Add time:09/04/2019 Source:sciencedirect.com
SummaryAlkyl chloroformates (RCF) in aqueous alcohol-pyridine (PYR) media enable amino acids (AAs) to be converted into derivatives amenable to gas chromatography (GC) analysis in seconds. There is no requirement for a dry residue, multiple reaction steps and sample heating. Moreover, the fast conversion of hydrophilic compounds to the organophilic ones has proved to be of general use for most carboxylic acids with the potential to become an integral part of sample work-up. Compatibility of the derivatization with novel sample preparation methods such as solid-phase extraction (SPE) and solid-phase microextraction (SPME), the design of a commercial kit for AA analysis and synthesis of novel fluorinated reagents – all this and more show that RCF-mediated derivatization has become a mature method in GC.Numerous papers by researchers from various countries and application fields have confirmed the significance and attraction of such a simple and rapid way of changing an analyte's structure prior to GC or even liquid chromatography-mass spectrometric (LC-MS) analysis. Reports published over more than a decade on the analysis of AAs in various materials following treatment with RCF are also summarized in this chapter.Several methodological lapses and false conclusions in the published papers are discussed throughout the text. In general, the “classic” ECF-ethanol procedure, refined for the whole group of protein AAs, was in most cases simply copied without any optimization of the reaction as were the extraction conditions for targeted applications. It is strange that mainly acidification of the medium, for promoting cyclization of Glu to pyroGlu, was almost always applied in studies where no GLU occurred. It was then clearly useless or even contra-productive.Further, employment of polar extractants such as chloroform was essential to obtain good yields of polar derivatives, i.e. Ser and Thr (free alcoholic groups) and Gin (free amide) following MCF or ECF treatment. Chloroform also promoted yields of some other analytes a bit. Otherwise, less polar hydrocarbon solvents with a small portion of a polar one were better to handle as an upper and higher-boiling phase. Extraction of, e.g., S- and Se-containing AAs would be feasible in such systems as shown for plasma tHcy and aromatic acids. The more hydrophobic the alkyl of the RCF agent, the less polar solvent was required. Despite this obvious fact, chloroform was used in the follow-up studies almost exclusively. In some papers a molar excess of ECF over PYR was described, but no reason for this was given. In our earlier reports it was shown that by reversing the molar ratio, formation of the mixed (carboxylic-carbonic) anhydrides was promoted, which was undesirable. The importance of clean GC injection port liners was highlighted in one report. In evaluating GC conditions for the derivatives it was found that the choice of the liner used for splitless injections was critical. We can affirm the importance of liner choice from our own studies, especially when MC-ME or EC-EE of the polar and prone to sorption analytes like Ser, Thr and Gin were to be analyzed. The same is true for quality of the GC capillary column.Regarding the stability of MC-ME and EC-EE, the published reports were somewhere contradictory. Some reported, rather surprisingly, a good stability of EC-EE of GLN (free amide) over 5 days, others reported that IBC-ME were more stable than MC or EC-ME as both the latter tended to decrease upon storage. In our own findings this was especially the case with HIS, the yield of which declined progressively over time if not refrigerated. However, more important for obtaining a good yield of it, and also of SER and GLN, was the quality of the GC column and injection port, as mentioned.Concerning SPE on exchangers, the possibility to elute directly with a basified reaction medium made the process an integral part of sample pretreatment, simplifying and accelerating it. At the same time, it pointed to robustness of the RCF-derivatization since the reaction proceeded smoothly in presence of the sorbent, sometimes even with improved yields of some analytes.Contrary to that, SPME did not seem to bring any special advantages in comparison to LLE. Unlike the latter smooth process, requiring seconds and giving high yields, with SPME additional time was required for sorption/desorption of the analytes with more variables. The technique might be beneficial in cases where minute concentrations of compounds of interest occur in large volumes of fluids to be examined.Introduction of fluorinated alcohols into the process, described first in a study by Wang et al., brought some obvious advantages. It enhanced volatility of the derivatives and lowered their retention in the column, which was in particular desirable in separating AA-enantiomers on the Chirasil-Val column. Furthermore, the strongly acidic fluorinated alcohols were eager to esterify carboxylic groups, leaving thus a minute chance for another alcohol, i.e. to that liberated from the reagent (ECF, IBCF), to compete by forming reaction by-products. Their absence is apparent on Figure 6, Figure 7. Eventually, the strongly electron-capturing fluorine atoms allowed substantial enhancement of LOD/LOQ in NICI GC-MS analysis.As fluorinated RCF (FCF) are commonly not available, they had to be synthesized in the lab. They were made and described for TFECF in AA-enantiomer analysis as TFEC-TFE esters. However, FCF with longer alkyls were synthesized especially by Italian researchers and applied for ultratrace determination (3-30 fmol injected) of highly hydrophilic compounds with multiple carboxylic, hydroxylic, or aminic groups in aqueous solution. Studies on the reactivity differences among four FCF are underway.Our current projects deal with synthesis of novel, mostly fluorinated agents, too. As a result, novel simplified procedures for treating AAs and other carboxylic acids in biological fluids, beverages and other fluids under optimized conditions are the subject of present tuning studies. Moreover, the reaction mechanism of the processes is being studied in more detail. The employment of FCF is giving interesting insights.
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