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)

2

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

3

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

4

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

5

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

6

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

7

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

8

AIMS

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

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


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


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



(mg/kg)

33

VOOs ANALYSIS



(mg/kg)

34

VOOs ANALYSIS



(mg/kg)

35

VOOs ANALYSIS



(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

FOOD SERVICES

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THANKS FOR

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SPME-GC-MS: Advantages of an internal multistandard method ...

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