Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 260–264 (2008)DOI: 10.1002/bmc

260 T. Zhou et al.ORIGINAL RESEARCH ORIGINAL RESEARCH

Copyright © 2007 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 22: 260–264 (2008)Published online 15 October 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.921

Determination of iguratimod in rat plasma by highperformance liquid chromatography: methodand application

Ting Zhou,1,2 Li Ding,1* Xiaomin Li,2 Fei Zhang,2 Qian Zhang,3 Bing Gong1 and Xiaofeng Guo1

1Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China2Jiangsu Simcere Pharmaceutical R & D Co., Ltd., Nanjing 210042, People’s Republic of China3Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, 1 Shennong Road, Nanjing 210009, People’s Repub-

lic of China

Received 12 June 2007; accepted 2 August 2007

ABSTRACT: A high-performance liquid chromatographic method with UV detection has been developed for the determinationof iguratimod (T-614) in rat plasma. Plasma was precipitated with acetonitrile after the addition of the internal standard (IS), N-[4-(2-formylaminoacetyl)-5-methoxy-2-phenoxyphenyl]-methanesulfonamide. The chromatographic separation was achieved on areversed-phase C18 column with the mobile phase acetonitrile–acetic acid aqueous solution, pH 4.5 (40:60, v/v), at a flow rate of1 mL/min, and the UV detection wavelength was set at 257 nm. The calibration curve was linear over the range 0.10–50.0 μg/mL,and the lower limit of quantification was 0.10 μg/mL. The intra- and inter-day relative standard deviations were all less than11.5%. The method has been successfully applied to study the pharmacokinetics of iguratimod in rats. A single 10 mg/kg dose ofiguratimod was given to the rats by intragastric administration. The mean maximum plasma concentration of iguratimod for thesix rats was 14.5 μg/mL, and the mean elimination half-life of iguratimod was 4.0 h. Copyright © 2007 John Wiley & Sons, Ltd.

KEYWORDS: iguratimod; pharmacokinetics; HPLC

INTRODUCTION

Iguratimod, also known as T-614 [N-(3-formylamino-4-oxo-6-phenoxy-4H-1-benzopyran-7-yl)-methanesulfonamide,Fig. 1(A)], is one of a series of 4H-1-benzopyran-4-oneswhich has potent anti-inflammatory, analgesic and anti-pyretic activities (Tanaka et al., 1992). It is being devel-oped by Toyama Chemical Company for the treatmentof rheumatoid arthritis and is awaiting regulatory ap-proval in Japan as a disease modifying anti-rheumaticdrug (DMARD). This drug significantly inhibits theproduction of inflammatory cytokines in culturedhuman synovial cells and human monocytic leukemiacell line (Kohno et al., 2001; Aikawa et al., 2002). Italso reduces immunoglobulin (Ig) production by actingdirectly on B lymphocytes in both mice and humans(Tanaka et al., 2003). A double-blind randomized clini-cal study showed that the efficacy of T-614 was superiorto the placebo and was not inferior to salazosul-fapyridine, an established DMARD (Hara et al., 2007).To control the quality of the bulk and tablets of T-614, an analytical method has been developed by Li et al.

(2005). There is no analytical method reported for thedetermination of T-614 in biological samples. This paperdescribes the development and validation of a reliableand specific HPLC method for the determination ofT-614 in rat plasma. The method is validated over therange 0.10–50.0 μg/mL, and has been successfully appliedto study the pharmacokinetics of T-614 in rats.

*Correspondence to: Li Ding, Department of Pharmaceutical Ana-lysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing210009, People’s Republic of China.E-mail: dinglidl@hotmail.com

Abbreviations used: DMARD, disease modifying anti-rheumaticdrug; Ig, immunoglobulin.

Figure 1. Chemical structures of T-614 (A) and the IS (B).

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 260–264 (2008)DOI: 10.1002/bmc

Determination of iguratimod in rat plasma 261ORIGINAL RESEARCH

EXPERIMENTAL

Chemicals and reagents

T-614 and N-[4-(2-formylaminoacetyl)-5-methoxy-2-phenoxyphenyl]-methanesulfonamide [the internal standard(IS), Fig. 1(B)] were provided by Jiangsu Simcere Pharma-ceutical R&D Co. Ltd (Nanjing, China). Acetonitrile was ofHPLC grade (Tedia Company, USA). Acetic acid was of ana-lytical grade and was purchased from Sinopharm ChemicalReagent Co. Ltd (Shanghai, China).

Apparatus and chromatographic conditions

HPLC analysis was carried out on an Agilent TechnologiesSeries 1200 HPLC with a UV detector (Agilent Technologies,Palo Alto, CA, USA). Chromatographic separations wereperformed on a reversed-phase Alltima C18 column (5 μm,250 × 4.6 mm i.d., Alltech Associates Inc., USA), equippedwith a SecurityGuard C18 guard column (4 × 3 mm i.d.,Phenomenex Inc., USA) at 25°C. The mobile phase com-posed of acetonitrile–acetic acid aqueous solution of pH 4.5(40:60, v/v). The flow rate was 1.0 mL/min and the detectionwavelength was set at 257 nm. The chromatographic datawere recorded and processed with HP Chemstation software(version B.02.01) supplied by Agilent.

Preparation of stock and working solutions

The stock solutions of T-614 (300 μg/mL) and the IS (600 μg/mL) were prepared in acetonitrile. The working solutions ofT-614 with concentrations of 150, 30.0, 6.0, 1.50 and 0.30 μg/mL were prepared by serial dilutions of the 300 μg/mL stocksolution with acetonitrile. A solution containing 60.0 μg/mLIS was obtained by further dilution of the 600 μg/mL stock solu-tion with acetonitrile. All the solutions were stored at −20°C.

Preparation of calibration standards and qualitycontrol samples

Calibration standards of T-614 were prepared by spikingappropriate amounts of the working solutions in 30 μL blankrat plasma. Standard curves were prepared at concentrationsof 0.10, 0.30, 0.50, 2.0, 5.0, 10.0, 25.0 and 50.0 μg/mL. Thequality control samples were prepared in 30 μL blank plasmaat concentrations of 0.20, 4.0 and 45.0 μg/mL, and wereanalyzed with processed test samples at intervals.

Sample preparation

Each plasma sample (30 μL) was deproteinated with 200 μLacetonitrile after the addition of 10 μL IS solution (60.0 μg/mL). The sample was vortexed for 4 min and then the result-ing mixture was centrifuged at 10,000 rpm for 10 min. Sub-sequently, the supernatant was evaporated to dryness undera gentle stream of nitrogen in a water bath of 40°C. Theresidue was reconstituted in 100 μL of the mixture ofacetonitrile–acetic acid aqueous solution of pH 3.0 (40:60,v/v), and a 30 μL aliquot of the reconstituted solution wasinjected into the HPLC system for analysis.

Method validation

Specificity. The specificity of the assay was checked by com-paring the chromatograms of six batches of blank rat plasmasamples with the corresponding spiked plasma. Each blankplasma sample was tested to insure no interference with T-614 and the IS from the plasma.

Linearity of calibration curve and lower limit of quantifica-tion. Calibration standards of eight concentration levels at 0.10,0.30, 0.50, 2.0, 5.0, 10.0, 25.0 and 50.0 μg/mL were assayed. TheT-614 calibration curve was constructed by plotting the peak-area ratios of T-614 to the IS vs the concentrations of T-614,using weighed least squares linear regression (the weighingfactor was 1/C). The lower limit of quantification (LLOQ) isdefined as the lowest concentration on the calibration curveat which the precision, expressed as relative standard deviation(RSD), is less than 20% and the accuracy is within ±20%, andit is established using five independent samples (US Departmentof Health and Human Services, Center for Drug Evaluationand Research and Center for Veterinary Medicine, 2001).

Precision and accuracy. The accuracy and precision of theproposed method were determined by analysis of the QCsamples. The intra-day accuracy and precision were assessedfrom the results of five replicates of the QC samples (0.20, 4.0and 45.0 μg/mL) on a single assay day. The inter-day accuracyand precision were determined from the QC samplesanalyzed on three consecutive days. Precision is expressed asRSD (%), while accuracy is defined as the relative deviationin the calculated value (E) of a standard from that of its truevalue (T) expressed as a percentage. It was calculated usingthe formula: RE (%) = (E − T)/T × 100.

Recovery. The recovery of T-614 was evaluated by analyzingfive replicates at 0.20, 4.0 and 45.0 μg/mL of T-614. Recoveryof T-614 was assessed by comparing the peak areas of theprocessed sample containing the known amount of T-614 withthe peak areas obtained from the direct injection of the stand-ard solutions containing the same concentrations of T-614.

Stability. The stability of T-614 in plasma was studied undera variety of storage and handling conditions at the low(0.20 μg/mL) and high (45.0 μg/mL) concentration levels. Theshort-term stability was assessed by analyzing three aliquotsof each of the low- and high-concentration samples that werethawed at room temperature and kept at this temperature for6 h. Freeze–thaw stability (−20°C in plasma) was checkedthrough three cycles. Three aliquots at each of the low andhigh concentrations were stored at −20°C for 24 h and thawedat room temperature. When completely thawed, the sampleswere refrozen for 24 h under the same conditions. Thefreeze–thaw cycles were repeated three times, and thenanalyzed on the third cycle. The long-term stability was deter-mined by analyzing three aliquots of each of the low and highconcentrations stored at −20°C after 6 weeks.

Application

The study in animals was in accordance with the guidelinesfor the Care and Use of Laboratory Animals in Jiangsu

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 260–264 (2008)DOI: 10.1002/bmc

262 T. Zhou et al.ORIGINAL RESEARCH

province, China. Sprague–Dawley rats (half of which weremale, n = 6, 200–250 g) were purchased from the experimen-tal animal center of Nanjing Medical University. The ratswere fasted overnight before administration of the drug withfree access to water. A single 10 mg/kg dose of T-614 wasgiven to the rats by intragastric administration. Blood(0.1 mL) were sampled from oculi chorioidea vein of the ratspredose and at 0.17, 0.5, 1, 2, 3, 4, 6, 8, 12, 18 and 24 hpostdose. Plasma was separated by centrifugation and storedat −20°C until analysis. Aliquots of 30 μL plasma sampleswere processed and analyzed for T-614 concentration. Thepharmacokinetic parameters of T-614 were determined usingthe plasma concentration-time data. The maximum plasmaconcentration (Cmax) and the time to reach it (tmax) were noteddirectly. The elimination rate constant (ke) was calculated bylinear regression of the terminal points of the semi-log plot ofplasma concentration against time. The elimination half-life(t1/2) was calculated from the formula t1/2 = 0.693/ke.

RESULTS AND DISCUSSION

Conditions of chromatography

In preparing the mobile phase, the pH value of theacetic acid aqueous solution was investigated. The testresults showed that the interferences from the endo-genous substances would be encountered at the reten-tion time of T-614 and the IS while the pH < 4.0 or thepH > 5.5, and a satisfactory separation and the sharpchromatographic peaks of the analytes were achievedat the pH value of 4.5. Finally a mixture of acetonitrile–acetic acid aqueous solution of pH 4.5 (40:60, v/v) waschosen as the mobile phase. The maximum UV absorp-tion wavelengths of T-614 in the mobile phase were 256and 264 nm. In this study, the UV detection wavelengthof the HPLC was set at 257 nm.

Sample preparation

Sample preparation is an important step for accurateand reliable HPLC assays. Currently, the most widelyemployed biological sample preparation techniques areliquid–liquid extraction (LLE), protein precipitation(PPT) and solid-phase extraction (SPE). SPE is limitedby the cost of the apparatus. In this study, both LLEand PPT were tested for sample preparation. However,LLE using different solvents including ethyl acetate,

ethyl ether and cyclohexane directly gave the lowrecoveries. Further test results of LLE showed thatadjustment of the plasma pH to a weakly acetic valuewith hydrochloric acid could improve the extractionefficiency of T-614. However, the addition of acidresulted in serious interferences with T-614 and the IS.PPT with acetonitrile showed high recoveries and nosignificant interference caused by endogenous com-pounds of the analytes. Therefore, the protein precipita-tion with acetonitrile was finally adopted in the samplepreparation procedures. After precipitation, the plasmasample was centrifuged, and the supernatant was evapor-ated to dryness. The residue was reconstituted andanalyzed by HPLC. While selecting the reconstitutionsolution of the residues, an interesting finding was that,if the residues were reconstituted in the mobile phase,the retention time of T-614 was not stable and variedfrom 10.6 to 12.3 min. If the residues were reconstitutedin the mixture of acetonitrile–acetic acid aqueous solu-tion of pH 3.0 (40:60, v/v), the retention time of T-614and the IS could be stable at 12.9 and 8.5 min, respec-tively. Therefore, the residues of the plasma sampleswere reconstituted in a mixture of acetonitrile–aceticacid aqueous solution of pH 3.0 (40:60, v/v).

Method validation

Representative chromatograms of rat blank plasma andrat blank plasma spiked with T-614 (5.0 μg/mL) and theIS are shown in Fig. 2(A and C), respectively. Figure 2(D)shows the chromatogram of a plasma sample obtainedfrom a rat at 3 h after a single intragastric dose of 10 mg/kg T-614. There was no significant interference fromendogenous substances observed at the retention timesof the analytes. Typical retention times for T-614 andthe IS were 12.9 and 8.5 min, respectively.

The calibration curves, which related the concentra-tions of T-614 to the area ratios of T-614 to the IS,showed good linearity over the range 0.10–50.0 μg/mL.The typical calibration curve for T-614 is f = 0.0015 +0.2001C, where the f represents the peak area ratio ofT-614 to the IS and the C represents the plasmaconcentration of T-614. The correlation coefficientsof the calibration curves were more than 0.99. TheLLOQ for T-614 in rat plasma is 0.10 μg/mL [Fig. 2(B),Table 1]. These LLOQ data show that the assay is

Table 1. Accuracy and precision for the analysis of LLOQ (n ===== 5)

Added to plasma (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) RSD (%) RE (%)

0.10 0.0967 −3.30.10 0.0999 −0.10.10 0.0861 0.0963 8.2 −13.90.10 0.0916 −8.40.10 0.1069 6.9

Note: RSD, relative standard deviation; RE, relative error; n, number of replicates.

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 260–264 (2008)DOI: 10.1002/bmc

Determination of iguratimod in rat plasma 263ORIGINAL RESEARCH

Figure 2. Typical chromatograms of blank plasma (A), LLOQ for T-614 inplasma (0.10 μg/mL) and the IS (B), plasma spiked with T-614 (5.0 μg/mL)and the IS (C), and plasma obtained from a rat at 3 h after a single 10 mg/kgintragastric dose of T-614 (D). This figure is available in colour online atwww.interscience.wiley.com/journal/bmc

sensitive enough for the determination of T-614 in therat plasma.

The intra- and inter-run precision and accuracies aresummarized in Table 2. The standard deviation was cal-culated using one-way-ANOVA. The results in Table 2demonstrate that the method is precise and accurate.The recovery values of the method determined at threeconcentration levels of 0.20, 4.0, and 45.0 μg/mL were90.4 ± 4.6, 88.7 ± 2.7 and 87.1 ± 2.1% (n = 5), respectively.

The stability test data are shown in Table 3. Theresults showed that no significant changes for the T-614

plasma samples were observed after being kept at roomtemperature for 6 h and during the three freeze–thawcycles for the T-614 plasma samples. T-614 in plasma at−20°C was stable for at least 6 weeks.

Application

The present HPLC method was successfully applied todetermining the plasma concentration of T-614 inSprague–Dawley rats. After a single intragastric admini-stration of 10 mg/kg T-614 to rats, the concentration–time

Table 2. Accuracy and precision for the analysis of T-614 in rat plasma (three runs, five replicates per run)

Added to plasma Mean measured Intra-assay Inter-assay(μg/mL) concentration (μg/mL) RE (%) RSD (%) RSD (%)

0.20 0.2012 0.6 11.5 7.14.0 4.128 3.2 4.6 1.245.0 45.39 0.9 6.0 1.7

Table 3. Stability data of T-614 in rat plasma under various storage conditions (n ===== 3)

Added concentration Measured concentration Inter-runStorage conditions (μg/mL) (μg/mL) RSD (%) RE (%)

Room temperature for 6 h 0.20 0.2046 2.5 2.345.0 46.44 0.3 3.2

Three freeze–thaw cycles 0.20 0.2070 1.1 3.545.0 46.23 0.4 2.7

6 weeks at −20°C 0.20 0.2048 3.4 2.445.0 45.43 0.9 1.0

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 260–264 (2008)DOI: 10.1002/bmc

264 T. Zhou et al.ORIGINAL RESEARCH

profile was constructed for up to 24 h. Figure 3 shows themean concentration–time profile of T-614 in the rat plasma.The mean maximum plasma concentration of T-614 forthe six rats was 14.5 μg/mL, and the mean Tmax was2.8 h. The mean elimination half-life of T-614 was 4.0 h.

CONCLUSION

A simple and reliable HPLC method has been developedand validated for the determination of T-614 in rat plasma.No significant interferences caused by endogenous com-pounds was observed. The method is accurate, precise andsuitable for the pharmacokinetics study of T-614 in rats.

Figure 3. Mean concentration–time profile of T-614 in plasmaafter a single 10 mg/kg intragastric dose of T-614 to the rats.

REFERENCES

Aikawa Y, Yamamoto M, Yamamoto T, Morimoto K and Tanaka K.An anti-rheumatic agent T-614 inhibits NF-κB activation in LPS-and TNF-α-stimulated THP-1 cells without interfering with IκBαdegradation. Inflammation Research 2002; 51: 188–194.

Hara M, Abe T, Sugawara S, Mizushima Y, Hoshi K, Irimajiri S,Hashimoto H, Yoshino S, Matsui N, Nobunaga M and Nakano S.Efficacy and safety of iguratimod compared with placebo andsalazosulfapyridine in active rheumatoid arthritis: a controlled,multicenter, double-blind, parallel-group study. Modern Rheumato-logy 2007; 17: 1–9.

Kohno M, Aikawa Y, Tsubouchi Y, Hashiramoto A, Yamada R,Kawahito Y, Inoue K, Kusaka Y, Kondo M and Sano H. Inhibi-tory effect of T-614 on tumor necrosis factor-alpha inducedcytokine production and nuclear factor-kappaB activation in cul-tured human synovial cells. The Journal of Rheumatology 2001; 28:2591–2596.

Li HY, Ma C and Wu JM. Determination of iguratimod by HPLC.Chinese Pharmaceutical Journal 2005; 40: 222–224.

Tanaka K, Shimotori T, Makino S, Aikawa Y, Inaba T, Yoshida Cand Takano S. Pharmacological studies of the new anti-inflammatoryagent 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran- 4-one. 1st communication: antiinflammatory, analgesicand other related properties. Arzneimittel-Forschung 1992; 42: 935–944.

Tanaka K, Yamamoto T, Aikawa Y, Kizawa K, Muramoto1 K,Matsuno H and Muraguchi A. Inhibitory effects of an anti-rheumatic agent T-614 on immunoglobulin production by culturedB cells and rheumatoid synovial tissues engrafted into SCID mice.Rheumatology 2003; 42: 1365–1371

US Department of Health and Human Services, Food and DrugAdministration, Center for Drug Evaluation and Research andCenter for Veterinary Medicine. Guidance for Industry, Bioanaly-tical Method Validation. http://www.fda.gov./cder/guidance/4252fnl.htm, May 2001.