Hydrophilic interaction liquid chromatography for biologically important metal complex speciation and low molecular weight ligand analysis

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Alsaeedi, Majidah
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University College Cork
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Metal ions play crucial roles both from a biological and an environmental perspective. They circulate around the human body by coordinating with selected biological molecules to generate stable and strong bonding to reach other organs in the body, such as the brain, liver, tissues and kidney. Therefore, understanding their forms distribution and concentration fluctuations in a biological fluids and tissues is of great interest. This research provides an overview of the roles of selected metal ions as well as their toxicity and their transportation within the human body. The significance of the labile metal pools concept and the role of low molecular weight compounds as metal chelators are discussed. Analytical techniques used for metal speciation and metallomics are reviewed, with a particular emphasis on the use and potential of hydrophilic interaction liquid chromatography (HILIC). Post chromatographic detection methods are compared including inductively coupled plasma-mass spectrometry (ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS). The potential of the zwitterionic HILIC stationary phases for the fast and efficient separation of labile metal is investigated. Specifically the aqueous metal complex species distribution analysis for the low molecular weight Ni(II)-histidine was investigated at various metal-ligand ratios and as a function of pH. While selective species separation and detection was not achievable using silica bonded reversed phase or on non-bonded silica particle HILIC stationary phases, the two stepwise species, Ni(II)-His1 and Ni(II)-His2, as well as free His, were rapidly separated within 120 seconds on the Z-cHILIC phosphocholine bonded. The identities of Ni(II)-His1 and Ni(II)-His2 species were confirmed using HILIC electrospray ionization- mass spectrometry (HILIC-ESI-MS) at negative mode supported with offline analysis of collected fractions by inductively coupled plasma-mass spectrometry (ICP-MS). In addition, sensitive, rapid and efficient separation of three selected catecholamines (CAs), namely dopamine (DA), epinephrine (EPI), and norepinephrine (NE), known to be aluminium (III) and iron (III) chelators was achieved in a time of 40 seconds using a poroshell Z-HILIC column coupled to electrochemical detection based on a boron-doped diamond electrode. The applicability of the proposed method was investigated in the determination of the urinary CAs in a real urine sample following phenylboronic acid (PBA) solid-phase extraction (SPE). Al(III) and Fe(III)-CA speciation was also investigated using poroshell Z-HILIC and core-shell RP-amide columns. The chromatographic behaviours of both metal complexes are discussed. Also, the production of aminochrome forms of each CAs during the chromatographic separation was observed and carefully monitored. Attention was then placed on the separation and detection of urinary selected monoamine neurotransmitters (MNTs) including serotonin, dopamine, norepinephrine, homovanillic acid , 5-hydroxyindoleacetic acid, and vanillylmandelic acid that are of importance as biomarkers for neuroblastoma and carcinoid tumors. The chromatographic separation was carried out using the Z-cHILIC column, with isocratic elution is followed by amperometric detection at a BDD electrode. LODs between 40 to 150 nM are obtained for the MNTs. The method was applied for the analysis of the selected MNTs in urine samples following phenylboronic acid (PBA) solid-phase extraction. The research outcomes presented represent an advancement in our knowledge and application of HILIC for the separation of biologically important biological ligands and selected metal complexes of importance in metal-related health conditions such as in neurodegenerative diseases and allergy.
Hydrophilic interaction liquid chromatography , Rapid separation , Low molecular weight ligands , Metal speciation
Alsaeedi, M. 2022. Hydrophilic interaction liquid chromatography for biologically important metal complex speciation and low molecular weight ligand analysis. PhD Thesis, University College Cork.