Aroma Profiling What? Why? How?

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

55 22.26

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

55 22.26

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

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

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

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.

Step 3: Analytes are transferred from refocusing trap into column. Trapping efficiency will depend on temperature, flowrate, and packing material (inert or adsorbent). TDU and CIS

Thermal Desorption Unit

TDU liner No Transferline! TDU

CIS liner CIS CIS

Cooled Injection System TDU

• Universal Thermal Desorption Unit TDU liner with frit for thermal extraction of solid samples

TDU liner packed with adsorbent

TDU liner for Twister desorption

TDU liner for thermal extraction in µ-vials ATEX – Automatic Tube Extraction -look at volatiles in a nonvolatile matrix

• In combination with GC-QTOF Highly selective and sensitive Mass spectrometer • Complex, dirty matrix, low level target analytes Sample Preparation; Part 2

Headspace techniques • SPME (Solid Phase Micro Extraction)

• HSSE (Headspace Sorptive Extraction)

• Static Headspace

• Dynamic Headspace • SPME (Solid Phase Micro Extraction)

• HSSE (Headspace Sorptive Extraction)

• Static Headspace

• Dynamic Headspace Solid Phase Micro Extraction (SPME)

Plunger

Barrel

Color-coded Screw Hub

Sealing Septum Retaining Nut

Fiber Sheath Fiber (pierces septum Attachment Rod of sample vial and GC injector) SPME Fiber SPME: sampling mode

Direct immersion Headspace SPME SPME (HS-SPME) • SPME (Solid Phase Micro Extraction)

• HSSE (Headspace Sorptive Extraction)

• Static Headspace

• Dynamic Headspace Headspace Sorptive Extraction (HSSE) Twister (Stir Bar Sorptive Extraction) 4 types: - l = 10 mm, 0.5 mm thickness - l = 10 mm, 1.0 mm thickness - l = 20 mm, 0.5 mm thickness - l = 20 mm, 1.0 mm thickness Magnet Polydimethylsiloxane (PDMS) Amount of PDMS phase is substantially greater than SPME Thermal Desorption set up

Thermal Desorption Unit - TDU

No Transfer line !

Cooled Injection System - CIS HS Twister Applications

• Volatiles in chocolate

%RSD (Headspace Analyte twister) Methylpyrazine 8.2 2,5-Dimethylpyrazine 3.9 2,3-Dimethylpyrazine 9.0 2-Nonanone 6.1 Trimethylpyrazine 2.8 3-Ethyl-2,5dimethylpyrazine 2.4 Tetramethylpyrazine 1.9 Butyrolactone 6.2 3-Methylbutanoic acid 8.5 Hexanoic acid 9.0 Maltol 9.8

Precision based on 5 chocolate extractions HS Twister Applications

• Volatiles in Bee Hives

• Well suited to GC/Q-TOF – Complex matrix • Deconvolute with sub-unit mass resolution

Mass accuracy 3ppm n-hexadecanoic acid HS Twister Applications

• Volatiles in Bee Hives

PDMS

EG • SPME (Solid Phase Micro Extraction)

• HSSE (Headspace Sorptive Extraction)

• Static Headspace

• Dynamic Headspace Static Headspace Static Headspace

Heated Syringe

Heated Vial Multiple Headspace Sample Enrichment (MHSE)

Several samples are taken from the same vial.

The analytes are focused on a packed bed liner in the cold PTV liner during multiple sample introductions.

Benefit: Increased sensitivity Multiple Headspace Sample Enrichment (MHSE) MHSE on Herbal Based Liqueur • SPME (Solid Phase Micro Extraction)

• HSSE (Headspace Sorptive Extraction)

• Static Headspace

• Dynamic Headspace Dynamic Headspace (DHS) Dynamic Headspace (DHS) Thermal Desorption

Thermal Desorption Unit

Cooled Injection System Dynamic Headspace (DHS)

DHS traps:

TDU Liner with frit packed with:

- Tenax TA (ca. 60 – 70 mg) - Tenax GR - Carbopack B, Carbopack X - Carbopack B, Carbopack X, Carboxen 1000 - Carbopack C, Carbopack B, Carbosieve SIII

Dynamic vs Static Headspace Gin, split 10:1

3000000 100 mL

2500000

2000000

1500000 1000000 60°C 500000

0 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

3000000

2500000 1 mL

2000000

1500000 1000000 60°C 500000

0 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 Dynamic Headspace (DHS): Multi-Volatile Method

Sequential DHS sampling Different trapping conditions

New DHS trap for very volatile compounds: Shincar-bon X/Carbopack B/Carbopack X (available since mid March) Dynamic Headspace (DHS): Multi-Volatile Method

Journal of Chromatography A Multi-volatile method for aroma analysis using sequential dynamic headspace sampling with an application to brewed coffee Nobuo Ochiaia,∗, Jun Tsunokawaa, Kikuo Sasamotoa, Andreas Hoffmann Dynamic Headspace (DHS): Multi-Volatile Method

• Soap Dynamic Headspace (DHS): Multi-Volatile Method

• Whisky, reproducibility (n=5) Ethyl dodecanoate * * Butyl acrylate Benzyl 2,3,4,5,6 d5 alcohol hexadecenoate MS off - Ethyl 9 Ethyl acetate Ethyl lactate Ethyl myristate Diethyl succinate

RSD = 2.3% for the butyl acrylate RSD= 4.7 % for the benzyl 2,3,4,5,6 d5 alcohol Dynamic Headspace (DHS): Multi-Volatile Method

• Whisky

SPME extraction DVB/CAR/PBMS fiber 35ᵒC

DHS single extraction Tenax extraction 80ᵒC

MVM extraction Shincarbon X/Carbopack B+X 30ᵒC Tenax 80ᵒC Dynamic Headspace (DHS): Multi-Volatile Method

• Whisky

SPME extraction DVB/CAR/PBMS fiber 35ᵒC

DHS single extraction Tenax extraction 80ᵒC

MVM extraction Shincarbon X/Carbopack B+X 30ᵒC Tenax 80ᵒC MultiPurpose Cooled Automated easy Liner Sampler MPS Injection Liner Exchange for GC/MS 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

Selectable Olfactory Preparative MultiPurpose 1D/2D Detection Fraction Sampler MPS GC/MS Port ODP Collector PFC for LC/MS

Disposable LC/MS MPS Solid Phase Pipette Effluent Workstation Extraction SPE Optimizer LEO Extraction DPX

MultiPosition MAESTRO MAESTRO Evaporation PrepAhead Software Station mVAP

And more … The ultimate detector

Your lab needs you! The ultimate detector ODP with the QTOF

TIC

ODP Vanilla Aroma Perception

Olfactory bulb

Orthonasal Retronasal Sensory Descriptors and Thresholds

Compound Descriptor (Taste) Threshold (ppb) 2,4-Dichloroanisole Sweet, fruity, scented 0.4 2,6-Dichloroanisole Musty, medicinal, phenolic 0.04

2-Chlorophenol Disinfectant, medicinal 0.1 2,6-Dibromophenol Iodoform 5x10-4 Styrene Hydrocarbon, plastic 37 2,4,6-Tribromoanisole Musty 8 x 10-6

Geosmin Earthy, musty 0.05 Guaiacol Smoky, phenolic, medicinal 50

6-Chloro-o-cresol Disinfectant, medicinal 0.08 p-Cresol Phenolic, horse manure 2 Sensory Methods

• Informal sensory assessment – Untrained/consumer – Trained panels– specific attributes

• Formal sensory methods – Discrimination tests – Triangle tests – Flavour omission tests – Descriptive sensory profiling

• Aroma Extract Dilution Analysis (AEDA) Which sensory method? Data analysis - omics

• Finding differences between multiple chromatograms can be challenging • Software deconvolution can identify components • Subsequent statistical analysis can indicate trends or cluster groups/categories Statistical Analysis: Mass Profiler Professional

Designed for Mass Spectrometry data (component list imported directly) Import, store, and visualize Agilent LC/MS TOF, QTOF, and QQQ Agilent GC/MS Quad, QQQ, and QTOF Generic file format import

Performs many types of statistical analysis ANOVA, clustering, PCA, class prediction tools

ID Browser for compound identification Samples from different groups Interrogate loadings