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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
Ist Teil von
Journal of Chromatography A, 2005-08, Vol.1084 (1), p.24-32
Ort / Verlag
Amsterdam: Elsevier B.V
Erscheinungsjahr
2005
Link zum Volltext
Quelle
Alma/SFX Local Collection
Beschreibungen/Notizen
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.