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Development and validation of an ion-pair reversed-phase high-performance liquid chromatographic method for the separation of phosphonodipeptides.

Journal of chromatography. B, Biomedical sciences and applications (1997-04-11)
N Surendran, C J Stankovic, B H Stewart
ABSTRACT

The objective of this research was to develop a rapid, sensitive and reliable method for the separation of phosphonodipeptide prodrugs and parent compounds to facilitate the evaluation of cell permeation using in vitro cell culture models. Separation was accomplished isocratically within 10.0 min using a C18 (150x4.6 mm I.D., 3 microm) reversed-phase 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 standard curves were linear (r2>0.999) over the concentration range 1-100 microM. The method was reliable and reproducible, with the limit of quantitation being 1 microM (25 ng on column).

MATERIALS
Product Number
Brand
Product Description

Supelco
Tetrahexylammonium hydrogensulfate, suitable for ion pair chromatography, LiChropur, ≥99.0% (T)
Sigma-Aldrich
Tetrahexylammonium hydrogensulfate, ≥98.0% (T)
Sigma-Aldrich
Tetrahexylammonium bromide, 99%
Sigma-Aldrich
Tetrahexylammonium chloride, 96%
Supelco
Tetrahexylammonium bromide, suitable for ion pair chromatography, LiChropur, ≥99.0% (AT)
Sigma-Aldrich
Tetrahexylammonium hydroxide solution, ~40% in H2O (T)
Sigma-Aldrich
Tetrahexylammonium iodide, ≥99.0% (AT)
Sigma-Aldrich
Tetrahexylammonium hydroxide solution, ~10% in methanol (T)

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