Levels of benzene, toluene, ethylbenzene, xylenes and styrene (BTEXS) find their way into olive trees and hence into the olives and olive oil mainly as a result of the presence of vehicle exhaust in ambient air. Although there is widespread concern about the presence of these carcinogenic compounds in olive oil, no definitive methods or limits have yet been prescribed. Various methods have been developed to detect and quantify these compounds down to levels of 5ng/g (5 ppb w/w). In this work, we have developed a simple method to determine these components in olive oil using headspace (HS) extraction and gas chromatography/mass spectrometry (GC/MS). Sample preparation simply comprises dispensing and sealing 10g of olive into a standard 22-mL headspace vial and sampling the headspace vapor after being equilibrated at 90°C. The vapor is introduced into a Carbowax capillary column for chromatographic separation. Detection, identification and quantification is performed using a quadrupole MS system with a novel ionizer and detection system that enables detection limits, in single ion monitoring (SIM) mode, down to below 0.5ng/g without the need for headspace vapor preconcentration. The analysis is fully automated and takes just 10.5 minutes for the chromatography and an additional 3.5 minutes for cool-down and equilibration between analyses. Excellent quantitative performance has been demonstrated and the system is easily able to see concentrations of these compounds in the range 0.9ng/g to 126.1ng/g in olive oil bought from a local supermarket. Examples of the chromatography and quantitative performance will be presented.

1. The Determination of
Benzene, Toluene, Ethyl
Benzene, Xylenes and
Styrene in Olive Oil using
HS Extraction and GC/MS
Andrew Tipler
Chromatography R&D Manager
PerkinElmer Inc., Shelton, CT
© 2009 PerkinElmer
2012

2. Olive Oil
Olive oil is produced by grinding olives and
extracting the oil by mechanical (pressing) or
chemical (solvent extraction) means.
Olive oil is composed mainly of the mixed
triglyceride esters of oleic acid and palmitic
acid and of other fatty acids, along with traces
of squalene and sterols.
It is commonly used in cooking, cosmetics,
pharmaceuticals, and soaps and as a fuel.

3. Olive Oil Production
Annual per
Production in Production % Consumption capita
Country
tons (2009) (2009) (2005) consumption
(kg)
World 2,907,985 100% 100% 0.4
Spain 1,199,200 41.2% 20% 13.6
Italy 587,700 20.2% 30% 12.4
Greece 332,600 11.4% 9% 23.7
Syria 168,163 5.8% 3% 7.0
Tunisia 150,000 5.2% 2% 11.1
Turkey 143,600 4.9% 2% 1.2
Morocco 95,300 3.3% 2% 1.8
Portugal 53,300 1.8% 2% 7.1
France 6,300 0.2% 4% 1.3
United States 2,700 0.1% 8% 0.6
Others 169,122 5.8% 18% 1.2
Source: Wikipedia.com

4. Effects of Air Pollution
Olive trees are good at absorbing air pollutants and passing them on to the
olives and thus into the oil

5. Acceptable limits for BTEXS in olive oil
“Concentrations in olive oil were found to be around several tens of µg/kg for
benzene; a few hundred µg/kg for toluene and many hundreds of µg/kg for the
sum of ethyl benzene and xylenes. Substantial concentrations of styrene were
found which however was probably of natural origin. The situation was
considered to be sufficiently serious that a definition of provisional limits for
these components was proposed (benzene: 50µg/kg; toluene: 180µg/kg; ethyl
benzene: 50µg/kg; xylenes: 280µg/kg). However, these were not imposed as a
consequence of disagreement in the European Union” . Maurus Biedermann,
Konrad Krob, Gianni Morchio, Z Lebensm Unters Forsch (1995) 200; 266-272.
Despite the strong concerns, even today, there still appears to be no
agreement on the acceptable limits of BTEXS in olive oil.
On 10 June 2011, the US National Toxicology Program has described styrene
as "reasonably anticipated to be a human carcinogen".

6. BTEXS (~0.6ng) separation using GC/MS
Styrene
30m x 0.25mm x 1.0µm Elite-Wax
o-Xylene
Scan EI+
9.92 TIC
100
35(1)-10-130(0)
Ethylbenzene
1.17e8
p-Xylene
m-Xylene
Toluene
8.85
7.94 8.07
Benzene
6.63
5.24
%
4.92 5.00
4.62
4.72
6.49
0 Time
5.05 5.55 6.05 6.55 7.05 7.55 8.05 8.55 9.05 9.55 10.05

7. Sampling Olive Oil
Olive oil is a heavy sample
matrix and is not very GC-
friendly. Direct injection will
cause problems with
breakdown and Don’t do this in your lab!
contamination.
Some form of extraction
technique is required to
separate the BTEXS from
the sample matrix prior to
introduction into the GC.

8. Headspace Sampling
Headspace sampling provides a very easy
way to extract the volatile BTEXS
compounds from the olive oil matrix.
10g of sample are placed in a vial which is
then sealed with a cap and maintained at
90°C for 20 minutes. The volatile
components (including BTEXS) will
partition into the vapor phase
0.15mL of the vapor phase is transferred
to the GC column for analysis.

9. Analytical System
Clarus SQ8 GC/MS TurboMatrix HS Sampler

10. Pressure-Balanced Headspace Sampling
Equilibration Pressurization Sampling
V2
V1 V1 V1
PHS
Valve
closed for
just a few
seconds
Olive oil
sample

11. Total Ion Chromatogram from Spiked Olive Oil
1: Scan EI+
Ethanol
100 TIC
10g olive oil spiked with 17ng/g BTEXS 1.17e8
30m x 0.25mm x 1.0µm Elite-Wax
35(1)-10-130(0)
Ethylbenzene
p-Xylene
Toluene
m-Xylene
Benzene
o-Xylene
Styrene
%
6.57
7.21
4.62
4.63 4.91
5.70
7.97 8.34
5.91 6.29
10.43
8.21
6.06 8.91
0 Time
5.05 5.55 6.05 6.55 7.05 7.55 8.05 8.55 9.05 9.55 10.05

12. Single Ion Monitoring Improves Selectivity and Detectability
9.93
100 2.61e4
Styrene
SIM @ 104 Daltons
%
0
Ethylbenzene
p-Xylene
8.17
m-Xylene
100 9.38e3
SIM @ 106 Daltons
o-Xylene
7.94 8.07 8.86
%
0
6.62
Toluene
100 4.80e4
SIM @ 92 Daltons
%
0
5.24
100
Benzene
6.95e4
SIM @ 78 Daltons
%
0 Time
5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10

13. SiFi Enables ‘Simultaneous’ Full and Single Ion Data collection

14. 0
%
100
5.10
OliveOilAStd_060
Benzene @ 78 Da
5.60
6.10
6.60
Toluene @ 92 Da
7.10
7.60
Ethylbenzene @ 106 Da
8.10
p-Xylene @ 106 Da
m-Xylene @ 106 Da
8.60
o-Xylene @ 106 Da
9.10
SIM of Olive oil spiked with 17ng/g BTEXS
9.60
9.95
Styrene @ 104 Da
10.10
Time

15. Extracted Calibration Plot for Benzene
18000
y = 178.38x - 60.006
16000
R² = 0.9998
14000
12000
Peak Area (Counts)
10000
8000
6000
4000
2000
0
0 10 20 30 40 50 60 70 80 90 100
Concentration (ppb w/w)

16. Extracted Calibration Plot for Styrene
1200
y = 12.021x - 3.8872
R² = 0.9997
1000
800
Peak Area (Counts)
600
400
200
0
0 10 20 30 40 50 60 70 80 90 100
Concentration (ppb w/w)

17. Quantitative Performance
RSD r2 Limit of Detection
Compound
(% at 45ng/g, n=10) (from 4.5 to 90ng/g) (ng/g)
Benzene 1.7 0.9998 0.1
Toluene 3.8 0.9986 0.2
Ethylbenzene 2.3 0.9995 0.3
p-Xylene 3.5 0.9997 0.3
m-Xylene 3.7 0.9998 0.3
o-Xylene 3.3 0.9995 0.3
Styrene 3.5 0.9997 0.3
Standard mixtures prepared in 10g ‘clean’ olive oil

18. Low-Level Determination of BTEXS in Olive Oil
S/N:RMS=23.81 TIC
100 1.84e3
Styrene, 3.1ng/g
10.03
9.71 9.80 10.17
%
0
S/N:RMS=39.67 TIC
100 2.47e3
m-Xylene, 5.4ng/g
8.85
% 8.34
7.93 8.06 8.57 8.93 9.05
7.73 7.80
0
S/N:RMS=71.15 TIC
100 1.10e4
Toluene, 5.9ng/g
%
0
S/N:RMS=14.84 TIC
100 3.90e3
Benzene, 0.9ng/g
5.01
5.42
%
0 Time
5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10

19. Background from ‘Clean’ Olive Oil
Toluene @ 92 Da
Benzene @ 78 Da
‘Clean’ Olive Oil spiked with 17ng/g BTEXS
OliveOilAStd_060
100
Ethylbenzene @ 106 Da
m-Xylene @ 106 Da
Styrene @ 104 Da
p-Xylene @ 106 Da
o-Xylene @ 106 Da
%
9.95
0 5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10 Time
‘Clean’ Olive Oil
OliveOilA_061
100
%
0 Time
5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10

20. BTEXS in Olive Oil from Local Supermarket (ng/g)
Ethylbenzene
Sample Source
m-Xylene
p-Xylene
o-Xylene
Benzene
Toluene
Styrene
California 0.89 5.86 1.66 1.45 5.24 3.77 3.07
Italy, Greece, Spain Tunisia 2.86 27.55 6.12 5.86 16.73 8.75 41.34
Italy, Spain, Greece, Tunisia 3.07 24.22 13.47 7.85 23.64 13.97 39.59
Italy, Spain, Tunisia, Turkey,
2.99 17.03 3.74 3.44 9.35 6.14 40.09
Argentina
Spain, Argentina 2.43 34.99 7.22 7.42 18.97 10.65 126.11
Italy, Spain, Greece, Tunisia,
4.09 35.71 19.13 17.10 59.31 28.10 61.05
Morocco, Syria, Turkey
Italy, Greece, Spain, Tunisia 1.25 2.79 ND 1.80 3.74 3.17 7.39

21. Conclusions
The sensitivity of the MS detector in SIM mode enables detection of low levels
of BTEXS with standard headspace sampling. Detection limits of 0.1 to 0.3ng/g
have been demonstrated which compare favorably against those in other
published papers (~5ng/g).
The HS sampling system simplifies sample preparation and eliminates the
effects from a heavy sample matrix entering the chromatographic column and
the MS detector.
Good quantitative performance has been shown.
Significant levels of BTEXS were found in olive oils purchased at a local
(Connecticut) supermarket.