Validation of a technique by Chromatography High Resolution Liquid for determination quantitative hemin in QF-012

Abstract

Hemin, Fe(III) protoporphyrin IX, is a model for the hemoglobin,
myoglobin, cytochromes and other enzymes, of which is the prosthetic group. Hemin
complexes are also medically and physiologically important compounds for the
treatment of acute porphyria attacks and correct the iron deficiency in those cases
when there are problems in the iron oral absorption. During the development of
QF-012, a new hemin complex, a simple, rapid and convenient technique was
needed for both the qualitative and quantitative determination of hemin. A
High-Performance Liquid Chromatographic technique was developed and validated
for the determination of hemin in QF-012. This method used a LiChrosorb
RP-18 column and the mobile phase is 0,04 % of piridine in acetonitriletrifluoracetic
acid 0,3 % [55 : 45 (v/v)]. Detection was at 400 nm . The method
was selective, even when degradation products were present. It can be seen
that the degradation products elutes separately and was detected without apparent
interference with the peak of interest. The calibration curve in the range
from 10,0 to 60.0 μg/mL was lineal, with a coefficient of correlation equal to
0.994. The intercept and slope statistical test was not significant. The variation
coefficients in the repeatability and reproducibility were less than 2 %.
No significant statistical differences were found between the repeatability and
reproducibility.