A rapid and sensitive CE method with field‐enhanced sample injection and in‐capillary derivatization for selenomethionine metabolism catalyzed by flavin‐containing monooxygenases

Abstract

A rapid and sensitive electrophoretically mediated microanalysis method with field-enhanced sample injection (FESI) for in-capillary derivatization was developed to determine selenomethionine (SeMet) and selenomethionine selenoxide (SeOMet). Phthalic anhydride (PA) was selected as the derivatization reagent due to the fast reaction at room temperature and the stability of derivatives. The in-capillary derivatization was accomplished by electrophoretically mixing PA and sample plugs. PA reagent was introduced hydrodynamically into the capillary, whereas the sample solution was injected electrokinetically, thus allowing a selective preconcentration of the analytes by FESI. For FESI, the optimum sample solvent was 2 mM borate solution. The borate buffer was suitable for both in-capillary derivatization and separation of the derivatives. The combination of electrophoretically mediated microanalysis with FESI for in-capillary derivatization was successfully achieved with about 800-fold concentration sensitivity enhancement compared to direct CE-UV detection in the same setup. The present method is miniaturized and fully automated, which ensures the on-line derivatization, stacking, separation and detection in 10 min. Finally, the developed method was successfully applied to measure enzyme activities by analyzing the reaction mixtures of SeMet with human flavin-containing monooxygenases (FMO). The results showed that both FMO1 and FMO3, but not FMO5 could catalyze the Se-oxygenation of SeMet.

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…An alternative for sensitive assays is the use fluorescence-labeled substrates in combination with laser-induced fluorescence (LIF) detection [22][23][24][25][26][27]. When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed.…”

“…Assay sensitivity was increased by field-amplified sample injection techniques in CE methods for sirtuin enzymes [19,21] and flavin-containing monooxygenases [22]. Furthermore, the reversed electrode polarity stacking mode was employed in a MEKC method for the monitoring of purine nucleoside phosphorylase and adenosine deaminase activity [23].An alternative for sensitive assays is the use fluorescence-labeled substrates in combination with laser-induced fluorescence (LIF) detection [22][23][24][25][26][27]. When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed.…”

“…Huang and Tzeng used polyethylene glycol 4000 in the BGE as the EOF suppressor for sample stacking by transient isotachophoresis when analyzing the activity of ribonuclease reductase [20]. Assay sensitivity was increased by field-amplified sample injection techniques in CE methods for sirtuin enzymes [19,21] and flavin-containing monooxygenases [22]. Furthermore, the reversed electrode polarity stacking mode was employed in a MEKC method for the monitoring of purine nucleoside phosphorylase and adenosine deaminase activity [23].…”

“…Carbohydrate substrates were derivatized by reductive amination with 7-aminonaphthalene-1,3-disulfonic acid [35]. The group of Van Schepdael used an interesting approach combining pre-capillary incubation of flavin-containing monooxygenases with in-capillary analyte derivatization and field-amplified sample injection [22]. The oxidation of selenomethionine to selenomethionine selenoxide by the oxygenases served as model reaction.…”

Simultaneous Determination of Pyridine-Triphenylborane Anti-Fouling Agent and Its Degradation Products in Paint-Waste Samples Using Capillary Zone Electrophoresis with Field-Amplified Sample Injection

“…However, CE has a low concentration sensitivity from minute sample volumes (at the nL level), and a short optical path length (typically 25 -100 μm) available with UV detection. 1 Different types of in-line sample concentration techniques have been proposed to enhance the CE sensitivity: large volume sample stacking, 2 large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP), 3,4 sweeping, [5][6][7] dynamic pH junction, 8,9 field-amplified sample injection (FASI), [10][11][12][13][14][15] two-end field amplified sample injection (TE-FASI), 16 acetonitrile (ACN)-mediated stacking, 17 isotachophoresis (ITP) and transient ITP, [18][19][20] electrokinetic supercharging (EKS), [21][22][23] counter-flow electrokinetic supercharging (CF-EKS), 24 pressure-assisted injection techniques, 25,26 and simultaneous electrokinetic and hydrodynamic injection (SEHI). 27,28 These procedures are useful, but some were somewhat complicated in their application to real samples.…”