High‐throughput method for the analysis of venlafaxine in pharmaceutical formulations and biological fluids,using a tris(2,2′‐bipyridyl) ruthenium(II)–peroxydisulphate chemiluminescence system in a two‐chip device

A simple, rapid and sensitive chemiluminescent (CL) method for the assay of venlafaxine (VEN) in pharmaceutical formulations and serum samples by a two‐chip device is proposed. The method is based on the reaction of this drug with a tris(2,2′‐bipyridyl) ruthenium(II)–peroxydisulphate CL system. The optimum chemical conditions for CL emission were investigated. The calibration graph was linear for the concentration range 0.02–8.0 µg/mL. The detection and quantification limits were found to be 0.006 and 0.018 µg/mL, respectively, while the relative standard deviation (RSD) was <2.0%. The present CL procedure was applied to the determination of VEN in pharmaceutical formulations and serum samples; the recovery levels were in the range 96.5–101.2%. The results suggest that the method is unaffected by the presence of common formulation excipients found in these samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Electrochemiluminescence of tris(2,2′‐bipyridyl)ruthenium and its applications in bioanalysis: a review

Electrochemiluminescence (ECL) of tris(2,2’‐bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺] is an active research area and includes the synthesis of ECL‐active materials, mechanistic studies and broad applications. Extensive research has been focused on this area, due to its scientific and practical importance. In this mini‐review we focus on the bio‐related applications of ECL. After a brief introduction to Ru(bpy)₃²⁺ ECL and its mechanisms, its application in constructing an effective bioassay is discussed in detail. Three types of ECL assay are covered: DNA, immunoassay and functional nucleic acid sensors. Finally, future directions for these assays are discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of fexofenadine in pharmaceutical formulations using tris(1,10‐phenanthroline)–ruthenium(II) peroxydisulphate chemiluminescence system in a multichip device

A simple, rapid and sensitive method has been developed for the analysis of fexofenadine (FEX) in pharmaceutical formulations, using a tris(1,10‐phenanthroline)–ruthenium(II) [Ru(phen)₃²⁺] peroxydisulphate chemiluminescence (CL) system in a multichip device. Various parameters that influence the CL signal intensity were optimized. These included pH, flow rates and concentration of reagents used. Under optimum conditions, a linear calibration curve in the range 0.05–5.0 µg/mL was obtained. The detection limit was found to be 0.001 µg/mL. The procedure was applied to the analysis of FEX in pharmaceutical products and was found to be free from interference from concomitants usually present in these preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

Flow‐injection determination of vitamin A in pharmaceutical formulations using Tris(2,2′‐bipyridyl)Ru(II)–Ce(IV) chemiluminescence detection

A flow injection method with chemiluminescence detection is reported for the determination of vitamin A. The method is based on the enhancement effect of vitamin A on chemiluminescence of tris(2,2′‐bipyridyl)Ru(II)–Ce(IV) in acidic medium. The proposed procedure is used to quantitate vitamin A in the range 1.0–100 × 10^?6 mol/L with a correlation coefficient of 0.9991 (n = 9) and relative standard deviation in the range 1.2–2.3% (n = 4). The limit of detection (3 × blank) was 8.0 × 10^?8 mol/L with a sample throughput of 100/h. The effect of common excipients used in pharmaceutical formulations and some clinically important compounds was also studied. The method was applied to determine vitamin A in pharmaceutical formulations and the results obtained were in reasonable agreement with the amount quoted. The results were compared using spectrophotometric method and no significant difference was found between the results of the two methods at 95% confidence limit. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatographic determination of acetylcholine based on pre‐column alkaline cleavage reaction and post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection

A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre‐column alkaline cleavage of ACh to form trimethylamine (TMA) and the post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180°C in the presence of lithium hydroxide, and the produced TMA was separated on a cation‐exchange/reversed‐phase dual‐functional column using a mixture of 0.2 m potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2′‐bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal‐to‐noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 µL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations. Copyright © 2009 John Wiley & Sons, Ltd.     

Flow‐injection analysis for meloxicam based on tris(2,2′‐bipyridine) ruthenium(II)–Ce(IV) chemiluminescent system

It was found that meloxicam could enhance the chemiluminescence (CL) of the tris(2,2'‐bipyridine) ruthenium(II)–Ce(IV) system in the medium of sulfate acid. Based on this phenomenon a new flow‐injection system with chemiluminescent detection has been proposed for determination of meloxicam. Under optimum conditions, meloxicam had a good linear relationship with the CL intensity in the concentration range of 6.0  10^?4 to 1.0 µg/mL and the detection limit was 3.7 × 10^?4 µg/mL. The proposed method was applied to detect meloxicam in tablets and a satisfactory recovery was obtained. The possible mechanism for this CL system is also discussed in this paper. Copyright © 2009 John Wiley & Sons, Ltd.     

Nepem‐211 ion exchange conductive membrane immobilized tris(2,2´‐bipyridyl) ruthenium(II) electrogenerated chemiluminescence flow sensor for high‐performance liquid chromatography and its application

We developed a sensitive and robust electrogenerated chemiluminescence (ECL) flow sensor based on Ru(bpy)₃²⁺ immobilized with a Nepem‐211 perfluorinated ion exchange conductance membrane, which has robustness and stability under a wide range of chemical and physical conditions, good electrical conductivity, isotropy and a high exchange capacity for immobilization of Ru(bpy)₃²⁺. The flow sensor has been used as a post‐column detector in high‐performance liquid chromatography for determination of erythromycin and clarithromycin in honey and pork, and tricyclic antidepressant drugs in human urine. Under optimal conditions, the linear ranges were 0.03–26 ng/μL and 0.01–1 ng/μL for macrolides and tricyclic antidepressant drugs, respectively. The detection limits were 0.02, 0.01, 0.01, 0.06 and 0.003 ng/μL for erythromycin, clarithromycin, doxepin, amitriptyline and clomipramine, respectively. There is no post‐column reagent addition. In addition to the conservation expensive reagents, the experimental setup was simplified. The flow sensor was used for 2 years with high sensitivity and stability. Copyright © 2013 John Wiley & Sons, Ltd.     

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)₃²⁺] electrogenerated chemiluminescence (ECL) detection. Ru(bpy)₃²⁺ 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 μM 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 μM, respectively, for 5 μl injected on a 150×1 mm I.D. C₁₈ column.     

Flow injection chemiluminescent determination of tetracycline using a tris(2,2'-bipyridine)ruthenium(II)-cerium(IV) sulphate system.

A flow-injection chemiluminescence method for the determination of tetracycline was developed. The method is based on an enhancement by tetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine)ruthenium(II). In sulphuric acid medium, the chemiluminescence is generated by the continuous oxidation of tris(2,2'-bipyridine)ruthenium(II) by cerium (IV) sulphate. The light-emission intensity is greatly enhanced in the presence of tetracycline. Under the optimum conditions, the calibration curve is linear over the range 3.75 x 10(-8) g/mL-1.5 x 10(-5) g/mL for tetracycline with the linear equation: deltaINT = 205.898 x C - 20.442 (R2 = 0.9974). The detection limit is 3.27 x 10(-8) g/mL. The proposed method was also successfully used to determine tetracycline in pharmaceutical formulation (mean recovery of tetracycline, 100.7%).     

Flow‐injection chemiluminescent determination of estrogen benzoate using the tris(1,10‐phenanthroline) ruthenium(II)–permanganate system

Chemiluminescence (CL) detection for the determination of estrogen benzoate, using the reaction of tris(1,10–phenanthroline)ruthenium(II)–Na₂SO₃–permanganate, is described. This method is based on the CL reaction of estrogen benzoate (EB) with acidic potassium permanganate and tris(1,10–phenanthroline)ruthenium(II). The CL intensity is greatly enhanced when Na₂SO₃ is added. After optimization of the different experimental parameters, a calibration graph for estrogen benzoate is linear in the range 0.05–10 µg/mL. The 3 s limit of detection is 0.024 µg/mL and the relative standard deviation was 1.3% for 1.0 µg/mL estrogen benzoate (n = 11). This proposed method was successfully applied to commercial injection samples and emulsion cosmetics. The mechanism of CL reaction was also studied. Copyright © 2011 John Wiley & Sons, Ltd.     

Perturbation of the tris(2,2′‐bipyridine) ruthenium(II)‐catalyzed Belousov–Zhabotinsky oscillating chemiluminescence reaction by l‐cysteine and its application

Perturbation of the tris(2,2′‐bipyridine)ruthenium(II) [Ru(bpy)₃²⁺]‐catalyzed Belousov–Zhabotinsky (BZ) oscillating chemiluminescence (CL) reaction induced by l ‐cysteine was observed in the closed system. It was found that the CL intensity was decreased in the presence of l ‐cysteine. Meanwhile, oscillation period and oscillating induction period were prolonged. The sufficient reproducible induction period was used as parameter for the analytical application of oscillating CL reaction. Under the optimum conditions, the changes in the oscillating CL induction period were linearly proportional to the concentration of l ‐cysteine in the range from 8.0 × 10^?7 to 5.0 × 10^?5 mol L^?1 (r = 0.997) with a detection limit of 4.3 × 10^?7 mol L^?1. The possible mechanism of l ‐cysteine perturbation on the oscillating CL reaction was also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemiluminescence determination of chlorpheniramine using tris(1,10‐phenanthroline)–ruthenium(II) peroxydisulphate system and sequential injection analysis

A sequential injection (SI) method was developed for the determination of chlorpheniramine (CPA), based on the reaction of this drug with tris(1,10‐phenanthroline)–ruthenium(II) [Ru(phen)₃²⁺] and peroxydisulphate (S₂O₈^2–) in the presence of light. The instrumental set‐up utilized a syringe pump and a multiposition valve to aspirate the reagents [Ru(phen)₃²⁺ and S₂O₈^2–] and a peristaltic pump to propel the sample. The experimental conditions affecting the chemiluminescence reaction were systematically optimized, using the univariate approach. Under the optimum conditions linear calibration curves of 0.1–10 µg/ml were obtained. The detection limit was 0.04 µg/ml and the relative standard deviation (RSD) was always < 5%. The procedure was applied to the analysis of CPA in pharmaceutical products and was found to be free from interferences from concomitants usually present in these preparations. Copyright © 2008 John Wiley & Sons, Ltd.     

A two‐electrode system‐based electrochemiluminescence detection for microfluidic capillary electrophoresis and its application in pharmaceutical analysis

A two‐electrode configuration powered by batteries was designed for a microchip capillary electrophoresis–electrochemiluminescence system. A home‐made working electrode for end‐column mode detection and wall‐jet configuration was made up of a platinum wire (0.3 mm diameter) and a quartz capillary (320 µm internal diameter). The platinum wire served as a pseudoreference electrode. The configuration of the detection power supply comprised two D‐size batteries (connected in series), a switch, and an adjustable resistor. The microchip consisted of two layers: the bottom layer was a glass sheet containing injection and separation channels; the upper layer was polydimethylsiloxane block. In order to reduce the loss of electrochemiluminescence signal, a coverslip (0.17 mm thickness) was used as the floor of the detection reservoir. The performance of the system was demonstrated by separation and detection of atropine, anisodamine and proline. The linear response for proline ranged from 5 µm to 100 µm (r = 0.9968), and the limit of detection was 1.0 µm (S/N = 3). The system was further applied to the measurement of atropine in atropine sulfate injection solutions with the limit of detection 2.3 µm . This new system is a potential tool in pharmaceutical analysis. Copyright © 2013 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence and electrogenerated chemiluminescence determination of escitalopram oxalate in tablet form

Rapid, simple and highly sensitive flow‐injection (FI) chemiluminescence (CL) and flow‐injection electrogenerated chemiluminescence (ECL) methods were developed for the determination of escitalopram oxalate (ESC), a selective serotonin reuptake inhibitor used as an antidepressant drug. The CL method was based on the CL reaction of ESC with acidic cerium(IV) and tris(2,2'‐bipyridyl)ruthenium(II) (Ru). Various experimental parameters affecting CL intensity were carefully studied and optimised. The method enabled the determination of 0.001‐50 µg/mL of ESC in bulk form with a correlation coefficient r = 0.9999. The limit of detection (LOD) was 0.01 ng/mL (S/N = 3). The ECL method was based on the ECL reaction of Ru with the drug in an acidic medium, permitting the determination of ESC in the range of 0.00001‐70 µg/mL with r = 0.9999 and LOD of 1 x 10^‐4 ng/mL. The proposed methods were applied to the determination of ESC in commercial tablets. The results were compared statistically with those obtained from a published method using t‐ and F‐tests. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of the pseudoephedrine content in pharmaceutical formulations and in biological fluids using a microbore HPLC system interfaced to a microfluidic chemiluminescence detector

A novel automated precolumn derivatization followed by separation using liquid chromatography for the determination of pseudoephedrine (PSE) by a microfluidic chemiluminescence detector has been developed. An on‐line derivatization procedure was utilized by converting PSE into a highly light emitting species in a Ru(bipy)₃²⁺‐peroxydisulphate chemiluminescence (CL) system by derivatizing it with a 1.0 M formaldehyde solution. The derivatized analyte was directly injected into a microbore high‐performance liquid chromatography (HPLC) system coupled to an on‐chip chemiluminescence detector. The newly developed highly selective, sensitive and fast HPLC‐CL method was validated and successfully applied for the analysis of PSE in pharmaceutical formulations and a human urine sample. The selectivity of the method is not only due to the HPLC separation but is also due to the highly selective detection principle of the Ru(bipy)₃²⁺‐peroxydisulphate CL system used. There was no interference observed from the common preservatives and excipients used in pharmaceutical preparations, which did not show any significant CL signal. The retention time of PSE was less than 3 min, and the detection limits and quantification limits were found to be 5.7 and 26.0 µg L^–1, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative electrochemiluminescence detection of proteins: Avidin-based sensor and tris(2,2'-bipyridine) ruthenium(II) label

Quantitative electrochemiluminescence (ECL) detection of a model protein, bovine serum albumin (BSA) was achieved via biotin–avidin interaction using an avidin-based sensor and a well-developed ECL system of tris(2,2′-bipyridine) ruthenium(II) derivative as label and tri-n-propylamine (TPA) as coreactant. To detect the protein, avidin was linked to the glassy carbon electrode through passive adsorptions and covalent interaction with carboxylate-terminated carbon nanotubes that was used as binder to immobilize avidin onto the electrode. Then, biotinylated BSA tagged with tris(2,2′-bipyridine) ruthenium(II) label was attached to the prepared avidin surface. After binding of BSA labeled with tris(2,2′-bipyridine) ruthenium(II) derivative to the surface-immobilized avidin through biotin, ECL response was generated when the self-assembled modified electrode was immersed in a TPA-containing electrolyte solution. Such double protein labeling protocol with a biotin label for biorecognition and ruthenium label for ECL detection facilitated the detection of protein compared to the classical double antibody sandwich format. The ECL intensity was linearly proportional to the feed concentration of BSA over two orders of magnitude in the range of 15 nM to 7.5 μM. The detection limit was estimated to be 1.5 nM. Further application to the lysozyme analysis was carried out to validate the present approach for an effective and favorable protocol for the quantitative detection of proteins. The dynamic range of lysozyme was from 0.001 g L⁻¹ to 0.1 g L⁻¹ and the detection limit was 0.1 mg L⁻¹. Electrochemical impedance and cyclic voltammetric measurements along with some necessary control experiments were conducted to characterize the successful formation of self-assembled modified electrodes and to grant the whole detection process.     

Chemiluminescence method for the determination of piroxicam by the enhancement of the tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–cerium(IV) system and its application

A simple, rapid chemiluminescence (CL) method was described for the determination of piroxicam, a commonly used analgesic agent drug. A strong CL signal was detected when cerium(IV) sulphate was injected into tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–piroxicam solution. The CL signal was proportional to the concentration of piroxicam in the range 2.8 × 10^–8–1.2 × 10^–5 mol/L. The detection limit was 2 × 10^–8 mol/L and the relative standard deviation (RSD) was 3.7% (c = 7.0 × 10^–7 mol/L piroxicam; n = 11). The proposed method was applied to the determination of piroxicam in pharmaceutical preparations in capsules, spiked serum and urine samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis and copper-mediated nuclease activity of a tetracationic tris(2,2′-bipyridine) ligand

We report herein the synthesis of a novel tetracationic tris(2,2′-bipyridine) ligand 4. We show that this ligand metalated with copper(II), and in the presence of ascorbate as a reducing agent, strongly damages pUC18 plasmid DNA. Copper complex formation was demonstrated by ESI-MS (electrospray ionization-mass spectrum) at a 1:3 ligand to metal ratio. Binding of both 4 and its copper(II) complex to CT-DNA (calf thymus DNA) was characterized by viscosimetry, thermal denaturation and fluorescence-based competition assays. The viscosimetric data indicated that 4 and its copper(II) complex bind DNA through partial intercalation and thermal denaturation studies revealed a significant increase of duplex DNA stability in the presence of these species (ΔT_m = 16.4 and 18.3 °C, respectively). Moreover, 4 and its copper(II) complex were found to effectively compete with ethidium bromide for the intercalative binding sites of DNA. Overall, the copper(II)-4 complex constitutes a very efficient DNA cleaving agent in the presence of ascorbate. Experiments with scavengers further suggest that the generation of Cu(I), hydrogen peroxide, superoxide, hydroxyl radical and singlet oxygen-like species contributes to the DNA breakage induced by the Cu(II) complex of 4.     

Highly sensitive electrochemiluminescence determination of etamsylate using a low‐cost electrochemical flow‐through cell based on a tris(2, 2'‐bipyridyl)ruthenium(II)–Nafion‐modified carbon paste electrode

A simple and sensitive electrochemiluminescence (ECL) method for the determination of etamsylate has been developed by coupling an electrochemical flow‐through cell with a tris(2,2'‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)–Nafion‐modified carbon electrode. It is based on the oxidized Ru(bpy)₃²⁺ on the electrode surface reacting with etamsylate and producing an excellent ECL signal. Under optimized experimental conditions, the proposed method allows the measurement of etamsylate over the range of 8–1000 ng/mL with a correlation coefficient of r = 0.9997 (n = 7) and a limit of detection of 1.57 ng/mL (3σ), the relative standard deviation (RSD) for 1000 ng/mL etamsylate (n = 7) is 0.96%. The immobilized Ru(bpy)₃²⁺ carbon paste electrode shows good electrochemical and photochemical stability. This method is rapid, simple, sensitive and has good reproducibility. It has been successfully applied to the determination of the studied etamsylate in pharmaceutical preparations with satisfactory results. The possible ECL reaction mechanism has also been discussed. Copyright © 2013 John Wiley & Sons, Ltd.     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.