Application of HS-SPME-GC-MS-derived variables for monitoring ...
SPME-GC-MS: Advantages of an internal multistandard method ...
Transcript of SPME-GC-MS: Advantages of an internal multistandard method ...
SPME-GC-MS: Advantages of an internal
multistandard method for quantification of VOCs
in olive oils Dr.ssa Martina Fortini Dr. Lorenzo Cecchi
Analytical Group – Firenze Università di Firenze
29 settembre 2017/Sanremo (IM)
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AIM OF THE METHOD: Quantitation of volatile organic compounds (VOCs) of virgin olive oils (VOOs)
HS-SPME Internal standard
method
1. Extraction
of VOCs 3. Quantittion
of VOCs
GC-MS
2. Separation and
detection
71 VOCs quantified
11 ISTD used
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Partner of AROMOLIO:
- C.R.A. Istituto Sperimentale per la Elaiotecnica
- Laboratorio Chimico Merceologico, Azienda Speciale della CCIAA di
Firenze.
- Università degli studi di Roma di Tor Vergata, Dipartimento di Ingegneria
Elettronica
- Firenze Tecnologia, Azienda Speciale della CCIAA di Firenze.
A.R.S.I.A. Agenzia Regionale per lo Sviluppo e l’Innovazione nel settore Agricolo forestale della
regione Toscana
AROMOLIO – Caratterizzazione analitica degli attributi sensoriali degli oli vergini di oliva, 2006-2009
Aim: setting up a simple, fast, reliable and
reproducible method to help and support
the «panel test» during its activities
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AROMOLIO
Lab. CCIAA Firenze HS-SPME-GC-MS
Quantification of VOCs
• Internal standard method
• 4-methyl-2-pentanol
Results: correlation
between some VOCs and
olive oil defectness
In the following years:
Improvements in
knowledge regarding
SPME
Need to improve the
method
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MULTIPLE EQUILIBRIA*
1. liquid /headspace
2. headspace/sorbent fiber
phase
3. competition among
analytes for
absorption/absorption
sites on SPME fiber
METHOD
QUANTITATION OF VOOCs via HS-SPME-GC-MS ANALYSIS
Henry’s Law
different affinities of some chemical classes to
the various polymer films result in analyte
sampling amounts that depend on the affinity
constants in addition to the concentration
headspace
this occurs when the concentrations exceed the
upper limit of linear range (concentration
headspace, fiber coating wear)
* Calamai et al. «Sample preparation for direct MS analysis of food», in:
Pawliszyn at al. «Comprehensive Sampling and Sample Preparation» Vol.4,
Elsevier 2012 – ISBN:9780123813732
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ISSUES REGARDING QUANTIFICATION BY HS-SPME SAMPLING TECHNIQUE
• different absorption capacity of different fiber
• fiber wearing upon usage
• competition of molecules at different concentration in different samples
• different affinities of different molecules for the coating material of the fiber
BIASED QUANTIFICATION
RISK
APPLIED SOLUTION
USE OF A SUITABLE INTERNAL STANDARD FOR AREA
NORMALIZATION
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PROBLEM:
Volatile composition of VOOs includes
• A very high number of molecules
• Compounds at ppm and ppb concentrations
• Molecules belonging to different chemical classes
• Molecules in a wide range of molecular weight
DIFFICULTY IN
QUANTIFYING THEM EVEN
BY THE USE OF AN
INTERNAL STANDARD
IDEAL INTERNAL STANDARD: a molecule that is absent in the volatile profile and that has a
behavior similar to the molecule to quantify during absorption on the fiber
BUT
CONSEQUENCE
WHAT
SOLUTION?
IDEA
Multiple internal standard for data
normaliztion
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AIMS
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AIMS
• To select several internal standards belonging to all the chemical classes and covering
all the molecular weights of VOO-VOCs
• To identify the suitable internal standard for each identified VOC
• To expand the linear working range of all the identified VOC, for covering the range
of concentration of almost all the VOOs from different provenance and characteristic
• To validate the method
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METHOD
QUANTITATION OF VOOCs via HS-SPME-GC-MS ANALYSIS
MULTIPLE EQUILIBRIA*
1. liquid /headspace
2. headspace/sorbent fiber
phase
3. competition among
analytes for
absorption/absorption
sites on SPME fiber
PARAMETER TO CHOOSE TO
QUANTITATE VOCs
AGITATION/STIRRING OF THE SAMPLE
TEMPERATURE OF FIBER,
SAMPLE AND HEADSPACE
CHOISE OF SUITABLE FIBER COATING
EXPOSURE TIME OF THE FIBER
CHOISE OF SUITABLE INTERNAL
STANDARD → MULTIPLE INTERNAL STANDARD
MATRIX MATCHED APPORACH
* Calamai et al. «Sample preparation for direct MS analysis of food», in:
Pawliszyn at al. «Comprehensive Sampling and Sample Preparation» Vol.4,
Elsevier 2012 – ISBN:9780123813732
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THE SELECTED INTERNAL STANDARD
• Deuterium labeled molecules
• Molecules absent in the virgin olive oils
O
Cl
6-chloro-2-hexanone
O
3-octanone
H
O
trimethylacetaldehyde
O
O D3C
D D
D D
D D
D D
ethyl hexanoate d11
D3C
O
O CD3
D D
ethyl acetate d8
D3C
O
OH
acetic acid 2,2,2-d3
OH
O
D3C
D
D D
D D
D D
D
hexanoic acid d11
D3C OD
D D
D D
D D
1-butanol d10
OH
4-methyl-2-pentanol
CD3
D
D
D
D
D
toluene d8
OH
3,4-dimethyl phenol
ISTD mix: all ISTDs in concentration 75 ppm in a ROO free from VOCs
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THE SELECTED INTERNAL STANDARDS
Time (min)5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
Ab
un
da
nce
(Mco
un
ts) HS-SPME-GC-MS
TIC - ISTDs
RT
=4
4.5
7 -
3,4
-dim
eth
ylp
hen
ol
RT
=3
7.0
6 -
Hex
anoic
acid-d
11
RT
=30.0
0 -
6-ch
loro
-2-h
exan
one
RT
=2
5.7
6 -
Acetic
acid-d3
RT
=1
8.2
6 -
3-o
ctanone
RT
=1
7.0
7 -
Eth
ylh
exan
oate-d
11
RT
=1
2.6
0-
Bu
tanol-d
10
RT
=2
.63
-T
rimethyl
acetaldehyde
RT
=6
.76
–T
olu
ene-d8
RT
=3
.29
-E
thylac
etate-d
8
RT
=1
4.4
0–
2-m
ethyl-4-pentanol
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METHOD
HS-SPME-GC-MS ANALYSIS
FIBER AND SAMPLE TREATMENT
• orbital shaking of the sample during fiber exposure
• fiber length: 1cm
• fiber coating: 50/30µm DVB/CAR/PDMS*
• temperature: 60ºC
• time exposure: 20 min
* Vichi et al. Journal of Chromatography A, 983 (2003) 19-33
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THE EXTERNAL STANDARDS MIXTURE
• 71 VOCs typical of VOOs, associated to sensory
defects and fruity notes, according to literature and
our previous work
•Six levels of calibration scale
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POINTS OF CALIBRATION CURVES
to 100 g with refined olive oil
STD 1 STD 2 STD 3 STD 4 STD 5 STD 6
EXT-
STD
mix (g)
X1
X2
X3
X4
X5
X6
ISTD
mix (g) K K K K K K
METHOD MATRIX MATCHED APPROACH
EXT-STD mix
Stock external standard solution mixture of 71
EXT-STDs in refined olive oil after veryifying
the absence of any analyte or ISTD
DIFFERENT FINAL CONCENTRATION EACH
EACH POINT OF
CALIBRATION CURVE
4.4 g in a 20 ml screw cup vial for
HS-SPME
→ EACH STD x1-x6 mg/kg
→ EACH ISTD 1,7 mg/kg
ISTD mix
Stock internal standard solution mixture of 11
ISTD in refined olive oil after veryifying the
absence of any analyte or ISTD
FINAL CONCENTRATION 75 mg/kg EACH
STORED IN THE DARK AT -20ºC UNTIL THE CHROMATOGRAPHIC ANALYSIS
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METHOD MATRIX MATCHED APPROACH
SAMPLES PREPARATION
4.3 g sample + 0.1 g ISTD mix
in a 20 ml screw cup vial for HS-SPME
→ EACH ISTD 1,7 mg/kg
ISTD mix
Stock internal standard solution mixture of 11
ISTD in refined olive oil after veryifying the
absence of any analyte or ISTD
FINAL CONCENTRATION 75 mg/kg EACH
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METHOD
HS-SPME-GC-MS ANALYSIS
THE GC-MS system
• GC-MS TraceDSQ Thermo Fisher Scientific equipped with a
CombiPal autosampler
• splitless injection mode at 260ºC
• ZB-FFAP capillary column 30 m x 0.25 mm, 0.25 µm
• Quadrupole mass detector – scan mode within 30-330 Th mass
range at 1500 Th/s – IE energy of 70eV
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METHOD
Time (min)5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
Ab
un
da
nce
(Mco
un
ts)
A
HS-SPME-GC-MS
TIC – EXT STD
CHROMATHOGRAPHIIC PROFILE
Matrix matched standard + ISTDs
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METHOD
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
Ab
un
da
nce
(Mco
un
ts)
A. Matrix matched standard + ISTDsB. Refined Olive oil + ISTDs
RT
=4
4.5
7 -
3,4
-dim
ethylp
hen
ol
RT
=3
7.0
6 -
Hexanoic
acid-d1
1
RT
=3
0.0
0 -
6-ch
loro
-2-h
exan
on
e
RT
=2
5.7
6 -
Acetic
acid-d3
RT
=1
8.2
6 -
3-o
ctanone
RT
=1
7.0
7 -
Ethy
lhex
anoate-d
11
RT
=1
2.6
0-
Bu
tanol-d10
RT
=2
.63
-T
rimethyl
acetaldehyde
RT
=6
.76
–T
olu
ene-d8
RT
=3
.29
-E
thylac
etate-d
8
RT
=1
4.4
0–
2-m
ethyl-4-pentanolA B
Time (min)
CHROMATHOGRAPHIC PROFILE
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METHOD
CHROMATHOGRAPHIC ANALYSIS
Extract ion chromatograms Total ion current chromatogram
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
4.0e+06
8.0e+06
1.2e+07
1.6e+07
2.0e+07
2.4e+07
2.8e+07
TIC 3.50 – 5.50 min
Are
a (co
un
ts)
Time (min)
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
5.0e+05
1.0e+06METHYL PROPANOATE rt 3.62 min;
quantifier 59 Th (58.70 to 59.70)
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
1.0e+06
2.0e+062-METHYL BUTANAL rt 3.74 min; quantifier 41 Th (40.70 to 41.70)
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
1.0e+06
2.0e+06
3.0e+06 ISOVALERALDEHYDE rt 3.75 min; quantifier 44 Th (43.70 to 44.70)
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
2.0e+054.0e+05
ETHYL PROPANOATE rt 4.52 min;
quantifier 102 Th (101.70 to 102.70)
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
4.0e+06
8.0e+06
3-PENTANONE rt 4.96 min; quantifier 57 Th (56.70 to 57.70)
VALERALDEHYDE rt 5.02 min;
quantifier 44 Th (43.70 to 44.70)
Time (min)
Are
a (co
un
ts)
3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40
1.0e+06
2.0e+06
3.0e+06
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METHOD
QUANTIFICATION OF THE VOLATILE ORGANIC COMPOUNDS
100
300
500 ISTD: 4-METHYL-2-PENTANOL
Y = 0.0145+9.17*X
R2 = 0.9992
0 10 20 30 40 50 60
E-2- HEXENAL
Area VOC
Area ISTD
Concentration Ratio
Are
a R
atio
Conc. VOC
Conc. ISTD ~ Conc. VOC
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SELECTION OF THE SUITABLE ISTD FOR EACH VOCs
ISOTOPE
ANALOGUE
0.0
0.4
0.8
1.2
ETHYL ACETATE-d8 RT:3,30’
Y = 0.00420+1.128*X
R2 = 0.9996
0.0 0.2 0.4 0.6 0.8 1.0
Are
a r
atio
Ethyl acetate RT:3,35’
Concentration ratio
5.0e+06
1.0e+07
1.5e+07
2.0e+07
0
NO ISTD
Y = 80942.7+2.20081e+07*X
R2 = 0.9985
0.0 0.2 0.4 0.6 0.8 1.0
Are
a (
co
un
ts)
STD6 = 125 x STD1 STD5 = 65 x STD1
RESULTS
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TRIAL-AND-ERROR APPROACH
CRITERIA
• chemical classes similarity
• chromatographic retention time
• larger width of the linear working range
• better linear correlation
• intercept close to zero
RESULTS
SELECTION OF THE SUITABLE ISTD FOR EACH VOCs
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E-2-Hexenal RT: 16,6’
RESULTS
Concentration ratio
Are
a R
atio
0
20
40
60
Y = -0.301383+0.696899*X
R2 = 0.9955
0 10 20 30 40 50 60
ETHYL ACETATE-d8 RT:3,30’
0
5
15
25
Y = -0.0776+0.444*X
R2 = 0.9971
10 20 30 40 50 60
ACETIC ACID-d3 RT:25,77’
0
1600
2400
Y = -28.261+44.923*X
R2 = 0.9877
0 10 20 30 40 50 60
3,4-DIMETHYLPHENOL RT:44,58’
800
0 10 20 30 40 50 60
0
100
200
300
Y = -2.598+5.0184*X
R2 = 0.9953
6-CHLORO-2-HEXANONE RT:30,00’
0
100
200
300
400
Y = 1.401+6.938*X
R2 = 0.9923
0 10 20 30 40 50 60
ETHYL HEXANOATE-d11 RT:17,11’
0
2.0e+08
4.0e+08
6.0e+08
8.0e+08
0 10 20 30 40 50
area (counts)
Y = -4.734e+06+1.339e+07*X
R2 = 1.0000
NO ISTD
60
0
1
2
3
4
0 10 20 30 40 50 60
Y = -0.0439+0.0680*X
R2 = 0.9959
TOLUENE-d8 RT:6,76’
0
20
40
60
Y = -1.064+0.580*X
R2 = 0.9887
0 10 20 30 40 50 60
TRIMETHYLACETALDEHYDE RT:2,63’
0
100
300
500
Y = -4.719+8.594*X
R2 = 0.996
10 20 30 40 50 60
3-OCTANONE RT:18,27’
30
90
0
60 Y = -0.719+1.211*X
R2 = 0.9994
0 10 20 30 40 50 60
BUTANOL-d10 RT:12,60’
20
60
100
Y = 2.131+1.620*X
R2 = 0.9968
0 10 20 30 40 50 60
HEXANOIC ACID-d11 RT:37,08’
100
300
500
4-METHYL-2-PENTANOL RT:14,40’
Y = 0.0145+9.17*X
R2 = 0.9992
0 10 20 30 40 50 60
25
E-2-Hexenal RT: 16,6’
RESULTS
Concentration ratio
Are
a R
atio
0
20
40
60
Y = -0.301383+0.696899*X
R2 = 0.9955
0 10 20 30 40 50 60
ETHYL ACETATE-d8 RT:3,30’
0
5
15
25
Y = -0.0776+0.444*X
R2 = 0.9971
10 20 30 40 50 60
ACETIC ACID-d3 RT:25,77’
0
1600
2400
Y = -28.261+44.923*X
R2 = 0.9877
0 10 20 30 40 50 60
3,4-DIMETHYLPHENOL RT:44,58’
800
0 10 20 30 40 50 60
0
100
200
300
Y = -2.598+5.0184*X
R2 = 0.9953
6-CHLORO-2-HEXANONE RT:30,00’
0
100
200
300
400
Y = 1.401+6.938*X
R2 = 0.9923
0 10 20 30 40 50 60
ETHYL HEXANOATE-d11 RT:17,11’
0
2.0e+08
4.0e+08
6.0e+08
8.0e+08
0 10 20 30 40 50
area (counts)
Y = -4.734e+06+1.339e+07*X
R2 = 1.0000
NO ISTD
60
0
1
2
3
4
0 10 20 30 40 50 60
Y = -0.0439+0.0680*X
R2 = 0.9959
TOLUENE-d8 RT:6,76’
0
20
40
60
Y = -1.064+0.580*X
R2 = 0.9887
0 10 20 30 40 50 60
TRIMETHYLACETALDEHYDE RT:2,63’
0
100
300
500
Y = -4.719+8.594*X
R2 = 0.996
10 20 30 40 50 60
3-OCTANONE RT:18,27’
30
90
0
60 Y = -0.719+1.211*X
R2 = 0.9994
0 10 20 30 40 50 60
BUTANOL-d10 RT:12,60’
20
60
100
Y = 2.131+1.620*X
R2 = 0.9968
0 10 20 30 40 50 60
HEXANOIC ACID-d11 RT:37,08’
100
300
500
4-METHYL-2-PENTANOL RT:14,40’
Y = 0.0145+9.17*X
R2 = 0.9992
0 10 20 30 40 50 60
Range of linear calibration > 50 mg/kg
26
RESULTS
0.0
0.4
0.8
1.2
ETHYL ACETATE-d8
Y = 0.00420+1.128*X
R2 = 0.9996
0.0 0.2 0.4 0.6 0.8 1.0
Are
a r
atio
Ethyl acetate
Concentration ratio
5.0e+06
1.0e+07
1.5e+07
2.0e+07
0
NO ISTD
Y = 80942.7+2.20081e+07*X
R2 = 0.9985
0.0 0.2 0.4 0.6 0.8 1.0
Are
a (
co
un
ts)
5.0e+06
1.5e+07
2.5e+07 NO ISTD Y = 531859+2.473e+07*X
R2 = 0.9944
0.0 0.5 1.0
Are
a (
co
un
ts)
Isovaleraldehyde
Are
a R
atio
Concentration ratio
0.3
0.9
1.5
Y = -0.0273+1.099*X
R2 = 0.9964
0.0 0.2 0.4 0.6 0.8 1.0
TRIMETHYLACETALDEHYDE
0.0
1.0
2.0
1-BUTANOL d10
Y = 0.1004+2.485*X
R2 = 0.9910
0.0 0.2 0.4 0.6
Are
a (
co
un
ts)
Propanol
Are
a R
atio
Concentration ratio
0
1.0e+07
2.0e+07 NO ISTD
Y = 632245+3.113e+07*X
R2 = 0.9960
0.0 0.2 0.4 0.6
0
1.0e+08
2.0e+08
Are
a (
co
un
ts)
NO ISTD
Y = 2.407e+06+1.246e+08*X
R2 = 0.9991
0.0 0.5 1.0 1.5
0
5
10
Are
a R
atio
ETHYL ACETATE d8 Y = 0.0490+6.630*X
R2 = 0.9981
0.0 0.5 1.0 1.5
3-pentanone
Concentration ratio
0
INTERNAL STD vs EXTERNAL STANDARD CALIBRATION
27
DATA NORMALIZATION ON A SINGLE ISTD:
THE CASE OF 4-METHYL-2-PENTANOL (RT: 14,40’)
0.05
0.10
0.15BUTIRRIC ACID
Y = 0.108*X
R2 = 0.9914
0.0 0.5 1.0 1.5
Concentration ratio
Are
a R
atio
0.02
0.06
0.10
HEXANOIC ACID
Y = 0.00999+0.0465*X
R2 = 0.9953
0.0 0.5 1.0 1.5 2.0
0.1
0.3
0.5BUTHYL_ACETATE
Y = 0.0111+0.283*X
R2 = 0.9953
0.0 0.5 1.0 1.5
0.00
0.05
0.10
0.15
Y = 0.00149+0.153*X
R2 = 0.9991
0.0 0.2 0.4 0.6 0.8 1.0
ETHYL_ACETATE
100
300
500 E-2-HEXENAL
Y = 0.0145+9.17*X
R2 = 0.9992
0 10 20 30 40 50 60
0.00
0.01
0.02
0.03 LIMONENEY = -0.000151+0.0278*X
R2 = 0.9899
0.0 0.5 1.0
0.5
1.5
2.5 2-BUTANONE
Y = -0.193+2.406*X
R2 = 0.9947
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0.0
0.5
1.0
1.5ETHYL VINYL KETONE
Y = 0.00399+0.843*X
R2 = 0.9984
0.0 0.5 1.0 1.5
0.0
0.5
1.0
1.5
2.0HEXANOL
Y = -0.0391+0.2765*X
R2 = 0.9961
0.0 1.0 2.0 3.0 4.0 5.0
0.001
0.003
0.005 OCTANOLY = -2e-05+0.0074*X
R2 = 0.9980
0.0 0.1 0.2 0.3 0.4
0.000
0.002
0.004
0.006
4-ETHYLPHENOLY = 0.000100+0.00206*X
R2 = 0.9537
0.0 0.5 1.0 1.5
0.005
0.015
0.025
GUAIACOLY = 0.000125+0.00891*X
R2 = 0.9796
0.0 0.5 1.0 1.5
RESULTS
RT: 31,42’ RT: 37,43’
RT: 3,35’
RT: 22,42’
RT: 6,30’
RT: 29,30’
RT: 8,64’
RT:3,35’
RT: 16,59’
RT:43,97’ RT: 37,83’ RT: 15,15’
28
Linearity of the calibration, by squared adjusted regression coeff. ≥ 0.95
LOQ and upper end of calibration
Accuracy: in terms of trueness and precision on 6 replicates
of two levels spiked samples
Selectivity: it was assured by the use of suitable ions
Linear range of calibration: 10-100 fold the LOQ
METHOD VALIDATION
29
VOOs ANALYSIS
LVOO supplied by the International Olive Council and qulified:
RANCID – median intensity 9.5
MUSTY – median intensity 4.7
Analysis of LVOOs: 32-fold dilution factor
EVOOs from different provenance and cv
30
All the 71 analytes were present in at least 1 sample, with the only exceptions
of ethyl propanoate, 2-butanol and 2-pentanol (96%)
The total volatile compounds in lampante virgin olive oils were higher than
extra virgin olive oils
Lampante virgin olive oils were characterized by high concentration of
volatiles different from extra virgin olive oils
VOOs ANALYSIS:
characterization of VOOs from different provenance, cv and classification
31
VOOs ANALYSIS
Concentration of the analytes quantified:
mean ± standard deviation of triplicates
(mg/kg)
32
VOOs ANALYSIS
Concentration of the analytes quantified:
mean ± standard deviation of triplicates
(mg/kg)
33
VOOs ANALYSIS
Concentration of the analytes quantified:
mean ± standard deviation of triplicates
(mg/kg)
34
VOOs ANALYSIS
Concentration of the analytes quantified:
mean ± standard deviation of triplicates
(mg/kg)
35
VOOs ANALYSIS
Concentration of the analytes quantified:
mean ± standard deviation of triplicates
(mg/kg)
36
• The proposed method allows to quantify a large number of volatile
compounds of virgin olive oils
• The proposed method allows the characterization of VOOs from different
provenance, cv and classifications
• Use of multiple ISTD is necessary for quantify VOO-VOCs in a wide
range of concentration
• The choice of the most suitable ISTD for each analyte is a key step for
obtain wider linear calibration
• The method requires an initial preparation of STD solutions and
optimization, but then it allows simple routine analysis
• The proposed method is a starting point from which an olive oil company
can build its own database: the most suitable one for requirements of its
oils
CONCLUSION