Georges Mahuzier

4-bromomethyl-6,7-dimethoxycoumarin as a fluorescent label for carboxylic acids in chromatographic detection

Abstract The UV absorbance, corrected fluorescence spectra, quantum yields and lifetimes of 4-bromomethyl-6,7-dimethoxycoumarin derivatives of carboxylic acids as well as of a few fatty esters are presented. The fluorescence quantum yields in methanol are not affected by the number of carbon atoms (C 2 C 5 ) of the carboxylic acid. Among various solvents, water gives the highest quantum yield (0.64), whereas in non-hydrogen-bonding solvents the yields are less than 0.23. The yield from the C 2 derivative rises rapidly from 0.43 to 0.60 as 20% water is added to methanol, and from 0.23 to 0.61 if 50% water is added to acetonitrile. The fluorescence spectra and quantum yields are only slightly affected by the pH, ionic strength and the nature of electrolytes used in the mobile phase. The results show that fatty acid esters of 4-hydroxymethyl-6,7-dimethoxycoumarin have higher quantum yields and intrinsic fluorescence sensitivities than homologous 7-methoxycoumarin derivatives. Furthermore they offer a better possibility for gradient elution chromatography. Derivatization of fatty acids with 4-bromomethyl-6,7-dimethoxycoumarin is a very sensitive method for the evaluation of picomole amounts by liquid chromatography using fluorescence detection.

Determination of alkylphosphonic acids by capillary zone electrophoresis using indirect UV detection

Abstract Capillary zone electrophoresis with indirect UV detection was used for the determination of a series of alkylphosphonic acids. For this purpose, a few UV-absorbing background electrolytes were tested and phenylphosphonic acid, which has a mobility close to that of the analysed compounds, was shown to be the most suitable. The influence of several parameters such as concentration of the UV-absorbing background electrolyte and concentration of borate on both sensitivity and efficiency was investigated. An increase in the borate concentration produced an improvement of the signal-to-noise ratio. Conversely, the sensitivity decreased with increasing concentration of the phenylphosphonic acid. The reproducibility of the method was very satisfactory and limits of detection were less than 0.21 pmol injected.

Simultaneous high-performance liquid chromatographic determination of ochratoxin A and citrinin in cheese by time-resolved luminescence using terbium

Abstract A simultaneous reversed-phase HPLC determination of two major mycotoxins, ochratoxin A and citrinin, in soft cheese is proposed. Both mycotoxins are eluted on a C18 RP support (25 × 4.6 mm I.D.) using an isocratic eluent consisting of methanol-water (70:30, v/v) containing tetrabutylammonium hydroxide (10−3 M), acidified to pH 5.5 with HCl, and pumped at a flow-rate of 0.8 ml/min. Prior to detection, a butanolic solution of 5·10−3 M terbium-5 · 10−4 M trioctylphosphine oxide (TOPO)-2.5 · 10−2 M triethylamine (TEA) was pumped in a postcolumn mode at a flow-rate of 0.2 ml/min to perform time-resolved luminescence (TRL) detection of the corresponding terbium chelates (λex = 331 nm/λem = 545 nm). The method is linear from 3.5·10−6 to 2·10−5 M for citrinin and from 1·10−5 to 5·10−5 M for ochratoxin A. The repeatability and reproducibility (R.S.D.) are 1.9 and 2.4% for citrinin (c = 3.5·10−6 M; n = 10), and 7.2 and 8.3% for ochratoxin A (c = 1.0·10−5 M; n = 10). The limits of detection, for a signal-to-background ratio of 3, are 2·10−6 and 3·10−6 M for citrinin and ochratoxin A, respectively. With the proposed method, ochratoxin A and citrin are easily determined in soft cheeses, with a significative increase in selectivity in comparison with direct fluorescence detection.

Postcolumn excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis

Measurement of fluorescence increase was used for the comparative quantification of the effect that several cyclodextrins (alpha-, beta-, heptakis-2,6-beta-omicron-dimethyl- and gamma-) produce on the fluorescent response of aflatoxins B1 and G1. This constitutes a new chromatographic method with stability of the mobile phase, and shows general improvements in the chromatographic conditions with respect to other methods (especially those using an iodine reservoir as a postcolumn reactor). A C18-type column was used, with methanol-water (60:40, v/v) as the mobile phase. The excitation phase of the natural fluorescence of aflatoxins, a 10(-2) M solution of each cyclodextrin, was introduced postcolumn. The determination of the elution order aflatoxin G2 > G1 > B2 > B1 was performed for each phase in less than 15 min. As expected using an aqueous-alcoholic medium, an increase in the fluorescence response of aflatoxins with an unsaturated furanic ring was found to occur with all the cyclodextrins studied, except gamma-cyclodextrin. The observed increase was larger for heptakis-2,6-beta-omicron-dimethyl- than for beta-cyclodextrin (to our knowledge, the only cyclodextrin previously described in the literature to serve for the determination of aflatoxins). The difference is of the order of 70.1-fold in the case of aflatoxin G1 and 45.2-fold in the case of aflatoxin B1. The detection limit in the mobile phase used was determined (for aflatoxin B1) for beta-cyclodextrin and 2,6-beta-omicron-dimethylcyclodextrin (signal-to-noise ratio 1:3) to be 4 and 9 mg 1(-1), respectively.

Cyclodextrins as modifiers of the luminescence characteristics of aflatoxins

Abstract The fluorescence properties in solution of the four main aflatoxins, B 1 , B 2 , G 1 and G 2 , in the presence of various cyclodextrin derivatives were studied. A preliminary study was made in order to determine the absorbance and fluorescence spectra, relative quantum fluorescence yields (φ F ), Stokes shifts, E S1 (0-0) energy levels and low-temperature (77 K) data for these aflatoxins. The effect of cyclodextrins on the fluorescence emission was then investigated. A substantial enhancement of the fluorescence emission of aflatoxins with an unsaturated furan ring (B 1 and G 1 ) in the presence of aqueous solutions of α,β-heptakis-di- O -methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin was observed. Surprisingly, γ-cyclodextrin was inefficient in this respect. Conversely, neither aflatoxin B 2 or G 2 , with a saturated furan ring, showed such an interaction and their emission characteristics remained constant in the presence of cyclodextrins. From a mechanistic point of view, the selectivity of the interaction seems to involve partially a non-inclusion process, because of the high molar ratio of cyclodextrin to aflatoxin needed for the fluorescence enhancement. The correlation between the behaviour in the presence of cyclodextrins and the solvent effect on the furan-unsaturated aflatoxins is discussed.

Liquid chromatographic study of the interaction between aflatoxins and β-cyclodextrin

Abstract The interaction between the four main aflatoxins (G 2 , G 1 , B 2 and B 1 ) and β-cyclodextrin (β-cyd) was studied using reversed-phase liquid chromatography (RP-LC). These aflatoxins are structurally very similar compounds that exhibit in RP-LC, and with methanol-water as eluent, a characteristic elution profile, i.e., k′ G2 k′ G1 k′ B2 k′ B1 . In the presence of β-cyd in the eluent, this elution order remains constant and the decrease in all the capacity factors ( k′ ) is used calculate the respective complex formation constant ( K f ) of each compound with β-cyd, which are 263, 327, 215 and 258 1 mol −1 for aflatoxins G 2 , B 2 and B 1 , respectively. The enthalpy (Δ H β ) and entropy (Δ S β changes associated with the interaction of the C 18 support with the complexed solutes were extracted from the temperature dependance of k′ . All the established thermodynamic data strongly suggest the occurrence of a 1:1 stoichiometry inclusion process between the aflatoxins and the introduced β-cyd. ON the other hand, the discrepancy between some of the thermodynamic parameters (Δ S ) and some of the chromatographic parameters ( K′ ) suggests that the driving forces involved in the chromatographic partition process and in the inclusion in the β-cyd cavity may be due to different parts of the aflatoxin molecule. Finally, the results obtained with the proposed method indicate that RP-LC can be successfully used to calculate complex formation constants and to contribute to a better insight into the β-cyd-aflatoxin interaction.

Effects of pH and solvent on the fluorescence properties of biomedically important benzamides. Application to determination in drugs and in human urine

The fluorescence properties of five substituted benzamides, including alizapride, metoclopramide, sulpiride, sultopride and tiapride, were investigated at several pH values and in various solvents (dimethyl sulfoxide, ethanol, ethylene glycol, methanol, propan-2-ol, tetrahydrofuran and water). Except for alizapride, the fluorescence intensities were found to be higher at acidic (1-6) than at alkaline (8-12) pH values. Using the optimum solvent (aqueous solutions) and appropriate pH conditions, linear spectrofluorimetric calibration curves were established over a concentration range of about two orders of magnitude, with correlation coefficients larger than 0.996. Limits of detection were between 1 and 13 ng ml-1, depending on the compound. The method was applied to the determination of benzamides in pharmaceutical preparations and in human urine, with recoveries ranging from 94 to 108% and from 93 to 104%, respectively.

LUMINARIN 4 AS A LABELLING REAGENT FOR CARBOXYLIC ACIDS IN LIQUID CHROMATOGRAPHY WITH PEROXYOXALATE CHEMILUMINESCENCE DETECTION

Abstract Luminarin 4 is a labelling reagent with a quinolizinocoumarin structure, which reacts with carboxylic acids after their activation by N-hydroxysuccinimide for 12 h at 20°C and dicyclohexylcarbodiimide for 60 min at 70°C. Small fatty acids were derivatized and separated by reversed-phase liquid chromatography. The fluorescence detection threshold was 300 fmol injected. The limit of chemiluminescence detection, which required a post-column reaction with an oxalic ester and hydrogen peroxide, was 50 fmol injected. The reaction was used to measure prostaglandin E2, whose detection limit was 32 fmol injected, but the derivatization limit was 60 pmol. Linearity was observed in the range 0.1–10 nmol of prostaglandin E2.

Precolumn derivatization of amines in liquid chromatography using luminescent probes: Comparison of several reagents with luminarin 1

Luminarin 1 is a labelling reagent with quinolizino coumarin structure and a N-hydroxysuccinimide ester reactive function, which reacts with primary amines in 20 min at 50°C, and with secondary amines in 180 min at 70°C. Several amines were derivatized and separated by liquid chromatography (pentylamine, pyrrolidine, tyramide, proline). The limits of detection with fluorescence detection were between 160–300 fmol injected, the limit of chemiluminescence detected with bis(2,4,6-trichlorophenyl) oxalate (TCPO) and hydrogen peroxide (H2O2) were between 15–100 fmol injected. Derivatization limits with luminarin 1 were in the range 180–300 pmol of amine. Luminarin 1 was compared with different derivatizing reagents like FMOC, OPA and fluorscamine using fluorescence and chemiluminescence detection. FMOC and luminarin appeared to have shown the best performances in terms of derivatization and detection limits, respectively.

Application of ion chromatography with indirect spectrophotometric detection to the sensitive determination of alkylphosphonic acids and fosfomycin

Abstract Indirect spectrophotometric detection was investigated for application in the anion-exchange chromatography of alkylphosphonic acids and fosfomycin. Four chromophoric eluent anions were studied: phthalate, bezoate, phenylphosphonate and p -toluenesulphonate. Phthalate showed the best displacing power and the larger dynamic reserve whereas benzoate exhibited the highest absorptivity and transfer ratio. The optimal experimental conditions were found to be 0.4 mM phthalate (pH 8.5) as mobile phase, a flow-rate of 1 ml min −1 and a detection wavelength of 272 nm. Selectivity (between 1.02 and 1.85, depending on the pair of vicinal compounds), linearity over a 5–100 μg ml −1 concentration range ( r = 0.999), precision (R.S.D. 0.6–5.3%) and sensitivity (0.4–1.0 μg, ml −1 according to the phosphonic acid) were determined. The minimum detectable concentrations were improved by an average factor of 50 in comparison with direct non-suppressed conductimetric detection. The application of the technique to the determination of fosfomycin in plasma samples is described.