Determination of Polycyclic Aromatic Hydrocarbons (PAH) in Food with GC-MS/MS Thorsten Bernsmann, Peter Fürst Chemical and Veterinary Analytical Institute Münsterland-Emscher-Lippe Joseph-König-Str. 40, 48147 Münster/Germany (thorsten.bernsmann@cvua-mel.de) 24 th CRC; Melbourne 12.11.2013
Outline background/ legislation sample preparation GC-MSD versus GC-MS/MS / PAH source results conclusion 12.11.2013 / 2
EU data collection on PAH in food PAH concentration (µg/kg) in 9,714 food products PAH N >LOD Concentration in µg/kg P05 Median Mean P95 Maximum LB UB LB UB LB UB LB UB LB UB BaP 9714 47.3% 0 0.007 0 0.12 1.02 1.08 3.60 3.60 270 270 BaA 5706 49.4% 0 0.010 0 0.15 1.97 2.06 6.00 6.00 1064 1064 BbFA 6728 47.4% 0 0.003 0 0.15 1.48 1.55 6.00 6.00 690 690 BkFA 6630 41.5% 0 0.001 0 0.10 0.57 0.62 2.44 2.44 150 150 BghiP 6406 35.8% 0 0.001 0 0.10 0.77 0.84 2.90 2.90 220 220 CHR 5421 60.8% 0 0.020 0.13 0.30 3.20 3.27 10.00 10.00 590 590 DBahA 6119 14.0% 0 0.010 0 0.09 0.12 0.20 0.53 0.54 37 37 IP 6908 24.8% 0 0.010 0 0.10 0.56 0.69 2.49 2.49 100 100 BjFA 2184 38.8% 0 0.010 0 0.05 0.58 0.66 2.43 2.43 57 57 CPP 1641 40.0% 0 0.010 0 0.09 1.00 1.36 1.96 6.00 112 112 DBaeP 2538 12.4% 0 0.050 0 0.10 0.16 0.28 0.9 1.00 27 27 DBahP 2341 2.2% 0 0.010 0 0.10 0.02 0.15 0 0.49 3 3 DBaiP 2471 4.9% 0 0.033 0 0.10 0.04 0.17 0 0.91 4 4 DBalP 2702 8.1% 0 0.010 0 0.10 0.06 0.16 0.30 0.68 14 14 MCH 2227 6.9% 0 0.010 0 0.04 0.07 0.15 0.10 0.45 18 18 BcFL 1148 46.5% 0 0.010 0 0.10 1.60 1.65 6.53 6.53 231 231 12.11.2013 / 3
Opinion on PAH of the European Food Safety Authority (EFSA) Opinion on PAH in food adopted on 9 June 2008. In this opinion EFSA concluded that benzo[a]pyrene is not a suitable marker for the occurrence of PAH in food and that a system of four specific PAH would be the most suitable indicators of PAH in food. Benz[a]anthracene Benzo[b]fluoranthene Benzo[a]pyrene Chrysene 12.11.2013 / 4
EU maximum levels for PAH in food Regulation (EC) No 1881/2006 (EC No 835/2011) 12.11.2013 / 5
Requirements for PAH analysis: Regulation (EC) No 333/2007 (836/2011) 12.11.2013 / 6
Performance criteria for methods of analysis for PAH: Regulation (EC) 333/2007 (836/2011) table 7 METHOD VALIDATION AND QUALITY CONTROL PROCEDURES FOR PESTICIDE RESIDUES ANALYSIS IN FOOD AND FEED SANCO/12495/2011 Limit of detection (LOD) will be defined by the lowest trace 12.11.2013 / 7
Determination of PAH Nowadays there are the two main analytical techniques to determine PAH in foods: HPLC FLD GC-MS They are sticky, not active Difficult to vaporize and keep from depositing high temperatures are important Minimize surface contact Tailing peaks Subject to desublimation (deposition) LODs and recoveries for PAH are compound, food matrix and method dependent. The LODs for PAH typically range from 0.1 to 1 µg/kg 12.11.2013 / 8
Method used at the * * German Research Foundation 2 g fat or 2-4 g solid food matrix extraction and saponification with potassium hydroxide in methanol liquid/liquid extraction with cyclohexane liquid/liquid extraction with dimethylformamide liquid/liquid extraction: dimethylformamid + NaCl + water/ cyclohexane Other method: Clean up with florisil, phenyl-spe MIP(molecule imprinted polymer) -SPE GPC silica gel clean up 12.11.2013 / 9
GC-MS/MS Method 7890 / 7000B GC-MS/MS System 12.11.2013 / 10
GC- Configuration for PAH 2 ul splitless injection Multi Mode Inlet 7000B TQ 30 m x 0.25 mm ID x 0.15 µm Agilent Select PAH Helium MS-Tee 2.0 ml/min Constant flow 7890A Option: Select PAH 15 m x 0.15 mm ID (0,10 µm) note: temperature between 180 and 230 C is important for the separation Inlet temp program : 80 (0.5) 400 C/min 330 C! Oven temp program : 80 (0.5) - 120 (0) 40 C/min 180 (0) 3 C/min 280(0) 330 (10) C 12.11.2013 / 11
PAH product ion scan Benzo(b)fluoranthene CID CID CID CID 50 520 V30 V V PAH were destroyed only by high CE energy not enough product ions with a necessary response use of [M]+ as precursor and quantifier: [M]+ -> [M]+ [M]+ -> [M-2]+ as qualifier ion the collision energy is used to destroy matrix/interfering ions M+ M-2 M-2 12.11.2013 / 12
Compounds /Triphenylene 12.11.2013 / 13
Multiple reaction monitoring (MRM) EI: 70 ev; Dwell 50 msec.; Q1 and Q2 resolution: unit 12.11.2013 / 14 [M]+ -> [M]+ [M]+ -> [M-2]+
How does it work? Comparison between MS and MS/MS GC-MSD SIM chromatogram of a 1 µg/l standard solution: Benzo[a]pyrene 12.11.2013 / 15
Comparison between MS and MS/MS GC-MS/MS chromatogram of a 1 µg/l standard solution: Benzo[a]pyrene Benzo[a]pyrene 252.0 -> 252.0 Benzo[a]pyrene 252.0 -> 250.0 13 C 4 -Benzo[a]pyrene (ISTD) 12.11.2013 / 16
Benzo[a]pyrene in a food sample: 0.03 µg/kg Benzo[a]pyrene 252.0 -> 252.0 Benzo[a]pyrene 252.0 -> 250.0 13 C 4 -Benzo[a]pyrene (ISTD) 12.11.2013 / 17
Benzo[b/k/j]fluoranthene Concentrations in a food sample: b: 0.09 µg/kg; k: 0.020 µg/kg; j: 0.015 µg/kg 252.0 -> 252.0 b 252.0 -> 250.0 b k j k j b k ISTD 12.11.2013 / 18
GC-MSD: matrix interference food sample spiked with 1 µg/kg TIC TIC MSD Important compounds like: Benzo[c]fluorene, Benzo[a]anthracene, Cyclopenta[c,d]pyrene, Chrysene, Methylchrysene, Benzo[b,k,j]fluoranthene eluted in this region 12.11.2013 / 19
Benzo[a]anthracene SIM-chromatogram:benzo[a]anthracene (food sample spiked with 1 µg/kg) the matrix interference suppressed the signal of benzo[a]anthracene (spiked with 1 µg/kg) completely Only the compounds which eluted after 25 min, such as benzo[a]pyrene can be determined reliably 12.11.2013 / 20
GC-MS/MS: matrix interference food sample spiked with 1 µg/kg TIC-MRM Important compounds like: Benzo[c]fluorene, Benzo[a]anthracene, Cyclopenta[c,d]pyrene, Chrysene, Methylchrysene, Benzo[b,k,j]fluoranthene eluted in this region 12.11.2013 / 21
Benzo[a]anthracene/Chrysene in a food sample (spiked with 1 µg/kg) normalized chromatogram of the different MRM Benzo[a]anthracene Chrysene 13 C 6 -Benzo[a]anthracene 13 C 6 -Chrysene ISTD 12.11.2013 / 22
Chrysene Triphenylene critical pair C 18 H 12 : same elemental formula; same transition separation mandatory for GC-MS/MS Benz(a)anthracene Triphenylene Chrysene 12.11.2013 / 23
Chrysene/triphenylene in a food sample: conc. (chrysene) = 0.5 µg/kg ratio between chrysene and triphenylene in all samples analysed so far range from 0.1 up to 1.5 12.11.2013 / 24
PAH analyzer PAH Automated Offline Source Cleaning Offline Cleaning Mode: 0.4 ml/min, Source 350C, Filament On Page 25 12.11.2013 / 25 2013.06.18
PAH analyzer Automated Continuous Source Cleaning Continuous Cleaning Mode: 0.075-0.2 ml/min during acquisition Page 26 12.11.2013 / 26 2013.06.18
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7000B MS/MS systems, stock configuration Dibenzo(a,l)pyrene, a late eluter Optimized PAH Analyzer Page 28 12.11.2013 / 28 2013.06.18
Standard solution: blue without H 2 cleaning; green with H 2 cleaning Standard solution: black with H 2 cleaning; red without H 2 cleaning and green H 2 cleaning before and after the run 12.11.2013 / 29
Standard solution 1; the lowest calibration point without H 2 : Cal 1: 0.5 pg/µl; injection 1 µl Continuous Cleaning Mode H 2 : Cal 1: 0.5 pg/µl; injection 1 µl 12.11.2013 / 30
conclusion: GC-MS/MS: LODs of 0.01 µg/kg were reached which is 20 times lower than with our single quadrupole and without changing the sample preparation and the sample concentration with selective GC columns like Select PAH it is possible to separate critical pairs, such as chrysene/triphenylene or benzo[b,k,j]fluoranthenes analyze larger amounts of sample without losing sensitivity due to increased interference from matrix use of 13C-labelled PAH as internal standard possible and advisable PAH analyzer improved peak shapes, less tailing, higher sensitivity; Source stays clean 12.11.2013 / 31
Many thanks to the people behind the work and you for your attention! 12.11.2013 / 32