A biochemometrics strategy for tracing diuretic components of crude and processed Alisma orientale based on quantitative determination and pharmacological evaluation.

We proposed a biochemometrics strategy for tracing diuretic components of herbs based on quantitative determination and pharmacological evaluation. First, a sensitive and robust liquid chromatography coupled with tandem mass spectrometry approach was established for simultaneous quantification of six major triterpenoids in crude and salt-processed Alisma orientale. The separation of triterpenoids was achieved on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid. Six major triterpenoids were detected by multiple reaction monitoring in the negative ion mode. Glycyrrhetinic acid was used as the internal standard. The approach showed good linearity. Intra- and inter-day precisions were all within 2.9%. The recovery rates of each triterpenoid ranged from 97.9% to 103.2%. The approach was then successfully employed for quantitative analysis of six triterpenoids in ten batches of crude and salt-processed A. orientale. Second, the diuretic effects of crude and salt-processed A. orientale were evaluated in mice. Third, principal component analysis and canonical correlation analysis were used to uncover the relationship between the contents of six major triterpenoids and the diuretic effect of different crude and salt-processed samples. Alisol B, alisol F, and alisol A have a close positive correlation with the diuretic effect.

Pharmacokinetics of cucurbitacin B from Trichosanthes cucumerina L. in rats.

BACKGROUND: Cucurbitacin B is the major bioactive constituent in Trichosanthes cucumerina L. fruits, which the pharmacological properties have been studied for decades particularly an anti-tumor activity. The pharmacokinetic profile of this compound is still limited and investigation is needed for further phytopharmaceutical product development. This study aimed to investigate the pharmacokinetic profile of cucurbitacin B after administering the compound at different doses and routes to rats. METHODS: Male Wistar rats (n = 6) were treated by cucurbitacin B extracted from Trichosanthes cucumerina L. The cucurbitacin B was administered at 0.1 mg/kg intravenously or by oral gavage at 2-4 mg/kg. Blood samples and internal organs were collected serially within 24 h after administration. Urine and feces were collected from time 0 to 48 h. The level of cucurbitacin B in biological samples was determined by liquid chromatography-tandem mass spectrometry. RESULTS: The absolute oral bioavailability of cucurbitacin B was approximately 10%. The maximum concentration in plasma after normalization by dose ranged from 4.85-7.81 µg/L and the time to reach maximum value was approximately within 30 min after oral dosing. The level of cucurbitacin B in plasma increased proportionally to the given dose. After intravenous administration, cucurbitacin B had a large volume of distribution of about 51.65 L/kg and exhibited a high tissue to plasma concentration ratio, approximately 60 to 280-fold in several organs. Negligible amount of unchanged cucurbitacin B could be detected in urine and feces and accounted less than 1% of administered dose. CONCLUSION: Cucurbitacin B had low oral bioavailability, but could be distributed extensively into internal organs with a high volume of distribution and tissue to plasma ratio. Only negligible amounts of unchanged cucurbitacin B were excreted via urine and feces suggesting that the compound might be biotransformed before undergoing an excretion. Further studies of the metabolic pathway and tissue uptake mechanism are required to strategize the future development of cucurbitacin B into clinical studies.

Profiling the metabolism of astragaloside IV by ultra performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry.

Astragaloside IV is a compound isolated from the Traditional Chinese Medicine Astragalus membranaceus, that has been reported to have bioactivities against cardiovascular disease and kidney disease. There is limited information on the metabolism of astragaloside IV, which impedes comprehension of its biological actions and pharmacology. In the present study, an ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS)-based approach was developed to profile the metabolites of astragaloside IV in rat plasma, bile, urine and feces samples. Twenty-two major metabolites were detected. The major components found in plasma, bile, urine and feces included the parent chemical and phases I and II metabolites. The major metabolic reactions of astragaloside IV were hydrolysis, glucuronidation, sulfation and dehydrogenation. These results will help to improve understanding the metabolism and reveal the biotransformation profiling of astragaloside IV in vivo. The metabolic information obtained from our study will guide studies into the pharmacological activity and clinical safety of astragaloside IV.

Identification of metabolites of ganoderic acid D by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Ganoderic acid D (GD) is the major active triterpenoid in Ganoderma lucidum, a medicinal fungus used daily. However, the metabolic fate of GD remains unknown. To know whether GD is extensively metabolized, we first investigated the metabolism of GD in vitro and in vivo. The metabolic profiles of the bile samples obtained from rats in vivo were almost the same as those obtained in vitro. Using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry, a total of 25 metabolites were identified from the bile sample. Few metabolites were found in the urine samples. These results indicated that biliary rather than renal clearance was the major route of excretion. The major metabolites were identified by comparison with the standard reference compounds. Metabolites at low concentrations were identified by interpreting the mass spectra. Both phase I and phase II metabolites were observed. The metabolic transformation included reduction, monohydroxylation, dihydroxylation, trihydroxylation, oxidation, desaturation, sulfation, and glucuronidation. The main metabolic soft spots in the chemical structure of GD were the 3-carbonyl group, angular methyl groups, the 7-hydroxy group, and the 26-carboxylic acid moiety. Overall, this study gives us an insight into the metabolism of GD, an active oxygenated tetracyclic triterpenoid.

Synthesis and pharmacokinetic profile of rhein- boswellic acid conjugate.

Rhein, an active metabolite of diacerein, down-regulates the gene-expression and production of pro-matrix metalloproteinases and up-regulates the tissue inhibitors of metalloproteinase-1 production. The therapeutic effects of diacerein on osteoarthritis are, at least in part, due to the chondroprotective effect of rhein. Boswellic acid is a specific, non-redox inhibitor of leukotriene synthesis. It is claimed to possess good anti-inflammatory, anti-arthritic, analgesic, and anti-ulcer activities. It prevents the destruction of articular cartilage by decreasing degradation of glycosaminoglycans. Therefore, rhein and boswellic acid were linked chemically through a bioreversible ester linkage to synthesize their mutual prodrug by reported procedure. In vitro release profile of this prodrug was extensively studied in aqueous buffers of varied pH, upper GIT homogenates and 80% human plasma. In vivo release studies were undertaken in blood, urine and feces of rats. The prodrug was stable in HCl buffer (pH 1.2) and stomach homogenates of rats. However; in phosphate buffer (pH 7.4) and in intestinal homogenates the prodrug exhibited 91% and 96% release of rhein and 27.5% and 38% release of boswellic acid respectively over a period of 6h following first order kinetics. In 80% human plasma (in vitro) and rat blood (in vivo) also 96.35% and 91% release of rhein and 78% and 86.41% release of boswellic acid respectively was observed. The 24 h pooled samples of rat urine revealed presence of 6.2% intact prodrug, 7.1% of rhein and 8.9% of boswellic acid indicating their renal excretion. Samples of rat feces pooled over a period of 24 h showed absence of rhein and presence of 3.1% of intact boswellic acid and 4.6% of boswellic acid emphasizing their intestinal excretion. The in vivo release kinetics of prodrug in rat clearly indicated activation of prodrug to be occurring in blood, being catalyzed by the weak alkaline pH of blood (7.4) in combination with esterases present therein.

Systematic screening and characterization of the major bioactive components of Poria cocos and their metabolites in rats by LC-ESI-MS(n).

Poria cocos is a well-known medicinal plant widely used in China and other East Asian countries owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of Poria cocos and their metabolites in vivo are still unclear to date. The aim of the present study was to develop a practical method based on the combined use of the liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS(n) ) for the comprehensive and systematic separation and characterization of the bioactive constituents of Poria cocos extract and their metabolites in rats. Based on the proposed strategy, a total of 34 compounds were characterized from the extract of Poria cocos. Among them, eight were unambiguously identified by comparing their retention times and mass spectra with those of reference standards, and 26 were tentatively identified on the basis of their MS(n) fragmentation behaviors and molecular weight information from literatures. In vivo, seven compounds were successfully detected in rat urine whereas one was found in rat plasma. This study proposed a series of potential bioactive components and provided helpful chemical information for further research on the action mechanism of traditional Chinese medicine.

Pharmacokinetics of maslinic and oleanolic acids from olive oil - Effects on endothelial function in healthy adults. A randomized, controlled, dose-response study.

To date, pharmacokinetics of maslinic (MA) and oleanolic (OA) acids, at normal dietary intakes in humans, have not been evaluated, and data concerning their bioactive effects are scarce. We assessed MA and OA pharmacokinetics after ingestion of olive oils (OOs) with high and low triterpenic acid contents, and specifically the effect of triterpenes on endothelial function. We performed a double-blind, dose-response, randomized, cross-over nutritional intervention in healthy adults, and observed that MA and OA increased in biological fluids in a dose-dependent manner. MA bioavailability was greater than that of OA, and consumption of pentacyclic triterpenes was associated with improved endothelial function. To the best of our knowledge, this is the first time MA pharmacokinetics, and effects on endothelial function in vivo, have been reported in humans.

Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry.

An accurate and selective method for the simultaneous determination of triptolide, tripdiolide and tripterine in human urine using hydrocortisone as an internal standard (IS) by high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization mass spectrometry in negative ion mode has been developed. After triptolide, tripdiolide and tripterine in human urine were extracted with ethyl acetate and cleaned by solid-phase extraction with C(18) cartridges, a satisfactory separation was achieved on an XDB C(18) short column (30 x 2.1 mm i.d., 3 microm) using the mobile phase of acetic acid-ammonium acetate (5 mmol/L, pH = 4.5)-acetonitrile-methanol in gradient elution. Detection was operated by APCI in selected ion monitoring mode. The target ions m/z 359, m/z 375, m/z 449 and m/z 419 were selected for the quantification of triptolide, tripdiolide, tripterine and IS, respectively. The linear range was 1.0-100.0 ng mL(-1), and the limits of quantification in human urine were found to be 0.1-0.5 ng mL(-1) for the three compounds. The precisions (CV%) and accuracies were 6.6-12.9 and 85.1-97.0%, respectively. The developed method could be applied to the determination of triptolide, tripdiolide and tripterine in human urine for diagnosis of the intoxication and for forensic purposes.

In vivo metabolic profiles of Bu-Zhong-Yi-Qi-Tang, a famous traditional Chinese medicine prescription, in rats by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been extensively used for conditioning sub-health status and diseases caused by spleen-qi deficiency in China for over 700 years. BZYQT is prevalent not only in China, but also in Japan and South Korea for the clinical treatment of chronic diseases, such as fatigue, tuberculosis and loss of appetite after surgery. However, due to a lack of research on the holistic metabolism of BZYQT, the in vivo bioactive components of BZYQT remain unclear, hindering further study of its in vivo mechanism of action and quality control. In the present study, a four-step integrated strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) was established to systematically screen the in vivo xenobiotics of BZYQT. Ultimately, a total of 162 xenobiotics (59 prototypes and 103 metabolites) were identified or tentatively characterized, including 48 in plasma, 147 in urine and 58 in feces, while the in vivo metabolic profile of atractylenolide III (a major component of BZYQT) was elucidated for the first time. The xenobiotics of BZYQT mainly included flavonoids from Astragali Radix, Glycyrrhizae Radix et Rhizoma and Citrus reticulatae Pericarpium; lactones from Angelicae Sinensis Radix and Atractylodis Macrocephalae Rhizoma; and triterpenoid saponins from Cimicifugae Rhizoma. After oral administration, BZYQT-related components underwent diverse metabolic pathways. Among them, flavonoids mainly underwent glucuronidation, sulfation and demethylation, while lactones mainly underwent hydroxylation and acetylcysteine conjugation, and deglycosylation was the major metabolic reaction of saponins. Our investigation gives a comprehensive analysis of the metabolic characteristics of BZYQT and will provide an important basis for further studying the pharmacokinetics of BZYQT to explore its in vivo disposal features and discover its in vivo bioactive components.

Determination of triptonide by cloud point extraction combined with MEKC.

A method has been developed for the determination of triptonide in the traditional Chinese herb Tripterygium wilfordii Hook F. by micellar electrokinetic capillary chromatography combined with cloud point extraction. The analyte was extracted at pH 3.0 by micelles of the nonionic surfactant polyoxyethylene 7,5-octylphenyl ether (Triton X-114). A 250-muL aliquot from the extracted surfactant-rich phase was diluted to 400 muL with ethanol to reduce its viscosity before separation by MEKC. Under optimum conditions, an enrichment factor of 25 is obtained and the determination limit of triptonide is found to be 3.15 x 10(-7) mol/L. The proposed method has been successfully applied to the determination of triptonide in T. wilfordii tablet and spiked urine matrix, demonstrating the feasibility and reliability of the proposed method.

Identification of rotundic acid metabolites after oral administration to rats and comparison with the biotransformation by Syncephalastrum racemosum AS 3.264.

The objective of this study was to identify the metabolites of rotundic acid after oral administration to rats and compare the similarities with its biotransformation by Syncephalastrum racemosum AS 3.264 using ultra-high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. A total of fourteen metabolites were determined based on the mass spectrometry and chromatographic behaviors, among which eleven (M1-M3, M7-M14) and six (M2, M4-M8) metabolites were identified in rats and S. racemosum, respectively. Three identical metabolites (M2, M7 and M8) were found in rats and S. racemosum, indicating that there were metabolic similarities. Moreover, to confirm the results of mass spectrometry, three (M2, M4 and M7) metabolites were obtained by the means of amplifying incubation and their structures were determined by various spectroscopic analyses, and M4 was proved to be a previously undescribed compound. This results showed that in vitro assisted preparation by microbial transformation is a feasible and effective method of obtaining metabolites which are in low amounts and difficult to be prepared in vivo.

HPLC determination of four triterpenoids in rat urine after oral administration of total triterpenoids from Ganoderma lucidum.

A sensitive and simple high-performance liquid chromatography (HPLC) method was applied for the quantitative determination of four major triterpenoids (ganoderic acids C(2), B, K and H) in rat urine after oral administration of total triterpenoids from Ganoderma lucidum. The urine sample was extracted with dichloromethane-ethyl acetate (90:10) after acidification by hydrochloric acid (0.2 mol/ml). Chromatographic separation was achieved on a Zorbax SB-C(18) column (250 mm x 4.6 mm, 5 microm) at 35 degrees C, with a linear gradient of acetonitrile and 0.03% aqueous phosphoric acid (v/v), at a flow rate of 1.2 ml/min. The four triterpenoids and internal standard (hydrocortisone) were detected at a wavelength 252 nm. The within- and between-day assay coefficients of variation for the four triterpenoids in urine were less than 9% and the extraction recovery of this method was higher than 90%. Using this method, the excretion profile of the triterpenoids in rat urine after oral administration of total triterpenoids of G. lucidum was revealed for the first time.

Immunochromatographic determination of bacopaside I in biological samples.

We describe a novel immunochromatographic method for qualitative and quantitative analyses of bacopaside I, a bioactive constituent in Bacopa monnieri (L.) Wettst in biological samples. The assay was performed on polyethersulfone membrane using a polyclonal antibody raised against bacopaside I. The finalised method could quantitatively determine bacopaside I in the range of 31.3-1000.0ng and the detection and quantification limits were 1.0 and 31.3ng, respectively. The percentage recoveries of bacopaside I in blood and urine were nearly 100% indicating the accuracy of the extraction. The method was then applied for the determination of this compound in rat serum, urine and feces after an oral dose of 15mg/kg body weight. About 4% of the ingested dose of bacopaside I was detected in rat feces but none was detected in serum and urine which accorded with results from liquid chromatography tandem mass spectrometry. The accuracy, selectivity, sensitivity of the method are appropriate for in vivo pharmacokinetic studies.

Simultaneous determination of cucurbitacin B, E, I and E-glucoside in plant material and body fluids by HPLC-MS.

A selective and sensitive analytical method for the simultaneous determination of cucurbitacin B, E, I and E-glucoside in plant material and body fluids by HPLC-MS was developed. After liquid-liquid extraction with dichlormethane, separation was achieved on a Phenomenex Luna Pentafluorophenyl Column (150mm×2mm, 5µm) using acetonitrile-water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis Q1 Scans with negative ionisation were chosen. The method was validated for serum as the matrix of choice. Limits of detection are in the picogram range, limits of quantification are between 0.05 and 0.42ng/mL, recoveries are above 50%. The assay was linear in the calibration range from 1.0 to 50ng/mL for cucurbitacin E and from 0.10 to 50ng/mL for the cucurbitacins B, I and E-glucoside. The applicability of the method was demonstrated by the determination of cucurbitacins in zucchini plant material and body fluids from intoxication cases.

Optimization of solid-phase extraction and liquid chromatography-tandem mass spectrometry for simultaneous determination of capilliposide B and its active metabolite in rat urine and feces: Overcoming nonspecific binding.

Capilliposide B, a novel oleanane triterpenoid saponin isolated from Lysimachia capillipes Hemsl, showed significant anti-tumor activities in recent studies. To characterize the excretion of Capilliposide B, a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of Capilliposide B and its active metabolite, Capilliposide A in rat urine and feces. Sample preparation using a solid-phase extraction procedure was optimized by acidification of samples at various degrees, providing extensive sample clean-up with a high extraction recovery. In addition, rat urinary samples were pretreated with CHAPS, an anti-adsorptive agent, for overcoming nonspecific analytes adsorption during sample storage and process. The method validation was conducted over the curve range of 10.0-5000ng/ml for both analytes. The intra- and inter-day precision and accuracy of the QC samples showed ≤11.0% RSD and -10.4-12.8% relative error. The method was successfully applied to an excretion study of Capilliposide B following intravenous administration.

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry.

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.

Pharmacokinetics of Astragaloside IV in rats by liquid chromatography coupled with tandem mass spectrometry.

Pharmacokinetics of Astragaloside IV (AGS-IV) in rats was studied by high performance liquid chromatography coupled with tandem mass spectrometry. The concentration in plasma was determined after i.v. administration of 1, 2, 4 mg/kg and p.o. administration of 20 mg/kg of AGS-IV. The AUC were linearly correlated to doses. Recoveries of AGS-IV in bile, urine and feces were also analyzed following i.v. dose of 2 mg/kg. Cumulative recovery of AGS-IV in bile reached 30.8% in 24h. Cumulative recovery of AGS-IV in urine and feces was 52.14%, which indicates that about 50% of AGS-IV was metabolized in vivo. The bioavailability of AGS-IV after p.o. administration was found to be 3.66%. These findings provide useful information for the research and development of AGS-IV and other potential agents.

In vivo disposition and metabolism of madecassoside, a major bioactive constituent in Centella asiatica (L.) Urb.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urb. herb is frequently used in traditional Chinese medicine for many indications, such as traumatic injuries, keloid and scar. Madecassoside is the main active ingredient of this herb drug with higher content than other triterpenoid constituents. Understandings of pharmacokinetic profiles of madecassoside should be beneficial for its development as a therapeutic agent. MATERIALS AND METHODS: Sprague-Dawley rats were intravenously and orally administered madecassoside (100 mg/kg), respectively. Plasma, heart, liver, spleen, lung, kidney, brain, bile, urine and feces were collected at the designed time points. Madecassoside concentrations in biological samples were determined by a sensitive and well-validated liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method. A liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOF-MS) method was established to identify its major metabolites in feces. To further pursue the disposition characteristics of madecassoside in rats, the involvement of the hepatobiliary efflux transporters in biliary elimination were studied by combination with digoxin (P-glycoprotein inhibitor) or probenecid (multidrug resistance-associated protein inhibitor). A linked-rat model was also used to assess the role of enterohepatic circulation in the pharmacokinetics of madecassoside. RESULTS: After a single oral dosing, madecassoside was widely distributed in heart, liver, spleen, lung and kidney of rats, and the levels of madecassoside in liver and kidney were relatively higher than other organs. The excretions of madecassoside in bile, urine and feces were 7.16% (0-12 h), 0.25% (0-72 h) and 24.68% (0-72 h), respectively. The findings suggested that madecassoside might excrete mainly by metabolites. The combination with either digoxin or probenecid significantly attenuated the excretion of madecassoside as parent from bile, indicating that P-glycoprotein and multidrug resistance-associated protein might contribute to the hepatobiliary elimination of madecassoside. The presence of enterohepatic circulation, as implied by double-humped profiles in plasma and tissue concentration-time curves, was confirmed by a linked-rat model. Furthermore, three metabolities of madecassoside were indentified in rat feces and the possible metabolic pathways were proposed. CONCLUSIONS: These findings provide valuable information regarding in vivo process of madecassoside, and help us to recognize the efficacy and safety of madecassoside itself, the relevant herbs or herbal preparations.

Ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction coupled with high performance liquid chromatography for sensitive determination of trace celastrol in urine.

Ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction (UA IL-DLLME) coupled with high-performance liquid chromatography (HPLC) has been developed for the determination of celastrol in human urine samples. In the microextraction procedure, ionic liquid (IL) was used as extraction solvent and dispersed into the aqueous sample solution as fine droplets by means of dispersive solvent and ultrasonication which promoted the analyte to migrate into IL phase more easily. Several important parameters affecting the extraction efficiency were studied and optimized, including the type and volume of extraction solvent and dispersive solvent, sample pH, ultrasonication time, cooling time, centrifugation time and salting-out effect. Under the optimized conditions, 110-fold enrichment factor was obtained and the limit of detection (LOD) was 1.6 µg/L at a signal-to-noise ratio of 3. The calibration curve was linear over the range of 10-1000 µg/L for celastrol in human urine sample, with a correlation coefficient of 0.9980. Intra- and inter-assay precision were 0.43% and 2.78%, respectively. The proposed method was successfully applied to the real human urine samples and good spiked recoveries in the range of 93.2-109.3% were obtained.

Determination of a new asiatic acid derivative, AS 2-006A in rat plasma and urine, and human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a new wound healing agent, AS 2-006A (ethoxymethyl 2-oxo-3, 23-O-isopropylideneasiatate), in rat plasma and urine, and human plasma. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto the reversed-phase column. The mobile phase employed was acetonitrile : H2O (9:1, v/v) and run at a flow rate of 1.1 ml/min. The column effluent was monitored by a UV detector set at 205 nm. The retention time for AS 2-006A was approximately 29.5 min. The detection limits for AS 2-006A in rat and human plasma were both 1 microg/ml, and in rat urine was 2 microg/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 10.8%) for rat plasma and urine, and human plasma. No interferences from endogenous substances were found.