Liquid chromatography electrospray-mass spectrometry of urinary aflatoxin biomarkers: Characterization and application to dosimetry and chemoprevention in rats

A liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the measurement of aflatoxin biomarkers in urine has been developed and validated. The two major aflatoxin-DNA adducts formed in rat tissues, aflatoxin N⁷-guanine and its imidazole ring opened derivative, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-aflatoxin B₁, were detected and quantified in urine by the LC-ESI-MS/MS technique. Other metabolites derived from the conjugation and/or oxidation of aflatoxin B₁ measured in the urine of dosed rats included aflatoxin P₁, aflatoxin P₁-glucuronide, aflatoxin Q₁, aflatoxin M₁, 8,9-dihydro-8,9-dihydroxy aflatoxin B₁, aflatoxin B₁-mercapturic acid, the aflatoxin-cysteine glycine adduct derived from the aflatoxin-glutathione conjugate, aflatoxin M₁P₁ and the aflatoxin B₁-dialcohol. For in vivo studies to determine the dosimetry of certain aflatoxin metabolites, aflatoxin B₂ was used as an internal standard for recovery since this compound is not naturally produced in rats. In the final method using the internal standard, the coefficient of variation of six replicate analyses of in vivo rat urine samples for aflatoxin N⁷-guanine, aflatoxin B₁-mercapturic acid, and aflatoxin M₁ was 12.5, 12.8, and 5.8%, respectively. Further, the LC-ESI-MS/MS method to detect aflatoxin N⁷-guanine in in vivo rat urine samples was at least 20-fold more sensitive than prior techniques. Using the LC-ESI-MS/MS technique, the dosimetry, on a weekly basis, of major urinary aflatoxin metabolites was assessed in animals chronically dosed over a 5-week period. Of particular importance was the application of this method to determine the modulation of levels of urinary aflatoxin metabolites by treatment with oltipraz, a chemopreventive agent that can completely ablate aflatoxin hepatocarcinogenesis in the rat. After 1 week, oltipraz administration diminished urinary aflatoxin N⁷-guanine, aflatoxin B₁-mercapturic acid and aflatoxin M₁ levels by 83, 92, and 82%, respectively. The magnitude of this reduction was persistent at the day 14, 21, 28, and 35-day time points with the average decrease of aflatoxin N⁷-guanine, aflatoxin B₁-mercapturic acid and aflatoxin M₁ being 73, 92, and 90%, respectively. Importantly, even under circumstances where the oltipraz intervention was most efficient in reducing aflatoxin metabolite levels, the LC-ESI-MS/MS method was still sensitive enough to detect the reduced biomarker content. This outcome has important translational implications for the application and analysis of the efficacy of primary and secondary prevention interventions in human populations where ambient exposure levels are low, but the toxicologic hazards of these exposures remain high.