Andries P. Bruins

Atmospheric pressure photoionization (APPI): a new ionization method for liquid chromatography-mass spectrometry

Atmospheric pressure photoionization (APPI) has been successfully demonstrated to provide high sensitivity to LC-MS analysis. A vacuum-ultraviolet lamp designed for photoionization detection in gas chromatography is used as a source of 10-eV photons. The mixture of samples and solvent eluting from an HPLC is fully evaporated prior to introduction into the photoionization region. In the new method, large quantities of an ionizable dopant are added to the vapor generated from the LC eluant, allowing for a great abundance of dopant photoions to be produced. Because the ion source is at atmospheric pressure, and the collision rate is high, the dopant photoions react to completion with solvent and analyte molecules present in the ion source. Using APPI, at an LC flow rate of 200 microL/min, it is possible to obtain analyte signal intensities 8 times as high as those obtainable with a commercially available corona discharge-atmospheric pressure chemical ionization source.

In vitro mimicry of metabolic oxidation reactions by electrochemistry/mass spectrometry

The aim of these studies was to investigate the scope and limitations of electrochemistry on-line with mass spectrometry as a quick and convenient way to mimic phase I oxidative reactions in drug metabolism. A compound with previously reported in vitro and in vivo metabolism, the dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin, was examined in an electrochemistry/mass spectrometry (EC/MS) system. The previously reported N-dealkylation was mimicked by the electrochemical cell while the oxidation of the phenol function was not fully mimicked by the EC/MS system, since the catechol and p-hydroquinone formed were immediately oxidized to the corresponding quinones. Since cytochrome P450 isoenzymes are the most important enzymes in phase I oxidative metabolism, two standard substrates used for the characterization of those enzymes, lidocaine and 7-ethoxycoumarin, were tested in the EC/MS system. The electrochemical cell was capable of mimicking the N-dealkylation of lidocaine but, under the conditions used in our experiments, the O-deethylation of 7-ethoxycoumarin could not be simulated in the electrochemical cell.

Electrochemistry-mass spectrometry in drug metabolism and protein research

The combination of electrochemistry coupled on-line to mass spectrometry (EC-MS) forms a powerful analytical technique with unique applications in the fields of drug metabolism and proteomics. In this review the latest developments are surveyed from both instrumental and application perspectives. The limitations and solutions for coupling an electrochemical system to a mass spectrometer are discussed. The electrochemical mimicking of drug metabolism, specifically by Cytochrome P450, is high-lighted as an application with high biomedical relevance. The EC-MS analysis of proteins also has promising new applications for both proteomics research and biomarker discovery. EC-MS has furthermore advantages for improved analyte detection with mass spectrometry, both for small molecules and large biomolecules. Finally, potential future directions of development of the technique are briefly discussed.

Reconstruction of the proteolytic pathway for use of ß-casein by Lactococcus lactis

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from β‐casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP‐generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase‐proficient and fivefold peptidase‐deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl‐terminal end of β‐casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo ; (iii) use of oligopeptides of 5–10 residues becomes only possible after uptake via Opp; (iv) only a few (10–14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.

Electrochemical oxidation and cleavage of tyrosine- and tryptophan-containing tripeptides.

Electrochemical oxidation of peptides and proteins has been shown to lead to specific cleavage next to tyrosine (Tyr) and tryptophan (Trp) residues which makes the coupling of electrochemistry to mass spectrometry (EC-MS) a potential instrumental alternative to chemical and enzymatic cleavage. A set of Tyr and Trp-containing tripeptides has been studied to investigate the mechanistic aspects of electrochemical oxidation and the subsequent chemical reactions including peptide bond cleavage, making this the first detailed study of the electrochemistry of Trp-containing peptides. The effect of adjacent amino acids was studied leading to the conclusion that the ratios of oxidation and cleavage products are peptide-dependent and that the adjacent amino acid can influence the secondary chemical reactions occurring after the initial oxidation step. The effect of parameters such as potential and solvent conditions showed that control of the oxidation potential is crucial to avoid dimer formation for Tyr and an increasing number of oxygen insertions (hydroxylations) for Trp, which occur above 1000 mV (vs Pd/H(2)). While the formation of reactive intermediates after the first oxidation step is not strongly dependent on experimental conditions, an acidic pH is required for good cleavage yields. Working under strongly acidic conditions (pH 1.9-3.1) led to optimal cleavage yields (40-80%), whereas no or little cleavage occurred under basic conditions. Online EC-MS allowed determining the optimal potential for maximum cleavage yields, whereas EC-LC-MS/MS revealed the nature and distribution of the reaction products.

Effect of solvent on dynamic range and sensitivity in pneumatically-assisted electrospray (ion spray) mass spectrometry

Mass-analyzed detector signal and spray current have been measured in pneumatically-assisted electrospray mass spectrometry. The sample was tetrabutylammonium bromide dissolved in water, methanol, acetonitrile, chloroform, dichloromethane or toluene. At low sample concentrations (less than or equal to 5 x 10(-6) M) the ion signal rose with increasing sample concentration. Above 10(-5) M the ion signal was fixed and independent of sample concentration, Comparison of signals with spray currents for tetrabutylammonium bromide at 2 x 10(-6) M in different solvents revealed a strong correlation between ion signal and spray current. Apparently, the abundance of the tetrabutylammonium ion at m/z 242 is fully controlled by the amount of charge on droplets, while other solvent properties such as volatility, surface tension and polarity do not play a role at low tetrabutylammonium bromide concentrations. Thus, water is a poor solvent for electrospray because it does not allow efficient droplet charging, not because it is less volatile and more difficult to spray than organic solvents. The ion signal at 2 x 10(-6) M tetrabutylammonium bromide in different solvents is highest for dichloromethane. At high sample concentrations (greater than or equal to 10(-5) M) the dependence of the ion signal on spray current is lost. It appears impossible to convert a high charge on droplets into sample ions. Creation of droplets having a surface fully covered with sample is assumed to be the cause of ion signal saturation. Increasing the sample concentration will only increase the number of ions inside a droplet. The number of sample ions at the surface escaping into the gas phase is fixed and independent of sample concentration above 10(-5) M.

Lidocaine Oxidation by Electrogenerated Reactive Oxygen Species in the Light of Oxidative Drug Metabolism

The study of oxidative drug metabolism by Cytochrome P450s (P450) is important in the earlier stages of drug development. For this purpose, automated analytical techniques are needed for fast and accurate estimation of oxidative drug metabolism. Previous studies have shown that electrochemistry in combination with mass spectrometry is a versatile analytical technique to generate drug metabolites that result from direct electron transfer. Here we show that electrochemical generation of reactive oxygen species (ROS), a process reminiscent of the catalytic cycle of P450, extends the applicability of electrochemistry in drug metabolism research. Oxidation products of lidocaine from one and two-compartment electrochemical cells, operated under various conditions were analyzed by LC-MS and metabolite structures were elucidated by collision-induced (LC-MS/MS), and thermally induced (APCI) fragmentation. Direct oxidation of lidocaine at the anode resulted in N-dealkylation, whereas reaction with H(2)O(2), generated at the cathode, produced the N-oxide, both known in vivo lidocaine metabolites. Catalytic activation of hydrogen peroxide, using the Fenton reaction, resulted in benzylic and aromatic hydroxylations thus covering all of the known in vivo phase-I metabolites of lidocaine. This study extends the applicability of electrochemistry combined with mass spectrometry as a valuable technique in assessing oxidative drug metabolism related to P450.

Oxidative protein labeling in mass-spectrometry-based proteomics.

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade.

Electrochemistry in the Mimicry of Oxidative Drug Metabolism by Cytochrome P450s

Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the oxidation reactions initiated by charge transfer, has shown promise in the mimicry of certain CYP-mediated metabolic reactions. The electrochemical approach may further be utilized in an automated manner in microfluidics devices facilitating fast screening of oxidative drug metabolism. A wide range of in vivo oxidation reactions, particularly those initiated by hydrogen atom transfer, can be imitated through the electrochemically-assisted Fenton reaction. This reaction is based on O-O bond activation in hydrogen peroxide and oxidation by hydroxyl radicals, wherein electrochemistry is used for the reduction of molecular oxygen to hydrogen peroxide, as well as the reduction of Fe(3+) to Fe(2+). Metalloporphyrins, as surrogates for the prosthetic group in CYP, utilizing metallo-oxo reactive species, can also be used in combination with electrochemistry. Electrochemical reduction of metalloporphyrins in solution or immobilized on the electrode surface activates molecular oxygen in a manner analogous to the catalytical cycle of CYP and different metalloporphyrins can mimic selective oxidation reactions. Chemoselective, stereoselective, and regioselective oxidation reactions may be mimicked using electrodes that have been modified with immobilized enzymes, especially CYP itself. This review summarizes the recent attempts in utilizing electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by CYP.

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 12 s (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.