Abstract: This paper reports the separation and determination of iron (Ⅱ), cobalt (Ⅱ) and nickel (Ⅱ) by RP-HPLC with 1-(2-pyridylazo)-2-6-sulphonic acid (PAN-S)as a precolumn derivatization reagent. On Nova-PakTM C18 column, methaol-water (50:50,V/V) containing 10 mmol/L acetic acid-sodium acetate buffer (pH=5) was used as mobile phase. Flow rate of the mobile phase was 1.0mL/ min and the wavelength for detecion was 550um. Under there conditions Fe(Ⅱ), Co(Ⅱ) and Ni(Ⅱ) were separated and determined, within 11min. A new method for rapid separation and determination of trace iron, cobalt and nickel by reverse phase ion-pair HPLC is thus proposed. When signal noise ratio was 2, detection limit of Fe(Ⅱ), Co(Ⅱ) and Ni(Ⅱ) were 0.043, 0.007 and 0.012μg/mL respectively. This method has ho applied to determine trace Fe, Co and Ni simultaneously in hydrochloric add, nitric acid and a the Sample with satisfactory results.