Determination of Aflatoxin B1-Lysine in Pig Serum and Plasma by Liquid Chromatography-Tandem Mass Spectrometry

Aflatoxin B-1 (AFB(1)) is a hepatocarcinogen produced by certain Aspergillus species growing on crops. After biotransformation in the liver, AFB(1) generates several metabolites, one of which is AFB(1) bound to lysine on serum albumin. AFB(1)-lysine (AFB(1)-lys) is a digest product of AFB(1)-albumin and is considered a biomarker of exposure to AFB(1) in humans and animals. The objectives of this paper were to evaluate the performance characteristics of a new analytical method for determination of AFB(1)-lys levels in pig serum, heparinized and ethylenediaminetetraacetic acid (EDTA) plasma and to evaluate the interference of these anticoagulants in AFB(1)-lys quantification. Blank blood samples were obtained from eight crossbreed 91-day-old barrows fed AFB(1)-free diets. Pooled samples (n = 3) and individual samples of serum, EDTA and heparinized plasma collected from five pigs were enzymatically digested with pronase at 37 degrees C for 4 h. AFB(1)-lys was isolated by solid-phase extraction and quantified by liquid chromatography coupled to tandem mass spectrometry. The analytical method was applied for determination of AFB(1)-lys in serum and EDTA plasma collected from five 49-day-old crossbreed barrows fed ad libitum diets containing 1.1 mg of AFB(1) per kg of feed during 7 days (three animals) or 42 days (two animals). Samples of heparinized plasma were only available from animals intoxicated for 42 days. All animals had lower levels of AFB(1)-lys in EDTA plasma samples (24.78-37.40 ng/mL), when compared to serum (49.32-252.07 ng/mL(-1)) or heparinized plasma (176.81 and 264.24 ng/mL(-1)). EDTA did not interfere in AFB(1)-lys standard detection, but our findings suggest that EDTA should be avoided during blood collection since it affects the pronase activity in AFB(1)-albumin adduct digestion and, consequently, causes a reduction in the AFB(1)-lys levels. Hence, determination of AFB(1)-lys in serum and heparinized plasma is an approach to assess an individual's exposure of swine to AFB(1).