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.
Elbarbry
Fawzy
F
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Sask., Canada S7N5C9.
Wilby
Kyle
K
Alcorn
Jane
J
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