Enantioselective separation of thalidomide on on immobilized alpha(l)-acidglycoprotein chiral stationary phase

An easy and rapid enantioselective separation for assay of racemic thalidom ide on an immobilized alpha (1)-acid glycoprotein chiral stationary phase ( GPA CSP) is described. The effects of tetrahydrofuran (THF) as organic modi fier, buffer concentration to control the ionic strength, and mobile phase pH were studied. These variations have consequences in terms of chromatogra phic retention (k), resolution (R-s), selectivity (alpha), and peak asymmet ry (USP tailing factor). The main condition affecting chromatographic reten tion was mobile phase pH. At pH 4.5, no separation of thalidomide enantiome rs wets achieved whereas at pH 7.0 chiral separation was optimum. Peak tail ing was directly related to changes in pH and to addition of THF as mobile phase modifier. Results also indicated that the resolution factor is THF co ncentration-dependent, and that the separation factor (alpha) is the best p arameter for evaluating enantioselectivity. The best mobile phase was pH 7. 0, 30 mM ammonium acetate containing 0.3% THF. Under these conditions valid ation including linearity recovery, and precision was performed. The suitab ility of this method has been successfully proved in a limited in-vivo stud y after intravenous administration of thalidomide to a New Zealand male rab bit.