DEVELOPMENT AND VALIDATION OF AN ION-PAIR REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF PHOSPHONODIPEPTIDES

The objective of this research was to develop a rapid, sensitive and r eliable method for the separation of phosphonodipeptide prodrugs and p arent compounds to facilitate the evaluation of cell permeation using in vitro cell culture models. Separation was accomplished isocraticall y within 10.0 min using a C-18 (150x4.6 mm I.D., 3 mu m) reversed-phas e column. The mobile phase consisted of 5 mM tetrahexyl ammonium (ion- pair reagent) in 0.02 M phosphate buffer pH 6.5-acetonitrile (48.5:51. 5, v/v). The flow-rate was 1.1 ml/min with detection at 221 nm. The st andard curves were linear (r(2)>0.999) over the concentration range 1- 100 mu M. The method was reliable and reproducible, with the limit of quantitation being 1 mu M (25 ng on column).