Abstract
Glutamine (Gln) is converted to excitatory (glutamate, aspartate) and inhibitory (γ-amino butyric acid) amino acid neurotransmitters in brain, and is a source of energy during glucose deprivation. Current research utilized an Analytical Quality by Design approach to optimize levels and combinations of critical gas pressure (sheath, auxiliary, sweep) and temperature (ion transfer tube, vaporizer) parameters for high-sensitivity mass spectrometric quantification of brain tissue glutamine. A Design of Experiments (DOE) matrix for evaluation of relationships between these multiple independent variables and a singular response variable, e.g. glutamine chromatogram area, was developed by statistical response surface methodology using central composite design. A second-order polynomial equation was generated to identify and predict singular versus combinatory effects of synergistic and antagonistic factors on chromatograph area. Predicted versus found outcomes overlapped, with enhanced area associated with the latter. DOE methodology was subsequently used to evaluate liquid chromatographic variable effects, e.g. flow rate, column temperature, and mobile phase composition on the response variable. Results demonstrate that combinatory AQbD-guided mass spectrometric/liquid chromatographic optimization significantly enhanced analytical sensitivity for Gln, thus enabling down-sized brain tissue sample volume procurement for quantification of this critical amino acid.
Highlights
Glutamine (Gln) is converted to excitatory and inhibitory (γ-amino butyric acid) amino acid neurotransmitters in brain, and is a source of energy during glucose deprivation
In order to avoid disadvantages of available colorimetric, amperometric, and fluorescence Gln detection methods[14,15,16,17,18,19], including issues arising from matrix interference, prolonged analysis duration, and analyte instability, we developed a combinatory high-resolution micropunch dissection/UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) approach for quantification of the fluorenylmethyloxycarbonyl (FMOC) derivative of Gln, e.g. Gln-FMOC in discrete brain structures
Table 5) reveal www.nature.com/scientificreports that factors sheath gas pressure (SGP), vaporizer temperature (VT), ion transfer tube temperature (ITT), and SGP have a significant impact on area
Summary
Glutamine (Gln) is converted to excitatory (glutamate, aspartate) and inhibitory (γ-amino butyric acid) amino acid neurotransmitters in brain, and is a source of energy during glucose deprivation. Current research utilized an Analytical Quality by Design approach to optimize levels and combinations of critical gas pressure (sheath, auxiliary, sweep) and temperature (ion transfer tube, vaporizer) parameters for high-sensitivity mass spectrometric quantification of brain tissue glutamine. The initial phase of this research utilized CCD methodology, involving performance of DOE-recommended experiments, DOE-generated quadratic equation-based validation of predicted responses, and statistical comparison of design versus desirability in brain tissue samples, to assess five critical mass spectrometric process variables, e.g. sheath gas pressure (SGP), auxiliary gas pressure (AGP), sweep gas pressure (SWGP), ion transfer tube temperature (ITT), and vaporizer temperature (VT), on Gln-FMOC chromatographic area. CCD methodology was employed to evaluate effects of critical liquid chromatographic process variables, such as column temperature, mobile phase flow rate, and mobile phase composition, in order to further optimization of analytical sensitivity for Gln quantification in neural tissue
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