Carmen Palermo

Determination of aflatoxins in cereal flours by solid-phase microextraction coupled with liquid chromatography and post-column photochemical derivatization-fluorescence detection.

A new method for the determination of aflatoxins B(1), B(2), G(1), and G(2) (AFB(1), AFB(2), AFG(1), AFG(2)) in cereal flours based on solid-phase microextraction (SPME) coupled with high performance liquid chromatography with post-column photochemical derivatization and fluorescence detection (SPME-HPLC-PD-FD) has been developed. Aflatoxins were extracted from cereal flour samples by a methanol:phosphate buffer (pH 5.8, I=0.1) (80:20, v/v) solution, followed by a SPME step. Different SPME and HPLC-PD-FD parameters (fiber polarity, temperature, pH, ionic strength, adsorption and desorption time, mobile phase) have been investigated and optimized. This method, which was assessed for the analysis of different cereal flours, showed interesting results in terms of LOD (from 0.035 to 0.2 ng g(-1)), LOQ (from 0.1 to 0.63 ng g(-1), respectively), within and inter-day repeatability (2.27% and 5.38%, respectively) linear ranges (up to 20 ng g(-1) for AFB(1) and AFG(1) and 6 ng g(-1) for AFB(2) and AFG(2)), and total raw extraction efficiency (in the range 55-59% at concentrations in the range 0.3-1 ng g(-1) and 49-52% at concentrations in the range 1-10 ng g(-1)). The results were also compared with the purification step carried out by conventional immunoaffinity columns.

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.

Multi-residue method for the determination of organochlorine pesticides in fish feed based on a cleanup approach followed by gas chromatography–triple quadrupole tandem mass spectrometry

A multi-residue method for the determination of organochlorine pesticides in fish feed samples was developed and optimized. The method is based on a cleanup step of the extracted fat, carried out by liquid-liquid extraction on diatomaceous earth cartridge with n-hexane/acetonitrile (80/20, v/v) followed by solid phase extraction (SPE) with silica gel-SCX cartridge, before the identification and quantification of the residues by gas chromatography-triple quadrupole tandem spectrometry (GC-MS/MS). Performance characteristics, such as accuracy, precision, linear range, limits of detection (LOD) and quantification (LOQ), for each pesticide were determined. Instrumental LODs ranged from 0.01 to 0.11 microg L(-1), LOQs were in the range of 0.02-0.35 microg L(-1), and calibration curves were linear (r2>0.999) in the whole range of explored concentrations (5-100 microg L(-1)). Repeatability values were in the range of 3-15%, evaluated from the relative standard deviation of six samples spiked at 100 microg kg(-1) of fat, and in compliance with that derived by the Horwitzs equation. No matrix effects or interfering substances were observed in fish feed analyses. The proposed method allowed high recoveries (92-116%) of spiked extracted fat samples at 100 microg kg(-1), and very low LODs (between 0.02 and 0.63 microg kg(-1)) and LOQs (between 0.05 and 2.09 microg kg(-1)) determined in fish feed samples.

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 kg−1 for AFB1, 0.02 µg kg−1 for AFB2, 0.16 µg kg−1 for AFG1 and 0.04 µg kg−1 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.

Comparative analysis of gluten proteins in three durum wheat cultivars by a proteomic approach.

The gluten protein composition and expression level influence dough properties and are cultivar and environment dependent. To broaden the knowledge of the durum wheat gluten proteome, three cultivars were compared in two different growing seasons by a proteomic approach. Cultivar-specific and differentially expressed spots in the two years were identified by mass spectrometry. Significant differences were observed among the cultivars: Ofanto showed the lowest protein spot volumes in the high molecular weight (HMW) and low molecular weight (LMW) <35,000 regions and the highest in the LMW 48,000-35,000 region, Latino the lowest in the LMW 48,000-35,000 region, and Simeto an intermediate expression level in both LMW regions. In the warmer year the up-regulation of HMW glutenins, α-gliadins, and a globulin 3 protein and the down-expression of LMW glutenins and γ-gliadins were observed. Among the cultivars, Simeto showed the highest stability across the environments.

Measurement of Histamine in Seafood by HPLC, CE, and ELISA: Comparison of Three Techniques

Among biogenic amines, histamine represents a very important freshness indicator in seafood quality and sanitary controls. In fact the presence of histamine at certain levels can cause anaphylactic poisoning episodes (scombroid poisoning). However, the state of food preservation involves not only histamine but also other biogenic amines (cadaverine, putrescine, spermidine, and spermine). Therefore, a Biogenic Amines Index (BAI) has been defined for the evaluation of quality of proteic foods (Malle and Valle’, 1996):

Strategies in protein sequencing and characterization: multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry.

A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis.

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”.

Differential Expression of Durum Wheat Gluten Proteome under Water Stress during Grain Filling.

Environmental stress during grain filling may affect wheat protein composition, thus influencing its final quality. A proteomic approach was used to evaluate changes in storage protein composition under water stress of two Italian durum wheat (Triticum turgidum ssp. durum) cultivars, Ciccio and Svevo. The high-molecular-weight glutenin region increased progressively in both cultivars and under two water regimens. The L48-35 region, corresponding to low-molecular-weight (LMW) glutenin subunits, increased slightly during grain development and decreased under water stress in both cultivars. In particular, an s-type LMW related to superior technological quality was down-expressed in the early-mid period in Svevo and in the mid-late period in Ciccio. Finally, the L<35 region, corresponding to gliadin-like proteins, decreased slightly during grain development and increased under stress in both cultivars. Several α-gliadins, associated with immunological potential, increased their expression under water stress, especially in Svevo in the early-mid stage of grain filling.

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.