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664 

S

chneider

&

A

ndersen

:

J

ournal of

AOAC I

nternational

V

ol

. 98, N

o

. 3, 2015

the composition of acetonitrile from 90 to 40% from 6–7 min

and held at 40% for a total run time of 12 min. Compared to

Method

2012.25

(20 min run time), overall chromatographic

run times for the six laboratories that adjusted the mobile phase

ranged from 7 to 25 min. Many participants made adjustments

to the injection volume, with eight laboratories reducing the

injection volume from 20 to 2, 3, 5, or 10 μL. Two laboratories

reduced the column flow rate from 250 to 200 μL/min, one

increased flow rate to 300 μL/min, and three laboratories used

column temperatures of 25 or 35°C instead of the method stated

30°C. Twelve participants used the exact column specified in

the protocol, and the remaining laboratories used a Phenomenex

(Torrance, CA) Prodigy ODS-3 100 × 2 mm, 3 μm or a Waters

Corp. Atlantis dC

18

100×2.1 mm, 3.5 μm column. Most

participants used a guard column, although four did not.

Avarietyof triple quadrupolemass spectrometerswere usedby

the participating laboratories, includingAB Sciex (Framingham,

MA) 5500 QTrap (5), AB Sciex 4000 QTrap (2), AB Sciex

3000, Agilent 6490 (2), Agilent 6460, Thermo Scientific (San

Jose, CA) TSQ Vantage, Thermo Scientific Quantum Discovery

Max, Waters Corp. Micromass Quattro Micro API, Waters

Corp. Acuity TQD, and Waters Corp. Quattro Premiere XE. All

LC-MS/MS systems used in this study provided suitable data.

Participants were requested to optimize mass spectrometer

source and ionization parameters to yield acceptable response

for the desired analyte range. Two participants analyzed

extracts from each test sample on both Agilent and AB Sciex

systems. These laboratories obtained comparable study results

from their Agilent 6460/AB Sciex 4000 or Agilent 6490/AB

Sciex 5500 QTrap analyses, further illustrating the suitability

of this method for varied instrumentation. Mass spectrometric

transitions provided in the method worked well for most analytes

(Table 2). In the case of CV, some participants found that the

qualitative transition (

m/z

 372→251) provided a peak with low

signal. As a result, four participants used a substitute transition

for qualitative purposes (

m/z

372→340). One laboratory

inadvertently used this alternate transition for quantification

of the catfish and shrimp matrixes. Transition

m/z

372→340,

when used, provided acceptable results. Although MS/MS

parameters and retention times were listed in the First Action

method and in Table 2 for the metabolite leucobrilliant green

(LBG), this analyte was not specifically included in Method

2012.25

LC-MS/MS validation nor required for inclusion in

the collaborative study. Analytical standards of LBG are not

commercially available and have limited stability (12, 13).

Nevertheless, three of the participating laboratories submitted

data for the LBG transitions. None of the study samples were

fortified with LBG, but as the expected metabolite from BG

exposure, LBG may be present in incurred samples. On review

of the data from three laboratories, potential responses for the

LBG transitions for incurred salmon and catfish were not large

enough to be distinguished from nonincurred samples. Without

an LBG standard, MS/MS optimization and retention time

comparison could not be performed by these laboratories. The

First Action method authors detected the LBG metabolite in the

concentration range 8 to 18 μg/kg for trout placed in a 100 µg/L

Table 3. (

continued

)

Analyte Matrix

Analyte

added,

µg/kg

Statistical

outlier

lab No.

a

No of

labs

No. of

replicates

b

Trueness

(recovery, %)

Mean

concn,

µg/kg

s

r

s

R

RSD

r

,

%

RSD

R

,

% HorRat

Shrimp

0.42

None

14

28

104.8

0.44

0.03 0.03 6.48 7.81

0.15

0.90

None

14

28

106.7

0.96

0.04 0.06 3.83 6.11

0.13

1.75

None

14

28

102.3

1.79

0.08 0.12 4.36 6.52

0.16

Inc:1.18

None

14

28

98.3

1.16

0.04 0.06 3.39 5.40

0.12

BG Salmon

0.42

10

c

, 2

d

12

24

104.8

0.44

0.04 0.08 8.02 19.07 0.37

0.90

None

14

28

101.1

0.91

0.07 0.15 7.55 16.96 0.37

1.75

None

14

28

96.0

1.68

0.09 0.22 5.31 12.98 0.31

Incurred

None

14

28

1.48

0.12 0.31 8.21 20.75 0.49

Catfish

0.42

12

c

, 11

e

12

24

104.8

0.44

0.02 0.09 5.17 21.51 0.42

0.90

None

14

28

101.1

0.91

0.05 0.36 5.90 39.36 0.86

1.75

None

14

27

97.7

1.71

0.14 0.59 8.23 34.46 0.83

Incurred

2

e

13

26

1.07

0.05 0.25 4.98 23.66 0.53

Shrimp

0.42

None

13

26

107.1

0.45

0.04 0.14 9.57 30.92 0.61

0.90

None

13

26

107.8

0.97

0.10 0.28 10.13 28.90 0.64

1.75

None

13

26

99.4

1.74

0.18 0.70 10.57 40.48 0.97

Inc:1.5

None

13

26

102.7

1.54

0.07 0.42 4.31 27.57 0.65

a

Laboratory numbers were randomly assigned and do not correspond to the sequence listed on either the title page or in the

Acknowledgments

.

b

No. of replicates after removal of invalid data and outliers.

c

Outlier by the Cochran test.

d

Outlier by Grubbs 1 test.

e

Outlier by Grubbs 2 test.