Activity of neolignans isolated from Piper regnellii (MIQ.) C. DC. var. pallescens (C. DC.) YUNCK against Trypanosoma cruzi

The in vitro antiproliferative effects of 4 neolignans purified from the ethyl-acetate extract from leaves of Piper regnellii (MIQ.) C. DC. var. pallescens (C. DC.) YUNCK against Trypanosoma cruzi were investigated. These isolated compounds were identified through spectral analyses of UV, EI-MS, 1H-, 13C-NMR, H-H COSY, gNOE, HETCOR, and HMBC. The compounds eupomatenoid-5, eupomatenoid-6, and conocarpan showed considerable activity against epimastigote forms of T. cruzi, with 50% inhibition concentrations (IC50) of 7.0, 7.5, and 8.0 microg/ml respectively. After methylation, these compounds showed a lessened inhibitory activity to the growth of the protozoan, suggesting that loss of the hydroxyl group from their molecules reduces the activity. The compound eupomatenoid-3 showed lower activity than the hexane fraction. Eupomatenoid-5 was significantly more active than benznidazole, the antiparasitic drug of choice for treatment of Chagas' disease. The crude extract, hexane fraction, and eupomatenoid-5 caused no lysis in sheep blood at concentrations which inhibit the growth of epimastigote forms. The compound eupomatenoid-5 showed low cytotoxic effects against Vero cells. These results provide new perspectives on the development of novel drugs obtained from natural products with trypanocidal activity. However, the extracts and active compound isolated from P. regnellii var. pallescens should be further studied in animal models for in vivo efficacy.     

Evaluation of antifungal activity of Piper solmsianum C. DC. var. solmsianum (Piperaceae)

We have studied the crude methanolic extract (CME), some fractions (hexane, dichloromethane and ethyl acetate) and four pure compounds: eupomatenoid-3 (1), eupomatenoid-5 (2), conocarpan (3) and orientin (4), from Piper solmsianum, for possible antifungal activity against 12 pathogenic fungi. The minimal inhibitory concentration (MIC) was determined and the experiments showed that the CME exhibited antifungal action against all the dermatophytes tested, with MIC values of between 20 microg/ml to 60 microg/ml. Similar activity also was verified for the hexane, dichloromethane and ethyl acetate fractions. However, the starting material (CME), and all the fractions, did not exert inhibitory effect against hyaline hyphomycetes and were only discretely active against the zigomycetes and yeasts. Compounds 2, 3 and 4 also exhibited pronounced activity against all the dermatophytes tested (MIC< or =1 to 9 microg/ml) with potency as high as the standard antifungal drug (ketoconazole). Compound 3 also exhibited activity against all the yeasts tested. In conclusion, the antifungal activity of P. solmsianum seems to be related mainly to the presence of compounds 2, 3 (neolignans) and 4 (flavonoid), however it was verified that another active compound, as yet unidentified, exists in the plant.     

Trypanocidal constituents in plants 1. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in Guaco,roots of Aristolochia taliscana

Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 43 kinds of methanolic and other organic extracts from 39 plants which were examined by the preliminary screening test to see immobilization of epimastigotes of T. cruzi in vitro. Eighteen of them showed activity at the concentration of 2 mg/ml after incubation for 2 h, while 13 showed activity at the concentration of 1 mg/ml after incubation for 48 h. Among them, the MeOH extract of roots of Aristolochia taliscana (Aristolochiaceae), locally known as "Guaco," immobilized all the epimastigotes even at lower concentration of 0.5 mg/ml (48 h). In order to identify principal compounds for this activity, the MeOH extract of Guaco was subjected to bioassay-guided fractionation. From the active fractions, four neolignans, eupomatenoid-7 (1), licarin A (2), eupomatenoid-1 (5) and licarin B (6), and two lignans, austrobailignan-7 (3) and fragransin E1 (4) were isolated. Compounds 1-4 immobilized all the epimastigotes at the minimum concentration of 25-75 microg/ml after incubation for 48 h, while compounds 5 and 6 were inactive. Corresponding concentration of gossypol, berberine chloride and harmine was 280 microg/ml, 300 microg/ml and >500 microg/ml, respectively.     

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.     

An enantiospecific HPLC method for the determination of (S)-enantiomer impurities in (R)-tolterodine tartarate

A high-performance liquid chromatographic method was developed for the separation of the enantiomers of tolterodine tartarate. The proposed method was applied to the determination of (S)-isomer in (R)-tolterodine tartarate, and satisfactory results were obtained. The enantiomers of tolterodine tartarate were separated on a Chiralpak AD-H (250 mm x 4.6 mm) column containing amylose tris-(3,5-dimethylphenyl-carbamate) at room temperature. The mobile phase consisted of n-hexane and isopropyl alcohol in the ratio of 85:15 (v/v) with 0.075% triethylamine (TEA) and 0.05% trifluoroacetic acid (TFA) as the additive. The flow rate was kept at 0.5 ml/min, and UV detection wavelength was set at 283 nm. The calibration curves of (S)-enantiomer in the concentration range from 0.05 microg/ml to 1 microg/ml range were linear. The relative standard deviations of within-day and between-day were less than 2% (n = 3). The limit of detection (LOD) was 0.75 ng (S/N = 3) and the limit of quantification (LOQ) was 0.05 microg/ml (RSD < 4.1%, n = 3). The determination recoveries of the (S)-enantiomer were in the range of 98.2-104.8%. The results demonstrated that the developed HPLC method was a reliable, simple technique and was applicable to the purity determination of (R)- tolterodine tartarate.     

Determination of atazanavir in human plasma using solid-phase extraction and high-performance liquid chromatography

Atazanavir is a new HIV-1 protease inhibitor. A simple high-performance liquid chromatographic method using UV detection was developed and validated for the analysis of atazanavir in human plasma. The sample clean up was carried out using solid-phase extraction with OASIS MCX cartridge. The chromatographic separation was achieved on a Kromasil C18 (150 mm x 3 mm, 5 microm) column with a mobile phase consisting of acetonitrile and water (38:62 v/v) delivered isocratically. The effluent of the column was monitored at a wavelength of 210 nm. The assay was linear over the concentration range of 0.156 to 10 microg/ml and the limit of quantification was 0.156 microg/ml. The method was also validated with respect to recovery, precision, accuracy and specificity. This method is suitable for therapeutic drug monitoring of atazanavir and can be easily reproduced with standard equipment.     

Antioxidant and photoprotective activity of a lipophilic extract containing neolignans from Krameria triandra roots

The antioxidant/photoprotective potential of a standardized Krameria triandra (KT) root extract (15% neolignans) has been evaluated in different cell models, rat erythrocytes and human keratinocytes cell lines, exposed to chemical (cumene hydroperoxide, CuOOH) and physical (UVB radiation) free radical inducers. The extract was significantly more active (IC50 0.28 +/- 0.04 microg/ml) than the typical chain-breaking antioxidant alpha-tocopherol (IC50 = 6.37 +/- 0.41 microg/ml) in inhibiting the CuOOH-induced hemolysis in rat blood cells. The KT constituent 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran, was the most active (IC50 = 0.03 +/- 0.005 microg/ml), followed by eupomatenoid 6 (IC50 = 0.29 +/- 0.06 microg/ml) and conocarpan (IC50 = 0.77 +/- 0.08 microg/ml). The same order of potency was observed in red blood cells exposed to UVB irradiation in continuo, with IC50 values 0.78 +/- 0.08 microg/ml for KT extract, 0.18 +/- 0.02 microg/ml for 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran, 0.95 +/- 0.11 microg/ml for eupomatenoid 6, and 3.8 +/- 0.39 microg/ml for conocarpan. In cultured human keratinocytes exposed to UVB radiation (50 mJ/cm2), KT extract (2.5-20 microg/ml) significantly and dose-dependently restrained the loss in cell viability and the intracellular oxidative damage: glutathione (GSH) depletion and the rise in dichlorofluorescein (DCF), marker of peroxide accumulation, were suppressed by 20 microg/ml KT and in parallel cell morphology maintained. The cytoprotective effect of the extract was confirmed in a more severe model of cell damage: exposure of keratinocytes to higher UVB doses (300 mJ/cm2), which induce a 50% cell death. In keratinocyte cultures supplemented with 10 microg/ml, cell viability was almost completely preserved and more efficiently than with (-)-epigallocatechin 3-gallate and green tea. The results of this study indicate the potential use of Rhatany extracts, standardized in neolignans, as topical antioxidants/radical scavengers against skin photodamage.     

HPLC assay and pharmacokinetic study of atorvastatin in beagle dogs after oral administration of atorvastatin self-microemulsifying drug delivery system

A specific and accurate reversed-phase HPLC with UV detection was developed for the assay of atorvastatin in beagle dog plasma. Indomethacin was used as the internal standard. Atorvastatin was extracted by protein precipitation, the extracts were injected into a Kromasil C8 column (150 mm x 4.6 mm, 5 microm) with UV wavelength set at 270 nm. The mobile phase consisted of acetonitrile:0.1 mol/L ammonium acetate buffer (pH 4.0) (65:35% v/v) at a flow rate of 1.0 ml/min. The column was at ambient temperature (25 degrees C). The injection volume was 25 microl. The blank plasma did not interfere with the determination of atorvastatin and indomethacin. A good linear relationship was obtained between the peak area ratio of atorvastatin to indomethacin and the concentration of atorvastatin over the range of 0.05 to 2.5 microg/mL. The limit of quantification was 25 ng/mL, the limit of detection was 8 ng/ml. The total chromatographic analysis time was within 9 min. The method is accurate, precise and fast for the assay of atorvastatin in plasma following oral administration of an atorvastatin SMEDDS to healthy beagle dogs.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

Validated HPLC method for determination of PAT-5A, an insulin sensitizing agent, in rat plasma

A high performance liquid chromatographic method for the determination of PAT-5A (a potent insulin sensitizer) using DRF-2095 (a thiazolidinedione) as internal standard (I.S.) is described. A 1:1 v/v ethylacetate and dichloromethane solvent mixture was used for extraction of PAT-5A from plasma. A Kromasil KR100-5C18-250A, 5 microm, 4.6 x 250 mm SS column was used for the analysis. Mobile phase consisting of sodium dihydrogen phosphate (pH 4.0, 0.05 M) and methanol mixture (25:75, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector set at 345 nm. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. Using this method the absolute recovery of PAT-5A from rat plasma was > 90% and the limit of quantification was 0.05 microg/ml. The intra-day relative standard deviation (RSD) ranged from 2.19 to 4.98% at 1.0 microg/ml, 1.05 to 3.68% at 10.0 microg/ml and 3.14 to 5.08% at 50 microg/ml. The inter-day RSD were 1.6, 2.24 and 1.54% at 1, 10 and 50 microg/ml, respectively. The method was applied to measure the plasma concentrations of PAT-5A in pharmacokinetic and bioavailability studies in male Wistar rats.     

Rapid and simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatographic method was developed for simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in human plasma. The method entailed injection of the samples after deproteination with perchloric acid and subsequent neutralizing. Primidone was used as internal standard. Chromatography was performed on a C(18) column (250 mm x 4.6 mm, 5 microm) under isocratic elution with 50 mM aqueous sodium dihydrogen phosphate-acetonitrile-triethylamine (100:25:0.5, v/v), pH 5.9. Detection was made at 240 nm and analyses were run at a flow-rate of 1.5 ml/min at a temperature of 35 degrees C. The recovery was 83.4, 88.5 and 98.2% for TMP, SMX and internal standard, respectively. The precision of the method was 2.6-9.8% over the concentration range of 0.125-2 microg/ml for TMP and 0.39-50 microg/ml for SMX. The limit of quantification (LOQ) in plasma was 0.125 and 0.39 microg/ml for TMP and SMX, respectively. The method was used for a bioequivalence study.     

Determination and pharmacokinetics of isoferulic acid in rat plasma by high-performance liquid chromatography after oral administration of isoferulic acid and Rhizoma Cimicifugae extract

A simple and specific high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance detection has been developed for the determination of isoferulic acid in rat plasma. The plasma samples were deproteinized with methanol after the addition of internal standard (IS) tinidazole. The analysis was performed on a Kromasil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size) with acetonitrile-0.05% phosphoric acid (25:75, v/v) as mobile phase. The linear range was 0.0206-5.15 microg ml(-1) and the lower limit of quantification (LLOQ) was 0.0206 microg ml(-1). The intra- and inter-day relative standard deviations (R.S.D.s%) were less than 11.4 and 12.3%, respectively, and accuracy as relative error (R.E.%) between -6.7 and -1.1%. Mean extraction recovery was above 80%. The validated method was successfully applied to the pharmacokinetic study of isoferulic acid in rat plasma after oral administration of isoferulic acid and Rhizoma Cimicifugae extract.     

The determination of glycyrrhizic acid in Glycyrrhiza uralensis Fisch. ex DC. (Zhi Gan Cao) root and the dried aqueous extract by LC-DAD

A rapid, sensitive and specific reversed phase high-performance liquid chromatographic (LC) method with photodiode array detection (DAD) has been developed for the determination of glycyrrhizic acid in both the raw herb and a commercially prepared dried aqueous extract of Glycyrrhiza uralensis Fisch. ex DC. root (Zhi Gan Cao, liquorice). It was determined that extracting the raw herb in aqueous methanol (50:50 v/v) by sonication for 2 x 30 min was the most efficient sample preparation. Baseline resolution of the glycyrrhizic acid peak was achieved on a Varian Polaris RP C18-A (250 mm x 4.6 mm, 5 microm packing) column using an isocratic mobile phase consisting of 0. v aqueous phosphoric acid and acetonitrile in the ratio 60:40 v/v. Chromatograms were monitored between 200 and 400 nm for peak purity assessments, with quantitation performed at 254 nm. Glycyrrhizic acid calibration curves in the concentration range of 14-558 microg/ml were prepared on the day of analysis. Curve fitting was by the least-squares method, with correlation coefficients of >0.9998 obtained each time. The average recovery at three spike levels (50, 100, 200%) was of 95.91+/-1.05% and 98.36+/-3.45% (+/-S.D., n=7) for the spiked raw herb and dried aqueous extract respectively. The limit of detection and limit of quantitation was 0.52 and 1.72 mg/g respectively for the raw herb, and 0.75 and 2.51 mg/g respectively for the dried aqueous extract. Identity confirmation of the chromatographic peak was achieved by (-) electrospray ionisation tandem mass spectrometry. The concentration of glycyrrhizic acid in the root and dried aqueous extract was found to be 31.1+/-0.2 and 40.4+/-0.3mg/g (+/-S.D., n=7) respectively.     

Analysis of clonazepam in a tablet dosage form using smallbore HPLC

A stability indicating, reversed phase high-performance liquid chromatographic method utilizing a smallbore HPLC column has been developed for the determination of clonazepam in a commercial tablet dosage form. The use of a small bore column results in a substantial solvent savings, as well as a greater mass sensitivity, especially in the identification of degradation peaks in a chromatogram. The method involves ultraviolet detection at 254 nm and utilized a 150 x 3.0 mm i.d. column packed with 3 microm octyldecylsilane particles with a mobile phase of water methanol acetonitrile (40:30:30, v/v/v) at a flow rate of 400 microl min(-1) at ambient temperature, with and without the use of 1,2-dichlorobenzene as the internal standard. The current USP method for the analysis of clonazepam using a 300 x 3.9 mm i.d. conventional octyldecylsilane column was utilized as a comparison to the smallbore method. The retention times for clonazepam and the internal standard on the 3.0 mm i.d. column were 4.0 and 12.5 min, respectively. The intra- and interday RSDs on the 3.0 mm i.d. column were < 0.55% (n =4) using the internal standard, and < 0.19% (n = 4) without the internal standard at the lower limit of the standard curve, 50 microg ml(-1) and had a limit of detection of 24 ng ml(-1). The assay using the 3.0 mm i.d. column was shown to be suitable for measuring clonazepam in a tablet dosage form.     

Sensitive LC determination of piroxicam after in vitro transdermal permeation studies

A direct, very sensitive, simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of piroxicam, with tenoxicam as internal standard, has been developed and validated. Samples were chromatographed on a 5 microm Scharlau C(18) column. The mobile phase was a mixture of acetonitrile-acetic acid 4% (pH 2.8) (45:55, v/v). Detection was at 354 nm and the run time was 7 min. The limit of detection was 0.025 microg/ml. The detector response was found to be linear in the concentration range 0.05-9 microg/ml. This HPLC assay has been applied to measure the 'in vitro' percutaneous permeation of piroxicam through abdominal hairless rat skin, using Franz-type diffusion cells, in order to obtain the concentration-time profiles of piroxicam.     

Capillary gas chromatographic determination of phenylpropanolamine in pharmaceutical preparation

Analytical procedure has been developed for the gas chromatographic determination of phenylpropanolamine (PPA) using trifluoroacetylacetone (FAA) as derivatizing reagent. Elution is carried out from the column HP-5 (30 mx0.32 mm i.d.) with film thickness 0.25 microm at initial column temperature 70 degrees C for 5 min, followed by heating rate 10 degrees C/min up to 120 degrees C. Injection port temperature was maintained at 270 degrees C. Nitrogen flow rate was 2 ml/min and detection was by FID. The linear calibration curve was obtained with 30-150 microg/ml PPA with detection limit of 6.0 microg/ml. The method was used for the determination of PPA from Sinutab and Tavegyl-D tablets. The relative standard deviation (R.S.D.) for the analysis of pharmaceutical preparation was obtained within 0.4-0.9%.     

Simultaneous LC determination of tizanidine and rofecoxib in tablets

A reverse phase high performance liquid chromatographic method to determine tizanidine (TZ) and rofecoxib (RF) in combination is proposed and applied to the pharmaceuticals. This method allows the determination of 0.1-0.5 microg/ml of TZ and 1.2-6.0 microg/ml of RF along with 10 microg/ml of nimesulide (internal standard), in a mobile phase consisting of 1% (v/v) triethylamine (pH adjusted to 2.5 using dilute orthophosphoric acid):acetonitrile in the ratio 55:45% (v/v). Detection wavelength of 303 nm and flow rate of 0.8 ml/min were fixed for the study. The limit of detection (LOD) for TZ and RF were found to be 10 and 1 ng/ml, respectively. The limit of quantification (LOQ) for TZ and RF were found to be 80 and 12 ng/ml, respectively. The amount of drug present in the tablet and the recovery studies were also carried out. The % R.S.D. of recovery studies for TZ and RF were found to be 0.0673 and 0.0146, respectively. The method is validated for accuracy, precision, ruggedness and robustness.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma

A high-performance liquid chromatographic method was developed and validated for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma. The coumarin components and the internal standard isopsoralen were extracted from plasma samples with the mixture of tert-butyl methyl ether and n-hexane (4:1, v/v). Chromatographic separation was performed on a C(18) column (200 mm x 4.6mm, 5 microm) with the mobile phase acetonitrile-methanol-water-acetic acid (20:15:65:2, v/v/v/v) at a flow-rate of 1.0 ml/min. Only the peak of oxypeucedanin hydrate and byak-angelicin could be detected in dog plasma after oral administration of ethanol extracts of A. dahurica mainly containing xanthotoxol, osthenol, imperatorin, oxypeucedanin hydrate and byak-angelicin. The calibration curves of oxypeucedanin hydrate and byak-angelicin were linear over a range of 22.08-8830.00 and 6.08-2430.00 ng/ml in dog plasma, respectively. The quantification limit of oxypeucedanin hydrate and byak-angelicin in dog plasma was 22.08 and 6.08 ng/ml, respectively. The intra- and inter-day precision was less than 7.6% and 8.5% and the accuracy was from 91.9% to 106.1%. The lowest absolute recoveries of oxypeucedanin hydrate and byak-angelicin were 85.7% and 87.0%, respectively. The method was successfully applied to the pharmacokinetic studies of oxypeucedanin hydrate and byak-angelicin in dog plasma after oral administration of ethanol extracts from A. dahurica.     

Bioavailability of salvianolic acid B in conscious and freely moving rats

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 microl) was vortex-mixed with 50 microl of internal standard solution (chloramphenicol 10 microg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile-methanol-20mM NaH(2)PO(4) (adjusted to pH 3.5 with H(3)PO(4)) (20:10:70 v/v/v) containing 0.1mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-200 microg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC-MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030+/-565 and 582+/-222 min microg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.