1993 Volume 22 Issue 4 Pages 252-257
We describe a method using high-performance liquid chromatography (HPLC) for the simultaneous determination of uroporphyrin (UP)-I and -III in urine and whole blood. HPLC was performed on a reversed-phase (Zorbax-ODS) column with fluorometric detection (Ex 394nm/Em 624nm) and with a column switching system. Mobile phase (1mol/l ammonium acetate buffer, pH 5.2: acetonitrile, 865: 135, v/v) was isocratically eluted at a flow rate of 0.8 ml/min. The within-assay reproducibilities (CV) for urine and whole blood were 1.6-2.0% (n=21) and 5.8-5.9% (n=6), respectively.
Between-day reproducibilities were 4.9-5.7% (n=7) for urine and 9.4-9.9% (n=6) for whole blood. The calibration curves for UP-I and -III were linear over the range of 2-500ng/ml. The rates of recovery of UP-I and -III in urine and whole blood were 100-112% (1-160ng/ml) and 91-94% (2-40ng/ml), respectively. The lower limit of detection of UP-I or -III in urine or whole blood was estimated to be 2ng/ml.
In conclusion, the present method is simple and quick. Moreover, precision, accuracy and sensitivity are allowable for the routine clinical examination.
