DOI: 10.17344/acsi.2016.2534 Acta Chim. Slov. 2016, 63, 661-669 661 Scientific paper Simultaneous GC-MS Determination of Free and Bound Phenolic Acids in Slovenian Red Wines and Chemometric Characterization Milena Ivanovi},1 Ma{a Islam~evi} Razborsek1 and Mitja Kolar2* 1 University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17 SI-2000 Maribor, Slovenia 2 University of Ljubljana, Faculty of Chemistry and Chemical Engineering, Vecna pot 113 SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: mitja.kolar@fkkt.uni-lj.si Tel.: (+386)-1-4798-694 Received: 20-04-2016 Abstract Several phenolic acids (PAs), caffeic, vanillic, syringic, p-coumaric and ferulic acid, found in Slovenian red wines were studied using gas chromatography and mass spectrometry. For isolation of the PAs from wine samples, solid phase extraction using hydrophilic modified styrene - HLB cartridges was used. The bound PAs were extracted after basic hydrolysis and o-coumaric acid was used as the internal standard. The developed method was validated and the linear concentration range for all analytes was from 1 to 100 mg L-1 with correlation coefficients above 0.999. We show that the method is repeatable (RSD<2%), recoveries were above 96%, and LOD and LOQ values were acceptable. In all of the wine samples tested, caffeic and p-coumaric acid were determined to be the predominant PAs (17-72 mg L-1), while other compounds were found in lower concentrations. Principal Component Analysis and Cluster Analysis were used to study differences between wines related towards varieties and Slovenian wine regions. The results demonstrate that variety has more influence on PAs content than wine regions in Slovenian red wines. Keywords: Phenolic acids, Slovenian red wines, gas chromatography, mass spectrometry, PCA, CLU 1. Introduction Antioxidant activity of plant materials and natural products has received a great deal of interest over the past years both in the public and scientific communities.1-4 Generally, it is believed that consumption of plant phenolics decreases the risk of diseases related to oxidative stress.5 Wine, as a complex matrix containing several hundreds of different chemical compounds,6,7 presents an analytical challenge, especially for identification and quantification of compounds in low concentrations. The chemical composition of red wines includes minerals, vitamins, proteins, sugars and phenolic compounds, among them PAs. Red wines are considered to have more protective function than white or rosé wines, because of their higher content in antioxidant substances released from the grape skin and seeds.8 The total amount of polyphenols in red wines has been estimated in the range from 2000 to 6000 mg L-1.9 Polyphenols are usually responsible for wi- ne colour and contribute to the bitter flavour of wine.10 From the literature it is known that lactic acid bacteria (LAB) are responsible for the occurrence of malolactic fermentation (MLF), a secondary fermentation which is considered to be beneficial in most red wines.11 The phenolic acids content of grapes and wines can positively or negatively affect the rate of MLF.12 For example, gallic acid at low concentrations has stimulatory effects on the growth and malolactic activity of LAB.13 On the other hand, some phenolic acids, especially those from the hydroxycinnamic class, delayed the conclusion of the malolactic fermentation by these bacteria.14 Hydroxycinnamic acids (particularly p-coumaric acid) are also known to inhibit growth of a variety of microorganisms including wine-spoilage strains of L. collinoides, L. brevis and L. hilgardii.15 Although Slovenia is a small country, its wine production has a significant role in the economy. Altogether, 22,000 hectares of vineyard area is divided among three major regions (Drava Valley-Podravje, Lower Sava Val-ley-Posavje and the Littoral-Primorska) with further division into sub-regions.1617 The Slovenian Littoral is Slovenia's most widely known and prominent wine region of both white and red wines. Slovenian vineyards are planted with different vine varieties, including Merlot, Cabernet Sauvignon, Chardonnay, Pinot Noir, Syrah, Barbera, and many others. PAs are present in their free forms or as glycosylated and esterified derivatives.18-20 Acidic, basic and enzymatic hydrolysis are the most commonly used methods for the extraction of PAs from natural materials.21-25 From the scientific literature it is obvious that the most commonly used techniques for the determination of PAs are high-performance liquid chromatography (HPLC) with UV or DAD detection or liquid chromatography coupled with mass spectrometry (LC-MS).26-29 Because of the longer sample preparation process for analysis, using gas chro-matography with mass spectrometry (GC-MS) in analysis of phenolic compounds is relatively rare, but in comparison with the other methods mentioned, GC-MS offers several advantages, including complete and high-resolution separation, sensitive detection, unambiguous identification and quantitation of a wide range of phenolics (including all isomers) in one chromatographic run.30-32 The aim of our study was to develop a simple and quantitative extraction method of selected PAs to ensure clean extracts in order to obtain a much more sensitive, selective and accurate GC-MS method for identification and quantitation of both free and bound PAs in red wine samples. For extraction of target compounds from the wine samples, solid-phase extraction (SPE) using hydrophilic modified styrene (HLB) cartridges was used. The bound PAs were determined after basic hydrolysis using NaOH in the presence of L-ascorbic acid and EDTA as stabilizers. The applicability of the developed method was tested on Slovenian red wines. Statistical and chemometric analyses were performed and the wines were classified. 2. Experimental 2. 1. Chemicals All reagents and solvents used were minimally of analytical purity. Standard compounds, trans-caffeic acid (99%), vanillic acid (97%), syringic acid (97%), trans-p-coumaric acid (98%), trans-o-coumaric acid (98%) and trans-ferulic acid (98%) and solvents, tetrahydrofuran-THF (99.5%) and pyridine (99.9%), were supplied by Merck (Germany). Derivatization reagent N-Methyl-N-(trimethyl-silyl)trifluoroacetamide (MSTFA), HPLC-grade methanol (MeOH) and sodium hydroxide-NaOH (99%) were purchased from Sigma (USA). GC-grade toluene (99.5%) and hydrochloric acid-HCl (36.5%) were purchased from Carlo Erba (Italy). Dichloromethane-DCM was purchased from JT Baker (Germany), L-ascorbic acid (99.7%) was purcha- sed from Alkaloid (Macedonia) and EDTA was purchased from Kemika (Croatia). The water used was obtained from a Milli-Q water purification system. 2. 2. Preparation of Standard Solutions and Calibration Curves Standard stock solutions of caffeic acid, vanillic acid, syringic acid, p-coumaric acid and ferulic acid, as well as of o-coumaric acid (ISTD) were prepared by accurately weighing 10 mg of each into a 10 ml volumetric flask, and then dissolving in THF. Five calibration standard solutions were prepared by combining various volumes of PAs stock solutions with 50 pl of ISTD in a 50 mL conical glass flask. Each solution was derivatized by treating it with 100 pL of MSTFA and 50 pL of pyridine for 1 h at 80 °C in a sand bath. After derivatization was finished, TMS derivatives were quantitatively transferred to 1 mL flasks and filled up to the mark with toluene. Five calibration standard solutions in concentration range from 1 to 100 mg L-1 were injected in triplicates. The calibration curves were constructed by linear regression of the peak-area ratio of individual PA standard to the ISTD (y), versus the concentration (mg L-1) (x). 2. 3. GC-MS Instrumentation and Working Conditions TMS derivatives of PAs were analyzed with a Varian 3900 gas chromatograph (GC), coupled to MS/MS Saturn 2100 ion trap mass spectrometer. GC separation was performed using a Varian capillary column VF-5ms CP8944 (30 m x 0.25 mm, with the stationary phase 0.25 pm). 1 pL of the sample was injected in split mode (split ratio 1:10). Carrier gas was He (6.0 UHP) at a flow rate of 1.0 mL min-1. The initial oven temperature was 40 °C, held for 1 min, and then the temperature was raised to 320 °C at a rate of 10 °C min-1, and finally, held for 3 min. The total run time was 32 min. The injection-port and transfer-line were set to 250 °C and 170 °C, respectively. Mass spectra were recorded in SCAN or SIM mode in a range from 50 to 650 m/z using electron ionization energy at 70 eV. Peak identification was done by comparing retention times (tR) and spectral properties with those of standard compounds or by library matching from NIST MS library containing the mass spectra of TMS derivatives of PAs. 2. 4. Validation Parameters for the GC-MS Method The method was validated for linearity, precision as repeatability, limit of detection (LOD) and limit of quanti-tation (LOQ). For linearity determination, all calibration curves were constructed using the internal standard method. The curves were fitted to linear least-squares regression. The precision was evaluated through the within-day (WD) and between-days (BD) repeatability, and expressed as relative standard deviation (RSD). The limit of detection (LOD) was calculated using the equation (3.3 + sy)/bx and the limit of quantitation (LOQ) was calculated from the equation (10 + sy)/b: (where sy is standard deviation of linear regression and b1 is slope of the calibration line).33 2. 5. Wine Samples The developed method was tested using Slovenian red wine samples. Twelve red wines from different Slovenian wineries and different varieties were purchased from local supermarkets. All the tested wine samples orginated from four vintages (2011-2015). Table 1 shows the varieties, wineries, year of production and percentage of alcohol. Wines were stored in a refrigerator at the temperature of +4 °C until analyzed. 2. 6. Preparation of the Wine Samples and Optimization of the Extraction Procedure 2. 6. 1. Extraction of Free PAs A standard solution of PAs mixture (in a concentration of 1000 mg L-1) was prepared in MeOH. Solutions of PAs mixture in synthetic wine (hydroalcoholic solution of 5 g L-1 tartaric acid, 12% of ethanol, and pH 3.2),34 were prepared by pipetting 30 and 100 pL of standard solution, respectively, in a 10 mL volumetric flask, and diluted with synthetic wine up to the mark. 1 mL of each solution was transferred into a 50 mL conical flask, spiked with 50 pL of ISTD (1000 mg L-1), diluted with 1 mL of ultra-pure water and acidified with 6 M HCl to a pH value of 2. Prepared samples were added to pre-conditioned HLB Supel- co® SPE cartridges (3 mL, 60 mg stationary phase made from hydrophilic modified styrene). A schematic procedure of the sample extraction is shown in Table 2. The free PAs fraction was eluted with 2 x 2 mL of THF. The eluate was collected and dried in a rotary evaporator (at 40 °C) to absolute dryness. Then the sample was derivatized by adding 100 pl of MSTFA and 50 pl pyridine, heated at 80 °C for 1 h, diluted with toluene, and analyzed by GC-MS. The analyses were carried out in triplicate. The accuracy of the extraction process was determined through the recovery value in % of the PAs. Table 2. Sample extraction by SPE (using HLB Supelco® cartridges). Sample extraction by SPE 1. pre-washing of cartridge with 2 x 2 mL DCM 2. column conditioning: 2 x 2 mL of MeOH and 2 x 2 mL acidified water (pH = 1-2) 3. sample application: 2 mL of the acidified sample 4. column washing: 2 x 2 ml ultra-pure water 5. elution: 2 x 2 ml THF For the determination of free PAs in selected red wines, the samples were prepared according to the same procedure. 1 mL of homogenized wine sample was spiked with 50 pL of ISTD, diluted with 1 mL of ultra-pure water and acidified with 6 M HCl to a pH value of 2, followed by the previously described steps. 2. 6. 2. Alkaline Hydrolysis of PAs The stability of the compounds and their recovery percentage after alkaline hydrolysis was first determined Table 1. Characteristics of the analyzed wine samples. Sample code Variety Variety code Winery Wine region Year of production % alcohol* SW1 Cabernet Sauvignon 1 „Vina Koper" Primorska 2014 13 SW2 Modra Frankinja 2 „Stari Hram" Posavje 2014 10.5 SW3 Cabernet-Sauvignon 1 „Vipava" Primorska 2014 11 SW4 Modri Pinot (Pinot noir) 2 Štajerska Slovenia-Ptuj Podravje 2011 12.5 SW5 Cabernet Sauvignon 1 Goriška Brda Primorska 2013 12.5 SW6 Refošk 3 Srednje Škofije Primorska 2014 11 SW7 Refošk 3 „Vina Koper" Primorska 2014 12.5 SW8 Modra Frankinja 2 Štajerska Slovenia-Ptujska Klet Podravje 2011 11.5 SW9 Modri Pinot (Pinot Noir) 2 „Vipava" Primorska 2013 12 SW10 Portugalka 3 Bela Krajina Posavje 2015 11 SW11 Cabernet Merlot 1 Jeruzalem-Ormož Podravje 2013 12.5 SW12 Metliška Črnina 2 Bela Krajina Posavje 2012 11.5 'According to the declaration on the wine bottle. with the standard compounds and later an optimized procedure was used on the real wine samples. Standard solution of PAs mixture (at a concentration of 1000 mg L-1) was prepared in MeOH. Solutions of PAs mixture in synthetic wine were prepared by pipetting 30 and 100 pL of standard solution, respectively, into the 10 mL volumetric flask, and diluted with the synthetic wine up to the mark. 1 mL of each solution was transferred into a 50 mL conical flask, spiked with 50 pL of ISTD, and exposed to alkaline hydrolysis, according to the previously described method with some modifications.35 1 mL of the spiked synthetic wine was treated by adding 9 mL of 2 M NaOH (which contained 1% L-ascorbic acid and 10 mM EDTA as stabilizers) for 2 h at room temperature. Then the sample was acidified to pH 2 using 6 M HCl, and PAs were extracted with SPE HLB cartridges. The whole procedure with alkaline hydrolysis was repeated also without stabilizers. 2. 7. Quantitation of PAs The contents of free and total PAs were determined from the corresponding calibration curves using the ISTD method, taking into account the recovery of the extraction procedure. PAs from the cinnamic group exist in transand cis-forms, both found in plants. Trans-forms of PAs are naturally predominant isomers. Therefore, for quantitative determination, the peak areas of the trans- and cis- forms of caffeic acid, p-coumaric acid and ferulic acid were summed. 2. 8. Statistical Analysis Chemometrical data analysis was carried out in order to discover any statistically or other significant differences between the samples grouped according to two categorical variables - wine variety and wine region. Microsoft Excel was used for the data preparation and result outputs. Statistical data treatment was performed using SPSS Statistics version 22. 3. Results and Discussion Our study tested isolation and quantitative determination of five target PAs (caffeic acid, vanillic acid, syrin-gic acid, p-coumaric acid and ferulic acid) in red wine samples using the GC-MS method. All GC-MS SCAN parameters for trimethylsilylated standard compounds, together with their retention times (tR) and characteristic fragment ions, are listed in Table 3. Linear regression analysis proved that the responses for all of the investigated compounds were linear over the tested concentration range (1-100 mg L-1), and correlation coefficients (r2) were above 0.999. The results of the regression analysis and calibration data are shown in Table 4. Table 4 Table 3. Retention times and fragmentation parameters for trimethylsilylated PAs obtained after trimethyl-silylation using the ion-trap mass detector. Compound tD Characteristic fragmentation ions m/z (relative intensity %) cis-o-Coumaric acid Vanillic acid cis-p-Coumaric acid trans-o-Coumaric acid Syringic acid cis-Ferulic acid trans-p-Coumaric acid cis-Caffeic acid trans-Ferulic acid trans-Caffeic acid 16.65 17.70 17.94 18.18 19.11 19.32 19.49 19.99 20.95 21.38 147(100), 293, 308 253, 267, 282, 297(100), 312 219, 249, 293 (100), 308 147, 219, 293 (100), 308, 381 298, 312, 328, 342 (100) 249, 293, 308, 323, 338(100) 219, 250, 293 (100), 308, 381 219, 381, 396 (100), 397 249, 293, 323, 338 (100) 73, 219, 381, 396 (100) Table 4. Validation parameters for investigated PAs. PA Linear correlation r2 1WD-RSD 2BD-RSD LOD* LOQ* Vanillic acid y = 0.0477x + 0.0771 0.9999 0.11 0.72 0.05 0.09 Syringic acid y = 0.0231x + 0.0921 0.9999 0.95 1.81 0.06 0.12 ^-Coumaric acid y = 0.0398x + 0.0555 0.9996 0.38 1.47 0.06 0.13 Caffeic acid y = 0.0558x + 0.0986 0.9999 1.36 1.97 0.07 0.15 Ferulic acid y = 0.0324x + 0.0718 0.9996 1.01 1.81 0.03 0.09 1 Within-day PA/ISTD peak-area ratio repeatability of individual PAs at the concentration 10 mg L expressed as %RSD. 2 Between-days PA/ISTD peak-area ratio repeatability of individual PAs at the concentration 10 mg L-1, expressed as %RSD. * LOD and LOQ are in mg L-1. also shows the within-day (WD) and between-days (BD) repeatability expressed as relative standard deviation (RSD), and it gives RSD below 2% in all cases. The determined values of LODs and LOQs for all selected PAs are also shown in Table 4. From the literature it is well known that anthocyanin-type pigments can cause great interference in the chromatographic separation and identification of non-anthocyanin phenolic compounds.36 In our study, anthocyanins were successfully removed using HLB cartridges. Another ad- vantage of HLB cartridges over conventional C18 columns in the separation of phenolic compounds are that more polar interferences (e.g. sugars) can be eliminated with water without losing analytes, higher sensitivity, good repeatability, reproducibility, and high percentages of recovery were reported by Perez-Magarino et al., 2008.37 Accuracy of SPE in determining free PAs was evaluated by spiking a synthetic wine with the standard solution at two different concentrations levels (30 and 100 mg L1). The recovery of free PAs ranged from 93% to 114% RIC MnH cr iOtit ikn.au 100" 6 i rfc 2 e Is; 1? 1 II La V IS » JO 1 - " " II ■nullit? Srz 2. lilt. Tunc: $.00 31.00. Fl Auto KitU. 50 650 in i . L . J.. .1. L. Figure 1. Chromatograms of standard solutions after; a) hydrolysis in presence of stabilizer (1. vanillic acid; 2. trans-o-coumaric acid; 3. syringic acid; 4. «s-ferulic acid; 5. trans-p-coumaric acid; 6. trans-ferulic acid; 7. trans-caffeic acid; 8. «s-p-coumaric acid; «s-caffeic acid (minimal peak at tR 19.99 min); b) hydrolysis without stabilizer (1. vanillic acid; 2. trans-o-coumaric acid; 3. syringic acid; 4. «s-ferulic acid; 5. trans-p-couma-ric acid; 6. trans-ferulic acid; 7. trans-caffeic acid (missing peak); 8. as-p-coumaric acid. Table 5. Determination of the method accuracy expressed as recovery (%). Recovery of extraction procedure (%) Bound PAs Phenolic acid Free PAs In the presence Without of of stabilizer a stabilizer 30 100 Concentration (mg L-30 4) 100 100 Vanillic acid 105.5 98.9 114.2 105 113.7 Syringic acid 106.8 93.6 107.5 101 105.4 p-Coumaric acid 101.7 106 110.5 103 135.1 Ferulic acid 94.5 96 97.4 104.4 133.7 Caffeic acid 102.7 106 105.9 96.32 NQa a NQ-not quantified. Concentration (mg L ')