Three chicken breasts were cut into six pieces of approximately 20 g each (18 pieces) and placed into the five marinades, leaving 3 unmarinated.  After marinating ten hours, chicken was placed directly on grill and cooked until chicken decreased in mass by approximately 60%.  A triple beam balance was used to determine mass reduction.  Chicken was removed from grill, mass was recorded, and labeled samples were placed into refrigerator.
       The samples were placed into cooled mortar and pestle, liquid nitrogen was added, and chicken was ground thoroughly to create a homogeneous mixture which was then placed into labeled jar.  To insure lack of cross contamination, the cutting board and knife were wiped with cleaning solution.
       Water was added to each sample to return to aqueous phase and then decantered into the labeled centrifuge tube.  0.5 mL of 5 M ammonium hydroxide was added to each sample in order to adjust the pH of the ground chicken to make it basic.  This allowed compounds to partition better into the ethyl acetate.  5 µL of a 666 µg/mL solution of D3 PhIP was added to each sample as well as 10 mL of ethyl acetate.  Ethyl acetate was used as a solvent and allowed compounds to separate from chicken for analysis.  The dissolved compounds in the ethyl acetate moved to the top because they are less dense than water, and the rest of the chicken remained below.  The vials were shaken thoroughly for thirty seconds using Vortex Genie 2 and allowed to settle for 1 minute. Samples were centrifuged at 3000 rpm for ten minutes using Eppendorf 5810R to aid phase separation.  2 x 1 mL aliquots of ethyl acetate were removed from the top portion of the chicken solution and placed into labeled plastic vials.  Samples were then taken to dryness at ambient temperature in SALVANT Speed-Vac centrifugal evaporator for two hours.  One set of the samples was reconstituted with 50 µL of water containing 0.1% formic acid and transferred to a LC autosampler vial with a low volume insert.  The other set was archived.
        The LC-MS/MS analysis was carried out on a 3200 QTrap LC-MS/MS system, Appled Biosystems MDS/Sciex and a C18 HPLC Column (Varian 50 x  2 mm) was used.  Samples were separated using a linear gradient elution program 5% methanol to 90% methanol in 15 min., at a flow rate of 0.25ml/min for twenty-five minutes.  The PhIP had a retention time of 4.5 min.  The mass spectrometer operated in MRM (multiple reaction monitoring) mode.  For PhIP the principal transition was m/z 225 to m/z 210 and for the deuturated analogm/z 228 to m/z 210.  The software program ANALYST graphed each sample and calculated area of peaks for PhIP and deuterium PhIP.  The ratio of the area of PhIP peak and deuterium peak was then determined.  This ratio and the known mass of the deuterium were used to calculate the mass of PhIP.  The mass of PhIP was divided by the mass of chicken after grilling to determine the number of micrograms of PhIP present in each gram of grilled chicken.




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