DETERMINATION OF TOTAL AND D-AMINO ACID CONTENT IN MICE BRAIN TISSUE BY ACHIRAL-CHIRAL HEART-CUTTING TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY: DIAGNOSTIC AND CLINICAL RELEVANCE AND A COMPARISON BETWEEN NON-PERFUSED AND PERFUSED TISSUE
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A heart-cutting two dimensional liquid chromatographic method for the quantification of free amino acid enantiomers from mouse brain tissue is demonstrated. Evidence to support the occurrence of D-amino acids in significant concentrations in physiological samples has spurred studies toward the elucidation of their importance and function. In this work, the level and regional distribution of select amino acids were determined by achiral-chiral LC/LC. Additionally, the effect of tissue perfusion on these levels was investigated. The free amino acid extracts were analyzed as their 9-fluorenylmethyloxycarbonyl derivatives and separated in the reverse phase mode on superficially porous C18. In the second dimension, enantiomers of selected amino acids were separated using a house prepared column featuring superficially porous silica derivatized with teicoplanin, a chiral selector known to exhibit excellent selectivity toward isomers of native and N-blocked amino acids. Total concentrations of free amino acids were determined through the use of an internal standard and enantiomeric ratios for 11 standard amino acids were determined with high sensitivity using fluorometric detection. Because some D-amino acids have been suggested to play a role in neurodevelopment and neurotransmission, and may have potential as disease biomarkers, the interest in their occurrence and disposition has recently grown.