An Effect-Driven Fractionation Approach for the Isolation and Characterization of CYP1A Inducing Components of Crude Oils
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Exposure to crude oils has been shown to induce CYP1A enzymes and cause chronically toxic effects in aquatic organisms. Earlier studies indicated that polycyclic aromatic hydrocarbons (PAHs) present in crude oil are primarily responsible for the chronic toxicity. Crude oil contains a variety of PAHs; the majority of them are alkyl substituted. In this work, we have used an effects-driven fractionation and analysis approach (EDFA) to isolate and characterize PAHs present in Alaskan North Slope and Scotian Light crude oils that are toxic to fish. The crude oil components were first fractionated into four fractions using a low temperature vacuum distillation technique. Among them, the heavy gas oil fraction (boiling range 287°C – 461°C) of both oils caused highest toxicity to fish. To isolate the PAHs from waxes present in this fraction, a low temperature wax precipitation method was developed and optimized. CYP1A induction results showed that the extract contained a large number of CYP1A inducers while the residue contained none. Chemical analyses confirmed that most of the PAHs were partitioned into the extract fraction. Alkyl PAHs present in the extract were further fractionated into five fractions based on the number of aromatic rings using a normal phase HPLC method. Chemical analysis and the toxicity testing of these fractions indicated that alkyl PAHs belonging to classes such as phenanthrene, fluorene, naphthobenzothiophene, and chrysene are likely responsible for the observed toxic effects. To aid the EDFA scheme, a new HPLC-DAD method for the analysis of alkyl PAHs was developed. The alkyl PAHs were first fractionated based on the number of aromatic rings using a normal phase column followed by their analysis using reverse phase HPLC–DAD technique. The reverse phase analysis involved classifying the alkyl PAH peaks into different PAH classes based on their DAD spectra. Then, alkyl carbon numbers for each peak were assigned based on their retention time. To analyze co-eluting alkyl PAH isomers an offline multi-dimensional HPLC method was developed. Orthogonal separation was achieved by first fractionating the alkyl PAHs on a normal phase column followed by the RP-HPLC-DAD analysis. Using these data a 2D contour plot was developed and used for the detailed analysis of alkyl PAHs isomers. Analysis results showed good agreement with a gas-chromatography-mass-spectrometric (GC-MS) analysis method, and the new method was able to distinguish some PAH types which could not be identified by GC-MS.