Patrice Prognon

Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis

HPLC using fluorescence detection has already become the most accepted method for the determination of aflatoxins due to its several advantages over other analytical methods. Both normal- and reversed-phase HPLC can be used. However the reversed-phase HPLC methods are more popular. Liquid chromatographic determination of aflatoxins using fluorescence detection and its application in food analysis is reviewed in this article.

Evaluation of drug administration errors in a teaching hospital

BackgroundMedication errors can occur at any of the three steps of the medication use process: prescribing, dispensing and administration. We aimed to determine the incidence, type and clinical importance of drug administration errors and to identify risk factors.MethodsProspective study based on disguised observation technique in four wards in a teaching hospital in Paris, France (800 beds). A pharmacist accompanied nurses and witnessed the preparation and administration of drugs to all patients during the three drug rounds on each of six days per ward. Main outcomes were number, type and clinical importance of errors and associated risk factors. Drug administration error rate was calculated with and without wrong time errors. Relationship between the occurrence of errors and potential risk factors were investigated using logistic regression models with random effects.ResultsTwenty-eight nurses caring for 108 patients were observed. Among 1501 opportunities for error, 415 administrations (430 errors) with one or more errors were detected (27.6%). There were 312 wrong time errors, ten simultaneously with another type of error, resulting in an error rate without wrong time error of 7.5% (113/1501). The most frequently administered drugs were the cardiovascular drugs (425/1501, 28.3%). The highest risks of error in a drug administration were for dermatological drugs. No potentially life-threatening errors were witnessed and 6% of errors were classified as having a serious or significant impact on patients (mainly omission). In multivariate analysis, the occurrence of errors was associated with drug administration route, drug classification (ATC) and the number of patient under the nurses care.ConclusionMedication administration errors are frequent. The identification of its determinants helps to undertake designed interventions.

Simple and sensitive high-performance liquid chromatography-fluorescence method for the determination of citrinin application to the analysis of fungal cultures and cheese extracts☆

A new and highly sensitive method for the detection of the important mycotoxin, citrinin, has been developed. Spectroscopic studies demonstrate that the fluorescence of this metabolite is influenced by the pH of the environment. This fact was exploited in the chromatographic determination of citrinin with fluorescence detection. The proposed method, based on the addition of 1 M hydrochloric acid as an acidic post-column reagent, has a limit of detection of 0.9 center dot 10(-7) M. Analytical validation shows that linearity can be assumed from 2 center dot 10(-7) to 10(-4) M citrinin. The repeatability and reproducibility are satisfactory, with R.S.D. = 5.1% (n = 9, c = 10(-5) M) and R.S.D. = 7.2% (n = 9, c = 10(-5) M). The method was also applied to the determination of this mycotoxin produced by mould cultures isolated from soft cheese and also from soft cheese and also from cheese extracts spiked with citrinin. The specificity of the method is demonstrated and the necessity for post-column acidification is illustrated on real samples.

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.

Screening patients with hypertrophic cardiomyopathy for Fabry disease using a filter-paper test: the FOCUS study

Background Patients with Fabry disease (FD) show left ventricular hypertrophy (LVH) mimicking hypertrophic cardiomyopathy (HCM) of sarcomeric origin and might benefit, if detected early, from specific enzyme replacement therapy. The prevalence of FD in patients with LVH of 13 mm or greater, screened using the leucocyte alpha-galactosidase A (α-gal A) activity test, a technique that is difficult to apply routinely, ranged from 0% to 6%. Objective To screen systematically for FD in patients with a diagnosis of HCM (LVH ≥15 mm) in primary cardiology practice, a validated, physician-friendly α-gal A assay was used on dried blood spots using a filter paper test. Design and patients A cohort of 392 adults (278 men) followed for HCM were screened for FD. A standard blood test was used for confirmation in nine men in whom the α-gal A result was 40% or less. Results Four men (1.5%; 1.8% of men ≥40 years vs 0% <40 years; all with α-gal A <30%), but no women, were diagnosed with FD. Index cases presented with diffuse but asymmetric LVH, with severe obstruction in one case and frequent high-grade atrioventricular conduction block necessitating a pacemaker in three cases. Family screening identified eight additional cases. Genotyping was performed successfully on DNA extracted from the filter papers. Conclusion In male patients diagnosed as having HCM, pure FD cardiac variants are not exceptional and can be specifically identified using a simple filter-paper test. The sensitivity of this test is low in female patients.

Interaction between cyclodextrins and aflatoxins Q1, M1 and P1 fluorescence and chromatographic studies

The fluorescence properties of the aflatoxins M1, Q1, P1 in solution and the effect of various cyclodextrins (alpha-, beta-, gamma-, hydroxypropyl-beta- and alpha-beta-heptakis-di-O-methyl-beta-) on their fluorescence emission were studied. Among the aflatoxins, a substantial enhancement of the fluorescence emission of aflatoxin Q1 in the presence of aqueous solutions of alpha-, beta-, hydroxypropyl-beta, and alpha-beta-heptakis-di-O-methyl-beta-cyclodextrin, was observed. On the contrary, gamma-cyclodextrin proved to be inefficient to enhance the fluorescence properties of this compound. No important fluorescence enhancement was found for aflatoxins P1 or M1 for any of the cyclodextrin derivatives tested. The complex formation constant (Kf) of these compounds with beta-cyclodextrin was chromatographically determined, and from the results obtained, we can conclude that Kf cannot be used alone to explain the fluorescence increase. Thermodynamic studies showed that delta-H and delta-S parameters, associated with the partition of aflatoxins in RP-HPLC, increased when beta-cyclodextrin was added to the eluent.

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.