Enzymatic Derivatization

Enzymatic derivatization offers major advantages over alternative procedures. The selectivity of an enzyme for its substrate along with the sensitivity offered by modern analytical instrumentation makes enzymatic derivatization a powerful tool in the field of drug residue analysis. 

Enzymes can be used in several ways in chromatographic applications to improve selectivity or to enhance the detector response. Applications may involve enzymes with either a broad specificity toward a group of related compounds or a high specificity toward a particular compound. In the field of drug residue analysis, most current applications concern enzymatic reactions taking place in separate reactors incorporated in LC systems before or after the analytical column. Reactors with immobilized enzymes have proven to be suitable in such continuous flow systems. 

The enzyme catalyzes the conversion of the analyte to a corresponding product. Determination of the amount of this product or of a specific coformed byproduct or, alternatively, measurement of the concentration decrease of a coreactant agent by means of a suitable LC detector can allow estimation of the analyte level in the injected sample. 

Changes in the activity of enzymes may also occur by a variety of other parameters including temperature, pH, and ionic strength. Temperature can affect both the activity and the stability of the enzymes. For most enzymes, the reaction velocity doubles with a temperature increase of 10 C but potential enzyme denaturation may also occur (265). 

Another important parameter is the acidity of the system. Since the relation between enzyme activity and pH often shows an optimum, buffers are regularly used in enzymatic derivatization to control the pH. Use of buffers, however, can cause increase of the ionic strength of the system, thus changing the tertiary structure of the enzyme and either making it more accessible for the substrate or blocking its active sites. Therefore, optimization of the chromatographic system is recommended for each specific system. 

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Regioselective enzymatic acylation of large, insoluble polysaccharides is still a quite difficult task and therefore it is not surprising that only scant data have been reported up to now, most of them describing reaction outcomes which met with limited success. Nevertheless, enzymatic derivatization of polysaccharides has been performed in nonpolar organic solvents using insoluble polysaccharides with soluble [51] or suspended enzymes [52]. Chemically modified celluloses with either enhanced solubility or more readily accessible hydroxyl groups, like cellulose acetate or hydroxypropyl cellulose, were acylated by CalB, as reported by Sereti and coworkers [53]. However, the same authors failed to modify crystalline cellulose under the same reaction conditions. 

The wide variety of enzymes available gives for promise enzymatic derivatization to become a potent analytical tool in the future. Better understanding and theoretical formulations will lead to commercial availability of immobilized enzymes and consequently to more ready use of them. Since in such systems a low content of organic cosolvent in the mobile phase can only be tolerated (whereas a compromise has to be made as far as the optimum mobile phase pH is concerned), artificial enzymes, which are synthetic polymer chains having functional groups that mimic the biocatalytic activity of natural enzymes, are currently being synthesized and investigated as a means to overcome such limitations (276). 

Assays of isotopic and chiral phosphoryl compounds have been greatly facilitated by 31P NMR substitution with 180 causes a shift to higher field,28,29 the magnitude of the isotope shift being related to the bond order 30 170, with its spin of and nuclear electronic quadrupole moment, rapidly relaxes the 31P resonance and hence washes out its signal.30"32 The methods will not be detailed in this chapter. Many of the assignments have involved some complex chemical and/or enzymatic derivatization. 

There are several advantages to performing digestions using enzymatically-derivatized... 

In rare instances, the derivatization with a CDA was carried out enzymatically (see below). Clearly, the requirements for optimization of such reactions are different from those of nonenzymatic chemical transformations and should be carefully examined before an enzymatic derivatization reaction is routinely applied for quantification. 

Detection with MS is particularly important for the isolation of compounds that lack useful chromophores or are found only in complicated mixtures such as lactose [56]. The lack of a UV-absorbing chromophore in the saccharide structure limits the mode of detection. Refractive index detection requires precise control of the mobile phase and often does not meet the demands of trace-level analysis needed concerning sensitivity and selectivity. Even chemical derivatization, for example, postcolumn derivatization and enzymatic derivatization, which greatly improves the selectivity and sensitivity of a chromatographic detection systan, does not meet the needed trace levels for the detection of the fine particle dose of lactose. The reason... 

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