Analysis of the stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography and mass spectrometry

Drew P. Manica, Julie A. Lapos, A. Daniel Jones, Andrew G. Ewing

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34 Scopus citations

Abstract

The stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) was investigated using a combination of high-performance liquid chromatography, solid-phase extraction, photodiode array spectrophotometric detection, and mass spectrometric (MS) characterization. The degradation of amino acid derivatives, generated using β-mercaptoethanol as a nucleophile, was characterized under a variety of environmental influences, with a focus on understanding the degradation kinetics and identifying the degradation products. The predominant degradation product observed under most reaction conditions was the nonfluorescent lactam form of the originally fluorescent isoindole derivative. First, the time-dependent degradation of the isoindole derivative L-serine-NDA-β -mercaptoethanol was found to follow pseudo-first order kinetics with a half-life of 2.0min at pH 9.2 and room temperature. The isoindole derivative was observed to react further with methanol to form a more stable fluorescent methoxy-isoindole, shedding new light on the basis for enhanced stability of these derivatives in methanol. Tandem mass spectrometry (MS/MS) experiments were used to demonstrate unimolecular degradation of the protonated isoindole in the absence of solvent or atmosphere, suggesting an intramolecular reaction mechanism involving the hydroxyethylthio group. Finally, in photobleaching studies, NDA derivatives rapidly degraded into a variety of products within the first 2min of photobleaching versus timed controls, with the predominant product being the lactam. These results suggest that the degradation pathway for NDA derivatives is similar to the previously reported pathway for o-phthalaldehyde derivatives and clearly identifies the reaction and degradation products under a variety of conditions.

Original languageEnglish (US)
Pages (from-to)68-78
Number of pages11
JournalAnalytical Biochemistry
Volume322
Issue number1
DOIs
StatePublished - Nov 1 2003

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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