S chneider & A ndersen : J ournal of AOAC I nternational V ol . 98, N o . 3, 2015  667

HorRat of 2.1. The exposure of salmon to a low concentration mixture of the analytes for 1 h was not sufficient to provide a significant concentration of CV residue in the salmon muscle. The higher HorRat for CV incurred salmon is a clear indication that the mean measured concentration of 0.03 µg/kg of CV is below the LOQ for this method. In the single-laboratory validation of the First Action method in trout matrix, the authors determined the decision limit (CC α ) and the detection capability (CC β ) for each analyte (12). CC α ranged from 0.13 to 0.42 µg/kg for the five analytes in trout matrix, and CC β ranged from 0.17 to 0.54 µg/kg. In trout matrix, the method was determined to have the greatest sensitivity for CV and the least for LCV. From collaborative study data, CC α and CC β were determined from the quantitative product ion transition 1 for each analyte in each matrix from the extracted calibration curves according to ISO 11843-2 (16) and Commission Decision 2002/657/EC (10). The medians of individual CC α and CC β values determined for each of the 14 laboratories are reported in Table 4a, as recommended in ISO 11843-2 for a multilaboratory validation (16). Individual values of CC α were >1 µg/kg only for one salmon analysis by one laboratory, where calibration data for MG and BG yielded CC α values of 1.23 and 1.16 µg/kg, respectively. For the overall median data, CC α ranged from 0.14 to 0.42 µg/kg for the five analytes in salmon, catfish, and shrimp, and CC β ranged from 0.16 to 0.47 µg/kg. Collaborative study results for salmon, catfish, and shrimp were consistent with those reported in the First Action method validation for trout (12). In addition to CC α and CC β , the method detection level (MDL) and LOQ were calculated from the 0.42 μg/kg fortified sample data at the 99% confidence level (17). The MDL was calculated as the SD of the 0.42 µg/kg sample results ( n  = 28, 14 laboratories with duplicate samples) multiplied by the Students t -value at the 99 % confidence interval (one tailed) for that number of samples. The LOQ was determined as 10 times the SD of the 0.42 μg/kg sample results. Results for the MDL and LOQ for the different analytes and matrixes are summarized in Table 4b. Samples were excluded from the MDL and LOQ determinations if they had been identified as statistical outliers or excluded for cause, and the total number of samples (degrees of freedom) was adjusted accordingly. All MDLs were less than the lowest concentration level for the fortified samples, and the majority of the LOQs were determined to be below the 1.0 µg/kg level of concern. Considering these data are compared across 14 different laboratories using different analytical instrumentation, the low MDLs and LOQs highlight the sensitivity and robustness of this method. MDLs and LOQs calculated from validation data produced by a single laboratory would be expected to be significantly lower; however, for the collaborative study, each laboratory only generated results for two samples/matrix at the 0.42 μg/kg concentration, which calculations, quantitative product ion transition data was calculated as the peak area ratio relative to the internal standard. At the zero calibration level, most participating laboratories reported numerical peak area data for small peaks or noise detected at the retention time of the analyte, while other labs reported the value “0”. It was beyond the scope of the study to obtain non-zero noise measurements from each laboratory. Of the 210 individual CC α calculations, 30% were based on calibration data sets that included the value of “0” for the peak area ratio at the zero calibration level. 1  For CC α

Repeatability SDs (s r ), reproducibility SDs (s R ), reproducibility RSDs (RSD R ), and number of statistical outliers are presented in Table 3. HorRat values are also presented in this table and are calculated as RSD R (observed)/RSD R (predicted), where the RSD R (predicted) is calculated using the equation RSD R = 2C –0.1505 , where C is the measured analyte concentration in decimal mass units. Cochran, Grubbs, and double Grubbs tests were used to remove statistical outliers where appropriate. When one data point was deemed to be an outlier, both replicates for that concentration level for that laboratory were excluded from the data set. The n = 14 number of participating laboratories permitted data from up to three laboratories to be excluded at each concentration level. Table 7. Analytical screening results for samples compared only to a single 0.5 μg/kg extracted matrix calibrant ( n = 28 a ; 14 laboratories with duplicate samples at each concentration level) Samples with peak area response >0.5 µg/kg calibrant, % MG LMG CV LCV BG Salmon Negative control 0 0 0 0 0 Spike level 0.42 µg/kg 7 0 7 4 18 Spike level 0.90 µg/kg 100 100 100 100 100 Spike level 1.75 µg/kg 100 100 100 100 100 Incurred 100 100 0 b 0 b 100 Catfish Negative control 0 0 0 0 0 Spike level 0.42 µg/kg 14 11 5 32 25 Spike level 0.90 µg/kg 79 100 100 100 96 Spike level 1.75 µg/kg 100 100 100 100 100 Incurred 100 100 4 b 100 100 Shrimp Negative control 0 0 0 0 0 Spike level 0.42 µg/kg 31 0 21 4 23 Spike level 0.90 µg/kg 96 100 100 100 100 Spike level 1.75 µg/kg 100 100 100 100 100 Incurred 96 c 100 100 100 100 a Data excluded in the case of reported cause and for calibration curve nonlinearity; statistical outliers were not excluded. b Mean concentrations of incurred samples were <0.4 µg/kg (0.03 for CV in salmon, 0.15 for CV in catfish, and 0.39 for LCV in salmon). c Mean concentration found for MG incurred shrimp was 0.71 µg/kg. ), repeatability RSDs (RSD r Overall, the analytical results in all matrixes were excellent for the test samples fortified at 0.42, 0.90 and 1.75 µg/kg, as can be seen in Table 3. Trueness ranged from 88 to 108% recovery for analytes in all matrixes except for MG in catfish, which yielded lower recoveries (78–79%). RSD r values were generally ≤10%, except in the case of low level incurred samples (CV in salmon and catfish). HorRat values were uniformly very low (<1). The sole exception was CV incurred salmon, with a Analyte Quantification

Made with FlippingBook flipbook maker