Determination of phenolic compounds using high-performance liquid chromatography with Ce-Tween 20 chemiluminescence detection

A novel method for the simultaneous determination of phenolic compounds such as salicylic acid, resorcinol, phloroglucinol, p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and m-nitrophenol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed. The procedure was based on the chemiluminescent enhancement by phenolic compounds of the cerium(IV)-Tween 20 system in a sulfuric acid medium. The separation was carried out with an isocratic elution or with a gradient elution using a mixture of methanol and 1.5% acetic acid. For six phenolic compounds, the detection limits (3σ) were in the range 1.40-5.02 ng/ml and the relative standard deviations (n=11) for the determination of 0.1 μg/ml compounds were in the range 1.9-2.9%. The CL reaction was well compatible with the mobile phase of HPLC, no baseline drift often occurred in HPLC-CL detection was observed with a gradient elution. The method has been successfully applied to the determination of salicylic acid and resorcinol in Dermatitis Clear Tincture and p-hydroxybenzoic acid in apple juices.     

Determination of polyamines by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the determination of polyamines (PA) by high-performance liquid chromatography with chemiluminescence detection. It is based on the unsaturated complex of PA with Cu(II) which had a strong catalytic effect on the luminol-H₂O₂ chemiluminescence reaction. The separation of PA was carried out on a reveres phase C18 column using methanol/water (25/75, v/v) as a mobile phase. The method was applied to the analysis of putrescine and the total amount of spermine and spermidine in apple leaves and strawberry fruit. The results indicated that the method is practical and useful.     

Determination of atmospheric nitrobenzanthrones by high-performance liquid chromatography with chemiluminescence detection.

A method using high-performance liquid chromatography with chemiluminescence detection was developed for analyzing mutagenic nitrobenzanthrone (NBA) isomers in airborne particulates. The method was a modification of our previously described method for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs). The pretreatment and reducing conditions for 1-, 2-, 3- and 10-NBAs were the same as those for NPAHs. In order to separate these NBA isomers, we used a polymeric-type ODS column (Cosmosil 5C-18MS); a mixture of 40% acetonitrile and 60% 10 mM imidazole-HClO4 buffer was employed as the mobile phase at a flow rate of 1 mL/min. The isomers of 1-, 2-, 3- and 10-NBA were determined in chemiluminescence with linear calibration graphs from 0.1 to 4 pmol, from 200 to 4000 pmol, from 1 to 50 pmol and from 10 to 400 pmol, respectively. The detection limits (S/N = 3) of 1-, 2-, 3- and 10-NBA isomers were 0.02 pmol, 35 pmol, 0.3 pmol and 3 pmol, respectively. The method was used to analyze airborne particulates at a heavy traffic site in Kanazawa. 2- and 3-NBAs were detected in the extracts of the particulates, while 1-NBA and 10-NBA were not detected. The atmospheric concentrations of 2- and 3-NBAs were 1.83 pmol/m3 and 24.7 fmol/m3, respectively.     

Determination of 1-nitropyrene metabolites by high-performance liquid chromatography with chemiluminescence detection

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H(2)O(2) CL system. The detection limits (signal-to-noise ratio=3) were in the range from 5fmol (6-OHNP and 8-OHNP) to 12fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.     

Determination of myoglobin in human serum by high-performance liquid chromatography with chemiluminescence detection

A highly sensitive method for the determination of myoglobin in serum is described, based on high-performance size-exclusion chromatography with chemiluminescence detection. Serum proteins are separated according to their molecular masses on columns packed with TSK-SW gel and those containing haem are detected selectively by post-column chemiluminescence reaction with luminol using a conventional fluorimetric detector. The method is rapid (30 min) and sufficiently sensitive for the diagnosis of myocardial infarction. The minimum detectable myoglobin concentration is 10 ng/ml.     

Determination of polyphenols by high-performance liquid chromatography with inhibited chemiluminescence detection.

A chemiluminescence reaction detector was developed for the detection of polyphenols separated by HPLC based on the inhibition of chemiluminescence from the luminol-potassium hexacyanoferrate(III) reaction by polyphenols. The separation was carried out on a RP-C18 column at 37 degrees C by using stepwise gradient elutions. The detection limits are in the range of 6.8 x 10(-7)-2.0 x 10(-9) g/ml for catechol, protocatechuic acid, chlorogenic acid, rutin, resorcinol, hydroquinone and p-tert.butylpyrocatechol. The method is sensitive, selective, fast and simple. It has been successfully applied to the determination of chlorogenic acid and rutin in real tobacco samples.     

Determination of paraquat in rat brain using ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive procedure for the measurement of N-methylisoquinolinium ion (NMIQ+), a putative neurotoxin, was devised using high-performance liquid chromatography (HPLC) with fluorescence detection. Separation of NMIQ+ was carried out by gel filtration and reversed-phase HPLC on a column of hydrophilic polymer gels (Asahipak GS-302H). The method was sensitive enough to measure 50 fmol of NMIQ+. Uptake of NMIQ+ into rat striatal slices was confirmed by this method.     

Determination of cobalt at picogram levels by high-performance liquid chromatography with chemiluminescence detection

A simple cation-exchange chromatographic system was used to separte cobalt from other metal ions. The cobalt thus separated was detected by the luminol chemiluminescence reaction using a spectrofluorimeter as the detector. A 3σ detection limit of 0.5 ng l^?1 was achieved, without the need for preconcentration, with a 200-μl sample. The system provided a linear analytical working range from 5 ng l^?1 to 10 μg l^?1. The cobalt content of a rice flour certified reference material was determined using the system and gave good agreement with the certificate value.     

Determination of serum estradiol by normal-phase high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

Estradiol extracted with an ODS minicolumn from serum (500 μl) is derivatized with 5-dimethylamino-1-naphthalenesulfonyl (dansyl) chloride at room temperature for 80 min, purified on the ODS minicolumn, separated on silica gel columns (150 × 1.9 mm i.d. and 100 × 1.9 mm i.d.) with n-hexane/chloroform/ethanol (70:30:0.1) as eluent at 3 ml min^?1 and detected by the chemiluminescence emission produced in a post-column reaction with bis-(2,4,6-trichlorophenyl) oxalate (8 mM in chloroform containing 100 mM triethylamine) and hydrogen peroxide (600 mM in methanol containing 6% (v/v) sodium acetate buffer at pH 4.0). The overall recovery of estradiol from serum is ca. 90% and the detection limit is ca. 50 pg.     

Aryl oxalate chemiluminescence detection for high-performance liquid chromatography

Aryl oxalate chemiluminescence detection for high-performance liquid chromatography offers ultrahigh sensitivity for interactive fluorophores. With optimal analytes, attomole limits of detection can be obtained with conventional instrumentation     

Determination of idebenone in rat serum and brain by high-performance liquid chromatography using platinum catalyst reduction and electrochemical detection.

A high-performance liquid chromatographic determination of idebenone, a new cerebral metabolism-improving agent, in rat serum and brain has been developed. After separation of idebenone on a reversed-phase column, idebenone was reduced once in a platinum catalyst reduction column connected on-line, then monitored quantitatively by electrochemical detection. A linear relationship between the peak-height ratio of idebenone to the internal standard and idebenone concentration was observed in the range 0.015-50 ng with a detection limit of 5 pg (signal-to-noise ratio = 5). This method was satisfactorily rapid and sensitive, and was successfully applied to the determination of idebenone in rat serum and brain tissues.     

Determination of naproxen in human urine by high-performance liquid chromatography with direct electrogenerated chemiluminescence detection

A simple and sensitive liquid chromatographic method coupled with electrogenerated chemiluminescence (ECL) was described for the separation and quantification of naproxen in human urine. The method was based on the ECL of naproxen in basic NaNO₃ solution with a dual-electrode system. Factors affected the ECL emission were investigated. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of naproxen in the range of 4.0 × 10⁻⁸ g mL⁻¹ to 2.0 × 10⁻⁶ g mL⁻¹ and the detection limit was 1.6 × 10⁻⁸ g mL⁻¹ (S/N = 3). Application of the method to the analyses of naproxen in human urine proved feasible.     

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)     

Evaluation of aryl oxalates for chemiluminescence detection in high-performance liquid chromatography

The chemiluminescence reaction between an aryl oxalate, hydrogen peroxide and a fluorescent compound is well known for use in h.p.l.c. post-column reactors. Here, several aryl oxalates are evaluated for this purpose in terms of intensity, rate of chemiluminescence decay, solubility in different solvents, and stability in the presence of hydrogen peroxide. Five oxalates are selected for different pH ranges of column eluates: bis(pentafluorophenyl) oxalate for pH < 2, bis(2,4-dinitrophenyl) oxalate for pH 2–4, bis(2-nitrophenyl) oxalate for pH 4–6, bis(2,4,6-trichlorophenyl) oxalate for pH 6–8, and bis(2,4,5-trichlorophenyl-6-pentyloxycarbonyl) oxalate for pH > 8.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Determination of bisphenol A in rat brain by microdialysis and column switching high-performance liquid chromatography with fluorescence detection

A sensitive column switching HPLC-fluorescence detection for determination of bisphenol A (BPA) in rat brain by coupling with microdialysis was developed. A microdialysis probe was inserted into the hypothalamus of rat brain and an artificial cerebrospinal fluid was used for perfusion. BPA in brain dialysate was subjected to a fluorescent derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl), and the excess reagent was removed by a column-switching technique. Separation was carried out on two ODS semimicro-columns with the mobile phase of acetonitrile-H(2)O-methanol-tetrahydrofuran (55:10:35:2.5, v/v) and acetonitrile-0.1 M acetate buffer (pH 3.0)-methanol (35:10:55, v/v) at a flow rate of 0.10 and 0.15 mL/min for a precolumn and a separation column, respectively. Fluorescence intensity was monitored at 475 nm with excitation of 350 nm. BPA could be sensitively detected at 0.3 ppb in 60 micro L brain microdialysate at a signal-to-noise ratio of 3. By the proposed method, concentrations of BPA in rat brain and plasma were monitored for 8 h after single i.v. or oral administration. It is proved that BPA is capable of penetrating the blood-brain barrier. The ratio of the area under the concentration-time curve of BPA in rat brain to that in blood was estimated to be about 3.0-3.8%.