Application of
PROTON TRANSFER REACTION-MASS
SPECTROMETRY
to alcoholic beverages
Where Did We Come From?
Where Are We?
Where Are We Going?
Iuliia Khomenko1,2), Luca Cappellin1), Michele Pedrotti1,3),
Vittorio Capozzi4), Franco Biasioli1)
1)
2)
3)
4)
2
 raw material quality
aroma
and
flavour
of
alcoholic
beverage
 geographical origin
 fermentation process
 technological regimen
 aging
 spoilage phenomena
 adulteration
3
Electronic sensing
GC-based methods
Direct injection mass
spectrometry
3
Electronic sensing
•
•
•
•
GC-based methods
Direct injection mass
spectrometry
Broad screenings of raw materials
Online monitoring of processes
Quality control
Product development
3
Electronic sensing
•
•
•
•
GC-based methods
Direct injection mass
spectrometry
Broad screenings of raw materials
Online monitoring of processes
Quality control
Product development
High-throughput mass spectrometric technique
3
Electronic sensing
•
•
•
•
GC-based methods
Direct injection mass
spectrometry
Broad screenings of raw materials
Online monitoring of processes
Quality control
Product development
High-throughput mass spectrometric techniques
4
PROTON TRANSFER REACTION MASS
SPECTROMETRY TIME OF FLIGHT
VOC+ H3O+ → VOCH+ + H2O
H3O+
VOCH+
H2O
VOC
VOCH+
4
PROTON TRANSFER REACTION MASS
SPECTROMETRY TIME OF FLIGHT
VOCH+
5
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
6
ISSUES WITH ALCOHOLIC BEVERAGES
High concentration of ethanol
Primary ion depletion
Previous solutions
Our solutions
Protonated ethanol as a primary ion 1)
Headspace dilution
Modification of operational conditions
WHERE WE WERE
1)
Dilution with Argon in a drift tube 4)
2)
Boscaini et al., 2004
2) Spitaler et al., 2007
3) Fiches et al., 2013
3)
fastGC add-on 5)
WHERE WE ARE
4)
Campbell-Sills et al., 2016
5) Romano et al., 2014
7
WHERE WE ARE
WINE YEAST EXPERIMENT
Online monitoring VOCs emissions
during yeast colony growth
A
Dilution with Argon in a drift tube
A
fastGC add-on
Iuliia Khomenko, Irene Stefanini, Luca Cappellin, Valentina Cappelletti, Pietro Franceschi,
Duccio Cavalieri, Tilmann D. Märk, Franco Biasioli. Submitted to Metabolomics.
P3 WINE YEAST VOLATILOME INVESTIGATED BY PTR-MS AND FAST GC ANALYSIS
8
Yeast selected for the experiment
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.
smoothed
M28-1A
M28-1B
M28-1C
M28-1D
filigreed
8
Yeast selected for the experiment
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
M28-1A
smoothed
filigreed
M28-1B
M28-1C
M28-1D
resistant
sensitive
5,5,5-Trifluoroleucine
4
YEAST SELECTED FOR THE EXPERIMENT
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.
M28-1A
smoothed
filigreed
M28-1B
M28-1C
M28-1D
resistant
sensitive
5,5,5-Trifluoroleucine
2. BY4741 and BY4742 the widely used laboratory strains of Saccharomyces cerevisiae
MAT a
BY4741 BY4742
MAT alpha
8
YEAST SELECTED FOR THE EXPERIMENT
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.
M28-1A
smoothed
filigreed
M28-1B
Is it possible to see the
differences in VOC release
M28-1C
of these different M28-1D
yeast during their growing?
resistant
sensative
5,5,5-Trifluoroleucine
2. BY4741 and BY4742 the widely used laboratory strains of Saccharomyces cerevisiae
MAT a
BY4741 BY4742
MAT alpha
9
SCHEMA OF THE ENTIRE EXPERIMENT
Stationary Phase
Death Phase
Exponential Growth Phase
Lag phase
Yeast cultures, medium, blanks
Day 5
Day 0
PTR-ToF-MS
Day 11
PTR-ToF-MS
fastGC #1
fastGC #2
10
SELECTION OF AN APPROPRIATE TECHNIQUE
GC-MS
injection
acquisition
waiting
1 sample
5 min
47 min
10 min
62 min
60 sec
1 min 30 sec
120 sec
2 min 34 sec
PTR-ToF-MS
fastGC PTRToF-MS
30 sec
4 sec
130 sec
10
SELECTION OF AN APPROPRIATE TECHNIQUE
GC-MS
injection
acquisition
waiting
1 sample
5 min
47 min
10 min
62 min
60 sec
1 min 30 sec
130 sec
120 sec
2 min 34 sec
12 replicas
6 blanks
PTR-ToF-MS
fastGC PTRToF-MS
30 sec
4 sec
BY4741
BY4742
M28-1A
M28-1B
M28-1C
M28-1D
medium YPD
(Yeast Peptone Dextrose)
90 samples
10
SELECTION OF AN APPROPRIATE TECHNIQUE
GC-MS
injection
acquisition
waiting
90 samples
5 min
47 min
10 min
~90 hours
60 sec
4 hours
120 sec
8hours 16 min
PTR-ToF-MS
fastGC PTRToF-MS
30 sec
4 sec
130 sec
BY4741
BY4742
M28-1A
M28-1B
M28-1C
M28-1D
medium YPD
(Yeast Peptone Dextrose)
automatization
multipurpose headspace automated sampler
PTR-ToF-MS
fast GC-ToF-MS
+
+/-
10
PTR-ToF-MS
dilution: Argon 120 sccm : sample 40 sccm
drift tube pressure: 2.30 mbar
E/N value: 140 Td
acquisition time: 1 sec
measurement time: 60 sec
VOC
VOC
clean air
darkness
at 30°C
11
PRINCIPAL COMPONENT ANALYSIS
70 m/z, centered and log-scaled
measurement
with fastGC
12
C13CH4OH+
isotope of
acetaldehyde
C13CH6OH+
isotope of
ethanol
C5H11+ - fragment of
3-methyl-1-butanol and
2-methyl-1-butanol
CH4OH+
methanol
C3H6OSH+
S-Methyl
thioacetate
12
FASTGC PTR-TOF-MS
dilution: NO NEED
drift tube pressure: 2.30 mbar
E/N value: 140 Td
acquisition time: 201.5 msec
measurement time: 140 sec
Column: MXT®-WAX (Siltek®treated stainless steel)
Length: 6m
13
EXAMPLE OF A CHROMATOGRAM OF A YEAST
SAMPLE
fragment of
3-methyl-1-butanol and
2-methyl-1-butanol
PTR-ToF-MS extracted: 279
Meaningful curves: 95
fastGC PTR-ToF-MS extracted: 203
Meaninful peaks: 25
ethyl acetate
fragment
methanol
ethanol
acetaldehyde
butanol
fragment
Fragment of
isovaleric acid
Comparison of butanol and
3-methyl-1-butanol and 2-methyl-1-butanol
measured by fastGC
15
14
WHERE WE ARE
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 3500
measurements 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
15
WHERE WE ARE GOING
FUTURE PROSPECTIVE
• fastGC add-on induce the loss of sensitivity
ion funnel
ion guide
• automation of fastGC measurements
and data analysis
Thank you for your
attention
[email protected]