Stopped-flow analysis of Ru(bpy)3(3+) chemiluminescent reactions.

The stopped-flow technique was employed to measure chemiluminescent emission from the reaction of a mixture of oxalate and proline with a chemiluminescence reagent, tris(2,2'-bipyridine) ruthenium(III), or Ru(bpy)3(3+) is a versatile reagent and is often used in bioanalytical applications, including the detection of certain drugs and their metabolites, for example. Unfortunately, Ru(bpy)3(3+) has not yet been fully examined as a possible chemiluminescence reagent for simultaneous kinetic determinations. In this work, a differential reaction rate method, based on simple least squares regressions of the pseudo-first order decay data, was used to resolve two compounds, oxalate and proline, reacting simultaneously with Ru(bpy)3(3+). Our results indicate that stopped-flow analyses with Ru(bpy)3(3+) could provide a viable method for simultaneous determinations of unresolvable analytes of environmental and pharmaceutical importance.

Determination of erythromycin in urine and plasma using microbore liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II) electrogenerated chemiluminescence detection.

Erythromycin is determined in both urine and plasma samples using microbore reversed-phase liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrogenerated chemiluminescence (ECL) detection. Ru(bpy)3(2+) is included in the mobile phase thus eliminating band broadening caused by post-column reagent addition. Extra column band broadening is an important concern in microbore liquid chromatography due to the small peak volumes. Erythromycin was studied in both water and biological samples. The detection limit for erythromycin in standards is 0.01 microM or 50 fmol injected with a S/N of 3 and a linear working range that extends four orders of magnitude. Human urine and blood plasma were also studied. Urine samples were diluted and filtered before injection. Ultrafiltration was used to remove protein from blood plasma samples prior to injection. Erythromycin was selectively detected in the body fluid samples without any further sample preparation. The detection limits obtained for erythromycin in urine and plasma are 0.05 and 0.1 microM, respectively, for 5 microl injected on a 150x1 mm I.D. C18 column.

Determination of Dansyl Amino Acids and Oxalate by HPLC with Electrogenerated Chemiluminescence Detection Using Tris(2,2'-bipyridyl)ruthenium(II) in the Mobile Phase.

A new electrogenerated chemiluminescence detection method is investigated for use in detection in reversed-phase and reversed-phase ion-pair HPLC with Ru(bpy)(3)(2+) in the mobile phase. In this method, different concentrations of Ru(bpy)(3)(2+) are dissolved in the mobile phase and the HPLC column flushed with the mobile phase for 1 h until the column is saturated with Ru(bpy)(3)(2+). The separated analytes along with Ru(bpy)(3)(2+) pass through an optical-electrochemical flow cell which has a dual platinum electrode held at a potential of 1250 mV vs a Ag/AgCl reference electrode. On the surface of the electrode, Ru(bpy)(3)(2+) is oxidized to Ru(bpy)(3)(3+) which reacts with the analytes to emit light. The retention times, retention orders, detection limits, and linearity in working curves are compared to those obtained with the conventional postcolumn Ru(bpy)(3)(2+) addition method. The retention times for dansyl amino acids with Ru(bpy)(3)(2+) in the mobile phase are longer than those obtained with the postcolumn addition approach. This may be caused by π-to-π interactions between the aromatic groups of the dansyl derivatives and the bipyridyl groups of Ru(bpy)(3)(2+) in the Ru(bpy)(3)(2+)-saturated reversed-phase column. Similarly, oxalate is separated from urine and blood plasma samples by reversed-phase ion-pair HPLC. Plasma samples are obtained using ultrafiltration to remove proteins from whole blood. Retention times for oxalate with the two detection techniques are identical, and detection limits for these techniques are compared.

Determination of oxalate in urine and plasma using reversed-phase ion-pair high-performance liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II)-electrogenerated chemiluminescence detection.

Oxalate is quantitated in both urine and plasma samples using reversed-phase ion-pair high-performance liquid chromatography (HPLC) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)2+(3)]-electrogenerated chemiluminescent (ECL) detection. Underivatized oxalate was separated on a reversed-phase column (Zorbax ODS) using a mobile phase of 10% methanol in 100 mM phosphate buffer at pH 7.0. The eluted compounds were combined with a stream of 2 mM Ru(bpy)2+(3) at a mixing tee before the ECL flow-cell. In the flow-cell, Ru(bpy)2+(3) is oxidized to Ru(bpy)3+(3) at a platinum electrode, and reacts with oxalate to produce chemiluminescence (CL). Urine samples were filtered and diluted prior to injection. Plasma samples were deproteinized before injection. A 25-microliters aliquot of sample was injected for analysis. Possible interferants, including amino acids and indole-based compounds, present in biological samples were investigated. Without the separation, amino acids interfere by increasing the total observed CL intensity; this is expected because they give rise to CL emission on their own in reaction with Ru(bpy)3+(3). Indole compounds exhibit a unique interference by decreasing the CL signal when present with oxalate. Indoles inhibit their own CL emission at high concentrations. By use of the indicated HPLC separation, oxalate was adequately separated from both types of interferants, which thus had no effect on the oxalate signal. Urine samples were assayed by both HPLC and enzymatic tests, the two techniques giving similar results, differing only by 1%. Detection limits were determined to be below 1 microM (1 nmol/ml) or 25 pmol injected. The working curve for oxalate was linear throughout the entire clinical range in both urine and plasma.

Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition.

Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k'1[AE] + k''1[AE][OH-]0.5, where k'1 = 0.020 +/- 0.006 s-1 and k''1 = 2.1 +/- 0.8 (L/mol)-0.5 s-1. Irreversible decomposition of the pseudobase is described by rate = k'2[AE][OH-], where k'2 = 20.1 +/- 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.

Chemiluminescence detection using regenerable tris(2,2'-bipyridyl)ruthenium(II) immobilized in Nafion.

The development of a detection method based on the electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+], immobilized in a Nafion film coated on an electrode is discussed. Control of the electrode potential controls creation of the reactive reagent Ru(bpy)3(3+) which reacts with certain analytes to yield chemiluminescence emission of intensity proportional to the analyte concentration. The reaction results in Ru(bpy)3(3+) being converted to Ru(bpy)3(2+), which then is recycled to Ru(bpy)3(3+) again at the electrode. This sensor has been used in flow injection to determine oxalate, alkylamines, and NADH. Detection limits are 1 microM, 10 nM, and 1 microM, respectively, with working ranges extending over 4 decades in concentration. Sensitivity is constant over the wide pH range from 3 to 10. With oxalate, and to a small extent with amines, emission intensities increase with increasing ionic strength; this was shown to be a phenomenon related to the Nafion film and not to the chemiluminescence reaction. Emission intensities increase with temperature. The sensor remains stable for several days with suitable storage conditions. Significant amounts of Ru(bpy)3(3+) are shown to be capable of storage within the film.

Conductometric monitoring of the amino acid oxidase reaction as a detector for high-performance liquid chromatography.

The deamination of L-amino acids by L-amino acid oxidase, which creates a change in the ionic strength of an HPLC eluent, is used to allow conductometric detection of L-amino acids. An example separation is given, and the conductometric method is compared to other methods of amino acid detection in the area of detection limits. Suggestions are also given for using this principle to create detectors based on other class selective enzymes.

Chemiluminescent reaction of lucigenin with reducing sugars.

The chemiluminescent reaction, in alkaline solution, of lucigenin with the reducing sugars sorbose, fructose, lactose, glucose, xylose, galactose, arabinose and mannose has been studied. There is a linear relationship (correlation coefficient = 0.996) between the emission intensity and the second-order rate constant for the alkaline oxidation of these sugars. The emission intensity is linearly related to the sugar concentration; at high sugar concentrations (5mM) it is independent of the lucigenin concentration, but at low concentrations (<0.05mM) is linearly related to the lucigenin concentration. These facts support the view that the rate-limiting step is the tautomerization of the sugars to the 1,2-enediol form; the enediol then undergoes reaction with lucigenin.