Quantification of coproporphyrin isomers I and III in urine by HPLC and determination of their ratio for investigations of Multidrug Resistance Protein 2 (MRP2) function in humans

We describe here the development of a high-performance liquid chromatography (HPLC) method for quantitative determination of the ratio of isomers I and III of urinary coproporphyrin [the UCP I/(I + III) ratio], which is used for the diagnosis of Dubin–Johnson syndrome (DJS). This technique could also be used for research applications, such as investigations of the function of Multidrug Resistance Protein 2 (MRP2) in humans. Chromatographic separation was achieved on a reverse-phase C18 Symmetry® column (5 μm; 4.8 mm × 250 mm), using a mobile phase consisting of a mixture of acetonitrile and acetate buffer (0.015 M, pH 4), with fluorescence detection based on excitation at 365 nm and emission at 624 nm. The method was validated over a concentration range of 10–400 nmol/l for UCP I and 30–560 nmol/l for UCP III, yielding calibration curves with correlation coefficients greater than 0.998. The lower limit of quantification (LLOQ) was 7 nmol/l for UCP I and 10 nmol/l for UCP III. Inter- and intra-day precision (CV < 5%) and accuracy (95–99%) complied with ICH guidelines. We also demonstrated that samples could be stored for 3 days at +4 °C and for 12 months at −20 °C with no change in UCP ratio (CV < 5%), providing a basis for storage recommendations for future clinical studies based on this analysis. Our method is simple, rapid and universal and is suitable for quantitative determinations of each isomer and their ratio for routine and research purposes.