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

Short Communication

Received: 15 January 2008,

Revised: 22 April 2008,

Accepted: 23 April 2008

Published online 10 October 2008 in Wiley Interscience

(http://www.emqy0h.com) DOI 10.1002/bmc.1084

Identification of key metabolites of

tectorigenin in rat urine by HPLC-MS

n

Wei-Dong Zhang,a Xiao-Lin Yang,a Lei-Xu,a Jun Cao,a Ping Lia and Zhong-Lin Yanga*

ABSTRACT:This is a report about the identification of key metabolites of tectorigenin in rat urine using high-performanceliquid chromatography?electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MSn). Six healthy ratswere administered a single dose (80mg/kg) of tectorigenin by oral gavage. Urine was sampled for 0?24h and centrifuged at12,000 rpm for 10min to obtain the supernatants, then the supernatants were purified by solid-phase extraction with a Ccartridge. The chromatographic separation was carried out on a reversed-phase C1818 column with a gradient elution programwhereas acetonitrile?0.1% formic acid water was used as mobile phase. Mass spectra were acquired in negative ionizationmode 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-MSn; metabolite; rat urine

Introduction

and NMR spectral data with the literature (Lee et al., 1989).Methanol and acetonitrile were of HPLC grade (Tedia, USA).As a traditional Chinese medicine, Rhizoma Belamcandae in AsianDeionized water was produced in our own department.countries is frequently used to treat disorders such as inflamma-tion, asthma and throat troubles (Shin et al., 1999). The bioactivecomponents of this herbal drug are mainly isoflavones, of whichApparatus and Chromatographic Conditions

tectorigenin is one of the main active components (Kim et al.,HPLC/DAD analysis was carried out on an Agilent 1100 series1999). Modern pharmacological studies have demonstrated aHPLC system (Agilent Technologies MA, USA) equipped with avariety of activities of tectorigenin, such as anti-inflammatory,quaternary pump, thermostatted column compartment andtreatment of prostate cancer (Thelen et al., 2006), aldose reduct-an online degasser. Chromatographic separation was carriedase inhibitory (Moon et al., 2006) and hypoglycemic (Jung et al.,out on a Kromasil C18 analytical column (150×4.6mm i.d.,2002).

5μm) at 25°C and the detection wavelength for UV detectorCompared with the comprehensive investigations for thera-was set at 264 nm. The mobile phase consisted of two sol-peutic purposes, the study of metabolism has been limited. Tovents: 0.1% aqueous formic acid (solvent A) and acetonitrileour knowledge, there has been no report on the metabolites of(solvent B). The proportion of acetonitrile was increased fromtectorigenin in rat urine. High-performance liquid chromatography?10 to 30% in the 60min. The mobile phase flow rate waselectrospray ionization ion trap tandem mass spectrometric method0.8mL/min and 20μL of sample solution was injected in each(HPLC-ESI-MSn) technique has been widely used to analyze drugrun.

metabolites in biological materials (Chan et al., 2006) with greatHPLC-MSn analysis was performed with an LC/MSD Trap XCTadvantages in being less time-consuming and less labor-intensivemass spectrometer (Agilent Technologies) equipped with ESI source.and having high sensitivity and specificity. The identificationThe mass spectrometer analysis worked in negative mode usingand structural elucidation of drug metabolites using HPLC-full-scan mode and the mass range was set at m/z 50?1000. TheESI-MSn method are based on the similarity of structural featuresMS conditions were as follow: drying gas (N2) flow rate, 9.0L/min;between the parent drug and its metabolites. Using the productdrying gas temperature, 325°C; nebulizer, 35 psig; capillary volt-ion mass spectrum of the parent drug as a substructural tem-age, 3000 V. All the operations, acquisition and analysis of dataplate, the structure of metabolites may be rapidly character-were controlled by Agilent LC/MSD Trap XCT Software (Agilentized even without standards of metabolites (Appolpnova et al.,Technologies).

2004).

Experimental

Chemicals and Reagents

*Correspondence to: Zhong-Lin Yang, Key Laboratory of Modern ChineseMedicines (China Pharmaceutical University), Ministry of Education; Nan-Tectorigenin with purity >98% was separated and purified fromjing, 210009, People’s Republic of China. E-mail: [email protected]://www.emqy0h.com

Rhizoma Belamcandae using silica gel chromatography, and itsa

structure was confirmed by direct comparison of their UV, IR, MS

Key Laboratory of Modern Chinese Medicines (China Pharmaceutical Univer-sity), Ministry of Education; Nanjing, 210009, People’s Republic of China

Biomed. Chromatogr. 2009; 23: 219?221Copyright © 2008 John Wiley & Sons, Ltd.

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