Quantification of aflatoxin-B1-N7-guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry

We report validation of the first isotope dilution mass spectrometry method for determination of aflatoxin B-1-N-7-guanine (AFB(1)-N-7-Gua), a major human aflatoxin - DNA adduct that is excreted in the urine. Measurement of urinary AFB(1)-N-7-Gua, a biomarker of the biologically effective dose following dietary aflatoxin B-1 (AFB(1)) exposure, has helped identify AFB(1) as a risk factor in the development of hepatocellular carcinoma, a common cancer worldwide. Triple-quadrupole mass spectrometry, coupled with the use of a stable isotope-labeled internal standard (AFB(1)-N-7-N-15(5)-Gua) and better solid phase extraction and immunoaffinity column chromatography, have enabled us to greatly improve accuracy, precision, specificity, and sensitivity over previously published determinations. The limit of quantitation for AFB(1)N(7)-Gua was 0.8 pg/20 mL urine (0.07 pg/mg creatinine). The method was validated for accuracy and precision over the range of 0.8-25 pg/20 mL urine, with between-day and within-day reproducibility for analysis of six aliquots of a human urine sample containing 6.0 pg/20 mL measured at < 6% coefficient of variation. AFB(1)-N-7-Gua concentrations were measured in 20 human urine samples collected in a region with known aflatoxin exposure. The mean concentration of AFB(1)-N-7-Gua, measured in 16/20 urine samples with levels above the method's limit of quantitation, was 2.9 pg/20 mL urine (0.28 pg/mg creatinine) with a range of < 0.8-7.2 pg/20 mL urine (0.04-65 pg/mg creatinine). With improved accuracy and precision, this sensitive biomarker for recent human exposure to AFB1 will be especially useful for measuring the efficacy of planned interventions to reduce aflatoxin-related liver cancer in AFB(1)-exposed populations.