High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]+ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MSn analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well

The Methodology of Forensic Neuroscience

Insanity is a distinctive element of criminal law because it brings together two very different disciplines, psychiatry and psychology on the one hand and the law on the other. It might strongly benefit from the introduction of structural neuroimaging, that, however, had so far a limited translational impact. Brain imaging purports to demonstrate functional status and thereby it can be useful to provide a scientific explanation for the clinical symptoms strenghtening the medico-legal reasoning. Despite international cases using brain imaging to support diminished responsibility, in Italy there are still a lot of controversies. Is the neuroscientific logic deterministic? How may the classic psychiatric/neurologic examination and neuroscientific evidence work side by side? Are the symptoms not legally relevant really not relevant? Could the study of the brain inform the clinical diagnosis? Could the study of the brain inform the expert opinion on responsibility and insanity? In this chapter, we describe the cognitive and behavioral profile of a defendant charged with murder, as well as his brain imaging correlates. Through the analysis of this real forensic case, we address the above questions and conclude that neuroscience may strengthen the results of psychiatric evaluations, thus reducing uncertainty in the forensic settings. We claim that besides the clinical diagnosis, the study of the brain allows a better understanding of the individual acts