Contribution of sample processing to variability and accuracy of measured residues
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1 Contribution of sample processing to variability and accuracy of measured residues Árpád Ambrus 1, Etelka Solymosné Majzik 2, Henriett Szemanné Dobrik National Food Chain Safety Office, ÉKI2 Tábornok u; Budapest, Hungary; 2 National Food Chain Safety Office; Velence, Hungary; 3 National Food Chain Safety Office; Miskolc, Hungary
2 Objectives -highlights Testing the efficiency of sample processing and stability of residues during sample homogenization Test matrices: tomato, lettuce and corn, surface treated with test mixture at 0.1 mg/kg and 0.2 mg/kg level Homogenization: at room temperature (RT) and deepfrozen sample material with dry ice (DI) Test analytes: mixture of pesticide active substances "Stable reference compounds: buprofezin, cyprodinil and chlorpyrifos
3 Basic relationships Combined uncertainty of the laboratory phase of the determination of pesticide residues Uncertainty of duplicate analyses of the split extracts (CV' A ) of test portions: d 2 =1.128
4 Determination of CV L from the analysis of replicate test portions: The uncertainty of sample processing (variability of residues in test portions): Actual average concentration of the reference compound Actual concentration of the analytes (adjusted): The proportion of j th analytes remained in the processed sample (average of result obtained with 1g and 10 g test portions:
5 Day 1 2 kg lettuce Day 2 0,6 kg saláta 1,2 kg lettuce Kept it in deep-freezer for minimum few hours Weigh 0,6 kg (accurately) Weigh 0,6 kg (accurately) Day 1 Extract 2 10 g with QuEChERS CL1, CL2 Add dry ice and chop it Day 2. screening results available Spike 5 10 g with mix "A": CL/10/1, CL/10/2, CL/10/3, CL/10/4, CL/10/5, and mix B 5 1 g*- CL/1/1, CL/1/2, CL/1/3, CL/1/4, CL/1/5 Surface treatment with C Chop at room temperature Surface treatment with C Deep-freeze for 1 night 3. nap Screening for potential residues present (2 samples) Proceed with QuEChERS Remove 5 10 g és 5 1 g* RTL/10/1, RTL/10/2, RTL/10/3 RTL/10/4, RTL/10/5 ill. RTL/1/1, RTL/1/2, RTL/1/3, RTL/1/4, RTL/1/5 Mix thoroughly Chop in dry ice LC-MS és GC-MS analyses (10 samples) Proceed with QuEChERS shake for 2 minutes Let it stand for 30 min at room temperature Remove frozen 5 10 g és 5 1 g* DIL/10/1, DIL/10/2, DIL/10/3, DIL/10/4, DIL/10/5 ill. DIL/1/1, DIL/1/2, DIL/1/3, DIL/1/4, DIL/1/5 Prepare 2 replicates for LC- MS/MS and GC-MS/MS Take portions from random positions Proceed without delay Proceed with QuEChERS shake for 15 minutes (20 minta) 2-2 párhuzamos fiolázása mindkét műszerre Flowchart for processing lettuce LC-MS és GC-MS vizsgálat (20 minta)
6 Flowchart for processing corn 2,5 kg Corn grain well mixed 200 g 100 g Grind to < 1 mm Mix thoroughly with 10 ml standard mix 'C' in a round bottom flask, evaporate solution at 35 o C Measure 3 10 g portions of ground corn for determining residues in untreated sample C1, C2, C3 Add Spike A : to 5 10 g-ground corn S10/1, S10/2, S10/3, S10/4, S10/5 Add sike B 5 1 g-ground corn S1/1, S1/2, S1/3, S1/4, S1/5 Mix it thoroughly with 900 g untreated corn Grind to < 1 mm Immediately spread the ground material on the clean glass surface, take test portions from different positions and extract them. Analyse test portions with Method B Take 5 10 g T10/1, T10/2, T10/3, T10/4, T10/5 and 5 1 g T1/1, T1/2, T1/3, T1/4, T1/5 Analyse spiked test portions with Method B Take up purified extracts in 0,5 g/ml eluent for LC/MS in 1 g/ml hexane:acetone 4:1 for GC/MS Remove aliquots for duplicate LC/MS and GC/MS analyses
7 RESULTS
8 Efficiency of chopping Tomato Average LC-MS/MS CV rec CV' Atestp CV L CV Sp CV Sp10 /CV Sp10 RT 1 g >4.93 Rt10g <0.03 DI 1 g DI10 g Lettuce Average GC-MS/MS CV rec CV' Atestp CV L CV Sp CV Sp1 /CV Sp10 RT1 g RT 10g DI1 g DI 10 g
9 Efficiency of grinding of corn mm 68.8 % ,5 mm 27.1 % < 0.25 mm 4.1 % Average LC-MS/MS CV rec CV' A testp CV L CV Sp CV Sp1 /CV Sp10 1 g g GC-MS/MS 1 g g
10 Performance characteristics of reference compounds LC-MS/MS 1 g Rec CV rec 10g Rec CV rec Buprofezin Tomato Cyoridinil Tomato Buprofezin Lettuce Buprofezin Corn Cyprodinil Corn GC-MS/MS 1 g Rec CVrec 10g Rec CVrec Buprofezin Tomato Chlorpyrifos Tomato Buprofezin Lettuce Chlorpyrifos Lettuce Cyprodinil Corn Recovery studies were performed at room temperature.
11 Proportion of analytes remained average of 1 g & 10g Matrix: tomato, lettuce, corn; Composition of test mixture: Buprofezin, Chlorothalonil, Chlorpyrifos, Cyprodinil, Dichlofluanid, Dichlorvos, Etridiazol, Fenhexamid, Heptenophos, Hexaconazole, Chlozolinate, Imidacloprid, Pyrimethanil, Prochloraz, Spiroxamine, Tebuconazole, Tecnazene, Thiacloprid, Trifloxystrobin Reference compounds: Buprofezin, Cyprodinil
12 Compounds degraded during chopping of tomato: Tomato LC-MS/MS GC-MS/MS RT DI RT DI Chlorothalonil nd nd Chlorpyrifos Dichlofluanid nd nd Dichlorvos Etridiazol Fenhexamid Chlozolinate Prochloraz Tecnazene
13 Compounds degraded during chopping of lettuce: LC-MS/MS GC-MS/MS RT DI RT DI Dichlorvos Etridiazol Chlozolinate
14 Compounds degraded/evaporated: during grinding of corn Corn LC-MS/MS GS-MS/MS Buprofezin 1 1 Chlorpyrifos Cyprodinil Dichlorvos Dichlofluanid NA Fenhexamid Heptenophos Spiroxamine 0.761
15 CONCLUSIONS
16 Contribution of extraction step to the combined uncertainty of the results There was no significant difference between the repeatability of recovery tests (CV rec ) and duplicate analyses of split extracts (CV' A ), indicating that the contribution of extraction to the variability of the results is not substantial, and it does not affect the CV Sp estimated from the average residues in test portions and CV rec.
17 Efficiency of chopping, grinding The ratio of the CV SP1g /CV SP10g ranged from 3.7 to 8.5. It was influenced by the actual CV L and CV rec obtained in the tests. One set of experiment is not sufficient to obtain typical CV L values. Nevertheless, the results indicate that decreasing the test portion size significantly increase the CV SP and the combined uncertainty (CV L ) of the determination of pesticide residues. Reducing test portion below g should only be done after verification of the efficiency of sample processing which depends on the equipment used and the matrix analysed, but independent from the analytes.
18 Efficiency of chopping, grinding (2) Note that neither the recovery studies nor the proficiency tests results provide information on the CV L of the laboratory procedure! The best approach would be, as part of the internal quality control, to regularly reanalyze retained test portions containing detectable residues in various matrices, and calculate the average CV L based on >15 corresponding tests.
19 Stability of residues Buprofezin, cyprodinil and chlorpyrifos remained >95% in tomato, lettuce and corn. Their performance characteristics were stable under the test conditions. These substances can be used as reference compounds in further studies. The difference between the theoretical concentration (100%) and the proportion remained < 15% could not be statistically confirmed (apply double t-test, and not testing the difference between two mean values). Proportion of analytes <85% indicated degradation.
20 Stability of residues (2) The results confirm previous studies that several pesticides undergo degradation during the homogenization process. The proportion remaining varied from <LOQ (dichlofluanid) chlozolinate ~ 25-30% to 75-83% (dichlorvos). The decomposition of analytes strongly depends on the matrix and temperature of processing. Good recoveries do not guaranty stability of analytes, which should be tested for each analyte-matrixhomogenization combinations as part of the validation of the methods.
21 Closing remarks Our limited tests only highlight some of the potential sources which may affect the accuracy and uncertainty of determination of analytes at trace level. Several tests performed during the application of a procedure should be performed to obtain more robust information for specific analytematrix and test procedure.
22 Acknowledgement The tests reported were carried out in two laboratories of the National Food Chain Safety Office. The indispensible contribution of the staff of the laboratories is emphasised and highly appreciated.
Contribution of sample processing to variability and accuracy of
Contribution of sample processing to variability and accuracy of measured residues Árpád Ambrus, Gabriella Kötelesné Suszter Etelka Solymosné Majzik Henriett Szemanné Dobrik Objectives -highlights Testing
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