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. ( 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 B. Apparatus

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




hydroxylamine/L) .—Dissolve 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, 5.0 g

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