Peroxyoxalate Chemiluminescence Research Articles

Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyl-oxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performance liquid chromatography to obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescence yield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol l-1 potassium acetate (pH 3.20)-50 mmol l-1 potassium phosphate (pH 3.20)-acetonitrile (90.15 + 4.85 + 3 v/v/v) containing 1 mmol l-1 sodium hexanesulfonate (40 degrees C) and flow rate, 0.5 ml min-1. Fluorogenic reagent solution, 105 mmol l-1 ED and 175 mmol l-1 imidazole in acetonitrile-ethanol (90 + 10 v/v) and flow rate, 0.25 ml min-1. Reaction coil (15 m x 0.5 mm i.d.) heated at 80 degrees C. Chemiluminogenic reagent solution, 0.25 mmol l-1 TDPO, 150 mmol l-1 hydrogen peroxide and 110 mmol l-1 trifluoroacetic acid in dioxane-ethyl acetate (50:50 v/v) and flow rate, 1.4 ml min-1. The detection limits for all the catecholamines were 1 fmol (signal-to-noise ratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of urinary oxalate and porphyrins by peroxyoxalate chemiluminescence

The application of the chemiluminescence system oxalic acid-fluorescer-peroxy compound-carbodiimide to biochemical analysis in protic solvents is described. By measuring the integrated chemiluminescence emission generated by the above reaction, very low concentrations of oxalate (down to 10 nmol l −1 can be determined. Optimized reaction conditions and preanalytical factors permit direct and rapid measurements of urinary oxalate without calcium oxalate precipitation. The system also permit rapid quantitative analysis for urinary porphyrins down to about 200 μg l −1.

Kinetic study of background emission from peroxyoxalate chemiluminescence reaction and application to the improvement of detection limits in liquid chromatography

Kinetic study of background emission from peroxyoxalate chemiluminescence reaction and application to the improvement of detection limits in liquid chromatography

Laser photolysis study of the kinetics and mechanism of photoinitiated peroxyoxalate chemiluminescence

Kinetics of photoinitiated peroxyoxalate chemiluminescence have been studied in order to further elucidate the mechanism of the reaction.

Determination of the fluorescent drugs dipyridamole and benzydamine in rat plasma by liquid chromatography with peroxyoxalate chemiluminescence detection

Determination of the fluorescent drugs dipyridamole and benzydamine in rat plasma by liquid chromatography with peroxyoxalate chemiluminescence detection

Flow-Injection Analysis with Chemiluminescence Detection of Glucose and Uric Acid Using Immobilized Enzyme Reactor

A rapid and sensitive flow-injection method with peroxyoxalate chemiluminescence (CL) detection for the determination of glucose and uric acid by using an immobilized enzyme reactor is described. CL produced in a flow cell by the reaction of hydrogen peroxide with bis(2, 4, 6-trichlorophenyl)oxalate and a fluorescent compound, 2, 4, 6, 8-tetrathiomorpholinopyrimido[5, 4-d]pyrimidine, was monitored. Linear responses between the concentration and the CL intensity for glucose and uric acid were obtained up to 3×10-4M (6nmol per 20μl injection, r=0.999) and 1×10-5M (0.2nmol per 20μl injection, r=1.000), respectively; both limits of detection were the same (5×10-7M, 10pmol per 20μl injection, S/N>3). The relative standard deviations for ten replicate measurements were 1.1% for glucose (2×10-5M, 0.4 nmol/injection) and 2.2% for uric acid (5×10-6M, 0.1nmol/injection). Good correlations between the results of serum glucose or uric acid assay by the proposed methods and by the conventional colorimetric methods (r=0.994for glucose and r=0.986 for uric acid) were obtained.

Peroxyoxalate Chemiluminescence Assay of Hydrogen Peroxide and Glucose Using 2,4,6,8-Tetrathiomorpholuiopyrimido[5,4-d]-pyrimidine as a Fluorescent Component

Peroxyoxalate chemiluminescence (CL) assay of hydrogen peroxide (H2O2) or glucose was developed by using 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine as a fluorescent component and bis(2,4,6-trichlorophenyl)oxalate (TCPO) as an oxalate. Linear relationships between CL intensity and final concentration of H2O2 from 10-8 to 10-4 M were obtained. The detection limit at the ratio of CL intensities for sample and blank (S/B) of 3 was 10 nM. The precision for five replicate measurements at 10-5 and 10-6 M of H2O2 were 17.6 and 15.7% of relative standard deviations, respectively. α-D-Glucose was transformed to β-D-glucose with mutarotase and converted to H2O2 and D-gluconic acid with glucose oxidase, which was detected by using peroxyoxalate CL reaction. A linear calibration graph was obtained up to 1.5×10-4 M of glucose solution. The method was applied to the assay of glucose in human serum. The recovery was 98.2% (N=4). The method correlated well with the conventional colorimetric method (r=0.968).

Determination of the fluorescent drugs dipyridamole and benzydamine in rat plasma by liquid chromatography with peroxyoxalate chemiluminescence detection

A facile and sensitive method for the determination of the fluorescent drugs dipyridamole (coronary vasodilator) and benzydamine hydrochloride (anti-inflammatory) in rat plasma is presented. The method consists of the addition of an internal standard (DNS- l-phenylalanine for dipyridamole and dipyridamole for benzydamine hydrochloride), deproteinization of plasma (1 μl for dipyridamole and 10 μl for benzydamine hydrochloride) with 13 volumes of acetonitrile, direct injection of the supernatant into an ODS column, separation by liquid chromatography and peroxyoxalate chemiluminescence detection using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and hydrogen peroxide. Calibration graphs were linear over the ranges 2.5–200 nM and 2.5–100 μM for dipyridamole and benzydamine hydrochloride in plasma, respectively. The detection limits (signal-to-noise ratio = 2) were 345 pM for dipyridamole and 147 nM for benzydamine hydrochloride in plasma. The relative standard deviation for four determinations of dipyridamole at 2.5 nM and benzydamine hydrochloride at 2.5 μM in plasma were 3.7% and 2.6%, respectively. The method was applied to real samples and the time courses of the plasma concentration after oral administration of the two drugs [0.50 mg kg −1 (0.99 μ mol kg −1) and 10 mg kg −1 (29 μ mol kg −1), respectively] were obtained.

Flow-injection determination of proteins using enhanced peroxyoxalate chemiluminescence applied to the determination of immunoglobin G and albumin in serum

The intensity of the chemiluminescence (CL) signal from an aqueous peroxyoxalate CL reaction can be significantly enhanced in the presence of various proteins with hydrophobic sites. A flow-injection measurement for various hydrophobic proteins based on this CL enhancement was developed. The enhancement is due to the inclusion of the CL species in the favorable environment provided by the protein's hydrophobicity, which results in efficient light production. Various protein structures were evaluated; the degree of enhancement depends on the protein structure and CL reaction conditions. The CL enhancement measurement in the flow-injection system is made after the introduction of the protein solution to the main phosphate buffer stream followed by the addition of the CL reagent streams: (1) hydrogen peroxide in water and (2) 8-anilino-1-naphthalene sulfonic acid and 4,4′-oxalylbis-(trifluoromethylsulfonylimino)ethylene bis(4-methyl morpholinium trifluoromethane sulfonate) in acetonitrile. Although prior separation of proteins is required before the measurement, the advantage of this approach is increased sensitivity without derivatization of the protein. The enhancement was demonstrated for several proteins, including antibodies, which suggests that this approach may be generally applicable to a variety of measurements, including immunoassay determinations. This CL enhancement was used to develop a simple and accurate flow-injection measurement for the determination of albumin and IgG in human serum.

Sensitive liquid chromatographic determination of alkyl-, nitro- and chlorophenols by precolumn derivatization with dansyl chloride, postcolumn photolysis and peroxyoxalate chemiluminescence detection

A liquid chromatographic method is described for the determination of alkyl-, nitro- and chlorophenols at sub-ppb levels using a very sensitive and selective detection system. The phenols are labelled by two-phase dansylation. The deprotonated phenolic anions are extracted as tetrabutylammonium ion pairs into an organic phase in which dansyl chloride is dissolved. After derivatization, the excess of reagent is removed on an amino-bonded column; dansyl chloride reacts with the amino groups whereas the phenol derivatives are not retained. Chromatography is carried out with a methanol-water gradient followed by photolysis of the derivatives. The strongly quenching electronegative nitro- and chlorophenol groups are photochemically removed from the derivative and the products, dansyl hydroxide and dansyl methoxide, are sensitively detected by peroxyoxalate chemiluminescence. Chemical excitation is carried out by adding 2-nitrophenyl oxalate and hydrogen peroxide dissolved in acetonitrile to the column eluate. Detection limits of about 0.01–0.1 ng/ml have been achieved and the method has been applied to the determination of several phenolic compounds in surface water.

Determination of carbonyl compounds in air by HPLC using on-line analyzed microcartridges, fluorescence and chemiluminescence detection

Sensitive and selective detection of dansylhydrazones of atmospheric carbonyl compounds (aldehydes and ketones) can be achieved using high performance liquid chromatography (HPLC) with fluorescence or chemiluminescence detection. The carbonyl compounds are derivatized by drawing air through small glass cartridges packed with porous glass particles impregnated with dansylhydrazine. After sampling, the contents of the cartridges are analyzed on-line by using a small plug of water (200 μL) to transfer and focus the hydrazone derivatives at the head of a HPLC column. Greatly increased sensitivity over traditional methods derives from 1) analysis of the entire contents of the sampling cartridge, and 2) detection by fluoresence or peroxyoxalate chemilum-inescence. Results are compared for photo-initiated and H2O2-initiated peroxyoxalate chemiluminescence. This novel and practical system enables the detection of sub-ppbv concentrations of formaldehyde, acetaldehyde, acetone and higher carbonyls in air using relatively short sampling times.

Effect of a dual‐head short‐stroke pump on post‐column peroxyoxalate chemiluminescence detection

A dual-head short-stroke pump has two advantages in the post-column peroxyoxalate chemiluminescence (PO-CL) detection system. The first is to increase the mixing efficiency of solutions. The second is to increase the stability of the PO-CL reaction by keeping the aryl oxalate and hydrogen peroxide solutions separate. The detection sensitivities for six polycyclic aromatic hydrocarbons (PAHs) increased in the present system by using bis(2,4-dinitrophenyl) oxalate (DNPO) or bis(2,3,4,5,6-pentafluorophenyl) oxalate (PFPO) instead of such popular aryl oxalates as bis(2,4,6-trichlorophenyl) oxalate (TCPO) and bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl] oxalate (TDPO). Both DNPO and PFPO increased the sensitivities by factors of 4.1-10.2 and 3.5-8.1, respectively. In addition, DNPO was more stable than PFPO in acetonitrile. These results suggest that DNPO is the most useful aryl oxalate for the sensitive PO-CL detection of PAHs.

4‐(N,N‐Dimethylaminosulphonyl)‐7‐Fluoro‐2,1,3‐Benzoxadiazole as chemilumigenic reagent for high performance liquid chromatographic peroxyoxalate chemiluminescence detection

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), presented as a fluorogenic labelling reagent for amines and amino acids, is preferred for peroxyoxalate chemiluminescence (PO-CL) detection in high performance liquid chromatography. Amino acids and epinephrine derivatized with DBD-F were separated on a reversed phase column and detected at the femtomole level by the PO-CL detection system.

Determination of Phenolic Herbicides in Soil by Peroxyoxalate Chemiluminescence

Abstract Peroxyoxalate chemiluminescence was used for the determination of bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), and bromofenoxim (3,5-dibromo-4-hydroxybenzaldehyde-0-(2,4-dinitrophenyloxime). Both compounds are potent herbicides and the presence of phenolic hydroxy group makes them amenable to analysis by peroxyoxalate chemiluminescence. Determinations were carried out in the soil, without prior separation. In contrast to previous analytical work, homogeneous solutions were used. Calibration curves are linear over a concentration range spanning three orders of magnitude and the limit of detection is 10 ppm in soil. para-Cyanophenol was also determined by peroxyoxalate chemiluminescence, and a limit of detection of 1 × 10−12M was found.

Investigation of the quenching of peroxyoxalate chemiluminescence by amine substituted compounds

The role of amine compounds in quenched peroxyoxalate chemiluminescence was investigated. The mechanistic steps examined included (1) fluorescence quenching; (2) base hydrolysis of the oxalate; and (3) a competitive interaction between the quencher and fluorophore for the peroxyoxalate reaction intermediate(s). The results showed no evidence of amines causing fluorescence quenching. Base hydrolysis of the oxalate is significant only at high concentrations of amines. When the concentration of amines is greater than or equal to the level of oxalate, the amines can compete with the fluorophore for reaction with the intermediate(s). Because competitive effects were demonstrated, one analytical implication is that caution must be exercised in applications where more than one fluorophore is present. At low concentrations of fluorophores relative to the oxalate, chemiluminescence emission of both fluorophores will be observed. At higher levels, the fluorophores can compete for reaction with the intermediate(s). Finally, an important analytical implication of this study is that the quenching response can be used to quantify amines without the need for derivatization. The limitation is that the linear response is approximately one order of magnitude.

N-(bromoacetyl)-N′-[5-(dimethylamino)naphthalene-1-sulphonyl]piperazine as a sensitive labelling reagent for the determination of carboxylic acids by liquid chromatography with peroxyoxalate chemiluminescence and fluorescence detection

The synthesis and application of a labelling reagent for the column liquid chromatographic determination of carboxylic acids using chemiluminescence (CL) detection is described. Dansyl chloride is first reacted with an excess of piperazine and then with bromoacetic acid. The resulting label, N-(bromoacetyl)-N′-[5-(dimethylamino)naphthalene-1-sulphonyl]piperazine (dansyl-BAP), which contains a reactive bromoacetamide group, is stable for at least 6 months. Pre-column derivatization is carried out in polar aprotic media in 30 min at room temperature for aliphatic acids, and in 30 min at 55 °C for aromatic acids, in the presence of 18-crown-6 and potassium hydrogen carbonate as a base catalyst. After derivatization, excess of dansyl-BAP is converted into a relatively polar product by reaction with thymine and is subsequently separated from the derivatized analytes on a silica cartridge. High-performance liquid chromatography is performed on C18 and C8 columns with acetonitrile–water mixtures as the eluent. Fluorescence detection is carried out at an excitation wavelength of 255 nm using a 470 nm emission cut-off filter. Chemiluminescence detection is carried out by adding bis(2-nitrophenyl) oxalate and hydrogen peroxide dissolved in acetonitrile to the column effluent. The fluorescence detection limit is 0.8–1 pmol for acids such as substituted benzoic acids and the drugs naproxen and ibuprofen; the CL detection limit is 25 fmol for retinoic acid, a vitamin A precursor.

Evaluation of peroxyoxalate chemiluminescence postcolumn detection of fluphenazine in urine and blood plasma using high performance liquid chromatography

Peroxyoxalate chemiluminescence may be used for sensitive postcolumn detection of phenothiazine analytes separated by high performance liquid chromatography with appropriate optimization of measurement conditions such as solvent, pH and oxalate ester. Detectability of fluorescent analytes by chemical excitation varies greatly, but analytes with low oxidation potentials are generally more readily detected at low levels, as demonstrated for phenothiazines, an important class of fluorescent drugs. Some improvement in detection limits is observed for fluphenazine when chemiluminescence detection is compared to conventional fluorescence detection. Because of the specificity of chemical excitation, fewer interferences from fluorescent impurities in a urine matrix are observed.

Flow-injection chemiluminescent method for an enzyme-labelled DNA probe

A flow-injection chemiluminescent (CL) method was investigated for the determination of hydrogen peroxide generated from an enzyme-labelled DNA probe. Biotinylated DNA probes were immobilized onto a membrane and labelled with biotinylated glucose oxidase by a streptavidin bridge and placed in a microtitre well. The glucose oxidase-labelled sample was incubated in a solution of glucose and the enzymatically produced hydrogen peroxide was determined using flow injection with a peroxyoxalate chemiluminescence reaction. The peroxide generated is related to the amount of the labelled DNA probe. The important factors for increasing the sensitivity of the CL measurement are to maximize the production of hydrogen peroxide and to reduce the non-specific binding of the enzyme label on the membrane. This CL-flow-injection measurement using biotinylated glucose oxidase-labelled DNA probes was applied to a DNA dot-blot hybridization assay and compared with a visual colorimetric method using 5-bromo-4-chloroindolyl phosphate and nitro blue tetrazolium as a chromogenic substrate for detection of an alkaline phosphatase label. The CL method was quantitative over the range 100 pg-100 ng of target DNA with a precision (r.s.d.) of 4–11%. In comparison, the colorimetric method was useful for the range 10 pg-1 ng.

Luminarin 4 as a labelling reagent for carboxylic acids in liquid chromatography with peroxyoxalate chemiluminescence detection

Luminarin 4 is a labelling reagent with a quinolizinocoumarin structure, which reacts with carboxylic acids after their activation by N-hydroxysuccinimide for 12 h at 20°C and dicyclohexylcarbodiimide for 60 min at 70°C. Small fatty acids were derivatized and separated by reversed-phase liquid chromatography. The fluorescence detection threshold was 300 fmol injected. The limit of chemiluminescence detection, which required a post-column reaction with an oxalic ester and hydrogen peroxide, was 50 fmol injected. The reaction was used to measure prostaglandin E 2, whose detection limit was 32 fmol injected, but the derivatization limit was 60 pmol. Linearity was observed in the range 0.1–10 nmol of prostaglandin E 2.

Liquid chromatographic determination of oestradiol in serum by pre-column derivatization with dansyl chloride or laryl chloride and peroxyoxalate chemiluminescence detection

Column liquid chromatography with chemiluminescence detection is used to determine oestradiol in serum. Oestradiol is labelled by means of a two-phase derivatization procedure with either dansyl chloride or laryl chloride. After derivatization the excess reagent is removed on a short amino-bonded column: the sulphonyl chloride functional group reacts with the amino groups causing removal of the label; the oestradiol derivatives are not retained. Chromatography of oestradiol derivatives is performed with methanol-water mixtures as eluent. Chemical excitation is carried out by adding bis(2-nitrophenyl)oxalate and hydrogen peroxide dissolved in acetonitrile to the column eluate. Linearity is observed (r = 0.9998; n = 7) over at least three decades (10(-6)-10(-9) M) for both dansyl chloride and laryl chloride. A detection limit of 5 x 10(-10) M (50 fmol injected) has been achieved and the dansyl derivatization method has been applied to the determination of oestradiol in spiked serum.