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S

chneider

&

A

ndersen

:

J

ournal of

AOAC I

nternational

V

ol

.

98, N

o

. 3, 2015 

667

Repeatability SDs (s

r

), reproducibility SDs (s

R

), repeatability

RSDs (RSD

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.

Analyte Quantification

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

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

1

 For CC

α

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.

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.