TY - JOUR
T1 - Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4′-hydroxydiclofenac in rat serum
AU - Kaphalia, Lata
AU - Kaphalia, Bhupendra S.
AU - Kumar, Santosh
AU - Kanz, Mary F.
AU - Treinen-Moslen, Mary
N1 - Funding Information:
Supported by National Institutes of Health grant DDK 56494, National Institute of Environmental Health Sciences Center grant ES06676; Texas Golf Coast Digestive Disease Center grant DK56338, and a Meadows Foundation Equipment Award. Mary Treinen-Moslen is the William C. Levin Professor of Environmental Toxicology. We are very grateful to anonymous reviewers for their constructive suggestions to rule out the possible confounding co-elution of other metabolites with the peak for 4′-hydroxydiclofenac. Dr James R. Halpert kindly provided the human cytochrome P450 3A4 expressed in Escherichia coli . We thank Dr John R. Peterson for analytical advice, Thomas Bednarek for figure preparation, and Laura L. Lemley for careful assistance with the animal experiments.
PY - 2006/1/18
Y1 - 2006/1/18
N2 - 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 μg/ml, respectively. Average extraction efficiencies of diclofenac, 4′-hydroxydiclofenac, and the internal standard were ≥76%. The method was applied to serum collected at 3 h after rats were treated with an experimentally useful dosage range of 3, 10 and 50 mg/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.
AB - 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 μg/ml, respectively. Average extraction efficiencies of diclofenac, 4′-hydroxydiclofenac, and the internal standard were ≥76%. The method was applied to serum collected at 3 h after rats were treated with an experimentally useful dosage range of 3, 10 and 50 mg/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.
KW - 4-Hydroxydiclofenac
KW - Cytochrome P450 3A4
KW - Diclofenac
KW - Metabolism
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U2 - 10.1016/j.jchromb.2005.10.045
DO - 10.1016/j.jchromb.2005.10.045
M3 - Article
C2 - 16301007
AN - SCOPUS:30444449007
SN - 1570-0232
VL - 830
SP - 231
EP - 237
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
IS - 2
ER -