High-performance Liquid Chromatography Chemiluminescence Research Articles

Determination of Glutathione and Cysteine in Human Breast Milk by High-Performance Liquid Chromatography with Chemiluminescence Detection for Evaluating the Oxidative Stress and Exposure to Heavy Metals of Lactating Women

Exposure to heavy metals may affect a vast array of biochemical and nutritional processes in the human body. The toxic manifestations of these metals are caused primarily due to the imbalance between pro-oxidant and antioxidant homeostasis which is termed as oxidative stress. A high-performance liquid chromatography–chemiluminescence (HPLC-CL) method was developed for the simultaneous determination of two antioxidants, glutathione and cysteine, in human breast milk. The HPLC system included a C18 analytical column with a mobile phase composed of 1.0% methanol. The aminothiols were eluted from the C18 column, mixed with formaldehyde and potassium permanganate solutions, and subsequently monitored by a chemiluminescence detector. The new method was validated by monitoring glutathione and cysteine in human breast milk of volunteers from the Gusu and Industrial Districts of Suzhou, China. Four heavy metals in atmospheric particulate matter collected from the two districts were also determined by inductively coupled plasma–mass spectrometry (ICP-MS). The results demonstrated that the concentrations of cysteine and glutathione were significantly (p < 0.05) lower in human breast milk from the subjects living in the Gusu region of Suzhou, Jiangsu Province, China compared to an industrial district. These results were inversely correlated with the concentration levels of the toxic metals in collected atmospheric particulate matter. The decrease in the glutathione and cysteine concentrations in human breast milk appeared to be due to oxidative stress in the breast-feeding mothers.

Determination of monoamine neurotransmitters and metabolites by high-performance liquid chromatography based on Ag(III) complex chemiluminescence detection

A novel, simple and efficient chemiluminescence system has been developed for the determination of monoamine neurotransmitters and metabolites. By using the Ag (III)-luminol chemiluminescence system as a detector, a high performance liquid chromatography chemiluminescence method (HPLC-CL) was established and used to detect seven monoamine neurotransmitters. Under the optimized conditions, the detection limits (3S/N) of epinephrine (E), levodopa (l-DOPA), dopamine (DA), serotonin (5-HT), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxypentylacetic acid (5-HIAA) were 20.0 μg dm−3,15.0 μg dm−3, 15.0 μg dm−3, 8.0 μg dm−3, 2.0 μg dm−3, 2.0 μg dm−3 and 3.0 μg dm−3, respectively. Moreover, they were well within the linear range of 50–1000 μg dm−3, 50–1000 μg dm−3, 50–1000 μg dm−3, 25–1000 μg dm−3, 5–25 μg dm−3, 5–25 μg dm−3 and 10–30 μg dm−3, respectively. The average recovery varied between 84.82% and 110.4%. The method has the attributes of simplicity, high sensitivity, and high efficiency. The sensitization and inhibition mechanisms for luminol-[Ag(HIO6)2]5–- analytes CL system were proposed by CL spectra and free-radical capture experiment.

An inter-laboratory comparison of different analytical methods for the determination of monomethylmercury in various soil and sediment samples: A platform for method improvement

An inter-laboratory study was conducted to compare results from different analytical methods for monomethylmercury (MeHg) concentrations in 17 soil and sediment samples. The samples were collected from mercury-contaminated areas, including Minamata Bay and Kagoshima Bay in Japan, the Idrija mercury mine in Slovenia, and an artisanal small-scale gold mining area in Indonesia. The Hg in these samples comes from several different sources: industrial waste from an acetaldehyde production facility, volcanic activity, Hg mining activity, and artisanal and small-scale gold mining activity (ASGM). MeHg concentrations in all the samples were measured in four separate laboratories, using three different determination methods: Kagoshima University (Japan), using high-performance liquid chromatography–chemiluminescence detection (HPLC-CL); National Institute for Minamata Disease (Japan), using gas chromatography–electron capture detection; and Metropolitan Council Environmental Services (USA) and Jozef Stefan Institute (Slovenia), both using alkylation-gas chromatography–atomic fluorescence spectrometry detection. The methods gave comparable MeHg results for most of the samples tested, but for some samples, the results exhibited significant variability depending on the method used. The HPLC-CL method performed poorly when applied to samples with elevated sulfur concentrations, producing MeHg concentrations that were much lower than those from the other methods. Additional analytical work demonstrated the elimination of this sulfur interference when the method was modified to bind sulfur prior to the analytical step by using Hg2+ as a masking agent. These results demonstrate the value of laboratory intercomparison exercises in contributing to the improvement of analytical methods.

Rapid method for monitoring N-nitrosodimethylamine in drinking water at the ng/L level without pre-concentration using high-performance liquid chromatography-chemiluminescence detection

As a contaminant in drinking water, N-nitrosodimethylamine (NDMA) is of great concern because of its carcinogenicity; it has been limited to levels of ng/L by regulatory bodies worldwide. Consequently, a rapid and sensitive method for monitoring NDMA in drinking water is urgently required. In this study, we report an improvement of our previously proposed HPLC-based system for NDMA determination. The approach consists of the HPLC separation of NDMA, followed by NDMA photolysis to form peroxynitrite and detection with a luminol chemiluminescence reaction. The detection limit for the improved HPLC method was 0.2ng/L, which is 10 times more sensitive than our previously reported system. For tap water measurements, only the addition of an ascorbic acid solution to eliminate residual chlorine and passage through an Oasis MAX solid-phase extraction cartridge are needed. The proposed NDMA determination method requires a sample volume of less than 2mL and a complete analysis time of less than 15min per sample. The method was utilized for the long-term monitoring of NDMA in tap water. The NDMA level measured in the municipal water survey was 4.9ng/L, and a seasonal change of the NDMA concentration in tap water was confirmed. The proposed method should constitute a useful NDMA monitoring method for protecting drinking water quality.

Selective and Sensitive Chemiluminescence Determination of MCPB: Flow Injection and Liquid Chromatography.

Two new chemiluminescence (CL) methods are described for the determination of the herbicide 4-(4-chloro-o-tolyloxy) butyric acid (MCPB). First, a flow injection chemiluminescence (FI-CL) method is proposed. In this method, MCPB is photodegraded with an ultraviolet (UV) lamp and the photoproducts formed provide a great CL signal when they react with ferricyanide in basic medium. Second, a high-performance liquid chromatography chemiluminescence (HPLC-CL) method is proposed. In this method, before the photodegradation and CL reaction, the MCPB and other phenoxyacid herbicides are separated in a C18 column. The experimental conditions for the FI-CL and HPLC-CL methods are optimized. Both methods present good sensitivity, the detection limits being 0.12 µg L(-1) and 0.1 µg L(-1) (for FI-CL and HPLC-CL, respectively) when solid phase extraction (SPE) is applied. Intra- and interday relative standard deviations are below 9.9%. The methods have been satisfactorily applied to the analysis of natural water samples. FI-CL method can be employed for the determination of MCPB in simple water samples and for the screening of complex water samples in a fast, economic, and simple way. The HPLC-CL method is more selective, and allows samples that have not been resolved with the FI-CL method to be solved.

Improved blood–brain barrier distribution: Effect of borneol on the brain pharmacokinetics of kaempferol in rats by in vivo microdialysis sampling

Ethnopharmacological relevanceKaempferol (KA) exists in a variety of herbal medicines. In vitro and in vivo studies have focused on the anti-Alzheimer effect of KA. However, little is known about its brain pharmacokinetic profile. The accumulated amount of KA in brain is very low because of the protection of blood–brain barrier (BBB). Borneol (BO) is a classical aromatic refreshing traditional Chinese medicine and commonly used as an adjuvant component of traditional Chinese medicines (e.g. compound Danshen dropping pills) in the treatment of cardiovascular and cerebrovascular diseases. According to the basic theories of traditional Chinese medicine, BO is called an “upper guiding drug”, which can guide other components to the targeting tissues or organs in the upper part of the body, especially in the brain. Materials and methodsThe probes for blood and brain sampling were implanted within the jugular vein/right atrium and right hippocampus of SD rats, respectively. Rats were intravenous administered of KA (25mg/kg) alone or combined with BO (15, 30mg/kg) via caudal vein. The blood and brain microdialysates were collected every 15min for 180min and every 30min for 180−300min. A selective and sensitive high performance liquid chromatography–chemiluminescence method was developed for the determination of unbound KA in rat blood and brain microdialysates, which can be converted to their actual free-form concentrations based on the in vivo relative recoveries of KA across microdialysis probes. ResultsKA quickly crossed the BBB to enter the extracellular fluid of hippocampus and reached the maximum concentration of 0.11μg/mL within 30min. The brain bioavailability and brain delivery of KA evidently increased with the co-administration of 15 and 30mg/kg of BO. The AUC0–inf of KA in brain increased 1.84 and 2.19 times, and the Cmax of KA in brain increased 2.09 and 3.18 times than that without BO, respectively. In addition, the brain-to-blood distribution ratio of KA increased by 48.68% and 57.97% compared with that without BO. However, no significant difference in the T1/2 of unbound KA in blood aserved between three groups. ConclusionsBO can enhance the BBB permeability and improve the transportation of KA to brain. The dose-dependent effect of BO on the brain pharmacokinetic parameters of KA was observed. This co-administration strategy can be designed to enhance the brain accumulation of other neuropsychiatric medications.

Selective determination method for measurement of methylmercury and ethylmercury in soil/sediment samples using high-performance liquid chromatography–chemiluminescence detection coupled with simple extraction technique

A method for the simultaneous determination of monomethylmercury (MeHg+) and monoethylmercury (EtHg+) in soil/sediment samples was developed. The method involves eluting mercury species from the soil/sediment samples using 5M HCl containing 5mM Pd2+ and 0.1M Cu2+ and then extracting MeHg+ and EtHg+ into toluene as chlorides. These alkylmercury chlorides are then back-extracted into an aqueous EDTA solution, creating EDTA complexes. Finally, an emetine-dithiocarbamate (emetineCS2) solution is added to the EDTA solution to form emetineCS2–alkylmercury complexes. EmetineCS2–MeHg and emetineCS2–EtHg were separated using reverse-phase HPLC and then detected by the chemiluminescence reaction with tris(2,2′-bipyridine)ruthenium(III). The MeHg+ and EtHg+ calibration curves, using the peak height, were linear from 0.5 to 20ng (as Hg). The detection limit was 0.16ng/g (analyzing 1g soil or sediment). The procedure was validated by analyzing a certified reference material (ERM CC580, estuarine sediment). The MeHg+ concentration determined using the proposed method was in good agreement with the certified value, and EtHg+ was detected in the reference material. A preliminary study of the relationship between environmental mercury concentrations and MeHg+ production was performed.

Simultaneous determination of amphetamine-type stimulants in abusers’ hair: clinical usefulness of hair analysis in prehospitalization for abusers

Simultaneous determination of amphetamine-type stimulants (ATSs), such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and p-methoxymethamphetamine (PMMA), has been made for hair of 32 female abusers from different ethnic backgrounds. One milligram of abuser's hair spiked with 1-methyl-3-phenylpropylamine as internal standard was used for each determination of the ATSs. After digestion with 1 M NaOH, the samples were extracted with n-heptane; the organic layer was evaporated to dryness, dissolved in 75 mM of borate buffer solution (pH 8.5), and labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The determination of the ATSs in hair was performed by the semi-micro high-performance liquid chromatography-chemiluminescence method. Thirty-two subjects who had self-report histories of ATS use for more than 1 week prior to hair sample collection participated in this study. Concentration ranges of ATSs in abusers’ hair were 0.07–6.73 ng/mg for MA (n = 24), 0.18–2.47 ng/mg for AP (n = 13), 0.05–0.86 ng/mg for MDMA (n = 12), 0.52–1.38 ng/mg for MDA (n = 3), and 0.09–1.88 ng/mg for PMMA (n = 6). Several discrepancies between detected drugs and information obtained from the interviewed abusers were found. Most of the drugs determined in this study could not be detected by the urine test performed at hospitalization. Correlation between ATS concentration and psychiatric symptoms on the basis of Brief Psychiatric Rating Scale (BPRS) was also studied. Simultaneous determination of ATSs in hair of outpatients and inpatients at psychiatric clinics and hospitals should be effective in providing proper diagnosis and treatment. This is the first report to demonstrate the presence of PMMA in hair of human subjects.

Development of HPLC Determination Method for Trace Levels of 1-, 2-Nitropyrenes and 2-Nitrofluoranthene in Airborne Particulates and Its Application to Samples Collected at Noto Peninsula

1-Nitropyrene (1-NP), 2-NP and 2-nitrofluoranthene (2-NFR) are useful markers for studying the atmospheric behaviors of polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs). However, present methods for measuring trace levels of these compounds are lesssensitive and laborious. Here we describe several improvements to a previously reported high-performance liquid chromatography-chemiluminescence detection system that allows it to determine trace levels of 1-, 2-NPs and 2-NFR. The proposed system was equipped with a reducer column packed with Pt/Rh instead of zinc whose life-time was limited. The combination of Cosmosil MS-II (monomeric ODS) and AR-II (polymeric ODS) columns was used instead of polymeric ODS columns as the separator column to improve the separation. An ethanol mixture with acetate buffer (pH 5.5) was used in place of an acetonitrile mixture with the same buffer to activate the reducer column. The same ethanol mixture was used as the mobile phase for the clean-up column. The switching time of the column switching valve was optimized to concentrate the amino-derivatives of above NPAHs quantitatively on the concentrator column. The concentrations of bis(2,4,6-trichlorophenly) oxalate and hydrogen peroxide in the chemiluminescence reagent solution were optimized to 0.4 mM and 30 mM, respectively, to increase the sensitivity. Under the above conditions, the detection limits (S/N=3) of 1-, 2-NPs and 2-NFR were 1 fmol (0.25 pg), 10 fmol (2.5 pg) and 4 fmol (1 pg), respectively. The proposed system was effectively used to determine trace levels of 1-, 2-NPs and 2-NFR in airborne particulates collected at Noto Peninsula. The atmospheric concentrations of 1-, 2-NPs and 2-NFR were not more than sub pg <TEX>$m^{-3}$</TEX> levels. They were higher in winter (January) than in summer (July). In both seasons, the concentrations were in decreasing order, [2-NFR]>[1-NP]>[2-NP].

High performance liquid chromatography with immobilized Ru(bpy)32+–KMnO4 chemiluminescence detection and its application in metabolism of repaglinide in pig liver microsomes

Abstract A novel high performance liquid chromatography–chemiluminescence (HPLC–CL) method for investigation of in vitro metabolism of repaglinide in pig liver microsomes with microdialysis sampling technique was developed. The chromatographic separation was performed on a Hypersil BDS-C18 column with an isocratic mobile phase consisting of methanol and 0.01 mol/L KH 2 PO 4 (pH 3.0) (volume ratio 75:25) at a flow rate of 1.0 mL/min. The detection was based on the chemiluminescence reaction of repaglinide with acidic potassium permanganate (KMnO 4 ) and tris (2,2′-bipyridyl)ruthenium(III) (Ru(bpy) 3 3+ ), which was immobilized on the cationic ion-exchange resin for obtaining high sensitivity and reducing consumption of expensive reagent.

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (−)-epicatechin and (−)-epigallocatechin gallate solutions, were linear over the range of 0.1–50 μM. The detection limits (signal-to-noise ratio = 3) were 0.8 pmol for (−)-epigallocatechin gallate and 1.2 pmol for (−)-epicatechin. The high-performance liquid chromatography–chemiluminescence (HPLC–CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.

Determination of phenolic compounds using high-performance liquid chromatography with Ce 4+-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 (3 σ) 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.

Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress.

To explore the role of lipid peroxidation (LPO) products in the initial phase of stress mediated signaling, we studied the effect of mild, transient oxidative or heat stress on parameters that regulate the cellular concentration of 4-hydroxynonenal (4-HNE). When K562 cells were exposed to mild heat shock (42 degrees C, 30 min) or oxidative stress (50 microM H2O2, 20 min) and allowed to recover for 2 h, there was a severalfold induction of hGST5.8, which catalyzes the formation of glutathione-4-HNE conjugate (GS-HNE), and RLIP76, which mediates the transport of GS-HNE from cells (Awasthi, S., Cheng, J., Singhal, S. S., Saini, M. K., Pandya, U., Pikula, S., Bandorowicz-Pikula, J., Singh, S. V., Zimniak, P., and Awasthi, Y. C. (2000) Biochemistry 39, 9327-9334). Enhanced LPO was observed in stressed cells, but the major antioxidant enzymes and HSP70 remained unaffected. The stressed cells showed higher GS-HNE-conjugating activity and increased efflux of GS-HNE. Stress-pre-conditioned cells with induced hGST5.8 and RLIP76 acquired resistance to 4-HNE and H2O2-mediated apoptosis by suppressing a sustained activation of c-Jun N-terminal kinase and caspase 3. The protective effect of stress pre-conditioning against apoptosis was abrogated by coating the cells with anti-RLIP76 IgG, which inhibited the efflux of GS-HNE from cells, indicating that the cells acquired resistance to apoptosis by metabolizing and excluding 4-HNE at a higher rate. Induction of hGST5.8 and RLIP76 by mild, transient stress and the resulting resistance of stress-pre-conditioned cells to apoptosis appears to be a general phenomenon since it was not limited to K562 cells but was also evident in lung cancer cells, H-69, H-226, human leukemia cells, HL-60, and human retinal pigmented epithelial cells. These results strongly suggest a role of LPO products, particularly 4-HNE, in the initial phase of stress mediated signaling.

Effect of n-3 polyunsaturated fatty acid supplementation on lipid peroxidation of rat organs.

The present study was undertaken in order to reexamine the effect of n-3 polyunsaturated fatty acid (PUFA)-rich diet supplementation on lipid peroxidation and vitamin E status of rat organs. Male Wistar rats were fed a diet containing safflower or fish oil at 50 g/kg diet and an equal amount of vitamin E at 59 mg/kg diet (1.18 g/kg oil; and 1.5 g/kg PUFA in safflower oil diet, and 4.3 g/kg PUFA in fish oil diet) for 6 wk. Fatty acid composition of total lipids of brain, liver, heart, and lung of rats fed fish oil was rich in n-3 PUFA, whereas that of each organ of rats fed safflower oil was rich in n-6 PUFA. The vitamin E levels in liver, stomach, and testis of the fish oil diet group were slightly lower than those of the safflower oil diet group, but the levels in brain, heart, lung, kidney, and spleen were not different between the two diet groups. The levels of phospholipid hydroperoxides were determined by the high-performance liquid chromatography-chemiluminescence method and the levels of thiobarbituric acid-reactive substances (TBARS) were determined at pH 3.5 in the presence of butylated hydroxytoluene with or without EDTA. Levels of phospholipid hydroperoxides and TBARS in the brain, liver, heart, lung, kidney, spleen, stomach and testis of the fish oil diet group were similar to those of the safflower oil diet group. The results indicate that high fish oil intake does not induce increased levels of phospholipid hydroperoxides and TBARS in rat organs.

Morphine Determination by HPLC with Improved Chemiluminescence Detection Using a Conventional Silica Based Column

A new high performance liquid chromatography-chemiluminescence analysis system for morphine is described. This system has an on-column limit of detection of 20 pg for morphine, allowing morphine determination down to 1 ng/mL, with a 20 μL injection size, without any concentration during sample preparation. Simultaneous determination of monoacetylmorphine to 15 ng/mL (300 pg on-column) is also achieved. The internal standard for both compounds is nalorphine and total analysis time is under 5 minutes.

Simultaneous Determinations of Histamine and Nτ-Methylhistamine by High-Performance Liquid Chromatography-Chemiluminescence Coupled with Immobilized Diamine Oxidase

A method for the simultaneous determinations of histamine and its metabolite Nτ-methylhistamine by HPLC-chemiluminescence coupled with immobilized diamine oxidase was developed. The method was based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture in alkaline medium with hydrogen peroxide which is one of the metabolic products of histamine and Nτ-methylhistamine formed by diamine oxidase. HPLC with postcolumn derivatization resulted in good separation of the two amines and gave linear relationships between the concentrations of both and their chemiluminescence intensities. The lower limits of chemiluminescent detection of histamine and Nτ-methylhistamine were 5 and 10 pmol, respectively. The immobilized column showed good operational stability for more than 1 month, during which period 200 samples were analyzed. With this system, the histamine contents of the cerebral cortex, forestomach, glandular stomach, and kidney of Wistar rats were found to be 0.30, 58, 396, and 2.4 nmol/g wet wt, respectively. These values are very similar to those determined by HPLC-fluorometry. The Nτ-methylhistamine contents of these tissues were 0.36, 0.40, 0.72, and 3.8 nmol/g wet wt, respectively. This method will be useful for studying the roles of histamine in both brain and peripheral tissues.

Evidence for accumulation of lipid hydroperoxides during the aging of human red blood cells in the circulation.

Young and old human red blood cells (RBC) were separated from freshly collected human blood by Percoll density gradient centrifugation. The amounts of lipid peroxidation products in the lipid fractions of these RBC were determined by high performance liquid chromatography-chemiluminescence, thiobarbituric acid and LPO-586 methods. The levels of phosphatidylcholine and phosphatidylethanolamine hydroperoxides in old RBC were higher than in young RBC. The products reflecting the advanced stage of lipid peroxidation, malonaldehyde (free and bound forms), 4-hydroxyalkenals (free and bound forms) and other aldehydes, were also higher in old RBC than in young RBC. The levels of these lipid peroxidation products increased when whole RBC were mildly oxidized with ferric ion. These results indicate that oxidative damage of RBC in the circulation takes place during the aging process, and lipid hydroperoxides and other lipid peroxidation products accumulate in old RBC.

Use of stop-flow oxalate ester chemiluminescence as a means to determine conditions for high-performance liquid chromatography chemiluminescence detection of retinoids using normal-phase chromatography

Stop-flow chemiluminescence (CL) has been used to determine the conditions necessary for the oxalate ester CL detection of selected retinoids after separation by normal-phase HPLC. Also, the detection of the selected retinoids by bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen peroxide (H2O2) chemiluminescence and fluorescence (FL) are compared. Stop-flow CL was performed on a prototype uit from High-Tech Scientific, Ltd (Salisbury, UK). Detection limits were determined for retinyl palmitate, retinyl acetate, retinol, etretinate, acitretin and tretinoin, after separation on a YMC PVA-sil, 25 cm x 4.6 mm column using hexane-tetrahydrofuran-acetic acid (75:25:0.01, v/v/v) as eluent at 1.5 ml min-1. A Schoeffel 970 detector was used for both CL and FL detection. Detection by FL was determined with lambda ex 355 nm and lambda em greater than 418 nm. CL detection was performed with the deuterium lamp off and no emission filter. CL was induced by mixing the reagents post-column. Pump A--hexane--THF--AcOH (75:25:0.01, v/v/v) at 1.5 ml min-1, pump B--70 mM TCPO in THF at 0.5 ml min-1, pump C--THF--50% aq H2O2 (75:25, v/v) 1.0 mg ml-1 imidazole at 1.0 ml min-1.

Photolytic interface for high-performance liquid chromatography--chemiluminescence detection of non-volatile N-nitroso compounds.

A photolytic interface between high-performance liquid chromatography (HPLC) and a chemiluminescence detector has been developed for the trace detection of non-volatile N-nitroso compounds in biological matrices. A chromatographic effluent containing separated N-nitrosoamino acids and N-nitrosamides is introduced into a glass coil with a purge stream of He and irradiated with ultraviolet light. Nitrogen oxide, cleaved by photolysis, is separated rapidly from the solvent through a series of cold traps and carried by the He into the reaction chamber of a chemiluminescence detector. The method is compatible with most types of HPLC, especially reversed-phase, and yields low-nanogram sensitivity for underivatised N-nitrosoamino acids and N-nitrosamides. The detection of a model N-nitrosamide, trimethylnitrosourea, in spiked porcine gastric fluid (42 micrograms l-1), and of N-nitrosoproline and N-nitroso-1,3-thiazolidine-4-carboxylic acid, in spiked human urine (7-8 micrograms l-1), is demonstrated.

Application of a microbore high-performance liquid chromatography—chemiluminescence detection system to the N-terminal amino acid analysis of bradykinin

Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) (230 pmol) was dansylated with 5-N,N-dimethylaminonapthalenesulphonyl chloride (Dns-Cl), followed by isolation in a reversed-phase high-performance liquid chromatography system with fluorescence detection (emission 530 nm, excitation 350 nm(. The N-Dns-bradykinin was acid-hydrolysed and 1/500 of the liberated N-Dns-arginine was analysed on Hypersil ODS by gradient elution microbore high-performance liquid chromatography (5 μm, 150 × 2.1 mm I.D.) and postcolumn reaction with bis-(2,4,6-trichlorophenyl) oxalate0hydrogen peroxide. The generated chemiluminescence was detected by a photomultiplier. The technical porblems of achieving sensitive detection by this procedure are discussed.