Application of the QuEChERS extraction method for the analysis of pyrethrin and pyrethroid pesticides in fin and non-fin fish.
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1 Application of the QuEChERS extraction method for the analysis of pyrethrin and pyrethroid pesticides in fin and nonfin fish. Veronica Roscoe, Judy Judge, Dorothea F. K. Rawn 2 Health Products and Food Program, Winnipeg 2 Bureau of Chemical Safety, Food Research Division, Ottawa Presentation: Western Canada Trace Organic Workshop, 2008 Overview Background Aquaculture industry Goals of project Sampling details Method of analysis Validation data Results to date Conclusions Next Steps 2
2 Background Aquaculture Industry Aquaculture is the managed production of aquatic organisms Includes finfish, shellfish (molluscs and crustaceans) and aquatic plants. Considered to be one of the fastest growing agrifood production activities Top 0 aquaculture producers in the world (2004) include China, India, Philippines, Indonesia, Japan, Vietnam, Thailand, Korea, Republic of Bangladesh and Chile According to 2006 Statistics Canada report, Canada s aquaculture industry reported record revenues of >$950 million, up 24.7% from Background Sea Lice Sea lice are small, saltwater crustaceans. Commonly found in all oceans and on many species of fish around the world. Can transfer to farmed fish and then back into the marine environment. L. salmonis have been detected in both Pacific and Atlantic salmon. Azamethiphos was registered for use for the control of sea lice in Canada between Although not registered for use in Canada, the natural pyrethrins, cypermethrin and deltamethrin are used in other countries for the control of sea lice. 4
3 Project: Organic and Inorganic Contaminants in Retail Freshwater Fish and Seafood Products Establish if trace levels of the natural pyrethrins and synthetic pyrethroids are present in fish and if differences in concentration levels exist between fish collected from the wild relative to levels in fish raised in farm situations. The data developed will be used for the determination of Canadian exposure of these pesticides through consumption of fish and nonfin fish grown domestically and internationally. 5 Survey: Sample collection and preparation Samples of fresh and salt water fin and nonfin fish (4) were collected at retail stores in BC, ON and NS during winter and spring of 2002 Country of origin Canada, Chile, China, Ecuador, India, Jamaica, Taiwan, Thailand and US Farmed and wild Majority of samples represented farmed products Fresh, frozen, previously frozen or live Edible portions were homogenized 6
4 Types of Samples Domestic Imported Totals Farmed Wild Farmed Wild Arctic Char 6 5 Crab Marlin Mussels Oysters Prawns Salmon Shark Shrimp 4 6 Tilapia Trout 9 9 Tuna Totals (%) 79 (56%) 30 (2%) 22 (6%) 0 (7%) 4 7 Analytical challenges Achieve subppb to ppb detection limits GC/MS in negative CI mode Avoid multiple extraction/cleanup steps QuEChERS method for extraction and cleanup Eliminate or reduce response variation Addition of surrogate internal standard Eliminate or reduce matrix effects Standards prepared in matrix 8
5 Surrogate Internal standard Cypermethrin Pyrethrin I cispermethrin 9 QuEChERS Extraction Method Weigh 5 g sample into 50 ml FEP centrifuge tube Add 250 ng cispermethrin (phenoxy 3 C 6 ) surrogate standard Add 5 ml % acetic acid in acetonitrile Add 2 g anhydrous MgSO 4 and 0.5 g NaAc Shake and centrifuge 0
6 QuEChERS Cleanup Method Transfer ml of the extract to the QuEChERS dispersive tubes* 50 mg MgSO 4 50 mg PSA 50 mg C8 Shake and centrifuge * Chromatographic Specialties p/n UCTCUMPSC8CT or Restek p/n 2624 Final cleanup Transfer 0.5 ml extract to graduated tube and evaporate solvent justtodryness Add 50 ng transpermethrin (phenoxy 3 C 6 ) and bring to 0.5 ml with TMP Add MgSO 4 to reach the 0.2 ml mark on tube and vortex. Inject 2 µl 2
7 Instrumental Analysis Thermo Trace GC/MS in negative CI mode GC Conditions HP5, 30m X 0.32 mm with 0.25 µm film Splitless, 240 C, Oven program 80 C, hold min 50 C/min to 200 C 5 C/min to 285 C 50 C/min to 325 C, hold 5 minutes Transfer line 250 C MS Conditions Source 50 C Methane reagent gas 3 SIM Analysis Ions Monitored Analyte QM RM Other ions monitored Cinerin I , 62, 69 Jasmolin I , 62, 69 Pyrethrin I , 68, 69 Cinerin II Jasmolin II Pyrethrin II Cypermethrin Deltamethrin ,37,295 cispermethrin (phenoxy 3 C 6 ) , 356, 362 transpermethrin (phenoxy 3 C 6 ) , 356, 362 4
8 Extracted Ion Chromatogram: Standard at 20 ng/ml 5 Validation data Selectivity Limits of detection (LOD) & quantification (LOQ) Instrument response Bias and repeatability Precision 6
9 Selectivity Three reagent blanks and matrix blanks (salmon) were prepared and analyzed along with a lowlevel standard The reagent blanks and matrix blanks were found to be free of quantifiable peaks at the retention times of the analytes. 7 Limits of Detection and Quantification Standards were prepared in matrix at 4 ng/ml and ng/ml. n=7 injections of 4 ng/ml std, n=3 injections of ng/ml std LOD = 3 x S/N LOQ = 0 x S/N Analyte Total pyrethrins Cypermethrin Deltamethrin Method LOD/LOQ in Salmon Matrix LOD (ng/g) LOQ (ng/g) 2 8
10 Measuring Instrument Response Prepared a series of standards in salmon matrix at 5 concentration levels ranging from 50 ng/ml with cispermethrin (phenoxy 3 C 6 ) added as IS Randomly injected, in triplicate Evaluated calibration curve Evaluate Response Factor (Rf) relative to concentration, RSDs of area counts from triplicate injections and residuals Expect to observe no trends RSDs <5% above the LOQ Compared calibration models: unweighted, weighted (/x) and average response factor 9 Summary of Calibration Data in Matrix Calibration coefficients All >0.998 for unweighted linear regression Response factor plot No trends above the LOQ relative to analyte level except for Jasmolin I, which exhibited a slight increase RSD plot No trends exhibited %RSD For the three injections at each level of standard, the %RSDs were typically 56% at levels above the LOQ except for Deltamethrin, which was <8%. Residual Plot Most compounds exhibited increasing residuals with increasing concentration (ie heteroscedastic data) suggesting other calibration models be evaluated. 20
11 Evaluation of Calibration Models Analyte Actual Conc n ng/ml Unweighted Calculated Amt (% diff) Weighted /x Calculated Amt (% diff) Avg Rf Calculated Amt (% diff) Cinerin I Jasmolin I (56%) (48%) 0.077(4%) (4%) 0.067(%) (3%) Pyrethrin I (37%) (9%) (3%) Cinerin II (3%) (8%) (7%) Jasmolin II (2%) (7%) (4%) Pyrethrin II Cypermethrin (50%).73 (80%) (23%).4 (8%) (8%).0 (5%) Deltamethrin (90%).7 (3%).02 (%) 2 Evaluation of Calibration Models Analyte Actual Conc n ng/ml Unweighted Calculated Amt (% diff) Weighted /x Calculated Amt (% diff) Avg Rf Calculated Amt (% diff) Cinerin I Jasmolin I (9%) 0.34 (9%) 0.27 (0.2%) 0.23 (0%) 0.27 (0.%) 0.25 (2%) Pyrethrin I (6%).82 (%).87 (%) Cinerin II (8%) 0.28 (2%) (%) Jasmolin II (0%) 0.05 (9%) 0.07 (%) Pyrethrin II Cypermethrin (5%) 4.59 (20%).34 (3%) 4.4 (7%).4 (9%) 4.6 (8%) Deltamethrin (0%) 3.87 (6%) 3.93 (5%) 22
12 Assessing Bias and Reproducibility salmon samples containing no detectable amount of analyte were fortified at three levels each sample was additionally fortified with 50 ng/g surrogate internal standard (cispermethrin (phenoxy 3 C 6 )) prior to extraction transpermethrin (phenoxy 3 C 6 ) was added to each sample prior to preparing at final volume (performance internal standard) Mean Recovery of cispermethrin (phenoxy 3 C 6 ) Sample Set 4 ng/g (n=5) 20 ng/g (n=7) 40 ng/g (n=7) % Recovery % RSD Assessing Bias and Reproducibility cont d Analyte Spike level (ng/g) Mean Calc d Amt (ng/g) % RSD Corrected Recovery (%) Pyrethrins Cypermethrin Deltamethrin
13 Assessing Precision, within and betweenrun 3 salmon samples were spiked at 4 ng/g and at 20 ng/g. sets completed on 5 different days Analyte Pyrethrins Spike level (ng/g) 3.89 Mean Calc d Amt (ng/g) 3.95 Withinrun %RSD 6 Betweenrun %RSD Cypermethrin Deltamethrin Results to date Analysis of arctic char, mussels, oysters, trout, tuna, shark, marlin and salmon completed (82/4 samples). No analytes detected except in salmon 7 of 22 samples of salmon were found to contain cypermethrin Domestic Farmed 3 of 7 indicated Atlantic salmon (other 4 purchased in Halifax) Range: ng/g Mean =.87 ng/g 26
14 Mean Spike Recovery data (20 ng/g) Matrix Pyrethrins (%) Cypermethrin (%) Deltamethrin (%) cispermethrin ( 3 C 6 ) (%) Salmon (n=0) Arctic Char (n=) Trout (n=2) Mussel (n=) Marlin (n=) Oysters (n=2) Mean (w/o marlin) RSD (w/o marlin) Conclusions QuEChERS extraction method has been successfully demonstrated to apply to the analysis of the natural pyrethrins and synthetic pyrethroid pesticides in fin and nonfin fish tissue. GC/MS in negative Chemical Ionization mode provided the required sensitivity to detect at low ppb levels. Limits of detection were established at 0.3 ng/g for cypermethrin and deltamethrin and at 0.5 ng/g for total pyrethrins. The use of standards prepared in matrix and a surrogate internal standard provided reproducible results however, for some matrices, quantification using standards prepared in the exact same matrix would be required. Average recoveries of the total pyrethrins, cypermethrin and deltamethrin ranged from 705% with relative standard deviations of 2% or less. 28
15 Conclusions cont d Of the residues studied in this project, Cypermethrin was the only detected compound, at concentrations ranging from 0.26 ng/g to 6.4 ng/g with a mean level of.9 ng/g. Cypermethrin was detected in domestic farmed salmon samples, generally from the Atlantic region. In all cases, cypermethrin residues were well below the General Maximum Residue Limit (MRL) of 00 ng/g established in the Canadian Food and Drug Regulations for chemicals not listed in Table 2 of Division Next Steps Remaining 59 samples will be analyzed and final data and validation report completed. These data will be shared with the PMRA for inclusion in dietary exposure estimates. The method and data will be published in a scientific journal. 30
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