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Voltammetric Determination of Tumor Biomarkers using Flow Injection Analysis with Amperometric Detection
Němečková-Makrlíková, Anna ; Matysik, F.-M. ; Navrátil, Tomáš ; Barek, J. ; Vyskočil, V.
Three tumor biomarkers (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindole-3-acetic acid) have been determined by flow injection analysis with amperometric detection at screen-printed carbon electrodes in optimum medium of Britton-Robinson buffer (0.04 mol l(-1), pH = 2.0). Dependences of the peaks current on the concentration of biomarkers were linear in the tested concentration region from 0.05 to 100 mu mol l(-1), with the limits of detection of 0.065 mu mol l(-1) for homovanillic acid, 0.053 mu mol l(-1) for vanillylmandelic acid, and 0.033 mu mol l(-1) for 5-hydroxyindole-3-acetic acid (calculated from heights), and 0.024 mu mol l(-1) for homovanillic acid, 0.020 mu mol l(-1) for vanillylmandelic acid, and 0.012 mu mol l(-1) for 5-hydroxyindole-3-acetic acid (calculated fromareas), respectively.
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HPLC-ED/UV for determination of vanillylmandelic acid in human urine after solid phase extraction
Němečková-Makrlíková, Anna ; Dejmková, H. ; Navrátil, Tomáš ; Barek, J. ; Vyskočil, V.
HPLC with electrochemical and spectrophotometric detection (ED/UV) after solid phase extraction (SPE) was used for determination of vanillylmandelic acid in human urine. HPLC-ED was performed at a glassy carbon electrode in a “wall-jet” arrangement in acetate-phosphate buffer at pH = 2.5 and gradient elution (increasing content of\nacetonitrile from 5 to 25% in 10 minutes) was used. Optimized parameters were following: flow rate of mobile phase 1 mL min−1, detection potential +1.1 V, detection wavelength 279 nm, injected volume 20 μL. Dependence of the peak current on the analyte concentration was linear in the concentration range from 10 to 150 μmol L−1, with obtained limits of detection 2.6 μmol L−1 (calculated from peak height) and 1.9 μmol L−1 (calculated from peak area) for HPLC-ED, and 11.0 μmol L−1 (calculated from peak height) and 9.8 μmol L−1 (calculated from peak area) for HPLC-UV.
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HPLC-ED/UV with Solid Phase Extraction for the Determination of 5-Hydroxyindole-3-acetic Acid
Němečková-Makrlíková, Anna ; Dejmková, H. ; Navrátil, Tomáš ; Barek, J. ; Vyskočil, V.
Determination of 5-HIAA in human urine was successfully performed using HPLC-ED/UV with SPE. HPLC-ED is a very sensitive technique, presented method does not need any sample pre-treatment apart from SPE procedure, which replaced filtration of samples of human urine to avoid problems with clogging a HPLC column. The found native\nconcentrations of 5-HIAA in human urine using HPLC-ED with SPE fully corresponds with published normal concentrations in urine (17.8 - 58.3 micromol l-1). The found native concentrations of 5-HIAA were 18.4 micromol L-1 (calculated from peak height). The presented method is fast and could be used for simple\nscreening of human urine, because HPLC-ED/UV allows simultaneous determination of 5-HIAA, HVA, and VMA as important tumour biomarkers.
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