Jack C. Cappozzo
Separation Science Webinar
March 12, 2013
Research/Analytical Purpose
 Adulteration (economical/food safety)
 Authenticity (economical/quality)
 Quality
 Sensory…Analytical Instrumentation
Adulteration
 Nineteen Arrested Over Alleged Olive Oil Fraud
(Spain, Olive Oil Times)
 Spanish Police Say Palm, Avocado, Sunflower Was
Passed Off as Olive Oil (Spain, Olive Oil Times)
 Two Plead Guilty in Olive Oil Fraud Scheme,
Sentenced to Two Years in Jail (Spain, 2011, Olive Oil
Times)
 Toxic Oil Syndrome (TOS), Spain 1981
Adulteration
 Quality
 Health
 Nutritional Loss
 Illness
 Economics
Economics
 At 10% adulteration (soybean oil), the producer saves
about $0.20/Kg……commodity pricing
 Metric Ton of Olive Oil….@$2900
 Metric Ton….$200
 3 M Metric Tons in Spain
 10% = 300,000 Metric Tons @ $60 million (in Savings)
 Retail…10% oil (100 Kg) savings…$5000-10,000
Analytical Tests Needed for
EVOO/OO
 23 tests (reduce that to about 20 tests)
 $$$$$$$$
Olive Oil (Extra Virgin)
(Standards of Identity)
 Sterol composition and
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content
Fatty acid composition
Saturated fatty acid content
in position 2 of the
triglyceride
Unsaponifiable material
Wax content
Erythrodiol and uvaol
ECN 42 content
UV Light Absorbency
Moisture and Volatiles
Metal Traces
 Hydrocarbons:
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stigmastadiene, stigmasta-3,
5 diene, and campesta-3, 5
diene
Trans fatty acid isomer
Total aliphatic alcohols
DNA characterization
Color
Aspect
Free Acidity
Peroxide Value
Insoluble Impurities
Flash Point
Olive Oil
(Standards of Identity)
 Halogenated Solvents
 Additives
 Heavy Metals &
Pesticide Residues
 Sensory
Olive Oil
(IFSH Focus)
 Technology for Olive Oil  LC-MS/MS and QTOF
Authenticity &
Adulteration
 Rapid Technology for
Olive Oil Quality
(Sensory)
 Collaborators
 Agilent
 Alpha Mos
 AOCS
 GC-MS
 ICP-MS
 Electronic Nose (FID)
 Statistical Analysis Software
 Goal
 Rapid Techniques to relate to
sensory quality
 (Rapid) Technology to
confirm Authenticity and
Adulteration
Technology
 LC-Q-TOF
 Electronic Nose/GC-MS
 PCA
 Statistical Mapping
Mass Spectroscopy
 LC-MS-TOF (Q-
TOF)
 LC-MS/MS
LC-QTOF Sample Prep and
Chromatographic Parameters
 Method: 5 mL oil diluted with 5 mL hexane extracted twice with 10 mL
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methanol, evaporated and reconstituted in 50% methanol in water.
(Polar Fraction)
Chromatographic separation achieved on a Poroshell 120 EC-C18 2.7
µm column kept at 45oC
Gas temp was 325oC with a flow of 10 L/min
Nebulizer was 45 psig
Analysis was conducted in positive ionization mode
Injection volume 5 µL
The mobile phase was a gradient of 0.1% acetic acid in water and
acetonitrile starting at 5% ACN and increasing to 100% ACN over 25
min with a post time of 3 min.
Italian Olive Oil/Soybean Oil
Overlay
Oil Analysis Using QTOF Technology
Chromatograms Produced Using MFE (Molecular Feature
Extraction) and Contain Several Thousands of Compounds
Olive Oil
from
Palestine
Soybean
Oil
Mass Profiler Professional
MPP software allows multivariance
statistical analysis of MS data
Data can be filtered based on set
parameters and organized into
entity lists.
Mass, Retention Time
PCA Italian Olive Oil
Olive Oil Varieties
PCA Analysis of Olive Oils and Olive
Oils Combined with Vegetable Oils
Venn Diagram of Olive Oils and
Olive Oils Overlap with Soybean
Specific entity lists can be displayed
in many different ways. An example
is A Venn diagram representing
similarities and differences among
various groups of samples.
ID Browser for Identification of
Compounds included in Entity Lists
An entity list can be also identified based on available
databases and libraries using the ID browser
Table of representative 38 compounds identified in EVOO. Over 200
compounds identified.
Name
Formula
Mass
RT
12-amino-octadecanoic acid
C18 H37 N O2
299.282
21.911
2-Oxo-4E-hexenoic acid
C6 H8 O3
128.0478
1.657
Vanillin
C8H8O3
152.0473
1.298
3,4-bis(methylene)-Hexanedioic acid
C8 H10 O4
170.0577
1.657
2,3-Dihydroxy-3-methylvaleric acid
C6 H12 O4
148.0733
4.995
Oleamide
C18 H35 N O
281.2714
21.911
2,2-dimethyl-4-pentenoic acid
C7 H12 O2
128.0831
8.676
(R)-3-methyl-2-oxo-Pentanoic acid
C6 H10 O3
130.0628
3.276
2,2-dimethyl-3-butenoic acid
C6 H10 O2
114.0691
3.919
2-Hydroxycinnamic acid
C9 H8 O3
164.0465
10.404
3-oxo-4-pentenoic acid
C5 H6 O3
114.031
3.61
(5R)-5-Hydroxyhexanoic acid
C6 H12 O3
132.079
16.458
(R)-3-methyl-2-oxo-Pentanoic acid
C6 H10 O3
130.0627
5.296
2-Octenedioic acid
C8 H12 O4
172.0729
2.709
15.79
(5R)-5-Hydroxyhexanoic acid
C6 H12 O3
132.079
2-keto-n-caprylic acid
C8 H14 O3
158.0938
1.716
2,2-dimethyl-4-pentenoic acid
C7 H12 O2
128.0825
10.621
Isovalerylglucuronide
C11 H18 O8
278.1018
4.495
2E,4E,6E,8E-Decatetraenedioic acid
C10 H10 O4
194.0573
14.721
11-amino-undecanoic acid
C11 H23 N O2
201.1724
1.248
2-ethyl-3E-hexenoic acid
C8 H14 O2
142.0984
1.949
12-amino-dodecanoic acid
C12 H25 N O2
215.1878
2.166
6,8-dihydroxy-octanoic acid
C8 H16 O4
176.1056
9.803
2,2-dimethyl-4-pentenoic acid
C7 H12 O2
128.083
10.32
13-methyl-pentadecanoic acid
C16 H32 O2
256.2393
0.931
20.984
10,12,14-octadecatrienoic acid
C18 H30 O2
278.2254
3,4,5-Trimethoxycinnamic acid
C12 H14 O5
238.0826
15.314
10-oxo-5,8-decadienoic acid
C10 H14 O3
182.093
10.629
10.629
3,6-dioxo-decanoic acid
C10 H16 O4
200.1035
2S-amino-pentanoic acid
C5 H11 N O2
117.0795
6.372
5-oxo-7-octenoic acid
C8 H12 O3
156.0798
11.306
(+)-catechin
C15 H14 O6
290.0809
8.108
Tyrosol
C8H10O2
138.0681
11.306
(+)-pinoresinol
C20H22O6
358.1416
12.684
Syringic acid
C9H10O5
198.0536
10.195
Caffeic acid
C9H8O4
180.0423
9.752
Elenolic acid
C11 H14 O6
242.0788
4.578
p-coumaric acid
C9H8O3
164.0465
10.404
Blue=phenolic compounds
Name
Formula
Mass
RT
12-amino-octadecanoic acid
C18 H37 N O2
299.282
21.911
2-Oxo-4E-hexenoic acid
C6 H8 O3
128.0478
1.657
Vanillin
C8H8O3
152.0473
1.298
3,4-bis(methylene)-Hexanedioic acid
C8 H10 O4
170.0577
1.657
2,3-Dihydroxy-3-methylvaleric acid
C6 H12 O4
148.0733
4.995
Oleamide
C18 H35 N O
281.2714
21.911
2,2-dimethyl-4-pentenoic acid
C7 H12 O2
128.0831
8.676
(R)-3-methyl-2-oxo-Pentanoic acid
C6 H10 O3
130.0628
3.276
2,2-dimethyl-3-butenoic acid
C6 H10 O2
114.0691
3.919
2-Hydroxycinnamic acid
C9 H8 O3
164.0465
10.404
3-oxo-4-pentenoic acid
C5 H6 O3
114.031
3.61
(5R)-5-Hydroxyhexanoic acid
C6 H12 O3
132.079
16.458
(R)-3-methyl-2-oxo-Pentanoic acid
C6 H10 O3
130.0627
5.296
2-Octenedioic acid
C8 H12 O4
172.0729
2.709
(5R)-5-Hydroxyhexanoic acid
C6 H12 O3
132.079
15.79
2-keto-n-caprylic acid
C8 H14 O3
158.0938
1.716
2,2-dimethyl-4-pentenoic acid
C7 H12 O2
128.0825
10.621
Isovalerylglucuronide
C11 H18 O8
278.1018
4.495
2E,4E,6E,8E-Decatetraenedioic acid
C10 H10 O4
194.0573
14.721
11-amino-undecanoic acid
C11 H23 N O2
201.1724
1.248
2-ethyl-3E-hexenoic acid
C8 H14 O2
142.0984
1.949
12-amino-dodecanoic acid
C12 H25 N O2
215.1878
2.166
6,8-dihydroxy-octanoic acid
C8 H16 O4
176.1056
9.803
2,2-dimethyl-4-pentenoic acid
C7 H12 O2
128.083
10.32
13-methyl-pentadecanoic acid
C16 H32 O2
256.2393
0.931
10,12,14-octadecatrienoic acid
C18 H30 O2
278.2254
20.984
3,4,5-Trimethoxycinnamic acid
C12 H14 O5
238.0826
15.314
10-oxo-5,8-decadienoic acid
C10 H14 O3
182.093
10.629
3,6-dioxo-decanoic acid
C10 H16 O4
200.1035
10.629
2S-amino-pentanoic acid
C5 H11 N O2
117.0795
6.372
5-oxo-7-octenoic acid
C8 H12 O3
156.0798
11.306
(+)-catechin
C15 H14 O6
290.0809
8.108
Tyrosol
C8H10O2
138.0681
11.306
(+)-pinoresinol
C20H22O6
358.1416
12.684
Syringic acid
C9H10O5
198.0536
10.195
Caffeic acid
C9H8O4
180.0423
9.752
Elenolic acid
C11 H14 O6
242.0788
4.578
p-coumaric acid
C9H8O3
164.0465
10.404
Green=fatty acids
PCA
3-D PCA
PCA Analysis of Italian (P) Greek (G)
Olive Oils and Soybean Oil(B).
Cluster analysis based on five
sensors with the highest
discrimination power.
Palestinian Olive Oil Adulterated
with 2%, 5% and 10% Soybean Oil
IFSH Direction
 Oxidation correlated to Analytical Methods
 PV, FFA, …
 Sensory correlated to Analytical Methods
 Continue with Adulteration and Authenticity
 ICP-MS
 HS-GC-MS
 Statistical Analysis
 PCA
 Mass Profiler Professional
IFSH
 Institute for Food Safety & Health (IFSH) Bedford Park, IL
 National Center for Food Safety and Technology
(NCFST)
 At the Illinois Institute of Technology (IIT) Chicago
 50:50 IIT:FDA
 Collaborative Model of Industry:Academia:FDA
 Food Safety…Chemistry, Process Engineering,
Microbiology (molecular biology)
 Nutrition (biological/clinical)
 In existence for 23 years
Thank you
Questions?