Spectrometric methods, enzymes

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

A novel mass spectrometric method has been described that permits the identification of the C-terminal peptide of a protein (68). The technique involves the incorporation of into all a-C02H groups released during enzyme-catalysed partial hydrolysis e. g. with trypsin) of the protein in 0-enriched water ( 0 0 50 50). Individual peptides were analyzed by GC/EIMS as their trifluoroacetyl permethyl derivatives and the C-terminal peptide was characterized as the one that did not incorporate (The non-... 

The technique of gas chromatography linked to mass spectrometry is an important tool in the study of volatile insect substances and also for the study of their biosynthesis. The subject of linked chromatographic-mass spectrometric methods, and the newer techniques that can be achieved with mass spectrometry are beyond the subject of this book. We can expect to see much wider use of high resolution mass spectrometry in the study of enzymes and biosynthetic pathways. 

Moriya S, Iwasald K, Samejima K, Takao K, Kohda K, Hiramatsu K, Kaweikita M (2012) A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism. Anal CWm Acta 478 45-52... 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

Very little is known about the biosynthesis of the three most recently proposed endocannabinoids, 2-AGE, virodhamine and NADA. Regarding 2-AGE (noladin ether), this compound was previously identified in pig brain (Hanus et al. 2001) and in some rat tissues and brain areas (Fezza et al. 2002) by using mass-spectrometric (MS) methods coupled to chromatographic separations. However, a recent study cast some doubt on the actual existence of 2-AGE in mammalian brain tissue (Oka et al. 2003). At the time of this study it was already known that (1) the only acyl ethers to have been detected in animals before the discovery of 2-AGE were 2-acyl ethers (e.g. alkenyl ethers such as platelet activating factor and plasmalogens) (2) there was no evidence for the existence of any enzyme catalysing the formation... 

The methods for determination of blood cholinesterases inhibition (AChE and BuChE) do not allow identification of the OP and do not provide reliable evidence for exposure at inhibition levels less than 20%. Moreover, they are less suitable for retrospective detection of exposure due to de novo synthesis of enzymes. A method has been developed which is based on reactivation of phosphylated cholinesterase and carboxylesterase (CaE) by fluoride ions. Treatment of the inhibited enzyme with fluoride ions can inverse the inhibition reaction, yielding a restored enzyme and a phos-phofluoridate which is subsequently isolated and quantified by gas chromatography and phosphorus-specific or mass spectrometric detection (Dll, Pll). Human (and monkey) plasma does not contain CaE but its BuChE concentration is relatively high [70-80 nM (M25, D8)], much higher than the concentration of AChE in blood [ca. 3 nM (H5)]. The plasma of laboratory animals, such as rats and guinea pigs, contains considerable concentrations... 

Off-Line Reaction Monitoring For slow enzyme reactions and long-lived intermediates, off-line reaction monitoring is more convenient. In these methods, the known amounts of an enzyme and a substrate are mixed together and incubated at physiological conditions. Aliquots are withdrawn at predetermined intervals, and the reaction is quenched immediately. The time course of the reaction can be monitored with mass spectrometric analysis immediately or later, at a more convenient time, by using either fast atom bombardment (FAB) [7], continuous-flow (CF)-FAB [7], matrix-assisted laser desorption/ionization (MALDI) [8], or electrospray ionization (ESI) [9-11]. Established quantitation procedures can be employed to monitor the concentration of the reactant or product (usually, the latter) (see Chapter 14). As an example, an appropriate internal standard that has no affinity for the enzyme can be added to the reaction mixture to improve... 

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