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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,