treatment of intestinal problems such, as diarrhea and nausea. AIM It was the aim of this study to fingerprint the volatile fraction derived from two winter savory (Satureja montana) samples of different origin, to identify the main components contained therein, and to compare the efficiency of the different extraction methods. An additional effort was undertaken to identify phenolic acids contained in water sample remaining from the steam distillation procedure. EXPERIMENTAL Herbal material: Two different lots of winter savory (Satuteja montana) were investigated in this study. Lot 1 was harvested in Pharmacognosy Garden of the Medical University, Lublin, Poland. The plant material comprised all parts of the plant (i.e., roots and the aerial parts) and it was dried for 40 h in an oven with a forced air flow at 35 to 40 o C.. Lot 2 originated from the farm market in Belgrade, Serbia, and it was not pretreated in any way in our laboratory. Volatile fraction Headspace gas chromatography – mass spectrometry of the volatile fraction: The headspace gas chromatography–mass spectrometry (HS–GC–MS) analyses were carried out with use of a TRACE 2000 model GC with an MS TRACE model mass detector (ThermoQuest, Waltham, MA, USA), equipped with a CTC Analytics model autosampler (Combi PAL, Basel, Switzerland), used in the headspace mode. Temperatures and time of the headspace desorption were, respectively, 80 and 100 o C, and 15 min. The 0.5 mL volume of the headspace phase was introduced on to the DB-5 capillary column (30 m ↔ 0.25 mm i.d., 0.25-μm film thickness; Agilent Technologies, Palo Alto, CA, USA). Helium (p = 100 kPa) was used as carrier gas. Gradient analysis was run using the following temperature program: 40ºC (1 min); 40–180ºC (12ºC/min); and 180ºC (20 min). The temperature of the injector was kept constant at 180ºC. Mass spectrometer was fitted with an EI source operated at 70 eV. Steam destillation modes: Mode 1: using the Deryng apparatus (recommended by Polish Pharmacopoeia VI applied to the 50-g plant samples from lot 1 and lot 2 (Fig 1.) [1]; Mode 2.1: using the Clevenger apparatus (recommended by European Pharmacopoeia ) applied to the 50-g plant samples from lot 1 and lot 2 (Fig 2.) [2]; Mode 2.2: using the Clevenger apparatus like in Mode 2.1, yet applied to the 20-g plant samples from lot 1; and Mode 3: using the Clevenger apparatus like in Mode 2.2 (lot 1), yet with the herbal/aqueous mixture additionally ultrasonicated in the round-bottomed flask, in order to enhance maceration of herbal material (Fig 3.) [3]. In the first set of the experiments valid for modes 1 and 2.1, the dried plant material (50 g) was placed in the round-bottomed flask, 400 mL water was added, and steam distillation was performed for 3 h. In the second set of the experiments valid for modes 2.2 and 3, the 20-g amounts of the plant material with 400 mL water were used. In each individual experiment, the 1-μL aliquots of the 5 % distillate solutions in methanol (v/v) were analyzed by GC–MS. Solid-phase extraction (SPE) and derivatization of phenolic acids: Liquid left from the steam distillation of lot 1 in mode 1 (i.e., with use of the Deryng apparatus) underwent the acidic and the basic hydrolysis in parallel, in order to liberate phenolic acids most probably present in the ester and the glycoside form. The acidic hydrolysis was performed in the following way: Liquid was adjusted with 36% HCl to pH  2 and the entity was kept boiling for 2 h under the reflux. In the case of the basic hydrolysis, the second portion of the liquid was adjusted with 4M NaOH to pH  10 and the entity was also kept boiling for 2 h under the reflux. The obtained hydrolysates were neutralized and filtered to separate the particulate matter and finally they underwent the solid phase extraction.The SPE tubes were first conditioned with 5 mL methanol and 10 mL redistilled water. After passing the hydrolyzed and filtered samples through the SPE tubes, elution of the adsorbed compounds was carried out with 6 mL methanol. The eluates were condensed to dryness in the air stream and then underwent the derivatization procedure. Derivatization was carried out by adding 100 μL BSTFA+TMCS and 1 mL pyridine to the dry residues and heating the obtained solutions at 80 o C for 2 h on the oil bath. After cooling, samples underwent the GC-MS analysis. Scheme 1. Presentation of the ASE extraction and acidic hydrolysis of bonded phenolic acids [4] Accelerated solvent extraction (ASE) and derivatization of phenolic acids (Scheme 1) RESULTS GC/MS: Samples were analyzed with use of a TRACE 2000 model GC with an MS TRACE model mass detector (ThermoQuest), equipped with an autosampler (Combi PAL), and the TR-35MS capillary column (30 m ↔ 0.25 mm i.d., 0.5-μm film thickness; Thermo Scientific, Northumberland, UK). Helium (p = 100 kPa) was used as carrier gas. Gradient analysis was run using the following temperature program: 80ºC (3 min); 80–160ºC (20ºC/min); 160– 280ºC (20 o C/min); and 280 o C (20 min). The temperature of the injector was kept constant at 280ºC. Mass spectrometer was fitted with an EI source operated at 70 eV. HPLC – DAD: High performance liquid chromatographic analysis was carried out using a Gyncotek liquid chromatograph (Gyncotek, Macclesfield, UK) equipped with a Gyncotek Gina 50 model autosampler, Gyncotek P 580A LPG model pump, Gyncotek DAD UVD 340U model diode array detector, and Chromeleon Dionex v. 6.4 software for data acquisition and processing. The analyses were carried out with the acetonitrile (A) – water modified with 1% glacial acetic acid (B) mobile phase at a flow rate of 0.7 mL min -1 in the gradient mode, using an RP-18 Hypersil GOLD (5  m particle size) column (250 mm  4.6 mm i.d.; Thermo Scientific, Waltham, MA, USA; cat. no. 0694830N). The following gradient was applied (v/v): 0–7 min, A + B, 20 + 80; 8–13 min, A + B, 40 + 60; 14–19 min, A + B, 60 + 40; 20–36 min, A + B, 100 + 0; and 37–40 min, A + B, 20 + 80. The chromatographic column was thermostated at 35 o C with use of the Varian Pro Star 510 model column oven. Table 1. Volatile compounds in Satureja montana (lot 1 and lot 2), their respective retention times (t R ), and semi-quantitative evaluation of their relative contributions (%) to the overall volatile fraction * depending on the applied extraction method (HS-80: headspace at 80 o C; HS-100: headspace at 100 o C; Deryng: mode 1; Clevenger: mode 2.1) * = estimated from the respective peak height + = data between limit of detection (LOD) and limit of quantification (LOQ) ++ = lack of adequate (i.e., enabling quantification peak separation) - = not detected Table 2. Volatile compounds in Satureja montana (lot 1), their respective retention times (t R ), and semi-quantitative evaluation of their relative contributions (%) to the overall volatile fraction * depending on the applied extraction method (mode 2.2 and mode 3) * = estimated from the respective peak height ** = numbering of the volatile compounds in conformity with Table 1 + = data between limit of detection (LOD) and limit of quantification (LOQ) Figure 4. Fingerprint GC-MS chromatograms of the volatile Satureja montana fraction for the following samples: A: Lot 1, HS-80; B: Lot 1, HS-100; C: Lot 1, Deryng; D: Lot 1, Clevenger; E: Lot 2, HS-80; F: Lot 2, HS-100; G: Lot 2, Deryng. Figure 5. Fingerprint GC-MS chromatograms of the volatile Satureja montana (lot 1) fraction vapor distilled with use of the Clevenger apparatus A: without ultrasonication (mode 2.2) and B: with ultrasonication (mode 3). Figure 6. Fingerprint GC-MS chromatograms of the non-volatile Satureja montana fraction obtained from the hydrolyzed, SPE-condensed, and finally trimethylsilylated water residue steam distilled in the Deryng apparatus from lot 1. A: basic hydrolysis; B: acidic hydrolysis. 1: 2,3-dihydroxyphenylacetic acid; 2: syringic acid; 3: α-hydroxydihydrocaffeic acid; and 4: caffeic acid Figure 7. Fingerprint GC-MS chromatograms of the non-volatile Satureja montana fraction obtained from the trimethylsilylated ASE extracts, lot 1 (samples E0 and E1). 5: caffeic acid methyl ester; 6: 3,4-dihydroxyphenylacetic acid methyl ester; and 7: 2-hydroxyphenylpropionic acid Figure 8. Fingerprint HPLC-DAD chromatogram of the non-volatile Satureja montana fraction contained in the diethyl ether extract E2 after the acidic hydrolysis. Analysis of the phenolic acids contents in Satureja montan a An attempt was made to analyze phenolic acids contained in the residue water from the steam distillation in the Deryng apparatus (lot 1, mode 1). In general, it can be stated that the SPE technique applied to the samples after both, the acidic and the basic hydrolysis, resulted in the low phenolic acids yields, which might probably be natural characteristics of winter savory (Satureja montana). In the extract originating from basic hydrolysis, then condensed by means of SPE, and finally derivatized by means of the trimethylsilylation reagent, the four phenolic acids were identified (in the form of the respective trimethylsilyl derivatives), i.e.: 2,3-dihydroxyphenylacetic, syringic, caffeic, and α-hydroxydihydrocaffeic (Fig. 6A). In the extract originating from the acidic hydrolysis and then analogically preprocessed, two phenolic acids were identified (also in the form of the respective trimethylsilyl derivatives), i.e., caffeic and o-hydroxydihydrocaffeic (Fig. 6B). The results of the accelerated solvent extraction (ASE) of the winter savory samples (lot 1) largely confirmed the above findings. In the trimethylsilylated extract E0, methyl esters of caffeic acid and 3,4-dihydroxyphenylacetic acid were identified. In the trimethylsilylated extract E1, methyl esters of 2-hydroxyphenylpropionic acid and 3,4- dihydroxyphenylacetic acid were found. In the trimethylsilylated extract E2, no phenolic acids were detected. T he HPLC-DAD analysis was performed for underivatized (i.e., non- trimethylsilylated) extracts E1 and E2 from lot 1 obtained with use of the ASE procedure described in sub-section 2.7, and extract E0 was not analyzed. In extract E1, cinnamic acid and p-hydroxybenzoic acid were identified, and in extract E2, chlorogenic acid and elagic acid were found. Fingerprinting and identification of the volatile fraction from Satureja montana The most frequently appearing volatile compounds were α- pinene, p-cymene, γ-terpinene, terpineol, ß-linalool, borneol and carvacrol,. These results remain in good agreement with those known from the literature[5,6]. From the obtained results it is clear that the origin of the plant material plays a meaningful role in chemical composition of the volatile fraction. A comparison of analytical results dealing with the headspace extraction of the volatile fraction carried out at 80 and 100 o C allowed drawing very practical conclusions. Respective chromatograms illustrating this particular issue are given in Fig. 4 (chromatograms A vs. B valid for lot 1 and chromatograms E vs. F valid for lot 2). A comparison of the aforementioned chromatograms showed that at the higher headspace temperature (100 o C) a higher number of the volatile compounds were isolated than at the lower one. Then the two steam distillation techniques were compared for lot 1 (Fig. 4, chromatograms C vs. D). Essential oil derived with use of the Deryng apparatus (mode 1, Fig. 4C) was richer in the chemical species than that derived with use of the Clevenger apparatus (mode 2.1, Fig. 4D). A consecutive comparison was made of the analytical results valid for the headspace extraction of the volatile fraction carried out at 100 o C with those obtained for the steam distilled fraction.This comparison allows a conclusion that the headspace extraction carried out at 100 o C is more effective particularly with the more volatile compounds than the steam distillation. The last comparison dealt with the contents of the volatile compounds in the samples belonging to lot 1 and originating from the steam distillation in the Clevenger apparatus without and with ultrasonication (derivation modes 2b and 3). Surprisingly enough, steam distillation combined with ultrasonication of the mixture of the herbal material and water resulted in lowering of practically all concentrations of the essential oil components (see Table 2). This can probably be explained by too long ultrasonication time (equal to 180 min), because according to the literature the best ultrasonication effects are observed for the shorter periods of time (lasting up to 20 min) [7]. CONCLUSIONS The methodical aspects of the analysis of the volatile fraction and the phenolic acids fraction contained in winter savory (Satureja montana) were discussed in this study. It was demonstrated that the headspace desorption of the volatile fraction run at 100 o C outperforms the advised pharmacopoeial steam distillation modes, and especially with the most volatile essential oil components. This is very likely due to an uncontrolled evaporation (and loss) of these volatile components both from the Deryng apparatus and the Clevenger apparatus. In the case of the phenolic acids analysis, usefulness of the complementary approach was shown, combining the GC-MS and HPLC-DAD technique. A convincing proof of this usefulness was that each of these two techniques allowed detection of the different phenolic acids. REFERENCES [1] Polish Pharmacopoeia VI, Polish Pharmaceutical Society, Warsaw, 2002 [2] European Pharmacopoeia, Vol. 3, Maisonneuve SA, Sainte Ruffine, France, 1975; p, 68 [3] Polish Patent No 208058, (2008.10) [4] R.K. Ibrahim, G.H. Towers. Identification by chromatography of plant phenolic acids. Arch. Biochem. Biophys. 87: 125-127 (1960) [5] G.S. Cetković, A.I. Mandić, J.M. Canadanović-Brunet, S.M. Djilas, V.T. Tumbas. HPLC screening of phenolic compounds in winter savory (Satureja montana L.) extracts. J. Liq. Chromatogr. Relat. Technol. 30: 293–306 (2007) [6] J. Mastelić, I. Jerković. Gas chromatography–mass spectrometry analysis of free and glycoconjugated aroma compounds of seasonally collected Satureja montana L. Food Chem. 80: 135-140 (2003) [7] R. Kowalski, J. Wawrzykowski. Effect of ultrasound-assisted maceration on the quality of oil from the leaves of thyme Thymus vulgaris L. Flavour Fragrance J. 24: 69–74 (2009) Non-volatile fraction Figure 1. Deryng apparatus Figure 2. Clevenger apparatus Figure 3. Clevenger apparatus with ultrasonication