The challenging extraction of non-polar

contaminants out of a non-polar vegetable oil

sample
Presented by Michael Ye
ExTech, 2014

sigma-aldrich.com/analytical 1

2012 Sigma-Aldrich Co. All rights reserved.

 Agenda

1. Background; why and how analyze PAHs in edible oils

2. A new approach dual layer SPE; EZ-POP NP
3. Summary of method using EZ-POP NP for olive oil
4. Experimental data
GC-MS background
PAH recoveries
GC Method ruggedness
Comparison to other SPE methods
5. Conclusions

2012 Sigma-Aldrich Co. All rights reserved.

The Issue
Why analyze PAHs in edible oils?
Contamination from environmental exposure and processing
Air pollution
Combustion gases produced during processing/production of oils

EU Commission Regulation No. 835/2011:

2 ng/g MCL for benzo[a]pyrene
10 ng/g total benzo[a]pyrene, benzo[b]fluoranthene, chrysene and
benzo[a]anthracene combined

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 How to analyze PAHs in edible oils?

ISO methods
Benzo[a]pyrene only, uses large alumina column
15302 (30 cm x 1.5 cm) for extraction
16 PAHs (light to heavy), uses LLE and 2-step
15753
cleanup with C18 and Florisil
Other methods
LLE followed by GPC
Silica gel or florisil SPE using large, glass columns
Molecularly imprinted polymer (MIP) SPE

Final analysis done by GC-MS/SIM or HPLC-FLD

Issues with current methods

GPC expensive, time consuming
Silica gel/Florisil (lg. column SPE) expensive, inadequate cleanup for GC
MIP SPE Poor recoveries of lighter PAHs

2012 Sigma-Aldrich Co. All rights reserved.

How to analyze PAHs in edible oils?
A new approach - EZ-POP NP SPE

Dual-layer SPE cartridge containing Florisil and Z-Sep/C18 mix.

Florisil - retains background constituents with polar functionality such as fatty
acids.
Z-Sep/C18 mixture - retains fatty matrix through both Lewis acid/base and
hydrophobic interactions.

Easy sample preparation methodology using minimal volume of solvent.

Final sample extracts compatible with GC or HPLC.

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What is Z-Sep / C18 ?
A mixture of a proprietary HybridSPE zirconia-coated silica and C18
functionalized silica:
ZrO C18
ZrO C18
Zr C18
ZrO C18
ZrO C18
ZrO C18
ZrO C18
ZrO C18

Zirconia acts as a Lewis acid, binding electron donating groups such as OH

found in mono and diacylglycerols.
C18 binds fats through hydrophobic interaction. Used with zirconia, it
produces a synergistic effect in enhancing fat retention.

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 Extraction of PAHs from Olive Oil Using EZ-POP NP

Condition SPE cartridge with 10 mL acetone using

gravity elution. Dry cartridge at -10 to -15 Hg for 10 min.

Weigh 0.5 mL olive oil onto top of SPE cartridge. Add

internal standard.

Add 15 mL of acetonitrile in 2 x 7.5 mL increments. Pull

through SPE cartridge at a drop rate of approximately 1
drop/second. Collect all eluent.
Florisil layer

Evaporate eluent at 40C under 5 psi nitrogen

Z-Sep&C18
(do not allow to go dry). layer

Adjust the final volume of the extracts to required final

volume using acetonitrile

EZ-POP NP dual-layer cartridge

Proceed with HPLC/FLD and/or GC-MS/SIM analysis.
during extraction of PAHs from
olive oil. 7

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Background extract of 0.5 gm olive oil sample
Background remaining in a 1mL olive oil extract; evaluated by GC-MS
in scan mode 2.00E+07

Silica gel SPE (5 gm/10 mL),

FV=1
abundance
1.00E+07
0.00E+00

10 20 30
Time (min)
2.00E+07

EZ-POP NP SPE, FV=1

1.00E+07
0.00E+00

10 20 30
Time (min)
8

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 Recoveries of PAHs Using EZ-POP NP
Olive oil spiked at 2 ng/g with PAHs from 2-6 rings, including those
listed in the EU regulation.
The sample set contained 3 spiked replicates and one unspiked
olive oil blank.
5-Methylchrysene used as internal standard.
Final extracts concentrated to 0.2 mL for analysis.
Extracts analyzed by GC-MS/SIM and HPLC-FLD.
Matrix-matched standards used for quantitation.

2012 Sigma-Aldrich Co. All rights reserved.

Example of GC-MS/SIM analysis of olive oil extract 1. Naphthalene
2. Acenaphthylene (GC only)
3. Acenaphthene
4. Fluorene
5. Phenanthrene
6. Anthracene
7. Fluoranthene
8. Pyrene
9. Benzo[a]anthracene
10. Chrysene
11. 5-methyl Chrysene (IS)
12. Benzo[b]fluoranthene
13. Benzo[k]fluoranthene
13 14. Benzo[a]pyrene
14
5
12 15. Dibenzo[a,h]anthracene
1 4 910 1715
23 6 7 8 11 16 16. Benzo[g,h,i]perylene
17. Indeno[1,2,3-cd]pyrene
2 4 6 8 10 12 14 16 18 20
Time (min)
Single quadrupole MS
GC-MS: Agilent 7890/5975, selected ion mode (SIM)
column: SLB-5ms, 20 m x 0.18 mm I.D. x 0.18 m
oven: 60 C (1 min.), 15 C/min. to 250 C, 8 C/min. to 330 C (7 min.) Higher MS temps to
inj. temp.: 300 C minimize tailing of
MS Temps: interface 330 C, source 250 C, quads. 200 C heavy PAHs
carrier gas: helium, 1 mL/min constant flow
injection: 0.5 L pulsed splitless (50 psi until 0.75 min, splitter open at 0.75 min.)
liner: FocusLiner with taper and quartz wool Pulsed injection to
increase response of
heavy PAHs 10

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Example of HPLC-FLD analysis of olive oil extract, 2 ng/g spike
1. Naphthalene
2. Acenaphthylene (GC only)
3. Acenaphthene
4. Fluorene
5. Phenanthrene
3 4
6. Anthracene
10
7. Fluoranthene
5 8. Pyrene
1 7 11 13
8
9. Benzo[a]anthracene
14
9 12 15
10. Chrysene
6 11. 5-methyl Chrysene (IS)
16 17 12. Benzo[b]fluoranthene
13. Benzo[k]fluoranthene
10 12 14 16 18 20 22 24 26 28
14. Benzo[a]pyrene
Time (min)
15. Dibenzo[a,h]anthracene
16. Benzo[g,h,i]perylene
17. Indeno[1,2,3-cd]pyrene
Instrument: Agilent 1200 / 1260 FLD
column: Supelcosil LC-PAH, 25 cm x 4.6 mm I.D., 5 m
mobile phase: (A) water (B) acetonitrile
gradient: 40% B for 5 min, to 100% in 15 min; held at 100% B
for 10 min
flow rate: 1.4 mL/min
column temp.: 30 C
detector: fluorescence, programmed
injection: 20 L

11

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 PAH Recoveries; GC-MS/SIM analysis
2 ng/g spike level % RSD
% Recovery (n=4) Recoveries >80% for most PAHs
Naphthalene 63 12
Acenaphthene 96 4 RSD values <15% for most PAHs
Acenaphthylene 95 12 Method was acceptable for detection
Fluorene 119 15
Phenanthrene 154 49 at 2 ng/g on single quadrupole MS
Anthracene 99 8 instrument
Fluoranthene 100 6
Pyrene 99 6
Benzo[a]anthracene 98 7
Chrysene 98 9
Benzo[b]fluoranthene 94 5
Benzo[k]fluoranthene 95 2
Benzo[a]pyrene 86 3
Indeno[1,2,3-cd]pyrene 85 2
Dibenz[a,h]anthracene 94 10
Benzo[g,h,i]perylene 82 4

Regulated PAHs per EU 835/2011

12

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 Comparison of GC-MS to HPLC-FLD results

180%
GC-MS; matrix stds. HPLC-matrix stds
160%
140%
120%
100%
80%
60%
40%
20%
0%

Does not fluoresce

Larger difference due to interference in HPLC analysis

13

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 Comparison of GC and HPLC Data- Summary

Analysis of 2 ng/g olive

oils spikes GC-MS/SIM HPLC-FLD

Recovery >80% for all except >80% for all except

naphthalene naphthalene
Reproducibility < 20% for all except < 20% for 12 of 16
phenanthrene compounds
Background No interference in SIM Interference with
fluorene

14

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 GC Method Ruggedness Test

Loss of response in GC can occur as a result of

residue buildup in the inlet liner
residue buildup in the column
contamination of the detector

Contaminated GC inlet
liner

Tested cleanliness of the olive oil extract

Made 110 injections of an olive oil extract into GC-MS system
Spiked with 28 different PAHs (2-6 rings)
Monitored absolute response of PAHs

15

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 GC Method Ruggedness Test- Results
100000
90000
80000
70000
Area counts

60000
50000
40000
30000 start

20000 110 inj

10000
0

Decrease in absolute response after 110 injections

12

10

Some loss of response was expected. # Compounds 8

17 of 28 PAHs decreased in response by 6

<20%. 4

0
16
< 10% 10-20% 21-30% 31-40% 41-50%

2012 Sigma-Aldrich Co. All rights reserved.

 Inlet liner after
110 injections
The cleanliness of the extract was of olive oil
acceptable for GC-MS analysis extract

Pattern left by
autosampler in vial caps
after 110 injections
17

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How does EZ-POP NP compare to other SPE methods?
1. Silica gel SPE
2. MIP SPE
Experiment
Olive oil samples spiked at 20 ng/g with 28 PAHs
Samples extracted using EZ-POP NP, silica gel SPE, and MIP
SPE; 3 replicates with each method plus a blank
0.5 gm sample size per SPE cartridge for each method
Internal standards used (deuterated PAHs)
GC-MS/SIM Analysis

Methods
Silica Gel SPE
Condition: 20 mL hexane
Load: 1 mL of 5 gm oil diluted to FV10 mL in hexane.
Add internal standard directly to cartridge.
Wash: 8 mL hexane:methylene chloride (70:30)
Elute: 8 mL hexane:methylene chloride (70:30)
Concentrate: FV=1 mL under nitrogen at 40C
MIP SPE
Condition: 1 mL cyclohexane
Load: 0.5 gm oil diluted to 1 mL in cyclohexane
Wash: 1 mL cyclohexane
Elute: 3 x 1 mL ethyl acetate
Concentrate: < 1 mL (not dryness) under nitrogen at
40 C. Adjust final volume to 1 mL with ethyl
acetate.
18

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Results- PAH Recoveries, 2-3 rings
EZ-POP NP Silica Gel MIP

120%

100%

80%

60%

40%

20%

0%

For Silica gel and MIP, matrix interference prevented accurate

quantitation for most of these PAHs.
Low recovery of naphthalene and biphenyl using MIP.
19

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Results- PAH Recoveries, 4 rings
EZ-POP NP Silica Gel MIP

120%

100%

80%

60%

40%

20%

0%

Low recoveries using silica gel

Matrix interference with chrysene & triphenylene using MIP

20

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Results- PAH Recoveries, 5-6 rings
EZ-POP NP Silica Gel MIP

120%

100%

80%

60%

40%

20%

0%

Lower recoveries using silica gel

MIP recoveries very good
MIP SPE suitable for heavier PAHs (>4 rings)

21

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 How does GC-MS background compare?
2.00E+07

Silica gel SPE

abundance

1.00E+07
0.00E+00

10 20 30
Time (min)
2.00E+07

EZ-POP NP SPE
1.00E+07
0.00E+00

10 20 30
Time (min)
2.00E+07
1.00E+07

MIP SPE
glycerides
0.00E+00

10 20 30 22
Time (min)

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 In Conclusion
EZ-POP NP is suitable for
Extraction of full range of PAHs from 2-6 rings from olive oil.

Detection of PAHs at levels specified in EU Regulation 835/2011.

Producing an extract that can be analyzed by GC-MS/SIM or

HPLC-FLD without the need for a solvent exchange.

Very rugged GC-MS method on a single quadrupole instrument

Very clean extract; will not prematurely foul GC-MS system

23

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 In Conclusion
Compared to other SPE methods
EZ-POP NP will give better recoveries for PAHs of 2-4 rings than
MIP SPE and overall better recoveries than silica gel SPE.
EZ-POP NP produces a cleaner extract than silica gel and MIP
SPE.
The EZ-POP method is easier and more versatile than silica gel or
MIP SPE.
Less steps in SPE method

Less solvent usage than silica gel

Produces an extract in acetonitrile; which can be run by GC or LC.

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