Subject derivatizing agents

A variety of methods are also available when the compound under investigation can be converted with a chiral reagent to diastereomeric products, which have readily detectable differences in physical properties. If a derivatizing agent is employed, it must be ensured that the reaction with the subject molecule is quantitative and that the derivatization reaction is carried out to completion. This will ensure that unintentional kinetic resolution does not occur before the analysis. The derivatizing agent itself must be enantiomerically pure, and epi-merization should not occur during the entire process of analysis. 

Derivatization with halogenated compounds offers the advantage that the derivatives are subject to extremely sensitive detection with an electron capture detector (BCD). The disadvantage is that excess derivatization agent remains in the sample solution after the reaction, necessitating its removal prior to gas chromatographic determination because of the potential for interference during BCD detection. 

Fatty acids can be derivatized with p-bromophenacyl (73, 85, 86], phenacyl [87-89], naphthacyl [90, 91] or p-nitrophenacyl [74] bromide. To prepare p-bromophenacyl esters [73], fatty acids (0.001—0.5 mM) were dissolved in methanol or water and neutralized to a phenolphthalein end-point by methanolic KOH. The solvent was removed by either a rotary evaporator or lyophilization. A 3—10-fold molar excess of alkylating agent, p-bromophenacyl bromide/18-crown-6 (20 1) in acetonitrile, was then added. The mixture was stirred continuously in a sealed Reacti-Vial at 80 °C for 15 min. After cooling, the solution containing the derivatives was directly subjected to RP-HPLC with UV detection at 260 nm. The mobile phase was usually acetonitrile/ water [87, 92, 93], methanol/water [73, 88, 94, 95] or acetonitrile/methanol/water (86, 89], The separation of phenacyl derivatives of palmitoleic (Ci, ) and arachi-donic ( 20 4) acids was not achieved with the acetonitrile/ water system [87, 89], and elution with methanol/water could not resolve linolenic (Cig.3) and myristic (C]4.q) acids [88, 89]. A ternary mobile phase [89] containing a mixture of acetonitrile, methanol and water seemed to be best for the separation of phenacyl derivatives of fatty acids. 

Proteins in foods such as milk have been the subject of much fluorimetric study involving measurement of the intrinsic fluorescence of tryptophan and tyrosine however, since proteins vary in their content of these amino acids, the fluorescence intensity also varies markedly from protein to protein and, for a single protein, with the experimental conditions. Also, the natural fluorescence of peptides is limited to peptide-containing tryptophans and tyrosines. However, the joint use of LC and derivatizing fluorogenic agents such as dansyl chloride, ninhydrin, fluorescamine, and o-phthaldialdehyde (OPA) has allowed the development of a number of methods for the determination of proteins, peptides, amino acids, and amines in food samples. 

Due to the need of extensive derivatization for GC-MS analysis (see Chapter 3), P-receptor blocking agents such as atenolol, bupranolol, metoprolol, and propranolol (Fig. 4.23,1-4) have been early subjects of LC-MS/MS analysis. The presence of a secondary amino function in all P-blockers provides sufficient proton affinity for positive ESI, and core structure-specific as well as individual product ions are derived from target analytes using low energy CID as summarized in Table 4.7. 

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