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

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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

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PRINCIPAL COMPONENT ANALYSIS

70 m/z, centered and log-scaled

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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

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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

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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

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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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Thank you for your

attention

iuliia.khomenko@fmach.it