Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration.

The mechanisms underlying the signal changes observed with pharmacological magnetic resonance imaging (phMRI) remain to be fully elucidated. In this study, we obtained microdialysis samples in situ at 5-min intervals during phMRI experiments using a blood pool contrast agent to correlate relative cerebral blood volume (rCBV) changes with changes in dopamine and cocaine concentrations following acute cocaine challenge (0.5 mg/kg iv) in the rat over a duration of 30 min. Three brain areas were investigated: the dorsal striatum (n = 8), the medial prefrontal cortex (mPFC; n = 5), and the primary motor cortex (n = 8). In the striatum and mPFC groups, cocaine and dopamine temporal profiles were tightly correlated, peaking during the first 5-min period postinjection, then rapidly decreasing. However, the local rCBV changes were uncorrelated and exhibited broader temporal profiles than those of cocaine and dopamine, attaining maximal response 5-10 min later. This demonstrates that direct vasoactivity of dopamine is not the dominant component of the hemodynamic response in these regions. In the motor cortex group, microdialysis revealed no local change in dopamine in any of the animals, despite large local cocaine increase and strong rCBV response, indicating that the central hemodynamic response following acute iv cocaine challenge is not driven directly by local dopamine changes in the motor cortex. The combination of phMRI and in situ microdialysis promises to be of great value in elucidating the relationship between the phMRI response to psychoactive drugs and underlying neurochemical changes.

Characterization of dehydroascorbic acid solutions by liquid chromatography/mass spectrometry.

The composition of a commercial dehydroascorbic acid (DA) solution at pH 2 was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to establish the nature of its different forms and its decomposition products. In freshly prepared solutions, dimeric forms of DA and the hydrated bicyclic hemiketal of DA are the species mainly present in solution. In the presence of light, the initial dimeric species disappears over time to give other dehydrated dimers some of which decompose to the monomer. The comparison of these data with similar data obtained for ascorbic acid (AA) solutions under the same experimental conditions revealed that, in the presence of light, the aging of such AA solutions gives rise to only the hemiketal form of DA, and that no dimeric species of DA were formed. The presence of the hemiketal form of DA was not revealed by analysis of the same AA solutions using the conventional LC/UV technique. The natural form of DA from the oxidation of AA is the hydrated bicyclic form.

Synthesis and SAR of new 5-phenyl-3-ureido-1,5-benzodiazepines as cholecystokinin-B receptor antagonists.

A series of 5-phenyl-3-ureidobenzodiazepine-2,4-diones was synthesized and evaluated as cholecystokinin-B (CCK-B) receptor antagonists. Structure-activity relationship (SAR) studies revealed the importance of the N-1 substituent for potent and selective CCK-B affinity. Addition of substituents at the urea side chain provided in some cases more potent compounds. Moreover the introduction of bulky substituents such as adamantylmethyl at N-1 and resolution of the racemic ureas resulted in our lead compound GV150013.

Cycloalkyl indole-2-carboxylates as useful tools for mapping the "north-eastern" region of the glycine binding site associated with the NMDA receptor.

A novel series of indole-2-carboxylate analogues of GV 150526 (1) in which the terminal phenyl ring belonging to the side chain present in the position C-3 has been replaced with a bridged cycloalkyl group was synthesized and evaluated for its pharmacological profile. Modelling studies on this class of novel glycine antagonist allowed us to identify an asymmetric lipophilic pocket present in the "North-Eastern" region of the pharmacophoric model of the glycine binding site associated to the NMDA receptor. Among the derivatives prepared, 3-[2-(1-adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2 -carboxylic acid 6b and 3-[2-(norbornylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-c arboxylic acid 6l were found to be antagonists acting at the strychnine-insensitive glycine binding site, showing nanomolar affinity for the glycine binding site (Ki = 63 and 19 nM, respectively), coupled with high glutamate receptor selectivity (IC50 > 10(-5) M at the NMDA, AMPA, KA binding sites) and high in vivo potency after systemic administration by inhibition of convulsion induced by NMDA in mice.

Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine beta-lactoglobulin B.

Complexation of acrylamide with bovine beta-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine-acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S-S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.

Investigation of newly synthesized peptides by capillary zone electrophoresis/electrospray mass spectrometry.

A series of newly synthesized peptides (M(r) = 1600-2250 Da), corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit have been examined by capillary zone electrophoresis/electrospray-mass spectrometry (CZE/ES-MS). The separation efficiency of CZE combined with the specificity of mass spectrometry allowed rapid and reliable identification of the target peptides and a number of associated side products. In solid-phase synthesis of peptides and small proteins such side products are inevitable, therefore the use of CZE and/or high-performance liquid chromatography combined with mass spectrometry is gaining a central role in such synthesis procedures.

Investigation of crudes of synthesis of [Leu31, Pro34]-neuropeptide Y by capillary zone electrophoresis/mass spectrometry.

[Leu31, Pro34]-Neuropeptide Y is a thirty-six amino-acid peptide which has a measured relative molecular mass of 4222. Solid-phase synthesis of this peptide resulted in complex crudes of synthesis which were examined by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry (ES-MS). The separation efficiency of CZE combined with the mass specificity of mass spectrometry yielded rapid and reliable information on the target peptide and a number of associated side products, which were mainly acetylated peptide sequences having relative molecular masses in the range 2240-4101. Such incomplete or, as they are commonly called, difficult sequences are provoked by problems of swelling and aggregation of the growing peptide chains in the course of synthesis. The use of mass spectrometry is indispensable for obtaining reliable information on the inevitable side products. Initial tuning of the ion source and optimization of the coupling between the CZE system and the mass spectrometer were achieved by performing a number of measurements pertaining to artificially made mixtures of commercial neuropeptides.

Investigation of complexes between some glycopeptide antibiotics and bacterial cell-wall analogues by electrospray- and capillary zone electrophoresis/electrospray-mass spectrometry.

Complexation in aqueous solutions between vancomycin, ristocetin A, teicoplanin and two bacterial cell-wall analogues, Ac2-L-Lys-D-Ala-D-Ala (tripeptide) and Ac-D-Ala-D-Ala (dipeptide) has been examined by positive-ion electrospray mass spectrometry (ES-MS) and capillary zone electrophoresis (CZE)/ES-MS. The ES-MS data demonstrate that as well as complexes between monomeric antibiotics and either peptide, the investigated solutions contained complexes ranging from the simple homodimer of the antibiotic to a more complex association giving ions of the type [2(antibiotic) + 3(tripeptide) + 3H]3+. The same data also demonstrate that the homodimers of the investigated antibiotics are significantly suppressed in the presence of the tripeptide. The use of CZE/ES-MS made it evident that the complexes between the antibiotic and the tripeptide were present in the solution prior to their introduction into the ion source. The two sets of data are compared with existing UV difference spectroscopy and nuclear magnetic resonance data on complexation and dimerization.

Collision-induced dissociation of some protonated peptides with and without mass selection.

Collision-induced dissociation (CID) of several protonated peptides formed by positive-ion electrospray (ES+) and by Cs+ fast-ion bombardment are reported. The CID spectra of ions formed by the first ionization method were effected at elevated extraction cone voltages within the ion source of a single quadrupole instrument, while linked-fields-scan measurements at constant B/E of ions formed by the latter technique were performed within the collision cell of a trisector instrument. For molecular masses below 1000 Da, the two sets of spectra yielded identical sequencing information; however, for higher molecular masses the similarity was less evident. This divergence between the two sets of measurements is partially attributed to dominant doubly charged ions and less efficient dissociation of sodium adduct ions at elevated cone voltages.

Effects of target identity and collision energy on the fragmentation of some mass-selected protonated neuropeptides.

A number of protonated neuropetides formed by 10 keV Cs+ ion bombardment are mass selected and their collision-induced dissociations (CID) with helium, argon, xenon and isobutane are monitored using a collision energy of 200 eV. Data obtained by fixing the collision gas and using translational energies in the range 50-250 eV are also presented. The present data imply that optimization of the CID spectra presented can be achieved through a judicious choice of both the target gas and the translational energy of the mass-selected ion. The combined effect of the two experimental parameters on the relative abundances of the major sequence ions and on the loss of side-chain moieties is discussed.

Characterization of the molecular species of glycerophospholipids from rabbit kidney: an alternative approach to the determination of the fatty acyl chain position by negative ion fast atom bombardment combined with mass-analysed ion kinetic energy analysis.

An alternative approach to identifying fatty acid chain position in the molecular species of glycerophospholipids has been studied and developed. The fatty acyl groups esterified to the glycerol backbone in isomeric glycerophosphatidyl-choline, -serine and -ethanolamine as well as glycerophosphatidic acid can be detected by the presence of a pair of anions derived from phosphatidic acid parent ions (M minus the polar head groups in glycerophospholipids), designed to be [M--polar head--R2COOH]- and [M--polar head--R2CO--H]-, produced by negative ion fast atom bombardment combined with mass-analysed ion kinetic energy analysis. Because of the significant abundance of [M--polar head--R2COOH]- anion, fatty acid chains differing by 2 Da can be distinguished by accurate measurements of the electrostatic voltage related to this ion. Three-volt differences can be evidenced. Using this approach, the molecular species of glycerophosphatidyl-choline, -serine, -ethanolamine and -inositol from rabbit kidney were characterized after the separation of both class and species by normal and reversed-phase high-performance liquid chromatography, respectively. We identified 11 arachidonoyl-containing molecular species of glycerophospholipids and the other 17 lipid molecules in this biological material. A couple of 1- alkenyl-2-arachidonoyl-sn-glycerol-3-phosphoethanolamine species, identified as plasmalogen GPE 16:0-20:4 and plasmalogen GPE 18:0-20:4, were found for the first time in rabbit kidney.

Melanin biosynthesis from dopamine. II. A mass spectrometric and collisional spectroscopic investigation.

Electron impact (EI) and fast atom bombardment (FAB) mass spectrometry together with collisional activation (CA) experiments were applied to the study of the oxidation pathway of dopamine by tyrosinase. In order to prevent attachment of the protein to the highly reactive intermediates, ultrafiltration was employed to remove the enzyme at different reaction times. FAB, privileging molecular species formation, was successfully used for identification of transient intermediates and their relative concentrations with respect to time, directly in the reaction mixture. The presence of isobaric molecular species made chromatographic separation necessary. Further EI mass spectrometry and collision spectroscopy led to structural identification of pure components. Of these, dopamine-o-quinone, leucoaminochrome, and aminochrome semiquinone were characterized for the first time as real intermediates in dopamine melanogenesis, in agreement with previous hypotheses. This approach elucidated the pathway of dopamine melanogenesis.

Ion-trap mass spectrometry applications in forensic sciences. I. Identification of morphine and cocaine in hair extracts of drug addicts.

Daughter-ion spectra obtained by ion-trap mass spectrometry have been successfully employed in the field of drug abuse investigation. Selection and collision-induced fragmentation of molecular-ion species of morphine and cocaine lead to an easy identification of such molecules in hair extracts of heroin and cocaine addicts.

Fast atom bombardment mass spectrometry in the study of dopamine melanogenesis intermediates.

Fast atom bombardment was applied to the study of the intermediates of the reaction dopamine-tyrosinase after treatment with diazomethane. The identification of trimethoxyindoline, dopamine-o-quinone and dimethoxyindole was easily achieved by this ionization method, together with accurate mass measurements and collision experiments. The structures of these compounds are in agreement with those already hypothesized in studies on melanogenesis.