John Wiley & Sons, Ltd. Identification of key metabolites of

Short CommunicationReceived: 15 January 2008,Revised: 22 April 2008,Accepted: 23 April 2008Published online 10 October 2008 in Wiley Interscience() DOI 10.1002/bmc.1084Biomed. Chromatogr. 2009; 23: 219–221Copyright © 2008 John Wiley & Sons, Ltd.219Identification of key metabolites oftectorigenin in rat urine by HPLC-MSnWei-Dong Zhang,a Xiao-Lin Yang,a Lei-Xu,a Jun Cao,a Ping Li a and Zhong-Lin Yang a *ABSTRACT:This is a report about the identification of key metabolites of tectorigenin in rat urine using high-performance liquid chromatography–electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MS n ). Six healthy rats were administered a single dose (80mg/kg) of tectorigenin by oral gavage. Urine was sampled for 0–24h and centrifuged at 12,000 rpm for 10min to obtain the supernatants, then the supernatants were purified by solid-phase extraction with a C 18cartridge. The chromatographic separation was carried out on a reversed-phase C 18 column with a gradient elution program whereas acetonitrile–0.1% formic acid water was used as mobile phase. Mass spectra were acquired in negative ionization mode and a data-dependant scan was used for the identification of the key metabolites of tectorigenin in the urine samples.As a result, four phase II metabolites and the parent drug tectorigenin were found and identified in rat urine for the first time.Copyright © 2008 John Wiley & Sons, Ltd.Keywords:tectorigenin; HPLC-ESI-MS n ; metabolite; rat urineIntroductionAs a traditional Chinese medicine, Rhizoma Belamcandae in Asian countries is frequently used to treat disorders such as inflamma-tion, asthma and throat troubles (Shin et al ., 1999). The bioactive components of this herbal drug are mainly isoflavones, of which tectorigenin is one of the main active components (Kim et al .,1999). Modern pharmacological studies have demonstrated a variety of activities of tectorigenin, such as anti-inflammatory,treatment of prostate cancer (Thelen et al ., 2006), aldose reduct-ase inhibitory (Moon et al ., 2006) and hypoglycemic (Jung et al .,2002).Compared with the comprehensive investigations for thera-peutic purposes, the study of metabolism has been limited. To our knowledge, there has been no report on the metabolites of tectorigenin in rat urine. High-performance liquid chromatography–electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MS n ) technique has been widely used to analyze drug metabolites in biological materials (Chan et al ., 2006) with great advantages in being less time-consuming and less labor-intensive and having high sensitivity and specificity. The identification and structural elucidation of drug metabolites using HPLC-ESI-MS n method are based on the similarity of structural features between the parent drug and its metabolites. Using the product ion mass spectrum of the parent drug as a substructural tem-plate, the structure of metabolites may be rapidly character-ized even without standards of metabolites (Appolpnova et al .,2004).ExperimentalChemicals and ReagentsTectorigenin with purity >98% was separated and purified from Rhizoma Belamcandae using silica gel chromatography, and its structure was confirmed by direct comparison of their UV, IR, MSand NMR spectral data with the literature (Lee et al ., 1989).Methanol and acetonitrile were of HPLC grade (Tedia, USA).Deionized water was produced in our own department.Apparatus and Chromatographic ConditionsHPLC/DAD analysis was carried out on an Agilent 1100 series HPLC system (Agilent Technologies MA, USA) equipped with a quaternary pump, thermostatted column compartment and an online degasser. Chromatographic separation was carried out on a Kromasil C 18 analytical column (150×4.6mm i.d.,5μm) at 25°C and the detection wavelength for UV detector was set at 264 nm. The mobile phase consisted of two sol-vents: 0.1% aqueous formic acid (solvent A) and acetonitrile (solvent B). The proportion of acetonitrile was increased from 10 to 30% in the 60min. The mobile phase flow rate was 0.8mL/min and 20μL of sample solution was injected in each run.HPLC-MS n analysis was performed with an LC/MSD Trap XCT mass spectrometer (Agilent Technologies) equipped with ESI source.The mass spectrometer analysis worked in negative mode using full-scan mode and the mass range was set at m/z 50–1000. The MS conditions were as follow: drying gas (N 2) flow rate, 9.0L/min;drying gas temperature, 325°C; nebulizer, 35 psig; capillary volt-age, 3000 V. All the operations, acquisition and analysis of data were controlled by Agilent LC/MSD Trap XCT Software (Agilent Technologies).*Correspondence to: Zhong-Lin Yang, Key Laboratory of Modern Chinese Medicines (China Pharmaceutical University), Ministry of Education; Nan-jing, 210009, People’s Republic of China. E-mail: yzl1950@aKey Laboratory of Modern Chinese Medicines (China Pharmaceutical Univer-sity), Ministry of Education; Nanjing, 210009, People’s Republic of ChinaW.-D. Zhang et al./journal/bmc Copyright © 2008 John Wiley & Sons, Ltd.Biomed. Chromatogr. 2009; 23: 219–221220Sample PreparationSix male Sprague–Dawley rats (200 ± 20g, Academy of Military Medical Sciences, Beijing) were housed in metabolic cages for the collection of urine. The rats were fasted for 24h but with access to water, then administered a single dose of tectorigenin (80mg/kg) by oral gavage and urine samples were collected at a period of 24h. Rat urine samples were centrifuged at 12,000g for 10min. An aliquot of 1 mL of mixed 0–24h urine was loaded onto a C 18 solid-phase extraction cartridge which was precondi-tioned with 2 mL of methanol and 1 mL of water. Then, the SPE cartridge was washed with 2 mL of water and the analytes were eluted with 3 mL of methanol. The elution solutions were filtered through 0.22μm membrane filters, and an aliquot of 20μL was injected into HPLC for analysis.Results and DiscussionTo identify the metabolites, probable structures were first postu-lated in accordance with the metabolism rule of drugs. Then these probable metabolites were analyzed by LC-MS n for their reten-tion times, changes in observed mass (Δm ) and spectral patterns of product ions with those of parent drug. Comparing chromato-grams of urine sample oral administration of tectorigenin with the blank, five major compounds in all were screened and detected in the urine sample. Extracted ion chromatography of the urine sample is shown in Fig. 1; four phase metabolites (M1, M2, M3and M4) and the parent drug (M0) were identified and the data are listed in Table 1.The negative electrospray mass spectrum of tectorigenin (M0,T r =60.1) showed a [M −H]− ion at m/z 299. Loss of CH 3 (15 Da)and CH 3OH (32 Da) from this species leads to the formation of the ions at m/z 284 and 267 (MS 2). CO 2 (44 Da) loss from the m/z 284 yields the fragment ion at m/z 240 (MS 3). Successive loss of CO (28 Da) from the m/z 284 produced the ions at m/z 256 and228 (MS 3). The product ions and the corresponding neutral fragment loss are the characteristic structural information of tectorigenin,and are a sound basis to identify metabolites of tectorigenin.M1 (T r =30.5min) gave a deprotonated molecular ion [M −H]− at m/z 475. In the MS 2 spectrum of m/z 475, the dominant fragment ion at m/z 299 was originated by the loss of 176 Da (a glucu-ronide unit) from the deprotonated molecular ion. The ions at m/z 175 and 113 correspond to cleavage feature of glucuronide conjugates (Gu et al ., 1999). The MS 3 spectrum of M1 generates two abundant ions at m/z 284 and 240, which are similar to the characteristic fragment ions of tectorigenin. As a result, M1could be identified as a glucuronide conjugate of tectorigenin.The [M −H]− ion of m/z of M2 (T r =42.5min) fragmented to form a product ion of m/z 299 that corresponded to tectorigenin itself after the loss of 80 Da (a sulfate unit). The characteristic ions in the MS 2 and the MS 3 spectrum of M2 are similar to that of tectorigenin.M2 is then easily identified as the sulfate conjugate of tectorigenin.The [M −H]− ion of M3 (T r =28.1min) was at m/z 459. The CID spectrum of M3 yielded fragment ion at m/z 299 by successive loss of neutral fragment 80 and 80 Da corresponding to two sulfate units. The loss of 32 Da (CH 3OH) from m/z 459 led to the forma-tion of the ion at m/z 427. Furthermore, other fragment ions (e.g.284, 240) are the same as the characteristic fragment ions of tectorigenin. M3 is thus confirmed as the two sulfate conjugates of tectorigenin.The [M −H]− ion of M4 (T r =33.0min) was at m/z 505. The MS 2spectrum of m /z 505 gave an abundant daughter ion at m /z 329by the neutral loss of 176 Da (glucuronide unit). Loss of a CH 3from m/z 329 yielded m /z 314 which further produced the ion at m/z 270 by loss of 44 Da (CO 2). By contrast with tectorigenin, the fragment ions at m /z 329, 314 and 270 were all 30 Da (CH 2O)more than the characteristic fragment ions at m/z 299, 284 and 240, respectively. Therefore, M4 was tentatively assigned as the pre-methoxy metabolic product of tectorigenin conjugates con-taining one glucuronide unit.Figure 1.Extracted ion chromatography of urine sample after oral administration of tectorigenin (80mg/kg).Table 1.HPLC/MS n data of the tectorigenin and its proposed metabolites in rat urineCompound T r (min)MS [M −H]−MS 2 (m/z )MS 3 (m/z )M060.1299284, 267256, 240, 228M130.5475299, 284, 113, 175284, 240M242.5379299, 284, 256284, 240 M328.1459379, 284, 240, 427M433.0505329, 314, 113, 175314, 270Identification of key metabolites of tectorigeninBiomed. Chromatogr. 2009; 23: 219–221Copyright © 2008 John Wiley & Sons, /journal/bmc 221ConclusionFor the first time, the metabolites of tectorigenine in rat urine were analyzed by the presented method. The metabolites of tectorigenine in rat urine were identified through comparing their chromatographic retention times, changes in observed mass (Δm) and tandem MS spectra with those of the parent drug. As a result, four phase II metabolites and the parent drug tectorigenin were identified in rat urine. The proposed in vivo metabolic pathways of tectorigenin are shown in Fig. 2.AcknowledgementsWe greatly appreciate the financial support from Prof. Z.L. Yang.ReferencesAppolpnova SA, Shpak AV and Semenov VA. Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine. Journal of Chromatography B 2004; 800: 281–289.Chan W, Cui L, Xu GW and Cai ZW. Study of the phase I and phase IImetabolism of nephrotoxin aristolochic acid by liquidchromatography/ tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2006; 20: 1755–1760.Gu JK, Zhong DF and Chen XY. Analysis of O-glucuronide conjugates in urine by electrospray ion trap mass spectrometry. Journal of Analytical Chemistry 1999; 365: 553–558.Jung SH, Lee YS, Lee S, Lim SS, Kim YS and Shin KH. 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Planta Medica 1999; 65: 776–777.Thelen P, Seseke F, Ringert RH, Wuttke W and Seidlová-Wuttke D. Pharmacological potential of phytoestrogens in the treatment of prostate cancer. Urologe-Ausgabe A 2006; 45: 195–201. Figure 2.The proposed in vivo metabolic pathways of tectorigenin in rat urine.。