Highly sensitive analysis of catecholamines by counter-flow electrokinetic supercharging in the constant voltage mode.

Electrokinetic supercharging is one of the most powerful sample-stacking methods that combines field amplified sample injection and transient ITP. In counter-flow electrokinetic supercharging, a constant counter pressure is applied during sample injection in order to counterbalance the movement of the injected sample zone. As a result, there will be a pronounced increase in the amount of sample injected and the portion of the capillary available for electrophoresis. In this report, counter-flow electrokinetic supercharging optimization factors such as the electric field application in the constant voltage and constant current modes, the magnitude of counter pressure, and the terminating electrolyte concentrations were investigated. The enrichments obtained with a 30 min injection of 10 nM catecholamines in 5 mM terminating electrolyte solution in the constant voltage mode applying a counter pressure of 1.3 psi were 41,000-fold for dopamine, 50,000-fold for norepinephrine, and 32,000-fold for epinephrine, yielding detection limits of 1.3, 1.4, and 1.2 nM, respectively, with absorbance detection at 200 nm.

Direct chiral analysis of primary amine drugs in human urine by single drop microextraction in-line coupled to CE.

Three-phase single drop microextraction (SDME) was in-line coupled to chiral CE of weakly basic amine compounds including amphetamine. SDME was used for the matrix isolation and sample preconcentration in order to directly analyze urine samples with the minimal pretreatment of adding NaOH. A small drop of an acidic aqueous acceptor phase covered with a thin layer of octanol was formed at the tip of a capillary by simple manipulation of the liquid handling functions of a commercial CE instrument. While the saline matrix of the urine sample was blocked by the octanol layer, the basic analytes in a basic aqueous donor phase were concentrated into the acidic acceptor drop through the octanol layer by the driving force of the pH difference between the two aqueous phases. The enantiomers of the enriched amines were resolved by using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector for the subsequent CE separation. From 10 min SDME with the agitation of the donor phase by a small stirrer retrofit to the CE instrument, enrichment factors were about a 1000-fold, yielding the LOD of 0.5 ng/mL for amphetamine. This low LOD value as well as the convenience of in-line coupled SDME make the proposed scheme well suited for the demanding chiral analysis of amphetamine-type stimulants.

A bifunctional molecule as an artificial flavin mononucleotide cyclase and a chemosensor for selective fluorescent detection of flavins.

Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF, vitamin B(2)), play important roles in numerous redox reactions such as those taking place in the electron-transfer chains of mitochondria in all eukaryotes and of plastids in plants. A selective chemosensor for flavins would be useful not only in the investigation of metabolic processes but also in the diagnosis of diseases related to flavins; such a sensor is presently unavailable. Herein, we report the first bifunctional chemosensor (PTZ-DPA) for flavins. PTZ-DPA consists of bis(Zn(2+)-dipicolylamine) and phenothiazine. Bis(Zn(2+)-dipicolylamine) (referred to here as XyDPA) was found to be an excellent catalyst in the conversion of FAD into cyclic FMN (riboflavin 4',5'-cyclic phosphate, cFMN) under physiological conditions, even at pH 7.4 and 27 degrees C, with less than 1 mol % of substrate. Utilizing XyDPA's superior function as an artificial FMN cyclase and phenothiazine as an electron donor able to quench the fluorescence of an isoalloxazine ring, PTZ-DPA enabled selective fluorescent discrimination of flavins (FMN, FAD, and RF): FAD shows ON(+), FMN shows OFF(-), and RF shows NO(0) fluorescence changes upon the addition of PTZ-DPA. With this selective sensing property, PTZ-DPA is applicable to real-time fluorescent monitoring of riboflavin kinase (RF to FMN), alkaline phosphatase (FMN to RF), and FAD synthetase (FMN to FAD).

Single drop microextraction using commercial capillary electrophoresis instruments.

Single drop microextraction (SDME), a simple and efficient sample preconcentration method for capillary electrophoresis (CE), was performed using two different commercial CE instruments. With a CE instrument providing adjustable forward and backward pressures, a single drop of an aqueous acceptor phase covered with a thin layer of an organic phase was formed at the capillary tip by programming the sample-handling functions of the instrument to perform 3-phase SDME where the organic film is essentially a membrane. Analytes from an acidic donor phase were concentrated into a basic acceptor phase yielding 190-fold enrichment in 10 min. When the donor phase was agitated using a microstirrer, a 2000-fold enrichment was obtained in 10 min. For a less flexible CE instrument, 2-phase SDME was carried out with a large volume pentanol drop held by a Teflon sleeve, yielding 110-fold enrichments in 30 min. We demonstrate a practical and automated SDME-CE technique with accuracy and reproducibility that is easy to practice to attain matrix isolation and high concentration sensitivity.