Determination of helicidum and its metabolites in dog plasma by LC/UV/MS/MS and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-beta-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-beta-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid-liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C(18) column (5 microm, 4.6mm i.d. x 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 x 10(-4) to 70.4 x 10(-4) micromol/mL (0.050-2.00 microg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 microg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.     

High performance liquid chromatographic determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel organoselenium compound, in dog plasma using pre-column derivatization and its application in pharmacokinetic study

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.     

Determination of schizandrin in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography-mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C(18) column with the mobile phase of acetonitrile-sodium acetate (10 micromol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005-2.000 microg/mL) (r=0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 microL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 microg/mL. Intra- and inter-day relative standard deviations were 5.95-12.93% and 3.87-14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.     

Rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of fosfomycin in human plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for determination of fosfomycin was developed and validated. Following protein-precipitation, the analyte and internal standard (fudosteine) were separated from human plasma using an isocratic mobile phase on an Ultimate XB-CN column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 137-->79 and m/z 178-->91 was performed to quantify fosfomycin and fudosteine, respectively. The method was linear in the concentration range of 0.10-12.0 microg/mL using 50 microL of plasma. The lower limit of quantification was 0.10 microg/mL. The intra- and inter-day relative standard deviation over the entire concentration range was less than 10.6%. Accuracy determined at three concentrations (0.25, 1.00 and 8.00 microg/mL for fosfomycin) ranged from -1.0% to -4.2% in terms of relative error. Each plasma sample was chromatographed within 5.0 min. The method was successfully used in a bioequivalence study of fosfomycin in human plasma after an oral administration of capsules containing 1.0 g fosfomycin (approximately 1.3g calcium fosfomycin).     

Improved RP-HPLC method to determine neferine in dog plasma and its application to pharmacokinetics

Existing methods to determine neferine, a bisbenzylisoquinline alkaloid, either have no internal standard or lack selectivity, or take longer time. Here an improved reverse-phase high-performance liquid chromatographic (RP-HPLC) method was established in biological samples. The extraction recovery was 90.9% for neferine at concentration level of 0.2 microg/ml and 77.7% for dauricine (the internal standard) at 5 microg/ml in dog plasma, respectively. The linear quantification range of the method was 25-2000 ng/ml in dog plasma, with linear correlation coefficients greater than 0.999. The intra-day and inter-day relative standard deviations (R.S.D.s) for neferine at 50, 200 and 1000 ng/ml levels in dog plasma fell in the range of 3.0-5.4% and 4.3-9.5%, respectively. The RP-HPLC method was successfully applied to a pharmacokinetics study, in which experimental dogs received a single dose of neferine (5 mg/kg i.v. or 10 mg/kg p.o.). The pharmacokinetic result was presented.     

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

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.     

Liquid chromatographic method for the simultaneous determination of cefalexin and trimethoprim in dog plasma and application to the pharmacokinetic studies of a coformulated preparation

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C(18) column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0-100.0 microg/ml for cefalexin and 0.5-50.0 microg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were < or =14.0% for cefalexin and < or =11.4% for trimethoprim, and accuracy (RE) was -1.4% for cefalexin and -3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.     

Selective and rapid liquid chromatography/negative-ion electrospray ionization mass spectrometry method for the quantification of valacyclovir and its metabolite in human plasma

A rapid, sensitive and specific method was developed for the quantification of valacyclovir and acyclovir in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. Valacyclovir, acyclovir and ganciclovir (internal standard) were separated isocratically on a reversed-phase porous graphitized carbon analytical column (2.1 mm x 125.0 mm i.d., particle size 5 microm), using a mobile phase of acetonitrile/water with 0.05% (v/v) diethylamine (50:50, v/v) at a flow rate of 0.15 mL min(-1) in 4.0 min. Detection was performed by negative electrospray ionization using the selected ion monitoring mode of the deprotonated molecular ions at m/z 323.0 for valacyclovir, 224.0 for acyclovir and 254.0 for ganciclovir. The assay had linear calibration curves over the range 0.020-0.800 microg mL(-1) for valacyclovir and 0.100-20.00 microg mL(-1) for acyclovir. Accuracy and precision were within the acceptance limit of 15%. The method was successfully applied to the analysis of plasma samples obtained from patients after oral administration of valacyclovir.     

Measurement of amoxicillin in plasma and gastric samples using high-performance liquid chromatography with fluorimetric detection

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 microg mL(-1)) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid-liquid extraction. A Kromasil ODS 3 microm (150 x 3.2 mm I.D.) column was maintained at 40 degrees C and used with a mobile phase consisting of methanol-water (55:45, v/v). Fluorimetric detection was at an lambda(ex) of 365 nm and an lambda(em) of 445 nm. The limits of quantitation for amoxicillin were 0.1 microg mL(-1) for gastric juice aspirate (500 microL), 0.5 microg mL(-1) for plasma (50 microL) and 0.075 microg g(-1) for gastric tissue (250 mg). The method was linear up to at least 15 microg mL(-1) in gastric juice aspirate, up to 200 microg mL(-1) in plasma and up to 100 microg g(-1) in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5 mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100 mm, 1.7 μm). A gradient program was used, with a 10 mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4 mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Fran?aise des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500 ng/mL for nicotine, 1-1000 ng/mL for cotinine, 2-1000 ng/mL for trans-3'-hydroxycotinine and 1-500 ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.     

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.     

Determination of epimedin C in rat plasma by reversed-phase high-performance chromatography after oral administration of Herba Epimedii extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile -0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05-4.0 microg/mL. The lower limit of quantification was 0.05 microg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.     

A rapid and validated HPLC method to quantify racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction

A HPLC method with UV detection was developed and validated for the determination of thiorphan in human plasma. Nevirapine was used as the internal standard. Separation was performed by a Waters sunfire C18 reversed-phase column maintained at 35 degrees C. The mobile phase was a mixture of 0.05 M phosphate buffer with the pH adjusted to 2.6 and acetonitrile (74:26, v/v) at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with solid-phase extraction cartridges (Oasis HLB 3 mL, 60 mg). The extraction recovery for plasma samples of thiorphan at 0.1, 0.4 and 2.0 microg/mL was 93.5%, 98.2% and 97.8%, respectively. The calibration curve was linear with the correlation coefficient (r) above 0.9998. Linearity was verified over the range of 0.05-4 microg/mL thiorphan in plasma. The limit of quantification (LOQ) is 0.05 microg/mL. The mean accuracy was 92.7-99.6%. The coefficient of variation (precision) in the within- and between-batch was 2.2-8.4% and 4.1-8.1%, respectively. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of thiorphan.     

High-throughput determination of fudosteine in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A 96-well protein precipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 microL) by the methanol (150 microL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 microL supernatant and 100 microL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC-MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol-water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178-->91 and m/z 284-->91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 microg mL(-1), with good linearity (r>0.999) over the linear range of 0.02-10 microg mL(-1). The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 microg mL(-1). The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.     

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.     

Quantitative determination of atractylenolide III in rat plasma by liquid chromatography electrospray ionization mass spectrometry

Atractylenolide III is a major active component in Atractylodes macrocephala. This paper describes a simple, rapid, specific and sensitive method for the quantification of atractylenolide III in rat plasma using a liquid-liquid extraction procedure followed by liquid chromatography mass spectrometric (LC-MS) analysis. A Kromasil 3.5 microm C(18) column (150 mm x 2.00 mm) was used as the analytical column. Linear detection responses were obtained for atractylenolide III concentration ranging from 5 to 500 ng L(-1). The precision and accuracy data, based on intra-day and inter-day variations over 5 days were within 10.29%. The lower limit of quantitation for atractylenolide III was 5 ng mL(-1), using 0.1 mL plasma for extraction and its recoveries were greater than 85% at the low, medium and high concentrations. The method has been successfully applied to a pharmacokinetic study in rats after an oral administration of atractylenolide III with a dose of 20.0 mg kg(-1). With the lower limits of quantification at 5 ng mL(-1) for atractylenolide III, this method was proved to be sensitive enough for the pharmacokinetics study of atractylenolide III.     

Validation of a capillary electrophoresis method for the quantitative determination of free and total apigenin in extracts of Chamomilla recutita

A capillary electrophoretic method for the quantification of free and total apigenin in methanolic, ethanolic and glycolic extracts of Chamomilla recutita L. Rauschert (Asteraceae) is described. The method was validated for measurement of apigenin in the range 5.00-300 microg/mL (r2 = 0.993) and showed coefficients of intra-day (replicability) and inter-day (repeatability) variability of better than 2%. The limits of detection and quantification were 3.80 and 11.5 microg/mL, respectively, and the average recovery was 102.0 +/- 0.8% at three concentration levels of apigenin. Free and total apigenin contents in the extracts were, respectively, determined as 106 and 903 microg/g (methanolic extract), 77 and 817 microg/g (ethanolic extract) and 11.0 and 247 microg/g (glycolic extract).     

A capillary gas chromatography-selected ion monitoring mass spectrometry method for the analysis of atractylenolide I in rat plasma and tissues, and application in a pharmacokinetic study

The aim of this paper is to investigate the characteristics of atractylenolide I (AO-I) in the body by a GC-MS method. All bio-samples were cleared up with a liquid-liquid extraction procedure. The calibration curves were linear within a range of 5-1000 ng/mL for plasma samples, 0.06-16.00 microg/g for cerebellum samples, and 0.03-8.00 microg/g for other tissue samples. The limit of quantification (LOQ) for AO-I was 1.0 ng/mL or 1.0 ng/g (S/N>micro=10) in the bio-samples. In the applications, the main pharmacokinetic parameters were firstly obtained as follows: Tmax=0.37+/-0.19 h, Cmax=0.26+/-0.05 microg/mL, AUC=1.95+/-0.30 microgh/mL and ka=10.08+/-5.60 h(-1). The tissue distribution of AO-I in rats after the oral administration of 50.0mg/kg was from 0.225 to 0.031microg/g with a decreasing tendency in different tissues like liver>kidney>spleen>cerebellum>heart>cerebrum>lung. The protein binding in rat plasma, human plasma and bovine serum albumin was 80.8+/-3.9, 90.6+/-3.1 and 60.9+/-5.1%, respectively.     

An HPLC method for determination of oridonin in rabbits using isopsoralen as an internal standard and its application to pharmacokinetic studies for oridonin-loaded nanoparticles

A simple and sensitive HPLC method has been developed and validated for the determination of oridonin (ORI) in rabbit plasma. A simple liquid-liquid extraction (LLE) method was applied to extract ORI and the internal standard (IS), isopsoralen, from rabbit plasma. Chromatographic separation of ORI and the IS was achieved with a Kromasil C18 5-mum column (250 mm x 4.6 mm) using methanol-water (50:50, v/v) as mobile phase at a flow rate of 1 mL/min. The ultraviolet (UV) detection wavelength was set at 241 nm. The lower limit of quantification (LLOQ) was 0.02 microg/mL. The calibration curves were linear over a concentration range of 0.02-10 microg/mL. The assay accuracy and precision were within the range of 95.1-113.5% and 5.4-8.6%, respectively. This HPLC method was applied successfully to the pharmacokinetic study of ORI-loaded poly(caprolactone)-poly(ethylene oxide)-poly(caprolactone) copolymer nanoparticles (ORI-PCL-PEO-PCL-NP) in rabbits, given as a single intravenous injection at the dose equivalent to 2mg of ORI/kg, and the pharmacokinetic parameters for ORI were compared with a single intravenous injection of a ORI solution at the same dose.