Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization.
Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.
Ptolemy
Adam S
AS
Department of Chemistry, McMaster University, Hamilton, Ont. L8S 4M1, Canada.
Tran
Lara
L
Britz-McKibbin
Philip
P
D000111:Acetylcysteine; D000409:Alanine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods; D003470:Culture Media / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D009764:o-Phthalaldehyde
D000596:Amino Acids; D003470:Culture Media; D009764:o-Phthalaldehyde; D000409:Alanine; D000111:Acetylcysteine