Pier Antonio Biondi

A headspace solid-phase microextraction gas-chromatographic mass-spectrometric method (HS-SPME–GC/MS) to quantify hexanal in butter during storage as marker of lipid oxidation

A method using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC/MS) was developed and validated for the extraction and quantification of hexanal content in butter (at ngg(-1) level) during storage at 4°C. The variability of hexanal content among seasons of production and the influence of high extraction temperature on ex-novo formation of hexanal were also evaluated. The HS-SPME conditions were optimised and analytical parameters of the method (linearity, accuracy, and precision) demonstrate its usefulness. The reproducibility and accuracy of the quantitative analysis was assured by the use of D(12)-hexanal as internal standard. For the applications, the headspace was extracted using CAR/PDMS fiber for 180min at 4°C. Hexanal contents in samples during all storage period (shelf-life) and from butters produced in different seasons were analysed. Butter samples at the end of shelf-life and samples produced in August showed highest values of hexanal, confirming that the temperature both in storage and distribution phases represents a critical factor to maintain the quality of butter.

Tetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC–ESI-mass spectrometry

Interconversion of hormonally active cortisol (F) into the corresponding inactive 11-keto form, cortisone (E), is catalyzed by 11beta-hydroxysteroid dehydrogenases (11β-HSDs). With a view to estimating in vivo activities of some 11β-HSD isoforms, the measurement of urinary F and E and their tetrahydro metabolites (tetrahydrocortisol, THF, allotetrahydrocortisol, ATHF, tetrahydrocortisone, THE) has been suggested. The basic knowledge of THF, ATHF and THE levels in farm cattle is limited. Therefore the aim of this study was first to optimize a simple and quick method to determine F and E tetrahydro-metabolites in bovine urine by HPLC-mass spectrometry with electrospray ionization (HPLC-ESI-MS) and then to apply the method to real urine of bovines treated with prednisolone. The samples underwent filtration, deconjugation, solid-phase extraction (SPE) and the relevant analytes were measured by HPLC-ESI-MS. The method described in this paper is simple and efficient, featuring good linearity (up to 0.996) and reproducibility (6.8-12.5%, CV). Especially, good LODs were obtained, from 1.63 to 2.67 ppb, depending on the analyte. The chromatographic conditions were optimized in order to obtain a resolution which would allow to simultaneously measure two diastereoisomers, i.e. THF and ATHF. In our study, ATHF turns out to be below the detection limit, while for 18 samples tested the contents of examinated metabolites were as followed: THF (12.5±4.8 ppb), THE (10.9±5.5 ppb), F (11.6±3.3 ppb) and E (5.0±2.2 ppb). When the method was applied to the subject treated with prednisolone a major increase in the concentration of tetrahydro metabolites was observed before the slaughter, mainly due to stress conditions; prednisolone treatment, most presumably, influenced the 11β-HSD activity, as indicated by the decrease in the F/E ratio. This work may provide a useful methodological contribution to the future definition of F, E, THF, ATHF and THE urinary baseline values in order to obtain indirect evaluations of HSDs activity in farm cattle and possible applications in screenings for suspected abuse of synthetic corticosteroids in bovines.

Computer-assisted evaluation of isoelectric focusing patterns in electrophoretic gels: identification of smoothhounds (Mustelus mustelus, Mustelus asterias) and comparison with lower value shark species.

In this work, the most commercially important Selachian species, smoothhound (Mustelus mustelus) and starry smoothhound (Mustelus asterias), have been identified by polyacrylamide isoelectric focusing (IEF‐PAGE), along with several shark species of minor commercial value. In Italy, these two smoothhound species are commonly subjected to fraudulent substitution with lesser valued sharks. After the electrophoretic runs, the band patterns of the two Mustelus species were compared with those of dogfish (Scyliorhinus canicula), spurdog (Squalus acanthias), blue shark (Prionace glauca) and black‐mouthed dogfish (Galeus melanostomus), both visually and with gel analysis software. The actual isoelectric points were then submitted to cluster analysis to differentiate the single species, despite the possible occurrence of electrophoretic variations or protein polymorphism. Every shark showed species‐specific band patterns and could therefore be well differentiated, as confirmed by statistical analysis.

Gas chromatographic analysis of neutral monosaccharides as their O-pentafluorobenzyloxime acetates

Abstract In order to develop a new derivatization procedure for the gas chromatographic analysis of sugars, O-(2,3,4.5.6-pentafluorobenzyl)hydroxylamine was reacted with the neutral monosaccharides most commonly present in glycoproteins. The resulting oximes were subsequently acetylated, and the resulting volatile derivatives can be separated on both packed and capillary columns. The method appeared to be rapid, simple and reliable. As an application, the neutral sugars content of some known glycoproteins was analyzed.

Substitution of fish species detected by thin-layer isoelectric focusing and a computer-assisted method for the evaluation of gels.

Fourteen fish species susceptible to substitution were analysed by the thin-layer isoelectric focusing technique on polyacrylamide gels of pH 3.5-9.5. Four fish per species were run on the same gel to verify the possibility to differentiate them according to their protein banding patterns. The occurrence of intraspecific differences due either to electrophoretic variations or to protein polymorphism was also observed. In fact, some of them showed few dissimilarities among their protein profiles. However, the differentiation was possible for all species, even for those belonging to the same order, family and genus. Computer-based tools combined with statistical analysis were implemented and usefully applied to avoid a subjective evaluation of the isoelectric focusing gels, and to verify the reliability of the preliminary visual comparison of protein patterns.

New, stable halogenated derivative suitable for the gas Chromatographic determination of muramic acid

The formation of a new volatile derivative of muramic acid is described. The procedure entails a reaction with O-(pentafluorobenzyl)hydroxylamine in pyridine to give the corresponding oxime and a treatment with acetic anhydride in the presence of 4-N,N-dimethylaminopyridine. The structures of the obtained derivatives have been confirmed by MS. The influence of various catalyst amounts used during the acetylation step was studied and a mechanism was proposed in order to explain the different reaction courses.

Determination of 3,4-dihydroxyphenylethyleneglycol in urine by gas chromatography with a flame ionization detector: a new rapid method.

3,4-Dihydroxyphenylethyleneglycol, a major metabolite of noradrenaline in rat brain, is estimated alone or with 4-hydroxy-3-methoxyphenylethyleneglycol in rat and human urine by gas chromatography with a flame ionization detector. The samples are hydrolyzed and extracted at pH 2 with ethyl acetate. Then, to analyze only 3,4-dihydroxyphenylethyleneglycol the reaction with n-butaneboronic acid is carried out directly; if 4-hydroxy-3-methoxyphenylethyleneglycol also has to be estimated, preliminary acetylation in alkaline aqueous solution is performed. The advantages of the specificity due to the reagents used is discussed.

High-performance liquid chromatographic determination of d-amino acid oxidase activity

A new procedure for the assay of D-amino acid oxidase activity has been developed. alpha-Ketoisovaleric acid, derived from D-valine, was estimated by high-performance liquid chromatography after reaction with o-phenylenediamine to give the corresponding quinoxalinol derivative. alpha-Ketovaleric acid was used as an internal standard to ensure the reproducibility of the method. As an example of application, kidney cortex homogenates were analyzed for their D-amino acid oxidase activity. The advantages of the presented procedure for the determination of the enzymatic activity in biological samples compared with previously reported procedures are discussed.

DETERMINATION OF CORTISOL, CORTISONE, PREDNISOLONE AND PREDNISONE IN BOVINE URINE BY LIQUID CHROMATOGRAPHY–ELECTROSPRAY IONISATION SINGLE QUADRUPOLE MASS SPECTROMETRY

A quantitative LC–ESI single quadrupole MS method for the determination of cortisol (F), cortisone (E), prednisolone (PL), and prednisone (PN) in bovine urine has been developed and validated. After adding flumethasone as internal standard, the samples were subjected to filtration, deconjugation, and solid-phase extraction, while the chromatographic separation was achieved using a Restek Ultra II Allure Biphenyl column with isocratic mobile phase. The analytes were detected after negative electrospray ionization using SIM mode. In order to obtain spectra with maximum intensities of at least one of the three characteristic ions, (M + formate)−, (M − H)−, and [(M − H) – CH2O]−, an individual optimization of MS parameters for each corticosteroid was set up. MS data was acquired in the three-ion selected monitoring mode and the ion ratios between chosen diagnostic ions were used in order to increase the specificity. Calibration graphs were linear and the intra-day and intermediate precision was estimated as RSD values which were less than 17%. For F and E, obtained values indicated negligible absolute matrix effects (103% and 98%, respectively). The method was applied to real samples, and basal levels of F and E were preliminarily evaluated, while PL and PN were not detected.

Gas chromatographic determination of galactose in milk: Example of a switching valve used for the protection of the capillary column

Galactose, a marker of heat treatment, has been analysed in milk as pentafluorobenzyloxime acetate by gas chromatography with flame ionization detection using a simple switching valve system. The procedure did not entail any prederivatization clean-up for lactose elimination from the sample. In a short pre-column, reagent and lactose derivative excess were separated and the galactose and internal standard derivatives were transferred to the analytical column by a four-port valve. Thus, the analytical column was protected from overloading, so avoiding rapid deterioration.