INVESTIGADORES
RODRIGUEZ fernanda Mariana
congresos y reuniones científicas
Título:
High performance liquid chromatography of aflatoxin B1 in human serum
Autor/es:
LOPEZ CLARA EDER; RAMOS LAURA; GARCIA C; GIOLITO Y; YUJNOVSKY FABIANA; BULACIO LUCIA; RODRIGUEZ FERNANDA
Lugar:
Salsomaggiore Terme, Parma
Reunión:
Congreso; Congress of International Society for Human and animal Mycology; 1997
Resumen:
Aflatoxin B1, the most toxic of the aflatoxins, causes a variety of adverse effect in different animals species. These include liver damage, impaired productivity and reproductive efficiency; increase susceptibility to disease, etc. In recent studies of human carcinogenicity, naturally occurring mixtures of aflatoxins and aflatoxin B1 were classified as class 1 human carcinogens. It is clear that exposure to aflatoxins is hazardous to human health. In order to facilitate investigations on the role of ingested aflatoxin in human disease a sensitive method for detection of aflatoxin B1 in small volumes of human sera is required. Blood samples were drawn in fast conditions from 13 voluntary persons wit hepatic pathologies. Serum were separated by centrifugation at 3000 rpm and kept at -20C until analysis. Initially, 1 ml hexane was added to 10 ml serum and gently mixed for 2 min, the mixture was centrifuged for 5 min at 2000g and the upper hexane layer containing the serum lipids, removed. This procedure was repeated twice with further 1 ml aliquots of hexane, and hexane layer removed after each centrifugation. The serum was then extracted 4 times with 1 ml aliquots of chloroform, by addition of 1 ml of chloroform to the serum, vigorous shaking for 4 min, centrifugation for 10 min at 2000g and removal of the chloroform layer. The chloroform extract were pooled, evaporated to dryness under a stream of nitrogen. Sample residues were taken to 200ul of a mixture prepared with chloroform/methanol (1:1), mixed by rotation during 2 min and transferred to micrivials. 20 ul were injected onto the chromatograph. Aflatoxin B1 was measured in a Hewlett Packard HPLC series 1050 with auto-sampler and fluorescence detector. Chromatograph conditions were reverse-phase Hypersil C18 15 cm x 4.6, 5 um column, column temperature was 40C, fluorescence detection Ex=366 nm, Em=418nm, mobil phase was methanol-water-acetonitrile (25:25:50) and a flow rate of 0.8 ml/min. The complete run took 5 min. Spiked serum samples were prepared by adding known amounts of aliquots of standard Aflatoxin B1 to make a calibration curve with concentration of 100, 200, 400 and 800 pg/ml and processed accordingly. The chromatograms for 13 unknown samples shown a positive result for one person and the serum concentration of AFB1 was found to be 0.47 ng/ml. No interference peak was detected in the negative samples. The detection limit was better tan 250 pg/ml. The recovery percentage of AFB1 ranged from 61%-84% and the mean coefficient variation was 7.1.