Determination of OCs, PCBs, and Synthetic Pyrethroids in Animal Fat 

The approach described in this application note is the use of tandem quadrupole MS/MS for the quantification and unequivocal confirmation of OCs, International Council for the Exploration of the Seas (ICES) 7 PCBs and SPs at low μg/kg levels in animal fat without the need for an additional clean-up technique following gel-permeation chromatography (GPC).

Benefits

The Quattro micro GC provides high selectivity toreduce any matrix interferences, high sensitivity to reach the reporting levels required by European Union legislation and quantitative and confirmatory data in a single injection

The regulations governing permitted levels of persistent organic pollutants (POPs) in food products are becoming more stringent in response to increased awareness of the hazards they pose to humans. POPs, such as organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs), have been linked to adverse effects such as cancer, damage to the nervous system, reproductive disorders, and disruption of the immune system. They do not readily degrade in the environment, are lipophilic and tend to bioaccumulate as they move through the food chain. Therefore, these can potentially occur at high concentrations in fatty foods, such as meats and fish. Due to concerns about the effects of POPs, an international treaty restricting their use was adopted via the Stockholm Convention in 2004.¹ Currently in the UK, maximum residue limits (MRLs) of OCs and synthetic pyrethroids (SPs) in meat products are between 0.02–1.0 mg/kg on a fat basis.² To enforce these regulations, high quality analytical methods with adequate confirmation and limits of quantification must be used.

OCs, PCBs and SPs have all been analysed by conventional gas chromatography (GC) with electron capture detection (ECD). This is a sensitive technique for compounds containing halogens but suffers extensively from matrix interferences and confirmatory evidence cannot be provided as it is only a two dimensional technique with retention time and intensity. To add a third dimension, mass spectrometry (MS) can be used with selected ion monitoring (SIM). Usually three or four selected ions are monitored for each compound but confirmatory evidence is reduced if one or more of the selected ions are affected by matrix interferences.

The likelihood of interference will increase as the amount of sample preparation decreases. To minimize sample preparation and/or the likelihood of interference the selectivity of the detection technique should be increased.

The approach described in this application note is the use of tandem quadrupole MS/MS for the quantification and unequivocal confirmation of OCs, International Council for the Exploration of the Seas (ICES) 7 PCBs and SPs at low μg/kg levels in animal fat without the need for an additional clean-up technique following gel-permeation chromatography (GPC).

Extraction Method

1.25 g melted porcine fat was weighed into a 10 mL volumetric flask. For recovery, the samples were spiked at 0.00625 μg/mL or 0.05 mg/kg. The volume was adjusted with ethyl acetate/cyclohexane (1:1). A 1 mL extract was cleaned-up by GPC to give a final extract with a matrix equivalent of 0.125 g/mL in hexane. The internal standard, δ-HCH, was added at a concentration of 0.050 μg/mL equivalent to 0.4 mg/kg.

GC Method

The samples were injected by splitless injection (4 μL, 250 °C, purge at 30 mL/min after 2.1 min) into a carrier gas of helium at a constant flow rate of 1.0 mL/min delivered from an Agilent 6890 GC with a CTC CombiPal autosampler attached. The column employed was a J & W Scientific DB-17MS 30 m x 0.25 mm i.d., 0.25 μm. The following temperature ramp rate was used: 100 °C (1 min) to 200 °C (6 min) at 20 °C/min, to 280 °C/min (15 min) at 10 °C/min. The total run time was 35 min. The temperature of the interface was held at 260 °C during the chromatographic run.

The Waters Micromass Quattro micro GC Tandem Quadrupole Mass Spectrometer was used (Figure 1) in  electron impact (EI+) mode. The ion source was operated at 180 °C with an electron energy of 70 eV and a trap current of 200 μA. The mode of acquisition was multiple reaction monitoring (MRM) at an argon collision gas pressure of 3.0 x 10^-3 mBar.

The Quattro micro GC was tuned so that the precursor and product ions were resolved with a peak width at  half height of less than 0.7 Da. The list of pesticide residues and the MRM transitions, along with the collision energies and retention times for the method are listed in Table 1.

The data were acquired using Waters MassLynx Software and processed using the Waters TargetLynx Application Manager.

A typical reconstructed ion chromatogram (RIC) for the twenty OCs, seven PCBs and nine SPs is illustrated in Figure 2. The MRM transitions were arranged into thirteen function windows represented by the different colors in Figure 2. These functions can be overlapped slightly to allow for small changes in retention time. Use of function windows allows more time to be spent on each MRM transition, thereby, improving the signal to noise (S/N) ratio for the analytes that gave the lowest response. Dwell times were set so that approximately fifteen data points described each chromatographic peak.

Comparison of SIM and MRM

Two selected mass chromatograms are illustrated in Figure 3 for hexachlorobenzene (8.52 min) acquired in SIM mode at a concentration in matrix of 0.01 mg/kg. For complete confirmation, three or four selected masses would need to be monitored, so this could impact on the sensitivity shown here. In this example the S/N ratios of both chromatograms would allow for routine integration and quantification of hexachlorobenzene in SIM mode.

Two selected mass chromatograms are illustrated in Figure 4 for α-HCH (8.92 min) acquired in SIM mode at a concentration in matrix of 0.01 mg/kg. In this example, there is matrix interference (8.83 min) immediately preceding the confirmation peak. For this compound in this matrix, monitoring by SIM is unlikely to allow routine integration and quantification.

In contrast, changing to MRM mode the selectivity for α-HCH (8.92 min) is significantly improved at the same concentration in matrix (Figure 5). In this example, routine integration and quantification by MRM could easily be achieved.

In summary, some compounds will have acceptable selectivity for quantification and confirmation via SIM mode. However, as the matrix complexity is  increased and/or the sample clean-up is reduced,other compounds require the more selective MRM technique for routine quantification and confirmation.

Matrix matched standards were prepared in the concentration range 0.1 to 25 ng/mL. Equivalent mg/kg concentrations were at the 0.0008, 0.0016, 0.0040, 0.0060, 0.0100, 0.0200, 0.0400, 0.0800, and 0.2000 mg/kg. δ-HCH was used as an internal standard to correct for any volumetric errors. The standards were injected in a typical batch analysis. The data was then processed using Waters TargetLynx Application Manager. Correlation coefficients of r² > 0.9970 were obtained for all compounds in matrix. A representative curve for α-endosulfan with a correlation coefficient of r² = 0.9998 is illustrated in Figure 6.

To test the extraction method described, four recovery experiments were performed in porcine fat, spiked at 0.05 mg/kg. Each analyte was compared to its calibration curve of matrix-matched standards. The mean recovery and relative standard deviation (RSDs) of each analyte is listed in Table 2.

The mean recoveries were in the range 72 to 123%, with RSDs between 1 and 23%. Inclusion of SPs compromises the clean-up and results in higher concentrations of matrix co-extractives in the final extract. Further optimization of the extraction to improve precision is being carried out. The high response and selectivity provided by tandem quadrupole MS/MS ensured that additional clean-up of the GPC extracts was not necessary for the majority of pesticides.

Limits of detection (LOD) and confirmation (LOQ) were determined from the matrix matched standards, and are illustrated in Figure 7. The LOD was defined as the amount injected that gave a signal equivalent to three times the baseline noise. All the compounds have an LOD of 0.006 mg/kg or less. The LOQ was defined as the response for both the quantification and confirmation transitions sufficient to confirm the identity. As all the UK MRLs are greater than 0.02 mg/kg in animal products, this confirmatory method is able to meet the current legislation requirements.

Two MRM transitions were chosen so that quantification and confirmation could be performed in a single run assuming that the ion ratio between the transitions was consistent between standards and samples. The confirmation criterion chosen was that the ion ratio of the sample was within ±10% of the standard. The ratio was then used to confirm or reject compounds in the extracts.

To illustrate sensitivity, selectivity and confirmatory ability of tandem quadrupole MS/MS, heptachlor (Figure 8), PCB 52 (Figure 9) and bifenthrin (Figure 10) at the 0.01 mg/kg concentration level are compared to the matrix blank.

For both heptachlor and PCB 52, the expected ion ratio from the solvent standards is 0.62. For the confirmation criteria to be passed any concentrations in the extracts must have a ratio between 0.56 and 0.68. At 0.01 mg/kg the ion ratios for heptachlor and PCB 52 are 0.60 and 0.63 respectively, so the presence of both can be confirmed at this level.

For bifenthrin, the expected ion ratio from the solvent standards is 0.86. For the confirmation criteria to be passed, any concentrations in the extracts must have a ratio between 0.78 and 0.95. At 0.01 mg/kg the ion ratio is 0.89 so the presence of bifenthrin can be confirmed at this level.

A method has been described for the determination and confirmation of OCs, PCBs, and synthetic pyrethroids in animal fat using the Waters Micromass Quattro micro GC Tandem Quadrupole Mass Spectrometer.

The Quattro micro GC provides high selectivity to reduce any matrix interferences, high sensitivity to reach the reporting levels required by European Union legislation and quantitative and confirmatory data in a single injection.

The tandem quadrupole MS/MS technique should be applicable to a much larger range of analytes in a range of more complex food commodities.

The TargetLynx Application Manager provides advanced quantification with a range of automatic quality control checks.

1. http://www.pops.int/
   
2. Statutory Instrument No. 2591, The Pesticides (Maximum Levels in Crops, Food and Feeding Stuffs) Amendment No. 2 (2003).