Voltammetric Detection of Vanillylmandelic Acid and Homovanillic Acid Using Urea-Derivative-Modified Graphite Electrode.

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are diagnostic markers of neuroblastoma. The purpose of this study was to understand the reason for the discrimination of structural analogues (VMA and HVA) onto a graphite electrode coated with an electrochemically oxidized urea derivative. Density functional theory calculations (DFT), FTIR spectroscopic measurements, and electrochemical impedance spectroscopic measurements were used in this work. Density functional theory calculations (DFT) were used to identify the most suitable binding sites of the urea derivative and to describe possible differences in its interaction with the studied analytes. The FTIR measurement indicated the enhancement and disappearance of NH vibrations on graphite and platinum surfaces, respectively, that could be connected to a different orientation and thus provide accessibility of the urea moiety for the discrimination of carboxylates. Additionally, the higher the basicity of the anion, the stronger the hydrogen-bonding interaction with -NH-groups of the urea moiety: VMA (pKb = 10.6, KAds = (5.18 ± 1.95) × 105) and HVA (pKb = 9.6, KAds = (4.78 ± 1.58) × 104). The differential pulse voltammetric method was applied to detect VMA and HVA as individual species and interferents. As individual analytes, both HVA and VMA can be detected at a concentration of 1.99 × 10-5 M (RSD ≤ 0.28, recovery 110-115%).

Application of an LC-MS/MS Method for the Simultaneous Quantification of Homovanillic Acid and Vanillylmandelic Acid for the Diagnosis and Follow-Up of Neuroblastoma in 357 Patients.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.

Determination of tumour biomarkers homovanillic and vanillylmandelic acid using flow injection analysis with amperometric detection at a boron doped diamond electrode.

A new method for the simultaneous determination of two tumour biomarkers, homovanillic (HVA) and vanillylmandelic acid (VMA), using flow injection analysis (FIA) with amperometric detection (AD) at a commercially available boron doped diamond electrode (BDDE) was developed. It was found that this method is suitable for the determination of HVA (in the presence of VMA) and VMA (in the presence of HVA) in optimum medium of Britton-Robinson buffer (0.04 mol L-1, pH 3.0). Calibration dependences consist of two linear parts for both biomarkers, the first one being in the concentration range from 1 to 10 µmol L-1 and the second one from 10 to 100 µmol L-1 (with obtained LODs 0.44 µmol L-1 for HVA and 0.34 µmol L-1 for VMA, respectively). To minimize any negative effects related to the passivation of the working electrode, suitable cleaning pulses (+2.4 V for 30 s) were imposed on the working electrode after each measurement. An attempt to use FIA with multiple pulse amperometric detection to determine both analytes in one run was not successful. Changing potentials in short intervals in multiple pulse detection probably results in mutual interaction of analytes and/or products of their electrochemical oxidation, thus preventing the application of this approach.

Experimental and theoretical elucidation of structural and antioxidant properties of vanillylmandelic acid and its carboxylate anion.

Vanillylmandelic acid (VMA), an important metabolite of catecholamines that is routinely screened as tumor marker, was investigated by the various spectroscopic techniques (IR, Raman, UV-Vis, antioxidant decolorization assay and NMR). Structures optimized by the employment of five common functionals (M05-2X, M06-2X, B3LYP, CAM-B3LYP, B3LYP-D3) were compared with the crystallographic data. The M05-2X functional reproduced the most reliable experimental bond lengths and angles (correlation coefficient >0.999). The importance of intramolecular hydrogen bonds for structural stability was discussed and quantified by the NBO analysis. The most prominent bands in vibrational spectrum were analyzed and compared to the experimental data. The positions of the carbon and hydrogen atoms in NMR spectra were well reproduced. The differences in UV-Vis spectrum were investigated by adding the explicit solvent and by performing NBO and QTAIM analyses. The discrepancy in the two spectra of about 50nm could be explained by the solvent effect on carboxyl group. The most probable antioxidant activity mechanism was discussed for VMA and its carboxylate anion. The Molecular Docking study with the C - reactive protein additionally proved that variety of functional groups present in VMA and its anion allowed strong hydrogen and hydrophobic interactions.

Vanillin analog--vanillyl mandelic acid, a novel specific inhibitor of snake venom 5'-nucleotidase.

Snake venom 5'-nucleotidase (5'NUC) plays a very important role in envenomation strategies; however, apart from its modulation of hemostatic functions, its other pharmacological effects are not yet well characterized. Several studies have used specific inhibitors of enzyme toxins as a biochemical or pharmacological tool to characterize or establish its mechanism of action. We report here for the first time vanillin mandelic acid (VMA), an analog of vanillin, to potentially, selectively, and specifically inhibit venom 5'NUC activity among other enzymes present in venoms. VMA is much more potent in inhibiting 5'NUC activity than vanillyl acid (VA). The experimental results obtained are in good agreement with the in silico molecular docking interaction data. Both VA and VMA are competitive inhibitors as evident by the inhibition-relieving effect upon increasing the substrate concentration. VMA also dose-dependently inhibited the anticoagulant effect in Naja naja venom. In this study, we report novel non-nucleoside specific inhibitors of snake venom 5'NUC and experimentally demonstrate their involvement in the anticoagulant activity of N. naja venom. Hence, we hypothesize that VMA can be used as a molecular tool to evaluate the role of 5'NUC in snake envenomation and to develop prototypes and lead compounds with potential therapeutic applications against snake bites.

Self-quenching in the electrochemiluminescence of tris(2,2'-bipyridyl) ruthenium(ii) using metabolites of catecholamines as co-reactants.

Electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) using metabolites of catecholamines: homovanillic acid (HVA) and vanillylmandelic acid (VMA) as co-reactants were investigated in aqueous solution for the first time. When HVA and VMA were co-existent in the buffer solution containing Ru(bpy)(3)(2+), ECL peaks were observed at a potential corresponding to the oxidation of Ru(bpy)(3)(2+), and the ECL intensity was increased noticeably when the concentrations of HVA and VMA were at lower levels. The linear calibration range was from 8.0 × 10(-5) to 1.0 × 10(-9) M for HVA and VMA. The detection limit (S/N = 3) of HVA and VMA was 4.0 × 10(-10) M. The formation of the excited state Ru(bpy)(3)(2+*) was confirmed to result from the reaction between Ru(bpy)(3)(3+) and the intermediates of HVA or VMA radicals. Moreover, it was found that the ECL intensity was quenched significantly when the concentrations of HVA and VMA were relatively higher. The mechanism of self-quenching processes involved in the Ru(bpy)(3)(2+)-HVA and -VMA ECL systems are proposed in this study.

New copper(II) complexes with dopamine hydrochloride and vanillymandelic acid: spectroscopic and thermal characterization.

The dopamine derivatives participate in the regulation of wide variety of physiological functions in the human body and in medication life. Increase and/or decrease in the concentration of dopamine in human body reflect an indication for diseases such as Schizophrenia and/or Parkinson diseases. The Cu(II) chelates with coupled products of dopamine hydrochloride (DO.HCl) and vanillymandelic acid (VMA) with 4-aminoantipyrine (4-AAP) are prepared and characterized. Different physico-chemical techniques namely IR, magnetic and UV-vis spectra are used to investigate the structure of these chelates. Cu(II) forms 1:1 (Cu:DO) and 1:2 (Cu:VMA) chelates. DO behave as a uninegative tridentate ligand in binding to the Cu(II) ion while VMA behaves as a uninegative bidentate ligand. IR spectra show that the DO is coordinated to the Cu(II) ion in a tridentate manner with ONO donor sites of the phenolic-OH, -NH and carbonyl-O, while VMA is coordinated with OO donor sites of the phenolic-OH and -NH. Magnetic moment measurements reveal the presence of Cu(II) chelates in octahedral and square planar geometries with DO and VMA, respectively. The thermal decomposition of Cu(II) complexes is studied using thermogravimetric (TG) and differential thermal analysis (DTA) techniques. The activation thermodynamic parameters, such as, energy of activation, enthalpy, entropy and free energy change of the complexes are evaluated and the relative thermal stability of the complexes are discussed.

Electrooxidative decarboxylation of vanillylmandelic acid: voltammetric differentiation between the structurally related compounds homovanillic acid and vanillylmandelic acid.

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are the major end products of catecholamine metabolism. Abnoramally high levels in both plasma and urine may be indicative of a number of diseases including neuroblastoma and phaeochromocytoma. Commonly the VMA:HVA ratio is used as a disease marker, so that any measurement techniques need to be able to differentiate between these two structurally similar compounds. Electrochemistry is often limited in selectivity due to many organic molecules being oxidized or reduced at similar potentials. This work investigates the electrochemical oxidation mechanism of VMA at an edge-plane pyrolytic graphite electrode and highlights how, although structurally similar to HVA, their voltammetric responses may be differentiated through appropriate selection of the electrode material. The oxidation of VMA exhibits two clear peaks and the mechanism is shown to proceed through the decarboxylation of VMA to form vanillin, which is further oxidized resulting in the second peak. Modification of the electrode with a porous layer of multiwalled carbon nanotubes so as to change the mass transport to that of a thin layer system causes the voltammetric resolution between the two species to be enhanced. Differential pulse voltammetry is used to measure the limits of detection for VMA on an edge-plane pyrolytic graphite electrode and on commercially available multiwalled carbon nanotube screen printed electrode, with limits of detection of 1.7 and 1.0 microM, respectively. These limits of detection are well within the range of sensitivity required for clinical sample measurement.

Quantitation of homovanillic acid (HVA) and vanillylmandelic acid (VMA) in urine using gas chromatography-mass spectrometry (GC/MS).

Neuroblastoma, in most cases, is characterized by increased production of catecholamines and their metabolites. Laboratory diagnosis and clinical follow-up include the measurement of urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA). In the following procedure, urine samples are diluted to give a creatinine concentration of 2 mg/dL. Deuterated internal standards are added to the diluted urine samples followed by acidification using HCl. Ethyl acetate is used to extract HVA and VMA from the acidified samples, and the extract is dried. The residue is treated with bis-(trimethylsilyl)trifluoroacetamide (BSTFA), 1% trimethylchlorosilane (TMCS), and pyridine to prepare trimethylsilyl derivatives of HVA and VMA. The derivatized samples are injected to into gas-chromatograph mass spectrometer. The concentration of HVA and VMA is determined by comparing responses of unknown sample to the responses of calibrators using selected ion monitoring.