Results

Electrochemically assisted reduction of disulphide bonds

Conclusions

Using the ROXY™ EC system on-line with MS results in fast generation of metabolites (seconds vs. days or weeks using in-vitro and/or in-vivo methods ), access to phase II reactions as well as reactive metabo-lites. Amiodaquine was successfully used as model drug to mimic the oxidative metabolic pathway in the human liver by on-line EC/MS. Phase I and II metabolites, which were already known from the literature, were generated in the EC reactor cell and on-line identified by MS using either amo-diaquine alone or in the presence of glutathione. A long-lasting, stable and efficient lectrochemical synthesis of metabolites is possible by applying a square wave pulse. we demonstrated new, electrochemically-based technique for efficient reduction of disulfide bonds in proteins and pep-tides.

Acknowledgements

With special thanks to the division of Analytical BioSciences (Leiden University, The Nether-lands) for access to their MS facility.

References[1] Lohmann W. et al., LC-GC Europe, January (2010) 1[2] Faber H. et al., Anal. Bioanal. Chem., 403 (2012) 345[3] Rojas-Chertó M. et al., Bioinformatics, 27 (2011) 2376[4] Jahn S. et al., J. Chromatogr. A, 1218 (2011) 9210

Prediction of Phase I and II Drug Metabolism

Amodiaquine (AQ) was oxidized in the ReactorCell by applying scan 0-1500 mV with 50 mV/s rate. AQ underwent oxidation by dehydrogenation to quinine imine (AQQI). Figure 9 shows the ad-duct formation of electrogenerated AQQI with β-actalbumin. The shift in the m/z values after the reaction with the reactive drug metabolites clearly indicates the occurrence of covalent drug-protein adduct formation.

Electrochemically Initiated Reactions Upfront MS -EC/MS an Unknown Panacea?

Martin Eysberg, Agnieszka Kraj, Hendrik-Jan Brouwer, Nico Reinhoud, Jean-Pierre ChervetAntec, Zoeterwoude, The Netherlands

Introduction

Electrochemistry (EC) in combination with mass spectrometry creates a powerful platform to simulate various oxidation and reduction processes in life sciences. Electrochemistry is a complementary technique to traditional in vivo or in vitro metabolism studies, and delivers the oxidative metabolic fingerprint of a (drug) molecule in a very short time. Mass spectrometrydelivers selective and sensitive detection and allows for unambiguous identification of all products generated in the electrochemical cell. Additionally, automated data analysis by use of data bases (proteomics) or mass spectral trees (metabolomics) can shorten considerably the total time needed for the experiment. Furthermore, the electrochemical cell can be used for preparative synthesis of reactive metabolites in a short period of time. The cell can be hyphen-ated to MS or LC/MS to perform separation and identification of the created oxidative compounds i.e., intermediates, (reactive) metabolites. Alternatively, the cell can be used off-line and the gen-erated metabolites can then be collected for supplementary research such as NMR.

Methods

A preparative electrochemical cell (µ-PrepCell, Antec) equipped with a Glassy Carbon (GC) or ti-tanium based working electrode was used for synthesis of metabolites or reduction of disulfide bonds. Typically, 2-3µM concentration of the drug/peptides were used in experiments with MS detection. 250 µM solution of the Verapamil in 20mM ammonium formate, pH 7.4 in acetonitrile (50/50, v/v) was used as a model drug. The electrochemically synthesized metabolites of Verapamil were collected off-line, followed by MS analysis of the collected fractions (Figure 2C). The flow rate used in the synthesis experiments was 50 µL/min. For the formation of GSH ad-ducts, 50 µM GSH solution was added after the EC cell using a mixing coil. For disulfide bond re-duction mobile phase containing 1% formic acid was used. A square wave pulse was applied to achieve stabile continuous metabolite generation over long periods of time. The settings of the pulse mode are presented in further sections of this poster. Optimization of the metabolite syn-thesis was performed based on scanning voltammetry. An LTQ-FT (Thermo, USA) mass spec-trometer equipped with electrospray (ESI) source was used to monitor the oxidation or reductive products.

Figure 6: A: Optimized square wave pulses settings. B: Long term current stability experiment. C: An overlay of 8 mass spectra of the control samples. The control samples were collected in 15, 45, 75, and 100 minute of synthesis. The control samples were diluted 100x before injection to MS.

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Figure 4: An excerpt of the Verapamil metabolic pathway.Blue dotted ellipses are indi-cating other places of possi-ble loss of CH2 [4].

Figure 3: A: Instrumental set-up of used for metabolite synthesis. B: µ-PrepCell (left), comparison of working electrodes (middle) and ReactorCell (right).

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Figure 5: A: A single (full) scan cycle of Verapamil. Conditions: E1=0V; E2=1.5V; cycle: con-tinuous; rate: 20mV/s; flow rate: 50μL/min. B: MS Voltammogram of Verapamil. C: Mass spec-trum corresponding to grey area of EIC from A (background was subtracted). Maximum signal was detected at 750mV and was consistent with experiment shown in the figure 5A.

Figure 10: Reduction of Insulin on typically used MD electrode and a specially developed working electrode (Antec). The optimized pulse settings are applied.

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Figure 1: Application area of electrochemistry combined with mass spectrometry.

Figure 8A: MS Voltammogram of Amodiaquine (Scan mode) and mass spectra of metabolites of Amodiaquine.B: Example of EICs of Metabolite 1 (m/z 354) and its conjugate (m/z 661) and Me-tabolite 2 (m/z 326) and its conjugate (m/z 633). Conjugation was performed in DC mode. The spectrum with cell OFF confirms that the conjugates are formed ONLY if potential is applied.

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Figure 9: Mass spectra and deconvolution results of unmodified β-Lactalbumin (A) and after re-action with AQQI (B). Courtesy of Prof. Dr. Jerzy Silberring (AGH University of Science and Technology, Kraków, Poland).

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Figure 2: ROXY EC system (Antec).

Figure 7: Oxidation products and metabo-lites of Amodiaquine. Table: comparison of products formed by electrochemical oxida-tion and microsomal in-cubation [2].