High-performance liquid chromatographic determination of Levetiracetam in human plasma: comparison of different sample clean-up procedures

An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of the novel antiepileptic, Levetiracetam, has been developed. Three clean-up procedures for the analysis of Levetiracetam in human plasma were implemented and evaluated, namely solid-phase extraction, deproteinization by addition of organic solvents and formation of insoluble salts. Adenosine was used as the internal standard for all three sample pretreatment procedures. Among the several cartridges used for solid-phase extraction, the hydrophilic-lypophilic balance (Oasis) HLB) phase provides the best extraction yield of Levetiracetam, together with high precision. With the two other clean-up procedures involving plasma deproteinization by addition of methanol or zinc sulphate, lower sensitivity and precision of the assays were obtained. However, they are cheaper and faster when compared with the solid-phase extraction procedure.     

Mass spectrometric identification of a candidate biomarker peptide from the in vitro interaction of epichlorohydrin with red blood cells

The reaction products of epichlorohydrin with human alpha- and beta- globins, obtained through in vitro incubation of these compounds and red blood cells, were determined by using reversed-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization tandem mass spectrometry. The alpha-globin was much more reactive than the beta-globin. At low incubation ratios, approximating the order of magnitude of epichlorohydrin concentration as found in workplaces, the only modified peptide still detectable was the 62-90 belonging to the alpha-chain and carrying an incremental mass of 92 u on either His72 or His89. Given that the two peptides co-eluted in a single chromatographic peak during RP-HPLC separation, they could be chosen as suitable biomarkers for quantification in the setting up of a new methodology for the biological monitoring of persons occupationally exposed, replacing currently known procedures.     

Mass spectrometric approach for the analysis of food proteins

In the study of food proteins, the need for accurate protein structural analysis has been acknowledged because of the fact that nucleotide sequencing alone is of limited analytical value if not combined with relevant information regarding the specific protein expressed and the occurrence of phosphorylation, glycosylation and disulphide bridges, and with the modification induced by the technological treatment. Mass spectrometry, whether used alone or to complement the traditional molecular-based techniques has become fundamental to the structural analysis of proteins. It is, moreover, virtually irreplaceable in determining post-translational modifications as conventional methods cannot deliver reliable data. What lies at the root of this methodological breakthrough is the combination of high-resolution separation techniques such as two-dimensional electrophoresis or capillary reverse- phase high-performance liquid chromatography with mass spectrometric analysis, what is termed "proteomic" analysis. Thus, it appears appropriate to state that the new mass spectrometric techniques have been established as a valuable and efficient tool for protein and peptide analysis in complex mixtures, like those from food matrices, enabling us therefore to provide accurate information on molecular weight and also to put forth a structural assessment at a low-picomole level of material. Thus, a series of alternative approaches have been developed based on advanced mass spectrometric analysis in conjunction with classic protein chemistry in order to provide an in-depth view of food protein structure. This review outlines several of these novel methodologies as they apply to structural characterization of food products.     

Development of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in milk after alfa-1-acid glycoprotein affinity chromatography

In this work we present a method for confirmatory analysis of chloramphenicol (CAP) in bovine and buffalo raw milk. CAP is extracted in acetonitrile and purified by affinity chromatography on an alpha-1-acid glycoprotein (AAG) column, then is identified and determined by ion-trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified at the minimum required performance limit (MRPL) of 0.30 ppb, by monitoring the [M-H]- ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in Decision 2002/657/EC for confirmation of prohibited veterinary drugs. The trueness and within-day and between-day repeatability data are also reported. Moreover, the loading capacity of affinity columns towards CAP was tested. This method, based on the molecular recognition between drug and AAG during the purification step to improve sample cleanup, represents a quantitative and repeatable procedure for confirmatory analysis, and fits the requirements for the routine official control of CAP residues in raw milk.     

Separation and analysis of the major constituents of cloves by micellar electrokinetic chromatography

A micellar electrokinetic chromatographic method was developed and applied for the first time to the separation and quantification of the main components of cloves, namely eugenol and beta-caryophyllene. The analytes were separated in less than ten minutes in an unmodified fused silica capillary (effective length: 40.0 cm) using a background electrolyte composed of 25 mM carbonate buffer (pH 10.0) and 60 mM sodium dodecylsulfate. Analyte concentrations were determined in clove oil and in methanolic clove extracts after simple sequential dilution with methanol and background electrolyte, and the resulting electropherograms showed no interference due to other components. Precision assays gave satisfactory results, with mean standard deviation of peak areas lower than 4.7% (lower than 1.5% for migration times). The eugenol content of a commercial clove oil was about 70% (RSD% = 3.2), while that of beta-caryophyllene was about 15% (RSD% = 4.2); in clove extracts, on the other hand, eugenol was approximately 15% (RSD% = 4.7) and beta-caryophyllene 4% (RSD% = 5.1). Accuracy was also good (mean recovery >98%).     

Fully imidized soluble polyimides based on a novel dianhydride: 2,2′-dichloro-4,4′,5,5′-benzophenone tetracarboxylic dianhydride

We have synthesized a novel dianhydride, 2,2′-dichloro-4,4′,5,5′-benzophenone tetracarboxylic dianhydride (DCBTDA). Polyimides were synthesized with DCBTDA or 3,3′,4,4′-benzophenone tetracarboxylic dianhydride (BTDA) and several relatively rigid meta- and para- substituted mononuclear diamines. The BTDA based systems were insoluble in dipolar, aprotic solvents whereas the DCBTDA based polymers displayed enhanced solubility in these solvents. The thermal stability of these polyimides was excellent as measured by 5% weight loss decomposition. The T_g's of the polymers were all above 290°C.     

Synthesis of functionalized polyhedral oligomeric silsesquioxane (POSS) macromers by microwave assisted 1,3-dipolar cycloaddition

In this paper we report the first synthesis of isoxazolidine and isoxazoline-POSS macromers by 1,3-dipolar cycloaddition reactions of vinyl- and styryl-POSS with N-methyl-C-ethoxycarbonylnitrone and ethoxycarbonyl nitrile oxide, promoted by microwave irradiation. The nature of the resulting cycloadducts has been determined by NOE experiments and supported by computational studies at PM3 and DFT B3LYP/6-31G* levels.