Mass spectrometry as a new detector for capillary electrophoresis: applications in forensic science

The present thesis explores the potential and achievements of capillary electrophoresis-mass spectrometry (CE-MS) hyphenated technique for forensic science purposes. After an extensive overview of Capillary Electrophoresis coupled to Mass Spectrometry (a review of CE-MS applications of forensic and toxicological interest updated to 2008 is presented in Chapter 2), two new original CE-MS applications of forensic interest are proposed, differing in scope, mass detection mode and molecular mass range. The first application (Chapter 3) deals with protein analysis and posttranslational modifications of hemoglobin (Hb) induced by severe alcohol abuse. The fragmentation ability of MS tridimensional ion trap is exploited for structure investigation, and data dependency is used as experimental mode to improve the quality and quantity of information gained in each analytical run. Hb adducts with acetaldehyde (AcH) had been recognized as possible markers for alcohol abuse since 1980s, but the lack of sensitive and specific techniques has hampered for years both the recognition of AcH-Hb adducts in volunteer blood samples and their systematic structural investigation. With the available literature data in our hands, we have exploited the on-line coupling of CE (P/ACE MDQ, Beckman Instruments, Fullerton, CA-US) with ion trap mass detector (LCQ Deca XP-Max, Thermo Fisher Scientific, San Josè, CA-US) through a co-axial ESI interface, properly modified for CE coupling, in order to identify and characterize Hb adducts with AcH obtained by in vitro incubation experiments. Optimisation of CE separation and mass detection conditions led us to the identification of all peptides gained by the tryptic digestion of Hb incubated with AcH at the physiologically relevant AcH concentration (0.2 mM), for different incubation times. The aid of data dependent experiments - comprising three subsequent steps (Triple play®, ThermoFisher Scientific) – enabled us to accurately measure the mass value of the ions carrying AcH residues and to identify the site of addition through the analysis of the fragmentation patterns. The condensation products of AcH with the terminal Valine (Ach-α1 and Ach-β1) and two further adducts (Ach-α4 and Ach-β3) on both alpha and beta Hb chains have been identified. This is the first time that four different adducts are identified and characterized by means of in vitro experiments using a sub millimolar concentration of acetaldehyde. The second application presented in this thesis (Chapter 4) is aimed at the identification of low molecular weight molecules of toxicological interest and their metabolites in hair. The mass accuracy achievable with high resolution time of flight (TOF) spectrometer has enabled the identification of unknown molecules in a generic experimental set-up. Hair testing is widely employed in forensic toxicology to investigate chronic drug abuse, since it shows the advantages of easy sample manipulation, collection and storage, poor metabolism and molecule preservation from both internal and external degradation, allowing for long-term stability (even months) of the absorbed substances. The rapid identification of drugs, toxicants and their metabolites by generic search methods is crucial for the prompt diagnosis of intoxication cases, for the characterization of drug abuse or misuse and, often, for the elucidation of the cause of death. CE (P/ACE 550, Beckman Instruments, Fullerton, CA-US) combination with TOF mass spectrometer (MicrOTOF, Bruker Daltonics, Bremen, Germany) looks particularly promising for the aim of accomplishing generic toxicological analysis of unknown samples, due to TOF high resolution (typically 10000), mass accuracy (lower than 5 ppm for molecular weights up to 500 Da) and full-scan sensitivity. Moreover, being the high scan speed of this pulsed mass spectrometer suitable for coupling with the highly efficient CE technique, the combination produces also the advantages of rapid method development and minimal sample requirements - typical of electrophoretic techniques - which are of major importance in broad spectrum toxicological analysis. In the present work, generic search of illicit and therapeutic drugs and their metabolites was carried out in hair specimens collected from subjects under treatment in a therapeutic community for a history of drug or alcohol addiction. The real cases study demonstrates that the combination of CE separation with TOF mass detection provides a proficient, although not exhaustive, approach to the general unknown screening of drugs in hair, showing the advantages of speed of analysis, retrieval of all valuable information in a single analytical run and good confidence in molecular identification through the evaluation of exact mass and ion isotopic pattern. The proposed method can provide an effective diagnostic power for toxicant identification and semi-quantitative comparison towards a cut-off limit of admissibility, although scarce suitability is proved for accurate quantitation of analytes in biological matrices. The two mass detectors employed in the research work presented here have different features, strengths and weaknesses and they have shown to fit for different purposes. The hyphenation of a highly efficient separation technique - such as CE - with the unequally specific mass detector, provides a unique tool in the hand of forensic scientists for screening and confirmation tests in many circumstances, combining the advantages of easy experimental set-up, tiny sample and solvent requirements, fast analytical runs, sensitivity, identification power. Although attractive, the hyphenation of CE with MS is a technological challenge, for the intrinsic features of the two techniques. The issues, historical solutions and technological developments of CE-MS coupling are discussed in the first part of the Thesis (Chapter 1). In particular, the key elements involved in the design of a CE-MS hyphenated instrument (namely, the ion source and the interface) and the mode they are connected to the mass analyzer are described in details. The recent achievements of CE-MS coupling are demonstrated by the huge rate of publishing in the last five to ten years in several scientific fields, ranging from pharmaceutical to environmental to “omic” sciences. Despite the cautious and sometimes suspicious forensic scientists’ attitude towards new techniques and methods, the recent technological improvements of CE-MS set-up have promoted its increased use as daily and well-established technique for formal testing in various application fields.