Modes of Derivatization

The above brief review illustrates that chemical derivatization techniques have been used extensively for the confirmation of identity of organochlorine residues. In most instances, the lower limits of detectability of the derivatives are substantiaUy lower than the established tolerance values for the parent compounds. Taken in conjunction with the many other modes of derivatization—e.g., during or after gas chromatography (59)— the analyst has a vast array of modification procedures at hand to aid in residue identification. They can be employed for residues in soil, biological, fat, and nonfat extracts and can be successfuUy extended, especially the more specific tests, to the identification of crosscontaminants in pesticide formulations and also fertilizer mixtures. So far, these latter two cases have only been a fringe area of application (60,61). 

The stable nonvolatile intermediate phenylthiocar-bamoyl derivatives are formed in basic media and can be analyzed directly by reverse-phase high-performance liquid chromatography (RP-HPLC). Their cyclization into hydantoins requires acid catalysis. This mode of derivatization is a very important supplement to the Edman s method of N-terminated sequencing of polypeptides. Before GC analysis, any hydantoins can be converted into N-trifluoroacetyl or enol-O-trimethylsilyl derivatives, which increases the selectivity of their determination in complex matrices. 

The applications of mass spectrometry to carbohydrate chemistry have been re vie wed.In general, stereoisomeric acetates or methyl ethers of carbohydrates are not well differentiated by mass spectrometry, but when the mode of derivatization depends on the stereochemistry of the parent sugar and leads to new ring-systems, as in acetal formation, structural isomers are produced that are more apt to show significant mass-spectral differences. 

More importantly, some silylating reagents used for protection of functional groups with active hydrogen atoms can react with carbonyl fragments with the formation of trimethylsilyl (TMS) derivatives of enols. If such unpredictable mode of derivatization is undesirable, all carbonyl fragments should be protected before silylation, for example, by conversion into alkoxyimino groups (alkyl ethers of oximes, see below). 

Fluorescence detection offers the possibility of high sensitivity and, in the case of complex samples, improved selectivity. However, this mode of detection requires that the analyte exhibit native fluorescence or contain a group to which a fluorophore can be attached by chemical derivatization. Because only tryptophan and tyrosine exhibit significant native fluorescence, fluorescence detection of proteins usually requires derivatization. 

Gas chromatographic separation has not gained wide acceptance in spite of being quite sensitive and specific. This mode of separation is complicated by the need for derivatization of sulfonamide residues before gas chromatographic analysis. These drugs are subjected to derivatization via methylation with diazomethane (223, 224, 253, 254, 271), or double derivatization via methylation followed either by silylation with Ai-methyl-Ai-trimethylsilytrifluoroacetamide (261) or by acylation with A-methyl-bis(trifluoroacetamide) (256). This derivatization step is required not only to form the volatile derivatives of the sulfonamides but also to improve their chromatographic properties (thermal stability and decreased polarity). 

Major advances have been made in recent years in the development and optimization of chiral resolutions of derivatized CD-based CSPs [68]. It has been reported that CSPs based on CD derivatives were more enantioselective than CSPs obtained from native CDs [68]. An acetyl /CCD column exhibited enhanced separation for scopolamine in comparison to the native / -CD CSP in the reversed-phase mode [69]. The enantiomeric resolution of some drugs was compared on the native [l-CD and on CSPs based on (S)- and (i )-2-hydroxy-propyl /I-CD, and the best resolution was reported on the derivatized CSPs [44]. Five types of natural and chemically modified [>- or y-C D stationary phases were developed and used for the chiral resolution of dansyl amino acids. The best resolution of dansyl amino acids was provided by y-CD CSPs [70]. 

Faure et al. [22] described nanoelectrochromatography on poly (dimethyl)-siloxane microchips using organic monolithic stationary phases for analysis of derivatized catecholamines. Surface modification of the PDMS material was carried out by UV-mediated graft polymerization. The efficiency of the unit was ascertained by measuring theoretical plates, which were 200,000 per meter. Furthermore, the authors optimized the separation by using pinched and electrokinetic modes at different applied potential of l.OkV/cm (Fig. 7.6) and 30kV/cm (Fig. 7.7) for the pinched and electrokinetic... 

The mode of operation in SBSE resembles that in SPME but with a few distinctive differences. For instance, after carrying out an SBSE procedure, two Twisters (dual SBSE extraction) can be simultaneously desorbed in one thermal desorption tube in order to further enhance the sensitivity. In the so-called multishot mode, up to five Twisters were desorbed in one desorption tube with in situ derivatization for quantification of estrogens in river water.46... 

One of the major advantages of LC-MS/MS over GC-MS or GC-MS/MS is that steroid hormones may be analyzed directly by LC-MS/MS without derivatization procedures, which are time-consuming and tedious [22,50-53], However, a number of studies demonstrated that the chemically derivatized steroid hormones were significantly more sensitive to LC-MS/MS detection than the underivatized hormones, because the neutral molecules of estrogens and metabolites might not be effectively ionized under electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) modes [4, 21, 25, 54, 55], In order to enhance the steroid hormone molecules sensitivity for LC-MS/MS analysis at pg/mL level, chemical derivatization is an effective technique for analysis of steroid hormones and metabolites. A list of derivatization reagents and application examples for steroid hormone analyses by LC-MS/MS and GC-MS are presented in Table 3. 

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