GERSTEL Solutions No. 7 (pdf; 1,86 MB)

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GERSTEL Solutions Worldwide Application Samples are being extracted by Twister stir bars prior to GC/MS analysis at the Asahi R&D Centre. MPS-GC/MS system used by Asahi for the analysis of terpenoids in hops. found. Kishimoto and his colleagues attribute this to the poor solubility of terpenoids in wort. -citronellole was not detected in wort samples at all. It is formed from geraniole, but not until the fermentation step of the brewing process. It was observed Total Ion Chromatogram of Japanese Beer that the reduction in terpenoid concentrations follows two different patterns. The first pattern was observed for myrcene and linaloole, whose concentrations decreased rapidly during the wort boiling process. The rapid decline is mainly attributed to the relatively low boiling points of these compounds (myrcene 167 °C, linaloole 194 °C). For comparison, humulene and farnesene have boiling points of 266 °C and 260 °C respectively. Conclusion: „The hops should not be added to the wort until the very end of, or even until after the wort boiling process” according to Kishimoto et al. The second pattern they observed was seen for higher boiling compounds, for example, -eudesmole, humulene, humuleneepoxide I, -farnesene, caryophyllene and geraniole: The concentrations of these compounds decreased slowly and linearly as a function of time over the course of the wort boiling process. „Both observations support our contention, that the degree to which a beer has a hop-based character is directly dependent on the point in time at which the hops were added to the brew“, the scientists said. Comparison between chromatograms from beer samples using standard liquid extraction (top) and Stir Bar Sorptive Extraction (SBSE) with the GERSTEL Twister (bottom). The result is extremely clear, according to Toru Kishimoto: ”SBSE is an extremely sensitive analysis technique, providing us with very low detection limits. It operates without the use of solvents, is easy to handle, produces first rate results, and enables the determination of a large number of compounds in one analytical run.“ Terpenoids Terpenoids, sometimes referred to as isoprenoids, are extensively used for their aromatic qualities. Terpenoid profiles form important characteristics, even enabling the identification of plants by their profile. More than 30,000 terpenoids are known, 8000 of which also belong to the chemical compound group terpenes. Source: wikipedia 6 GERSTEL Solutions Worldwide – May 2007
GERSTEL Solutions Worldwide Application Materials, Reagents and Methods Reagents Linaloole, geraniole, myrcene, caryophyllene, -humolene, -damascenone and -damascone from Fluka (Steinhaus, Switzerland); - citronelle and cis-3-Heptene-1-ol from Sigma-Aldrich (St. Louis, MO) and from Avocado Research Chemicals Ltd. (Lancashire, U.K.). Eudesmol, -farnesene and synthetic humulene epoxide from Wako (Osaka, Japan). All reagents were Analytical Grade. SBSE Method: CV and Detection Limits CV (%) Detection Limit (ppb) (S/N=3) Linalool 4.3 0.049 Geraniol 5.7 0.099 Citronellol 6.8 0.278 Myrcene 7.4 0.001 Caryophyllene 8.2 0.031 a-Humulene 4.2 0.035 Humulene epoxide1 2.5 0.044 Eudesmol 5.5 0.013 b-Farnesene 6.7 0.023 b-Damascenone 2.0 0.019 Liquid extraction using methylene chloride A 350 mL beer sample was taken and 5 μL of a cis-3-heptene-1-ol solution added as internal standard. Liquid extraction was performed by adding 150 mL dichloromethane and allowing the extraction to proceed for 3 hours. After separating the dichloromethane, it was dried with anhydrous sodium sulphate for 30 minutes and concentrated to approximately 1 mL in a rotary evaporator at 40 °C under vacuum (750 hPa). GC/MS method used for the liquid extract An Agilent ® Technologies GC 6890 coupled with a 5973 N MSD quadropole mass spectrometer was used for the analysis. The column used was a DB-WAX, 60 m length x 0.25 mm i.d., 0.25 μm film thickness from Agilent Technologies. GC: 1 μL extract was introduced to the GC in splitless, pressure pulsed mode. Inlet temperature: 250 °C. Helium was used as carrier gas at constant flow of 1 mL/ min. Oven temperature program: Initial temperature 40 °C , held for 5 min. Ramped at 3 °C /min to 240 °C and held for 20 min. MSD: Scan range: 30 – 359 (m/z); EI mode, ionization energy: 70 eV. All compounds were identified on the basis of their mass spectra as well as on the retention time. Standard mixtures were used in order to compare spectra and retention times with those of authenticated compounds. Sample Preparation for Stir Bar Sorptive Extraction (SBSE) using the GERSTEL Twister ® The GERSTEL Twisters used were 20 mm long, coated with 47 μL Polydimethylsiloxane (PDMS). Prior to their use, the Twisters were conditioned under a flow of Helium for one hour at 300 °C. 0.1 ppb -damascenone was added to the sample (beer or wort) as internal standard. A 30 mL sample was diluted fourfold with distilled water and the Twister added. The vial was sealed and was stirred using the Twister for 2 hours at 40 °C. Following the extraction step, the Twister was removed from the sample, rinsed with distilled water and dabbed dry on lint-free paper cloth. The Twisters were desorbed in the Thermal Desorption Unit (TDU), mounted on the GERSTEL CIS inlet. Using the GERSTEL MultiPurpose Sampler (MPS) up to 196 Twisters can be processed in one automated sequence. Prior to GC/MS determination, analytes were concentrated in the Cooled Injection System (CIS) and subsequently transferred to the column using a CIS temperature program. Analyte quantitation was performed using the following m/z target ions: 69 (geraniole), 80 (-humulene), 85 (humulenole), 93 (linalo- Literature [1] Lam, K. C.; Foster, R. T.; Deinzer, M. L. Aging of hops and their contribution to beer flavor. J. Agric. Food Chem. 1986, 34, 763-779. [2] Steinhaus, M.; Schieberle, P. Comparison of the most odor-active compounds in fresh and dried hop cones (Humulus lupulus L. variety spalter select) based on GC-olfactometry and odor dilution techniques. J. Agric. Food Chem. 2000, 48, 1776-1783. [3] De Keukeleire, D.; David, F.; Haghebaert, K.; Sandra, P. Automated reporting on the quality of hops and hop products.; J. Inst. Brew. 1998, 104, 75-82. [4] Irwin, A. J. Varietal dependence of hop flavour volatiles in lager. J. Inst. Brew. 1989, 95, 185-194. [5] Goiris, K.; De Ridder, M.; De Rouck, G.; Boeykens, A.; Van Opstaele, F.; Aerts, G.; De Cooman, L.; De Keukeleire, D.; The oxygenated sesquiterpenoid fraction of hops in relation to the spicy hop character of beer. J. Inst. Brew. 2002, 108, 86-93. [6] Lermusieau, G.; Bulens, M.; Collin, S. Use of GC-olfactometry to identify the hop aromatic compounds in beer. J. Agric. Food Chem. 2001, 49, 3867-3874. Toru Kishimoto and co-workers in the Asahi R&D Centre laboratories. ole, myrcene, -caryophyllene and -farnesene), 123 (-citronellole and humulen epoxide I), 149 (-eudesmole), 177 (-damascenone, internal standard) and 190 (-damascone). Thermal desorption and GC/MS method parameters Flavor compounds concentrated in the Twister PDMS phase were subsequently thermally desorbed in the GERSTEL TDU using the following temperature program: Initial temp. 25 °C, hold time 0 min, ramp rate 2.5 °C/min to 240 °C, hold time 5 min. Analytes were refocused in the GERSTEL CIS at –100 °C using LN2 cooling; CIS Mode: splitless. At the start of the GC/MS run, the CIS temperature program was started, releasing the focused analytes to the GC column. CIS program: Initial temp. –100 °C; hold time 0 min; ramp rate 2 °C/min to 240 °C; hold time 5 min. GC/MS parameters were identical to those listed above under liquid extraction with dichloromethane. [7] David, F.; Sandra, P.; Hoffmann, A.; Harms, D.; Nietzsche, F.; Elucidation of the Hoppy Aroma in Beers by Stir Bar and Headspace Sorptive Extraction followed by Thermal Desorption – CGC-MS/PFPD: http://www.gerstel.com/an_2001_04.htm (accessed 30th May 2005), Gerstel Application Notes 4/2001. [8] Demyttenaere, J. C. R.; Sanchez Martinez, J. I.; Verhe, R.; Sandra, P.; De Kimpe, N. Analysis of volatiles of malt whisky by solid-phase microextraction and stir bar sorptive extraction. J. Chromatogr. 2003, 985, 221-232. [9] King, A. J.; Dickinson, J. R. Biotransformation of hop aroma terpenoids by ale and lager yeasts. FEMS Yeast Res. 2003, 3, 53-62. [10] Maylan, M. W.; Howard, P. H. Atom/Fragment contribution method for estimating octanol-water partition coefficients. J. Pharm. Sci. 1995, 84, 83-92. GERSTEL Solutions Worldwide – May 2007 7
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