Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum.
A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.
Kaphalia
Lata
L
Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX 77555-0632, USA.
Kaphalia
Bhupendra S
BS
Kumar
Santosh
S
Kanz
Mary F
MF
Treinen-Moslen
Mary
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000097:blood* / Q000302:isolation & purification; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D004008:Diclofenac; C108036:4'-hydroxydiclofenac