Chemiluminescence determination of ultramicro DNA with a flow-injection method

A high sensitive flow-injection chemiluminescence method for determination of calf thymus DNA and herring sperm DNA has been developed. The method is based on the chemiluminescence reaction of Rhodamine B-cerium(IV)-thermally denatured DNAs in sulfuric acid media. The proposed procedure allows quantitation of DNAs in the range 2.6×10⁻⁵ to 0.26 μg ml⁻¹ for calf thymus DNA and 5.0×10⁻⁸ to 5.0×10⁻⁵ μg ml⁻¹ for herring sperm DNA with correlation coefficients 0.9998 and 0.9996 (both n=11), respectively. The detection limits (3σ) are 6.5×10⁻⁶ μg ml⁻¹ for calf thymus DNA and 4.3×10⁻⁸ μg ml⁻¹ for herring sperm DNA. The possible mechanism of chemiluminescence in the system is discussed.     

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.     

Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0×10⁻⁸ to 0.1 μg ml⁻¹ for herring sperm DNA and 2.0×10⁻⁶ to 0.2 μg ml⁻¹ for calf thymus DNA with 3σ detection limits of 8.3×10⁻⁹ μg ml⁻¹ for herring sperm DNA and 3.5×10⁻⁷ μg ml⁻¹ for calf thymus DNA, respectively. The relative standard deviation for 1.0×10⁻⁴ μg ml⁻¹ herring sperm DNA was 0.99% and 2.0×10⁻³ μg ml⁻¹ for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper.     

Flow-injection chemiluminescence determination of aminomethylbenzoic acid and aminophylline based on N-bromosuccinimide-luminol reaction

A novel and sensitive chemiluminescence (CL) method for the determination of aminomethylbenzoic acid and aminophylline coupled with flow-injection analysis (FIA) technique is developed in this paper. It is based on the inhibition effect of the studied drugs on the chemiluminescence emission of N-bromosuccinimide-luminol (NBS-luminol) system. Under the optimum conditions, the decreased CL intensity is linear with the concentration of aminomethylbenzoic acid in the range of 2×10⁻⁸ to 1.0×10⁻⁶ g ml⁻¹ and with the concentration of aminophylline in the range of 1×10⁻⁷ to 7.0×10⁻⁶ g ml⁻¹, respectively. The detection limit is 7.0×10⁻⁹ g ml⁻¹ for aminomethylbenzoic acid (3σ) and 3.4×10⁻⁸ g ml⁻¹ for aminophylline (3σ). The relative standard deviations (R.S.D.) for 11 parallel measurements of 2.0×10⁻⁷ g ml⁻¹ aminomethylbenzoic acid and 1.0×10⁻⁶ g ml⁻¹ aminophylline are 2.6 and 3.0%, respectively. The proposed methods have been applied for the determination of the studied drugs in their pharmaceutical formulations with satisfactory results. The possible use of the proposed system for the determination of aminomethylbenzoic acid in plasma sample was also tested. The possible inhibition mechanism of aminomethylbenzoic acid and aminophylline on luminol-NBS system was discussed briefly.     

In vitro detecting ultra-trace novalgin in medicine and human urine by chemiluminescence

A sensitive chemilumimetric method for the determination of novalgin at the sub-nanogram level is presented. The method is based on immobilized luminol and dichromate chemiluminescence detection coupled with a flow injection system. The intensity of the chemiluminescence can be strongly inhibited by novalgin and the decrement of CL intensity was linear with the logarithm of novalgin concentration in the range of 5.0×10⁻¹¹ to 5.0×10⁻⁸ g ml⁻¹. The detection limit is 2.0×10⁻¹¹ g ml⁻¹ (3σ) and the relative standard deviation is 2.57% (n=5) for a 1.0×10⁻¹⁰ g ml⁻¹ novalgin sample. A typical analytical procedure, including sampling and washing, could be performed in 1 min at a flow rate of 2.0 ml min⁻¹, giving a throughput of 60 h⁻¹. The proposed procedure was applied successfully in pharmaceutical preparations and furthermore the monitoring of novalgin in human urine without any pre-treatment process during 10 h. It was found that the novalgin concentration reached its maximum after orally administrated for about 4 h, and the novalgin metabolism ratio in 10 h was 10.83% in the body of volunteers. The flow system offered reagentless procedures and remarkable stability in determination of novalgin, and could be easily reused over 600 times.     

Flow injection chemiluminescence determination of paracetamol

A simple chemiluminometric method using flow injection has been developed for the determination of paracetamol (acetaminophen), based on the chemiluminescence produced by the reduction of tris(2,2′-bipyridyl)ruthenium(III). The latter is obtained by oxidation of tris(2,2′-bipyridyl)ruthenium(II) by potassium permanganate in dilute sulphuric acid in the presence of paracetamol. A standard or sample solution was injected into the ruthenium(II) stream (flow rate 1.5 ml min⁻¹) which was then merged with potassium permanganate in dilute sulphuric acid stream (flow rate 0.5 ml min⁻¹). The chemiluminescence intensity is enhanced by the presence of manganese(II) ions. Under the optimum conditions, a linear calibration graph was obtained over the range of 0.3-50.0 μg ml⁻¹ and the detection limit was 0.2 μg ml⁻¹ (s/n = 3). The relative standard deviation of the proposed method calculated from 20 replicate injections of 5.0 μg ml⁻¹ paracetamol was 1.1%. The sample throughput was 90 h⁻¹. The method was successfully applied to the determination of paracetamol in commercial pharmaceutical formulations.     

Lanthanide sensitized chemiluminescence determination of grepafloxacin in tablets and human urine

A flow-injection chemiluminescence (CL) method, based on the luminescent properties of the Ce(IV)-Na₂SO₃-lanthanide(III)-grepafloxacin system, was developed for the determination of grepafloxacin {1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid}. La(III), Tb(III), and Eu(III) ions were tested as possible chemiluminescence sensitizers. The best results were achieved when Tb(III) was used as lanthanide ion, so the technique was optimised working with this ion. Under the optimum experimental conditions, the linear range was 0.05-2.00 μg ml⁻¹ grepafloxacin, with a 0.01 μg ml⁻¹ detection limit and 2.0% relative standard deviation (n=10). The proposed procedure has been applied to the determination of grepafloxacin in tablets and spiked human urine.     

Flow-injection chemiluminescence determination of dihydralazine sulfate based on hexacyanoferrate(III) oxidation sensitized by eosin Y

A novel flow-injection chemiluminescence (CL) method for the determination of dihydralazine sulfate (DHZS) is described. The method is based on the reaction between DHZS and hexacyanoferrate(III) in alkaline solution to give weak CL signal, which is dramatically enhanced by eosin Y. The CL emission allows quantitation of DHZS concentration in the range 0.02-2.8 μg ml⁻¹ with a detection limit (3σ) of 0.012 μg ml⁻¹. The experimental conditions for the CL reaction are optimized and the possible reaction mechanism is discussed. The method has been applied to the determination of DHZS in pharmaceutical preparations and compared well with the high performance liquid chromatography (HPLC) method.     

Flow injection kinetic spectrophotometric determination of trace amounts of Se(IV) in seawater

A simple, accurate, sensitive and selective flow injection catalytic kinetic spectrophotometric method for rapid determination of trace amounts of selenium is proposed in this paper. The proposed method is based on the accelerating effect of Se(IV) on the reaction of ethexlenediamine tetrecetic acid disodium salt (EDTA) and sodium nitrate with ammonium iron(II) sulfate hexahydrate in acidic media. The absorbance intensity was registered in this reaction solution at 440 nm. The calibration graph is linear in the range of 5 × 10⁻⁹-2 × 10⁻⁷ and 2 × 10⁻⁷-2 × 10⁻⁶ g ml⁻¹. The detection limit is 2 × 10⁻⁹ g ml⁻¹. The relative standard deviation was 3.4% for 5 × 10⁻⁸ g ml⁻¹ Se(IV) (n = 11), 2.7% for 5 × 10⁻⁷ g ml⁻¹ Se(IV) (n = 11). This method is very simple, rapid and suitable for automatic and continuous analysis. The presented system has been applied successfully to determination of Se(IV) of seawater samples.     

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.     

Chemiluminescence determination of citrate and pyruvate and their mixtures by the stopped-flow mixing technique

A novel kinetic chemiluminescent method using the stopped-flow mixing technique has been investigated for the rapid and sensitive determination of citrate and pyruvate. The method is based on a tris(2,2′-bipyridiyl)ruthenium(III) (Ru(bpy)₃³⁺) chemiluminescence (CL) reaction. Ru(bpy)₃³⁺ was generated in the mixing chamber by oxidising tris(2,2′-bipyridyl)ruthenium(II) with cerium(IV). After selecting the best operating parameters, calibration graphs were obtained over the concentration ranges 0.38-38 μg ml⁻¹ and 8.7-1300 ng ml⁻¹ for citrate and pyruvate, respectively. The limits of detection were 0.1 μg ml⁻¹ for citrate and 0.3 ng ml⁻¹ for pyruvate. Based on the differential rate of the chemiluminescent reaction corresponding to citrate and pyruvate, a very simple kinetic procedure was developed for the simultaneous determination of both compounds. Mixtures of citrate and pyruvate in ratios between 15:1 and 1.5:1 were satisfactorily resolved. The proposed method was successfully applied to the determination of citrate in pharmaceutical formulations, pyruvate in animal blood serum and both compounds in human urine.     

Catalytic adsorptive stripping voltammetry determination of ultra trace amount of molybdenum using factorial design for optimization

A highly sensitive procedure is presented for the determination of ultra-trace concentration of molybdenum by catalytic adsorptive stripping voltammetry. The method is based on adsorptive accumulation of the molybdenum (Mo)-pyrocatechol violet (PCV) complex on to a hanging mercury drop electrode, followed by reduction of the adsorbed species by voltammetric scan using differential pulse modulation. The reduction current is enhanced catalytically by chlorate. The influence of variables was completely studied by factorial design analysis. Optimum analytical conditions for the determination of molybdenum were established. Molybdenum can be determined in the range 1.0 × 10⁻³-100.0 ng ml⁻¹ with a limit of detection of 0.2 pg ml⁻¹. The influence of potential interfering ions on the determination of molybdenum was studied. The procedure was applied to the determination of molybdenum in mineral water and some analytical grade substances with satisfactory results.     

Flow-injection post chemiluminescence determination of atropine sulfate

A new post chemiluminescence (PCL) reaction was observed when atropine sulfate was injected into the reaction mixture after the finish of CL reaction of Ce(IV) and sodium sulfite. The possible mechanism for the PCL reaction was discussed via the investigation of the CL kinetic characteristics, the CL spectra, the UV absorption spectra and the fluorescence spectra of some related substances. The flow injection PCL method for the determination of atropine sulfate was established. The relative standard deviation (R.S.D.) was 2.8% (n = 11, c = 5.0 × 10⁻⁶ g mL⁻¹). The PCL intensity responded linearly to the concentration of atropine sulfate in the range 1.0 × 10⁻⁶ to 5.0 × 10⁻⁵ g mL⁻¹ with a linear correlation of 0.9947. The detection limit was 4 × 10⁻⁷ g mL⁻¹ atropine sulfate. The method had been applied to the determination of atropine sulfate in the tablets and the results were consistent with the method of Chinese pharmacopoeia.     

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 indole-3-acetic acid and indole-3-butyric acid in mung bean sprouts using high performance liquid chromatography with immobilized Ru(bpy)3-KMnO4 chemiluminescence detection

A novel method for determination of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in an extract from mung bean sprouts using high performance liquid chromatography (HPLC) with chemiluminescence (CL) detection is described. The method is based on the CL reaction of auxin (indole-3-acetic acid and indole-3-butyric acid) with acidic potassium permanganate (KMnO₄) and tris(2,2′-bipyridyl)ruthenium(II), which was immobilized on the cationic ion-exchange resin. The chromatographic separation was performed on a Nucleosil RP-C18 column (i.d.: 250 mm × 4.6 mm, particle size: 5 μm, pore size: 100) with an isocratic mobile phase consisting of methanol-water-acetic acid (45:55:1, v/v/v). At a flow rate of 1.0 mL min⁻¹, the total run time was 20 min. Under the optimal conditions, the linear ranges were 5.0 × 10⁻⁸ to 5.0 × 10⁻⁶ g mL⁻¹ and 5.0 × 10⁻⁷ to 1.0 × 10⁻⁵ g mL⁻¹ for IAA and IBA, respectively. The detection limits were 2.0 × 10⁻⁸ g mL⁻¹ and 2.0 × 10⁻⁷ g mL⁻¹ for IAA and IBA, respectively. The relative standard deviation (RSD) of intra-day were 3.1% and 2.3% (n = 11) for 2 × 10⁻⁶ g mL⁻¹ IAA and 2 × 10⁻⁶ g mL⁻¹ IBA; The relative standard deviations of inter-day precision were 6.9% and 4.9% for 2 × 10⁻⁶ g mL⁻¹ IAA and 2 × 10⁻⁶ g mL⁻¹ IBA. The proposed method had been successfully applied to the determination of auxin in mung bean sprouts.     

Micro determination of ascorbic acid using methyl viologen

A new simple and sensitive analytical spectrophotometric method is developed for the determination of ascorbic acid reduces methyl viologen to form a stable blue coloured free radical ion. This method has a sensitivity and lower limit detection of 0.1 μg ml⁻¹ of ascorbic acid (0.1 ppm) which is comparable to the flow injection analysis reported earlier. Beer's law is obeyed over the concentration range of 1.0-10 μg ml⁻¹ of ascorbic acid per 10 ml of the final solution (0.1-1.0 μg ml⁻¹) at 600 nm. The molar absorptivity and Sandell's sensitivity were found to be 1.5 × 10⁵ ± 100 l mol⁻¹ cm⁻¹ and 0.001 μg cm⁻², respectively. The method has been applied to the determination of ascorbic acid in food, pharmaceuticals and biological samples.     

Flow-injection chemiluminescent determination of metoclopramide hydrochloride in pharmaceutical formulations and biological fluids using the [Ru(dipy)(3)(2+)]-permanganate system

A flow-injection (FI) methodology using (2,2′-dipyridyl) ruthenium(II) [Ru(dipy)₃²⁺] chemiluminescence (CL) was developed for the rapid and sensitive determination of metoclopramide hydrochloride. The method is based on the CL reaction of metoclopramide with Ru(dipy)₃²⁺ and KMnO₄ in a sulfuric acid medium. Under the optimum conditions, a calibration graph was obtained over the concentration range 0.005-3.5 μg ml⁻¹ with a limit of detection (S/N=2) of 1 ng ml⁻¹. The correlation coefficient was 0.99993 (n=8) with a relative standard deviation of 0.48% for 10 determinations of 1 μg ml⁻¹ of drug. The method was successfully applied to the determination of metoclopramide in pharmaceutical preparations and biological fluids after IP administration of 25 mg kg⁻¹ dose to rats. The elimination half-life was 2.5±0.4 h.     

Highly sensitive catalytic determination of molybdenum

A novel, highly sensitive, selective, and simple kinetic method was developed for the determination of Mo(VI) based on its catalytic effect on the oxidation of 1-amino-2-naphthol-4-sulfonic acid (ANSA) with H₂O₂. The reaction was followed spectrophotometrically by tracing the oxidized product at 465 nm after 30 min of mixing the reagents. The optimum reaction conditions were: 10 mmol l⁻¹ ANSA, 50 mmol l⁻¹ H₂O₂, 100 mmol l⁻¹ acetate buffer of pH 5.0 ± 0.05 and at 40 °C. Addition of 200 μg ml⁻¹ diethylenetriaminepentaacetic acid (DTPA) conferred high selectivity for the proposed method. Following the recommended procedure, Mo(VI) could be determined with a linear calibration graph up to 2.5 ng ml⁻¹ and a detection limit, based on the 3S_b-criterion, of 0.027 ng ml⁻¹. The unique sensitivity and selectivity of the implemented method allowed its direct application to the determination of Mo(VI) in natural and industrial waste water. The method was validated by comparison with the standard ETAAS method. Moreover, published catalytic-spectrophotometric methods for the determination of molybdenum were reviewed.     

Determination of ternary mixtures of penicillin G, benzathine and procaine by liquid chromatography and factorial design study

In the present work, a rapid and sensitive method for simultaneous determination of penicillin G (PG), benzathine (BE) and procaine (PR) in drug and serum media is introduced. The polar hydro-organic (55/45) mobile phases containing an aqueous solution adjusted to pH = 3.7 and an organic solvent (MeOH) including triethylamine (TEA) and trifluroacetic acid (TFA) are used. The flow rate of 1 ml min⁻¹, a C₈ column (150 mm × 46 mm) with 5 μm i.d. and wavelength at 215 nm are selected for optimal separation condition. The limit of detection (LOD), linear concentration range and relative standard deviation (R.S.D.) of this method for the PG are 1.1 μg ml⁻¹, 10-2400 μg ml⁻¹ and 1.7% and for the BE are 1.2 μg ml⁻¹, 12-2100 μg ml⁻¹ and 1.8% and for the PR are 1.5 μg ml⁻¹, 20-2000 μg ml⁻¹ and 2%, respectively. The factorial design is used for the determination of main and interaction effects of pH, flow rate and concentration of MeOH, TEA and TFA in the separation at two levels. Also, the analysis of variance (ANOVA) table is obtained. The results show that TFA and TEA have higher effect than concentration of MeOH, pH and flow rate factors.     

A novel capillary microliter droplet sample injection-chemiluminescence detector and its application to the determination of benzoyl peroxide in wheat flour

A novel capillary microliter order droplet injection-chemiluminescence (CL) system is proposed. In this system, the liquid sample microdrops, automatically formed at the end of a capillary tip by the effect of the gravity and the gas pressure, repeatedly drop into the miniature reaction cell and reacts with CL reagent to generate CL signal. The phenomena of sample zone dilution and spreading are eliminated as the capillary is used as the sample channel and gas pressure is used as driving force without the liquid carrier stream. Therefore, a high sensitivity is obtained. To evaluate the applicability of the proposed method, a determination of benzoyl peroxide (BP) is examined. The system shows that the benzoyl peroxide is detected linearly in the concentration range from 5×10⁻¹⁰ to 1×10⁻⁶ g ml⁻¹. The detection limit (signal-to-noise ratio=3) is 1.4×10⁻¹⁰ g ml⁻¹ for benzoyl peroxide (mass concentration is 1.1 pg, i.e., 4.5 fmol), which is the best result reported so far. The relative standard deviation (n=11) is 1.5% for 2.0×10⁻⁸ g ml⁻¹ BP. The proposed detector for the detection of benzoyl peroxide offers the advantages of sensitivity, simplicity, rapidity, automation and miniaturization. The proposed method has been applied satisfactorily to the determination of benzoyl peroxide in wheat flour.