Discontinuous electrolyte systems for improved detection of biologically active amines and acids by capillary electrophoresis with laser-induced native fluorescence detection

Abstract

On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.

Capillary electrophoretic separation of biologically active amines and acids using nanoparticle‐coated capillaries

“…Because the EOF is toward the anodic end, anionic acids were detected earlier than the cationic amines. In the presence of different amounts of ACN, the EOF and the electrophoretic mobilities of the analytes varied, mainly because of different zeta potentials and slight changes in viscosity and their dissociation capabilities [8,37,38]. In addition, they are also due to changes in the interactions between the analytes and the capillary wall.…”

“…Although HPLC is still the most common method for the analysis of biogenic amines and their metabolites, CZE, MEKC, and CEC have recently become attractive [7][8][9][10][11]. CZE is easy but does not provide enough resolving power for the separation of the analytes.…”

“…To optimize separation resolution of amines and acids by CZE, buffer pH value close to 4.0 is favorable. In addition, the analytes are more stable and also possess strong intrinsic fluorescence characteristics at low pH when excited by using a UV laser at 266 nm [7,8]. In order to increase EOF mobility at low pH, strategies such as use of CTAB as well as anionic and cationic polymers to form bilayers or multilayers on the capillary wall have been suggested [12][13][14][15][16].…”

“…In order to increase EOF mobility at low pH, strategies such as use of CTAB as well as anionic and cationic polymers to form bilayers or multilayers on the capillary wall have been suggested [12][13][14][15][16]. Alternately, the separation of biogenic amines and their metabolites by CE in conjunction with LIF detection has been conducted under discontinuous conditions using a capillary treated with 0.5 M NaOH prior to each run [8].…”

“…We note that the compounds (especially acids) are relatively hydrophobic when compared to those at higher pH values. To minimize analyte adsorption, adding salt or organic solvent such as ACN to aqueous electrolytes or using polymer solution like poly(ethylene oxide) as the separation matrix is effective [8].…”