title: Quantitation of various indolinyl caged glutamates as their o-phthalaldehyde derivatives by high performance liquid chromatography coupled with tandem spectroscopic detections: derivatization, stoichiometry and stability studies.
abstract: Quantification, stability and unique spectroscopic properties of indolinyl-caged glutamates (ICGs), with the o-phthalaldehyde-3-mercaptopropionic acid (OPA-MPA) reagent, were described, at first. As new principle to the field, reactivity and stoichiometry of variously substituted OPA-MPA derivatized ICGs, such as 4-methoxy-7-nitroindolinyl-(MNI-Glu), 4-methoxy-5,7-dinitroindolinyl-(DNI-Glu), 2-dimethylamino-propoxy and dimethylamino-isobutoxy alternatives (2DMA-1PO-DNI-Glu, 1DMA-2P-DNI-Glu and 3DMA-1iBU-DNI-Glu), was demonstrated. Derivatives' stability was determined using high performance liquid chromatography (HPLC) applying simultaneous photodiode array (DAD) and fluorescence (Fl) detections, while their structural identity was confirmed by HPLC-time of flight mass spectrometry (HPLC-TOF-MS). The SH-additive of the reagents was also varied. ICGs react unequivocally, with one OPA-SH-group molecule, in the molar ratios of ([OPA-SH-additive]/[ICG]=1/1, resulting in species with the characteristic isoindole spectral property (EEx/EEm=337/454nm; λmax=337nm). ICGs' isoindole derivatives, due to their sandwich structure, are manifesting the π-π-stacking phenomenon: they fail to show fluorescence. ICGs' stability decreased in the order of MNI-Glu, 2DMA-1PO/1DMA-2PO, 3DMA-1iBU and DNI-Glu, correspondingly, resulting in increasing order of free glutamic acid (GA), as their decomposition product. GA and ICGs were determined as their OPA/MPA derivatives while uncaged species (MNI, DNI and its substituted alternatives) in their initial forms. The practical utility of the method was confirmed analyzing ICGs and their decomposition products, simultaneously. Quantifications' reliability and reproducibility were characterized with the relative standard deviation percentages of responses (RSDs%): for GA 0.41-12 RSD% for ICGs 0.057-7.0 RSD% were obtained. Stability properties of variously substituted, recently introduced ICGs, prepared in laboratories of Institute of Experimental Medicine, were defined.
authors.affiliation: Institute of Chemistry, Department of Analytical Chemistry, L. Eötvös University, Pázmány Péter sétány 1/A, Budapest 1117, Hungary. Electronic address: vasa@chem.elte.hu.
authors.forename: A
authors.initials: A
authors.lastname: Vasanits-Zsigrai
authors.affiliation: Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, Budapest 1083, Hungary.
authors.forename: O
authors.initials: O
authors.lastname: Majercsik
authors.affiliation: Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes Endre utca 7, Budapest 1092, Hungary.
authors.forename: G
authors.initials: G
authors.lastname: Tóth
authors.affiliation: Institute of Chemistry, Department of Inorganic Chemistry, L. Eötvös University, Pázmány Péter sétány 1/A, Budapest 1117, Hungary.
authors.forename: A
authors.initials: A
authors.lastname: Csámpai
authors.affiliation: Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, Budapest 1083, Hungary.
authors.forename: Cs
authors.initials: C
authors.lastname: Haveland-Lukács
authors.affiliation: Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, Budapest 1083, Hungary.
authors.forename: D
authors.initials: D
authors.lastname: Pálfi
authors.affiliation: Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, Budapest 1083, Hungary.
authors.forename: Z
authors.initials: Z
authors.lastname: Szadai
authors.affiliation: Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, Budapest 1083, Hungary.
authors.forename: B
authors.initials: B
authors.lastname: Rózsa
authors.affiliation: Institute of Chemistry, Department of Analytical Chemistry, L. Eötvös University, Pázmány Péter sétány 1/A, Budapest 1117, Hungary. Electronic address: perlne@chem.elte.hu.
authors.forename: I
authors.initials: I
authors.lastname: Molnár-Perl
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005971:Glutamates / Q000737:chemistry*; D007202:Indicators and Reagents; D007211:Indoles / Q000737:chemistry*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D009764:o-Phthalaldehyde / Q000737:chemistry*
chemical_list: D005971:Glutamates; D007202:Indicators and Reagents; D007211:Indoles; D009764:o-Phthalaldehyde
keywords: 3-Mercaptopropionic acid; Derivatization; Glutamic acid; HPLC-UV/Fl/TOF-MS; Indolinyl caged glutamates; o-Phthalaldehyde