Application of PROTON TRANSFER REACTION-MASS SPECTROMETRY to alcoholic beverages Iuliia Khomenko 1,2) , Luca Cappellin 1) , Michele Pedrotti 1,3) , Vittorio Capozzi 4) , Franco Biasioli 1) 1) 2) 3) 4) Where Did We Come From? Where Are We? Where Are We Going?
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Application of PROTON TRANSFER REACTION-MASS
SPECTROMETRY to alcoholic beverages
Iuliia Khomenko1,2), Luca Cappellin1), Michele Pedrotti1,3),
Vittorio Capozzi4), Franco Biasioli1)
1) 2) 3) 4)
Where Did We Come From?
Where Are We?
Where Are We Going?
raw material quality
geographical origin
fermentation process
technological regimen
aging
spoilage phenomena
adulteration
aroma and
flavourof
alcoholic beverage
2
Electronic sensingGC-based methods
Direct injection mass spectrometry
3
• Broad screenings of raw materials
• Online monitoring of processes
• Quality control
• Product development
Electronic sensingGC-based methods
Direct injection mass spectrometry
3
• Broad screenings of raw materials
• Online monitoring of processes
• Quality control
• Product development
High-throughput mass spectrometric technique
Electronic sensingGC-based methods
Direct injection mass spectrometry
3
• Broad screenings of raw materials
• Online monitoring of processes
• Quality control
• Product development
High-throughput mass spectrometric techniques
Electronic sensingGC-based methods
Direct injection mass spectrometry
3
PROTON TRANSFER REACTION MASS
SPECTROMETRY TIME OF FLIGHT
VOC
H2O
H3O+ VOCH+ VOCH+
VOC+ H3O+ → VOCH+ + H2O
4
PROTON TRANSFER REACTION MASS
SPECTROMETRY TIME OF FLIGHT
VOCH+
4
PROS/CONS OF PTR-TOF-MS
+ online (direct/real time)
+ fast
+ non-invasive
+ high sensitivity
+ most VOCs at a time
- only sum formula (compound identification is
mostly tentative)
- issues with alcoholic beverages
5
ISSUES WITH ALCOHOLIC BEVERAGES
High concentration of ethanol
Primary ion depletion
Previous solutions
Protonated ethanol as a primary ion 1)
Headspace dilution 2)
Modification of operational conditions 3)
Dilution with Argon in a drift tube 4)
fastGC add-on 5)
1) Boscaini et al., 20042) Spitaler et al., 20073) Fiches et al., 2013
4) Campbell-Sills et al., 20165) Romano et al., 2014
Our solutions
WHERE WE WERE WHERE WE ARE
6
AA
WINE YEAST EXPERIMENT
WHERE WE ARE
Online monitoring VOCs emissions during yeast colony growth
Dilution with Argon in a drift tube fastGC add-on
P3 WINE YEAST VOLATILOME INVESTIGATED BY PTR-MS AND FAST GC ANALYSIS
7
Iuliia Khomenko, Irene Stefanini, Luca Cappellin, Valentina Cappelletti, Pietro Franceschi,
Duccio Cavalieri, Tilmann D. Märk, Franco Biasioli. Submitted to Metabolomics.
Yeast selected for the experiment1. Four meiotic segregants of the M28 natural Saccharomyces cerevisiae strain
was isolated from wine grapes in a Tuscan vineyard. They share 99.99% genes.
M28-1A
M28-1B
M28-1C
M28-1D
filigreedsmoothed
8
Yeast selected for the experiment
M28-1A
M28-1B
M28-1C
M28-1D
filigreedsmoothed
sensitive resistant
5,5,5-Trifluoroleucine
1. Four meiotic segregants of the M28 natural Saccharomyces cerevisiae strain was isolated from wine grapes in a Tuscan vineyard. Between each other they have a variation of only 6% of entire genome (roughly 400 genes changed)* *Cavalieri et al, 2000 Proc Natl Acad Sci USA, 97(22): 12369-12374
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YEAST SELECTED FOR THE EXPERIMENT
M28-1A
M28-1B
M28-1C
M28-1D
filigreedsmoothed
sensitive resistant
5,5,5-Trifluoroleucine
2. BY4741 and BY4742 the widely used laboratory strains of Saccharomyces cerevisiae
MAT a BY4741 BY4742 MAT alpha
4
1. Four meiotic segregants of the M28 natural Saccharomyces cerevisiae strain was isolated from wine grapes in a Tuscan vineyard. They share 99.99% genes.
8
1. Four meiotic segregants of the M28 natural Saccharomyces cerevisiae strain was isolated from wine grapes in a Tuscan vineyard. They share 99.99% genes.
YEAST SELECTED FOR THE EXPERIMENT
M28-1A
M28-1B
M28-1C
M28-1D
filigreedsmoothed
sensative resistant
5,5,5-Trifluoroleucine
2. BY4741 and BY4742 the widely used laboratory strains of Saccharomyces cerevisiae
MAT a BY4741 BY4742 MAT alpha
Is it possible to see the differences in VOC release of these different yeast during their growing?
Yeast cultures, medium, blanks
Day 0 Day 11Day 5
fastGC #1
PTR-ToF-MS PTR-ToF-MS
fastGC #2
SCHEMA OF THE ENTIRE EXPERIMENT
Lag phase
Exponential Growth Phase
Stationary Phase
Death Phase
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SELECTION OF AN APPROPRIATE TECHNIQUE
injection acquisition waiting 1 sample
GC-MS 5 min 47 min 10 min 62 min
PTR-ToF-MS 30 sec 60 sec 1 min 30 sec
fastGC PTR-ToF-MS
4 sec 130 sec 120 sec 2 min 34 sec
10
BY4741BY4742
M28-1AM28-1BM28-1CM28-1D
medium YPD(Yeast Peptone Dextrose)
12 replicas
SELECTION OF AN APPROPRIATE TECHNIQUE
injection acquisition waiting 1 sample
GC-MS 5 min 47 min 10 min 62 min
PTR-ToF-MS 30 sec 60 sec 1 min 30 sec
fastGC PTR-ToF-MS
4 sec 130 sec 120 sec 2 min 34 sec
6 blanks 90 samples
10
SELECTION OF AN APPROPRIATE TECHNIQUE
injection acquisition waiting 90 samples
GC-MS 5 min 47 min 10 min ~90 hours
PTR-ToF-MS 30 sec 60 sec 4 hours
fastGC PTR-ToF-MS
4 sec 130 sec 120 sec 8hours 16 min
automatization
multipurpose headspace automated sampler
BY4741BY4742
M28-1AM28-1BM28-1CM28-1D
medium YPD(Yeast Peptone Dextrose)
PTR-ToF-MS +fast GC-ToF-MS +/-
10
VOC
VOC
darkness
at 30°C
drift tube pressure: 2.30 mbarE/N value: 140 Tdacquisition time: 1 secmeasurement time: 60 sec
PTR-ToF-MS
dilution: Argon 120 sccm : sample 40 sccm
clean air
10
PRINCIPAL COMPONENT ANALYSIS
70 m/z, centered and log-scaled
11
C13CH4OH+
isotope of acetaldehyde
measurement with fastGC
C13CH6OH+
isotope of ethanol
C5H11+ - fragment of
3-methyl-1-butanol and 2-methyl-1-butanol
CH4OH+
methanol
C3H6OSH+
S-Methyl thioacetate
12
drift tube pressure: 2.30 mbarE/N value: 140 Tdacquisition time: 201.5 msecmeasurement time: 140 sec
Column: MXT®-WAX (Siltek®-treated stainless steel)
Length: 6m
dilution: NO NEED
FASTGC PTR-TOF-MS
12
EXAMPLE OF A CHROMATOGRAM OF A YEAST
SAMPLE
PTR-ToF-MS extracted: 279Meaningful curves: 95fastGC PTR-ToF-MS extracted: 203Meaninful peaks: 25
acetaldehyde
methanol
ethanol
butanolfragment
fragment of 3-methyl-1-butanol and
2-methyl-1-butanol
ethyl acetate fragment
Fragment of isovaleric acid
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Comparison of butanol and 3-methyl-1-butanol and 2-methyl-1-butanol
measured by fastGC
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CONCLUSIONS
• We investigated two approaches to overcome issues of PTR-MS with alcoholic bevarages: Argon dilution and a fastGC add-on
• This set-up allowed to acquire more than 3500measurements in 11 day of a continuous experiment in a non-invasive way
• Selected yeast strains showed significantly different profiles
• A fastGC add-on coupled to PTR-ToF-MS added a chromatographic dimension without considerable increase in measurement time
WHERE WE ARE
14
FUTURE PROSPECTIVE
WHERE WE ARE GOING
ion funnel ion guide
• fastGC add-on induce the loss of sensitivity
• automation of fastGC measurements
and data analysis
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