Detection of sulphamethazine residues in cattle and pig hair by HPLC-DAD.

An HPLC method with diode array detection (DAD) is proposed for the detection of sulphamethazine (SMZ) residues in pig and cattle hair. Hair samples were extracted under alkaline conditions (NH4OH 0.2M for calf samples and NaOH 0.1M for piglet samples) and purified with a dual solid-phase extraction (SPE) cartridge system (reverse phase/strong-cation exchange). Recovery of SMZ in fortified samples varied from 70 to 85%, with a limit of quantification of 0.155 ng/mg. Residues of SMZ (7.2-59.2 ng/mg) were detected both in calf and piglet hairs after a therapeutic treatment with SMZ, while no interfering peak was observed in samples from untreated animals.

Hair analysis for veterinary drug monitoring in livestock production.

This review summarizes the basic information and applications concerning the use of hair analysis for the detection of misuse of therapeutic and anabolic agents in livestock animals. Hair biology, hair-shaft structure and the mechanisms of drug incorporation are described, considering the different factors which can affect the deposition. Sampling and extraction methods are reviewed with special attention to the particularities of this matrix, while the use of different analytical techniques is discussed, taking into account the concentration and the sensitivity required for drug detection. Advantages, drawbacks, promising prospects and possible applications of this technique in the future are also discussed.

Multiresidue analysis of cephalosporin antibiotics in bovine milk based on molecularly imprinted polymer extraction followed by liquid chromatography-tandem mass spectrometry.

This work reports the preparation of molecularly imprinted polymers (MIPs) selective to cephalosporin (CF) antibiotics, and their application as molecularly imprinted solid-phase extraction (MISPE) sorbents for the determination of these antimicrobials in milk samples. Several functional monomers and cross-linkers have been screened to select the best combination that provides high selectivity for the simultaneous multiresidue extraction of cefthiofur (THIO), cefazolin (AZO), cefquinome (QUI), cephapirin (API), cephalexin (ALE) and cephalonium (ALO) from the samples. The novel MIPs were prepared by a non-covalent imprinting approach in the form of spherical microparticles using the synthetic surrogate molecule sodium 7-(2-biphenylylcarboxamido)-3-methyl-3-cepheme-4-carboxylate, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea (VPU) as functional monomer, and divinylbenzene (DVB) as crosslinking agent in a 1:2:20 molar ratio. The optimized MISPE method allowed the extraction of the target antimicrobials from raw cow milk samples using a selective washing with 5mL methanol/2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (0.1M, pH 7.5) (2:98, v/v) to remove the non-specifically retained compounds, followed by elution with 1mL of trifluoroacetic acid (TFA) in methanol (0.1:99.9, v/v). The extracts have been analysed by UHPLC-MS/MS and the analytical method has been validated according to EU guideline 2002/657/EC. The limits of quantification (S/N=10) were in the 1.7-12.5µgkg-1 range, well below the maximum residue limits (MRLs) currently established for the quantified cephalosporins in milk samples. The developed MIP allows mutiresidual determination of the six cephalosporin antibiotics mentioned above, significantly broadening the application to food analysis of MISPE methods.

Detection of perfusion defects during coronary occlusion and myocardial reperfusion after thrombolysis by intravenous administration of the echo-enhancing agent BR1.

The purpose of this study was to detect myocardial perfusion defects as a result of coronary occlusion and myocardial reperfusion after thrombolysis with intravenous (i.v.) administration of the echo contrast agent BR1 (Bracco Research, Switzerland), which consists of microbubbles (median diameter 2.5 microm) containing sulfur exafluoride in a phospholipidic shell. To generate a coronary thrombosis, a copper coil was advanced into the left circumflex coronary artery in eight anesthetized dogs with opened chest cavities. Coronary occlusion occurred 18 +/- 10 minutes after the insertion of the coil and was documented both by an electromagnetic flow meter (as zero blood flow) and by radiolabeled microspheres (as myocardial perfusion defect). After 2 hours of occlusion, streptokinase was infused i.v.; reperfusion was documented by both the flow-meter and microspheres. Left ventricular cavity enhancement was apparent after all contrast injections. Peak cavity intensity did not increase with dose and was not affected by signal processing (suggesting signal saturation), whereas the duration of contrast effect significantly increased with the dose (from 26 +/- 16 to 147 +/- 74 seconds). Myocardial contrast intensity also increased after contrast (from 15 +/- 12 to 21 +/- 18 gray level/pixel, p < 0.001). Contrast echo detected myocardial perfusion defects (corresponding to 17% +/- 11% of LV cross-sectional area) in all the injections performed during coronary occlusion and detected myocardial reperfusion with a sensitivity of 50% versus microspheres. The extent of perfusion defects by contrast echo showed a good correlation with microspheres (r = 0.73). Myocardial reperfusion was not detected by changes in heart rate, aortic pressure, pulmonary arterial pressure, cardiac output, left ventricular fractional area change, or wall-motion score index. Hemodynamic parameters were not affected by contrast injections. Thus, the i.v. administration of BR1 allows us to accurately detect myocardial perfusion defects during coronary occlusion and, to a lesser extent, myocardial reperfusion after thrombolysis.

Comparison of different immunoaffinity clean-up procedures for high-performance liquid chromatographic analysis of ochratoxin A in wines.

Three immunoaffinity clean-up procedures to analyse ochratoxin A (OTA) in wines were compared. The direct wine clean-up with Ochraprep and OchraTest columns gave equivalent results in terms of recovery and precision if compared with the reference procedure involving a preliminary extraction of OTA with chloroform. OTA quantification limit in wine ranged from 0.020 to 0.045 microg/l. The 'on-flow' OTA emission spectrum (excitation 333 nm) showed a maximum at 460 nm and could be used to confirm the quantitative results. The analysis of 11 red and white wines gave no significant quantitative differences between the three clean-up techniques.

Influence of aging conditions on the quality of red Sangiovese wine.

A red Sangiovese wine was stored in barrels of different woods (oak and chestnut) and types (225-L "barriques" and 1000-L barrels) at 12 and 22 degrees C for 320 days to evaluate the effects of different aging conditions on wine quality. Chestnut barrels led to wines richer in phenolics, and which were more tannic, colored, and fruity. Oak barrels gave wines with more monomeric phenolics, but less astringent, with higher vanilla smell, and more harmonious. The type of barrel could be used as a parameter to regulate the extraction of wood components and the polymerization of monomeric phenolics. Storage at 22 degrees C favored the formation of polymerized phenolics and the increase of color density and color hue. The temperature produced less pronounced effects on aroma and taste, even if wines stored at 12 degrees C showed more harmony.

Removal of ochratoxin A in red wines by means of adsorption treatments with commercial fining agents.

The presence in wine of the fungal metabolite, ochratoxin A (OTA), represents a serious risk for consumer health. A variety of fining agents, including activated carbon, silica gel, potassium caseinate, egg albumin, and gelatin, was evaluated in relation to their abilities to remove OTA in fortified wines. Freundlich adsorption isotherms were used to model the adsorption behavior between ochratoxin A and the fining agent. Potassium caseinate and activated carbon were found to be the best fining agents that could be used to remove OTA in wine. Potassium caseinate removed up to 82% of OTA when used at 150 g/hL, whereas activated carbon showed the highest specific adsorption capacity due to a high surface area per mass and low adsorption of total polyphenols.

Oxidative stress in fulminant hepatic failure: comparison of two pig models with and without liver necrosis.

BACKGROUND/AIMS: No experimental study has clearly demonstrated how liver necrosis worsens the evolution of fulminant hepatic failure. Considering that several types of liver injury are associated with oxidative stress, we decided to measure plasma oxidative markers in two pig models of fulminant hepatic failure without and with liver necrosis. METHODOLOGY: Fulminant hepatic failure was produced in two groups of six pigs each by either total hepatectomy or complete hepatic devascularization. The following parameters were recorded before and during the course of hepatic failure: electrocerebral activity, plasma vitamin E, malondialdehyde and fluorescent protein-aldehyde adducts, total cholesterol, lactate-dehydrogenase, creatine phosphokinase, and ammonium. RESULTS: Despite comparable survival periods, hepatic necrosis was associated with earlier electrocerebral deterioration. Plasma concentration of malondialdehyde and fluorescent protein-aldehyde adducts rose and vitamin E content decreased in both groups. However, while in the group without liver necrosis the rates of cholesterol and vitamin E decay were identical, in the group with liver necrosis cholesterol concentration decreased less than vitamin E concentration, strongly indicating a true intravascular oxidation of vitamin E. Interestingly, in both models the rise of oxidative parameters preceded the development of cell injury. CONCLUSIONS: Oxidative stress, although present in both models, was significantly higher in the group with liver necrosis.

High-performance liquid chromatographic analysis of free amino acids in fruit juices using derivatization with 9-fluorenylmethyl-chloroformate.

A simple, rapid, and reliable reversed-phase high-performance liquid chromatographic method for the analysis of 16 amino acids of main interest in commercial fruit juices (pear, orange, grapefruit, pineapple, peach, and apricot) is described. No sample cleanup is required. The pH of the fruit juices is adjusted to alkaline value (8.5) using 200 mM borate buffer, then amino acid is converted to stable derivatives using 9-fluorenylmethyl-chloroformate. The excess of derivatization reagent is removed by a hydrophobic amine, 1-amino-adamantane hydrochloride. The derivatization procedure is simple, fast, and described in detail. Amino acids are detected at 263 nm and eluted within 35 min. The calibration, precision (< or = 6.1%), and recovery (102% +/- 4%) of the method are reported. The conditions of separation are optimized; however, serine partially overlapped with aspartic acid. The amino acid profile of fruit juices is consistent with data from the literature.

Determination of carboxylic acids, carbohydrates, glycerol, ethanol, and 5-HMF in beer by high-performance liquid chromatography and UV-refractive index double detection.

A high-performance liquid chromatographic method is proposed for the simultaneous separation of main carboxylic acids, carbohydrates, ethanol, glycerol, and 5-HMF in beer by direct injection. A column packed with a sulfonated divinyl benzene-styrene copolymer and an isocratic elution with 0.0045N sulfuric acid and acetonitrile (6%, v/v) are employed. UV and refractive index detectors connected in series are also used to reduce the matrix interference of phenolic compounds. In conditions described, nine compounds are quantitated in a single chromatographic run without any pretreatment except for sample dilution and filtration before injection. Precision, accuracy, linearity of response, limit of detection, and limit of quantitation are also evaluated for each compound. Satisfactory results are obtained to justify the application of this method to all phases of beer production for process and quality control.

Influence of enrofloxacin administration and alpha-tocopheryl acetate supplemented diets on oxidative stability of broiler tissues.

The objective of this study was to assess the oxidative stability and presence of antibiotic residues in tissues of broilers fed diets supplemented with alpha-tocopheryl acetate and treated with enrofloxacin. The activities of antioxidant enzymes and antibiotic concentrations in chicken breast, leg, and liver were determined. Iron-induced TBA-reactive substances (TBARS) and vitamin E were evaluated in muscles. The antioxidant effectiveness of vitamin E was reflected by TBARS values being lower in antioxidant-supplemented treatments than in the other dietary groups. On the other hand, antioxidant enzyme activities were not substantially affected by dietary treatments. The concentration of enrofloxacin in tissues was considerable, even after withdrawal 12 d before slaughter. Contrary to the findings in previous studies, enrofloxacin was not extensively metabolized to ciprofloxacin. Supplementation of the diet with 100 mg/kg of alpha-tocopheryl acetate did not have a significant effect on the level of antibiotic found in breast muscle samples. When comparing treatments without antibiotic withdrawal time, alpha-tocopheryl acetate supplementation led to a significant decrease in enrofloxacin level in leg and liver samples. These results showed that mutual interactions between different molecules could modify the drug residues in the tissue, which should be taken into account when considering the drug administration and the establishment of a correct withdrawal time.

[Lipoma of the colon. Apropos of a case report]. / Il lipoma del colon. A proposito di un'osservazione.

Lipoma of the large intestine is a rare, benign tumor. The clinical findings and the diagnostic studies are analysed and a case of giant submucosal colonic lipoma removed by colonoscopy is reported. Colonoscopic removal of submucosal lipomas is recommended, while the necessity of a surgical treatment in presence of intussusception or subserosal lipomas is confirmed.

[Tumoral calcinosis. A case report]. / In tema di calcinosi tumorale. Descrizione di un caso.

A case of tumoral calcinosis of the hand is reported. The lesion was localized at the fifth digit of the right hand and caused pain and disability. The clinical, diagnostic and therapeutic problems of this rare soft tissue disease are discussed.

Primary intra-abdominal malignant fibrous histiocytoma. Case report and literature review.

Malignant fibrous histiocytoma is a pleomorphic soft tissues sarcoma usually found in the extremity and in the retroperitoneum. The primary intra-abdominal localization of this tumour is very rare and only thirty-five previous reports are present in the world medical literature. The Authors report a case of giant malignant fibrous histiocytoma of the mesentery, which is treated by surgical removal plus adjuvant chemotherapy.

[The endovascular treatment of hemoptysis]. / Tratamiento endovascular de la hemóptisis.

We studied 18 patients, 9 women and 9 men, ranging in age from 18 to 76 years. The main symptom was hemoptysis and the underlying pathology was tuberculosis, actinomycosis, lung cancer, metastatic carcinoma and systemic lupus. Nonsurgical patients, with recurrent hemoptysis or massive bleeding were selected. The embolization substances were spongostan, avitene (R) and PVA; they all produce temporary as well as persistent hemostasis. The procedure was successful in 16 patients. In two patients the embolization was not performed, one for technical reasons and the other because the vessel to be treated was the source of an anterior spinal artery. It is considered that the endovascular treatment constitutes an alternative for hemoptysis, even during the acute period, mainly in the management of nonsurgical patients.

Multiresidue analysis of fluoroquinolone antimicrobials in chicken meat by molecularly imprinted solid-phase extraction and high performance liquid chromatography.

This paper describes the synthesis of novel molecularly imprinted polymer (MIP) micro-beads for the selective extraction (MISPE) of six fluoroquinolone (FQ) antibiotics (enrofloxacin, ciprofloxacin, lomefloxacin, danofloxacin, sarafloxacin and norfloxacin) from chicken muscle samples and further analysis by high-performance liquid chromatography (HPLC) with fluorescence (FLD) or mass spectrometry (MS) detection. A combinatorial screening approach has been applied to select the optimal functional monomer and cross-linker formulation for polymer synthesis. The MIP prepared using enoxacin (ENOX) as the template - a mixture of methacrylic acid (MAA) and trifluoromethacrylic acid (TFMAA) as functional monomers and ethylene glycol dimethacrylate (EDMA) as the cross-linker - showed superior FQ recognition properties than the rest of the materials generated. MIP spherical particles were prepared using silica beads as sacrificial scaffolds. The polymers were packed in solid phase extraction (SPE) cartridges. The optimized MISPE-HPLC method allows the extraction of the antimicrobials from aqueous samples followed by a selective washing with acetonitrile/water (0.005% TFA, pH=3.0), 20:80 (v/v) and elution with 5% trifluoroacetic acid in methanol. Optimum MISPE conditions led to recoveries of the target FQs in chicken muscle samples ranging between 68 and 102% and precisions in the 3-4% range (RSD, n=18). The method has been validated according to European Union Decision 2002/657/EC, in terms of linearity, accuracy, precision, selectivity, decision limit (CCα) and detection capability (CCß) by HPLC-FLD and HPLC-MS/MS. The limits of detection were improved using HPLC-MS/MS analysis and ranged between 0.2 and 2.7µgkg(-1) (S/N=3) for all the FQs tested.

Effect of pH(24h), curing salts and muscle types on the oxidative stability, free amino acids profile and vitamin B2, B3 and B6 content of dry-cured ham.

The effects of a curing salt composition (with and without nitrifying salts), and the pH at 24h postmortem (pH24>6.0, 5.5

Identification of 5-hydroxymethyl-2-furfural (5-HMF) in Cava sparkling wines by LC-DAD-MS/MS and NMR spectrometry.

The aim of this study was to elucidate the structure of a potential ageing marker for Cava sparkling wine. In order to clarify the structure of this compound, NMR spectroscopy and hyphenated UHPLC-DAD-MS/MS techniques were used. We identified the hitherto unknown compound as 5-hydroxymethyl-2-furfuraldehyde (5-HMF). This is the first time that this compound has been reported in sparkling wines. A survey, based on the analysis of 80 commercial sparkling wines, showed that 5-HMF is present between 0.25 and 12.81 mg/L, and is compared with those reported for other types of wine. Hypothetical origin of 5-HMF in Cava sparkling wine is discussed.

Vitamin (B1, B2, B3 and B6) content and oxidative stability of Gastrocnemius muscle from dry-cured hams elaborated with different nitrifying salt contents and by two ageing times.

The effect of the amount of added nitrate and nitrate plus nitrite to dry-cured hams on the vitamin (B1, B2, B3, B6) content, the antioxidant enzyme superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) activities and the thiobarbituric acid reactive substances (TBARS) was assessed in Gastrocnemius muscle at the end of two ripening processes. Five different curing mixtures (Hi-N: 600 KNO3; Lo-N: 150 KNO3; Hi-Mix: 600 KNO3+600 NaNO2; Lo-Mix: 150 KNO3+150 NaNO2; Hi-Mix/Asc: 600 KNO3+600 NaNO2+500 sodium ascorbate, expressed as mg of salts added on surface per kg of fresh ham) were evaluated in dry-cured hams aged for 11.5months (standard process, SP) and 22months (long process, LP). Minor differences in target parameters between the hams due to the process were found. The amount of nitrate when it was added alone or as a mixture of nitrate and nitrite, as well as the ascorbate addition to dry-cured hams did not affect vitamin B1, B2 and B3 contents. The level of vitamin B6 was affected by both the amount and the mixture of salts; the addition of nitrite reduced around 40% the content of vitamin B6, but it was not affected by nitrate or ascorbate. The activity of SOD and CAT decreased with the amount of nitrate and nitrite, while GSHPx and TBARS resulted unaffected.

Analysis of vitamin B1 in dry-cured sausages by hydrophilic interaction liquid chromatography (HILIC) and diode array detection.

A method based on hydrophilic interaction liquid chromatography (HILIC) and diode array detection (DAD) was developed to quantify thiamine (vitamin B1) concentration in Spanish dry-cured sausages ("chorizo," "fuet," and "salchichón"). Samples were extracted with diluted acid (HCl 0.1M) followed by an enzymatic hydrolysis to release vitamin B1 vitamers from food matrix. Crude extracts were purified on a weak cation exchange SPE cartridge and total thiamine concentration was determined by LC-HILIC-DAD with a limit of detection better than 0.01 mg/100g. The proposed conditions, that do not require the derivatization of the extracts nor the use of fluorescence or MS detectors, are suitable to provide chromatographic separation and identification of vitamin B1 within 8 min. Selectivity, repeatability and accuracy of the method were evaluated with both spiked samples and the reference material Pig Liver BCR® 487. Quantification of vitamin B1 was also carried out for different kinds of commercial samples of Spanish dry-cured products.