Preparation of a Major Metabolite of Iguratimod and Simultaneous Assay of Iguratimod and Its Metabolite by HPLC in Rat Plasma

Document Type : Research article


1 School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, No. 639 Longmian Avenue, Jiangning District, Jiangsu Province, PR China.

2 Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu Province, 215004, PR China.

3 Nanjing BRT-Biomed Co., Ltd. Jiangning District, Jiangsu Province, PR China.

4 School of Pharmacy, China Pharmaceutical University, No. 639 Longmian Avenue, Jiangning District, Jiangsu Province, PR China.

5 Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.


Iguratimod is a new synthetic disease-modifying antirheumatic drug intended to treat patients with rheumatoid arthritis. A new method using recombinant human CYP450s yeast cells containing c-DNA expressed P450s was applied to identify the metabolic pathways of iguratimod and to prepare its metabolite. The metabolite was isolated, and its structure was identified by quadrupole time-of-flight-mass spectrometry and nuclear magnetic resonance. Furthermore, a selective and sensitive high performance liquid chromatography (HPLC) method was developed for the simultaneous quantification of iguratimod and its major metabolite in rat plasma for the first time. The results indicated that iguratimod was mainly metabolized to a metabolite by CYP2C9 and CYP2C19 in in-vitro study. The structure of the metabolite was identified as M2 (N-[3-(acetamido)-4-oxo-6-phenoxy-4H-chromen-7-yl]methanesulfonamide). HPLC assay was achieved on a C18 column using methanol-water containing 0.1% trifluoroacetic acid (55:45 v/v) at a flow rate of 1 mL/min with UV detection at 257 nm. Standard calibration curves were obtained in the concentration range of 0.5–20 µg/mL for iguratimod and its metabolite M2. The lower limits of detection of iguratimod and M2 in rat plasma were 0.1 and 0.25 µg/mL, respectively. The intra- and inter-day precision (RSD%) were within 5% for the two analytes. The average recoveries of the analytes were greater than 90%. In conclusion, recombinant human CYP450s whole-yeast transformation system could be successfully used to identify and prepare the major metabolite of iguratimod. The HPLC method we developed could be successfully applied to evaluate pharmacokinetics of iguratimod and its metabolite M2 in rats.


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