Derivatization, increasing sensitivity

Certainly, a vast amount of experience has been gained by the widespread use of conventional amino acid analysers. They offer high reliability, accuracy, reproducibility and can separate complex samples. Because conventional analysers can be fully automated, they are widely used in routine analysis. However, the method is limited by the sensitivity which can be achieved using ninhydrin as the derivatizing agent. Sensitivity can be increased by using ortho-phthaldialdehyde (OPA) instead, but where extremely high sensitivity is required, HPLC is the method of choice. 

Endogenous and exogenous androgens can be derivatized with trimethylsilyl (TMS) for hydroxy functions and by 0-methylation for ketones, and analyzed with GC-FID or GC-MS (Shimada et al., 2001). MS is more prevalent due to unequivocal identification and greatly increased sensitivity but FID is still used in laboratories for some steroids. Sterols have typically been analyzed by GC-FID and GC-MS with derivatization to optimize peak shape (Shimada et al., 2001), and bile acids can be derivatized with M-butyl ester-TMS ether and analyzed by GC-FID from plasma samples (Batta et al., 1998). Juricskay and Telegdy (2000) reported an assay capable of analyzing 28 steroids in urine samples using GC-FID. 

The analysis of codeine, morphine, 6-monoacetylmorphine (6-MAM, a metabohte of heroin), and cocaine is important for many toxicology labs to determine illicit drug use. When analyzing opiates in urine samples, frequently the matrix chosen for drug screening, the conjugated metabolites must be hydrolyzed however, this process can break down 6-MAM (Christophersen et al., 1987). These compounds can be derivatized to increase sensitivity, and both BCD and NPD are used for these assays. Derivatizations used include reaction with N-methyl-N-trimethylsilyltrifluoroacetamide followed by GC-FID (Lin et al., 1994) or with N,0-bis(trimethylsilyl)trifluoroacetamide (Christophersen et al., 1987 Lee and Lee, 1991), PFPA (Christophersen et al., 1987), or heptafluorobutyric anhydride (HFBA) followed by GC-ECD. All these methods show good sensitivity and selectivity. 

Fluorescence detection was selected to increase sensitivity and selectivity. Histamine has no natural fluorescence and a post-column derivatization with OPT was found to be facile. The OPT reaction with histamine or any primary amine will only occur in an alkaline medium. The derivatization reagent, pumped into the system after the mixture has been separated on the column, must be strongly basic to neutralize the acid in the mobile phase. The structure of the OPT adduct has been found to be dependent upon the pH at which the reaction is carried out as wel1 as the sol vent system (15). 

Research has been done showing that rapid pressnre-driven LC analysis can be done with little solvent consumption, demonstrating this as a viable process analytical tool. Using electrokinetic nanoflow pumps LC can be miniaturized to the point of being a sensor system. Developments in terms of sampling to enable sampling directly from a process stream, to the separation channel on a chip are critical for the application of miniaturized process LC. The components (valves and pumps) required for hydrodynamic flow systems appear to be a current limitation to the fnll miniatnrization of LC separations. Detection systems have also evolved with electrochemical detection and refractive index detection systems providing increased sensitivity in miniaturized systems when compared to standard UV-vis detection or fluorescence, which may require precolumn derivatization. 

AA and lAA can be easily detected nsing UV or ED. Compared to AA, DHAA has a weak UV absorption and no response on ED. Therefore, derivatization prior to or after the chromatographic separation is needed to increase sensitivity. Different strategies can be applied (a) DHAA may be rednced to AA, prior to HPLC by L-cysteine [547] or dithiothreitol [548], or using a postanalyti-cal colnmn for a solid-state redaction [549] (b) DHAA may be derivatized with o-phenyldiamine to form the flnorophore 3(l,2-dihydroxy-ethyl)furo[3,4- ]quinoxaline-l-one (DFQ) [550] (c) AA may be oxidized to DHAA by an on-line postcolnmn oxidation with Cn + or Hg + followed by derivatization with o-phenyldiamine [551,552]. 

The method was subjected to a comparison between two different laboratories. Table 10.1 shows the results obtained and indicated that there was no significant difference between the values obtained in the two laboratories. The use of this method as opposed to some of the others reported in the literature deserves comment. Generally, most investigators describe derivatization as a necessary step in obtaining usable chromatograms. However, derivative formation is not always a reliable method, but appears necessary in biological samples with low steroid levels to increase sensitivity as well as selectivity. 

Detection of the B6 vitamers is complicated by the low levels at which they occur in foods (102,103). The sensitivity and specificity of the detection methods is therefore critical. All of the principal B6 vitamers are UV absorbers (70). Although their spectra are similar in 0.1 M hydrochloric acid, this is not the case at higher pH. These vitamers fluoresce naturally in slightly acidic to neutral solution and under strongly alkaline conditions (42,70). However, the individual vitamers exhibit some qualitative dissimilarities in their fluorescence spectra and significant differences in the intensities of their quantum fluorescence response PLP is significantly less fluorescent than the other five vitamers. In general, fluorescence is the preferred method of detection, due to its increased sensitivity and specificity relative to UV absorbance. Derivatization has been used to enhance and standardize the fluorescence properties of the B6 vitamers. Detailed reviews of the spectral properties of the B6 vitamers have been published (102,103). 

Amino acids are derivatized two ways to increase sensitivity. Free amino acids in solution are reacted with o-phthaldehyde (OPA) to form a fluorescent derivative that excites at UV,230nm, and emits at FL, 418 nm. These OPA derivatives are separated on Ci8 in a complex mixture of An/MeOU/ DMSO/water at pH 2.65. PTH amino acids are formed from the N-terminai end of peptides during Edman degradation for structure analysis of peptides and proteins. HPLC is used to identify which amino acids are released. PTH amino acids are separated at UV, 254 nm, on a Ci8 column with a gradient from 10% THF/water containing 5 mM acetic acid to 10% THF/AN.The separation with reequilibration takes 60min. Work with short 3-pm columns has reduced this separation to a 10-min gradient. 

Halogenated acyl derivatives of steroids have been applied in order to increase sensitivity of the analysis. It follows from a comparison of the ECD responses of haloacetates of steroids that the highest sensitivity can be obtained with the aid of monochloro-acetates [351]. Brownie et al. [352] applied them in the analysis of testosterone in blood. The method involves the extraction of blood plasma with diethyl ether, purification by TLC and derivatization. GC analysis is performed only after a preliminary separation on a thin layer. Preparation of the derivatives is carried out by treating a dried extract with... 

Derivatization of analytes and modification of the mobile phase can both contribute to markedly increased sensitivities for particular analytes [3]. It is probably the latter (mobile phase modification) that has the greater impact upon the LC-MS of alkaloids, since the need to derivatize to enhance ion formation is reduced by the presence of the ionizable nitrogen. 

Although derivatization increases detection sensitivity, it is usually undesirable, as it directly changes species. The selectivity of such reactions is much lower than expected by theory. These derivatization reactions... 

Since part of the ethos of SFC is to extend high resolution analysis to high molecular weight, reactive and thermolabile compounds not accessible to GC without derivatization, and to use universal detection, applications of derivatization in SFC have been sparse. They fall into two main groups derivatization to increase solubility in the supercritical mobile phase, and hence to extend further the molecular weight range of SFC and derivatization to provide selectivity and increase sensitivity in detection. 

For increased sensitivity, reagents that yield fluorescent products are employed. These include OPA, fluorescamine, and 4-fluoro-7-nitro-benzo-2-oxa-l, 3-diazole (NBD-F). Derivatization with OPA provides the best limits of detection for most amino acids. Although the products of OPA derivatization are unstable and decompose fairly rapidly to produce nonfluorescent 2,3-dihydro-lH-isoindol-l-ones, this is not a problem with postcolumn derivatization since the products are detected almost instantaneously. 

To achieve increased sensitivity and reduced analysis time it is necessary to produce derivatives of the amino acids which can be separated by reversed-phase HPLC and then detected using fluorescent, spectrophotometric, or electrochemical detection. Both precolumn and postcolumn derivatization methods have been described with a range of derivat-izing agents including ... 

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