Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) assay for biperiden in bulk form and pharmaceutical dosage forms

Current compendial (USP) methods of assay for the analysis of biperiden in bulk form and pharmaceutical dosage forms involve the use of titrimetric and spectrophotometric procedures, respectively. These are non-selective and non-stability-indicating techniques. In this work, a stability-indicating high performance liquid chromatographic assay procedure has been developed and validated for biperiden. The liquid chromatographic separation was achieved isocratically on a symmetry C8 column (150 mm x 3.9 mm i.d., 5 microm particle size) using a mobile phase containing methanol-buffer (50:50, v/v, pH 2.50) at a flow rate of 1 ml/min and UV detection at 205 nm. The buffer was composed of sodium dihydrogen phosphate (50 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The method was linear over the concentration range of 0.5-25 microg/ml (r=0.9998) with a limit of detection and quantitation 0.03 and 0.1 microg/ml, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay biperiden in bulk form and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of biperiden and the assay is thus stability-indicating.     

Simultaneous enantiomer determination of 20 (R)- and 20 (S)-ginsenoside-Rg2 in rat plasma after intravenous administration using HPLC method

20 (R,S)-Ginsenoside-Rg2, an anti-shock agent, is prescribed as a racemate. To analyze simultaneously the enantiomers of 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2 in plasma, a simple and reproducible high-performance liquid chromatographic (HPLC) method has been developed. The enantiomeric separation and determination were successfully achieved using a Diamonsil ODS C18 reversed-phase column (5 microm, 250 mm x 4.6 mm) with an RP18 (5 microm) guard column and a mobile phase of MeOH-aq. 4% H3PO4 (65:35, v/v, pH 5.1) with UV detection at 203 nm. Both enantiomers, 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2, were well separated at 14.5 min and 13.6 min, respectively. The linear ranges of the standard curves were 2.0-250 microg/ml. The intra- and inter-day precision (R.S.D.) were 相似文献   

Determination of paeoniflorin in rat hippocampus by high-performance liquid chromatography after intravenous administration of Paeoniae Radix extract

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of paeoniflorin in rat hippocampus was developed in this study. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Zorbax SB-C(18) column, a mobile phase of methanol-water (32:68, v/v), and detection by ultraviolet (UV) absorption at 233 nm. The lower limits of quantitation (LLQ) were 1 microg/ml for paeoniflorin. The calibration curve for paeoniflorin was linear (r = 0.9999) over the concentration range of 1-50 microg/ml. The coefficients of variation of intra- and inter-day assays were 7.00, 0.58, 1.46% and 5.48, 1.79, 1.70% at concentrations of 1, 10, 50 microg/ml, respectively. The recoveries of paeoniflorin from rat hippocampus were 98.28 +/- 2.14, 98.96 +/- 1.48, and 95.34 +/- 0.92 at concentrations of 1, 10 and 50 microg/ml, respectively. Stability studies showed that paeoniflorin was stable at temperatures of 2-8 degrees C in methanol for at least 20 days. The method was applied to determine the time course of paeoniflorin in rat hippocampus, following the administration of a 60 mg/kg i.v. dose of paeoniflorin in Paeoniae Radix extract to a male Wistar rat.     

Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of balofloxacin in human plasma and its pharmacokinetics

A selective and sensitive high performance liquid chromatography-electrospray ionisation-mass spectrometry method has been developed for the determination of balofloxacin (BLFX) in human plasma. The sample preparation was a liquid-liquid extraction, and chromatographic separation was achieved with an Agilent ZORBAX 300SB C18 2.1 mm x 150 mm column using a mobile phase comprised of methanol-water (10 mM CH(3)COONH(4), pH 3.0)=40:60 (v/v). Standard curves were linear (r=0.9992) over the concentration range of 0.03-3 microg/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation (R.S.D.). The limit of detection (LOD) was 0.02 microg/ml. The validated HPLC-electrospray ionization (ESI)-MS method has been used successfully to study balofloxacin pharmacokinetics in healthy volunteers.     

Liquid chromatography method for quantifying D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (D-threo-PPMP) in mouse plasma and liver

A high-performance liquid chromatography (HPLC) method was developed to measure levels of d-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (d-threo-PPMP) in mouse plasma and liver. d-threo-PPMP was measured by HPLC with a Luna Pheny-Hexyl column (5 microm, 250 mm x 4.6 mm) employing UV detection at 210 nm using a mobile phase of potassium phosphate buffer (20mM, pH 3.0)-acetonitrile in a 45:55 (v/v) ratio. d-threo-1-phenyl-2-pentadecanoylamino-3-morpholino-1-propanol (PC15MP) was employed as an internal standard (IS). The lower limit of quantitation (LLOQ) was 0.3 microg/ml. The assay was linear over a concentration range of 0.3-10 microg/ml, with acceptable precision and accuracy. Assayed in plasma, the intra- and inter-day validation for all coefficients of variation (R.S.D.%) were found less than 15%. The method was applied to samples from athymic (nu/nu) mice treated with d-threo-PPMP by intraperitoneal injection. d-threo-PPMP levels of approximately 10-20 microg/ml ( approximately 20-40 microM) in plasma and approximately 45 microg/g in liver were obtained. The present method can be used to quantify d-threo-PPMP in mice for bioavailability and dose-response studies.     

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.     

Determination of ascorbic acid and its related compounds in foods and beverages by hydrophilic interaction liquid chromatography

A new hydrophilic interaction liquid chromatography method for the simultaneous determination of ascorbic acid (AA), erythorbic acid (EA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG) was developed using a diol column with an isocratic solution of acetonitrile-66.7 mM ammonium acetate solution (85:15, v/v) at a detection wavelength of 260 nm. The calibration curves were found to be linear in the range of 1-50 microg/ml for AA and EA and in the range of 2.5-100 microg/ml for AA-2G and AA-2betaG. Detection limits of AA, EA, AA-2G and AA-2betaG were 0.3, 0.3, 0.03 and 0.03 microg/ml, respectively. This method was satisfactorily applied to the determination of AA, EA, AA-2G and AA-2betaG in a fruit, a food and beverages. The results show that the procedure is simple and sensitive and that it can be employed for the simultaneous determination of AA and its related compounds in foods and beverages.     

Validated HPLC method for the determination of gabapentin in human plasma using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and its application to a pharmacokinetic study

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C(18) column using a mixture of 50 mM NaH(2)PO(4) (pH=2.5)-acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05-5 microg/ml of gabapentin in plasma (r(2)>0.999). The within-day and between-day precision values were in the range of 2-5%. The limit of quantification of the method was 0.05 microg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.     

Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane-isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C(8) micro-bore column (250 mm x 2.1 mm i.d.; 3.5 microm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min(-1). The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml(-1) (r(2) > 0.996). The limit of quantitation was 0.50 ng ml(-1). Accuracy and precision were below the acceptance limits of 15%.     

Determination of betaxolol in human aqueous humour by high-performance liquid chromatography with fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the determination of betaxolol in human aqueous humour. Betaxolol and the internal standard metoprolol were extracted with cyclohexane and separated on a reversed-phase column (Luna C(18), 250 x 4.6 mm, 5 microm) with a mobile phase containing acetonitrile-phosphate buffer (40:60, v/v) at a flow-rate of 0.8 ml/min. The column effluent was monitored with a fluorescence detector at 227 nm (excitation) and 301 nm (emission). The retention times for metoprolol and betaxolol were 3.55 and 5.63 min, respectively. The recovery from aqueous humour was found to be 71.6% for betaxolol at 1.25 microg/ml. The within-day and day-to-day accuracy values were in the range of 96.17-105.2% for betaxolol at 0.1, 4 and 12 microg/ml (n=6), within-day and day-to-day precision values were less than 10% for betaxolol at the concentrations given above. The detection limit corresponding to the signal-to-noise ratio of 3:1 was 15 ng/ml. The presented method was suitable for measuring betaxolol levels in human aqueous humour samples obtained from patients after topical administration.     

Bioanalysis of tobramycin for therapeutic drug monitoring by solid-phase extraction and capillary zone electrophoresis

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis (CZE) for the analysis of tobramycin in human serum is presented. An off-line SPE employing a carboxypropyl bonded phase (CBA) cartridge was used for the extraction of tobramycin from human serum. Adsorbed tobramycin was eluted from the CBA cartridge using a mixture of NH(3) (25%, w/v)-methanol (30:70, v/v). After evaporation, the analyte was reconstituted and derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA). The resulting tobramycin-OPA/MPA derivative was purified, and then identified by mass spectrometry. The tobramycin-OPA/MPA derivative was then analysed by CZE with a background electrolyte (BGE) comprising of 30 mM sodium tetraborate pH 10.0-acetonitrile (ACN) (80:20, v/v) with ultraviolet detection at 230 nm. A linear response was observed in the range of 0.3-30 microg/ml with r(2) = 0.992. The sensitivity of the method was determined by its limit of quantitation (LOQ) and limit of detection (LOD) of 0.3 microg/ml and 0.1 microg/ml, respectively. SPE recovery ranged from 68 to 79% at the trough levels to 98% at the peak levels found in serum. Furosemide has been added as internal standard (IS) to improve precision. For the therapeutic range of tobramycin in serum (2-10 microg/ml) the relative standard deviation (R.S.D.) was less than 11% for the entire SPE/CE process. The method demonstrated excellent selectivity as shown by the lack of interference from a total of 20 drugs investigated. The method was then used in therapeutic drug monitoring of patients receiving the drug.     

Efficient HPLC method for the determination of nicarbazin, as dinitrocarbanilide in broiler liver

A simple, fast and reliable HPLC-UV method has been developed for the determination of dinitrocarbanilide residues in broiler liver. Liver samples (2 g) were extracted with two portions of acetonitrile (10 and 5 ml), defatted with hexane and evaporated to dryness under nitrogen. Extracts were reconstituted in acetonitrile-water (70/30, v/v, 500 microl), loaded onto C18 solid phase (SPE) cartridges and eluted with acetonitrile-water (70/30, v/v, 2.5 ml) into clean test-tubes. Extracts were evaporated to dryness and reconstituted in acetonitrile-water (80/20, v/v, 500 microl). An aliquot of the extract was assayed by high performance liquid chromatography (HPLC) with UV detection at 350 nm. The method was validated according to EU guidelines using liver tissues fortified at levels of 100, 200 and 300 microg/kg, with dinitrocarbanilide. The decision limit (CC(alpha)) and the detection capability (CC(beta)) were calculated from the within laboratory repeatability data to be 228 and 266 microg/kg, respectively. The mean recovery was typically >70% and the limits of quantitation was 12.5 microg/kg (based on the lowest standard on the calibration curve).     

The simultaneous separation and determination of six flavonoids and troxerutin in rat urine and chicken plasma by reversed-phase high-performance liquid chromatography with ultraviolet-visible detection

The method of high-performance liquid chromatography (HPLC) with UV-vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm x 4.6 mm, 5.0 microm) maintained at 35.0 degrees C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10-70.00 microg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50-350.00 microg/mL (troxerutin). The detection limits were 0.010-0.050 microg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.     

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.     

Development of simultaneous gas chromatography-mass spectrometric and liquid chromatography-electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine

To prove the intake of recently controlled designer drugs, N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a simple, sensitive and reliable method which allows us to simultaneously detect BZP, TFMPP and their major metabolite in human urine has been established by coupling gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). GC-MS accompanied by trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed after the enzymatic hydrolysis and the solid phase extraction with OASIS HLB, and BZP, TFMPP and their major metabolites, 4'-hydroxy-BZP (p-OH-BZP), 3'-hydroxy-BZP (m-OH-BZP) and 4'-hydroxy-TFMPP (p-OH-TFMPP), have found to be satisfactorily separated on a semi-micro SCX column with acetonitrile-40 mM ammonium acetate buffer (pH 4) (75:25, v/v) as the eluent. The detection limits produced by GC-MS were estimated to be from 50 ng/ml to 1 microg/ml in the scan mode, and from 200 to 500 ng/ml in the selected ion monitoring (SIM) mode. Upon applying the LC-ESI-MS technique, the linear calibration curves were obtained by using the SIM mode for all analytes in the concentration range from 10 ng/ml to 10 microg/ml. The detection limits ranged from 5 to 40 ng/ml in the scan mode, and from 0.2 to 1 ng/ml in the SIM mode. These results indicate the high reliability and sensitivity of the present procedure, and this procedure will be applicable for proof of intake of BZP and TFMPP in forensic toxicology.     

Development and validation of an HPLC-UV method for iodixanol quantification in human plasma

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.     

Simultaneous analysis of docetaxel and the formulation vehicle polysorbate 80 in human plasma by liquid chromatography/tandem mass spectrometry

An analytical procedure for the simultaneous determination of the anticancer agent docetaxel (Taxotere) and its formulation vehicle polysorbate 80 (Tween 80) in human plasma samples is described. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compounds of interest, including the internal standard paclitaxel, was achieved on a reversed-phase Waters X-Terra mass spectrometry (MS) column (50 x 2.1mm internal diameter) packed with a 3.5-microm octadecyl stationary phase, using isocratic elution. Detection of docetaxel and polysorbate 80 was performed using tandem MS detection with electrospray ionization. Validation results indicated that the method is accurate and precise and has lower limits of quantitation of 0.500 nM (approximately 0.4 ng/ml) and 1.00 microg/ml for docetaxel and polysorbate 80, respectively. The method was subsequently used to measure concentrations of docetaxel and polysorbate 80 in plasma samples in support of a project to assess the influence of polysorbate 80 concentrations on the disposition and toxicity profile of docetaxel in cancer patients receiving Taxotere.     

Comparison of a liquid chromatographic method with ultraviolet and ion-trap tandem mass spectrometric detection for the simultaneous determination of sulfadiazine and trimethoprim in plasma from dogs

A method for the simultaneous determination of sulfadiazine and trimethoprim in plasma from Beagle dogs was developed and validated. Samples were deproteinized with acetonitrile and extracted with ethyl acetate. Sulfachloropyridazine and ormethoprim were used as internal standards for the sulfadiazine and trimethoprim analysis, respectively. The chromatography was carried out both on an LC-UV (liquid chromatography-ultraviolet detection) and ion-trap LC-MS(n) (liquid chromatography-mass spectrometric detection) instrument, operating in the positive APCI mode (atmospheric pressure chemical ionization). The purpose of this work was to compare the quantification results of both methods. Both the LC-UV and LC-MS-MS methods were validated for their linearity, accuracy, precision, limit of detection and limit of quantification, according to the requirements defined by the European Community. Calibration curves using plasma fortified between 0.1 and 1 microg/ml of sulfadiazine, 0.1 and 2 microg/ml of trimethoprim, 1 and 20 microg/ml of sulfadiazine showed a good linear correlation (r> or =0.9990, goodness-of-fit< or =8.4%). The results for the accuracy and precision at 1 microg/ml of sulfadiazine and trimethoprim and at 20 microg/ml of sulfadiazine fell within the ranges specified. The limits of quantification of both methods were 0.1 microg/ml. The limits of detection were 0.019 microg/ml of sulfadiazine and 0.024 microg/ml of trimethoprim for the LC-UV method, and 0.020 microg/ml of sulfadiazine and 0.062 microg/ml of trimethoprim for the LC-MS-MS method. The methods have been successfully applied in a pharmacokinetic study to determine the drug concentrations in plasma samples from dogs. A good correlation between the results of both methods was observed (R=0.9724, slope=1.0239, intercept=-0.2080 microg/ml for sulfadiazine and R=0.9357, slope=1.0433, intercept=0.0325 microg/ml for trimethoprim). The precision of both methods was also tested on the results of the same samples using an F-test (alpha=0.05), indicating that both methods did not differ in precision.     

Simultaneous determination and pharmacokinetic studies of dihydromyricetin and myricetin in rat plasma by HPLC-DAD after oral administration of Ampelopsis grossedentata decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination of dihydromyricetin (1) and myricetin (2) in rat plasma after orally administrating the decoction of Ampelopsis grossedentata. Plasma samples were acidified with 0.375% phosphoric acid and extracted with ethyl acetate. Analysis of the extract was performed on reversed-phase C(18) column with a gradient eluent composed of acetonitrile and 0.04% phosphoric acid. The flow rate was kept at 1 ml/min and the detection wavelength was set at 290 and 370 nm for 1 and 2, respectively. The calibration curves were linear in the range of 0.247-4.114 microg/ml and 0.150-2.501 microg/ml for 1 and 2, respectively. The intra-day and inter-day precisions were better than 4.9 and 6.2%, respectively. The limits of detection (LOD) for 1 and 2 in plasma were 21.600 and 52.530 ng/ml, and the limits of quantification (LOQ) were 0.247 and 0.150 microg/ml, respectively. The mean recoveries for 1 and 2 were 92.0 and 93.3%, respectively. The accuracy and precision were well within the acceptable range and R.S.D. of measured rat samples was less than 7.5%. This validated method has been successfully applied in the pharmacokinetics study of dihydromyricetin and myricetin in vivo after orally administrating the decoction of A. grossedentata to rats.