Fluorescence quantification of aflatoxin N7-guanine adducts.
Cancer Epidemiol Biomark Prev 1994 Dec; 3(8):669-674
A method for determining aflatoxin N7-guanine adducts in urine was developed. The hydrolysis procedure was based on 8,9-dihydro-8-(N7- guanyl)-9-hydroxy-aflatoxin-B1 (AFB1-N7-Gua), the N7-guanine adduct of aflatoxin-B1 (1162658) (AFB1). The hydrolysis reaction involved the acidic cleavage of the AFB1-N7-Gua bond for the production of fluorescent derivatives. Urine samples were obtained from two male F344-rats which received a 0.8mg/kg AFB1 injection, and purified by passage through a C18 Sep-Pak cartridge followed by a monoclonal antibody immunoaffinity column. The samples were then injected into a high performance liquid chromatography (HPLC) system utilizing a fluorescence detector set at 365 nanometers (nm) for excitation and 428nm for emission to separate and collect the AFBG peak. The eluate was treated with concentrated hydrochloric-acid at 95 degrees- C for 60 minutes. This hydrolyzed the AFB1G to 8,9-dihydro-8,9- dihydroxyaflatoxin-B1 (AFB1diol). The samples were then quantitated by HPLC with fluorescence detection. Calibration plots of urine samples spiked with AFB1diol standards were linear over the range 39 to 5,000 picograms (pg) hydrolyzed AFB1-N7-Gua. The detection limit was 39pg. The precision obtained for four replicate determinations of urine samples spiked with 39 to 5,000pg AFB1diol expressed as the relative coefficient of variation averaged 11.8%. A well resolved fluorescent peak corresponding to AFB1diol was found. Experiments leading to optimization of the procedure including investigating synchronous fluorescence spectrophotometry (SFS) as an alternative technique were described. The HPLC method was found to be more sensitive than SFS. The authors conclude that the new procedure can detect AFB1G at concentrations that are 100 times lower than available with previously used methods.
NIOSH-Publication; NIOSH-Grant; Training; Mycotoxins; Analytical-methods; Chromatographic-analysis; Neoplastic-agents; Urinalysis; Sample-preparation; Hydroxylation-reactions; DNA-adducts; Fluorescence-spectrometry; In-vivo-studies; Laboratory-animals; Biochemical-indicators;
Environmental Health Sciences Johns Hopkins University 615 N Wolfe Street Baltimore, MD 21205
Cancer Epidemiology, Biomarkers & Prevention
Johns Hopkins University, Baltimore, Maryland