S chneider & A ndersen : J ournal of AOAC I nternational V ol . 98, N o . 3, 2015  661

and LCV-D6). To prepare each of these solutions, combine 1 mL of each individual stock solution required for the mixture and dilute to 100 mL final volume with acetonitrile. These solutions are stored in glass at –20°C and protected from light (stated stability = 1 month). ( c )  Working standard solutions .—Prepare five to six working standard solutions. Six will be described here (WS 1–6). These are prepared by diluting aliquots (0, 50, 100, 200, 500, and 1000 µL, respectively) of the mixed intermediate standard solution of analytes to a final volume of 10 mL with acetonitrile. The resultant working standard solutions thus contain 0, 5, 10, 20, 50, and 100 µg/L, respectively, of the analytes. Prepare a working standard solution of internal standards by taking 400 µL of the mixed intermediate internal standard solution and diluting to a final volume of 10 mL with acetonitrile (final concentration 40 μg/L). Prepare all working standard solutions daily. These solutions may be kept at room temperature but protected from light. F. Sample Preparation ( a )  Homogenization of samples .—Homogenize muscle tissue with dry ice in a food processor to produce a finely ground powder. Allow the dry ice to sublime at –20°C and then store the homogenized tissues at –80°C. Homogenize salmon with attached skin, but for catfish, homogenize only the skinless filet. Remove shells, legs, and heads from shrimp prior to homogenization. ( b )  Extracted matrix calibrant samples.— Accurately weigh 2.00 g (±0.02 g) portions of homogenized negative control tissue into each of six 50 mL disposable centrifuge tubes. Once thawed, fortify these samples (extracted calibrants 1–6) with 100 µL aliquots of WS1, WS2, WS3, WS4, WS5, and WS6, respectively. To each tube then add 100 μL internal standard working solution. The extracted matrix calibrant samples are thus fortified with 0, 0.25, 0.5, 1.0, 2.5, and 5.0 µg/kg of analytes and 2.0 µg/kg of internal standards. Allow calibrants to equilibrate 15 min protected from light before beginning the extraction with the addition of hydroxylamine solution. [ Note : Method 2012.25 specifies that five extracted matrix calibrants are prepared in the range 0 to 2 µg/kg with concentrations 0, 0.5, 1.0, 1.5, and 2.0 µg/kg of analytes (8). For the collaborative study, the range was extended from 0 to 5 µg/kg to ensure that residues found in incurred samples would fall within the calibration range.] ( c )  Extraction of samples .—Accurately weigh 2.00 g (±0.02 g) portions of homogenized tissue into 50 mL disposable centrifuge tubes and let thaw. Fortify thawed tissue with 100 μL internal standard working solution (2.0 µg/kg), and allow samples to equilibrate for 15 min while protected from light. Add hydroxylamine solution (9.5 g/L, 500 μL) to the samples, vortex mix briefly, and allow samples to stand in the dark for 10 min. Add acetonitrile (8 mL) and 1.0 g (±0.1) anhydrous magnesium sulfate to each tube. Vortex mix tubes (1 min, maximum speed), then shake tubes (10 min) using a rotary stirrer or a multitube vortexer. Centrifuge the tubes (2000 × g , 5 min, 4°C), and transfer all supernatant to a clean tube for evaporation. Evaporate the supernatant to dryness (50°C, N 2 ). For the salmon matrix, the point of dryness may be a viscous oil. Reconstitute the extracted matrix calibrant samples and test samples with 800 μL Reconstitution Solution.

Vortex mix all samples sufficiently to break up dried extracts; for example, vortex mixing on high speed for 30 s followed by 10 min of mixing on a multitube vortexer ensures complete dissolution of analytes and internal standards. Transfer extracts to microcentrifuge tubes, centrifuge at 20 000 × g for 5 min, and filter (PVDF, 0.45 μm) into autosampler vials for LC-MS/MS analysis. The extraction results in a 2.5X concentration factor; therefore, a calibrant or sample fortified at 1.0 µg/kg in the seafood matrix will produce an extract with an equivalent concentration of 2.5 µg/L in the LC vial. or comparable column is used, with or without a guard column. The mobile phase was made up of ammonium formate buffer (A, 0.05 M, pH 4.5) and acetonitrile (B). The gradient program is described in Table 1. The flow rate is 250 μL/min, the injection volume is 20 μL, and the column oven is set to 30°C. Potential carryover, particularly from CV can be reduced by injection of water between each test sample. ( b ) Triple quadrupole MS .—A Waters Corp. Quattro LCZ triple quadrupole, or comparable instrument is used. The mass spectrometer is operated in the positive ion mode using electrospray ionization. Two SRM transitions are collected for each analyte and one SRM transition is collected for each internal standard; these transitions are detailed in Table 2, along with instrument parameters for the Waters Quattro LCZ system. Conditions are optimized so that all SRM transitions for the lowest concentration solvent calibrant are present with an acceptable S/N (≥3). The method can be used to screen test samples against a single calibrant or to quantify samples using a full calibration curve. A screen is accomplished by extracting test samples along with a negative matrix control sample and a matrix sample fortified at 0.5 µg/kg. Concentration of the test sample is estimated by comparison of the quantification ion peak area ratio of sample:internal standard with the corresponding ratio for the fortified matrix sample. To confirm suspected positive samples, test samples should be extracted and analyzed in duplicate along with a range of fortified calibrants (including negative control). Test samples are then quantified using the calibration curve according to the quantification method described below in Section I (8). G. LC-MS/MS Analysis ( a )  LC .—A Waters Corp. Symmetry C 18 H. Screening

Table 1. LC elution gradient Time, min A, %

B, % (acetonitrile)

(ammonium formate buffer)

0 1

60 10 10 60 60

40 90 90 40 40

15 16 20

Made with FlippingBook flipbook maker