Marina M. Carrozzo

Chemoselective and enantioselective analysis of proteinogenic amino acids utilizing N-derivatization and 1-D enantioselective anion-exchange chromatography in combination with tandem mass spectrometric detection

D-Amino acid analysis in biological samples still poses a challenge to analytical chemists. In higher developed species trace amounts of d-amino acids have to be detected in vast excesses of the corresponding L-enantiomers. This method utilizes an easy-to-carry-out derivatization step on the amino group with an iron ferrocenyl propionate hydroxy succinimide ester followed by one-dimensional enantioselective anion exchange chromatography with cinchona alkaloid based chiral stationary phases (CSPs). MS detection is carried out in the highly sensitive SRM (selected reaction monitoring) mode, which allows a chemoselective differentiation of amino acid derivatives as well as their enantioselective separation in one step. Application of this method allows LOD (limits of detection) in the low μmol L(-1) range and baseline enantioseparation for all proteinogenic amino acids except for Pro, Arg and His. The D-enantiomers of isomeric Leu and Ile were separated chromatographically and pose an example for the complementary selectivities of LC and MS. A successful application of this procedure to unprocessed human urine indicated the eligibility to analyse biological samples.

Simultaneous measurement of adenosine, dopamine, acetylcholine and 5-hydroxytryptamine in cerebral mice microdialysis samples by LC–ESI-MS/MS

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous measurement of adenosine (ADE), dopamine (DA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) in mouse brain microdialysates. High method sensitivity (LLOQ of 0.05nM) was achieved by optimization of chromatographic and mass spectrometric parameters. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. The LC-MS/MS method was successfully applied to evaluate the effect of the systemic administration of cocaine or amphetamine on the extracellular levels of ADE, DA, ACh and 5-HT in the mouse nucleus accumbens by microdialysis.

Quantitative analysis of acetylcholine in rat brain microdialysates by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the quantitative analysis of acetylcholine in rat brain dialysates. The separation of acetylcholine (ACh), choline (Ch), acetyl-β-methylcholine (IS) from endogenous compounds and Ringers salts was achieved with cation exchange chromatography. Optimization of chromatographic and mass spectrometry parameters were perfomed in order to improve sensitivity of the method. The limit of detection were 0.05 and 3.75 fmol on column with S/N ratio of 3:1 for ACh and Ch, respectively. The limit of quantitation (LOQ) for ACh and Ch measured in Ringers solution were 0.05 nM (0.25 fmol) and 3.75 nM (18.75 fmol), respectively at S/N ratio of 10:1. Linearity of the method has been evaluated in the concentrations range between 0.05 and 5.00 nM and 3.75 and 200 nM for ACh and Ch respectively. The correlation coefficients were 0.999 and 0.995 for ACh and Ch respectively, indicating very good linearity. The LC/MS/MS method developed has been applied to evaluate the effect of oral administration of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21), a positive modulators of AMPA receptor, on the release of ACh in the rat prefrontal cortex by microdialysis.

Enantiomerization of chiral 2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide by stopped-flow multidimensional HPLC ☆

An on-column stopped-flow multidimensional HPLC (sfMDHPLC) procedure using two chiral stationary phases (CSPs) and one achiral C18 column was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide. Moreover, a stopped-flow HPLC (sfHPLC) method previously developed was applied to the determination of kinetic parameters of enantiomerization of the above compound in the presence of a CSP. The individual enantiomers of the studied compound were isolated in parallel by preparative HPLC and the rate constants and free energy barriers of enantiomerization were determined in different solvents (off-column method). The data obtained by sfMDHPLC, sfHPLC and off-column methods were compared. The (S) enantiomer of the studied compound (S18986) was prepared by asymmetric synthesis and subsequently purified by preparative HPLC, followed by the determination of rate constants and free energy barriers of enantiomerization in different buffer solutions at pH 2-9.3.

Enantiomerization and hydrolysis of (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide by stopped-flow multidimensional high-performance liquid chromatography.

A novel stopped-flow multidimensional HPLC (sf-MD-HPLC) procedure has been developed to investigate simultaneously the effect of the pH on the enantiostability and hydrolysis of (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide [(+/-)IDRA21]. It was possible to determinate the rate constants and free energy barriers of enantiomerization and hydrolysis rate constants of (+/-)IDRA21, by using two chiral stationary phases (CSPs) and one achiral C18 column. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis at the same temperature and in the same buffers used in sf-MD-HPLC. The good agreement of the results obtained validate the sf-MD-HPLC procedure. Furthermore, hydrolysis rate constants of (+/-)IDRA21 were calculated in a series of buffers over a pH range of 1.20-10.60 at 37 degrees C in order to evaluate the influence of the pH on hydrolysis.

5-Arylbenzothiadiazine Type Compounds as Positive Allosteric Modulators of AMPA/Kainate Receptors

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPAr) has led to the search for new AMPAr positive modulators. On the basis of crystallographic data of the benzothiadiazines binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-dihydrobenzothiadiazine type compounds has been synthesized and tested. Electrophysiological results suggested that 5-heteroaryl substituents on the benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the activity as positive modulators of AMPAr with respect to IDRA21 and cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-5-(3-furyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide crosses the blood-brain barrier after intraperitoneal injection. Biological results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-positive modulator candidates.

Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer

Benzothiadiazines differently substituted at the sulfonamidic nitrogen atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesized and fully characterized. Enantioseparation of these compounds has revealed rapid on-column enantiomerization. The recently developed software DCXplorer has been successfully applied to calculate enantiomerization kinetic parameters. Enantiomerization barriers of 3-phenyl substituted benzothiadiazines, calculated in this work, have indicated a higher enantiomerization rate suggesting that the aromatic substituent exerts a strong effect on the enantiomerization process. Methyl substitution on N(2) position led to higher free energy barriers of enantiomerization, suggesting negative influence of methyl in the N(2) position on enantiomerization kinetics. However, methylation on N(4) position increases the enantiomerization rates significantly. The results obtained have been employed to postulate an enantiomerization mechanism for chiral benzothiadiazine type compounds.

Simultaneous determination of enantiomerization and hydrolysis kinetic parameters of chiral N-alkylbenzothiadiazine derivatives.

On-column stopped flow multidimensional HPLC (sfMDHPLC) and dynamic high-performance liquid chromatography were applied to investigate the influence of alkyl substituents at the sulfonamidic and amino moieties of benzothiadiazine 1,1-dioxide derivatives on hydrolysis and enantiomerization rate constants. The data obtained indicate the presence of pyrrolo substituent at the 3,4 positions on benzothiadiazine rings inhibits the hydrolysis, whereas the enantiomerization occurs in acidic medium. Hydrolysis rates are quite similar for the two benzothiadiazines methyl substituted to nitrogen at 2- and 4-positions. Conversely, enantiomerization rate of 4-N-methyl substituted is significantly higher than 2-N-methyl substituted.

Evaluation of stereo and chemical stability of chiral compounds

A stopped-flow bidimensional recycle HPLC (sf-BD-rHPLC) configuration has been used to investigate simultaneously the stereo and chemical stability of labile chiral compounds. The single enantiomers of a racemate can be separated on chiral column (first dimension) and each one can be trapped in the achiral column (second dimension) that works as reactor.By filling the achiral column with the appropriate aqueous buffers it is possible to evaluate the stability of the trapped enantiomer toward aqueous buffer itself. It was possible to recycle the reaction products formed in the chiral column (first dimension) where they are separated by a second six valve port. The reaction rate constants were calculated for the different processes occurred in the achiral column by means of corresponding peak areas. The method was applied to a pharmacological active compound: (±)7-chloro-5-ethyl-3-methyl-3,4-dihydro-2H-benzo[1,2,4]thiadiazine 1,1-dioxide ((±)-1) to evaluate enantiostability and hydrolysis in conditions similar to those of biological fluid. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis and enantiomerization at the same temperature and in the same solvents used in sf-BD-rHPLC. The good agreement of the results obtained validate the novel procedure developed. Furthermore, the results generated off-line were used to determine the influence of solvents on the racemization of (±)-1.

On-line racemization by high-performance liquid chromatography

An on-column stopped-flow bidimensional recycling HPLC procedure was developed to obtain an enantiomeric enrichment starting from a racemic mixture. The method developed was applied to two chiral compounds of pharmaceutical interest, (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide (1) and (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-)IDRA21, (2)), since the pharmacological activity of the two benzothiadiazine derivatives investigated has been ascribed to only one enantiomer. Starting from a racemic mixture it was possible to obtain about 95% of pure enantiomer. The procedure was applied both in reverse-phase mode and in normal-phase mode. The scaled up and automatization of the novel analytical HPLC procedure represents a powerful tool to obtain pure enantiomer starting from racemic compounds without cumbersome stereoselective synthesis or expensive enantiopurification processes.