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To quantify metals in biological samples, we tried to find good conditions for wet-digested mineralization of the samples and for separation of metal ions in chromatography. A 500 microliters volume of aliquot was transferred to a glass tube, and was evapolated at 100 degrees C for 2 hours. A 5.5 ml volume of a mixture of concentrated nitric acid-70% perchloric acid (10: 1, v/v) was added and heated, consecutively, at 80 degrees C for 12 hours, at 140 degrees C for 2 hours, at 180 degrees C for 2 hours, and finally at 190 degrees C for 1 hour to evapolate the residual acids. After addition of 500 microliters of 10 mM nitric acid, the metals were extracted by a suspension mixer (32 r.p.m., 1 hour). One hundred microliters of the extracted solution was applied to the chromatographic system: cation-exchange column, TSKgel IC-Cation SW (Tosoh Co.); eluent, 0.35 M lactic acid-0.35 M sodium lactate (pH 3.0); flow rate, 0.7 ml/minute; column temperature, 30 degrees C. After adding a color-forming reagent (100 mg/l 4-(2-pyridylazo)-resorcinol in 40 g/l Na2CO3; flow rate, 0.7 ml/minute) to the effluent, five different metal ions of Cd2+, Co2+, Cu2+, Ni2+ and Zn2+ were detected at 520 nm. The peaks were separated in approximately 25 minutes, and were quantified even at the 1-10 ppb levels. The present procedures were considered to provide simultaneous detection and accurate quantitation of the above five metals in the biological samples.

journal of toxicological sciences

Application of biological samples treated by wetdigested mineralization to cation-exchange high-performance liquid chromatography with postcolumn reaction by 4-(2-pyridylazo)-resorcinol.