Isocratic separation of phenytoin metabolites by high-performance liquid chromatography from human and animal microsomes and urine

Deodutta Roy, Wayne R. Snodgrass

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

A rapid and simple high-performance liquid chromatography analytical method is described for the quantitative determination of phenytoin and five of its metabolites—phenytoin dihydrodiol, catechol, methoxycatechol, para-hydroxyphenytoin, and meta-hydroxyphenytoin—in biological materials. Following ethyl acetate extraction and incubation with betaglucuronidase, samples were passed through a C18 Sep-Pak cartridge. The eluate was chromatographed on a reverse-phase 10 cm C18 Radial Nova-Pak (5 µm) column using a mobile phase of isopropanol: Water (20:80) at a flow rate of 1.4 ml/min and monitored at 235 nm. Total chromatographic analysis time was 23 min, with complete baseline resolution of all metabolites. Five different columns and 10 different mobile phase conditions were studied to determine the best system. The 10-cm C18 Radial Nova-Pak (5 µm) gave the best resolution, reproducibility, and durability. This method should prove applicable for clinical use as well as for research purposes.

Original languageEnglish (US)
Pages (from-to)57-62
Number of pages6
JournalTherapeutic Drug Monitoring
Volume11
Issue number1
DOIs
StatePublished - Jan 1989

Keywords

  • HPLC
  • Isocratic
  • Phenytoin metabolites

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)

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