Evaluation After Derivatization

It would have been possible to combine Chapter 5 Evaluation without Derivatization with the present Chapter 7 to form a single Chapter Evaluation , as the earlier Chapter is mainly of general application. However, evaluation before derivatization in the course of the TLC process needed to be clearly emphasized. As the use of a TLC scarmer was described in detail in Section 5.2, it would be a good idea to reread this Section at this point in the book. 

Evaluation after derivatization does not only involve assessment of the chromatogram in daylight or measurement in light of wavelength 400 nm there are also a large number of reactions whose products are detected in 254-mn or 365-nm UV light. 

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Two steps have been added to the original Pesticide Analytical Manual method to increase the stability of the trimethylsilyl derivatives and to clean up the final extract prior to GC analysis, namely the use of a sodium sulfate mini-column to dry the extract after derivatization and the use of a Florisil Sep-Pak cartridge to remove matrix interferences. The advantages of the current method are the simultaneous evaluation of the four analytes, reproducibility and low matrix interference. 

To evaluate the enantiomeric purity of (S,S)/(R,R)-statine, racemic mixtures and en-antiomerically enriched samples are analyzed by HPLC after derivatization with 2,3,4,6-tetra-0-acetyl- 3-D-glucopyranosyl isothiocyanate (GITC, 51 Scheme 21 and Table 7).[49 The GITC method also allows the determination of the enantiomeric composition of ethyl esters of statine analogues.[44 491 Another assay to determine the optical purity of statine has been developed, and uses l-G1u-NCA to produce dipeptides with different retention times in HPLC.[18]... 

Sun et al. [59] used NCE-LIF to evaluate a derivatization method mediated by liposome for single cell analysis. According to these workers single cell analysis revealed that liposome-membrane fusion occurred after entrance of liposomes into the cells, with release of encapsulated fluorescence dyes and labeling of intracellular species. 

LC-MS plays an important role in the analysis of pesticides and related componnds, e.g., herbicides, insecticides, acaricides, as well as their degradation prodncts and metabolites. The analysis of pesticides is relevant for environmental studies, food safety, toxicology, and occupational health. Pesticides have to be analysed in environmental samples, such as different water compartments, soil, sediments, sludge, and animal tissue like fish, in food, especially fruit and vegetables, and in (human) body fluids and tissues. Many modem pesticides and related compounds are not amenable to GC-MS analysis, or only after derivatization. Therefore, LC-MS has been evaluated as an alternative. Various review papers on the analysis of pesticides and related compounds in various sample matrices were published [1-8]. 

A visual evaluation is only adequate for identity tests. However, in accordance with GMP guidelines, in the absence of any further documentation a second person should also inspect the TLC plate immediately after derivatization and the result should be entered in a database. The same applies to results to be submitted as evidence in court. 

The purity of protected amino acids is especially important for the synthesis of longer peptides. Standard techniques such as melting point determination, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and optical rotation are effective means of characterization. The optical purity can also be evaluated by high-performance liquid chromatography (HPLC) after derivatization with Marfey s reagent [216,217]. The advanced Marfey method refers to analysis by mass spectrometry after derivatization with Marfey s reagent [218-221]. Purification of side-chain protected amino acids by recrystallization is usually sufficient. 

Similar to Si, P as orthophosphate may be detected after derivatization with Sb(III) and Mo(VI) of the ternary heteropoly acid which is adsorbed onto the DME (detection limit being 5x10 mol 1 ). Phosphorus determination, like nitrite and nitrate determinations, is of importance for the evaluation of natural, waste, and potable water quality. 

More recently, liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) have been evaluated as possible alternative methods for carfentrazone-ethyl compounds in crop matrices. The LC/MS methods allow the chemical derivatization step for the acid metabolites to be avoided, reducing the analysis time. These new methods provide excellent sensitivity and method recovery for carfentrazone-ethyl. However, the final sample extracts, after being cleaned up extensively using three SPE cartridges, still exhibited ionization suppression due to the matrix background for the acid metabolites. Acceptable method recoveries (70-120%) of carfentrazone-ethyl metabolites have not yet been obtained. 

After the labeling experiment, the biomass is harvested, chemically fractionated, derivatized, and, finally, in most cases analyzed by GC-MS (see below and Section IV.C). Electron impact ionization is used, which leads to fragmentation of the metabolite, so that not only the molecular ion is measured but also several fragments, a circumstance that makes the measured information more valuable for subsequent data evaluation. 

Graham and Garrison (17) evaluated on-line trace enrichment for the determination of trace organic compounds in aqueous environmental samples. These workers were primarily interested in nonvolatile and thermally labile compounds that were not readily analyzed by GC methodology. A 2-mm i.d. X 70-mm long stainless steel precolumn was packed with 30-75 pm diameter octadecyl-derivatized silica. This precolumn was substituted for the sample loop in a conventional, high-pressure, six-port valve. Water samples, 10-100 mL, were pumped directly on the precolumn. After loading, the valve was switched to... 

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