Validation of a HPLC method for the determination of p-nitrophenol hydroxylase activity in rat hepatic microsomes.
We report a HPLC-UV method for determination of p-nitrophenol (PNP) hydroxylation to 4-nitrocatechol (4NC) as a marker for CYP2E1 activity in rat hepatic microsomes. Proteins were precipitated by addition of 50 microL phosphoric acid (50%, v/v in water) to 500 microL microsomal suspensions. Following vortex mixing and centrifugation the supernatant (20 microL) was injected onto a Supelcosil C(18) column (150 mm x 4.6 mm, 5 microm), and mobile phase (22% acetonitrile, 0.1% trifluoroacetic acetic acid, 0.5% triethylamine) delivered at 1.0 mL/min produced resolved peaks for internal standard, 4NC, and PNP in < 11 min. Calibration curves were linear (r(2) = 0.999) from 0.1 to 40 microM with intra- and inter-day precision < 12% and accuracy >90%. The method's improved sensitivity (LOQ = 0.1 microM) and minimal sample processing allowed rapid monitoring of PNP hydroxylase activity in fetal, neonatal, juvenile, and adult rat livers.
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Sask., Canada S7N5C9.
Fawzy
F
Elbarbry
Kyle
K
Wilby
Jane
J
Alcorn
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D019392:Cytochrome P-450 CYP2E1