Analysis of aflatoxin B1‐lysine adduct in serum using isotope‐dilution liquid chromatography/tandem mass spectrometry

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Abstract
A method for quantitative analysis of aflatoxin B1‐lysine adduct (B1‐Lys) in serum by liquid chromatography using tandem mass spectrometry (LC/MS/MS) is presented. The protein in a 250‐µL sample was digested in the presence of a stable‐isotope internal standard during a 4‐h incubation at 37°C with Pronase™. B1‐Lys and the internal standard were extracted using mixed‐mode solid‐phase extraction cartridges and eluted with 2% formic acid in methanol. Following evaporation and reconstitution, extracts were injected onto a Luna C‐18(2) column and eluted with a step gradient of acetonitrile and 0.06% formic acid. The B1‐Lys and the internal standard were detected in a positive ionization selective reaction monitoring mode with a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer. Calibration curves were linear for concentrations from 0.05–8.0 ng/mL. The method was validated with aflatoxin B1 dosed rat serum diluted to anticipated high and low concentrations. Total imprecision determined from 30 measurements over 15 days was 5.6% and 9.1%, respectively. Recoveries of 78.8 ± 6.4% for B1‐Lys and 85.4 ± 12.4% for the internal standard were based on the full extraction and reconstitution processes. The method can be used to quantitate B1‐Lys at the 0.5 pg/mg albumin level and is suitable for routine analysis. Copyright © 2005 John Wiley & Sons, Ltd.