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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.
Candidates for 2016 Method of the Year
291