Determination of Benzo[a]pyrene at low-levels in Olive Oil

Walter B. Wilsona, Jacolin A. Murray

a

, and Blaza Tomanb

a Chemical Sciences Division, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA

b Statistical Engineering Division, Information Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants originating from a wide variety of natural and anthropogenic

sources. Studies have shown that exposure to PAHs can lead to an increased risk of cancer and as such, PAHs are classified as potential carcinogens

and makes their presence in foods a health concern. Edible oils and fats are potentially a significant source of PAHs in the human diet due to their

lipophilic nature and prevalence in food. Edible oils are susceptible to contamination by various routes including environmental pollution of the raw

vegetable material and/or industrial processing steps prior to refining such as seed drying with smoke and solvent extraction. Benzo[a]pyrene (BaP) is

one of the markers for the occurrence of PAHs in foods, for which regulatory limits around the world have been set in edible oils to be from 2.0 μg/kg to

10 μg/kg. The determination of PAHs in lipids is difficult due to low concentration levels, the complexity of extraction and clean-up procedures, the need

for good chromatographic resolution, and the need for sensitive detectors. Several methods of extraction and clean-up of PAHs from edible oils have

been developed, which typically consist of a solvent extraction procedure followed by solid-phase extraction or column chromatography. PAH

determination is often carried out by reversed phase liquid chromatography with fluorescence detection (RPLC/FL) or gas chromatography/mass

spectrometry (GC/MS). In this study, a new analytical method combining size-exclusion chromatography (SEC) fractionation and RPLC/FL for the

determination of BaP in two olive oil samples. The new SEC-RPLC/FL method was compared to a traditional extraction method combined with GC/MS.

Both methods provided accurate and precise results; however, the SEC-RPLC/FL allowed for shorter sample preparation times, analysis times, and no

requirements of solid-phase extractions.

Materials and Methods

1. Materials and Reagents

Olive oil samples were purchased from a local supermarkets in Gaithersburg, Maryland. SRM 2260a (calibration solution) and SRM 2270 (Internal

Standard Mix) were obtained from the Office of SRMs at NIST. HPLC grade water (H2O), acetonitrile (ACN), dichloromethane (DCM), and n-hexane

were purchased from Fisher Scientific, MilliporeSigma, and/or J.T. Baker.

2. Sample Preparation

The two olive oil samples were spiked with SRM 2260a to have a mass fraction value of ≈ 2 μg/kg for BaP and homogenized by vibro-mixing at room

temperature for 24 h. Samples were stored in the dark at room temperature prior to analysis. Stock calibration solutions of SRM 2260a or SRM 2270

were prepared in HPLC grade n-hexane and kept in the dark at 4

oC. Working solutions containing both SRM solutions were prepared gravimetrically of

stock solutions in n-hexane. Approximately 100 μL aliquots of the working internal standard solution was mixed with the olive oil sample through

sonication for 30 s. The olive oil samples were allowed to settle for 5 min to remove any bubbles formed during the mixing procedure. The olive oil

sample was split into four subsamples for analysis.

3. Sample Clean-up via SEC Fractionation

SEC fractionation was performed using a Varian 9012 Solvent Delivery System coupled to Jasco UV-1570 Intelligent UV-vis detector. Samples were

fractionated using a semi-prep PLGel column purchased from NETA Scientific Inc. and manufactured by Agilent Technologies with the following

characteristics: 30 cm length, 25 mm diameter, 10 μm average particular diameter, and 100 Å pore size. Approximately 500 μL of olive oil was injected

into the SEC system and processed through the semi-prep column with a mobile phase of 100 % dichloromethane (DCM) at a flowrate of 10.0 mL/min.

The PAH fractions were collected, combined, and evaporated to near dryness under nitrogen. Additional n-hexane (2 mL) was added to the sample and

evaporated further to ≈ 250 μL and transferred to an autosampler vial.

Table 1: RPLC/FL operating conditions for BaP 

measurements in olive oil.

Time H2O (%) ACN (%) Excitation (nm) Emission (nm)

0.0 50 50 404 440

3.0 50 50

23.0 0 100

24.6 290 411

40.0 0 100

4. Reversed-Phase Liquid Chromatography with Fluorescence Detection

The RPLC/FL measurements were performed on an Dionex Ultimate 3000 LC system

equipped with a pump, autosampler, column compartment, diode array detector, and

FL detector. Separations were carried out on a polymeric (Zorbax Eclipse PAH) C18

column purchased from Agilent with the following characteristics: 25 cm length, 4.6

mm diameter, and 5 μm average particle diameter. Separation and detection was

achieved using flowrate of 1.5 mL/min, column temperature of 25 oC, and the mobile

gradient listed in Table 1. All sample injections were held constant at 2 μL. FL spectra

were recorded using a stop-flow function on the instrument at the apex of each

chromatographic peak.

Table 2: GC/MS operating conditions for BaP 

measurements in olive oil.

Time Temperature (

oC) Ramp (oC/min)

0.0 70 0.0

1.0 70 30.0

3.7 150 0.0

6.7 150 2.0

86.7 310 0.0

146.7 310 0.0

5. Gas Chromatography – Mass Spectrometry

The GC/MS measurements were performed on an Agilent HP 6890 Series GC system coupled to

a quadrupole mass spectrometer with electron ionization (HP 5973 MSD, Agilent). Separations

were carried out on a 50 % phenyl GC stationary phase (SLB-PAHms) purchased from

MilliporeSigma (Bellefonte, PA) with the following characteristics: 60 m x 0.25 mm i.d. x 0.25 μm

film thickness. The chromatographic conditions for analysis included an ultra-high purity helium

gas flow rate of 1.1 mL/min, transfer line temperature of 325 oC, ion source temperature of 230

oC, quadrupole temperature of 150 oC, and the oven temperature program described in Table 1.

All sample injections were on-column and held constant at 1 μL. A 5 m guard column from

Restek was used for all analysis.

Results and Discussion

1. RPLC/FL Analysis of BaP and six MM 252 PAH isomers in SRM 2260a

Previous studies at NIST developed a RPLC/UV method using a Pinnacle II PAH column (250 mm x 4.6 mm) with a 5 μm particle size for the

certification of SRM 1647e. The mobile phase gradient elution program was the following: 3 min hold at 50 % H2O/ACN; linear gradient to 100 % ACN

over 15 min; and hold for 15 min. The new RPLC/FL method established here for BaP and six MM 252 PAH isomers used a different column with the

200 250 300 350 400 450 500 550

0.0

8.0x105 Intensity (counts)

Wavelength (nm) 200 250 300 350 400 450 500 550 0.0

8.0x105 Intensity (counts)

Wavelength (nm)

Figure 1: Stop-flow fluorescence spectra.

Per BaP

same dimensions and a similar mobile phase gradient (Table 1). The

fluorescence wavelengths selected in this study were based on the

fluorescence spectra (Figure 1) recorded using the stop-flow function of the

RPLC system.

Quantitative analysis of PAHs in complex samples via RPLC/FL is normally

based on the internal standard calibration method to account for variations in

detection response and injection irreproducibility. Isotopically labeled internal

standards are preferred for quantitation and readily available. Two solution

SRMs are available with perdeuterated PAHs for use as internal standards

(SRM 2269 and SRM 2270).

SRM 2270 contains six isotopically labeled PAHs with a range of MM:

naphthalene-d8(136 g/mol), acenaphthene-d10(164 g/mol), pyrened10

(212 g/mol), BaP-d12(252 g/mol), perylene-d12(Per-d12, 252

g/mol), and benzo[ghi]perylene-d12(288 g/mol). SRM 2270 is

prepared in n-hexane/toluene mixture (96:4 volume fraction) and is

miscible with the calibration solution SRM 2260a.

The target PAH in the olive oil samples is BaP with a MM 252 g/mol,

so the internal standard of choice could be either BaP-d12 or Per-d12.,

which have identical fluorescence spectra to their parent PAHs shown

in Figure 1. The RPLC/FL chromatogram obtained for SRM 2270 and

for a mixture of SRM 2270/SRM 2260a are shown in Figure 2.

Of the 35 PAHs in SRM 2260a, six isomers of BaP are present in the

solution: Per, benzo[e]pyrene (BeP), benzo[a]fluoranthene (BaF),

benzo[b]fluoranthene (BbF), and benzo[k]fluoranthene (BkF). Per and

Per-d12 have significantly different fluorescence profiles than the coeluting MM 252 isomers with maximum excitation and emission

wavelengths of 406 nm and 440 nm, respectively. Under these

fluorescence conditions for the first 24.6 min, the four MM 252 isomers

in red are not detectable as shown in Figure 2.

22 23 24 25 26 27 28

0.0

3.0x106

22 23 24 25 26 27 28

0.0

5.0x106 Signal Counts Signal Counts

Time (min) BaP

BaP-d12

Per-d12

Per

BaP-d12

Per-d12

SRM 2270

SRM 2260a/2270

BaF

BbF

BeP

BjF

BkF

Figure 2: RPLC/FL chromatograms obtained using the separation conditions in Table 1.

The red labels refer to the four additional MM 252 PAHs present in SRM 2260a.

2. Determination of BaP in Olive Oil Samples via SECRPLC/FL

Previous work completed at NIST has used SEC fractionation to

isolate PAHs and polychlorinated biphenyl congeners from complex

fatty matrixes prior to GC/MS analysis. SEC fractionation removes

most of the lipid and biogenic material in the mussel tissue samples

and fish oil samples. More recently, Drabov and co-workers studied

the potential use of SEC for the isolation of PAHs in olive oil samples

with no prior sample extraction. These studies suggested that SEC

was insufficient to adequately clean-up olive oil samples and a silica

gel solid-phase extraction (SPE) step was implemented to remove any

remaining interferences prior to GC/MS measurements.

Within this context, the current study evaluated the use of a single

SEC fractionation step to clean up the olive oil sample prior to

RPLC/FL measurements. The SEC fractionation chromatogram

obtained for one olive oil sample spiked with SRM 2260a and SRM

2770 is shown in Figure 3. PAHs were collected between the time

interval of 9.5 to 11.5 min. The SEC fractionation procedure was

repeated four times, fractions were combined, and evaporated to near

dryness for analysis via RPLC/FL. The RPLC/FL chromatograms for a

calibrant, blank olive oil sample, and spiked olive oil sample are

shown in Figure 4. The stop-flow fluorescence spectra of BaP and Per

in Figure 1 were used to confirm peak identity in the calibrants and

spiked olive oil samples.

A summary of the determined BaP mass fraction values for the SECRPLC/FL method is reported in Table 3. The determined mass fraction

values for BaP in the olive oil samples is in good agreement with the

gravimetric value and GC/MS data not shown here for a conventional

extraction procedure. However, the mass fraction values were

significantly higher when Per-d12 was used as the internal standard.

The significantly different mass fraction values suggest that changes

in the labeled internal standards occur during sample processing. If

Per-d12is not fully recovered during cleanup (i.e., relative to the

recovery of BaP), this would positively bias the unknown compared

with full recovery, which is assumed for BaP-d12. To illustrate, the Per

mass fraction values were determined in the same spiked oil samples

to be almost identical to the gravimetric value (1.85 ± 0.04 μg/kg and

1.87 ± 0.03 μg/kg, respectively).

SEC-GC/MS was utilized to investigate these results because the

seven MM 252 isomers are baseline resolved using a 50 % phenyl

stationary phase. The MS sensitivity degraded immediately after the

first SEC fraction injection as shown in Figure 5. The lost in sensitivity

can be attributed to the oil sample not being cleaned up enough prior

to analysis indicating the need for an additional SPE step for GC/MS

analysis.

0 5 10 15 20

0

1010 Signal (mV)

Time (min)

Figure 3: SEC fractionation chromatogram of spiked olive oil sample.

PAHs

23 24 25 26 27 28

0.0

1.7x106

23 24 25 26 27 28

0.0

1.8x105

23 24 25 26 27 28

0.0

2.5x105 Signal (counts)

Time (min)

Figure 4: RPLC/FL chromatograms obtained for the calibrant, blank olive oil, and

spiked olive oil samples after SEC fractionation.

BaP-d12

BaP-d12

BaP-d12

BaP

BaP

Per-d12

Per-d12

Per-d12

Calibrant

Blank

Olive Oil

Spike

Olive Oil

20 30 40 50 60 70 80 90

0.0

2.5x104 Signal Abundance

Time (min)

Figure 5: GC/MS chromatogram in SIM mode for the spiked olive oil

sample after SEC fractionation.

Table 3: Mass Fraction values for spiked olive oil samples.

Gravimetric

(μg/kg)

SEC-RPLC/FL

(μg/kg)

Sample 1 1.99 2.07 ± 0.26

Sample 2 2.54 2.30 ± 0.17

Conclusion

The results presented here demonstrate the ability to use SEC fractionation prior to

RPLC/FL analysis. The SEC-RPLC/FL method allowed for the quantitation of BaP in

olive oil samples at very low concentration levels.