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Determination of hydroxyproline using ion-exchange liquid chromatography with VIS detector and high performance liquid chromatography with fluorescence detector
Zuzana Lacková, Natalia Cernei, Dagmar Štěrbová, Yazan Haddad, Veronika Rozíková, Tomáš Komprda, Ondřej Zítka
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Abstract
The first aim of the experiment was an optimization of the method for determination of hydroxyproline in a pig skin using high-performance liquid chromatography with fluorescence detector (HPLC-FLD) and ion-exchange liquid chromatography with VIS detector (IEC-VIS). On the basis of the experiments performed, it was found that HPLC-FLD method is three times more sensitive than IEC-VIS method. For IEC analysis of hydroxyproline, the limit of detection (LOD) was 4.10 g/ml whereas the limit of quantification (LOQ) was 13.50 g/ml. For HPLC analysis of hydroxyproline, the limit of detection (LOD) was 1 ng/ml whereas the limit of quantification (LOQ) was 3 ng/ml. The second aim was the testing of influence of different volume of 6M HCl on extraction of sample (50 mg) for analysis using HPLC-FLD. Here it was found that the best volume was 250 µl 6M HCl.