Electrochemistry (EC) is a mature scientific discipline aimed to study the movement of electrons in an oxidation–reduction reaction. EC covers techniques that use a measurement of potential, charge, or current to determine the concentration or the chemical reactivity of analytes. The electrical signal is directly converted into chemical information. For in-depth characterization of complex electrochemical reactions involving the formation of diverse intermediates, products and byproducts, EC is usually combined with other analytical techniques, and particularly the hyphenation of EC with mass spectrometry (MS) has found broad applicability. The analysis of gases and volatile intermediates and products formed at electrode surfaces is enabled by differential electrochemical mass spectrometry (DEMS). In DEMS an electrochemical cell is sampled with a membrane interface for electron ionization (EI)-MS. The chemical space amenable to EC/MS (i.e., bioorganic molecules including proteins, peptides, nucleic acids, and drugs) was significantly increased by employing electrospray ionization (ESI)-MS. In the simplest setup, the EC of the ESI process is used to analytical advantage. A limitation of this approach is, however, its inability to precisely control the electrochemical potential at the emitter electrode. Thus, particularly for studying mechanistic aspects of electrochemical processes, the hyphenation of discrete electrochemical cells with ESI-MS was found to be more appropriate. The analytical power of EC/ESI-MS can further be increased by integrating liquid chromatography (LC) as an additional dimension of separation. Chromatographic separation was found to be particularly useful to reduce the complexity of the sample submitted either to the EC cell or to ESI-MS. Thus, both EC/LC/ESI-MS and LC/EC/ESI-MS are common.

LC-MS represents an important technology for the qualitative and quantitative analysis of nucleic acids. For MS, ESI in negative ion mode is used. The chromatographic method of choice is ion-pair (IP) RP chromatography. Chromatographic separations are usually accomplished by gradients of an organic modifier in aqueous solutions of IP reagents. Commonly applied IP reagents are 2.3 mM triethylamine/400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (TEA/HFIP, pH 7.0) and 10-25 mM cyclohexyldimethylammonium acetate (CycHDMAA, pH 8.4). Direct comparison of mass spectrometric performance of the two solvent systems revealed that the TEA/HFIP system offers better detection sensitivity than the CycHDMAA system. This is mainly attributable to the depletion of HFIP during droplet formation and solvent evaporation. Removal of the anionic counterion facilitates oligonucleotide ionization, and the oligonucleotides are desorbed as highly charged ions into the gas phase. TEA/HFIP-based mobile phases are recommended for developing quantitative assays targeting defined oligonucleotides. The CycHDMAA system allows the formation of cyclohexyldimethylammonium adducts. These adducts are cleaved in the gas phase, and this decomposition gives rise to charge state reduction. Ammonium adduct formation is of particular importance in preventing adducting with metal ions. Thus, adducts with metal ions are efficiently suppressed with CycHDMAA. For the TEA/HFIP system, however, such adducting represents a severe problem particularly if large oligonucleotides are analyzed. Thus, CycHDMAA-based mobile phases are recommended for qualitative assays such as LC-MS-based genotyping.

Electrospray ionization (ESI) involves the dispersion of a liquid containing analytes of interest into a fine aerosol by applying a high potential difference to the sample solution with respect to a counter electrode. Thus, from the electrochemical point of view, the ESI source represents a two-electrode controlled-current electrochemical flow cell. The electroactive compounds part of the solvent sprayed may be altered by occurring electrolysis (oxidation in positive ion mode and reduction in negative ion mode). These reactions can be troublesome in the context of unknown identification and quantification. In the search for a simple, inexpensive, and efficient way to suppress electrochemical oxidation in positive ESI, the usability of ascorbic acid, hydroquinone, and glutathione for homogenous redox buffering was tested. Performance of the antioxidants was assessed by analyzing pharmaceutical compounds covering a broad range of functional groups prone to oxidation. Different emitter setups were applied for continuous infusion, flow injection, and liquid chromatography/mass spectrometry experiments. Best performance was obtained with ascorbic acid. In comparison to hydroquinone and glutathione, ascorbic acid offered superior antioxidant activity, a relatively inert oxidation product, and hardly any negative effect on the ionization efficiency of analytes. Furthermore, ascorbic acid suppressed the formation of sodiated forms and was able to induce charge state reduction. Only in the very special case of analyzing a compound isobaric to ascorbic acid, interference with the low-abundant [ascorbic acid+H]+ signal may become a point of attention.FigureAscorbic acid efficiently suppresses analyte oxidation and formation of sodiated forms in positive electrospray ionizationElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-012-6196-z) contains supplementary material, which is available to authorized users.

It is becoming increasingly evident that genetic variants contribute to the development of opioid addiction. An elucidation of these genetic factors is crucial for a better understanding of this chronic disease and may help to develop novel therapeutic strategies. In recent years, several candidate genes were implicated in opioid dependence. However, most study findings have not been replicated and additional studies are required before reported associations can be considered robust. Thus, the major objective of this study was to replicate earlier findings and to identify new genetic polymorphisms contributing to the individual susceptibility to opioid addiction, respectively. Therefore, a candidate gene association study was conducted including 142 well-phenotyped long-term opioid addicts undergoing opioid maintenance therapy and 142 well-matched healthy controls. In both study groups, 24 single nucleotide polymorphisms predominantly located in pharmacogenetic candidate genes have been genotyped using an accurate mass spectrometry based method. The most significant associations with opioid addiction (remaining significant after adjustment for multiple testing) were observed for the rs948854 SNP in the galanin gene (GAL, p = 0.001) and the rs2236861 SNP in the delta opioid receptor gene (OPRD1, p = 0.001). Moreover, an association of the ATP binding cassette transporter 1 (ABCB1) variant rs1045642 and the Mu Opioid receptor (OPRM1) variant rs9479757 with opioid addiction was observed. The present study provides further support for a contribution of GAL and OPRD1 variants to the development of opioid addiction. Furthermore, our results indicate a potential contribution of OPRM1 and ABCB1 SNPs to the development of this chronic relapsing disease. Therefore it seems important that these genes are addressed in further addiction related studies.

Abstract. Phosporothioate oligonucleotides represent an important class of therapeutic oligonucleotides, in which none-bridging oxygen atoms of the phosphate groups are replaced by sulfur. These oligonucleotides are designed to treat disease by modulating gene expression of an affected individual. As the development and application of these therapeutical oligonucleotides require analytical support, the development, validation, and application of an assay for the quantitative analysis of a phosporothioate oligonucleotide in rat plasma is described. The method employs ion-pair reversedphase chromatography on a monolithic capillary column with acetonitrile gradients in cyclohexyldimethylammonium acetate for separation and high-resolution tandem mass spectrometry for detection of nucleic acids. Chromatographic parameters (i.e. column temperature, mobile phase composition) as well as mass spectrometric parameters (i.e. spray voltage, gas flow, and capillary position, scan mode) have been optimized for sensitive oligonucleotide quantification. Furthermore, a solid-phase extraction method was developed which enabled processing of 10 μl of plasma. The five-point calibration curve showed linearity over the range of concentrations from 100 to 1,000 nM of the oligonucleotide. The limit of detection was 50 nM. The intra-and inter-day precision and accuracies were always better than 10.2 %. Using this assay, we performed a pharmacokinetic study of the phosporothioate oligonucleotide in rat treated with a single intravenous dose of 0.39 μmol/kg. The assay sensitivity was sufficient to study the early phase elimination of the oligonucleotide. Small amounts of the oligonucleotide were detectable up to 3 h after dosing.