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Frank Neugebauer , Cathrin Ast, Olaf Paepke Eurofins GfA Lab Service GmbH, Neulaender Kamp 1, 21079 Hamburg, Germany. THE TOTAL PCB-TASK: ANALYSIS OF ALL 209 PCB CONGENERS IN FISH OILS AND BIOTA HRGC-HRMS METHOD FOR ANALYSIS. PCB: History and Importance of analysis.
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Frank Neugebauer, Cathrin Ast, Olaf Paepke Eurofins GfA Lab Service GmbH, Neulaender Kamp 1, 21079 Hamburg, Germany THE TOTAL PCB-TASK: ANALYSIS OF ALL 209 PCB CONGENERS IN FISH OILS AND BIOTA HRGC-HRMS METHOD FOR ANALYSIS
PCB Analysis: How it started • Performed since the 1960s (LRGC on packed column); resolution of only about 10 signals for technical pattern. Quantification via technical mixtures as reference
PCB Analysis over the time • HRGC (since 1980s on capillary columns); detection with ECD and LRMS • first assignation of the majority of single compounds, detailed pattern analysis • Quantification methods for 6 (7) identified major compounds (“indicator PCB”) • Toxicity discussion of dl-PCB since 1990s; TEF assignation (WHO-TEF) • Improvement in quantification: standard substances and quantification methods for virtually all congeners in principle available • Further toxicity and limit value discussions since 2010s; adressing different modes of toxic action for dl-/ndl-PCB • EU Food/feed limit values for dl-PCB and ndl-PCB
PCB - Importance of Analysis • Minimization of human intake: Under US/California Drinking Water and Toxic Enforcement Act of 1986 (“Proposition 65”) definition of an overall “safe harbor level” of 0,09 µg/day total PCB in 2006. • Implication of analysis of a total PCB value • implicitly asking for more sophisticated analytical methods for all PCB congeners. • Still, present methods are often the old, inaccurate ones or rely upon approximations, e.g. quantification against technical mixtures or analysis of only few “marker compounds”. Method USEPA 1668 gives only a first approach towards a total PCB method
samples • analytical system • performance
Samples- analytical system -performance • Starting schedule: total 25 samples, arrived in our laboratory in 2012, • Sample type: presumably processed fish oil samples of different provenience, origin South America • Characterization : Tuna (n=6) and Anchovy (n=19). Both fish are at intermediate (Anchovy =3) to high trophic level (Tuna =4) • Reporting basis: pg/g original sample (=fat) • additional marine biota (i.e. oil) samples have meanwhile also been analysed
Samples - analytical system - performance • Analyses have been performed at the Eurofins GfA Lab Service GmbH Dioxin/POP competence centre, Hamburg, Germany • Analytical method: deduced from analytical EU reference method for dl/ndl-PCB with added elements from EPA1668 • Analysis for total PCB, adding up all single congeners as far as separable to congener group totals (Mono- to Decachlorobiphenyls) • 3 g oil matrix, diluted in n-hexane • Multistep cleanup: Column chromatography (silica, alumina)
Samples - analytical system - performance • HRMS (Thermo DFS) / R ≥ 10.000 • 8 function method • mass calibration via FC5311 • HRGC: SGE HT8PCB* (=altered) column, • 60m * 0.25 mm i.d. * 0.25 µm dF • Temperature program 90⁰C, 1 min – 20 ⁰/min • @180 ⁰ – 2 ⁰/min @260 ⁰ – 5 ⁰/min @300⁰C – 8 min • Isotope Dilution: 35 individual 13C12-quantification standards and 7 13C12-injection standards; • 209 native standards; 10-point initial calibration; multiple 1-point-calibration daily
Samples - analytical system - performance • QA/QC: Quantification standard recovery rates (40-130%), blanks, control samples. • Method LOQ at around 2pg/g per signal = compound (Mono-TriCB 5pg/g); Total PCB at 400pg total PCB/g (= 0.4 µg/kg).
Findings • Difficulties
Difficulties - interferences Int Hx Int Hp Int Hp Hp Hx Hp Hp Hx Int Sorry, no native #126 – employ Carbon cleanup!
Summary and conclusions • Analysis of all single PCB compounds resolved to about 180 separations • Analysis of total homologue groups is possible with reasonable method and quantification performance. • The results for the evaluated fish oil samples are well within the usual range of findings, as can be deduced from individual congener results • Even with an „altered“ HT8-PCB GC column, a reasonable number of PCB congener separations are possible; unique properties
Summary and conclusions • Increased baselines due to HRMS reference compounds • as well for FC5311 as for PFK • Mainly for Mono- to TriCB • Signals from Cl or 2Cl-loss of higher mass PCB congeners are sometimes a problem • Maintenance of chromatographic separation and mass separation of R≥10.000 important but not completely helpful • Especially of concern: Te #81; Pe #126 near Hp #185; • typically at 2-8% of the molecular ion signal intensities • Partially wide mass range for instrument data registration • (up to 4 chlorination degrees in a single function)
Take Home Analysis of 180 PCB signals instead of 18 dl/ndl-PCB gives you an unique opportunity for having 10 times the trouble!
Thank you for your attention! The R+D laboratoryteam
The HT8-PCB column: • Issues and separation
The HT8-PCB* Column - Issues • Polysiloxane with m-Carborane (three-dimensional • aromatic cage structure) in backbone • Unique properties for PCB separation due to selective • interactions with PCB congeners • Upon installing a HT8-PCB column, an altered elution • profile observed • Identification of new elution profile by native mix of all 209 PCB • Following communications: • HT8-PCB “sensitive to lower (storage) temperatures” (M. Oehme, pers. comm.) • Meanwhile solved problems with manufacturing (SGE, pers. comm.)
DetourSimilarities: HT8PCB* vs. Active carbon HeptaCB after preparative column chromatography on active carbon
Separation HT8PCB vs. HT8PCB*Example Hexa-/Heptachlorobiphenyls
The HT8-PCB* Column - Separation • All together 176 practically achievable separations (peaks) under our defined quality criteria (66% valley) • counting detectable peaktops 187 separations are achieved • Comparison: HT8PCB originally gives 192 peak tops = maximal possible separations • GC separation Quality criteria have been deduced by assigning 2 critical peak pairs (PCB 60/56; PCB 180/193) • Separation and elution order has been stable over 6 month and several hundred injections
155 150 152 145 148 136 154 151 155 140 143 134 133 142/131 146 168 137 130 163/164 138/160 158 129 166 161 132/153 163/164 167 156 159 162 128 157 169 The HT8-PCB* Column - Separation 135/144/147 149/139 165 141 37