Beatriz I. Vázquez

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.

Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese

In the present work we studied the antifungal effect of eugenol and thymol on the growth and production of citrinin from Penicillium citrinum (NRRL 2274 and NRRL 2269) in culture media and in different Spanish cheeses (Arzúa-Ulloa, Cebreiro and San Simón). The rate of growth was assessed by measuring colony diameters and the production of citrinin was measured using a rapid semi-quantitative fluorometric technique confirmed by RP-HPLC. A stronger inhibitory effect of eugenol than thymol was evident. 200 microg/ml of eugenol in solid culture medium increased the lag time of growth up to 9 days, and decreased the rate of colony growth. In liquid medium, a complete inhibition of fungal growth was observed. By contrast, thymol in the liquid culture medium only affected the growth rate. In Arzúa-Ulloa cheese, 200 microg/ml of eugenol fully inhibited fungal growth, while in Cebreiro cheese no effect was observed for this compound. Regarding the capacity to inhibit mycotoxin production 100 microg/ml eugenol delayed citrinin production until the sixth day, after which a limiting effect persisted. In Arzúa-Ulloa cheese, no citrinin was detected at a concentration of 150 microg/ml of eugenol, but citrinin was detected after 5 days in the case of thymol at the same concentration. In Cebreiro cheese, neither eugenol nor thymol prevented the production of citrinin at the concentrations applied.

Determination of quinolones in animal tissues and eggs by high-performance liquid chromatography with photodiode-array detection

A rapid, specific reversed-phase HPLC method is described, with solid-phase extraction, for assaying five quinolones (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin and marbofloxacin) with confirmative diode-array detection in samples of bovine kidney, muscle and eggs. The least efficient extraction was marbofloxacin from kidney tissue (64%). The lower detection limit for each quinolone was: enrofloxacin and ciprofloxacin, 1 ng; norfloxacin and difloxacin, 2 ng; marbofloxacin, 4 ng injected. The intra-day relative standard deviations were lower than 7.9% and lower than 8.6% for inter-day assays. These results indicate that the developed method had an acceptable precision.

New Additive for Culture Media for Rapid Identification of Aflatoxin-Producing Aspergillus Strains

ABSTRACT A new reliable, fast, and simple method for the detection of aflatoxigenic Aspergillus strains, consisting of the addition of a cyclodextrin (a methylated β-cyclodextrin derivative) to common media used for testing mycotoxin production ability, was developed. We propose the use of this compound as an additive for fungal culture media to enhance the natural fluorescence of aflatoxins. The production of aflatoxins coincided with the presence of a bright blue or blue-green fluorescent area surrounding colonies when observed under long-wavelength (365-nm) UV light after 3 days of incubation at 28°C. The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by high-pressure liquid chromatography with fluorescence detection.

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.

Quantitative LC-MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum

We have developed a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P(5)), progesterone (P(4)), 17hydroxyP(5), 17hydroxyP(4), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E(1)), 2, 4 and 16 hydroxyE(1), 2 and 4 methoxyE(1)]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.

Evolution of Resistance in Poultry Intestinal Escherichia coli During Three Commonly Used Antimicrobial Therapeutic Treatments in Poultry

The resistance rates of intestinal Escherichia coli populations from poultry were determined during treatment and withdrawal period with 3 antimicrobial agents commonly used as therapeutics in poultry medicine. A total of 108 chickens were considered: 18 were treated orally with enrofloxacin, 18 with doxycycline, and 18 with sulfonamides, whereas another 18 chickens were maintained as controls for each antimicrobial group. Fecal samples were taken during the treatment and after the withdrawal period, and E. coli were isolated through Fluorocult media plating. A total of 648 E. coli strains (216 per antimicrobial tested) were isolated and identified though biochemical methods. Minimal inhibitory concentrations to the antimicrobials used were also determined using a broth microdilution method. The resistance rates of intestinal E. coli to all of the antimicrobials tested significantly increased during the course of the therapeutic treatment. In addition, significant differences (P = 0.0136) in resistance rates persisted between the intestinal E. coli of the enrofloxacin-treated and control batches until the end of the withdrawal period, but this difference was not observed for the cases of doxycycline or sulfonamides treatments. Antimicrobial use in poultry medicine seems to select for antimicrobial-resistant strains of pathogenic bacterial species such as E. coli. In some cases, the higher frequencies of resistant strains may persist in the avian intestinal tract until the end of the withdrawal period, when it is legal to use these animals for human consumption.

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.

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.