Reversed-phase HPLC route

Two more recent routes to the detection and assay of ethanolamine and related compounds deserve mention here. Sundler and Akesson (1975) reported an elegant method for the analysis of ethanolamine and possible derivatives (e.g., O-phosphoethanolamine) using the dansyl(5-dimethylaminonaphtha-lene-l-sulfonyl) derivatives and their fluorescence characteristics, in the 0.05-5 xM range. McMasters and Choy (1992) outlined a procedure for the determination of ethanolamine, by reverse-phase HPLC, as the phenythio-carbamyl derivative. A quantitative evaluation could be obtained in the 0.1-10 nmol range. 

In the following, the synthesis of the most often employed stationary phase is discussed spherical silica with an n-octadecyl modification. The synthesis route has been chosen because all synthesis steps are well characterized and documented in standard operation procedure (SOP) protocols. The objective of this work was to develop a manufacturing process for a reversed phase C18-bonded silica column for HPLC according to standardized and validated procedures and to perform certification of the column, the tests and the mobile phases (du Fresne von Hohenesche et al., 2004). Figure 3.13 shows a scheme of the whole manufacturing process, and Table 3.7 summarizes the main steps. 

Examination of the synthetic route used in production allows for the prediction of potential residual synthetic impurities present in the drug substance. The API structure allows for the postulation of degradation pathways via hydrolytic, oxidative, catalytic, and other mechanisms. Both of these evaluations serve to facilitate the interpretation of (subsequent) identification tests. An examination of the physicochemical properties also allows for the rational establishment of method screening experiments by precluding certain conditions. For example, the use of normal-phase HPLC will be eliminated if the API is a salt or shows limited solubility in nonpolar organic solvents. Similarly, if the API (or suspected related substances) has no significant chromophore above 250 nm, the use of tetrahydrofuran (THE) and other solvents as mobile-phase components is severely limited. For compounds with an ionizable group, variation of pH will have considerable influence on elution behavior and can be exploited to optimize the selectivity of a reversed-phase separation. 

In view of the availability of published alternative procedures, as well as related information contained in companion chapters of this book, the synthetic method described here will be limited to the latest and most flexible high-yielding route to the phosphorothioates using conventional phosphoramidite chemistry (42), in conjunction with stepwise sulfurization by tetraethylthiuram disulfide (TETD) (43). For similar reasons, the recommended phosphorothioate purification methodology given here is limited to isolation of 5 -dimethoxytrityl (DMT) derivatives either by reversed-phase manual procedures or HPLC (44). Both routes for purification have proven to be effective... 

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