Hydrolysis prone analytes

It has been noted (Travenius 1982) that common adsorption methods of sampling BCME from air are prone to giving inaccurate results because of hydrolysis of the analyte by coadsorbed water. For this reason, most procedures for methods involving collection of BCME on solid adsorbents require that samples in collection tubes be processed within a few days and protected from humidity. 

NPC is ideally suited for the analysis of compounds prone to hydrolysis because it employs nonaqueous solvents for the modulation of retention. An example of the use of NPC in the analysis of a hydrolysable analyte was demonstrated by Chevalier et al. [28] for quality control of the production of benorylate, an ester of aspirin. A major issue in benorylate production is the potential formation of impurities suspected of causing allergic side effects therefore monitoring of this step is critical to quality control. The presence of acetylsalicylic anhydride prohibited the use of RPLC since it can be easily hydrolyzed in the water-containing mobile phase. However, an analytical method based on the use of normal-phase chromatography with alkylnitrile-bonded silica as the stationary phase provided an ideal solution to the analysis. Optimal selectivity was achieved with a ternary solvent system hexane-dichloromethane-methanol, containing 0.2 v/v% of acetic acid to prevent the ionization of acidic function and to deactivate the residual silanols. The method was validated and determined to be reproducible based on precision, selectivity, and repeatability. 

Another application that demonstrates the advantages of using NPC for the separation of analytes prone to hydrolysis is the reaction monitoring for the formation of 9,10-anthraquinone [29]. Anthraquinone is an important intermediate in the manufacturing of various dye products but also is used as a catalyst in the isomerization of vegetable oils. It is produced in large amount by Friedel-Crafts reaction of phthaUc anhydride with benzene in the presence of AICI3 catalyst. 

In addition, sometimes a normal-phase HPLC method at subambient temperature must be applied for analytes that are extremely prone to hydrolysis. In the synthesis of leukotriene D4 antagonist, accurate quantitation of mesylate intermediate is essential for process optimization. Owing to its inherent instability, analysis of mesylate intermediate must be carried out under normal-phase conditions with nonprotic solvents however, significant cycliza-tion of mesylation was stiU observed in such condition at room temperature. The authors concluded that the on-column reaction of the mesylate was silica-catalyzed cyclization. By conducting the normal-phase HPLC analysis at -30 C, it was demonstrated that on-column cyclization was adequately inhibited [30]. 

In normal-phase chromatography, polar stationary phases are employed and solutes become less retained as the polarity of the mobile-phase system increases. Retention in normal-phase chromatography is predominately based upon an adsorption mechanism. Planar surface interactions determine successful use of NPC in separation of isomers. The nonaqueous mobile-phase system used in NPC has found numerous applications for extremely hydrophobic molecules, analytes prone to hydrolysis, carbohydrates, and sat-urated/unsaturated compounds. In the future, with the advent of new stationary phases being developed, one should expect to see increasingly more interesting applications in the pharmaceutical industry. 

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