MEASUREMENT OF DECHLORANES IN FOOD AND FEED. ASSESSMENT OF DIETARY INTAKE. ULTRA-TRACE LEVELS OF. Calaprice Chiara

Transcription

1 MEASUREMENT OF ULTRA-TRACE LEVELS OF DECHLORANES IN FOOD AND FEED. ASSESSMENT OF DIETARY INTAKE. Calaprice Chiara PhD student at the University of Liege (Belgium) And at the Polytechnic University of Bari (Italy) L Homme B., Calvano C.D., Zambonin C., Leardi R., Focant J.-F

2 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Results on real food and feed samples, assessment of dietary intake 7. Conclusions

3 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Results on real food and feed samples, assessment of dietary intake 7. Conclusions

4 Dechloranes structures and uses Mirex (Dechlorane) pesticide and flame retardant banned: toxic, persistent and bioaccumulative Flame Retardants (not regulated) Pesticides impurities in Aldrin, Dieldrin (603) Chlordane, Chlordene (CP) Sverko, E., et al., Dechlorane plus and related compounds in the environment: A review. Environmental Science and Technology, (12): p

5 DPs isomers environmental occurrence Greenland Total Dechlorane Plus isomers concentrations in air Correlation with distance from the facilities distance from an e-waste recycling plant in China population density DPs are subject to long range transport* * Möller, A., et al., Large-scale distribution of dechlorane plus in air and seawater from the Arctic to Antarctica. Environmental Science and Technology, (23): p

6 Dechlorane analogues environmental occurrence Total DPs Dechlorane analogues concentrations in sediments from the Great Lakes Sverko, E., et al., Dechlorane plus and related compounds in the environment: A review. Environmental Science and Technology, (12): p

7 Biological occurrence DPs isomers were detected in Canadian trout, gull eggs and serum from bald eagles. DPs were detected in human serum and hair of workers in e-waste recycling facility in China. DPs and analogue compounds, such as Dec 602, Dec 603, CP and Mirex were detected in human serum samples from France*. * Brasseur, C., et al., Levels of dechloranes and polybrominated diphenyl ethers (PBDEs) in human serum from France. Environ Intern, : p

8 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Preliminary results on real food and feed samples 7. Conclusions

9 Novelty and aim of the present work The reason for the presence of dechloranes in human serum from Europe is not clear, since no production sources have been identified in Europe. Likely Dechlorane compounds are subject to long range transport as shown by environmental samples. And they are biopersistent and bioaccumulative, as shown by sample from biota, even if more information are needed. Biopersistence: Dechloranes enter the food chain and distribute by food

10 Novelty and aim of the present work The same sample preparation procedure used for routine Dioxins and PCBs analysis Recovery rates verified step by step to allow simultaneous and reliable analyses Development of GC-MS/MS method for trace level analysis. Performances comparable to HRMS PTV inlet parameters set by means of Experimental Design Usually HRMS instruments were used. Experimental parameters customized to optimize target compounds detection DPs isomers and Dec 602, Dec 603, CP and Mirex levels assessed on 88 samples of different food and feed matrices from Europe Assessment of average daily intake (pg/g fat or ww) of all the dechloranes for people from Europe (Belgium)

11 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Results on real food and feed samples, assessment of dietary intake 7. Conclusions

12 Sample preparation and clean-up ISO procedures used for routine analysis of PCDDs, PCDFs and PCBs* Simultaneous analysis of dioxins and Dechlorane compounds in routine food and feed controls Isotopic dilution for quantitation (labelled ISTDs not yet available for all compounds) Accelerated solvent extraction ASE TM Dionex 350 Manual acid silica column for fat degradation PowerPrep TM clean-up Dechloranes are not degraded by the acidic treatment Dechloranes elute in the MO/NDL-PCBs fraction Kim, J., et al., Assessment of Dechlorane compounds in foodstuffs obtained from retail markets and estimates of dietary intake in Korean population. Journal of Hazardous Materials, : p

13 Manual acidic silica column Sample clean-up recovery rates 22% acidic SiO 2 20 g 44% acidic SiO 2 20 g Neutral SiO 2 5 g Na 2 SO 4, 5 g Glass wool Hexane Manual acidic silica column PCDDs, PCDFs and PCBs Recovery > 95% Dechloranes %

14 Carbon/Celite Column Alumina Column Manual acidic silica column ABN Silica Column Sample clean-up recovery rates Manual acidic silica column Hexane 22% acidic SiO 2 20 g 44% acidic SiO 2 20 g Neutral SiO 2 5 g Na 2 SO 4, 5 g Glass wool Hexane Manual acidic silica column PCDDs, PCDFs and PCBs Recovery > 95% Dechloranes % Hexane Eluting FMS ABN silica Recovery Dechloranes %

15 Carbon/Celite Column Alumina Column ABN Silica Column Sample clean-up recovery rates Manual acidic silica column Hexane Hexane/DCM 50:50 Hexane (Waste) Loading alumina column Recovery Dechloranes 0-2 % Hexane/DCM 50:50 Eluting alumina column (without loading carbon column) Recovery Dechloranes 85-90%

16 Carbon/Celite Column Alumina Column ABN Silica Column Sample clean-up recovery rates Manual acidic silica column Hexane Forward elution of carbon column with Hex/DCM: MO fraction Recovery PCDDs and PCDFs 0-5% MO-PCBs, NDL-PCBs % Dechloranes % Hexane/DCM 50:50 Hexane/DCM 50:50 Hexane (Waste) Toluene backward elution of carbon column: Dioxins, furans and coplanar PCBs fraction Recovery PCDDs and PCDFs 60-90% NO-PCBs % Dechloranes 0-1% Toluene (backflush) Toluene (backflush)

17 GC-MS/MS parameters

18 GC-MS/MS parameters Column: Agilent DB-5m ultra inert for dioxin analysis (60m x 0.25mm x 0.25µm) Oven program temperature: 140 C for 2.6 min; ramp at 100 C to 320 C and hold for 21.1 min- Total run time: 25 min Carrier gas: He Flow rate 1.1 ml/min

19 GC-MS/MS parameters Transfer line temperature 320 C: set after the Experimental Design to prevent memory effect.

20 GC-MS/MS parameters Electron energy 70eV Ion source temperature 280 C

21 GC-MS/MS parameters MS1 and MS2 temperature: 150 C Collision gas: N 2 at 1,5 ml/min Quenching gas: He at 2,25 ml/min MRM mode, 2 transitions were recorded for each compound.

22 GC-MS/MS parameters PTV (programmed temperature vaporization) inlet, solvent vent mode Injection volume: 5µL (extracts in nonane) Inlet temperature program: 45 C for 1.3 min; ramp at 720 C to 320 C and hold until end run Solvent Vent: Vent flow 120 ml/min; Vent Pressure 10.5 psi Purge flow: 1200 ml/min to prevent memory effects in the inlet

23 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Results on real food and feed samples, assessment of dietary intake 7. Conclusions

24 Experimental Design for PTV Programmed Temperature Vaporizer inlet for Large Volumes Injection: Solvent removal (nonane) Sample transfer into the column increase Method sensitivity enhancement 3 main parameters identified as affecting PTV performance 1. Initial inlet temperature 2. Vent flow 3. Vent pressure Postulated model y b b x b x b3 x3 b12x1x2 b13x1x3 b23x2x3 b11x1 b22x2 b33x3 Central Composite Design Face Centered Design x i Experimental factor representing T (i=1), VF (i=2) or VP (i=3) xij Experimental factor representing the interaction between factor i and j, or the quadratic effect of a factor if i=j b 0 b i b ij Constant term, equal to the response in the centre of the domain (x i =0) Effect of the factor i Effect of the interaction between factor i and j, or the quadratic effect for i=j

25 Experimental Design for PTV 3 main parameters identified as affecting PTV performance 1. Initial inlet temperature 2. Vent flow 3. Vent pressure Postulated model y b b1 x1 b2 x2 b3 x3 b12x1x2 b13x1x3 b23x2x3 b11x1 b22x2 b33x3 17 experiments (3 replicates in the centre) Levels x 1 = T ( C) x 2 = VF(mL/min) x 3 = VP (psi) Responses Peak area Peak symmetry Goal To be maximized In the range

26 Significant effects peak area Factor Effect on peak area T (x 1 ) b 11 0 Negative quadratic effect VF (x 2 ) b 2 0 Positive linear effect VP (x 3 ) b 3 0 Negative linear effect (almost significant)

27 Significant effects peak symmetry Factor Effect on peak symmetry A s = B/A T (x 1 ) b 1 0 Positive linear effect 0.9 < A s < 1.1 VF (x 2 ) b 2 0 Positive linear effect VP (x 3 ) b 3 0 Positive linear effect (almost significant)

28 Levels to set optimum values Peak area Significant effects Goal Levels to set Values to set b 11 <0 To be T = 0 T = 45 C b 2 >0 maximized VF = +1 VF = 100 ml/min b 3 <0 VP = -1 VP = 1 psi

29 Levels to set optimum values Peak area Significant effects Goal Levels to set Values to set b 11 <0 To be T = 0 T = 45 C b 2 >0 maximized VF = +1 VF = 100 ml/min b 3 <0 VP = -1 VP = 1 psi Peak symmetry Significant effects Goal Levels to set Values to set b 1 >0 In the range T = +1 T = 60 C b 2 > VF = +1 VF = 100 ml/min b 3 >0 VP = +1 VP = 20 psi

30 VP = 1psi VP = 10.5 psi Levels to set optimum values Peak area Significant effects Goal Levels to set Values to set b 11 <0 To be T = 0 T = 45 C b 2 >0 maximized VF = +1 VF = 100 ml/min b 3 <0 VP = -1 VP = 1 psi Peak symmetry Significant effects Goal Levels to set Values to set b 1 >0 In the range T = +1 T = 60 C b 2 > VF = +1 VF = 100 ml/min b 3 >0 VP = +1 VP = 20 psi The optimum values for the three factor are the compromise leading to maximum peak area with symmetric peak shape

31 Peak Area Operative conditions VP = 10.5 psi Peak area increased with VF in the outlined direction, outwards the experimental domain replicates in the outlined direction: VF=120 ml/min T=45 C VP=10.5psi At 120 ml/min peak area is statistically higher (t-test) than at 100 ml/min, still showing acceptable peak symmetry Factor Best values T 45 C VF 120 ml/min VP 10.5 psi

32 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Results on real food and feed samples, assessment of dietary intake 7. Conclusions

33 Method validation criteria Criteria used to ensure proper identification, calculations and determinations of LOQs were the same as the ones listed in the EU Regulation for dioxin analysis (589/2014 and 709/2014), since at the present there is no regulation for Dechlorane compounds. Validation Criteria Selectivity Quantitation MRM transitions Chromatographic Separation Performed by Isotopic dilution (ID) with the two commercial standard available 13 C 10 -Dec 602 and 13 C 10 -DP syn 2 specific ion transitions for unlabelled compounds ;1 for labeled compounds; relative ion intensities max 15%; resolution MS quadrupoles = 1 Procedural blanks procedural blanks for each analytical procedure, depending on sample matrix. mloq Assessed either based on blank levels, or from iloq or the lowest calibration point when no signal was recorded in blanks. Recoveries range %

34 Selectivity Retention time Area Compound retention time 13C ISTD Mirex C 10 Dec 602 Dec C 10 Dec 602 CP C 10 Dec 602 Dec C 10 Dec 602 DP-syn C 10 DP-syn DP-anti C 10 DP-syn 13 C 10 Dec C 12 PCB-80 For each compound: t R shift (from expected t R ) must be the same as the reference 13 C ISTD t R shift All peaks are resolved 13 C 10 DP-syn C 12 PCB C 12 PCB recovery STD

35 Quantitation and MRM transitions Retention time Area Compound Quantitation transition Confirmation transition Mirex 272 > > Dec > > 239 CP 272 > > 239 Dec > > DP-syn 272 > > 239 DP-anti 272 > > 239 Compound Quantitation transition 13 C 5 Dec > C 5 DP-syn 277 > C 12 PCB > Relative ion intensity deviation = max 15% resolution MS quadrupoles = 1

36 Procedural blanks 88 samples from 9 different food and feed matrices analyzed. 16 procedural blanks prepared following the same procedure as for their corresponding real samples Matrix Milk 16 4 Chicken 8 7 Pork 8 7 Egg 8 2 Fat 18 4 Vegetable Oil 2 4 Salmon 8 2 n Sample amount (g) Blank n B_milk: Na 2 SO 4 and diatomaceous earth mixture B_fat: Hexane 3 9 Feed B_feed: Corn 3 30 Water 4

37 mloqs Method Limit of Quantification (mloq) assessed for each matrix, starting from values of procedural blanks. mloq = average blank value + 6σ If no detection in blank mloq = iloq iloq assessed by 8 replicate injections of the lowest calibration point*: evaluation of average value and its standard deviation σ. iloq = 10σ * lowest calibration point whose RRF gives acceptable = (RRF - ARRF) <30% If iloq < Lowest Calibration Point consistent = RRF RSD<15% mloq = Lowest Calibration Point deviation from the Average RRF (ARRF). Milk Chicken Pork Eggs Fat Vegetable Feed Corn Salmon oil additive (for feed) mloqs - pg/g fat mloqs - pg/g ww Mirex Dec CP Dec DP syn Dp anti

38 mloqs Method Limit of Quantification (mloq) assessed for each matrix, starting from values of procedural blanks. mloq = average blank value + 6σ If no detection in blank mloq = iloq iloq assessed by 8 replicate injections of the lowest calibration point*: evaluation of average value and its standard deviation σ. iloq = 10σ * lowest calibration point whose RRF gives acceptable = (RRF - ARRF) <30% If iloq < Lowest Calibration Point consistent = RRF RSD<15% mloq = Lowest Calibration Point deviation from the Average RRF (ARRF). Milk Chicken Pork Eggs Fat Vegetable Feed Corn Salmon oil additive (for feed) mloqs - pg/g fat mloqs - pg/g ww Mirex Dec CP Dec DP syn Dp anti

39 Outline 1. Introduction: chemical structures, uses and reasons for concern 2. Novelty and aim of the present work 3. Sample preparation and GC-MS/MS parameters 4. Experimental design to set PTV inlet parameters 5. Method validation 6. Results on real food and feed samples, assessment of dietary intake 7. Conclusions

40 Procedural blanks 88 samples from 9 different food and feed matrices analyzed. 16 procedural blanks prepared following the same procedure as for their corresponding real samples Matrix Milk 16 4 Chicken 8 7 Pork 8 7 Egg 8 2 Fat 18 4 Vegetable Oil 2 4 Salmon 8 2 n Sample amount (g) Blank n B_milk: Na 2 SO 4 and diatomaceous earth mixture B_fat: Hexane 3 9 Feed B_feed: Corn 3 30 Water 4

41 Results reporting approach Results were calculated according to the reporting method used for dioxin analysis under the EU Legislation in terms of lower-bound (lb) and upper-bound (ub). Lower bound approach: report 0 for the target whenever the level measured in the sample is <mloq. Sample levels are underestimated. target = 0 _mloq _ measure _0 Upper bound approach: report mloq for the target whenever the level measured in the sample is <mloq. Sample levels are overestimated. target = mloq _mloq _ measure _0

42 Average levels in food and feed Average values calculated with the upper-bound approach (overestimated values), and their standard deviation. TEQ levels of the same samples are also reported to show that selected samples were not particularly contaminated and can be representative of the Belgian food market. Milk (n=16) Chicken (n=8) Pork (n=8) Egg (n=8) Fat (n=18) Vegetable oil (n=2) Salmon (n=8) Feed additives (n=17) Corn (feed) (n=3) pg/g fat pg/g ww Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mirex Dec CP Dec DP syn DP anti Sum DPs Sum 6 Dechl TEQ

43 * Kim, J., et al., Assessment of Dechlorane compounds in foodstuffs obtained from retail markets and estimates of dietary intake in Korean population. Journal of Hazardous Materials, : p **Kakimoto, K., et al., Inhalation and dietary exposure to Dechlorane Plus and polybrominated diphenyl ethers in Osaka, Japan. Ecotoxicology and Environmental Safety, : p Average levels in food and feed Upper-bound (top of the box) and lower-bound (bottom of the box) levels of Dechloranes detected in real samples. The true concentration is in the range defined by the box. The maximum value for the upper-bound is 32 pg/g fat (egg) and 13 pg/g ww (feed additive, 2 samples showed very high levels). These levels are lower than the ones reported in Korea*, and similar to the one detected in Japan** Sum of 6 Dechloranes pg/g fat pg/g wet weight Milk Chicken Pork Egg Fat Vegetable oil (feed) 00 Salmon Feed additives Corn (feed)

44 Dechlorane distribution (upper-bounds) DP isomers are the major contributors for all matrices. Anyway this effect is be due to upper bound approach (mloq DP syn the highest = 50 pg absolute value) f anti = 0.3 ( in the technical mixture)

45 Dechlorane distribution (lower-bounds) This figure displays only the levels higher than mloqs. Mirex is still present in food even if it was banned. DP anti is the major contributor for several matrices. Dec 603 is present in almost all the matrices ( high levels found in human serum). DP isomers, Dec 603 and CP (pesticide related Dechloranes) in feed

46 Dietary intake of food Dechloranes Average daily intake of the Belgian population, based on upper-bound overestimated levels. The selected food matrices represented commonly consumed goods, to investigate food as possible route of exposure to Dechloranes for humans, after their detection in human serum. No class of food can be declared as strongly contributing to human exposure to Dechloranes. This could be the starting point for further studies aimed to investigate other route of exposure. Estimated dietary consumption Sum 6 Dechloranes Dechloranes sum intake pg/g fat g/day g fat/100g g fat/day or pg/g ww pg/day Salmon Chicken Pork Egg Milk Estimated Dechloranes dietary intake (pg/day) 136.2

47 Conclusions A GC-MS/MS Triple Quad method has been developed Sample extraction and clean-up are the same as for dioxin analysis Dioxin instrumental set-up in order to perform simultaneous analysis of Dechloranes and dioxins in routine labs Injection parameters have been settled by means of Experimental Design to increase method sensitivity The method has been validated according to applicable criteria from EU 589/2014 regulation, since it doesn t exist for Dechloranes yet. Real food samples show levels in the order of tenth pg/g for all the dechloranes. Average dietary intake of Dechloranes is ~136 pg/day

48 Further development Data about biodegradation, bioaccumulation and internal dose are needed to assess the passable levels for humans Proficiency test are needed to fully validate the developed method The method will be adapted for the analysis of environmental samples, in order to assess environmental route of exposure (house dust, car dust).

49 THANKS FOR YOUR KIND ATTENTION Calaprice Chiara PhD student at the University of Liege (Belgium) And at the Polytechnic University of Bari (Italy) Thanks to FMS for the instrumental support