An ultrasensitive and highly selective determination method for quinones by high-performance liquid chromatography with photochemically initiated luminol chemiluminescence

Quinones are a class of compounds of substantial toxicological and pharmacological interest. An ultrasensitive and highly selective chemiluminescence (CL) method was newly developed for the determination of quinones based on the utility of photochemically initiated luminol CL. The method involved ultraviolet (UV) irradiation of quinones to generate reactive oxygen species (ROS) through the unique photosensitization reaction accompanied with the photolytical generation of 3,6-dihydroxyphthalic acid (DHPA) from quinones. The photoproducts were detected by luminol CL reaction. Interestingly, it was noticed that DHPA had enhancement effect for the luminol CL. The generation of the enhancer (DHPA) in association with the oxidant (ROS) in the photochemical reaction greatly increases the sensitivity and selectivity of the proposed luminol CL method. In order to elucidate the type of ROS produced by the photosensitizaion reaction in relation to the proposed CL reaction, we investigated the quenching effect of selective ROS scavengers in the luminol CL. Although several ROS were generated, superoxide anion was the most effective ROS for the generated CL. Moreover, the enhancement mechanism of DHPA for luminol CL was confirmed. The enhancement was found to be through the formation of stabilized semiquinone anion radical that provided long-lived CL. The generation of the semiquinone radical was confirmed by electron spin resonance technique. Furthermore, we developed an HPLC method with on-line photochemical reaction followed by the proposed CL detection for the determination of four quinones. A luminol analogue, L-012, was used for its high sensitivity. The detection limits for quinones obtained with the proposed method (S/N = 3) were in the range 1.5–24 fmol that were 10–1000 times more sensitive compared with the previous methods. Finally, the developed HPLC-CL system was successfully applied for the determination of quinones in airborne particulate samples collected at Nagasaki city.     

Determination of thyroxine hormone by luminol chemiluminescence

A chemiluminescence (CL) flow system for determination of thyroxine (Thy) is presented. It is based on the catalytic effect of cobalt(II) on the CL reaction between luminol and hydrogen peroxide. The iodinated chemical structure of Thy causes a heavy atom effect. The luminol CL signals show significant quenching by Thy. The calibration graph for Thy is linear for 15-70 μg ml⁻¹ and the 3σ detection limits are 27 μg ml⁻¹ for d-Thy and 23 μg ml⁻¹ for l-Thy.     

Determination of bromide ions in seawater using flow system with chemiluminescence detection

A simple flow-based procedure with chemiluminescence (CL) detection is proposed for bromide ion determination in seawater. The procedure was based on the oxidation of bromide to bromine by chloramine-T followed by the reaction of bromine with luminol resulting in CL emission. Since no significant reaction within chloramine-T and luminol was observed, the detection was carried out without bromine extraction from the oxidant medium. The proposed flow system had a sampling rate of 40 determinations per hour, reagents consumption of 100 μg luminol and 60 μg chloramine-T per determination, a limit of detection of 0.5 mg l⁻¹ bromide ions, a linear concentration range (r = 0.999 and n = 7) between 0 and 100 mg l⁻¹, and a coefficient of variance better than 2.5% (for 10 measurements of a 10 mg l⁻¹ Br⁻ solution) were achieved. The analytical system was applied for the determination of bromide in seawater and estuarine-water samples, obtaining an analyte recovery ranging from 94 to 102% and comparing the results with a reference spectrophotometric method no significant difference was observed in 95% confidence level.     

Chemiluminescence determination of carbofuran at trace levels in lettuce and waters by flow-injection analysis

A simple, fast chemiluminescence (CL) flow-injection (FI) method based on the reaction of luminol with KMnO₄ in alkaline medium has been described for the direct determination of carbofuran. The method is based on the enhancing effect in the emission light from the oxidation of luminol produced in presence of carbofuran. The optimisation of instrumental and chemical variables influencing the CL response of the method has been carried out by applying experimental design, using the proposed flow-injection manifold. Under the optimal conditions, the CL intensity was linear for a carbofuran concentration over the range of 0.06-0.5 μg ml⁻¹, with a detection limit of 0.02 μg ml⁻¹. The method has been successfully applied to the determination of carbofuran residues in spiked water and lettuce samples.     

Detection of paracetamol by capillary electrophoresis with chemiluminescence detection

Indirect detection of paracetamol was accomplished using a capillary electrophoresis-chemiluminescence (CE-CL) detection system, which was based on its inhibitory effect on a luminol-potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) CL reaction. Paracetamol migrated in the separation capillary, where it mixed with luminol included in the running buffer. The separation capillary outlet was inserted into the reaction capillary to reach the detection window. A four-way plexiglass joint held the separation capillary and the reaction capillary in place. K₃[Fe(CN)₆] solution was siphoned into a tee and flowed down to the detection window. CL was observed at the tip of the separation capillary outlet. The CL reaction of K₃[Fe(CN)₆] oxidized luminol was employed to provide the high and constant background. Since paracetamol inhibits the CL reaction, an inverted paracetamol peak can be detected, and the degree of CL suppression is proportional to the paracetamol concentration. Maximum CL signal was observed with an electrophoretic buffer of 30 mM sodium borate (pH 9.4) containing 0.5 mM luminol and an oxidizer solution of 0.8 mM K₃[Fe(CN)₆] in 100 mM NaOH solution. Under the optimal conditions, a linear range from 6.6 × 10⁻¹⁰ to 6.6 × 10⁻⁸ M (r = 0.9999), and a detection limit of 5.6 × 10⁻¹⁰ M (signal-to-noise ratio = 3) for paracetamol were achieved. The relative standard deviation (R.S.D.) of the peak area for 5.0 × 10⁻⁹ M of paracetamol (n = 11) was 2.9%. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

Flow injection determination of cationic surfactants by using N-bromosuccinimide and N-chlorosuccinimide as new oxidizing agents for luminol chemiluminescence

Two highly sensitive chemiluminescence (CL) systems are described. The method is based on the CL generated during the oxidation of luminol by N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS) in alkaline medium. The emission intensity is reduced by the presence of some surfactants at concentrations lower than critical micelle concentration (cmc).A new, simple, rapid and selective flow injection CL method for the determination of cationic surfactants such as dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC) is proposed. Their determinations are based on the reducing effect on the emission intensity of NBS-luminol and NCS-luminol chemiluminescent reactions. The effect of analytical and flow injection analysis (FIA) variables on these CL systems and on the determination of the cationic surfactants are discussed. The optimum parameters for the determination of cationic surfactants were studied and were found to be the following: luminol, 1×10⁻⁶ M; NBS and NCS both, 5×10⁻² M; NaOH, 5×10⁻² M and flow rate, 3.5 ml min⁻¹.     

Pre-evaluation of metal ions as a catalyst on chemiluminometric sequential injection analysis with luminol-H2O2 system

Catalytic effect of metal ions on luminol chemiluminescence (CL) was investigated by sequential injection analysis (SIA). The SIA system was set up with two solenoid micropumps, an eight-port selection valve, and a photosensor module with a fountain-type chemiluminescence cell. The SIA system was controlled and the CL signals were collected by a LabVIEW program. Aqueous solutions of luminol, H₂O₂, and a sample solution containing metal ion were sequentially aspirated to the holding coil, and the zones were immediately propelled to the detection cell. After optimizing the parameters using 1 × 10⁻⁵ M Fe³⁺ solution, catalytic effect of some metal species was compared. Among 16 metal species examined, relatively strong CL responses were obtained with Fe³⁺, Fe²⁺, VO²⁺, VO₃⁻, MnO₄⁻, Co²⁺, and Cu²⁺. The limits of detection by the present SIA system were comparable to FIA systems. Permanganate ion showed the highest CL sensitivity among the metal species examined; the calibration graph for MnO₄⁻ was linear at the concentration level of 10⁻⁸ M and the limit of detection for MnO₄⁻ was 4.0 × 10⁻¹⁰ M (S/N = 3).     

Determination of N-methylcarbamate pesticides in water and vegetable samples by HPLC with post-column chemiluminescence detection using the luminol reaction

In this paper we proposed a reverse high performance liquid chromatography method for the simultaneous determination of three N-methylcarbamates (NMCs) named carbofuran, carbaryl and methiocarb, using the post-column chemiluminescence (CL) detection with the luminol reaction. This method is based on the enhancing effect of these analytes on the CL emission generated by the oxidation of luminol with potassium permanganate in alkaline medium. The separation was reached in less than 14 min using a C18 column and an isocratic binary mobile phase consisting of acetonitrile:water (50:50, v/v) pumped at a flow rate of 1 mL min⁻¹. CL reagents (luminol and KMnO₄) were incorporated by means of a peristaltic pump and were firstly mixed using a three-way connector. Then this stream was mixed with the eluate using another three-way connector just before reaching the detection cell. The optimization of variables affecting the CL reaction (reaction medium, concentration, flow rate of reagents and distance between both connectors) were optimized by means of experimental designs. Ethiofencarb, a NMC which has nowadays fallen into disuse was used as internal standard. For the analysis of theses pesticides in real water samples a pre-treatment step consisting of solid phase extraction (SPE) was conducted in order to reach sensitivity levels below the legal maximum concentration permitted. In the case of vegetable sample, SPE was used for matrix cleaning purpose.     

Quantum dot-enhanced chemiluminescence detection for simultaneous determination of dopamine and epinephrine by capillary electrophoresis

A sensitive method based on quantum dot (QD)-enhanced capillary electrophoresis-chemiluminescence (CE-CL) detection was developed for simultaneous determination of dopamine (DA) and epinephrine (E). In this work, CdTe QD was added into the running buffer of CE to catalyze the post-column CL reaction between luminol and hydrogen peroxide, achieving higher CL emission. Negative peaks were produced due to the inhibitory effects on CL emission from DA and E eluted from the electrophoretic capillary. The decrease in CL intensity was proportional to the concentration of DA and E in the range of 8.0 × 10⁻⁸-5.0 × 10⁻⁶ M and 4.0 × 10⁻⁸-5.0 × 10⁻⁶ M, respectively. Detection limits for DA and E were 2.3 × 10⁻⁸ M and 9.3 × 10⁻⁹ M, respectively. Using this method, the levels of DA and E in human urine from healthy donors were determined.     

CdTe nanocrystals sensitized chemiluminescence and the analytical application

It was found that the mixing of CdTe semiconductor nanocrystals (NCs) with luminol in the presence of KMnO₄ can induce a great sensitized effect on chemiluminescence (CL) emission. When the concentration of luminol, KMnO₄ and NaOH were fixed at 1 μM, 1 μM and 0.05 M, respectively, the most excellent performance can be obtained for the CdTe NCs sensitized CL. By means of CL and photoluminescence spectra, we suppose the enhanced CL signals resulted from the accelerated luminol CL induced by the oxidized species of CdTe NCs. Based on the finding, using thioglycolic acid-capped CdTe NCs as label and immunoglobulin G (IgG) as a model analyte, a CL immunoassay protocol for IgG content detection was developed. The strong inhibition effect of phenol compounds on luminol-KMnO₄-CdTe NCs CL system was also observed. All these findings demonstrated the possibility of semiconductor nanocrystals induced chemiluminescence to be utilized for more practical applications.     

Sub-picogram determination of Vitamin B12 in pharmaceuticals and human serum using flow injection with chemiluminescence detection

A sensitive chemiluminescence (CL) method, based on the enhancive effect of cobalt(II) on the CL reaction between luminol and dissolved oxygen in a flow injection (FI) system, was proposed for determination of Vitamin B₁₂. The increment of the CL intensity was proportional to the concentration of Vitamin B₁₂, giving a calibration graph linear over the concentration from 2.0×10⁻¹⁰ to 1.2×10⁻⁶ g l⁻¹ (r²=0.9992) with the detection limit of 5.0×10⁻¹¹ g l⁻¹ (3σ). At a flow rate of 2.0 ml min⁻¹, a complete determination of Vitamin B₁₂, including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations (R.S.D.) of less than 5.0%. The proposed method was applied successfully to the determination of Vitamin B₁₂ in pharmaceuticals, human serum, egg yolk and fish tissue.     

Sensitive determination of captopril by flow injection analysis with chemiluminescence detection based on the enhancement of the luminol reaction

This work reports a novel flow injection (FI) method for the determination of captopril, 1-[(2S)-3-mercapto-2-methylpropionyl]-l-proline (CPL), based on the enhancement CPL affords on the chemiluminescence (CL) reaction between luminol and hydrogen peroxide. For this purpose alkaline luminol and hydrogen peroxide solutions were mixed online, the sample containing CPL was injected into an aqueous carrier stream, mixed with the luminol-hydrogen peroxide stream and pumped into a glass flow cell positioned in front of a photomultiplier tube (PMT). The increase in the CL intensity was recorded in the form of FI peaks, the height of which was related to the CPL mass concentration in the sample. Different chemical and instrumental parameters affecting the CL response were investigated. Under the selected conditions, the log-log calibration curve was linear in the range 5-5000 μg l⁻¹ of CPL, the limit of detection was 2 μg l⁻¹ (at the 3σ level), the R.S.D., s_r was 3.1% at the 100 μg l⁻¹ level (n=8) and the sampling rate was 180 injections h⁻¹. The method was applied to the determination of CPL in pharmaceutical formulations with recoveries in the range 100±3%.     

Ultrasensitive chemiluminescence detection of aM vanadium(IV) by capillary electrophoresis

Ultrasensitive chemiluminescence (CL) detection of aM vanadium(IV) in capillary electrophoresis (CE) is first reported. In this work, inclusion of the luminol in the electrophoretic carrier electrolyte avoids the loss of light signal that occurs when luminol and hydrogen peroxide are mixed in advance, as in the conventional method in CE-CL detection. The detection limit (S/N ratio=3) for V(IV) is 2.4×10⁻¹⁷ M (24 aM), which has been improved by a factor of 10⁴ as compared with that of the most sensitive metal ion detection (Co²⁺ 0.5 pM) reported previously. In addition, the separation of V(IV) and V(V) has been performed successfully.     

The use of a micropump based on capillary and evaporation effects in a microfluidic flow injection chemiluminescence system

The performance of a micropump operating on evaporation and capillary effects, developed for microfluidic (lab-on-a-chip) systems, was studied employing it as the fluid drive in a microfluidic flow injection (FI) system, with chemiluminescence (CL) detection. The micropump featured simple structure, small dimensions, low fabrication cost and stable and adjustable flow-rates during long working periods. Using a micropump with 6.6 cm² evaporation area, with the ambient temperature and relative humidity fluctuating within 2 h in the ranges 20-21 °C and 30-32%, respectively, an average flow-rate of 3.02 μL/min was obtained, with a precision better than 1.2% R.S.D. (n = 61). When applied to the microchip FI-CL system using the luminol/hexacyanoferrate/H₂O₂ reaction, a precision of 1.4% R.S.D. (n = 11) was obtained for luminol at a sampling frequency of 30 h⁻¹.     

2D spatiotemporal visualization system of expired gaseous ethanol after oral administration for real-time illustrated analysis of alcohol metabolism

A novel 2-dimensional spatiotemporal visualization system of expired gaseous ethanol after oral administration for real-time illustrated analysis of alcohol metabolism has been developed, which employed a low level light CCD camera to detect chemiluminescence (CL) generated by catalytic reactions of standard gaseous ethanol and expired gaseous ethanol after oral administration. First, the optimization of the substrates for visualization and the concentration of luminol solution for CL were investigated. The cotton mesh and 5.0 mmol L⁻¹ luminol solution were selected for further investigations and this system is useful for 0.1-20.0 mmol L⁻¹ of H₂O₂ solution. Then, the effect of pH condition of Tris-HCl buffer solution was also evaluated with CL intensity and under the Tris-HCl buffer solution pH 10.1, a wide calibration range of standard gaseous ethanol (30-400 ppm) was obtained. Finally, expired air of 5 healthy volunteers after oral administration was measured at 15, 30, 45, 60, 75, 90, 105 and 120 min after oral administration, and this system showed a good sensitivity on expired gaseous ethanol for alcohol metabolism. The peaks of expired gaseous ethanol concentration appeared within 30 min after oral administration. During the 30 min after oral administration, the time variation profile based on mean values showed the absorption and distribution function, and the values onward showed the elimination function. The absorption and distribution of expired gaseous ethanol in 5 healthy volunteers following first-order absorption process were faster than the elimination process, which proves efficacious of this system for described alcohol metabolism in healthy volunteers. This system is expected to be used as a non-invasive method to detect VOCs as well as several other drugs [1] in expired air for clinical purpose.     

Microchip electrophoresis with chemiluminescence detection for assaying ascorbic acid and amino acids in single cells

A method based on microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed for the determination of ascorbic acid (AA) and amino acids including tryptophan (Trp), glycine (Gly) and alanine (Ala) present in single cells. Cell injection, loading, lysing, electrophoretic separation and CL detection were integrated onto a simple cross microfluidic chip. A single cell was loaded in the cross intersection by electrophoretic means through applying a set of potentials at the reservoirs. The docked cell was lysed rapidly under a direct electric field. The intracellular contents were MCE separated within 130 s. CL detection was based on the enhancing effects of AA and amino acids on the CL reaction of luminol with K₃[Fe(CN)₆]. Rat hepatocytes were prepared and analyzed as the test cellular model. The average intracellular contents of AA, Trp, Gly and Ala in single rat hepatocytes were found to be 38.3, 5.15, 3.78 and 3.84 fmol (n = 12), respectively.     

Gold nanorod-catalyzed luminol chemiluminescence and its selective determination of glutathione in the cell extracts of Saccharomyces cerevisiae

In this study, gold nanorods were firstly found to exhibit a tremendously higher catalytic activity towards luminol chemiluminescence (CL) than spherical gold nanoparticles. More importantly, ultra-trace aminothiols can cause a great CL decrease in the gold nanorod-catalyzed luminol system by the formation of Au-S covalent bonds on the ends of gold nanorods. Aminothiols can occupy the active sites of gold nanorods, and further interrupt the generation of the active oxygen intermediates. Other biomolecules including 19 standard amino acids, alcohols, organic acids and saccharides have no effect on gold nanorod-catalyzed luminol CL signals. Moreover, in order to evaluate the applicability and reliability of the proposed method, it was applied to the determination of glutathione in the cell extracts of Saccharomyces cerevisiae. Good agreements were obtained for the determination of glutathione in the cell extracts of S. cerevisiae between the present approach and a standard Alloxan method. The recoveries of glutathione were found to fall in the range between 96 and 105%. The calibration curve for glutathione was found to be linear from 0.05 to 100 nM, and the detection limit (S/N = 3) was 0.01 nM. The relative standard deviation (RSD) for five repeated measurements of 5.0 nM glutathione was 2.1%.     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

Development of integrated chemiluminescence flow sensor for the determination of adrenaline and isoprenaline

A novel integrated chemiluminescence (CL) flow sensor for the determination of adrenaline and isoprenaline is developed based on the enhancing effect of analytes on CL emission of luminol oxidized by periodate in alkaline solution. The analytical reagents luminol and periodate are immobilized on anion exchange resins, respectively, and packed in a glass tube to construct a reagentless sensor. The proposed sensor allows the determination of adrenaline and isoprenaline over the range from 2.0×10⁻⁸ to 1.0×10⁻⁵ g ml⁻¹ and 2.0×10⁻⁷ to 5.0×10⁻⁵ g ml⁻¹, respectively. The detection limits are 7.0×10⁻⁹ g ml⁻¹ for adrenaline and 5.0×10⁻⁸ g ml⁻¹ for isoprenaline with a relative standard deviation of 1.7% for the 1.0×10⁻⁷ g ml⁻¹ adrenaline (n=11) and 2.1% for 1.0×10⁻⁶ g ml⁻¹ isoprenaline (n=11). The sample throughput was 60 samples h⁻¹. The sensor has been successfully applied to the determination of adrenaline and isoprenaline in pharmaceutical preparations.