A New Concept for the Performance of Inter-Laboratory Method Validation Studies

Buchner, N.1, Stachel, C.S.1, Kunath, K.2, Uhlig, S.2

Introduction
In Germany numerous Official Field Laboratories (OFL) are involved in the official pesticide residue control and an expert group for pesticide residue analysis (short EPRA) coordinates inter-laboratory method validation studies. Currently the validation in Germany is carried out according to the AQC-document [1]. With this concept it is possible to determine the recovery and the repeatability with acceptable statistical uncertainty but the uncertainty of the reproducibility s.d. is quite high. Therefore an evaluation with regard to the ruggedness of the method is difficult. Also the number of samples analysed by each laboratory is quite high in relation to the still high uncertainty of the reproducibility s.d. A major drawback of this concept is also that the statistical parameters are calculated only for the one matrix tested per matrix group. They do not necessarily apply for other matrices of the matrix group! In the new validation concept eight matrices are selected at random from one commodity group ( example: eight randomly selected oils of the commodity group high fat content). Each of the eight participating laboratories ( - ) analyses only two different matrices in two replicates (e.g. lab analyses safflower oil and line seed oil, while lab analyses sesame oil and sunflower oil.) The calculated statistical parameters (recovery, matrix-comprehensive repeatability s.d. and reproducibility s.d.) give equivalent or lower uncertainties than the statistical parameters obtained with the former concept. As a main advantage these values hold valid for the whole matrix category. Also the sample number for each laboratory is reduced to 10 samples per matrix (4 samples + 1 blank for two concentration levels). This results in a total of 50 samples per lab and 400 samples for the validation of all five commodity groups including eight matrices per group. In order to prove the applicability of the new concept a pre-validation study was carried out in our lab. Current Validation Concept Two concentration Levels 0.01 and 0.1 mg/kg (x2) Five replicates and one blank (x6) 12 samples per matrix Five matrices per lab (x5) 60 samples in total for the validation in one laboratory Eight laboratories (x8) 480 samples for the complete validation
Parameter sr (repeatability s.d.) sR (reproducibility s.d.) Rec (recovery) Example 8.0 % 20.0 % 94.0 % Relative Bias 14.5 % 27.5 % 7.6 %
Raisins

Wheat

Olive Oil

Lemon Cucumber

Uncertainty (95%) 28.4 % 53.8 % 19.6 %

Safflower

New Validation Concept Two matrices (x2) Two replicates and one blank (x2+1) Two concentration Levels 0.01 and 0.1 mg/kg (x2) 10 samples per matrix Five matrices groups (x5) 50 samples in total for the validation in one laboratory Eight laboratories (x8) 400 samples for the complete validation
Parameter sr (repeatability s.d. matrix comprehensive) sR(reproducibility s.d. matrix comprehensive) Rec (recovery) Example 8.0 % 20.0 % 94.0 %

Sunflower Line Seed Sesame Olive

Grape Seed

Rape Seed

Pumpkin Seed

Relative Bias 17.7 % 22.7 % 6.0 %

Uncertainty (95%) 34.6 % 44.6 % 14.2 %

Materials & Methods

Random selection of 8 different native oils (grape seed oil, line seed oil, olive oil, pumpkin seed oil, rape seed oil, safflower oil, sesame oil, sunflower oil) Spiking with a pesticide mix of 50 analytes (analyte selection according to missing validation data and ranking points >150) Concentrations 0.02, 0.05 and 0.1 mg/kg (one blank each) Application of the Quechers Method [2] for oil

The uncertainties of the validation parameters were calculated using algorithms specifically provided by InterVal (following CD 2002/657/EC) and published in [3]. Rec [%] 99.1 100.1 89.6 72.8 81.7 90.0 87.8 64.6 95.7 82.3 94.8 93.1 89.2 72.5 92.0 53.8 95.3 92.6 44.9 88.0 94.5 92.0 95.8 75.1 73.5 65.9 97.8 89.4 98.0 96.2 91.1 77.9 91.3 86.6 80.6 81.8 91.3 74.4 53.6 26.0 21.6 94.9 75.0 80.0 93.7 78.1 100.6 90.9 97.2 85.7 sr [%] 11.9 10.6 14.0 12.2 13.8 9.18 11.0 11.1 11.5 13.4 8.9 11.9 11.7 15.7 11.3 14.6 9.2 12.8 19.3 11.8 10.7 14.2 10.8 13.7 18.5 19.6 12.4 11.8 10.3 12.4 9.6 11.4 10.9 13.5 12.0 9.3 9.4 10.5 15.3 > 20 > 20 11.6 10.5 10.5 10.8 13.7 14.0 10.5 11.5 10.4 MU [%] 26.2 23.7 30.9 34.6 30.4 46.7 24.9 26.9 25.5 29.5 20.8 26.5 26.2 35.4 25.1 34.8 21.3 28.1 46.9 26.3 25.5 30.5 24.0 30.8 39.8 46.1 27.1 27.6 23.2 27.0 21.8 26.3 24.5 29.7 27.2 22.1 21.9 25.0 36.0 > 50 > 50 25.6 24.8 24.5 24.5 30.8 30.0 23.7 25.4 23.9

Weight 2.0 g sample in a 50-mL centrifugal tube (with screw cap) Spike samples with pesticide mix and blanks with acetonitrile (ACN) Add 10 mL ACN Shake 15 min with shaker (first extraction)

Analyte Aldicarb Azoxystrobin Boscalide Carbendazim Chlorpyrifos-methyl Clofentezine Cyproconazole Cyprodinil Desmethylpirimicarb Difenoconazole Dimethomorph Epoxiconazole Ethoprophos Etofenprox Fenamidone Fenazaquin Fenbuconazole Fenoxycarb Fenpropimorph Fluquinconazole Flusilazole Flutriafole Fosthiazate Hexaconazole Hexythiazox Imazalil Indoxacarb Linuron Malathion Metalaxyl Myclobutanil Pendimethalin Phosalone Pirimiphos-methyl Prochloraz

Rank 195 333 374 324 209 167 308 415 212 358 229 366 296 171 163 178 270 201 200 282 430 304 218 299 175 359 266 319 191 266 414 181 173 199 277 274 176 174 210 210 174 260 191 315 256 196 256 255 165

RL [mg/kg] 0.02 0.02 0.02 0.02 0.02 0.025 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.04 0.02 0.02 0.02 0.02 0.02 0.02 0.05 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

Evaluation of the Pre-Validation Study

By means of the software InterVal (quo data GmbH, Dresden, Germany www.quodata.de) the required validation parameters by the AQC-document [1] (Rec-recovery 70-120%, sr-repeatability s.d. <20% and MU-extended measurement uncertainty <50%) were calculated. The RL-reporting level was determined for each analyte (see for obtained results). By using three concentration levels it was possible to generate concentration depended functions for the statistical parameters (e.g. the recovery function and the uncertainties contributing the MU for the analyte Aldicarb).

Centrifuge for 5 min at 3000 g Transfer 6 ml of the extract in a 12-mL centrifugal tube (with screw cap) Add 60 L of Internal Standard (e.g. TRIS) For Blanks 60 L ACN mix it Transfer extract in a centrifugal tube containing 150 mg PSA and 150 mg ODS C18 (shake briefly) Shake for 30 sec Centrifuge for 5 min at 3000 g Transfer 4 mL of extract in 12-mL centrifugal tube (with screw cap) add 40 L of 5% formic acid in ACN (10 L/mL extract) Fill in vials and spike extracts according to spiking scheme HPLC-MS/MS

Discussion and Outlook

50 Analytes were validated in our lab via a new validation concept. 43 analytes out of 50 possess a RL at 0.02 mg/kg with Rec 70-120%, sr <20% and an MU <50%. Seven analytes did not fulfill the AQC criteria and are therefore not valid at 0.02 mg/kg. The analyte Clofentezine proved to be unstable in the calibration solution and could therefore not be determined correctly at 0.02 mg/kg in sunflower oil and line seed oil. The RL could be set to 0.025 mg/kg. Six analytes were extracted badly during the sample preparation therefore the recovery was below 70%. For Cyprodinil and Fenazaquin the RL can be set to 0.02 mg/kg. For Fenpropimorph and Imazalil the RL can be set to 0.04 and 0.05 mg/kg. SpinosynA and SpinosynD no RL can be set due to an extreme low recovery which prevented the determination by means of low signal response. The applicability of the concept was proven. As a next step to fully implement the new concept a validation study in EPRA with the 43 successfully validated analytes will be performed.

The

343 new validaito 0.02

Propiconazole Pyraclostrobin Pyriproxifen Quinoxyfen SpinosynA SpinosynD Tebufenozid Tebufenpyrad Terbuthylazin Tetraconazole Thiabendazole Thiamethoxam Triadimefon Trifloxystrobin Triticonazole

References
[1] METHOD VALIDATION AND QUALITY CONTROL PROCEDURES FOR PESTICIDE RESIDUES ANALYSIS IN FOOD AND FEED - Document N SANCO/2009/10684 [2] Anastassiades, M., et. al. Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/ Partitioning and Dispersive Solid-Phase Extraction" for the determination of pesticide Residues in Produce. Journal of AOAC International 2003, 86 (2), 412-431. [3] Jlicher, B., Gowik, P. and Uhlig, S. A top-down in-house validation-based approach for the investigation of the measurement uncertainty using fractional factorial experiments. Analyst 1999, 124, 537-545.

1 Federal

Office of Consumer Protection and Food Safety (BVL) National Reference Laboratories for Pesticide Residues (NRL) P.O. Box 11 02 60 10832 Berlin Germany e-mail: NRL-Pestizide@bvl.bund.de 2 quo data - quality management and statistics GmbH Kaitzer Str. 135 01187 Dresden Germany