Prospects on the Hyphenated Electrochemistry and Mass Spectrometry as a Practical Analytical Technique in the Assessment of Oxidative Drug Metabolism

For several years, electrochemistry in combination with mass spectrometry has been the subject of attention and development for studying the oxidative drug metabolism at early stages of new drug development. Though the technique could successfully imitate the in vivo oxidative drug metabolism initiated by electron transfer, it lags in the imitation of reactions initiated by either hydrogen atom transfer or oxygen atom transfer. The prospect of using electrochemistry as a practical analytical technique in the imitation of oxidative drug metabolism, therefore, relies on the extension of its utility toward covering those reactions initiated by hydrogen atom transfer and oxygen atom transfer. In this brief critical review, I discuss potential electrochemical techniques that can benefit the application of electrochemistry beyond electron transfer reactions

Targeted Proteomics in Translational and Clinical Studies

This chapter provides a concise overview on the methods and applications of targeted proteomics in the context of translational and clinical studies. Mass spectrometry-based targeted proteomics has emerged as a promising technique for protein and peptide quantification, presenting a great potential for clinical applications. While significant amount of discovery works have been carried out in both genomics and proteomics for an assortment of diseases, it has been challenging in further characterizing individual protein targets for their biological significance and clinical value due to the lack of effective and “universal” techniques. The development of targeted proteomics approach opened a unique avenue to bridge the discovery-based genomics and proteomics with candidate-based protein analysis, which is clinically highly relevant. Targeted proteomics analysis has been implemented on a variety of instrument platforms, and applied for a wide range of studies, from blood biomarker detection to pathway-driven mechanistic investigations, with the triple quadrupole-based selected reaction monitoring (SRM) technique being the most widely used method. With a right combination of calibration approach, internal standards, and sample preparation strategies, mass spectrometry-based targeted analysis has proven to be of inter-laboratory reproducibility and sensitivity in analyzing many clinical specimens. More recently, the advent of mass spectrometry with high frequencies and resolutions yielded the data independent acquisition (DIA) techniques, such as sequential window acquisition of all theoretical fragment ion spectra (SWATH). The unbiased nature of DIA methods would enable a wider analytical scope and a greater robustness in targeted analysis, representing a paradigm shift in targeted proteomics

Evaluation of peroxidative stress of cancer cells in vitro by real time quantification of volatile aldehydes in culture headspace

Rationale Peroxidation of lipids in cellular membranes results in the release of volatile organic compounds (VOCs), including saturated aldehydes. The real‐time quantification of trace VOCs produced by cancer cells during peroxidative stress presents a new challenge to non‐invasive clinical diagnostics, which as described here, we have met with some success. Methods A combination of selected ion flow tube mass spectrometry (SIFT‐MS), a technique that allows rapid, reliable quantification of VOCs in humid air and liquid headspace, and electrochemistry to generate reactive oxygen species (ROS) in vitro has been used. Thus, VOCs present in the headspace of CALU‐1 cancer cell line cultures exposed to ROS have been monitored and quantified in real time using SIFT‐MS. Results The CALU‐1 lung cancer cells were cultured in 3D collagen to mimic in vivo tissue. Real‐time SIFT‐MS analyses focused on the volatile aldehydes: propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde (propanedial), that are expected to be products of cellular membrane peroxidation. All six aldehydes were identified in the culture headspace, each reaching peak concentrations during the time of exposure to ROS and eventually reducing as the reactants were depleted in the culture. Pentanal and hexanal were the most abundant, reaching concentrations of a few hundred parts‐per‐billion by volume, ppbv, in the culture headspace. Conclusions The results of these experiments demonstrate that peroxidation of cancer cells in vitro can be monitored and evaluated by direct real‐time analysis of the volatile aldehydes produced. The combination of adopted methodology potentially has value for the study of other types of VOCs that may be produced by cellular damage

A comparative study of synthetic winged peptides for absolute protein quantification

A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein

On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSn experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase

ElectrochemicalN-demethylation of tropane alkaloids

A practical, efficient, and selective electrochemicalN-demethylation method of tropane alkaloids to their nortropane derivatives is described. Nortropanes, such as noratropine and norscopolamine, are important intermediates for the semi-synthesis of the medicines ipratropium or oxitropium bromide, respectively. Synthesis was performed in a simple home-made electrochemical batch cell using a porous glassy carbon electrode. The reaction proceeds at room temperature in one step in a mixture of ethanol or methanol and water. The method avoids hazardous oxidizing agents such as H(2)O(2)orm-chloroperbenzoic acid (m-CPBA), toxic solvents such as chloroform, as well as metal-based catalysts. Various key parameters were investigated in electrochemical batch or flow cells, and the optimized conditions were used in batch and flow-cells at gram scale to synthesize noratropine in high yield and purity using a convenient liquid-liquid extraction method without any need for chromatographic purification. Mechanistic studies showed that the electrochemicalN-demethylation proceeds by the formation of an iminium intermediate which is converted by water as the nucleophile. The optimized method was further applied to scopolamine, cocaine, benzatropine, homatropine and tropacocaine, showing that this is a generic way ofN-demethylating tropane alkaloids to synthesize valuable precursors for pharmaceutical products

Electrochemistry in the mimicry of oxidative drug metabolism

The thesis commences in Chapter 1 with reviewing known electrochemical techniques and their advantages and limitations in the context of oxidative drug metabolism. The thesis continues with development of new electrochemical techniques to cover certain in vivo oxidative drug metabolism reactions which were not possible by constant potential oxidation. Electrochemically generated reactive oxygen species (ROS) in Chapter 2, electrochemical oxidation by square-wave potential pulses in Chapters 3 and 4, as well as electrocatalytic activation of hydrogen peroxide on a platinum electrode in Chapter 5 are among the electrochemical techniques developed throughout my research. Chapter 1 presents Cytochrome P450s (CYP), their discovery, structure, and role in in vivo oxidative drug metabolism by catalytic activation of molecular oxygen and the generation of ROS, mainly oxo-ferryl radical cations. Chapter 1 further reviews different electrochemical techniques that have been developed so far, including direct electrochemical oxidation (in combination with mass spectrometry), oxidation by electrochemically generated ROS, and oxidation with modified electrodes containing metalloporphyrines and enzymes. Direct electrochemical oxidation in combination with mass spectrometry, introduced in Chapter 1, has been used widely to imitate in vivo oxidative drug metabolism. The oxidation mechanisms were discussed in relation to in vivo oxidative drug metabolism. Direct electrochemical oxidation, accordingly, is capable of imitating oxidative reactions that are initiated by electron transfer, such as N-dealkylation and hydroxylation of substituted aromatic rings, whereas oxidation reactions which are initiated by hydrogen atom transfer (HAT) or oxygen atom insertion are difficult to imitate. Another major obstacle in imitation emerges when the oxidation products are oxidized more easily than the drug substrates, which prevents isolation of the latter.

Electrochemical Oxidation by Square-Wave Potential Pulses in the Imitation of Oxidative Drug Metabolism

Electrochemistry combined with mass spectrometry (EC-MS) is an emerging analytical technique in the imitation of oxidative drug metabolism at the early stages of new drug development. Here, we present the benefits of electrochemical oxidation by square-wave potential pulses for the oxidation of lidocaine, a test drug compound, on a platinum electrode. Lidocaine was oxidized at constant potential and by square-wave potential pulses with different cycle times, and the reaction products were analyzed by liquid chromatography-mass spectrometry [LC-MS(/MS)]. Application of constant potentials of up to +5.0 V resulted in relatively low yields of N-dealkylation and 4-hydroxylation products, while oxidation by square-wave potential pulses generated up to 50 times more of the 4-hydroxylation product at cycle times between 0.2 and 12s (estimated yield of 10%). The highest yield of the N-dealkylation product was obtained at cycle times shorter than 0.2 s. Tuning of the cycle time is thus an important parameter to modulate the selectivity of electrochemical oxidation reactions. The N-oxidation product was only obtained by electrochemical oxidation under air atmosphere due to reaction with electrogenerated hydrogen peroxide. Square-wave potential pulses may also be applicable to modulate the selectivity of electrochemical reactions with other drug compounds in order to generate oxidation products with greater selectivity and higher yield based on the optimization of cycle times and potentials. This considerably widens the scope of direct electrochemistry-based oxidation reactions for the imitation of in vivo oxidative drug metabolism