Rapid method for the quantification of 13 sulphonamides in milk by conventional high-performance liquid chromatography with diode array ultraviolet detection using a column packed with core-shell particles.

In the present study, a column packed with core-shell particles was used for the separation and the quantification of 13 sulphonamides in milk by conventional high-performance liquid chromatography coupled with diode array ultraviolet detection (HPLC/UV-DAD). Preliminary experiments were carried out to investigate selectivity of different stationary phases. Best results were achieved using a C18 column packed with 2.6µm core-shell particles (diameter 4.6mm, length 75mm). A binary gradient elution based on acetate buffer solution at pH 4.50 and a mixture of methanol acetonitrile 50:50 (v/v) was employed at the flow rate of 1.2mLmin-1 with an injection volume of 6µL. These chromatographic conditions allowed the efficient separation of 13 sulphonamides in about 8min. To evaluate the suitability of the method for official control analysis, the most important validation parameters were investigated according to the European Decision 657/2002/EC as established for analysis of drug residues in food. Sulphonamides were recovered from milk samples by a simple and quick preparation procedure consisting of an extraction step with chloroform/acetone and a purification step with n-hexane. Mean recoveries from raw milk ranged between 55% and 86% at the Maximum Residual Limit of 100µgkg-1, and RSDs% resulted lower than Thompson and Horwitz RSD% reference values for all sulphonamides. The LOQ value (2.7-15µgkg-1) was low enough to satisfy legal limits suggested by European Regulation 37/2010/EC.

Sarcoidosis in an Italian province. Prevalence and environmental risk factors.

BACKGROUND AND AIM: Sarcoidosis is a systemic granulomatous inflammatory disease whose causes are still unknown and for which epidemiological data are often discordant. The aim of our study is to investigate prevalence and spatial distribution of cases, and identify environmental exposures associated with sarcoidosis in an Italian province. METHODS: After georeferentiation of cases, the area under study was subdivided with respect to Municipality and Health Districts and to the altitude in order to identify zonal differences in prevalence. The bioaccumulation levels of 12 metals in lichen tissues were analyzed, in order to determine sources of air pollution. Finally, the analysis of the correlation between metals and between pickup stations was performed. RESULTS: 223 patients were identified (58.3% female and 41.7% male of total) and the mean age was 50.6±15.4 years (53.5±15.5 years for the females and 46.5±14.4 for the males). The mean prevalence was 49 per 100.000 individuals. However, we observed very heterogeneous prevalence in the area under study. The correlations among metals revealed different deposition patterns in lowland area respect to hilly and mountain areas. CONCLUSIONS: The study highlights a high prevalence of sarcoidosis cases, characterized by a very inhomogeneous and patchy distribution with phenomena of local aggregation. Moreover, the bioaccumulation analysis was an effective method to identify the mineral particles that mostly contribute to air pollution in the different areas, but it was not sufficient to establish a clear correlation between the onset of sarcoidosis and environmental risk factors.

Anticoagulant rodenticide poisoning in animals of Apulia and Basilicata, Italy.

This study evaluates the presence of anticoagulant rodenticides in animals with a diagnosis of suspected poisoning and in bait samples. The survey was carried out from 2010 to 2012, in 2 regions of South Italy (Puglia and Basilicata) on 300 organs of animals and 90 suspected bait samples. The qualitative and quantitative analyses were conducted using an analytical method based on high­performance liquid chromatography (HPLC) with fluorimetric detection (FLD) for the simultaneous determination of 8 anticoagulant rodenticides (bromadiolone, brodifacoum, coumachlor, coumafuryl, coumatetralyl, difenacoum, flocoumafen, and warfarin). The presence of anticoagulant rodenticides was detected in 33 organs of animals (11% of the total) and 6 bait samples (7% of the total). The most commonly detected compound was coumachlor (47% of 39 positive samples) followed by bromadiolone (24%), and brodifacoum (11%). The species mostly involved in anticoagulant rodenticide poisoning were dogs and cats. This study emphasizes the relevance of the determinations of anticoagulant rodenticides in cases of suspected poisoning in veterinary practice.

Core-Shell in Liquid Chromatography: Application for Determining Sulphonamides in Feed and Meat Using Conventional Chromatographic Systems.

A C18 column packed with core-shell particles was used for the chromatographic separation of sulphonamides in feed and meat by a conventional high performance liquid chromatography system coupled with a diode array detector. Two analytical methods, already used in our laboratory, have been modified without any changes in the extraction and clean-up steps and in the liquid chromatography instrumentation. Chromatographic conditions applied on a traditional 5-µm column have been optimized on a column packed with 2.6 µm core-shell particles. A binary mobile phase [acetate buffer solution at pH 4.50 and a mixture of methanol acetonitrile 50: 50 (v/v)] was employed in gradient mode at the flow rate of 1.2 mL with an injection volume of 6 µL. These chromatographic conditions allow the separation of 13 sulphonamides with an entire run of 13 minutes. Preliminary studies have been carried out comparing blanks and spiked samples of feed and meat. A good resolution and the absence of interferences were achieved in chromatograms for both matrices. Since no change was made to the sample preparation, the optimized method does not require a complete revalidation and can be used to make routine analysis faster.

Development of an analytical method for the determination of polyphenolic compounds in vegetable origin samples by liquid chromatography and pulsed amperometric detection at a glassy carbon electrode.

A sensitive and accurate method for the determination of polyphenolic compounds in artichoke bract extracts and olive mill wastewaters by liquid chromatography coupled with pulsed amperometric detection at a glassy carbon working electrode was developed. Preliminary experiments were carried out by cyclic voltammetry to investigate the electrochemical behavior of polyphenols under different mobile phase compositions, and to test the detection and cleaning electrode potentials. Chromatographic separations were performed by using a core-shell C18 column, eluted with acetic acid and acetonitrile, by combined concave-linear binary gradients. Under the optimized experimental conditions, a good column efficiency and peak symmetry were observed, also for stereo and positional isomeric compounds. The developed three-step potential waveform for pulsed amperometric detection was successfully applied for the sensitive chromatographic determination of polyphenols in artichoke extracts and olive mill wastewaters. Linearity, precision and sensitivity of the proposed method have been evaluated. A wide linear range of response (up to 20 mg/L) has been obtained for all the investigated compounds. Detection and quantification limits in the vegetable origin sample extracts were in the range 0.004-0.6 mg/L and 0.01-2mg/L, respectively, while the injection-to-injection repeatability (n=6) ranged from 5 to 13%. The obtained results confirmed the excellent sensitivity of the electrochemical detection, and its suitability for the determination of electroactive polyphenolic compounds at low concentration levels.

Development and validation of an HPLC/DAD method for the determination of 13 sulphonamides in eggs.

A simple, sensitive and selective multiresidue high-performance liquid chromatography with diode array detection method for determination of 13 sulphonamides in eggs was developed and validated. Sample extraction and clean-up conditions were carefully studied and factors as gradient elution and column temperature were found as key parameters to improve separation efficiency. The method was validated following the European Commission Decision 2002/657/EC criteria adopting spiking levels of 20, 40 and 60 µg kg(-1) "As Low As Reasonably Achievable". The necessary requirements for precision (RSDR% below 23%) and trueness (recovery ranging from 45.2% to 87.5%) were fulfilled. Decision limit (CCα) values below 18.5 µg kg(-1), comparable to those reported in LC-MS detection, demonstrated the suitability of the method in residues surveillance plans for the sulphonamides analysis in eggs at the carry-over level without the use of sophisticated and expensive instrumentation.

A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages.

In the present work a sensitive and accurate method by ion chromatography and conductimetric detection has been developed for the determination of biogenic amines in food samples at microgram per kilogram levels. The optimized extraction procedure of trimethylamine, triethylamine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine from real samples, as well as the separation conditions based on a multilinear gradient elution with methanesulfonic acid and the use of a weak ionic exchange column, have provided excellent results in terms of resolution and separation efficiency. Extended calibration curves (up to 200 mg/kg, r > 0.9995) were obtained for all the analyzed compounds. The method gave detection limits in the range 23-65 µg/kg and quantification limits in spiked blank real samples in the range 65-198 µg/kg. Recovery values ranged from 82 to 103 %, and for all amines, a good repeatability was obtained with precision levels in the range 0.03-0.32 % (n = 4). The feasibility and potential of the method were tested by the analysis of real samples, such as tinned tuna fish, anchovies, cheese, wine, olives, and salami.

Determination of deoxynivalenol and nivalenol by liquid chromatography and fluorimetric detection with on-line chemical post-column derivatization.

A rapid, sensitive and selective analytical method was developed for the quantitative determination of deoxynivalenol (DON) and nivalenol (NIV) in cereals intended for human and animal consumption. The method, based on liquid chromatography and fluorescence detection, involves an automated 2 channel post-column derivatization, performed with sodium hydroxide, methyl acetoacetate and ammonium acetate. The chromatographic separation was accomplished using a C18 column eluted in isocratic mode with a mixture of 0.01% acetic acid and acetonitrile. Optimal fluorescence detection was obtained by an excitation and emission wavelength of 360 nm and 470 nm, respectively. The sample preparation required a rapid extraction of mycotoxins with water and a purification step by hydrophilic-lipophilic balance column clean-up. Under the optimized experimental conditions, a complete separation of DON and NIV was obtained in less than 20 min. The on-line post-column derivatization ensures excellent results in terms of simplicity and sensitivity, with limits of detection down to 0.014 mg/kg. The proposed method was extensively validated and the analytical performances of linearity (correlation coefficient of 0.9998), selectivity, precision (intra-day precision lower than 8%) and recovery (ranging from 89% to 101%) were evaluated, demonstrating the method feasibility in accurate confirmation analyses.

Validation and application of multi-residue analysis of eight anticoagulant rodenticides by high-performance liquid chromatography with fluorimetric detection.

Poisoning of domestic animals is frequently caused by anticoagulant rodenticides. Validation and applications of a rapid and reliable method for the simultaneous determination of 8 anticoagulant rodenticides (bromadiolone, brodifacoum, coumachlor, coumafuryl, coumatetralyl, difenacoum, flocoumafen, and warfarin) in baits and animal livers using high-performance liquid chromatography with fluorescence detection are reported herein. The methodology was validated by an in-house validation model at 2.5 mg/kg, which is the level commonly found in the tissues of poisoned domestic animals. The 8 anticoagulants can be determined at the concentration range of 1.25-100 mg/kg with determination coefficients higher than 0.992. A recovery value from 70% to 109% was observed for all the studied molecules. The results of the validation process demonstrate suitability for application in official analysis and for monitoring purposes of animal poisoning by anticoagulant rodenticides.

Investigation on the presence of sulphites in fresh meat preparations: estimation of an allowable maximum limit.

Sulphiting agents are commonly used food additives. They are not allowed in fresh meat preparations. In this work, 2250 fresh meat samples were analysed to establish the maximum concentration of sulphites that can be considered as "natural" and therefore be admitted in fresh meat preparations. The analyses were carried out by an optimised Monier-Williams Method and the positive samples confirmed by ion chromatography. Sulphite concentrations higher than the screening method LOQ (10.0 mg · kg(-1)) were found in 100 samples. Concentrations higher than 76.6 mg · kg(-1), attributable to sulphiting agent addition, were registered in 40 samples. Concentrations lower than 41.3 mg · kg(-1) were registered in 60 samples. Taking into account the distribution of sulphite concentrations obtained, it is plausible to estimate a maximum allowable limit of 40.0 mg · kg(-1) (expressed as SO(2)). Below this value the samples can be considered as "compliant".

Determination of fumonisins B1 and B2 in maize food products by a new analytical method based on high-performance liquid chromatography and fluorimetric detection with post-column derivatization.

A sensitive and selective analytical method for the quantitative determination of fumonisins B(1) (FB(1)) and B(2) (FB(2)) in maize-based foods for direct human consumption is described. The method, based on high-performance liquid chromatography and fluorescence detection, presents a rapid and automated online post-column derivatization, performed with o-phthalaldehyde and N,N-dimethyl-2-mercaptoethylamine (Thiofluor™). A complete separation of fumonisins is achieved in less than 13 min by using a C18 column and a gradient elution. Fumonisins are extracted from the sample with a mixture of water, acetonitrile, and methanol. The filtered extract is purified by immunoaffinity column and FB(1) and FB(2) are eluted with methanol. The method has been successfully validated, and performances comply with -criteria of the Regulation EC No 401/2006.

A confirmatory method for aflatoxin M1 determination in milk based on immunoaffinity cleanup and high-performance liquid chromatography with fluorometric detection.

A sensitive and reliable analytical method based on immunoaffinity chromatography cleanup followed by HPLC separation and fluorimetric detection is described for the quantitative determination of aflatoxin M(1) in milk. The chromatographic separation is accomplished by using a C18 column and a gradient elution with methanol, acetonitrile, and water. No extraction solvent process is required and a minimal milk sample cleanup is performed by a direct loading of the immunoaffinity columns and elution with methanol. The method has been successfully validated according to Decision EC No 657/2002 by using the conventional validation approach. The results of the validation process demonstrate the agreement of the method with the provisions of Regulation EC No 401/2006.

Simultaneous determination of aflatoxins B1, B2, G1, and G2 in foods and feed materials.

A high-performance liquid chromatographic method with on-line post-column photochemical derivatization and fluorimetric detection for the simultaneous separation and quantitative determination of aflatoxin (AF) B(1), B(2), G(1), and G(2) in foodstuffs and feed materials is reported.The chromatographic separation is accomplished by using a C(18) column eluted with an isocratic mobile phase consisting of water, methanol, and acetonitrile. The sample preparation requires a simple extraction of aflatoxins with a mixture of water and methanol, and a purification step by immunoaffinity column clean-up. The total analysis time, including sample preparation and chromatographic separation, does not exceed 40 min with a run time of 10 min. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption has been extensively validated, in agreement with Regulation (EC) No. 882/2004, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006 in terms of sensitivity, linearity, selectivity, and precision.

Assessment of fumonisins B1 and B2 levels in commercial maize-based food products by liquid chromatography with fluorimetric detection and postcolumn chemical derivatization.

The occurrence of the fumonisins B(1) and B(2) in maize-based food products marketed in Italy was examined. A simply and reliable chromatographic method with fluorimetric detection and postcolumn o-phtalaldehyde derivatization was used for a monitoring of 100 samples (8 flours, 21 corn-meal, 16 snacks, 7 maize samples, 13 gluten-free products, and 35 corn-flakes) bought in local supermarkets during the years 2008 and 2009. The presence of both fumonisins B(1) and B(2), at a concentration higher than 15 µg/kg, was observed in all samples of corn-meal and maize-flour, in 75% of snacks, in 57% of maize samples, in 54% of gluten-free products, and in 29% of corn-flakes. A total of 7 samples including 4 corn-meals, 2 maize-flours, and 1 maize showed a value exceeding the maximum level fixed in the Regulation 1126/2007/EC; no positive sample was observed in corn-flakes, snacks, and gluten-free foods. Fumonisins contamination, on the whole range of maize-based food products analyzed, emphasizes the need of improve agricultural practices, and increase official control and monitoring studies.

Survey of benzoic acid in cheeses: contribution to the estimation of an admissible maximum limit.

Benzoic acid and its salts are commonly used additives in the food industry. Their use is not allowed in dairy products even though they can be found naturally. In this work, 100 cheese samples were tested to establish the maximum concentration that can be considered as "natural" and, therefore, permitted in cheeses. Analyses were carried out by a validated ion chromatography method and "positive" samples were confirmed by two other HPLC methods. Benzoic acid concentrations higher than the method LOQ (8.8 mg kg(-1)) were found in 18 samples, ranging from 11.3 to 28.7 mg kg(-1), with a mean value of 20.5 mg kg(-1). Taking into account the distribution of benzoic acid concentrations observed in "positive" samples, it is plausible to estimate a maximum admissible limit of 40.0 mg kg(-1) for benzoic acid in cheese. Below this value, samples can be considered "compliant".

Development of a new analytical method for the determination of sulfites in fresh meats and shrimps by ion-exchange chromatography with conductivity detection.

An accurate and reliable analytical method, based on ion chromatography and suppressed conductivity detection, has been developed and validated for the quantitative determination of sulfites in fresh meats and shrimps. The chromatographic separation was accomplished by using an anion-exchange column eluted with sodium carbonate and sodium hydroxide. The optimized step-change elution, followed by column re-equilibration at the initial mobile phase composition, guaranteed a good resolution even toward endogenous interfering peaks, and an excellent retention time repeatability (1.1%, n=6). Good results in terms of sample extract stability, recovery efficiency were achieved with an extraction solvent mixture based on sodium hydroxide, fructose and EDTA. The method validation, performed by an in-house model according to Decision 657/2002/EC and Regulation 882/2004/EC, provided excellent results with respect to linearity (correlation coefficient up to 0.9998), limits of detection and quantification (2.7 and 8.2 mg kg(-1), respectively, expressed as SO(2)), expanded measurement uncertainty (below 10%), recovery values (ranging from 85% to 92%) and repeatability (down to 8%), demonstrating the conformity of the proposed method with the European directives. Finally, by major changes ruggedness studies, the method applicability to the quantitative analysis of cow hamburger, pork and horse sausage, and shrimps was demonstrated.

Validation of a confirmatory analytical method for the determination of aflatoxins B1, B2, G1 and G2 in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection.

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of aflatoxins B1, B2, G1 and G2 in foodstuffs and materials for feed has been validated. The method is based on high performance liquid chromatography with on-line post-column photochemical derivatization and fluorescence detection. The chromatographic separation of aflatoxins was accomplished using a C18 column eluted with an isocratic mobile phase consisting of water, methanol and acetonitrile. The sample preparation required a simple extraction of aflatoxins with MeOH/H2O (80:20, v/v) and a purification step by immunoaffinity column cleanup. The total analysis time, including sample preparation and chromatographic separation, did not exceed 40 min with a run time of 10 min. The on-line photochemical derivatization ensures better results in terms of simplicity, sensitivity and reproducibility with respect to chemical derivatization techniques, and provides an increase of the peak resolution and an extent of automation in comparison with the electrochemical ones. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption was extensively validated following Regulation (EC) No. 882/2004. Detection limits in wheat bran samples of 0.08 µg kg1 for AFB1, 0.02 µg kg1 for AFB2, 0.16 µg kg1 for AFG1 and 0.04 µg kg1 for AFG2 were attained. The method allows high recovery with mean values ranging from 72 to 94% and it satisfies the necessary requirements for sensitivity, linearity, selectivity, precision and ruggedness, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006.

Development of a new analytical method for the determination of fumonisins B1 and B2 in food products based on high performance liquid chromatography and fluorimetric detection with post-column derivatization.

A sensitive and selective analytical method was developed for the quantitative determination of fumonisins B(1) and B(2) in maize-based foods for direct human consumption. The method, based on high-performance liquid chromatography and fluorescence detection, presents a rapid and automated on-line post-column derivatization, performed with o-phtalaldehyde and N,N-dimethyl-2-mercaptoethylamine. Several factors affecting the separation and detection of fumonisins were investigated, including mobile phase composition, column features, derivatization agent flow-rate and both the excitation and the emission wavelengths. Optimal fluorescence detection was obtained by using a lambda(exc) of 343 nm and a lambda(em) of 445 nm. Under the optimized experimental conditions, a complete separation of fumonisins was obtained in less than 13 min by using a C(18) column and a gradient elution at 0.8 mL/min with methanol and 0.1M phosphate buffer at pH 3.15. The limits of detection for FB(1) and FB(2) were 4 and 5 microg/L corresponding to 5 and 6 microg/kg in matrix. Each fumonisin was determined in the range 40-320 microg/L that correspond to 50-400 microg/kg in matrix. The necessary requirements for accuracy, reproducibility and sensitivity were fulfilled and recovery values ranged from 87 to 94% for FB(1) and from 70 to 75% for FB(2) in cornflake samples at three fortification levels in the range 100-300 microg/kg. The potential of this method, combined with a simple clean-up procedure, was assessed by the measurements of FB(1) and FB(2) in maize-based products, such as maize flour, "polenta", tortillas and cookies.

Validation according to European Commission Decision 2002/657/EC of a confirmatory method for aflatoxin M1 in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence detection.

A high performance liquid chromatographic method with fluorimetric detection for the determination of aflatoxin M1 (AFM1) in milk has been optimized and validated according to Commission Decision 2002/657/EC by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CC(alpha)), detection capability (CC(beta)) and ruggedness of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401/2006/EC. The mean recovery calculated at three levels of fortification (0.5, 1.0, and 1.5-fold the MRL) was 91% and the maximum relative standard deviation value for the within-laboratory reproducibility was 15%. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 microg kg(-1) and 0.015 microg kg(-1) while the CC(alpha) and CC(beta) values were 0.058 microg kg(-1) and 0.065 microg kg(-1), respectively. The relative expanded measurement uncertainty of the method was 7%. The method was not affected by slight variations of some critical factors (ruggedness minor changes) as pre-treatment and clean-up of milk samples, thermal treatment and different storage conditions, as well as by major changes valued in terms of milk produced by different species (buffalo, goat and sheep). The method allowed accurate confirmation analyses of milk samples, resulted positive by the screening method. In fact, the Z-score values attained in a proficiency test round were well below the reference value of 1, proving the excellent laboratory performances.