perkinelmer: the determination of benzene, toluene, ethyl benzene, xylenes and styrene in olive oil...

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The Determination of Benzene, Toluene, Ethyl

Benzene, Xylenes and Styrene in Olive Oil using HS Extraction and GC/MS

Andrew TiplerChromatography R&D Manager

PerkinElmer Inc., Shelton, CT

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.

Olive Oil Production

Country Production in tons (2009)

Production % (2009)

Consumption (2005)

Annual per capita

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

Effects of Air Pollution

Olive trees are good at absorbing air pollutants and passing them on to the olives and thus into the 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".

Acceptable limits for BTEXS in olive oil

5.05 5.55 6.05 6.55 7.05 7.55 8.05 8.55 9.05 9.55 10.05Time0

100

%

Scan EI+ TIC

1.17e89.92

8.85

7.94

6.63

5.24

5.004.92

4.62

4.72

6.49

8.07

Ben

zen

e

Tolu

ene

p-X

ylen

em

-Xyl

en

e

Eth

ylb

enze

ne

o-X

ylen

e

Sty

ren

e

BTEXS (~0.6ng) separation using GC/MS

30m x 0.25mm x 1.0µm Elite-Wax35(1)-10-130(0)

Sampling Olive Oil

Olive oil is a heavy sample matrix and is not very GC-friendly. Direct injection will cause problems with breakdown and contamination.

Some form of extraction technique is required to separate the BTEXS from the sample matrix prior to introduction into the GC.

Don’t do this in your lab!

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.

Analytical System

Clarus SQ8 GC/MS TurboMatrix HS Sampler

Pressure-Balanced Headspace Sampling

Pressurization Sampling

V1 V1

Equilibration

V1

PHS

V2

Valve closed for just a few seconds

Olive oil sample

5.05 5.55 6.05 6.55 7.05 7.55 8.05 8.55 9.05 9.55 10.05Time0

100

%

1: Scan EI+ TIC

1.17e8

4.62

4.914.63

6.57

5.70

6.295.91

6.06

7.21

7.97 8.34

8.21 10.43

8.91

Eth

ano

lB

enze

ne

Tolu

ene

p-X

ylen

em

-Xyl

en

e

Eth

ylb

enze

ne

o-X

ylen

e

Sty

ren

e

Total Ion Chromatogram from Spiked Olive Oil

10g olive oil spiked with 17ng/g BTEXS

30m x 0.25mm x 1.0µm Elite-Wax35(1)-10-130(0)

5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10Time0

100

%

0

100

%

0

100

%

0

100

%

2.61e49.93

9.38e38.17

8.077.94 8.86

4.80e46.62

6.95e45.24

Be

nze

ne

Tolu

en

e

p-X

yle

ne

m-X

yle

ne

Eth

ylb

en

zen

e

o-X

yle

ne

Sty

ren

e

SIM @ 78 Daltons

SIM @ 92 Daltons

SIM @ 106 Daltons

SIM @ 104 Daltons

Single Ion Monitoring Improves Selectivity and Detectability

SiFi Enables ‘Simultaneous’ Full and Single Ion Data collection

5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10Time0

100

%

OliveOilAStd_060

9.95

Ben

zen

e @

78

Da

Tolu

ene

@ 9

2 D

a

p-X

ylen

e @

106

Da

m-X

yle

ne

@ 1

06 D

a

Eth

ylb

enze

ne

@ 1

06 D

a

o-X

ylen

e @

106

Da

Sty

ren

e @

104

Da

SIM of Olive oil spiked with 17ng/g BTEXS

y = 178.38x - 60.006R² = 0.9998

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 10 20 30 40 50 60 70 80 90 100

Peak

Are

a (C

ount

s)

Concentration (ppb w/w)

Extracted Calibration Plot for Benzene

y = 12.021x - 3.8872R² = 0.9997

0

200

400

600

800

1000

1200

0 10 20 30 40 50 60 70 80 90 100

Peak

Are

a (C

ount

s)

Concentration (ppb w/w)

Extracted Calibration Plot for Styrene

Quantitative Performance

Compound RSD (% at 45ng/g, n=10)

r2

(from 4.5 to 90ng/g)Limit of Detection

(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

5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10Time0

100

%

0

100

%

0

100

%

0

100

%

TIC1.84e3

S/N:RMS=23.81

9.809.7110.03

10.17

TIC2.47e3

S/N:RMS=39.67

7.93

7.807.738.06

8.85

8.348.57 8.939.05

TIC1.10e4

S/N:RMS=71.15

TIC3.90e3

S/N:RMS=14.84

5.015.42

Benzene, 0.9ng/g

Toluene, 5.9ng/g

m-Xylene, 5.4ng/g

Styrene, 3.1ng/g

Low-Level Determination of BTEXS in Olive Oil

Background from ‘Clean’ Olive Oil

5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10 Time0

100

%

OliveOilA_061

5.10 5.60 6.10 6.60 7.10 7.60 8.10 8.60 9.10 9.60 10.10 Time0

100

%

OliveOilAStd_060

9.95

Ben

zen

e @

78

Da

Tolu

ene

@ 9

2 D

a

p-X

ylen

e @

106

Da

m-X

yle

ne

@ 1

06 D

a

Eth

ylb

enze

ne

@ 1

06 D

a

o-X

ylen

e @

106

Da

Sty

ren

e @

104

Da

‘Clean’ Olive Oil

‘Clean’ Olive Oil spiked with 17ng/g BTEXS

BTEXS in Olive Oil from Local Supermarket (ng/g)

Sample Source Benzene

Toluene

Ethylbenzene

p-Xylene

m-Xylene

o-Xylene

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, Argentina 2.99 17.03 3.74 3.44 9.35 6.14 40.09

Spain, Argentina 2.43 34.99 7.22 7.42 18.97 10.65 126.11

Italy, Spain, Greece, Tunisia, Morocco, Syria, Turkey 4.09 35.71 19.13 17.10 59.31 28.10 61.05

Italy, Greece, Spain, Tunisia 1.25 2.79 ND 1.80 3.74 3.17 7.39

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.

Conclusions