Extraction and Analysis of PAHs in Olive Oil using Molecularly Imprinted Polymer SPE and GC-MS

Olga Shimelis, Katherine Stenerson, Daniel VitkuskeSanja Beyowich¹, Brian Boyd¹

MIP Technologies AB, Scheelevagen 22, 220 07 Lund, Sweden

Reporter US Volume 27.5

Introduction

Polycyclic Aromatic Hydrocarbons (PAHs) are environmental carcinogenic compounds that can be present in food from many different sources – either naturally or from pollution, packaging, or food preparation procedures such as heating and grilling (1). PAHs consist of fused aromatic rings. Due to the lipophilic nature of PAHs, fats and oils can be especially susceptible to PAH contamination. Previously, levels as high as 26 μg/kg of PAHs were found in virgin olive oils (2). The structures of representative PAHs are shown in Figure 1. In 2005 the European Union Commission Regulation (EC) No 208/2005 set maximum limits for PAHs in food. For oils and fats intended for direct human consumption, the maximum residue limit as benzo(a)pyrene on wet weight is 2 μg/kg (3). Edible oils are particularly difficult to analyze, because the oil matrix overloads HPLC columns.

Figure 1. Representative Structures of PAHs

SPE is one of the most popular methods for extraction of PAHs from oil. We have evaluated the recently developed molecularly imprinted polymer (SupelMIP^® PAHs) for the extraction of PAHs from olive oil and subsequent analysis by GC-MS.

Molecularly Imprinted Polymers

Molecularly imprinted polymers are a class of highly cross-linked polymer-based molecular recognition elements engineered to bind one specific target compound or a class of structurally related compounds with high selectivity. The MIP material is designed with cavities that are sterically and chemically complementary to the target analyte(s). As a result, multiple interactions (e.g. hydrogen bonding, ionic, van der Waals, hydrophobic) can take place between the MIP cavity and the analyte.

Extraction and Analysis of PAHs in Olive Oil

A standard mix of PAH compounds was prepared in methylene chloride and contained benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(123-cd)pyrene, dibenzo(ah) anthracene and benzo(ghi)perylene.

Store-purchased olive oil was spiked with PAHs in duplicate at the level of 2 μg/kg. Chrysene-d₁₂ was spiked as the internal standard at the level of 20 μg/kg.

0.5 g of spiked and blank oil sample was mixed with 0.5 mL cyclohexane and was extracted using the SupelMIP SPE procedure described in Table 1 and analyzed via GC-MS conditions detailed in Table 2.

Table 1. SupelMIP PAHs SPE Procedure (52773-U)

SPE cartridge:	SupelMIP SPE -PAHs,50 mg/3 mL(52773-U)
1.	Condition with 1 mL cyclohexane
2.	Load diluted oil sample
3.	Wash with 1 mL cyclohexane
4.	Elute with 3 x 1 mL ethyl acetate
5.	Evaporate SPE eluate under nitrogen at 40 °C and reconsitute in 0.2 mL ethyl acetate for analysis.

Table 2. GC-MS Conditions for PAH Analysis (28471-U)

column:	SLB-5ms,30 m x 0.25 mm 1.D.,0.25  μm (28471-U)
Instrument:	Agilent® GC-MS
oven:	60 °C, 25 °C/min. to 275 °C (5 min.), 10°C/min to 300 °C (1 min.)
flow rate:	helium, 2 mL/min., constant
injector temp.:	300 °C
MS detection:	Autotune + EM offset of 300
scan range:	SIM
Injection:	1 μL, split

Recovery

The recovery of PAHs was evaluated in the spiked olive oil samples. First, the blank sample of oil was analyzed for the presence of background contamination. The recovery values were corrected for the background levels found in the blank sample. The results are shown in Table 3.

Table 3. Analysis of Blank and Spiked Olive Oil Samples

Compound	Blank μg/kg	Spiked 2(μg/kg) Recovery
Benzo(a)anthracene	3.4	65%
Chrysene	9.4	70%
Benzo(b)fluoranthene	2.2	82%
Benzo(k)fluoranthene	1.4	84%
Benzo(a)pyrene	3.0	87%
Indeno(123-cd)pyrene	2.4	95%
Olbenzo(ah)anthracene	1.8	82%
Benzo(ghi)perylene	3.4	87%

PAH background contamination was found in the blank olive oil sample. The most abundant compound was found to be chrysene at almost 10 μg/kg. This is consistent with previous studies that found light (2-4 rings) PAHs to be more predominant in the oils (2).

Using the SupelMIP SPE PAH approach, an average recovery of 82% was observed. All compounds contained at least 4 fused aromatic rings.

Conclusions

In this article we described the extraction of PAHs from olive oil using SupelMIP SPE PAHs and analysis by GC-MS. The extraction procedure is very simple and includes only a 5-step SPE cleanup process. The procedure is amenable to larger PAH molecules. Extraction of smaller PAH molecules (2-3 rings) may be possible, however, lower recoveries may be expected.

References

1. Purcaro G, Morrison P, Moret S, Conte LS, Marriott PJ. 2007. Determination of polycyclic aromatic hydrocarbons in vegetable oils using solid-phase microextraction?comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. Journal of Chromatography A. 1161(1-2):284-291. https://doi.org/10.1016/j.chroma.2007.05.105

2. Teixeira VH, Casal S, Oliveira MBP. 2007. PAHs content in sunflower, soybean and virgin olive oils: Evaluation in commercial samples and during refining process. Food Chemistry. 104(1):106-112. https://doi.org/10.1016/j.foodchem.2006.11.007

3. EU sets maximum levels of PAHs in food:. http://www.ihta.org/word_documents/EU_sets_ maximum_levels_of_PAHs_in_food.doc