Massimo Onor

Separation and determination of denatured αs1-, αs2-, β- and κ-caseins by hydrophobic interaction chromatography in cows’, ewes’ and goats’ milk, milk mixtures and cheeses

Abstract Caseins α s1 -, α s2 -, β- and κ- from raw cows’, ewes’ and goats’ milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 μ M . The detection limit for α-, β- and κ-caseins ranged between 0.35 and 0.70 μ M . The precision of the method was evaluated, the coefficient of variation for α-, β- and κ-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows’, goats’ and ewes’ milk (10 samples analyzed for each species), in one sample of unprocessed buffalos’ milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (α s1 /κ, α s2 /β, β/κ and α s2 /α s1 ) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.

Mercury speciation by liquid chromatography coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection (LC-CVGAFS)

Reverse phase chromatography (RPC) coupled on-line with UV-vis diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) is proposed for the speciation and determination of inorganic and organic mercury (methylmercury, ethylmercury and phenylmercury) in the form of cysteine, penicillamine and glutathione complexes. The mercury-thiol complexes are separated on a C(18) Reverse Phase column and oxidized on-line with bromine, generated in situ by KBr/KBrO(3) in HCl medium, in order to fast convert organic mercury species to inorganic mercury in less than 2.5s, at room temperature, in a 30cm knitted coil. Hg(II) is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg(0). Under optimized conditions, on-line bromine treatment gives recoveries of thiol-complexed methylmercury, ethylmercury and phenylmercury with respect to inorganic mercury ranging between 79 and 85%, 80 and 85%, 63 and 76%, respectively, depending on the complexing thiol employed. Optimized elution conditions were provided in the three complexing agents. The detection limits (LODc) for inorganic mercury, methylmercury, ethylmercury and phenylmercury, in the optimized conditions complexed with thiols were about 16, 18, 18 and 20pg (as mercury), respectively, a relative standard deviation (R.S.D) ranging between 1.5 and 2.0%, and a linear dynamic range between 0.1 and 100ng injected. LC-DAD-CVGAFS method has been validated by analysing two certificate reference material, DORM-2 and NIES CRM 13, obtaining 98+/-6 and 97+/-5% of methylmercury recovered, respectively.

Characterization of the Balm of an Egyptian Mummy from the Seventh Century B.C.

Abstract An analytical procedure for the characterization of plant resins, waxes, bitumen, oils and their degradation products in mummy balms is described. The method is based on the selective extraction of these compounds from 19 samples with CH2Cl2 and, after drying the residue, with n-hexane, followed by a Florisil chromatographic clean-up step of the non-polar extract and by the saponification of an aliquot of dried CH2Cl2 extract. Polar compounds were derivatized with t-butyl-dimethyl silyl trifluoroacetamide (MTBSTF A). Quantitative determinations were obtained by gas chromatography-mass spectrometry. The procedure, tested on suitable reference materials such as beeswax and plant resins, showed a recovery of about 100% for non-polar compounds. Using this method, pine resins (P. pinea and P. sylvester), mastic resin (Pistacia lentisca and P. terebinthus), sandarac (Tetraclinis articulata), elemi ( Canarium), dammar (Hopea), myrrh (Commiphera) and a pine resin from the second century A.D. were characterized. Identification was based on the characteristic pattern of the diterpenoid and triterpenoid compounds contained in these natural products. Results on Egyptian mummy samples from the seventh century B.C. were obtained and showed that the main components used in the embalming process were mastic resin from the genus Pistacia, an unidentified vegetable oil, beeswax and bitumen. Terpenoids degraded by a thermal treatment were also found; therefore a hot fluid was used for embalming, confirming the practice hypothesized in the Late Period by egyptologists.

Physico-chemical characterization of protein–pigment interactions in tempera paint reconstructions: casein/cinnabar and albumin/cinnabar

AbstractIn this work, we characterized paint reconstructions using ovalbumin and casein as binders, and cinnabar (HgS) as a pigment, before and after artificial ageing. Egg and casein are common paint binders that were used historically in the technique of tempera painting. Despite extensive research on the identification of proteinaceous binders in paintings, there is a substantial lack of knowledge regarding the ageing pathway of their protein content, and their chemical interaction with inorganic pigments. Thermogravimetric analysis, infrared spectroscopy and size-exclusion chromatography (SEC) were used to reveal the physico-chemical processes involved in the ageing of proteins in paintings. Taken together, the three techniques highlighted that proteins are subject to both cross-linking and hydrolysis upon ageing, and to a lesser extent, to oxidation of the side chains. Mercury–protein interactions were also revealed using a cold vapour generation atomic fluorescence spectrometer mercury-specific detector coupled to SEC. The study clearly showed that HgS forms stable complexes with proteins and acts as a sensitizer in cross-linking, hydrolysis and oxidation. FigureA multi-techinque approach to the study of protein/cinnabar tempera paint recontructions: thermogravimetric analysis, Fourier Transform Infrared Spectroscopy and size exclusion chromatography

Measurement of Warfarin in the Oral Fluid of Patients Undergoing Anticoagulant Oral Therapy

Background Patients on warfarin therapy undergo invasive and expensive checks for the coagulability of their blood. No information on coagulation levels is currently available between two controls. Methodology A method was developed to determine warfarin in oral fluid by HPLC and fluorimetric detection. The chromatographic separation was performed at room temperature on a C-18 reversed-phase column, 65% PBS and 35% methanol mobile phase, flow rate 0.7 mL/min, injection volume 25 µL, excitation wavelength 310 nm, emission wavelength 400 nm. Findings The method was free from interference and matrix effect, linear in the range 0.2–100 ng/mL, with a detection limit of 0.2 ng/mL. Its coefficient of variation was <3% for intra-day measurements and <5% for inter-day measurements. The average concentration of warfarin in the oral fluid of 50 patients was 2.5±1.6 ng/mL (range 0.8–7.6 ng/mL). Dosage was not correlated to INR (r = −0.03, p = 0.85) but positively correlated to warfarin concentration in the oral fluid (r = 0.39, p = 0.006). The correlation between warfarin concentration and pH in the oral fluid (r = 0.37, p = 0.009) confirmed the importance of pH in regulating the drug transfer from blood. A correlation between warfarin concentration in the oral fluid and INR was only found in samples with pH values ≥7.2 (r = 0.84, p = 0.004). Conclusions Warfarin diffuses from blood to oral fluid. The method allows to measure its concentration in this matrix and to analyze correlations with INR and other parameters.

Microwave-assisted photochemical reactor for the online oxidative decomposition and determination of p-hydroxymercurybenzoate and its thiolic complexes by cold vapor generation atomic fluorescence detection.

We developed a photochemical method for the online oxidation of p-hydroxymercurybenzoate (PHMB), an organic mercury species widely used for mercaptan and thiolic compound labeling. The method is based on a fully integrated online UV/microwave (MW) photochemical reactor for the digestion of PHMB, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the quantitative conversion of PHMB and thiol-PHMB complexes to Hg(II), with a yield between 91% and 98%, without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. This reaction was followed by the reduction of Hg(II) to Hg(0), performed in a knitted reaction coil with NaBH(4) solution, and AFS detection in an Ar/H(2) miniaturized flame. The low MW power applied (18 W) allowed us to keep constant the temperature of the photochemical reactor (21 ± 1 °C), using a flowing water bath. This avoided peak widening due to diffusion processes generally occurring at high temperatures and in the additional cooling coil. This method has been applied to the determination of thiols in human plasma, blood, and wine.

Oxidative decomposition of atrazine in water in the presence of hydrogen peroxide using an innovative microwave photochemical reactor.

The simultaneous application of microwave (MW) power and UV light leads to improved results in photochemical processes. This study investigates the oxidative decomposition of atrazine in water using an innovative MW and UV photochemical reactor, which activates a chemical reaction with MW and UV radiation using an immersed source without the need for a MW oven. We investigated the influence of reaction parameters such as initial H(2)O(2) concentrations, reaction temperatures and applied MW power and identified the optimal conditions for the oxidative decomposition of atrazine. Atrazine was completely degraded by MW/UV/H(2)O(2) in a very short time (i.e. t(1/2) = 1.1 min for 20.8 mg/L in optimal conditions). From the kinetic study, the disappearance rate of atrazine can be expressed as dX/dt = k(PH)[M](0)(b-X)(1-X), where b ≡ [H(2)O(2)](0)/[M](0)+k(OH)[·OH]/k(PH)[M](0), and X is the atrazine conversion, which correlates well with the experimental data. The kinetic analysis also showed that an indirect reaction of atrazine with an OH radical is dominant at low concentrations of H(2)O(2) and a direct reaction of atrazine with H(2)O(2) is dominant when the concentration of H(2)O(2) is more than 200 mg/L.

Human exposure to thallium through tap water: A study from Valdicastello Carducci and Pietrasanta (northern Tuscany, Italy).

A geological study evidenced the presence of thallium (Tl) at concentrations of concern in groundwaters near Valdicastello Carducci (Tuscany, Italy). The source of contamination has been identified in the Tl-bearing pyrite ores occurring in the abandoned mining sites of the area. The strongly acidic internal waters flowing in the mining tunnels can reach exceptional Tl concentrations, up to 9000μg/L. In September 2014 Tl contamination was also found in the tap water distributed in the same area (from 2 to 10μg/L). On October 3, 2014 the local authorities imposed a Do Not Drink order to the population. Here we report the results of the exposure study carried out from October 2014 to October 2015, and aimed at quantifying Tl levels in 150 urine and 318 hair samples from the population of Valdicastello Carducci and Pietrasanta. Thallium was quantified by inductively coupled plasma - mass spectrometry (ICP-MS). Urine and hair were chosen as model matrices indicative of different time periods of exposure (short-term and long-term, respectively). Thallium values found in biological samples were correlated with Tl concentrations found in tap water in the living area of each citizen, and with his/her habits. Thallium concentration range found in hair and urine was 1-498ng/g (values in unexposed subjects 0.1-6ng/g) and 0.046-5.44μg/L (reference value for the European population 0.006μg/L), respectively. Results show that Tl levels in biological samples were significantly associated with residency in zones containing elevated water Tl levels. The kinetics of decay of Tl concentration in urine samples was also investigated. At the best of our knowledge, this is the first study on human contamination by Tl through water involving such a high number of samples.

Determination of thiolic compounds as mercury complexes by cold vapor atomic absorption spectrometry and its application to wines.

We report on the application of a commercially available mercury analyzer, which is based on vapour generation of Hg(0) by NaBH(4) reduction and atomic absorption detection, to the quantification and characterization of -SH groups and its application to wine samples. The behaviour of Hg(II) and thiol-Hg(II) (RS-Hg) complexes at nanomolar level (RS=l-cysteine, dl-penicillamine, N-acetyl penicillamine, glutathione, cysteinylglycine, homocysteine) has been studied following their reduction with alkaline NaBH(4) to give Hg(0). In the absence of thiol-Hg(II) is quantitatively converted to Hg(0) by stoichiometric amount of NaBH(4) (reaction ratio 1/4mole NaBH(4)/mole Hg), while the complete reduction of Hg(II)-thiol complexes to Hg(0) requires molar excess of NaBH(4) up to six orders of magnitude, depending on the type of complex and on the pK(a) of the thiolic group. Under an appropriate excess of reductant, Hg(II) and its thiol complexes are not distinguishable giving the same response. These properties allow the discrimination of Hg(II) from Hg(II)-thiol complexes without any preliminary separation and the quantification of thiol groups. Instrumental detection limits are as low as 2.5pg, permitting sample dilution, therefore, minimizing the risk of possible interferences occurring with complex real matrices. The method has been applied to quantification of thiol groups in wine samples. Comparison with results obtained by HPLC coupled to atomic fluorescence detection confirmed the promising potentialities of the method.

Heterogeneous catalytic reaction of microcrystalline cellulose in hydrothermal microwave-assisted decomposition: effect of modified zeolite Beta

Zeolite Beta, modified with some salts of alkali and alkaline earth metals (K, Zn, Sn), was tested in the hydrothermal heterogeneous catalytic decomposition of microcrystalline cellulose. The reactions were microwave-assisted, where the microwaves were issued by an in situ coaxial applicator. Zeolites were subjected to an ion-exchange process which determines the loss of crystallinity in the following order: Sn-Beta-IE > K-Beta > Zn-Beta > acid form H-Beta. The interaction between zeolites and microwaves was studied by irradiating zeolite powder under constant power and the heating response was in the following order: K-Beta > NH4-Beta > Sn-Beta-IE ≈ Zn-Beta > H-Beta > alumina. These results show that the nature of the counterion strongly affects the absorption of microwaves. The catalytic activity of the different systems on the cellulose decomposition was studied, and resulted in the following order: H-Beta > K-Beta > Zn-Beta > Sn-Beta-IE > alumina, when the reaction medium contained 5 mM HCl. The most active catalyst was the acid zeolite Beta and the identified product distribution under the investigated conditions was (mol yield %): levulinic acid (22.3), glucose (12.1), lactic acid (4.1), formic acid (6.6), 5-(hydroxymethyl) furfural (14.6), acetic acid (15.2) and furfuraldehyde (3.1). The effect of temperature, time and the heterogeneous catalyst reuse (H-Beta) on the yields of different products was investigated. The use of MW radiation with a coaxial applicator instead of conventional heating gave clear advantages in the decrease of the reaction time (45 min) and in terms of yield enhancement (78.6% under the best conditions).