The concentration of selected key aroma compounds was measured by a range of approaches on five R&G coffees. The relative abundance of the key aroma compounds in the headspace above the R&G coffee (R&G SPME TOF) and above the coffee brew (Brew SPME TOF); and the concentration of select key aroma compounds in the R&G (MASE GC-MS) and in the coffee brew (LLE GC-MS) was measured. Analytical approaches were chosen to represent key user liking criteria (for example aroma on opening the pack, aroma on brewing, aroma in the coffee beans and aroma in-cup on consumption) and all were shown to reliably measure key volatile compounds present in R&G coffee and coffee brew.
R&G arabica coffee was purchased from a commercial source in the United Kingdom; their origins are defined as Costa Rica, Java, Brazillian Daterra, Colombian and an espresso preparation (country of origin not disclosed on packaging). These were chosen as R&G coffee beans from the named locations have previously been shown to offer repeatable discrimination by aroma chemistry profiles[23, 28]. Samples were frozen on day of purchase at −80°C for no longer than 90 days.
Key aroma compounds
Aroma compounds of interest were previously identified by odour-port analysis as per Ullrich (by the method of aroma extract dilution analysis) and are defined as key aroma compounds of R&G coffee[23, 30], compounds identified as having high seasonal variability (>10% CV inter-batch variation) or rapid destabilization over storage (for example oxidation or polymerization) were excluded from this study, In addition, other compounds were not included in this paper for confidentiality reasons.
Liquid-liquid sample preparation
Volatiles were extracted (20 min) from 4 g of R&G coffee brew using liquid-liquid extraction (LLE) with tertiary butyl methyl ether as the solvent (2 mL) above 2 g of anhydrous sodium sulphate, solvent was isolated by centrifugation (8000 RCF) and isolated solvent was analysed by direct injection GC-MS.
Membrane assisted solvent extraction sample preparation
A total of 1.5 g of R&G coffee was dispersed in 10 mL of distilled water and capped in a membrane assisted solvent extraction (MASE) vial, Gerstel (Mülheim, Germany). One millilitre of TMBE was injected into the cap and the sample allowed to extract (75 min). Samples were centrifuged (8000 RCF) and solvent isolated by aspiration and analysed by direct injection GC-MS.
Solid phase solvent extraction sample preparation
Samples (5 g in 25 mL vial) were incubated for 15 min at 60°C and exposed to a 50/30 DVB/Carboxen PDMS solid phase micro extraction (SPME) fiber for 15 min before direct thermal desorption within the GC-injector, with the inlet temperature set at 200°C.
GC × GC TOF MS
Chromatography was achieved with a Leco GC × GC (modified Agilent 7890A, MI, USA) equipped with a split/splitless injector containing a deactivated single tapered split liner and a liquid nitrogen, dual stage quad-jet thermal modulator (Leco, MI, USA). In the first dimension a Varian VF-5MS 15 m × 0.25 mm × 0.25 μm column (Middelburg, the Netherlands) was used. In the second dimension an Agilent DB-1701 column (1 m × 0.10 mm × 0.10 μm, Santa Clara, CA, USA) was used. A 20:1 split flow was used resulting in a total flow of 21 mL/min set to constant flow. The inlet temperature was set to 200°C and the transfer line temperature set to 250°C. Oven programming was set to an initial target temperature of 40°C for 30 s then increased at a rate of 10°C/min to a target temperature of 260°C. The secondary oven was set to an initial temperature of 50°C for 30 s then increased at a rate of 10°C/min to a target temperature of 270°C.
A dual stage quad-jet thermal modulator was used. The compounds reached the modulator and were trapped for 0.6 s then re-injected at a 30°C offset relative to the secondary oven. This temperature was held for 0.9 s with a total modulation time of 3 s.
Detection was by mass spectrometer (LECO Pegasus® 4D Time-of-Flight mass spectrometer, MI, USA): detection range 35–600 amu, acquisition rate 200 spectra/s, voltage 1550 V and a filament bias voltage of −70 V. The ion source was set to 200°C and the mass defect mode was set to manual.
Direct injection GC-MS
An Agilent 6890 gas chromatograph coupled with an Agilent 5975 mass spectrometer, equipped with Gerstel automated robot and a mid-polar Varian Factor Four™ (VF-1701 ms) column was used for the GC-MS analysis. Inlet temperature of the GC was set at 270°C and helium was the carrier gas with a column flow rate of 1.0 mL/min in splitless mode. The oven parameters used were: 40°C with no hold, rising to 270°C at a rate of 30°C/min, holding for 1.33 min. The injector temperature was constant at 280°C with an injection volume of 1 μl. The mass spectrometer operated in the electron ionization mode with an ion source temperature of 230°C and a quad temperature of 150°C. The full-mass range mode was used for the analysis of the standards with a mass range of m/z 40–200 amu run in SIM/SCAN mode.
Key aroma compounds of interest were identified using mass spectra, retention time and authentic standards. Concentrations were calculated against internal standards (1-pentanol, 4-heptanone) added prior to extraction, response factors were calculated for differential MS response and differential partition coefficients for each compound.
Calibration curves were generated in triplicate at five concentration points with authentic standards of all key aroma compounds, the concentrations varied depending on analytical approach but in all cases the upper calibration point exceeded the maximum analysis concentration by two-fold. In all cases analytical reproducibility across multiple samples from a single production batch was <10% CV.
The absolute mV response for each internal standard was tracked for each method and any deviation from normal distribution, trends towards abnormality or unexpected results resulted in machine clean down and recalibration.
Samples (2 g) were tested for moisture content as per Fisk et al. to ensure that any significant deviation between origins would not impact the evaluation; there was no significant difference between the batches, P < 0.05 by ANOVA.
Triplicate samples were prepared from within a single production code of each sample set, samples were then analysed in duplicate by each method. Absolute concentration data was then evaluated for its discriminatory ability using principle component analysis and multivariate factor analysis, XLSTAT 2011 (Addinsoft, Anglesey, Wales), for data illustration the results are normalized to the Costa Rica preparation for each analytical approach.
Partition coefficients were calculated by EPI suite (US Environment Protection Agency, New York, NY, USA).