High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes

J Chromatogr. 1993 Jun 23;616(1):73-8. doi: 10.1016/0378-4347(93)80473-h.


A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both > 90%. The assay, which has a limit of detection of 15 pmol injected (or an incubation 4NC concentration of 0.25 microM), is accurate, reproducible and straightforward. With a chromatographic time of 12 min, 40-50 samples may be analyzed per day. Rates of 4NC formation were linear with time and protein concentration to 50 min and 0.5 mg/ml, respectively. Preliminary studies of 4NP hydroxylation showed that this reaction followed single enzyme Michaelis-Menten kinetics in human liver microsomes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catechols / analysis
  • Catechols / metabolism
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 CYP2E1
  • Cytochrome P-450 Enzyme System / metabolism*
  • Glycerol / pharmacology
  • Humans
  • Hydroxylation
  • In Vitro Techniques
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology*
  • Nitrophenols / analysis*
  • Nitrophenols / metabolism
  • Nitrophenols / pharmacokinetics
  • Oxidoreductases, N-Demethylating / metabolism*
  • Quality Control
  • Solvents
  • Spectrophotometry, Ultraviolet


  • Catechols
  • Nitrophenols
  • Solvents
  • Cytochrome P-450 Enzyme System
  • Cytochrome P-450 CYP2E1
  • Oxidoreductases, N-Demethylating
  • Glycerol
  • 4-nitrocatechol
  • 4-nitrophenol