Download Authenticity testing of liquor samples using LC-MS/MS

Authenticity Testing of Liquor Samples using LC-MS/MS and
Statistical Data Processing
André Schreiber1, Christopher Borton2; and Anna Marques3
1
AB SCIEX Concord, ON (Canada), 2 AB SCIEX Golden, CO (USA), 3 AB SCIEX Framingham, MA (USA)
Overview
Various liquor samples (Brazilian cachaça, Cuban rum,
Canadian whisky, French cognac, Mexican tequila, Russian
vodka, Scotch whisky, etc) were analyzed after simple dilution
using Liquid Chromatography Tandem Mass Spectrometry (LCMS/MS). Spectra were acquired in Enhanced MS mode in
negative and positive polarity Electrospray Ionization (ESI) with a
3200 QTRAP® system. Full scan data were processed using
Principal Components Analysis (PCA) in MarkerView™ software
to find markers for authenticity and adulteration of liquor. In
addition, Enhanced Product Ion (EPI) spectra and high resolution
accurate mass MS and MS/MS data was collected using a
TripleTOF™ 5600 system to further characterize and identify the
detected marker compounds. Marker compounds identified
included different sugars, acids, and polyphenols.
Introduction
Experimental
Liquors are drinkable liquids containing ethanol that is produced
by distilling fermented grain, fruit or vegetable. Popular liquors
include brandy, cognac, gin, rum, schnapps, tequila, vodka,
whiskey, whisky, etc. Liquors are popular recreational and life
style enhancing beverages all over the world.
Samples and Sample Preparation
Authentic and regional liquors can be expensive. For that reason
local producers are interested to protect their products from
adulteration. Thus, there is a need for analytical methods to
prove the authenticity and quality of liquors.1-2
In this work, an LC-MS/MS method was used to analyze various
liquor samples (Brazilian cachaça, Cuban rum, Canadian whisky,
French cognac, Mexican tequila, Russian vodka, Scotch whisky,
etc) directly after simple dilution. Statistical data processing was
performed to detect markers for authenticity and adulteration.
Dilution of liquor samples by a factor of 50 with LC-MS grade
water before LC-MS/MS analysis.
All samples were injected in triplicates and in randomized order.
LC Separation
A Shimadzu UFLCXR system was used with a Restek Ultra II
Aqueous C18 2.2 µm (50 x 2.1 mm) with a fast gradient of water
and methanol containing 10 mM ammonium formate at a flow of
0.3 mL/min. The total run time was 10 min. The injection volume
was set to 10 µL.
MS/MS Detection
®
Analysis was performed using an AB SCIEX 3200 QTRAP
LC/MS/MS system equipped with Turbo V™ source and an ESI
probe. Samples were screened using EMS in positive and
negative polarity over a mass range of 100 to 1000 Da. The
Declustering Potential (DP) was set to ±40 V. In addition
Information Dependent Acquisition (IDA) was used to
automatically acquire EPI spectra molecular ions. Dynamic
Background Subtraction™ (DBS) algorithm was activated to
p1
allow EPI collection even in the case of co-elution. The Collision
Energy (CE) was set to ±35 V with Collision Energy Spread
(CES) of 15 V resulting in characteristic fragmentation patterns
for compound identification. All scan functions were used at a
speed of 4000 Da/sec with Dynamic Fill Time (DFT) being
activated to avoid possible space charge of the linear ion trap.
Statistical Data Processing
Principal Components Analysis (PCA) was performed on all EMS
data using MarkerView™ software. PCA finds combinations of
variables (LC-MS signals in this case) that explain most of the
variance present in the data set. For each principal component
(PC), every sample has a score and every variable has a loading
that represents its contribution to the combination. It is common
practice to plot the scores and loadings for the first two PCs.
Figure 2 illustrates PCA and the resulting PC of two simulated
sample clusters.
Results and Discussion
Representative chromatograms and EMS spectra of the entire
chromatogram of two liquor samples, one Scotch whisky and
one Brazilian cachaça are displayed in Figure 3.
Threshold
and DBS
Base Pe ak Chrom. of -EMS: Exp 1...
Ma x. 2.3e5 cps.
Base P eak Chrom. of -EMS: Exp 1 ...
Scotch whisky
2.3e5
3.0e5
2.0e5
0.5
3.0
10 .6
1.0e5
1 1.2
1.7
4 .4
5.0e4
5.4
6.7
10
12
14
1.7e4
1.6e4
255.5
2 83.4
1 71.2
5000 .0 213 .4
8 .9
1 1.2
9.8
6.5
13 .4
7.4
4 .9
10
12
14
Max. 1 .7e 4 cps.
1 12.9
255.5
283 .4
1.2e4 137.0
Inten sity, cp s
Inten sity, cp s
3.4
1.4e4
2 33.2
1.0e4 2 27.4
3 19.3
409.6
26 5.4 3 77.5
385 .3
0 .0
2.6
2
4
6
8
Time , min
-EMS: Exp 1 , 0.0 17 to 14 .89 7 min ...
Ma x. 2.6e4 cps.
2.0e4 155.0
In addition, the AB SCIEX TripleTOF™ 5600 LC/MS/MS system
was used to acquire high resolution accurate mass MS and
MS/MS data to characterize and identify the detected marker
compounds.
1.7
1.0e5
0 .0
1 12.9
2.5e4
1.5e4
1.5e5
10 .5
0.4
5.0e4
2
4
6
8
Time , min
-EMS: Exp 1, 0 .1 75 to 14.68 6 min...
Figure 1. Flowchart of the Information Dependent Acquisition (IDA)
experiment used to detect marker ions in EMS (top) and identify them
based on automatically acquired EPI (bottom) spectra
2.0e5
1 3.9
0 .0
Max. 3 .1e 5 cps.
Brazilian cachaça
2.5e5
9.8
Intensity, cps
Intensity, cps
8.4
1.5e5
0.1
200
345 .6
400
1.0e4
800 0.0
600 0.0
400 0.0
5 85.5 691 .7
60 0
m/z, Da
161 .2
8 00
10 00
0.0
3 31.7
227 .4
199.3
200 0.0
79 9.2 985.2
233.2
1 75.2
309 .4 353.5
744 .7
45 3.1
200
40 0
69 1.8
6 00
m/z, Da
67 6.3
867 .1
800
10 00
Figure 3. Chromatograms (top) and EMS spectra of the entire
chromatogram (bottom) of a Scotch whisky (Glenmorangie 10 years) and
a Brazilian cachaça (Espírito de Minas)
PC1 = 0.1 x + 0.3 y + 0.95 z
2D
3D
z
z
y
Rotated 3D
PC1
y
(0, 0, 0)
PC1 S core
x
x
y
x
Figure 2. Illustration of Principal Component Analysis (PCA) using two simulated sample clusters (red and blue), PCA “rotates” the three dimensional
plot of variables of samples to determine the variables that maximize the variation between groups and those which minimize the variation within a
group, a vector is calculated for each PC
p2
The example indicates that each liquor has its own
representative profile. However, differentiating between different
liquors based on the comparison of LC-MS chromatographic
profiles alone is not sufficient - further data mining is required at
the spectral level as well. Processing such data manually is
difficult and very time consuming; additionally, such a manual
procedure is incomplete and very likely will not allow
distinguishing between original and adulterated or not authentic
products.5
PCA was performed on all EMS data using MarkerView™
software. In the preliminary analysis, the various liquor samples
were plotted together. From the scores plots in Figure 4a and 5a,
the groupings between the different liquors are shown to be
clearly demonstrated.
French cognac
French brandy
Canadian whisky
Scotch whisky
Red wine
Brazilian cachaça, Cuban rum, Tequila, Vodka
Fruity and sweet liquor
Figure 4a. Scores plot of PCA of different liquor samples analyzed using
positive polarity LC-MS showing a clear grouping
From this analysis, it was evident that the LC-MS method,
developed with data processing based on PCA, was valuable for
making distinctions between different liquors. It can be expected
that the separation in Figure 4a is based on compounds ionizing
in positive polarity, such as amino acid, polyphenols, glucosides,
and sugars, while the separation in Figure 5a is based on
compounds ionizing in negative polarity, such as acids, sugars
etc.
The corresponding loading plots in Figure 4b and 5b show the
variables that make the most difference in separating liquor
samples. It can be used to identify the molecular ion and
retention time of marker compounds.
Characteristic marker compounds of a group of samples are
located in the same area of the loadings plot as the group is
located in the scores plot.
For example, red wine samples are located in the lower right and
French cognac samples are located in the upper left of the
scores plot (Figure 4a). Thus, characteristic markers for red wine
can be found in the lower right of the loadings plot (Figure 5a),
such as malvidin-3-glucoside (oenin), a compound primarily
responsible for the color of red wine. Characteristic markers for
French cognac can be found in the upper left of loadings plot.
These compounds were probably produced during distillation
from wine or were extracted from wood during aging. In addition,
there is a third group of markers which is located between the
markers for red wine and cognac. These compounds are
characteristic for both samples means they originate from grapes
and were not altered during distillation and aging.
Scores for D1 (14.2 %) v ersus D2 (14.2 %), Log | Pareto (DA )
Wi - Cabernet M erlot (Canada)
19
Wi - Dogajolo di Toscana (Italy)
Wi - Cabernet M erlot (Canada)
18
17
A pricot
Cachaca
Canadian w hiskey
Cognac
Cointreau
French brandy
Rum
Scotch Whisky
Tequila
V odka
Wine
Red wine
20
Wi - Dogajolo di Toscana (Italy)
16
15
Markers for French cognac
14
Wi - Dogajolo di Toscana (Italy)
13
Wi - Cabernet M erlot (Canada)
12
Markers for French cognac and red wine
11
10
9
Fruity and sweet liquor
8
Cuban rum
7
493: malvidin-3-glucoside
(oenin)
Markers for red wine
Li - A pricot
Brazilian cachaça
5
Li - Cointreau
vodka
tequila
2
1
0
-1
229: resveratrol
343, 360: sucrose
Ca - Pitu Co - St. Remy V SOP
Co - St. Remy V SOP
-5
-4
French cognac and brandy
Sc - A rdbeg
CW - Canadian Club Reserv e
Sc - Glenfiddich 18
Scotch whisky
Sc - Glenfiddich 15
Sc - Glenfiddich 12
-6
Co - St. Remy V SOP
Co - Remy M artin V SOP
Sc - M acallan 18
CW - Crow n Royal
CW - Crow n Royal
Sc - M acallan 12
Co - Hennessy XO
Ca - Nega Fulo
Sc - Highland Park 18
-4
116: proline
Ca - Pitu
Sc - M acallan 18
-2
-5
Ru - Hav ana
Canadian
whisky
-3
181, 198: glucose / fructose
Li - A pricot
4
3
Li - A pricot
Li - Cointreau
Li - Cointreau
6
CW - Canadian Club Reserv e
Sc - Glenmorangie 10
-3
-2
-1
0
1
2
3
4
5
6
D1 Score
7
8
9
10
11
12
13
14
15
16
17
18
Figure 5a. Scores plot of PCA of different liquor samples analyzed using
negative polarity LC-MS showing a clear grouping
Figure 4b. Loadings plot of PCA of different liquor samples analyzed
using positive polarity LC-MS showing identified marker ions
p3
Scotch whisky
114: proline
French cognac and brandy
191: C12H16O2 (C5 H10 benzoate)
179: glucose / fructose
Canadian whisky
149: tartaric acid
167: vanillic acid
195: ethyl vanillate
341: sucrose
169: gallic acid
197: ethyl gallate
Cuban rum
Brazilian cachaça
199: lauric acid
vodka
tequila
171: capric acid
Figure 5b. Loadings plot of PCA of different liquor samples analyzed
using negative polarity LC-MS showing identified marker ions
Analysis in negative polarity resulted in grouping of distilled
liquors. For further differentiation the wine and sweet liquor
samples were excluded from processing. The resulting scores
and loadings plot are presented in Figure 6a and 6b.
Figure 6a. Scores plot of PCA of different distilled liquor samples
analyzed using negative polarity LC-MS showing a clear grouping
171: capric acid
199: lauric acid
Three characteristic groups (1: whisky, 2: cognac, brandy, and
rum, 3: clear distilled liquors) were observed in the Scores plot
(Figure 6a).
The corresponding loadings plot (Figure 6b) was used to identify
capric acid and lauric acid as characteristic markers for Scotch
whisky. Both compounds were also detected in Canadian whisky
and French cognac and brandy but at lower concentration.
Capric acid and lauric acid are known markers for alembic
distillation using copper pot stills.3
Sucrose was identified as the characteristic marker for French
cognac, brandy, and Cuban rum.4 Although both Brazilian
cachaça and Cuban rum are produced from sugar cane, only
Cuban rum contains sucrose. This can be explained by the
difference in production. While cachaça is made from fresh cane
sugar juice that is fermented and distilled, rum is usually made
from molasses, a by-product from refineries that boil the cane
juice to extract as much sugar crystals as possible.
The unidentified ion of 367 Da was found to be characteristic for
French cognac and brandy. This compound was not present in
Cuban rum. In addition 645 Da was a unique marker for vodka
and 162 Da and 241 Da were unique for tequila, respectively.
341: sucrose
645
367
241
162
Figure 6b. Loadings plot of PCA of different distilled liquor samples
analyzed using negative polarity LC-MS showing identified marker ions
After initial PCA selected liquor samples were injected into the
AB SCIEX TripleTOF™ 5600 system to acquire high resolution
and accurate mass MS and MS/MS spectra for compound
identification.
The Formula Finder tool of PeakView™ software was used to
empirically calculate molecular formulae and to characterize
marker ions by comparing MS/MS fragments with a suspected
molecular structure (Figure 7a and 7b).
p4
Summary
TOF-MS
Various liquor samples (Brazilian cachaça, Cuban rum,
Canadian whisky, French cognac, Mexican tequila, Russian
vodka, Scotch whisky, etc) were analyzed using LC-MS/MS in
positive and negative polarity.
Figure 7a. TOF-MS analysis of Cuban rum in negative polarity, a
molecular formula of C6H12O6 was calculated based on the accurate
molecular ion (-0.6 ppm error) and the isotopic pattern
EMS data acquired on an AB SCIEX 3200 QTRAP® LC/MS/MS
system was processed using Principal Components Analysis
(PCA) to differentiate between different liquors and to identify
characteristic marker compounds. These ions were further
investigated by high resolution and accurate mass MS and
MS/MS using an AB SCIEX TripleTOF™ 5600 LC/MS/MS
system. Empirical formula calculation and software assisted
interpretation of MS/MS fragments allowed to identify different
acids, sugars, and polyphenols.
Further data acquisition and processing is planned to identify
marker compounds for other sample types, geography and
differences in production processes.
References
TOF-MS/MS
Figure 7b. TOF-MS/MS analysis of French cognac in negative polarity,
accurate mass MS/MS signals were automatically compared to a
predicted structure and 11 of 13 fragment ions were explained allowing a
maximum cleavage of 2 bonds confirming the identification of sucrose
1
J. K. S. Møller et al.: ‘Electrospray ionization mass
spectrometry fingerprinting of whisky: immediate proof of
origin and authenticity’ The Analyst 130 (2005) 890-897
2
P. P. de Souza et al.: ‘Electrospray Ionization nMass
Spectrometry Fingerprinting of Brazilian Artisan Cachaça
Aged in Different Wood Casks’ J. Agric. Food Chem. 55
(2007) 2094-2102
3
P. P. de Souza et al.: ‘Brazilian cachaça: “Single shot”
typification of fresh alembic and industrial samples via
Electrospray ionization mass spectrometry fingerprinting’
Food Chemistry 115 (2009) 1064-1068
4
P. P. de Souza et al.: ‘Differentiation of rum and Brazilian
artisan cachaça via Electrospray ionization mass
spectrometry fingerprinting’ J. Mass Spectrom. 42 (2007)
1294-1299
5
Chr. Borton et al.: ‘Authenticity Testing of Liquor Samples
using Liquid Chromatography tandem Mass Spectrometry
(LC-MS/MS) and Statistical Data Processing’ presentation at
AOAC conference (2010) Orlando, FL
Acknowledgements
The authors would like to thank Debora R. Santos, Takeo
Sakuma, and Brent Lefebvre for providing liquor samples for
analysis.
For Research Use Only. Not for use in diagnostic procedures.
© 2011 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
Publication number: 2170411-01
353 Hatch Drive Foster City CA
Headquarters
353 Hatch Drive Foster City CA 94404 USA
Phone 650-638-5800
www.absciex.com
International Sales
For our office locations please call the division
headquarters or refer to our website at
www.absciex.com/offices