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 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 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 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.     

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 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 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 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 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.     

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 vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

Determination of artemisinin in human serum by high-performance liquid chromatography with on-line UV irradiation and peroxyoxalate chemiluminescence detection

Artemisinin is an antimalarial drug containing an internal endoperoxide linkage in its structure. A simple, selective and sensitive high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence (PO-CL) method for the determination of artemisinin was developed. This method is based on the fact that endoperoxide in artemisinin structure can be converted to hydrogen peroxide (H(2)O(2)) under ultraviolet (UV) irradiation and the generated hydrogen peroxide can be measured using PO-CL detection. The HPLC-PO-CL system was optimized on a mobile phase, post column chemiluminescence reagent, UV source and irradiation time. In addition, the system was combined with simple liquid-liquid extraction using n-hexane that allowed selective and sensitive determination of artemisinin in serum. The limit of detection using 0.5 mL of blood was 0.062 micromol/L (17.5 ng/mL) at a signal-to-noise ratio of 3. Calibration curve obtained for artemisinin in human serum 4-80 micromol/L (1.1-22.6 microg/mL) showed a good linearity (r = 0.999).     

Sensitive determination of aliphatic amines in water by high-performance liquid chromatography with chemiluminescence detection

A sensitive method has been developed for liquid chromatographic determination of short aliphatic amines in water samples. Analytes are preconcentrated and dansylated on solid sorbents (C18 solid-phase extraction cartridges). The dansyl derivatives are chromatographed and post-column mixed with peroxyoxalate (TCPO) and H2O2 in order to perform chemiluminescence detection. Optimal results have been obtained using a sample volume of 5 ml. The method has been applied to the quantification or screening of several aliphatic amines: methylamine, ethylamine, butylamine, diethylamine, pentylamine and hexylamine. The screening procedure has been developed including also polyamines (putrescine, cadaverine, spermidine and spermine). The results obtained by using chemiluminescence (CL) detection have been compared with other detection systems (fluorescence and UV). The sensitivity can increase from 3 to 75 times respect UV detection and from 2 to 10 times respect fluorescence detection depending on the amine. The detection limits achieved were between 0.15 and 0.9 microg/l.     

Determination of tetracycline antibiotics by reversed-phase high-performance liquid chromatography with fluorescence detection.

A highly sensitive method for the determination of tetracycline antibiotics (TCs) using reversed-phase high-performance liquid chromatography with fluorescence detection is presented. This method was based on the use of disodium ethylenediaminetetraacetate (EDTA) and calcium chloride as fluorescence-increasing reagents in the mobile phase. The concentrations of each reagent in the mobile phase greatly influenced the fluorescence intensity of TCs. When the concentration of EDTA and calcium chloride were 25 and 35 mM, respectively, and the pH of the mobile phase was 6.5, the maximum fluorescence intensity was obtained. The column temperature hardly influenced the fluorescence intensity. At 3.75 ng of TCs injected, the precision (relative standard deviation) ranged from 1.12 to 2.20%. In the range 0.075-37.5 ng for tetracycline and oxytetracycline and 0.225-37.5 ng for chlortetracycline, a linear response was observed. The detection limits of this method were 49-190 pg for three different TCs. The proposed method was applied to the determination of one of the TCs in pharmaceuticals by the internal standard method using other TCs as internal standards and was also applied to determination of TCs added to fish tissue.     

Determination of nine pyrethroid insecticides by high-performance liquid chromatography with post-column photoderivatization and detection based on acetonitrile chemiluminescence

An HPLC method was developed to determine pyrethroids, including fenpropathrin, beta-cyfluthrin, lambda-cyhalothrin, deltamethrin, fenvalerate, permethrin, acrinathrin, tau-fluvalinate, and bifenthrin, by coupling HPLC, post-column irradiation with UV light and chemiluminescence detection of the resulting photoproducts. It is based on the observation that photolyzed pyrethroids take part in a chemiluminescent reaction in presence of K3Fe(CN)6 and NaOH, whose signal increases with the percentage of acetonitrile in the reaction medium. As the yield of the photoderivatization process and the chemiluminescent signals depend on the percentage of acetonitrile, the chromatographic column (a Gemini C18, Phenomenex 150 mm x 4.6 mm, 5 microm particle size) was chosen with the aim of using high percentages of this organic solvent in the mobile phase. Previous studies showed that the rate of the chemiluminescent reaction was very fast. Therefore, a modification was carried out in the detector in order to mix the analytes and reactives as near as possible to the measure cell. The optimised method was validated with respect to linearity, precision, limits of detection and quantification accuracy. Under the optimised conditions, linear working range extends three orders of magnitude with the relative standard deviation on intra-day precision below 10% and detection limits between 0.013 and 0.049 microg mL(-1), according to the compound. The proposed method has been successfully applied to the determination of pyrethroids in tomato with good results.