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 B₁-N⁷-guanine (AFB ₁-N⁷-Gua), a major human aflatoxin-DNA adduct that is excreted in the urine. Measurement of urinary AFB₁-N⁷-Gua, a biomarker of the biologically effective dose following dietary aflatoxin B₁ (AFB₁) exposure, has helped identify AFB₁ 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 (AFB₁-N⁷- ¹⁵N₅-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 AFB₁-N⁷-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. AFB₁-N⁷-Gua concentrations were measured in 20 human urine samples collected in a region with known aflatoxin exposure. The mean concentration of AFB₁-N⁷-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 AFB₁-exposed populations.