Tracking Chemical Changes in

Aging Beer using GC-TOFMS

Elizabeth Humston-Fulmer

Life Science and Chemical Analysis Centre

LECO Corporation, St. Joseph, MI

Outline

Objective: Investigate chemical changes related to beer aging using non-targeted discovery based tools

• Beer Aging – general background and analysis techniques

• Analytical Tools and Experimental Methods

• Review of data

Beer Aging

A complex dynamic system

• After bottling, beer continues change

• As it ages, flavors fade or appear, oxidation occurs, degradation of proteins and other molecules, continued yeast activity, etc.

Beer AgingUsually dark, malty beers with >8% ABV

Aim for consistent product that tastes

the same over the duration of shelf life

Maintain with age

Usually hop-forward beers

Beer Analysis Methods

Sensory

Panel

Trained human panel uses

senses to evaluate consumer

products

Target

Acquisition

Instrumental analysis to

screen for specific, pre-

selected analytes of interest

Non-target

Acquisition

Instrumental analysis to

acquire comprehensive data

and discover

Beer Analysis Methods

Sensory

Panel

Trained human panel uses

senses to evaluate consumer

products

Target

Acquisition

Instrumental analysis to

screen for specific, pre-

selected analytes of interest

Non-target

Acquisition

Instrumental analysis to

acquire comprehensive data

and discover

Beer Analysis Methods

Sensory

Panel

Trained human panel uses

senses to evaluate consumer

products

Target

Acquisition

Instrumental analysis to

screen for specific, pre-

selected analytes of interest

Non-target

Acquisition

Instrumental analysis to

acquire comprehensive data

and discover

LECO Pegasus BT with L-PAL 3 Autosampler

Generate Time Course Samples

• Samples: Commercially available craft IPA (canned) purchased from grocery store• Sample prep: Accelerated/simulated aging with elevated temperature[1]

[1] Marques. ASBC Presentation A30. Chicago, IL. June 2014.

Packaging to analysis = 8 weeks

General Aroma Profile Sampling Method

General profiling of aroma analytes. Headspace solid phase micro-extraction (HS-SPME) concentrates volatile and semi-volatile compounds in the headspace above a sample onto a fiber

Method Details:

1) Incubate 5 mL beer for 10 min at 60˚C to

drive compounds into the gas phase

2) Expose tri-phase SPME fiber

(DVB/CAR/PDMS) to headspace for 20 min

to collect volatile analytes

3) Expose fiber to GC inlet at 250˚C for 3 min to

desorb and inject analytes for analysis

GC

Automated with LECO’s L-PAL 3 Autosampler

Various models available to perform liquid, headspace, and/or SPME injections.

• Automatic Tool Exchange to switch between injection types

• Controlled by LECO’s ChromaTOF software

• Agitator for sample heating/agitation

• Needle conditioning port

Fully automates the HS-SPME process

GC-TOFMS for Non-target acquisition

GC Agilent 7890

Column Stabilwax, 30 m x 0.25 mm i.d. x 0.25 µm coating (Restek)

Carrier Gas He @ 1 ml/min

Oven Program 4 min at 35°C, ramp 5°C/min to 180°C, ramp 10°C/min to 220°C hold 5 min

MS LECO Pegasus® BT

Ion Source Temp 250 °C

Mass Range 35-650 m/z

Acquisition Rate 10 spectra/s

• Gas chromatography (GC) to

separate individual analytes

within the complex matrix

• Time-of-flight mass spectral

(TOFMS) with full mass range

detection for identification &

quantitation

GC-TOFMS Data

Overlay of replicate injections of all time points:

0, 1, 2, 3, 4, 5, 6, 8, 12, and 20

0

1

2

3

4

5

6

8

12

20

“Sensory”

Observations

Target Analysis

0

1

2

3

4

5

6

8

12

20

Target Analysis

0

1

2

3

4

5

6

8

12

20

Esters ethyl acetate and isobutyl acetate

Terpenes humulene and β-myrcene

Terpenoids linalool and geraniol

Ketone diacetyl

Sulfur dimethyl disulfide

Ester: ethyl acetate

0

1

2

3

4

5

6

8

12

20

Odor: ethereal, fruity, sweet, weedy,

and green

TIC

m/z 61.03

Ester: isobutyl acetate

Odor: Sweet, fruity, ethereal, banana,

tropical

m/z 56.07

Terpene: β-myrcene

Odor: peppery, terpene, spicy,

balsam, plastic

m/z 69.08

Terpene: humulene

Odor: Woody

m/z 93.07

Terpenoid: linalool

Odor: citrus, floral, sweet, bois de rose,

woody, green, blueberry

m/z 93.08

Terpenoid: geraniol

Odor: Sweet, floral, fruity, rose,

waxy, citrus

m/z 69.07

Ketone: diacetyl

Off odor: strong, butter, sweet,

creamy, pungent, caramel

m/z 86.08

Sulfur: dimethyl disulfide

m/z 93.99

Odor: Sulfurous, vegetable,

cabbage, onion

Target Analysis

0

1

2

3

4

5

6

8

12

20

Esters ethyl acetate and isobutyl acetate

Terpenes humulene and β-myrcene

Terpenoids linalool and geraniol

Ketone diacetyl

Sulfur dimethyl disulfide

Non-target Analysis

General peak finding and deconvolution provide information on hundreds of analytes:

1) Compile peak area information for 317 analytes Esters, terpenes, terpenoids, alcohols, aldehydes/ketones, sulfur containing, heterocyclic

2) Search for differences and trends with t-test (time 0 vs time 20) and regression statistics108 significantly differ at 95% confidence and exhibit a time course trend based on R2

3) Group analytes that trend up or down based on slopes50 increase and 58 decrease

Non-target Analysis

0 1 2 3 4 5 6 8 12 20

Summary of analytes that change:

Each row is an analyte and each column is a

time point. Color scale shows relative

intensity.

• Blue/green to yellow – increase

• Yellow to blue/green – decrease

Non-target Analysis

0 1 2 3 4 5 6 8 12 20

Esters ethyl acetate and isobutyl acetate

Terpenes humulene and β-myrcene

Terpenoids linalool and geraniol

Ketone diacetyl

Sulfur dimethyl disulfide

Non-target Analysis

humulene

0 1 2 3 4 5 6 8 12 20

*

107 non-targeted analytes to explore

Non-target Analysis

m/z 95.02*

0 1 2 3 4 5 6 8 12 20sweet, woody, almond, fragrant baked bread

Non-target Analysis

m/z 95.02

sweet, woody, almond, fragrant baked bread0 1 2 3 4 5 6 8 12 20

*

Non-target Analysis

m/z 93.08

herbal, fresh, camphor, sweet, pine, earthy, woody0 1 2 3 4 5 6 8 12 20

*

Non-target Analysis

m/z 93.08

herbal

TIC

0 1 2 3 4 5 6 8 12 20

*

Non-target Analysis

TIC

tR=324

tR=326

tR=328

tR=330

tR=332

Non-target Analysis

TIC

0 1 2 3 4 5 6 8 12 20

*

Non-target analysis: α-pinene and isobutyl acetate

TIC m/z 93.08m/z 56.07

Ester increases

Fruity, sweet, apple, pineapple, tutti fruitti0 1 2 3 4 5 6 8 12 20

*

m/z 88.06

Ester decreases

1212 1214 1216 1218 1220 1222 1224

0

1e6

2e6

3e6

4e6

Time (s)

XIC(89.06±0.05) a0A XIC(89.06±0.05) a0A_2

m/z 89.06

Oily, green, waxy, soapy, clean, fruity, creamy0 1 2 3 4 5 6 8 12 20

*

Furan increases

0 1 2 3 4 5 6 8 12 20

*

ethereal

Furan increases

0 1 2 3 4 5 6 8 12 20

*

chocolate, ethereal, acetone

Furan increases

0 1 2 3 4 5 6 8 12 20

*

spicy, fruity, wine, sweet, mild

Furan increases

0 1 2 3 4 5 6 8 12 20

*

fruity, green earthy, beany, vegetable

Furan decreases

0 1 2 3 4 5 6 8 12 20

*

woody

Terpene increases

Woody, terpene, lemon, herbal, medicinal, citrus0 1 2 3 4 5 6 8 12 20

*

Terpene decreases

0 1 2 3 4 5 6 8 12 20

*

Sweet, woody, spice, clove, dry

Terpenoid decreases

0 1 2 3 4 5 6 8 12 20*

Sweet, fresh, dry, woody, spicy

Aldehyde increases

0 1 2 3 4 5 6 8 12 20

*

pungent, cocoa, musty, green, malty, bready

More Information

Much more information present in data

- Analytes that change

- Analytes that are consistent

0 1 2 3 4 5 6 8 12 20

Summary

GC-TOFMS is a non-targeted analytical technique that provides comprehensive data that can be mined for specific target analytes of interest and also reviewed for inherent trends and differences in the data without specifying target analytes in advance of acquisition for discovery analyses

For More Information

Contact LECO at:

World Headquarters/United States

In United States: 800-292-6141 or 269-985-5496

Outside U.S.A.: 269-983-5531

Email: info@leco.com

www.leco.com