Aroma Profiling What? Why? How? Aroma profiling • What? - Aroma • From Wikipedia; An aroma is a volatilised chemical compound that humans or other animals perceive by the sense of olfaction (the sense of smell). • An aroma compound, also known as odorant, aroma, fragrance, or flavour, is a chemical compound that has a smell or odor. A chemical compound has a smell or odor when it is sufficiently volatile to be transported to the olfactory system in the upper part of the nose. • Volatile organic compounds (VOCs) are organic chemicals that have a high vapor pressure at ordinary room temperature. VOCs are numerous, varied, and ubiquitous. They include both human-made and naturally occurring chemical compounds. Most scents or odours are VOCs • NOT ALL Volatile compounds are Aroma active.. Aroma profiling • What? – Profiling • From Wikipedia; Profiling, the extrapolation of information about something, based on known qualities 10.13 39.97 45.83 70 40.81 65 8.93 43.59 60 55 22.26 50 45 12.18 36.98 40 14.16 35 48.74 30 4.00 25Relative Abundance 20 33.10 30.32 5.37 21.62 56.90 15 7.38 49.26 21.01 29.00 10 17.92 27.74 23.14 34.25 27.11 5 16.32 49.79 55.49 0 5 10 15 20 25 30 35 40 45 50 55 Time (min) Aroma profiling Aroma profiling • When? During the Before the raw During the During the storage, material processing/ Final storage of raw transport and enters the materials product retail of the process Product manufacture end-product Aroma profiling • How? Choice of methods • Liquid extractions – exhaustive and quantitative? • Headspace methods? • Aroma active/key compounds can have a range of chemical and physical properties and it is not always safe to assume that all compounds can be extracted using one analytical approach. Analytical Challenges • Extremely low levels can be relevant • Complex and variety of matrices – Understand what is ‘normal’ – Selectivity vs sensitivity – Trace contaminant vs matrix components • Homogeneity – Consider sampling • Potential for sample contamination (lab environment) • Sensory descriptors (consumer vs. experts) • Sample not changed during extraction • Screening or targeted analysis? • Does everything need to be identified? Flavour Volatiles - Key Aroma Compounds 10.13 39.97 45.83 70 40.81 65 8.93 43.59 60 55 22.26 50 45 12.18 36.98 40 14.16 35 48.74 30 4.00 25Relative Abundance 20 33.10 30.32 5.37 21.62 56.90 15 7.38 49.26 21.01 29.00 10 17.92 27.74 23.14 34.25 27.11 5 16.32 49.79 55.49 0 5 10 15 20 25 30 35 40 45 50 55 Time (min) Choice of instrument Choice of instrument GC-FID, retention time GC-MS, GC-MS/MS Retention time and Retention Mass spectra time, MRM transition GC-QTOF, Retention time, Mass spectra , accurate mass isotope ratios, Q to confirm product ion spectra Accurate mass QTOF • Complex TIC chromatogram Look for a key analyte - extract most abundant mass TOF vs Single Quad data x10 7 +EI TIC Scan KR240915_06.D 1 8 TIC 6 4 2 0 +EI EIC(152.0468) Scan KR240915_06.D x10 5 Noise (PeakToPeak) = 19422.08; SNR (22.54min) = 15.9 1 22.54 3 Nominal mass extracted ion 860561.53 2 Calculated s/n 15.9 1 0 +EI EIC(152.0468) Scan KR240915_06.D x10 5 Noise (PeakToPeak) = 5292.46; SNR (22.54min) = 58.8 1 22.54 3 Accurate mass (20ppm) extracted ion 891081.32 2 Calculated s/n 58.8 1 0 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Counts vs. Acquisition Time (min) Improved sensitivity and selectivity Mass accuracy and resolution Single Quad (Unit mass) ToF 113 114 115 116 Resolution = • Mass Accuracy= (Measured Mass/∆Mass Mass-Theoretical)/ (Theoretical / 1000000) =100/0.01 = (100.0005-100.000)/(100.000 / 99.99 100.01 =10,000 1000000) ∆M = 5 ppm • Typical MS Resolution High 100.00 12000 Low 7000 Aroma profiling Choice of sample preparation procedure • Why do sample Prep? • Removal of matrix interferences • Increased selectivity • Improved chromatography • Analyte enrichment • Increase sensitivity – achieve lower limits of detection • Reduce instrument maintenance • The ideal sample prep Selective(?) sensitive, minimum number of steps, environmentally friendly, robust, Automated? Automation options MultiPurpose Cooled Automated easy Liner Sampler MPS Injection Liner Exchange System CIS EXchange eLEX ALEX MultiFiber Thermal Thermal Automated TDU EXchange Desorption Desorption Liner Exchange MFX System TDS Unit TDU ATEX Twister Dynamic TDU PYRO µFlowManager Headspace DHS Disposable Selectable Olfactory Preparative Pipette 1D/2D Detection Fraction GC/MS Port OPD Collector PFC Extraction DPX MultiPosition MAESTRO Solid Phase Evaporation PrepAhead Extraction SPE SPME Station mVAP Filtration Balance mVorx Sample preparation; Part 1 Liquid extraction techniques Liquid extraction techniques • Extraction from a liquid sample (or extract) by partition into a liquid phase – Liquid-liquid/ Solvent extraction – Steam distillation extraction (SDE) – Solvent Assisted Flavour Extraction (SAFE) – Stir Bar Sorptive Extraction (SBSE) Liquid-liquid/ Solvent extraction • Liquid – Liquid/Solvent Extraction – Selectivity through choice of solvent – Traditionally uses a large volume of solvents – Often need for subsequent concentration step – Glassware- potential for contamination/losses Solvent Extraction using mVORX • Vortex and Orbital shaker for the MultiPurpose Sampler (MPS) • Automated Liquid/Liquid extraction – Extraction of solids with solvent Solid phase extraction • Uses liquid-solid partition - sorbent is the extracting phase • Extracts and concentrates analytes from liquid samples or solutions Step 1 Step 2 Step 3 Step 4 Conditioning Retention Rinsing Elution C B A B A D B C D A Instrument Top Sample Preparation • Small Scale Solid phase Extraction – 15-35 mg packing comprehensive range of sorbents (ITSP specials) – Typical particle size 30-60 micron (100 Amstrong) 34 mm Automated Sample Prep 10 ul Syringe 2.5 ml HS Syringe • Multiflex – Consists of Dual Head MPS •Thermal Desorption unit – Cold Inlet System - CIS Close up of Tray Application: Water industry (NDMA and Meltaldehyde) Coconut Charcoal ITSP cartridges (NDMA) ENV (Metaldehyde) Right MPS (2.5 ml Headspace syringe) Conditioned 750 µl dichloromethane 1000 µl of methanol Equilibrated 2000 µl of HPLC grade water Load 10 ml of sample (in water) Dried 15 minutes Eluted 400 ul dichloromethane X 25 concentration Left MPS (10 ul) Large Volume injection Large Volume Injection method– removing DCM boiling point 40 °C, Metaldehyde and NDMA both exceed 100 °C Inlet kept at 10 °C (peltier cooled) Slow injection speed at 0.5 ul/s (to remove DCM) ramped to 250 °C (12 °C /s) NDMA (similar for Metaldehyde) mVAP • Automated evaporative concentration of multiple samples or extracts in parallel – Improved detection limits through concentration of liquid samples or extracts – Enables automated solvent exchange prior to instrumental analysis Co-distillation extractions (SDE and SAFE) SDE SAFE Further concentration step required Twister™ (Stir Bar Sorptive Extraction) Twister SBSE – SBSE Stir Bar Sorptive Extraction Phase Polydimethylsiloxane - thickness 0.5 mm, 10 mm length (24 µl) (PDMS) - thickness 0.5 mm, 20 mm length (47 µl) - thickness 1.0 mm, 10 mm length (63 µl) - thickness 1.0 mm, 20 mm length (126 µl) Magnet Can be over 100 fold increase in concentration 100ml sample to <100µl extraction phase Method • Twisters pre-conditioned • Take an aliquot of water/sample (10-100 ml) • After stirring for 2 hours Theory of Twister SBSE – Example (Methylisoborneol) – Log K o/w = 3.31 Twister SBSE Applications • Over 400 publications since 1999 • Extraction of nonpolar compounds (LogKow > 2.0) • Food, flavour, natural products – Taints and off flavours – Profiling of alcoholic beverages – Plant stress volatiles – Fragrances in the environment • Biological fluids, tissues • Polymer/packaging- leachables, extractables, BPA, PBDE • Environmental Applications Twister SBSE – PAH solution • 100 ml water samples (2 hours) – Dried and placed in TDU tubes – SIM 16 PAH (0.02 ug/l to 1 ug/l) – Acenaphthene 0.999 (1-2%) Cypermethrin method • Target LOD 0.01 ng/L • 2 hour SBSE extraction with PDMS Twister™ • Thermal desorption with TDU/CIS followed by analysis by GC-MS/MS (EI) 163 100 Log Kow 5.3 181 Cl O Cl O 50 91 N O 77 127 51 209 152 39 65 115 27 55 83 99 141 191 15 215 224 235 244 254 265 280 289 310 319 343 353 379 415 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 (mainlib) Cypermethrin Initial results Reproducibility: Water @ 0.1 ng/L cypermethrin (n=6,)RSD 9% (without internal standard) Cypermethrin using Twister 16000 14000 R² = 0.9932 12000 10000 8000 6000 4000 2000 0 0 0.1 0.2 0.3 0.4 ng/L 0.5 0.6 EG-Silicone • Sorbent phase is a mixture of silicone and ethylene glycol – Efficient concentration of non-polar analytes similar to the PDMS Twister – Concentration of polar analytes that form hydrogen bonds acting as proton donors, for example phenols – Low limits of detection and good recovery due to large phase volume Twicester • Magnetic positioning of up to three Twisters using Twicester • Multiple (mSBSE) using two or more Twisters • Simultaneous thermal desorption of the Twisters, Cryofocusing in the CIS, and GC/MS analysis Thermal Desorption Step 1: Sample (in TDU tube) is placed in hot zone for desorption. Desorption process is most efficient at high temperature and high gas flow rate (20-100 mL/min). Step 2: Analytes are refocused in a cooler zone of smaller dimensions.