Problems and Pitfalls in Using Derivatization in Liquid Chromatography

There are some potential problems and pitfalls in the routine use of derivatizations in LC. Major amongst these is the need to remove the excess reagent and/or its hydrolysis and thermal degradation products from the final derivatization solution prior to detection. This can be accomplished by an initial sample cleanup offline, and/or by addition of a large amount of another reactive compound to consume all of the excess reagent to form a single known derivative easily separated from the analyte's derivative. Sometimes the LC conditions themselves may resolve the excess reagent and any of its hydrolysis/byproducts from the desired derivative. Other approaches utilize a derivatizing reagent that, together with its hydrolysis/by-products, does not appear in the final chromatogram because it has very different detector properties from those of the analyte's derivative.

Another possible problem in utilizing derivatiz-ation involves a low per cent conversion to the desired derivative. This can be improved by forcing the reaction conditions, working at elevated temperatures for longer periods of time, invoking a suitable catalyst and by increasing the concentrations of analyte and reagent. Sometimes isolating the analyte from the sample on a solid support, followed by reaction with the usual derivatization solution, can lead to a much faster and more efficient reaction and conversion. In general the higher the per cent conversion, the easier it is to detect trace levels of analyte in complex matrices, such as biofluids.

Another problematic area has to do with reactions from other components in the sample mixture, besides that of the desired analyte, leading to a complex mixture of derivatives difficult to resolve by LC and/or detection methods. This can be improved by selectively isolating the analyte of interest from the sample matrix prior to derivatization, followed by the desired reaction conditions and introduction of the derivative into the LC system. This can more easily be accomplished by combining affinity LC with another LC mode, such as reversed-phase, so that the affinity step isolates the analyte of interest. This is then followed by a derivatization on the affinity support with the analyte immobilized, or initial elution of the analyte from this support, solution reaction, and then introduction into the second LC system. A simple, solid-phase affinity extraction column can be used to isolate the desired analyte from the complex sample, and prepare it for the desired, homogeneous (solution) or heterogeneous (solid-phase) de-rivatization reaction.

Yet another possible pitfall has to do with the formation of several derivatives from the analyte, rather than the usual (desired) production of a single, homogeneously tagged derivative. It is usually desired to form a single, homogeneous derivative with good chromatographic and detector properties. However, if there are several possible reactive sites on the analyte, then it is always possible that more than one product will result. This can be avoided by using reaction conditions that force all sites to be tagged, leading to a single product, or by preventing some of the sites from reacting by using suitable reaction conditions or protecting groups that will then leave only a single site left to react. In the case of protein or biopolymer derivatizations, multiple products are usually formed, leading to several LC peaks that then raise detection limits and make identification of the original protein and quantitation more difficult, especially at trace levels. In general, homogeneous (uniform) tagging of biopolymers is always problematic, though conditions are currently being developed that may eliminate such difficulties.

It is possible that the reaction conditions required for derivatization may cause the analyte itself to degrade, even as it reacts with the reagents. The degradation products can also react with the very same tagging reagent. This leads to a multiplicity of products, rather than a single homogeneous derivative, again making quantitation at trace levels and identification of the original analyte more difficult. However, this complex mixture of products can be forced to elute as a single, sharp peak by using suitable LC conditions. This can then function as a suitable peak for quantitation and identification of the analyte of interest.

Finally, there are the issues of reagent stability, purity, uniformity and shelf-life, all important areas when using a reagent over a long period of time for numerous analyses. Conditions must be found that provide a pure reagent with good shelf-life, long-term stability during the course of the reaction and storage, available from several commercial vendors at reasonable cost and amounts, and available in high purity and consistency. In most cases, such commercial reagents are indeed available for many LC applications today.

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