EURL for Cereals and Feeding stuff National Food Institute Technical University of Denmark
Validation Report 21
Determination of pesticide residues in maize for livestock feed by GC-MS/MS and LC-MS/MS
(QuEChERS method)
Susan Strange Herrmann Mette Erecius Poulsen December 2016 Page 2 of 17
CONTENT:
1. Introduction ...... 3 2. Principle of analysis...... 3 3. Validation design ...... 4 4. Chromatograms and calibration curves ...... 4 5. Validation parameters...... 7 6. Criteria for the acceptance of validation results ...... 7 7. Results and discussion ...... 8 8. Conclusions ...... 8 9. References ...... 9 Appendix 1a. MRM transitions GC-MS/MS...... 10 Appendix 1b. MRM transitions for LC-MS/MS...... 12
Appendix 2. Recoveries, repeatability (RSDr), Combined Uncertainty (Comb. U.) and Limit of Quantification (LOQ) for pesticides validated on maize for livestock feed...... 14 Appendix 3: Principles of the QuEChERS method for feed extraction...... 17
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1. Introduction This report describes the validation of the QuEChERS method combined with GC-MS/MS and LC- MS/MS. The method was sought validated for 80 pesticides in maize for livestock feed (in-house standard mixture B). The QuEChERS method is an extraction method which has been developed to be Quick, Easy, Cheap, Efficient, Rugged and Safe. The method is most commonly used on fruit, vegetables and cereals1.
2. Principle of analysis Sample preparation: The samples were milled with a sieve size of 1 mm. Extraction: The sample was shaken and a salt and buffer mixture was added and the sample shaken again. Clean-up: After centrifugation the supernatant was transferred to a clean tube and put in -80 degree freezer. When the extract was almost thawed it was centrifuged and the supernatant was transferred to a tube containing PSA and MgSO4. After shaking and an additional centrifugation step the final extract was diluted 1:1 with acetonitrile to obtain the same matrix concentration as in the matrix matched calibration standards. The principle of the extraction and clean-up procedure is outlined in Appendix 3. Quantification and qualification: The final extract was analysed by GC/MS/MS and LC-MS/MS. GC-MS/MS: The pesticide residues were separated on a DB5-MS column and analysed by triple quadrupole operating in the multiple reaction monitoring mode (MRM) with electron energy at 70 eV, source temperature at 180°C and transfer line at 250°C. The injection volume was 4 µl. For each pesticide two sets of precursor and product ions were determined. One for quantification and one for qualification. The MRM transitions for the pesticides and degradation products are given in Appendix 1a. LC-MS/MS: The pesticide residues were separated on a reversed-phase column and detected by tandem mass spectrometry (MS/MS) by electrospray (ESI). The validation includes pesticides 13 determined with both positive and negative ESI. C6-carbaryl was used as internal standard. All pesticides were detected in the MRM mode. For each pesticide precursor ion and 2 product ions were determined. One product ion for quantification and one for qualification. The MRM transitions for the pesticides and degradation products sought validated are given in Appendix 1b.
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3. Validation design The method was south validated for 80 pesticides or degradation products in maize (see Table 1). The validation was performed on 5-6 replicates at each of four spiking levels; 0.005, 0.01, 0.02 and 0.1 mg/kg. A blank sample of maize was included in each analytical batch.
Table 1. Pesticides included in the recovery experiments.
Pesticides included in recovery experiments
Acetamiprid EPN Iprovalicarb Phoxim Acrinathrin Ethoprophos Isofenphos‐methyl Profenofos Aldicarb Etofenprox Lufenuron Propargit Aldicarb sulfon Fenamiphos Mepanipyrim Propyzamide Bitertanol Fenamiphos sulfoxide Methamidophos Prothiofos Bromopropylate Fenamiphos sulfone Methidathion Pyridaben Bromuconazole Fenarimol Methiocarb Pyriproxyfen Bupirimate Fenazaquin Methiocarb sulfone Spinosyn A Buprofezin Fenoxycarb Methiocarb sulfoxide Spinosyn D Cadusafos Fenpropathrin Monocrotophos Spirodiclofen Carbendazim Fenthion Myclobutanil Tau‐Fluvalinate Chlorfenapyr Fenthion oxon Oxadixyl Tebufenpyrad Chlorobenzilate Fenthion oxon‐sulfon Oxamyl Tefluthrin Clofentezine Fenthion oxon‐sulfoxid Paraoxon‐methyl Tetraconazole Dicloran Fenthion sulfone Parathion‐methyl Tetradifon Dimethomorph Fenthion sulfoxide Pencycuron Thiabendazole Diphenylamine Flufenoxuron Phenthoate Tolclofos‐methyl DMF Fosthiazate Phosalone Tolylfluanid DMPF Hexythiazox Phosmet Triflumuron DMST Indoxacarb Phosmet oxon Zoxamide
4. Chromatograms and calibration curves The calibration curves were based on at least 4 calibration levels, i.e. 0.003, 0.01, 0.033 and 0.1 µg/ml. The calibration curves were in general best fitted to a linear curve. The quantification was performed from the mean of two bracketing calibration curves. The majority of the correlation coefficients (R) were higher or equal to 0.99. Examples of chromatograms and calibration curves obtained when analysing extracts by GC-MS/MS and LC-MS/MS are presented in Figure 1-2 and Figure 3-4, respectively.
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150130st10 Smooth(Mn,2x2) F1:MRM of 6 channels,EI+ 6. EUPT-CF 9 Spk. 0.005 ppm C 169>168 Diphenylamine;9.73;2522.9190;35006 3.802e+004
%
9.41
2 min
150130st10 Smooth(Mn,2x2) F1:MRM of 6 channels,EI+ 6. EUPT-CF 9 Spk. 0.005 ppm C 168>167 Diphenylamine;9.73;2319.8440;29917 3.555e+004 100
%
9.41
0 min 9.500 9.600 9.700 9.800 9.900 10.000 Figure 1: Examples of GC-MS/MS chromatograms for diphenylamine in maize for livestock feed obtained when analysing extract of sample spiked with 0.005 mg/kg (Quantifier and qualifier MRM transitions are shown).
150114_13 Smooth(SG,1x2) F8:MRM of 8 channels,ES+ 6. EUPT-CF 9 spk 0.005 ppm C 511 > 158.1 Lufenuron 1.176e+003 99 22.91 180.646 1154
%
22.19 23.67 24.08 21.82 22.39 23.35 24.56 -1 min
150114_13 Smooth(SG,1x2) F8:MRM of 8 channels,ES+ 6. EUPT-CF 9 spk 0.005 ppm C 511>141 Lufenuron 6.067e+002 99 22.83 125.847 600
%
23.67 21.94 22.35 22.47 21.82 23.39 23.92 24.12 24.48 24.68
-1 min 22.00 22.50 23.00 23.50 24.00 24.50 25.00 Figure 2: Examples of LC-MS/MS chromatograms lufenuron in maize for livestock feed obtained in positive mode when analysing extract of sample spiked with 0.005 mg/kg (Quantifier and qualifier MRM transitions are shown).
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Compound name: Diphenylamine Correlation coefficient: r = 0.997576, r^2 = 0.995159 Calibration curve: 2.15397e+006 * x + -307.883 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
80000
70000
60000
50000
40000 Response 30000
20000
10000
-0 ug/ml -0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035
Figure 3. Examples of GC-MS/MS calibration curves for diphenylamine matrix matched with maize for livestock feed (concentrations from 0.001-0.100 µg/ml)
Compound name: Lufenuron Correlation coefficient: r = 0.997610, r^2 = 0.995225 Calibration curve: 23.6916 * x + 0.00116182 Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
8.00
6.00
4.00 Response
2.00
-0.00 Conc -0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350
Figure 4. Examples of LC-MS/MS calibration curves for lufenuron matrix matched with maize for livestock feed (concentrations from 0.001-0.333 µg/ml).
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5. Validation parameters Precision – repeatability
Repeatability was calculated for all pesticides and degradation products on four spiking levels (0.005, 0.01, 0.02 and 0.1 mg/kg). Repeatability is the relative standard deviation on the results from two or more analysis of replicate samples, prepared by the same technician, on the same instrument and within a short period of time.
Repeatability (RSDr) in this validation was calculated from the 5-6 replicate determinations as given in ISO 5725-22.
Accuracy – Recovery The accuracy was determined from recovery studies in which samples were spiked at three levels (0.005, 0.01 mg/kg, 0.02 and 0.1 mg/kg) with the relevant pesticides, isomers and degradation products.
Robustness The QuEChERS method has, in connection with the development of the method, been shown to be robust by Anastassiades et al. 20031.
Limit of quantification, LOQ The quantification limits (LOQ) was determined as the lowest spike level for which the acceptance criteria (se Section 6) were meet.
The obtained results including recovery, RSDr, combined uncertainty (Uc) and limit of quantification (LOQ), are presented in Appendix 2.
6. Criteria for the acceptance of validation results For the pesticides to be accepted as validated the following criteria for precision and trueness must to be fulfilled: 1. The relative standard deviation of the repeatability should be ≤20%3. 2. The average relative recovery must be between 70 and 120%3. If the above mentioned criteria have been meet, the quantification limits, LOQs is stated. The combined uncertainty given by: