Liquid chromatographic determination of cyclophosphamide enantiomers in plasma by precolumn chiral derivatization

On the basis of reactions described in the synthetic literature, a two-step chiral derivatization sequence was developed for the anticancer agent cyclophosphamide (CP). The sequence involves amidoalkylation of CP with anhydrous chloral containing 1% dimethylformamide followed by acylation of the resulting secondary alcohol with a chiral carboxylic acid chloride, (+)-6-methoxy-alpha-methyl-2-naphthaleneacetyl chloride, to form a diastereomeric pair. Derivatized (-)-CP and (+)-CP exhibited retention times of 17.2 and 20.7 min, respectively, when chromatographed on Hypersil ODS with acetonitrile/phosphate buffer (pH 6.8) as the mobile phase. Preparation of the individual diastereomers from enantiomerically pure CP enabled correlation of the chromatographically observed peaks with a particular enantiomer. Various aspects of the overall assay methodology have been systematically investigated (derivatization solvents, temperatures, reaction time, and work-up procedure) and optimized on the scale required for trace analysis in biological fluids. Calibration curves were established for each enantiomer in spiked human plasma over the therapeutically relevant concentration range of 0.99-49.94 micrograms/mL.