The challenging extraction of non-polar contaminants …€¦ · The challenging extraction of non-polar contaminants out of a non ... •Z-Sep/C18 mixture - retains fatty matrix

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The challenging extraction of non-polar contaminants out of a non-polar vegetable oil sample

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Presented by Michael YeExTech, 2014


Agenda

1. Background; why and how analyze PAHs in edible oils2. A new approach – dual layer SPE; EZ-POP NP3. Summary of method using EZ-POP NP for olive oil4. Experimental dataGC-MS backgroundPAH recoveries GC Method ruggednessComparison to other SPE methods

5. Conclusions

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The Issue

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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 methods15302

15753

Benzo[a]pyrene only, uses large alumina column (30 cm x 1.5 cm) for extraction16 PAHs (light to heavy), uses LLE and 2-step 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

GPC expensive, time consuming

Silica gel/Florisil (lg. column SPE)MIP SPE

expensive, inadequate cleanup for GCPoor recoveries of lighter PAHs

Issues with current methods


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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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• A mixture of a proprietary HybridSPE zirconia-coated silica and C18 functionalized silica:

What is Z-Sep / C18 ?

ZrO

ZrO

ZrZrO

ZrOZrO

ZrO

ZrO

• 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.

C18

C18

C18C18

C18C18

C18

C18


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Extraction of PAHs from Olive Oil Using EZ-POP NP

EZ-POP NP dual-layer cartridge during extraction of PAHs from olive oil.

Florisil layer

Z-Sep&C18 layer

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.

Adjust the final volume of the extracts to required final volume using acetonitrile

Evaporate eluent at 40°C under 5 psi nitrogen (do not allow to go dry).

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.

Proceed with HPLC/FLD and/or GC-MS/SIM analysis.


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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

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Silica gel SPE (5 gm/10 mL), FV=1

EZ-POP NP SPE, FV=1


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• 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 unspikedolive 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.

Recoveries of PAHs Using EZ-POP NP


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Example of GC-MS/SIM analysis of olive oil extract

GC-MS: Agilent 7890/5975, selected ion mode (SIM)column: SLB-5ms, 20 m x 0.18 mm I.D. x 0.18 µmoven: 60 °C (1 min.), 15 °C/min. to 250 °C, 8 °C/min. to 330 °C (7 min.)inj. temp.: 300 °CMS Temps: interface 330 °C, source 250 °C, quads. 200 °Ccarrier gas: helium, 1 mL/min constant flowinjection: 0.5 µL pulsed splitless (50 psi until 0.75 min, splitter open at 0.75 min.) liner: Focus™Liner with taper and quartz wool

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6 1615

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2 4 6 8 10 12 14 16 18 20Time (min)

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Higher MS temps to minimize tailing of heavy PAHs

Pulsed injection to increase response of heavy PAHs

Single quadrupole MS

1. Naphthalene2. Acenaphthylene (GC only)3. Acenaphthene4. Fluorene5. Phenanthrene6. Anthracene7. Fluoranthene8. Pyrene9. Benzo[a]anthracene10. Chrysene11. 5-methyl Chrysene (IS)12. Benzo[b]fluoranthene13. Benzo[k]fluoranthene14. Benzo[a]pyrene15. Dibenzo[a,h]anthracene16. Benzo[g,h,i]perylene17. Indeno[1,2,3-cd]pyrene


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Instrument: Agilent 1200 / 1260 FLDcolumn: Supelcosil LC-PAH, 25 cm x 4.6 mm I.D., 5 µmmobile phase: (A) water (B) acetonitrilegradient: 40% B for 5 min, to 100% in 15 min; held at 100% B for 10 minflow rate: 1.4 mL/mincolumn temp.: 30 °Cdetector: fluorescence, programmedinjection: 20 µL

1. Naphthalene2. Acenaphthylene (GC only)3. Acenaphthene4. Fluorene5. Phenanthrene6. Anthracene7. Fluoranthene8. Pyrene9. Benzo[a]anthracene10. Chrysene11. 5-methyl Chrysene (IS)12. Benzo[b]fluoranthene13. Benzo[k]fluoranthene14. Benzo[a]pyrene15. Dibenzo[a,h]anthracene16. Benzo[g,h,i]perylene17. Indeno[1,2,3-cd]pyrene

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Example of HPLC-FLD analysis of olive oil extract, 2 ng/g spike


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2 ng/g spike level% Recovery

% RSD (n=4)

Naphthalene 63 12Acenaphthene 96 4

Acenaphthylene 95 12Fluorene 119 15

Phenanthrene 154 49Anthracene 99 8

Fluoranthene 100 6Pyrene 99 6

Benzo[a]anthracene 98 7Chrysene 98 9

Benzo[b]fluoranthene 94 5Benzo[k]fluoranthene 95 2

Benzo[a]pyrene 86 3Indeno[1,2,3-cd]pyrene 85 2Dibenz[a,h]anthracene 94 10Benzo[g,h,i]perylene 82 4

Regulated PAHs per EU 835/2011

PAH Recoveries; GC-MS/SIM analysis

• Recoveries >80% for most PAHs• RSD values <15% for most PAHs• Method was acceptable for detection

at 2 ng/g on single quadrupole MS instrument


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20%

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180%GC-MS; matrix stds. HPLC-matrix stds

Comparison of GC-MS to HPLC-FLD results

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Larger difference due to interference in HPLC analysis

Does not fluoresce


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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 naphthalene

>80% for all except naphthalene

Reproducibility < 20% for all except phenanthrene

< 20% for 12 of 16 compounds

Background No interference in SIM Interference with fluorene


GC Method Ruggedness Test

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• 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


GC Method Ruggedness Test- Results

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0

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110 inj

• Some loss of response was expected.• 17 of 28 PAHs decreased in response by

<20%.

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

# C

ompo

unds

Decrease in absolute response after 110 injections


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Pattern left by autosampler in vial caps after 110 injections

The cleanliness of the extract was acceptable for GC-MS analysis

Inlet liner after 110 injections

of olive oil extract


How does EZ-POP NP compare to other SPE methods?

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1. Silica gel SPE2. 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

MethodsSilica Gel SPECondition: 20 mL hexaneLoad: 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 40°C

MIP SPECondition: 1 mL cyclohexaneLoad: 0.5 gm oil diluted to 1 mL in cyclohexaneWash: 1 mL cyclohexaneElute: 3 x 1 mL ethyl acetateConcentrate: < 1 mL (not dryness) under nitrogen at 40 °C. Adjust final volume to 1 mL with ethyl acetate.


Results- PAH Recoveries, 2-3 rings

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• For Silica gel and MIP, matrix interference prevented accurate quantitation for most of these PAHs.

• Low recovery of naphthalene and biphenyl using MIP.

0%

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120%

EZ-POP NP Silica Gel MIP


Results- PAH Recoveries, 4 rings

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• Low recoveries using silica gel• Matrix interference with chrysene & triphenylene using MIP

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120%



Results- PAH Recoveries, 5-6 rings

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• Lower recoveries using silica gel• MIP recoveries very good

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MIP SPE suitable for heavier PAHs (>4 rings)


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How does GC-MS background compare?

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Silica gel SPE

EZ-POP NP SPE

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MIP SPEglycerides


In Conclusion

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• 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

EZ-POP NP is suitable for …


In Conclusion

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• 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.

Compared to other SPE methods…

Documents

The challenging extraction of non-polar contaminants …€¦ · The challenging extraction of non-polar contaminants out of a non ... •Z-Sep/C18 mixture - retains fatty matrix