Sensitive and specific high-performance liquid chromatographic assay for 4'-hydroxyflurbiprofen and flurbiprofen in human urine and plasma.
A high-performance liquid chromatographic assay has been developed for the simultaneous quantitation of flurbiprofen and its major metabolite, 4'-hydroxyflurbiprofen, in urine and plasma. No extraction procedure was necessary for analysis of these compounds, which reduced time involved in sample preparation. The analytes were separated on a Brownlee Spheri-5 C18 column with a mobile phase of acetonitrile-20 mM dibasic potassium phosphate pH 3 buffer (40:60, v/v). Fluorescence detection was utilized with an excitation wavelength of 260 nm and an emission wavelength of 320 nm, providing excellent sensitivity. The limit of quantitation for 4'-hydroxyflurbiprofen and flurbiprofen was 0.25 microg/ml in urine and 0.05 microg/ml and 0.25 microg/ml, respectively, in plasma. All components were eluted within 16 min. Intra-day, inter-day, freeze-thaw, and in process stability were tested for both compounds and the coefficient of variation was less than 14% in all cases. This method provides a sensitive and specific assay for the detection of flurbiprofen and 4'-hydoxyflurbiprofen in urine and plasma and is suitable for use in in vivo studies evaluating the regulation of CYP2C9 activity.
Hutzler
J M
JM
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown 26506, USA.
Frye
R F
RF
Tracy
T S
TS
D001189:Aryl Hydrocarbon Hydroxylases; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005480:Flurbiprofen / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000652:urine; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism
C065522:4'-hydroxyflurbiprofen; D005480:Flurbiprofen; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase