61 / 71
660
S
chneider
&
A
ndersen
:
J
ournal of
AOAC I
nternational
V
ol
. 98, N
o
. 3, 2015
instrument conditions used, any variations in the experimental
procedure, raw data reports provided by the instrument, and
chromatograms for all transitions monitored for each analyte
and internal standard in all samples.
AOAC Official Method 2012.25
Residues of Three Triphenylmethane Dyes
and Their Metabolites (Malachite Green,
Leucomalachite Green, Crystal Violet,
Leucocrystal Violet, and Brilliant Green) in
Aquaculture Products
Liquid Chromatography/Tandem Mass Spectrometry
First Action 2012.25
[Applicable for the determination and confirmation of MG,
LMG, CV, LCV, and BG in fish and shrimp muscle.]
Caution:
Triphenylmethane dyes and leuco metabolites
are toxic and known or suspected mutagens, carcinogens,
and/or teratogens. Refer to Material Safety Data Sheets before
handling any chemicals, wear safety glasses and appropriate
personal protective equipment, and dispose of waste in an
environmentally responsible manner in accordance with
pertinent regulations.
A. Principle
Triphenylmethane dyes and their leuco metabolites, in
the presence of hydroxylamine and anhydrous magnesium
sulfate, are extracted from salmon, catfish, and shrimp tissue
with acetonitrile. After evaporation of the extract, the residue
is redissolved in acetonitrile/ascorbic acid and then analyzed
using LC-MS/MS. Quantitative analysis uses extracted matrix
calibrants and four isotopically labeled internal standards to
correct for matrix effects and extraction losses.
B. Apparatus
(
a
)
Vortex mixer
(
b
)
Rotary stirrer
.—Set to 100 rpm or multitube vortexer
(platform shaker) set to 2500 rpm, or equivalent.
(
c
)
Centrifuge
and tubes
.—Capable of accelerating 50 mL
polypropylene centrifuge tubes (or equivalent) to 2000 ×
g
and
refrigerated to 4°C.
(
d
)
Transfer pipets
.—Disposable.
(
e
)
Nitrogen evaporator
.—Capable of heating sample tubes
to 50°C.
(
f
)
Evaporation tubes
.—10–15 mL polypropylene tubes, or
equivalent.
(
g
)
Microcentrifuge
and tubes.—
Capable of accelerating
microcentrifuge tubes containing 800 μL of volume to
20000 ×
g
.
(
h
)
PVDF syringe filters
and syringes
.—0.45 μm, 13 mm
Millex-HV (EMD Millipore Corp., Billerica, MA) and 1 mL
disposable syringes, or equivalent.
(
i
)
Autosampler vials
.—Glass or polypropylene, with caps.
Amber colored vials recommended to protect light sensitive
compounds.
(
k
)
LC-MS/MS system
.—HPLC system equipped with pump,
solvent degasser, autosampler, and column oven (Waters Corp.
2695, Agilent 1200 series, or equivalent). Triple quadrupole
mass spectrometer system equipped with an electrospray
ionization source for operation in the positive ion mode and
capable of selected reaction monitoring (SRM) with at least two
transitions/analyte and one transition/internal standard (Waters
Corp. Quattro LCZ, Agilent 6490, or equivalent).
(
l
)
LC column
.—C
18
stationary phase (100×2.1 mm,
3.5 μm) with C
18
guard column (10 × 2.1 mm; Waters Corp.
Symmetry), or equivalent.
C. Reagents
Note
: All reagents should be, analytical HPLC or LC-MS
grade
(
a
)
Acetonitril
e.
(
b
)
Hydroxylamine hydrochloride.
(
c
)
Magnesium sulfate, anhydrous.
(
d
)
Ammonium formate.
(
e
)
Ascorbic acid.
(
f
)
Formic acid.
(
g
)
Water
.—Deionized, distilled.
(
h
)
MG oxalate
.—CAS No. 2437-29-8.
(
i
)
LMG
.—CAS No. 129-73-7.
(
j
)
CV chloride
.—CAS No. 548-62-9.
(
k
)
LCV
.—CAS No. 603-48-5.
(
l
)
BG
.—CAS No. 633-03-4.
(
m
)
D5-MG picrate
.—CAS No. 1258668-21-1.
(
n
)
D5-LMG
.—CAS No. 947601-82-3.
(
o
)
D6-CV trihydrate.
(
p
)
D6-LCV
.—CAS No. 1173023-92-1.
D. Preparation of Reagent Solutions
(
a
)
Hydroxylamine
solution
in
water
(9.5
g
hydroxylamine/L)
.—Dissolve
5.0
g
hydroxylamine
hydrochloride in deionized water and dilute to a final volume
of 250 mL.
(
b
)
Ascorbic acid solution in water (1 g/L)
.—Dissolve
100 mg ascorbic acid in deionized water and dilute to a final
volume of 100 mL.
(
c
)
Reconstitution solution
.—Combine 1 mL ascorbic acid
solution (1 g/L) with 100 mL acetonitrile and mix.
(
d
)
Formic acid solution in water (5%, v/v)
.—Add 5 mL
concentrated formic acid to approximately 90 mL deionized
water and dilute with deionized water to a final volume of
100 mL.
(
e
)
Ammonium formate buffer (0.05 M, pH 4.5)
.—Dissolve
3.15 g ammonium formate in approximately 900 mL deionized
water. Then add 5 mL formic acid solution (5%, v/v) and dilute
with deionized water to a final volume of 1000 mL.
E. Preparation of Standard Solutions
(
a
)
Stock standard solutions
.—Prepare individual stock
solutions of each dye, metabolite, and internal standard
compound at a concentration of 100 μg/mL in acetonitrile,
taking into account the purity and presence of counterions.
Store all solutions in glass at –20°C and protect from light
(stated stability = 1 year).
(
b
)
Mixed intermediate standard solutions
.—Prepare mixed
intermediate standard solutions (1.000 μg/mL each compound)
for the analytes (containing MG, LMG, CV, LCV, and BG) and
the internal standards (containing MG-D5, LMG-D5, CV-D6,