Chemiluminescence-high Performance Liquid Research Articles

Novel assay of antibacterial components in manuka honey using lucigenin-chemiluminescence-HPLC

Five components (hydrogen peroxide, methylglyoxal, dihydroxyacetone, fructose and glucose) of New Zealand manuka honey (Leptospermum scoparium) were analyzed using lucigenin chemiluminescence high-performance liquid chromatography (lucigenin-CL-HPLC). We focused on active oxygen species produced from the components in order to easily detect these five components contained in manuka honey. H2O2 and O2− generated from these components were identified by lucigenin-CL and electron spin resonance (ESR), and the bactericidal effect of ROS was confirmed using E. coli. The previously reported assays for Manuka honey components have low specificities and require complicated preprocessing methods. As our results, the detection and identification of these components were possible within 30 min in lucigenin-CL-HPLC system, without any special treatment. It is considered that lucigenin-CL-HPLC is useful for the quality control and the analysis of various honey.

Effect of Curcumin on the Increase in Hepatic or Brain Phosphatidylcholine Hydroperoxide Levels in Mice after Consumption of Excessive Alcohol

Curcumin is a bright yellow compound found in Curcuma longa L., a member of the family Zingiberaceae. In the present study, we determined whether curcumin protects against oxidative stress in liver and brain in mice fed excessive alcohol. BALB/c mice were administered 20% alcohol (16 g/kg body weight) with or without curcumin (0.016% in diet) for 12 weeks. The concentrations of phosphatidylcholine hydroperoxide (PC-OOH) in liver and brain samples were determined using chemiluminescence high-performance liquid chromatography. Mice treated with ethanol and curcumin significantly (P < 0.05) showed a lower hepatic PC-OOH level compared to mice treated with only ethanol. However, there was no significant difference in the brain PC-OOH level among all mice. Our finding indicates that the dosage of alcohol might increase the lipid peroxide level of liver but not of brain, and daily curcumin consumption might be protective for liver against alcohol-related oxidative stress in mice.

Detection of synthetic corticosteroids in bovine urine by chemiluminescence high‐performance liquid chromatography

The development of a black market of chemical cocktails for illegal growth promotion in food-producing animals includes substances that are potentially dangerous for human health, such as synthetic corticosteroids. The potential presence of these residues in food makes it necessary to develop rapid and sensitive analytical methodologies to detect such substances, preferably in live animals before they arrive at the market. A chemiluminescence (CL) detection method for the determination of four synthetic corticosteroids (prednisolone, betamethasone, dexamethasone and flumethasone) in bovine urine has been developed. The proposed system, which does not need any derivatization procedure, offers an easy method well suited for routine research. Urine samples were homogenized with methanol:water (50:50, v/v) and centrifuged. The upper layer was collected and Strata X cartridges were used for cleaning up. The purified residues were evaporated to dryness and then redissolved in the mobile phase. Analysis of the extracts was performed using high-performance liquid chromatography with chemiluminescence detection, employing luminol as the CL reagent. The recovery curves, obtained at four spiking levels (different for each corticosteroid), showed that recoveries of at least 70% could be obtained for urine. The chemiluminescence detection procedure afforded satisfactory results with respect to sensitivity and the LOD and LOQ, taken as the first point of the regression curve, ranged from 4 ppb to 65 ppb. The maximum mean RSD was below 13% and below 15% for intra- and inter-day assay, respectively, in all cases.

Increase of serum phosphatidylcholine hydroperoxide dependent on glycemic control in type 2 diabetic patients

In order to clarify the relationship between serum phosphatidylcholine hydroperoxide (PCOOH) levels and blood glucose control in type 2 diabetes patients (DM), DM ( n=61) and normal control ( n=11) were enrolled. High-density lipoprotein (HDL) was separated from serum by the addition of sodium phosphotungstate and magnesium chloride, and the precipitated fraction was prepared as non-HDL. Phospholipids were extracted from whole serum, non-HDL and HDL to estimate PCOOH level with chemiluminescence high performance liquid chromatography (CL-HPLC). PCOOH level (nmol/l, mean±S.D.) was higher in DM than in control (33.1±9.5 vs. 23.0±8.2 for serum; P<0.01, 17.0±5.5 vs. 10.6±3.8 for non-HDL; P<0.01, and 16.1±6.3 vs. 12.3±5.5 for HDL; not significant, respectively). DM was divided into five groups according to hemoglobin A 1c (HbA 1c) levels (%): (1) less than 6, (2) 6–6.4, (3) 6.5–6.9 (4) 7.0–7.4, and (5) over than 7.5. Increase of PCOOH levels was dependent on HbA 1c. We concluded that (1) serum and non-HDL PCOOH increased in DM, (2) the level was strongly correlated with diabetic control, and (3) approximately a half amount of serum PCOOH was present in HDL of both control and DM.

New approach for measurement of sympathetic nervous abnormality in conscious, spontaneously hypertensive rats.

We previously reported a highly sensitive chemiluminescence high-performance liquid chromatographic method to determine catecholamines in plasma. In this study, we employed this method to measure the cardiac function and plasma norepinephrine (NE) concentration in conscious rats. Benidipine, 1,4-dihydropyridine calcium antagonist (4 mg/kg), and beta-blocker (propranolol, 30 mg/kg) were administered orally to conscious spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats, and blood pressure, heart rate and plasma NE levels were measured. Plasma NE concentration was used as an index of sympathetic nervous system activity in conscious rats. The basal plasma NE levels were significantly higher in SHRs than in WKY rats (P<0.05), indicating the activity of the basal sympathetic nervous system in SHRs was elevated. The sensitivity of the baroreflex-mediated sympathetic nervous response was reduced in SHRs as compared to that in WKY rats. The concomitant administration of benidipine and a beta-blocker decreased heart rate without affecting the baroreflex-mediated sympathetic nervous response, indicating that propranolol might suppress mainly the cardiac beta-adrenoceptor. The present study suggested the high activity of the basal sympathetic nervous system and the reduced response of the baroreflex-mediated sympathetic nervous system in SHRs compared to WKY rats in the conscious condition.

Determination of dexamethasone in bovine liver by chemiluminescence high-performance liquid chromatography.

A new method for the determination of dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha -methylpregna-1, 4-diene-3,20-dione) in bovine liver was developed. This new liquid-liquid extraction method comprises the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract is evaporated to dryness and the residue dissolved in acetonitrile. The cleaning of the fat is performed with n-hexane, and the acetonitrile layer is evaporated. Analysis of the extracts is performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as CL reagent. A series of recovery curves performed at spiking levels of 50, 30, 10, 5, and 2.5 ppb show that at least 80% of DEX can be recovered from liver and that the chemiluminescence detection yields satisfactory results with respect to sensitivity (LOD 0.2 ppb), reproducibility (CV% 10.7) and repeatability (CV% 6.2-8.9).

Development of Electrogenerated Chemiluminescence HPLC for Ofloxacin Using Tris (2, 2′-bipyridine) ruthenium (II) and Its Application to Residual Analysis in Chicken Tissues

An electrogenerated chemiluminescence high performance liquid chromatography (ECL-HPLC) for ofloxacin (OFLX) using tris (2, 2′-bipyridine) ruthenium (II) was developed and applied for residue analysis in chicken tissues. OFLX was extracted from tissues by homogenizing with 0.1mol/L phosphate buffer (pH 12)-acetonitrile (1:3, v/v) and centrifuging the mixture. The supernatant was shaken with n-hexane saturated with acetonitrile. After having been centrifuged, the lower layer was evaporated to dryness under reduced pressure and the residue was dissolved in acetonitrile-0.05mol/L NaH2PO4 (pH 2.4) (20:80, v/v) and analyzed by ECL-HPLC. The mean recoveries of OFLX from chicken tissues were 66-102%. The detection limit of this method was 10ng/g (0.01ppm) of OFLX. The correlation between ECL-HPLC and FL-HPLC was excellent.

Chemiluminescence High Performance Liquid Chromatography of Corticosteroids and p-Nitrophenacyl Esters Based on the Luminol Reaction

Reducing agents, especially compounds having an α-hydroxycarbonyl group, give an intense chemiluminescence with luminol in the presence of catalyst in alkaline solution. Based on this reaction, the chemiluminescence detection of corticosteroids and p-nitrophenacyl esters of carboxylic acids has been developed. These compounds were separated by high performance liquid chromatography on a reversed phase column (Prodigy ODS) for corticosteroids with 30% acetonitrile and for p-nitrophenacyl esters with 50% acetonitrile as eluent, and detected by the chemiluminescence reaction with luminol-sodium hydroxide solution containing the catalyst as the post-column reagent. Hexacyanoferrate (III) and hexacyanoferrate (II) were used as catalyst of the reaction. The detection limits of cortisone and p-nitrophenacyl hippurate were 1.1 pmol and 0.8 pmol.

Chemiluminescence–High-Performance Liquid Chromatographic Determination of Tea Catechin, (−)-Epigallocatechin 3-Gallate, at Picomole Levels in Rat and Human Plasma

A method of chemiluminescence detection–high-performance liquid chromatography (CL–HPLC) for the highly specific determination of tea catechin, (−)-epigallocatechin 3-gallate (EGCg), present in rat and human plasma has been newly developed. The CL–HPLC system consists of reversed-phase HPLC and chemiluminescence detector, in which separated EGCg generates chemiluminescence at post column successively reacting with the following two chemiluminescence cocktails; 8.2macetaldehyde in 50 mmphosphate buffer (pH 7.4, contained 108 mg horseradish peroxidase/L) and 8.8mhydrogen peroxide aqueous solution. The plasma EGCg was extracted by methanol. This method enables the detection of EGCg in the free form selectively at as low as 2 pmol with recovery of 84%. The EGCg concentration in fasted rat plasma was initially below the detection limit (<2 pmol/ml), but increased to maximum level (2284 pmol/ml plasma, 1047 ng/ml; calculated 0.012% of ingested EGCg) 30 min after a single oral supplementation of 56 mg EGCg per rat. The EGCg concentration in fasted human plasma was also initially below the detection limit and increased to 341 pmol/ml (156 ng/ml; calculated 0.32% of ingested EGCg) at 60 min after a single oral intake of 97 mg EGCg per subject. The results indicated that tea catechin, EGCg, is absorbed from the digestive tract into the rat and human body and that the CL–HPLC method reported here should be a powerful tool for studying the metabolic fate and bioavailability of EGCg.

Behavior of Lipid Hydroperoxides During Mashing

Lipid hydroperoxides such as trilinolein hydroperoxides in barley, malt, and mash were analyzed using a chemiluminescence high-performance liquid chromatography method. During mashing in a laborato...

Detection of Squarene Hydroperoxide

Squalene hydroperoxide present in human skin surface lipids was examined by a chemiluminescence high performance liquid chromatography (CL-HPLC) system. A mixture of cytochrome c and luminol served as the hydroperoxide-specific luminescence reagent for post-column detection in reversed phase HPLC. Squalene hydroperoxide was previously detected in skin surface lipids of the human forehead following exposure to the sun. Therefore, in this study, squalene monohydroperoxide was prepared by dye-sensitized photooxidation and its chemical structure was determined by 1H-NMR, 13C-NMR, and FAB-MS. The detection limit of squalene monohydroperoxide in CL-HPLC system was as low as 1 pmol. Skin surface lipids from forehead, scalp, and dandruff were also studied. By exposure to sun light or after washing with shampoo, squalene monohydroperoxide could be detected in nearly all samples examined. Squalene may thus be considered the first target lipid on human skin surface that incurs oxidative stress by sun light exposure, etc.

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL—HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of α-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.

Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate

A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide—dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm × 4.0 mm I.D.) packed with amido-80 silica beads (5 μm diameter). For post-column elution in the chemiluminescence system, 1 m M bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3 m M hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.

Chemiluminescence high performance liquid chromatography of corticosteroids using lucigenin as post‐column reagent

A chemiluminescence high performance liquid chromatographic method for the determination of corticosteroids and tetrahydrocorticosteroids has been developed. Corticosteroids and their metabolites extracted from urine samples were separated using an ODS column and a mixture of methanol + water + 0.01 M sodium acetate solution (70:30:5) as eluent. The eluent from the column was mixed with the chemiluminescent solution containing lucigenin and Triton X-100 and a 0.28 M KOH solution by pumps and monitored by a chemiluminescence detector. No interference was encountered and the method is both precise and reproducible.

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC.

A combined system of chemiluminescence detection and high performance liquid chromatography (CL-HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL-HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL-HPLC method, a cytochrome c-luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide-O2. PCOOH in normal human blood was found to be 10-500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.