L. Puignou

Isolation by solid-phase extraction and liquid chromatographic determination of mutagenic amines in beef extracts

A solid-phase extraction method was successfully optimized for the isolation and preconcentration of five mutagenic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9H-pyrido[2,3-b]indole and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine, and two co-mutagens, 1-methyl-9H-pyrido[4,3-b]indole and 9H-pyrido[4,3-b]indole. Coupling of diatomaceous earth, propylsulphonyl silica gel, and octadecylsilane cartridges was used to separate selectively the imidazopyridine and indolpyridine derivatives from those of quinoxaline and quinoline. A method based on this sample preparation was applied to the determination of twelve heterocyclic amines and related substances in a commercial beef extract using HPLC with electrochemical and fluorescence detection. Good recovery values were obtained, ranging between 55 and 99%. The co-mutagens 1-methyl-9H-pyrido[4,3-b]indole (harman) and 9H-pyrido[4,3-b]indole (norharman) were found in the beef extract at levels of 110 and 53 ng g-1, respectively, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) were tentatively identified.

Solid-phase extraction for the selective isolation of polycyclic aromatic hydrocarbons, azaarenes and heterocyclic aromatic amines in charcoal-grilled meat

A method for the simultaneous analysis of 12 mutagenic and/or carcinogenic compounds is described; these substances belong to three different chemical groups: polycyclic aromatic hydrocarbons (PAHs), azaarenes, i.e., nitrogen-containing polycyclic aromatic hydrocarbons (PANHs), and heterocyclic aromatic amines (HAAs). The selective enrichment procedure includes coupling of solid-phase extraction (SPE) steps using diatomaceous earth, propylsulfonic acid, silica gel and octadecylsilane columns. The eluted fractions were analysed by high-performance liquid chromatography with UV and electrochemical detection. Levels measured were estimated to be 4-19 ng g-1. Peak confirmation was carried out by GC-MS for both PAHs and PANHs, and by LC with a photodiode array detector for HAAs. The method was applied to the analysis of charcoal-grilled meat and was judged to be generally applicable for detection of these mutagens at the ppb level in processed foods.

Comparison of capillary electrophoresis and reversed-phase ion-pair high-performance liquid chromatography for the determination of paraquat, diquat and difenzoquat☆

Abstract The conditions were established for the simultaneous determination of paraquat, diquat and difenzoquat by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). For the HPLC separation, the use of a reversed phase, heptanesulphonate as ion-pairing agent and an aqueous—acetonitrile mobile phase with stepwise elution from 93:7 to 70:30 was adopted. Acetic acid— sodium acetate (pH 4.0) with 100 m M sodium chloride as running buffer and electrokinetic injection were used in CE. The figures of merit were calculated and the two techniques were compared. Detection limits (signal-to-noise ratio = 3:1) ranged from 2.9 to 5.5 μg 1 −1 and were similar for both techniques when standards were dissolved in water, but when CE was used the response was greatly affected by the nature of the sample matrix. The run-to-run and day-to-day reproducibilities and the analysis times were similar for both techniques. However, CE did not require preconditioning and a long stabilization period was needed in ion-pair HPLC. The methods were applied to the determination of the herbicides in crop waters.

Comparison of different electroosmotic flow modifiers in the analysis of inorganic anions by capillary electrophoresis

Abstract The determination of inorganic anions using capillary electrophoresis with non-treated silica capillaries requires reversal or elimination of the cathodic electroosmotic flow (EOF). The behaviour of different monocharged [tetrabutylammonium (TBA), cetyltrimethylammonium (CTA) and tetradecyltrimethylammonium (TTA)] or multicharged [hexamethonium (HM) and hexadimethrine (HDM)] alkylammonium quaternary ions as electroosmotic flow modifiers has been compared. Indirect spectrophotometric detection using chromate as carrier ion has been used. The effect of EOF modifiers and chromate concentration, pH and applied potential on the separation of bromide, chloride, nitrite, nitrate, sulfate and phosphate has been studied. Optimal conditions for the separation of these anions have been established and resolution has been compared. Reproducibility in areas (R.S.D. 1.8–8.5%) and in migration times (R.S.D. 2.3–7.5%), and detection limits (200–1000 μg l −1 ) were similar for all the EOF modifiers. For HDM very low concentrations, down to 0.0001%, can be used successfully.

Evaluation of different clean-up procedures for the analysis of heterocyclic aromatic amines in a lyophilized meat extract.

Along with other mutagenic and carcinogenic contaminants in foods such as aflatoxins, and polycyclic aromatic hydrocarbons, heterocyclic aromatic amines (HAAs) have received considerable attention in recent years. A major drawback in the analysis of HAAs in foods is their very low level of concentration (0.1 50 ng g-1) as well as matrix interferences. Solid-phase extraction (SPE), forming an integral part of chromatographic analysis, is one of the procedures currently used for the extraction and purification of HAAs in food samples. In this paper a comparative study of several SPE procedures for HAAs determination was performed. Recoveries of the heterocyclic amines in the analysis of both a simple matrix such as a standard methanolic solution and a contaminated meat extract were established. HAAs were determined by HPLC analysis with photodiode-array detection (DAD) of the purified extracts, and the adequacy of different clean-up procedures for the analysis of a contaminated meat extract was discussed.

Sample stacking with matrix removal for the determination of paraquat, diquat and difenzoquat in water by capillary electrophoresis

Conditions for the simultaneous determination of paraquat, diquat and difenzoquat by capillary zone electrophoresis using a stacking technique in a chemically modified capillary have been established. To apply the stacking method with sample matrix removal for the analysis of cations, an anodic electroosmotic flow is mandatory. For quats, 50 mM acetic acid-ammonium acetate (pH 4.0) with 5% (v/v) methanol as electrophoretic buffer and the addition of 0.8 mM cetyltrimethylammonium bromide as wall capillary organic modifier was proposed. Field polarity reversal time was optimised for several sample matrices. Detection was carried out at 220 and 255 nm. Detection limits, based on a signal-to-noise ratio of 3:1, were lower than 15 microg l(-1) for standards in Milli-Q water and two to ten times higher for drinking water samples. Run-to-run and day-to-day reproducibility have been established. The method was successfully applied to the determination of the three herbicides in spiked drinking water.

Ion-trap tandem mass spectrometry for the determination of heterocyclic amines in food

Heterocyclic amines (HAs) are mutagenic compounds to which humans are regularly exposed through diet. Due to the high complexity of the sample matrix and the low level of concentration of HAs, sensitive and selective analytical methodologies are required. Here we describe a methodology based on liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry using an ion-trap to analyse HAs. The collision-induced dissociation parameters for tandem ion-trap spectrometric analysis of these mutagenic compounds were optimised, and the full scan MS-MS spectra were used for unequivocal identification of the analytes. For aminoimidazoazaarenes, the most abundant ions were derived from the loss of a methyl group and the breaking of the aminoimidazole moiety, while for carbolines the major product ions arose from the loss of ammonia and HCN. Moreover, the performance of the LC-atmospheric pressure chemical ionisation MS-MS method was evaluated. The good precision (RSD lower than 11%) and the low detection limits achieved (10-60 pg injected) allow the determination of HAs at low part-per-billion level (0.4-5.0 ng g(-1)) in a lyophilised meat extract.

Pressure-assisted capillary electrophoresis-electrospray ion trap mass spectrometry for the analysis of heparin depolymerised disaccharides.

A pressure-assisted capillary electrophoresis-ion trap mass spectrometry method was developed for the analysis of eight heparin-derived disaccharides. A 30 mM formic acid buffer at pH 3.20 was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of -30 kV and an overimposed pressure of 0.5 p.s.i. (3.45 kPa). The application of pressure assistance was needed to provide stable electrospray conditions for successful coupling. The linearity of the CE-MS and CE-MS-MS methods was checked under these conditions. Quality parameters such as run-to-run precision and limits of detection were established in both CE-MS and CE-MS-MS modes. Finally, enzymatically depolymerised bovine and porcine mucosal heparins were analysed in this CE-MS system and the characteristic relative molar percentages of major and minor disaccharides were calculated.

Use of reversed polarity and a pressure gradient in the analysis of disaccharide composition of heparin by capillary electrophoresis

A capillary electrophoresis method with reversed polarity, combining both the application of a voltage and a pressure gradient between the buffer vials, was developed for the analysis of eight heparin-derived delta-disaccharides obtained by enzymatic depolymerization. A 60 mM formic acid buffer at pH 3.40 was selected as running electrolyte, with an applied voltage of -15 kV and an over-imposed pressure gradient (3.45.10(-3) MPa) for 6 min from inlet to outlet starting at 20 min. Figures of merit such as run-to-run and day-to-day precision, and limits of detection were established. The electrophoretic method was applied to the analysis of depolymerization products of different kinds of heparins. The composition of the depolymerization buffer was selected in order to reduce baseline distortions in the electrophoretic separation, thus a buffer solution containing 20 mM Tris, 50 mM sodium chloride, and 3 mM calcium chloride at pH 7.10 was used. Percentages of molar disaccharide compositions for unfractionated heparins from porcine, bovine and ovine intestinal mucosa, and bovine lung were determined. In addition, low-molecular-mass heparins from bovine and porcine intestinal mucosa were analysed as well.

Liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry as a routine method for the analysis of mutagenic amines in beef extracts

A liquid chromatography-mass spectrometry (LC-MS) method using atmospheric-pressure chemical ionisation as interface was developed for the simultaneous determination of 14 heterocyclic aromatic amines and related compounds in beef extracts. The separation was performed on a conventional C18 column using a binary mobile phase composed of acetonitrile and 50 mM ammonium acetate at pH 5.7, and elution was carried out in gradient mode. Several parameters influencing the mass spectra were optimized, and the effect of the variation of cone voltage on the mass spectra was studied. The [M+H]+ ions and some fragments produced in the source were observed in the mass spectra when several extraction voltages were applied. Quality parameters (run-to-run and day-to-day reproducibility, intervals of linearity, and limits of detection) were studied in the optimum working conditions. The method was used to analyze the heterocyclic amines present in a commercial beef extract. Therefore, a solid-phase extraction clean-up procedure was performed prior the LC-MS analysis due to the complexity of the sample and the compounds Glu-P-1, Harman, Norharman and A alpha C were identified in the samples at ppb levels and successfully confirmed using in-source fragmentation.