Rapid analysis of porphyrins at low ng/l and μg/l levels in human urine by a gradient liquid chromatography method using octadecylsilica monolithic columns

Abstract

Rapid gradient RP-HPLC method with fluorimetric detection for trace analysis of diagnostically significant porphyrins in human urine was developed for clinical and diagnostic purposes. Results show that optimized high-pressure gradient elution and monolithic column Chromolith SpeedRod RP18e enabled separation of seven urine porphyrins including baseline separation of I and III positional isomers of uro- and coproporphyrins within 3.2 min. Problems associated with high metal cation complexing ability of the analytes and common stainless steel based instrumentation were substantially reduced by use of 0.1 mol/l ammonium citrate buffer (pH 5.47) and methanol as a mobile phase components. Good reproducibilities of retention times (within ±0.36% RSD) and peak areas (from ±0.6 to ±2.5% RSD) at 5–20 μg/l level of the analytes were achieved. Determined LOQ (10 × S/N) values of diagnostically important porphyrins using fluorimetric detection (ex.405 nm/em.620 nm) were 82 pmol/l (65 ng/l, 1.30 pg/injection) for uroporphyrin I, 44 pmol/l (33 ng/l, 0.66 pg/injection) for uroporphyrin III, 50 pmol/l (40 ng/l, 0.80 pg/injection) for coproporphyrin I and 47 pmol/l (39 ng/l, 0.78 pg/injection) for coproporphyrin III. Attained LOQ concentration level is approximately 20–120 times lower than concentration of porphyrins in a urine of healthy person. Calculated LOD's (3 × S/N) were at a low ng/l levels, what enabled quantification of carry-over effect to be from 2.0% to 0.2% in each of three consecutive blank runs and from 2.5% to 7% in total after injection of mixed standard of porphyrins with 5–20 μg/l concentrations. Recovery of porphyrins at low μg/l concentration levels was from 93% to 97.5%. Devised method increases productivity of clinical laboratory from 2 to 10 times in dependence of duration of currently used method.