Chemiluminescence quenching microarrays for high throughput screening of antioxidants and its application in evaluating herbal extracts and pure compounds.

In this paper, a novel high throughput screening method for antioxidants was described. The screening process was completed on a 5 cm × 5 cm silica gel 60 plate. Luminol was used as the fluorogenic substrate while hydrogen peroxide employed to excite the chemiluminescence. A dark spot was observed due to quenching effect of antioxidant. A linear model of the integral area of the spot as a function of analyte amount was fit by using vitamin C as positive control. Excellent linearity (R2 = 0.9978) was obtained in the range of 2.31-23.1 µg. The variation coefficients of intra- and inter-chip precisions were below 5% (n = 10) and 10% (n = 6) respectively. The new method was validated by comparing the analysis result of six flavonoids with that from a sequential injection method. The Pearson correlation coefficient was up to 0.98. A set of traditionally used herbal medicines was screened, and Rhei Radix et Rhizoma showed the strongest signal. The major bioactive ingredients were further assigned by flow injection and MS analyses. These results reveal the prospects of the proposed method to supply a promising tool in vitro for high throughput screening and activity evaluation of antioxidants in a fast, low-cost and reliable manner.

Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-κB signaling pathway with anti-inflammatory activity.

BACKGROUND: Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action. METHODS: In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope. RESULTS: A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 µg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation. CONCLUSION: In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.

Pharmacokinetics, Tissue Distribution, and Metabolism Study of Icariin in Rat.

Icariin is one of the predominant flavonoids contained in Herba Epimedii (Yin-yang-huo in Chinese), a well-known Chinese medicine for the treatment of cancers and immune system diseases. Although Herba Epimedii has been widely used in China and there are so many and various research reports on the herbal drug and its main flavones, very limited data is available on the tissue distribution and biotransformation of icariin. In the present study, a liquid chromatographic method combined with electrospray ionization tandem mass spectrometry was developed to quantify the concentration of icariin in rat plasma and various tissues collected at different time points after oral administration of the total flavonoid extract of Herba Epimedii at a dose of 0.69 g/kg (corresponding to 42 mg/g icariin). Biological samples were processed by simple protein precipitation. Genistein was chosen as internal standard. The method was successfully applied to plasma pharmacokinetic and tissue distribution studies of icariin in rat. As a result, it was worth noting that the tissue distribution characteristics of icariin exhibited a significant gender difference. Moreover, in vivo metabolism of icariin was also investigated. A total of 11 potential metabolites were found in rat feces collected in different time periods after oral and intramuscular administration of icariin. In vivo metabolic pathways were involved in hydrolysis, demethylation, oxidation, and conjugation. The preclinical data would be useful for fully understanding in vivo disposition of this compound and interpreting the mechanism of its biological response.

Determination of Epimedin B in Rat Plasma and Tissue by LC-MS/MS: Application in Pharmacokinetic and Tissue Distribution Studies.

A simple, sensitive, and specific liquid chromatography tandem mass-spectrometric method was developed and validated for the determination of epimedin B in rat plasma and tissue samples. After being processed with a protein precipitation method, these samples were separated on an Agilent Eclipse XDB-C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution (32 : 68, v/v). The calibration curve of epimedin B was linear over the concentration range from 1 to 500 ng/mL in plasma and tissue homogenate. The method was then applied to pharmacokinetic and tissue distribution studies after a single oral administration of Herba Epimedii extract to SD rats. Results showed that epimedin B reached the plasma peak concentration at 0.4 h and the terminal elimination half-life was 1.6 h in rat plasma, and the plasma area under the curve from time zero to infinity (AUC0-∞ ) was 14.35 µg/L·h. The concentration distribution of epimedin B in rat tissue was in the following order: liver > ovary > womb > lung > kidney > spleen > heart > brain, indicating that the compound could be widely distributed in rat, and the reproductive system may be the principal target of epimedin B for female rat.

[In vivo study of Chuankezhi metabolism in rat].

To study the metabolic products of main compounds of Chuankezhi injection in rat, 12 Sprague Dawley rats were classed into 2 groups, a blank control group and an intermuscular administration group, respectively. Rat feces and urine samples were collected from 0−24 h and 24−48 h after administration. All the samples were ultrasonically treated with methanol and then analyzed using LC-LTQ Orbitrap MSn. By comparison with the total ion chromatogram of samples from the blank control group, the metabolites in the samples of drug-treated group were screened. These metabolites were further analyzed by multistage product ion scanning and comparison of retention time with reference substances. As a result, a total of 12 flavonoid metabolites were tentatively identified from the rat feces and no metabolite was discovered in the rat urine. Epimedin C and icariin were detected in the rat blood samples after 30 min of administration, but their metabolites and other original flavones were not detected. Furthermore, no original flavones and their metabolites were detected in rat blood samples after 2 and 4 h of administration. The potential metabolism paths were further characterized and the principal in vivo transformation of flavones from Chuankezhi injection were deglycosylation, dehydration, methylation, oxidation and isomerization in rats.

Comparative Pharmacokinetics and Bioavailability of Epimedin C in Rat after Intramuscular Administration of Epimedin C, a Combination of Four Flavonoid Glycosides and Purified Herba Epimedii Extract.

Chuan-Ke-Zhi (CKZ), a purified Herba Epimedii extract, is a potent Chinese medicine preparation whose main bioactive components are a class of flavonoid glycosides such as epimedins A, B, and C and icariin. And epimedin C is far more abundant than other flavones in this extract. This study aims to investigate the pharmacokinetic and bioavailability of epimedin C and what effects, if any, other ingredients in CKZ have on its pharmacokinetics. Epimedin C, CKZ, and a combination of epimedins A, B, and C and icariin were, respectively, administrated to rats, and then the pharmacokinetic parameters of epimedin C in the three groups were calculated and compared. The result indicated that CLZ, MRT0-∞, and AUC0-∞ of epimedin C were significantly different among the three groups (P < 0.05), and compared with the epimedin C group, the absorption of epimedin C significantly increased in the CKZ group. Furthermore, in this study the absolute bioavailability of epimedin C was also investigated by comparing intramuscular and intravenous administration of epimedin C. As a result, epimedin C could be quickly absorbed with extremely high absolute bioavailability after intramuscular administration.

[Simultaneous determination of 4 prenylflavonoids of Chuankezhi injection in rat plasma by LC-MS/MS].

A quantitative method for epimedin A, B, C and icariin in rat plasma was established using LC-MS/MS after intermuscular administration of Chuankezhi injection to rat. Chromatographic separation was performed on an Agilent Eclipse XDB-C(18) column (150 mm × 2.1 mm, 5.0 µm) at 40 ℃. Mobile phase consisted of acetonitrile-0.1% formic acid in water（35∶65）, and the flow rate was 0.22 m L·min(-1). The LC effluent was detected and analyzed using an ESI-triple quadrupole tandem mass spectrometer under the multiple reaction monitoring (MRM) in the negative ion mode. The plasma samples were treated with solid phase extraction prior to LC-MS/MS analysis. As a result, all of the four analytes displayed a good linearity over the concentration of 1-1 000 ng·mL(-1). The RSDs of intra-day and inter-day assays were less than 5.99% and 10.16%, respectively. The relative recovery of each analyte was between 88.1%-101.1% with RSD < 7.9% and the absolute recovery was between 72.0%-86.6%（RSD < 6.3%）. In conclusion, the established method shows good specificity, sensitivity and efficiency for quantifying the four flavonoid glycosides contained in rat plasma.

[Pharmacokinetics of epimedin A, B, C and icariin of Chuankezhi injection in rat].

To study pharmacokinetic characteristics of epimedin A, B, C and icariin after intermuscular administration of Chuankezhi injection to rat. The established RRLC-MS/MS method was applied for simultaneous determination of four analytes in rat plasma and calculating their pharmacokinetic parameters. As a result, each analyte showed a good linear relationship in the concentration range of 1-1 000 µg•L⁻¹.The intra-day precise was 96.9%-107.5% with RSD<5.99%, inter-day precise was 92.3%-105.0% with RSD<10.16%. The relative recovery of four analytes was 88.1%-101.1% with RSD<7.9% and their absolute recovery was 72.0%-86.6% with RSD<6.3%. After intermuscular administration of Chuankezhi injection, the plasma concentration of four flavonoid glycosides rapidly arose to peaks at about 10 min, and then quickly declined in rat. Tmax of epimedin A, B, C and icariin was 0.21, 0.19, 0.16 and 0.49 h, respectively, and their mean elimination half-life(t1/2z) was 0.60, 0.62, 0.47 and 0.49 h. The established method was validated to be sensitive, rapid and specific for determination of the four analytes. Serum concentration of 4 species of epimedium flavonoids in Chuankezhi injection was low, and their absorption and elimination seem quickly, displaying similar pharmacokinetic characteristics in this study.

Quality analysis of commercial samples of Ziziphi spinosae semen (suanzaoren) by means of chromatographic fingerprinting assisted by principal component analysis.

Due to the scarcity of resources of Ziziphi spinosae semen (ZSS), many inferior goods and even adulterants are generally found in medicine markets. To strengthen the quality control, HPLC fingerprint common pattern established in this paper showed three main bioactive compounds in one chromatogram simultaneously. Principal component analysis based on DAD signals could discriminate adulterants and inferiorities. Principal component analysis indicated that all samples could be mainly regrouped into two main clusters according to the first principal component (PC1, redefined as Vicenin II) and the second principal component (PC2, redefined as zizyphusine). PC1 and PC2 could explain 91.42% of the variance. Content of zizyphusine fluctuated more greatly than that of spinosin, and this result was also confirmed by the HPTLC result. Samples with low content of jujubosides and two common adulterants could not be used equivalently with authenticated ones in clinic, while one reference standard extract could substitute the crude drug in pharmaceutical production. Giving special consideration to the well-known bioactive saponins but with low response by end absorption, a fast and cheap HPTLC method for quality control of ZSS was developed and the result obtained was commensurate well with that of HPLC analysis. Samples having similar fingerprints to HPTLC common pattern targeting at saponins could be regarded as authenticated ones. This work provided a faster and cheaper way for quality control of ZSS and laid foundation for establishing a more effective quality control method for ZSS.

Quality assessment of Fructus xanthii based on fingerprinting using high-performance liquid chromatography.

Because almost every traditional Chinese medicine (TCM) is a multicomponent system, QC of TCMs always involves various difficulties. As a current popular quality assessment approach, focusing on qualitative and quantitative analysis of certain compounds contained in herbal medicine has been widely used for the sake of expediency rather than being a practical and realistic way. However, this method does not take the existence of other constituents into account. Comparatively, the chromatographic fingerprint of the components is a more suitable approach to holistically assess the quality of herbal drugs. Fructus xanthii is a well-known herbal drug listed in all editions of the Chinese Pharmacopoeia. However, there is no quality evaluation method given in its monograph, even for the above-mentioned expediency. This paper reports an HPLC fingerprinting method for quality evaluation of F. xanthii. The HPLC profiles of 27 batches of commercial samples were further analyzed using chemometric methods, including similarity evaluation and principal component analysis. As a result, the established HPLC fingerprint contained 23 characteristic peaks; therein, 13 peaks were unambiguously assigned by comparing their retention times and UV spectra with those of reference compounds, and five peaks were tentatively identified on the basis of their MS/MS fragmentation patterns and UV spectra. Moreover, it could be clearly observed that caffeoylquinic acid and its analogs predominate in F. xanthii. Except for three samples identified as outliers, 24 other commercial samples displayed similar HPLC profiles, indicating that the quality of the herbs from different markets is stable and consistent.

[Simultaneous determination of 4 phenolic acids in cangerzi by ultra-performance liquid chromatography].

In this study, an analytical method was developed and used to quantify simultaneously protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and 1, 3-dicaffeoylquinic acid--four bioactive compounds contained in Fructus Xanthii using UPLC. The contents of four phenolic components of 28 batches of samples collected from different product areas and markets were determined and compared by means of this established method. The mobile phase was composed of methanol and water containing 0.1% phosphoric acid. Chromatography was monitored at dual-wavelengths--220 and 327 nm. Flow rate was 0.4 mL x min(-1) and column temperature was 35 degrees C. The correlation coefficient between concentration and chromatographic peak area of protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and 1, 3-dicaffeoylquinic acid was over 0.9999 in the range of 0.3570-35.70, 2.500-250.0, 1.060-106.1, 1.010-101.0 microg x mL(-1), respectively. The average recoveries of the four compounds were 97.68%, 99.55%, 97.92% and 100.4%, respectively. In conclusion, the established method can rapidly attain an accurate and reproducible result used to control the quality of Fructus Xanthii.

In vivo metabolism study of ginsenoside Re in rat using high-performance liquid chromatography coupled with tandem mass spectrometry.

Ginsenoside Re is one of the major the bioactive triterpene saponins in ginseng root, a well-known adaptogen in traditional Chinese medicine. It is believed that the lead compound may be further developed into a promising new drug for preventing hypertension and cardiovascular disease. To better understand the pharmacological activities of the component, an investigation of its in vivo metabolism was necessary. In the present study, a high-performance liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-TOF-MS/MS) has been applied to discover and identify the metabolites of ginsenoside Re in rat urine following intravenous and oral administration of the component, respectively. The rat urine samples were collected and pretreated through C(18) solid-phase extraction cartridges prior to analysis. Negative electrospray ionization mass spectrometry was used to discern ginsenoside Re and its possible metabolites in urine samples. The metabolites were identified and tentatively characterized by means of comparing molecular mass, retention time, and fragmentation pattern of the analytes with those of the parent compound, ginsenoside Re. As a result, eleven and nine metabolites together with Re were detected and identified in rat urine collected after intravenous and oral administration, respectively. A possible metabolic pathway of ginsenoside Re was also investigated and proposed. Oxidation and deglycosylation were found to be the major metabolic processes of the constituent in rat.

Quantitative and fingerprint analyses of Chinese sweet tea plant ( Rubus suavissimus S. Lee).

The quality of botanical food is increasingly assessed by the content of multiple bioactive compounds. This study reports, for the first time, an HPLC fingerprinting method for the quality evaluation of Rubus suavissimus leaves possessing multiple bioactivities. Five constituents, gallic acid, rutin, ellagic acid, rubusoside, and steviol monoside, were quantified and used in developing qualitative chromatographic fingerprints. The limits of detection and quantification ranged from 0.29 to 37.86 mug/mL. The relative standard deviations (RSDs) of intra- and interday precisions were no more than 3.14 and 3.01%, respectively. The average recoveries were between 93.1 and 97.5%. The developed method was validated in the analysis 14 leaf samples with satisfactory results. The contents of the five marker compounds accounted for an average of about 6% w/w with a variability of 16% among the 14 samples collected from a single site and year. Gallic acid was the least, whereas steviol monoside the most, variable compound among the 14 leaf samples. The characteristic compound rubusoside that is responsible for the sweet taste accounted for 5% of leaf weight. The validated method can now be used to quantitatively and qualitatively assess the quality of R. suavissimus leaves as traditional beverage or potential medicines.

Species differentiation and quality assessment of Radix Paeoniae Rubra (Chi-shao) by means of high-performance liquid chromatographic fingerprint.

There are two species under the monograph of Radix Paeoniae Rubrae ("Chi-shao" in Chinese) in Chinese Pharmacopoeia 2005 edition-Paeonia lactiflora Pallas and Paeonia veitchii Lynch. Due to different species and growing conditions, there are significant chemical differences between the two species, which may result in the improper clinical usage under the same name. Chemical pattern expressed by high performance liquid chromatographic (HPLC) fingerprint analysis can play an important role in species differentiation and quality control of Radix Paeoniae Rubra. In the present work, HPLC fingerprints of two kinds of Radix Paeoniae Rubra have been established and analysed with chemometric methods including similarity evaluation and principal component analysis. Both of the fingerprint common patterns of the two species comprise 13 characteristic peaks, nine of which were common peaks of the two species. However, significant differences between the roots of P. veitchii and P. lactiflora exist not only in the content of certain constituents, especially phenolic acids but also in peak-to-peak ratios expressed by the fingerprint patterns. According to the recent pharmacological studies on polyphenolic constituents, root originating from P. veitchii may possess better efficacy and quality than that from P. lactiflora. Our research reveals that further pharmacological investigation is very necessary to determine whether the two species should be embodied under the same monograph in Chinese Pharmacopoeia.

Determination of ginsenoside-Rg(1) in human plasma and its application to pharmacokinetic studies following intravenous administration of 'Shenmai' injection.

'Shenmai' injection is derived from traditional Chinese medicine 'Shenmaisan' and made from Radix ginseng Rubra and Radix Ophiopogonis. Ginsenoside-Rg(1), as a major constituent of Radix ginseng Rubra, is considered responsible for the efficacy of this injection. A rapid, simple and accurate method has been established for determination of ginsenoside-Rg(1) in Shenmai injection and human plasma using LC-ESI-MS/MS, and to study the pharmacokinetics of Rg(1) in ten healthy volunteers after intravenous single dosing of 60 mL of Shenmai injection. Following solid-phase extraction (SPE), samples were separated on a C(18) column coupled with electrospray ionization mass spectrometry. The protonated analyte was quantified by multiple reaction monitoring (MRM) with a quadruple mass spectrometer in positive mode. Linearity was confirmed in the concentration range of 1 to 1000 ng/mL for Rg(1), and the lower limit of quantification (LLOQ, S/N > 10) was 1 ng/mL. The intraday and interday RSDs were within 15% and mean extraction recoveries ranged from 98.6% to 104.9%. The pharmacokinetics of Rg(1) in healthy volunteers conforms to the two-compartment open model. The main pharmacokinetics parameters were as follows: t(1/2beta), 2.09 +/- 1.89 h; CL, 0.03 +/- 0.01 L kg(-1) h(-1); AUC (0 approximately infinity), 124.4 +/- 35.9 2 ng mL(-1) h and AUC (0 approximately infinity), 127.9 +/- 37.2 ng mL(-1) h, respectively.

[HPLC fingerprinting of radix paeoniae alba].

To establish a sensitive and specific HPLC method for quality control of Radix Paeoniae Alba, HPLC method was applied for quality assessment of Radix Paeoniae Alba. HPLC analysis was performed on a Symmetry C18 column (250 mm x 4. 6 mm ID, 5 microm, Waters, USA). The mobile phase consisted of acetonitrile (solvent A) and water containing 0.1% (v/v) phosphoric acid (solvent B) at a constant flow rate of 0.8 mL x min(-1). An increasing linear gradient (v/v) of solvent A was used (t/min, % A): (0,10), (5,10), (25,15), (45, 22), (46, 65), (50, 80) and (60, 80). The column temperature was set at 25 degrees C. The chromatograms were monitored at 230 nm and the on-line UV spectra were recorded in the range of 190 - 400 nm. The HPLC chromatographic fingerprinting of Radix Paeoniae Alba, showing 11 characteristic peaks, was established from 28 lots of Radix Paeoniae Alba. The areas of main chromatographic peaks were found to complied with the following rule: paeoniflorin > 1, 2, 3, 4, 6-penta-O-galloyl-glucos > albiflorin > methyl gallate > other compounds. The chromatographic fingerprinting of Radix Paeoniae Alba with high specificity can be used to control its quality and assure lot-to-lot consistency.

In vivo pharmacokinetic and metabolism studies of ginsenoside Rd.

A high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS(n)) method has been developed to determine ginsenoside Rd in human plasma and to identify its metabolites in rat urine. The plasma and urine samples were pretreated by solid phase extraction (SPE) prior to analyses. In this work, gentiopicroside was used as the internal standard. The lower limit of quantification (LLOQ) for Rd in human plasma was 3 ng/ml. The average half-life time in plasma was detected as 19.29 h, when 10 mg of ginsenoside Rd was administrated intravenously to the volunteers. Seven metabolites including three oxygenated, two combined and two hydrolyzed components were identified in rat urine samples by using LC-MS and MS-MS, when ginsenoside Rd administered either orally or intravenously.

[Rapid identification of compounds in cortex moutan by liquid chromatography-tandem mass spectrometry].

AIM: To analyze the chemical constituents of cortex moutan by liquid chromatography coupled with electrospray ionization mass spectrometry. METHODS: An on-line optimized HPLC-DAD/MS2 technique was employed. RESULTS: In the negative ion detection mode, 38 components such as monoterpene glucosides, galloylglucoses and acetophenones were isolated. Among them, over thirty compounds were identified, including paeonol, paeonilflorin, oxypaeoniflorin, benzoylpaeoniflorin, benzoyloxypaeoniflorin, galloylpaeoniflorin, galloyloxypaeoniflorin, mundanpioside A, C, D, E, H, etc. by the high performance liquid chromatography with diode-array detection in parallel with electrospray ionization and quadrupole-time of flight tandem mass spectrometry (HPLC-DAD/ESI-MS2). CONCLUSION: This method can be used to rapidly determine the constituents of cortex moutan.

Characterization of compounds in the Chinese herbal drug Mu-Dan-Pi by liquid chromatography coupled to electrospray ionization mass spectrometry.

Cortex Moutan is a well-known traditional Chinese medicine derived from Paeonia suffruticosa ANDREWS. However, root cortices of P. delavayi and P. decomposita also are used under the name of this drug in some regions such as Yunnan and Sichuan Provinces, respectively. In order to make a comparison of their chemical constituents, the compounds of the three Paeonia species were analyzed by high-performance liquid chromatography-diode array detection/electrospray ionization and quadrupole-time-of-flight tandem mass spectrometry (HPLC-DAD/ESI-MS2). A total of 50 compounds were observed in the 50% (v/v) methanolic extracts, including 17 monoterpenes, 14 galloyl glucoses, 10 acetophenones, 5 phenolic acids, 3 flavonoids and 1 triterpene. These chemical constituents were separated on a C18 column and identified or tentatively characterized based on UV spectra and MS fragmentation behavior. The chemical compositions of the three Paeonia species were found to have many differences. Paeonol was the predominant constituent of P. suffruticosa and P. decomposita, while P. delavayi contained albiflorin and more galloyl glucoses than the other two Paeonia species. Most of these identified compounds have been reported from P. delavayi and P. decomposita for the first time. The ESI-MS fragmentation behavior of monoterpene glycosides, acetophenones and galloyl glucoses was also investigated successively, and appropriate characteristic pathways were proposed. The large differences in chemical compounds among the three Paeonia species strongly encouraged further comparison of the bioactivities of these three species.

[Determination of ginsenoside Rd and its metabolites in rat urine by LC-MS].

AIM: To study the metabolic pathways of ginsenoside Rd in rats. METHODS: Urine samples were collected before and after 24 h of single oral administration of 150 mg and intravenous administration of 60 mg of ginsenoside Rd to six rats, separately. The samples were purified by SPE column and then were analyzed by liquid chromatography-ESI-mass spectrometry for putative metabolites. RESULTS: Parent drug and its seven metabolites were identified in rat urine based on comparing total ion chromatograms of the blank with the metalolic urine as well as mass spectra. Its main metabolic pathways and possible structures are elucidated. CONCLUSION: Oxidation, combination and deglucosylation were found to be the major metabolic pathway of ginsenoside Rd in rats.