High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.     

A simple HPLC method for the determination of apigenin in mouse tissues

The flavone apigenin occurs in many leafy vegetables and fruits. It has been reported to have cancer chemopreventive efficacy in rodents. An HPLC method described previously for the determination of tricin, the dimethoxy cogener of apigenin, was modified and validated for measurement of apigenin in mouse tissues. Separation was carried out on a Hypersil-BDS C(18) column (4.6 x 250 mm) with an isocratic mobile phase of 55% methanol in 0.1 m ammonium acetate, pH 5.10, containing 0.27 mm disodium ethylenediamine tetraacetic acid. UV detection was at 336 nm, without interference from endogenous tissue compounds. The assay was linear in the range 25-400 ng/mL, 0.25-4 microg/mL and 2.5-40 microg/mL, with r(2) > 0.99 in all cases, for mouse plasma, liver and intestinal mucosa, respectively. Apigenin in mouse plasma, liver and intestinal mucosa was efficiently extracted with 0.1 m acetic acid in acetone. The assay recovery at low, medium and high concentrations was between 94.6 and 131.7% for all biomatrices, with a relative standard deviation of <10%. The lower limit of quantification for plasma was 25 ng/mL with a relative standard deviation of <15%. The method was used to measure the steady-phase apigenin levels in tissues of mice receiving apigenin in their diet.     

Determination of antiviral nucleoside analogues AM365 and AM188 in perfusate and bile of the isolated perfused rat liver using HPLC

Development, validation and application of an HPLC assay for new antiviral nucleoside analogues AM365 and AM188 in isolated perfused rat liver perfusate and bile were performed. An analytical column (Phenosphere-NEXT, 250 x 4.6 mm, C(18), 4 microm, Phenomenex) was used in tandem with a guard column (4 x 3 mm, C(18), Phenomenex) and operated at 25 degrees C. The mobile phase [methanol:10 mmol/L sodium orthophosphate buffer (pH 7.0), 15:85, v/v] was pumped at 1 mL/min. The signal from a diode array detector was collected from 190 to 300 nm. The chromatogram was processed at 220 and 252 nm for AM365 and AM188, respectively. The HPLC method was validated by six intraday and seven interday runs. Standard curves were linear in the range 0.125-8.00 microg/mL for AM365 and AM188, and the lower limit of quantification for AM365 and AM188 was 0.125 microg/mL. Mean interday precision and accuracy of IPL perfusate quality control samples were within 8.8%, and mean intraday precision and accuracy were within 13.1%. The assay has been successfully used in the study of metabolism and disposition of AM365 in the isolated perfused rat liver.     

The determination of raloxifene in rat tissue using HPLC

We report a rapid and reliable HPLC-UV method for determination of raloxifene, a kind of selective estrogen receptor modulator (SERM), in rat tissue. Proteins were precipitated by adding 200 microL of acetonitrile and 50 microL of methanol to 100 microL of the tissue homogenates, following vortex mixing and centrifugation. Separation was carried out on a reversed-phase C(18) column (150 x 4.6 mm, 5 microm) with a mobile phase of acetonitrile:0.05 m ammonium acetate (pH 4.0 +/- 0.1; 33:67, v/v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 289 nm and the temperature of column was kept at 23 degrees C, without interference from endogenous tissue compounds. The calibration curve was linear from 0.0125 to 10.0 microg/mL with correlation coefficient of over 0.994, while the limit of quantification was 0.008 microg/mL. The intra- and inter-day coefficients of variation were less than 10% (RSD). The recovery of assay was between 95.8 and 104.5%. Furthermore, the method was used to measure the concentration of raloxifene in rat tissue after a simple oral dose. The highest level was observed in liver, lung, spleen, then heart and kidney. The lowest level was found in brain. These results suggest that raloxifene distributes rapidly and moderately into tissues such as liver, lung and spleen.     

Determination of zolmitriptan enantiomers in rat liver microsomes by chiral high performance liquid chromatography with fl uorescence detection

A selective chiral high performance liquid chromatographic method was developed and validated to separate and quantify the enantiomers of a new potent selective 5-HT(1B/1D) receptor partial agonist, S-zolmitriptan, and its antipode in rat liver microsomes induced with beta-naphtho flavone. S- and R-zolmitriptan were extracted from rat hepatic microsomal incubates with chloroform/isopropanol (75:25, v/v), and were separated on a narrow-bore enantioselective normal phase Chiralpak AD-H column (250 x 0.46 mm) with hexane-isopropanol-triethylamine (72/28/0.25, v/v/v) as mobile phase and fluorescence detection with emission at 350 nm and excitation at 291 nm. The calibration curves were linear for R- and S-zolmitriptan concentration over the range 0.1-5.0 microg/mL (r = 0.9996 and 0.9999), and the limits of quantitation were 0.1 microg/mL. The metabolism and interaction of the enantiomers of zolmitriptan in treated hepatic microsomes were investigated using chiral HPLC. There was significant difference between the disposition of the S- and R-zolmitriptan when racemic zolmitriptan or single enantiomers of zolmitriptan were incubated for 5, 10 and 20 min, suggesting that the metabolism of zolmitriptan in rat liver microsomes is enantioselective. In addition, there was also a significant difference between the IC(50) of R- to S-zolmitriptan and S- to R-zolmitriptan (IC(50S/R)/IC(50R/S) = 45.2). This indicated that the disposition process favored the S-form of zolmitriptan.     

高效液相色谱-荧光检测法测定罂粟籽和火锅汤料中的罂粟碱

用所建立的高效液相色谱-荧光检测法测定了罂粟籽和火锅汤料中的罂粟碱。采用的色谱柱为RP-C18柱(250 mm×4.6 mm i.d.,5 μm);检测激发波长为285 nm,发射波长为355 nm;流动相为甲醇-0.02 mol/L乙酸铵(体积比为70∶30),流速0.8 mL/min。实验结果表明,罂粟碱的进样量为1×10-4~0.1 μg时其质量浓度与相应峰面积有良好的线性关系,最低检测限(以信噪比大于3计)达到0.02 ng。罂粟籽中罂粟碱的回收率为99.0%～100.8%。方法快速准确,简便灵敏,分离度高,能够满足有关食品中罂粟碱的检测要求。     

Quantification of myrislignan in rat plasma by solid-phase extraction and reversed-phase high-performance liquid chromatography

A rapid and simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of myrislignan in rat plasma after intravenous administration. The analytes extracted from plasma samples by solid-phase extraction were successfully carried out on a Diamonsiltrade mark ODS C(18) column (250 x 4.6 mm i.d., 5 microm) with an RP(18) guard column (8 x 4.6 mm i.d., 5 microm) and a mobile phase of MeOH-H(2)O (4:1, v/v). The UV detector was set at a single wavelength of 270 nm. The linear ranges of the standard curves were 0.5-30.0 microg/mL with the correlation coefficients greater than 0.9992. The lower limits of detection and quantification were 0.1 and 0.3 microg/mL for myrislignan. Intra- and inter-day precisions were 2.4-7.5 and 1.3-5.7%, respectively. The extraction recovery from plasma was more than 90%. This assay method has been successfully used to study the pharmacokinetics of myrislignan in rats.     

Analysis of 3‐methoxypterostilbene in biological fluids by high‐performance liquid chromatography: application to pre‐clinical pharmacokinetics

A method of analysis for 3‐methoxypterostilbene [trans‐3,3′5‐trimethoxy‐4′hydroxystilbene] in biological fluids is necessary to study pharmacokinetics. A novel and simple high‐performance liquid chromatographic method was developed for the determination of 3‐methoxypterostilbene in rat serum and urine. The internal standard, pinosylvin, was added to 0.1 mL serum or urine (serum proteins were precipitated with cold acetonitrile at ?20°C). Separation was achieved with a Phenomenex® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 327 nm. The calibration curves in both matrices were linear ranging from 0.05 to 100 µg/mL, and the mean extraction efficiency was >99%. Precision of the assay for both matrices was <12% (RSD) and was within 13% for all points on the calibration curve. The limit of quantification for this method was 0.05 µg/mL. The assay was successfully applied to a preliminary study of 3‐methoxypterostilbene pharmacokinetics in a rat. Copyright © 2012 John Wiley & Sons, Ltd.     

HPLC method for determination of DRF-4367 in rat plasma: validation and its application to pharmacokinetics in Wistar rats

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of DRF-4367, a novel cyclooxygenase-2 inhibitor in rat plasma. The assay procedure involved simple liquid/liquid extraction of DRF-4367 and internal standard (IS, celecoxib) from plasma into dichloromethane. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C(18) column (4.6 x 250 mm, 5 microm). The mobile phase consisting of 0.01 M potassium dihydrogen ortho-phosphate (pH 3.2) and acetonitrile (40:60, v/v) was used at a flow rate of 1.0 mL/min. The eluate was monitored using an UV detector set at 247 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of DRF-4367 and IS were 6.6 and 11.2 min, respectively. The standard curve for DRF-4367 was linear (r(2) > 0.999) in the concentration range 0.1-20 micro g/mL. Absolute recovery was >86% from rat plasma for both analyte and IS. The lower limit of quantification of DRF-4367 was 0.1 micro g/mL. The inter- and intra-day precisions in the measurement of quality control samples, 0.1, 0.3, 8.0 and 15.0 microg/mL, were in the range 6.93-9.34% relative standard deviation (RSD) and 0.48-6.59% RSD, respectively. Accuracy in the measurement of QC samples was in the range 91.24-109.36% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze-thaw cycles. Stability of DRF-4367 was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats is described.     

A liquid chromatography-mass spectrometry assay method for simultaneous determination of amiodarone and desethylamiodarone in rat specimens

A liquid chromatographic-mass spectrometry (LC/MS) assay method was developed for the determination of amiodarone and desethylamiodarone in rat specimens. Analytes were extracted using liquid-liquid extraction in hexane. The LC/MS system consisted of a Waters Micromass ZQtrade mark 4000 spectrometer with an autosampler and pump. A C(18) 3.5 microm (2.1 x 50 mm) column heated to 45 degrees C was used for separation. The mobile phase consisted of methanol and 0.2% aqueous formic acid pumped at 0.2 mL/min as a linear gradient. Components eluted within 12 min. The concentrations of ethopropazine (internal standard), desethylamiodarone and amiodarone were monitored for m/z of 313.10, combination of 546.9 and 617.73, and 645.83, respectively. In plasma (0.1 mL), linearity was achieved between the peak area ratios and concentrations over the range of 2.5-1000 ng/mL for both amiodarone and desethylamiodarone (r(2) > 0.999). The intraday and interday CV were equal or less than 18%, and mean error was <12%. Similarly, in homogenates containing 0.1 g of rat tissue, linearity was observed in standards ranging from 5 to 5000 ng/g. The method was successfully used to measure tissue and plasma concentrations of drug. The validated lower limit of quantitation was 2.5 ng/mL for drug and metabolite, based on 0.1 mL of plasma.     

LC-MS/MS method for the determination of cynandione A in rat plasma and tissues

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the quantitative determination of cynandione A in rat plasma and tissues. The plasma samples were pretreated by liquid-liquid extraction with ethyl acetate after the internal standard (honokiol) had been spiked. The tissue samples were homogenized with physiological saline and treated further like the plasma samples. The separation was performed using a Zorbax SB-C(18) column (3.5 microm, 2.1 x 100 mm) and a C18 guard column (5 microm, 4.0 x 2.0 mm) with an isocratic mobile phase consisting of methanol-0.1% formic acid (78:22, v/v) at a flow rate of 0.2 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode using the electrospray ionization technique in negative mode. The nominal retention times for cynandione A and honokiol were 1.41 and 2.63 min, respectively. The method was validated within the concentration range 0.2-1000 ng/mL in plasma and homogenized tissue for cynandione A, and the calibration curves were linear with correlation coefficients >0.992. The lower limit of quantification of cynandione A was 0.2 ng/mL. The intra-day and inter-day precision and accuracy of the assay in plasma were less than 14.4%, while the intra-day and inter-day precision and accuracy of the assay in tissue homogenate were less than 14.2%. This method proved to be suitable for study of pharmacokinetics and tissue distribution of cynandione A in rat.     

Development of an HPLC multi-residue method for the determination of ten quinolones in bovine liver and porcine kidney according to the European Union Decision 2002/657/EC

A sensitive multi-residue analytical method was developed for the determination of ten quinolones: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine in bovine liver and porcine kidney. A simple liquid extraction step followed by a solid phase extraction clean up procedure was applied for the extraction of quinolones from liver and kidney tissues. Recoveries of the extraction varied between 82 and 88% for bovine liver and 92 and 95% for porcine kidney. Separation was performed on an ODS-3 PerfectSil Target (250 x 4 mm) 5 microm analytical column at 25 degrees C. The mobile phase consisted of a mixture of TFA 0.1%-CH(3)CN-CH(3)OH, delivered at a flow rate of 1.2 mL/min according to a gradient program. Elution of quinolones and the internal standard (caffeine, 7.5 ng/microL) was complete within 27 min. Photodiode array detection was used for monitoring the eluants at 275 and 255 nm. The method was fully validated according to the European Union Decision 2002/657/EC, determining linearity, selectivity, decision limit, detection capability, accuracy, and precision. The LODs of the specific method of quinolone determination in bovine liver varied between 3 and 7 microg/kg and in porcine kidney between 3 and 4 microg/kg.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

High-performance liquid chromatography analysis of curcumin in rat plasma: application to pharmacokinetics of polymeric micellar formulation of curcumin

A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of curcumin in rat plasma. Plasma was precipitated with acetonitrile after addition of the internal standard (IS), 4-hydroxybenzophenone. Separation was achieved on a Waters muBondapak C(18) column (3.9 x 300 mm, 5 microm) using acetonitrile (55%) and citric buffer, pH 3.0 (45%) as the mobile phase (flow rate = 1.0 mL/min). The UV detection wavelength was 300 and 428 nm for IS and curcumin, respectively. The extraction efficiencies were 97.08, 95.69 and 94.90% for 50, 200 and 1000 ng/mL of curcumin in rat plasma, respectively. The calibration curve was linear over the range 0.02-1 microg/mL with a correlation coefficient of r(2) > 0.999. The intra- and inter-day coefficients of variation were less than 13%, and mean intra- and inter-day errors were less than +/-6% at 50, 200 and 1000 ng/mL of curcumin. This assay was successfully applied to the pharmacokinetic studies of both solubilized curcumin and its polymeric micellar formulation in rats. It was found that polymeric micelles increased the half-life of curcumin 162-fold that of solubilized curcumin and increased the volume of distribution (Vd(ss)) by 70-fold.     

Determination of miglitol in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the determination of miglitol in human plasma using voglibose as internal standard has been developed and validated. Samples of plasma were deproteinated with acetonitrile and washed with dichloromethane before being analyzed by reversed-phase high-performance liquid chromatography (HPLC). Separation was carried out on a short Nucleosil C(18) column (5 microm, 50 x 4.6 mm i.d.) using 10 mmol/L ammonium acetate at 1.0 mL/min as mobile phase. The detector was an Applied Biosystems Sciex API 4000 mass spectrometer using atmospheric pressure chemical ionization (APCI) for ion production. The instrument was operated at unit resolution in the multiple reaction monitoring mode. The assay was linear over the range 5.00-2000 ng/mL with a limit of detection of 1.00 ng/mL. Intra- and inter-day precision were <2.82% and <2.92%, respectively, with accuracy of 93.3-106%. The assay was successfully applied to a clinical pharmacokinetic study of miglitol given as a single oral dose (50 mg) to healthy volunteers.     

Development and validation of a reversed-phase HPLC method for determination of nitrendipine in rat plasma: application to pharmacokinetic studies

A simple and sensitive method for the determination of nitrendipine in rat plasma was developed using high-performance liquid chromatography (HPLC). The procedure involves extraction of nitrendipine in dichloromethane/sodium hydroxide, followed by reversed phase HPLC using a Waters, Spherisorb ODS2 (250 x 4.6 mm, 5 microm) column and UV detection at 238 nm. The retention times of nitrendipine and internal standard (felodipine) were 5.0 min and 7.5 min, respectively. The calibration curves were linear over the range of 5 ng/mL (lower limit of quantification, LOQ) to 200 ng/mL for nitrendipine. The intra- and inter-day coefficients of variation for all criteria of validation were less than 15% over the linearity range. The sensitivity and precision of the method were within the accepted limits (< 15%) throughout the validation period. The present method was also successfully applied for the study of plasma pharmacokinetics of nitrendipine loaded solid lipid nanoparticles (SLN) in rats.     

Liquid chromatography/mass spectrometry for the quantitation of muraglitazar in monkey plasma

A high-performance liquid chromatographic-mass spectrometric (LC/MS) assay was developed and validated for the determination of muraglitazar, a novel alpha/gamma, dual PPAR activator, in monkey plasma. The method utilized trazodone as the internal standard (IS). The extraction scheme involved a simple protein precipitation procedure with the use of a mixture of acetonitrile and methylene chloride. Separation was carried out on a BDS Hypersil C(18) analytical column (2 x 50 mm, 3 microm) and an effective chromatographic separation of muraglitazar (3.31 min) and trazadone (2.27 min) was achieved at a ssow rate of 0.3 mL/min. The mobile phase, used in an isocratic mode, consisted of 90% A (acetonitrile: 0.1% formic acid, 50:50 v/v) and 10% B (acetonitrile: 0.1% formic acid, 95:5 v/v). Detection of muraglitazar and trazodone was by positive ion turbo-ion spray mass spectrometry in the SIM mode. The mass spectrometer was programmed to admit the protonated molecules at m/z 372.0 (IS) and m/z 517.1 (muraglitazar). The standard curve, which ranged from 2 to 500 ng/mL, was fitted to a 1/x weighted linear regression model. The between run precision and within-run precision values of the assay was within 6.2% RSD. The assay accuracy was within 10.0% of the nominal values of the range of QC samples (6.0-400 ng/mL). At the lower limit of quantitation (LLQ) of 2 ng/mL, the deviation of the predicted concentrations from the nominal value of LLQ samples (n = 6) were within +/-16.6%. Muraglitazar was stable in monkey K(3)EDTA plasma for at least three freeze-thaw cycles. The processed samples (spiked samples) were stable for 48 h in auto-sampler at 10 degrees C. The average extraction recoveries of muraglitazar and IS were 83.3 and 91.9%, respectively. The assay was applied to delineate the pharmacokinetic disposition of muraglitazar in monkeys following a single oral dose.     

Determination of bradykinin and arg-bradykinin in rat muscle tissue by microdialysis and capillary column-switching liquid chromatography with mass spectrometric detection

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.     

Simultaneous quantitative analysis of ketanserin and ketanserinol in plasma by RP-HPLC with fluorescence detection

A sensitive and selective high-performance liquid chromatographic assay for the quantification of ketanserin and ketanserinol in human plasma was developed and validated. The procedure involves extraction of ketanserin and ketanserinol from plasma using an Extrelut NT-1 solid-phase extraction column. The chromatograph was equipped with a Hypersil BDS column (100 x 4.5 mm, 3 micro m particle size). Separation was performed with a mixture of acetate buffer 0.01 M, pH 4.9-methanol-acetonitrile (52:40:8, v/v/v). Detection was performed with fluorescence detection (lambda(ex) = 332 nm and lambda(em) = 410 nm). Calibration curves were linear (r(2) = 0.999) in the range 0-400 ng/mL for both ketanserin and ketanserinol. The repeatability coefficient for ketanserin and ketanserinol was 3.1 and 3.0%, respectively. The reproducibility coefficient for ketanserin and ketanserinol was 10.5 and 9.1%, respectively. The limit of quantification for both ketanserin and ketanserinol was 2.0 ng/mL. The mean recovery yield for both ketanserin and ketanserinol was 60%. In an 8 h work day approximately 60 samples, including calibration and reference standards, could be processed.     

Simultaneous stereoselective analysis of naftopidil and O‐desmethyl naftopidil enantiomers in rat feces using an online column‐switching high‐performance liquid chromatography method

A novel online column‐switching chiral high‐performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O‐desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column‐switching system. Analyte extraction was performed on the Capcell Pak mixed‐functional column by acetonitrile–phosphate buffer (pH 7.4; 10 mm ; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol–acetonitrile–acetate buffer (pH 5.3; 5 mm ; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ_ex) and 340 nm (λ_em). The validated method showed a good linearity [22.5–15,000 ng/mL for (+)‐/(?)‐NAF; 35–25,000 ng/mL for (+)‐/(?)‐DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra‐ and inter‐day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)‐NAF. Nonstereoselective excretion of (+)‐ and (?)‐NAF was found in feces, while stereoselective excretion of (+)‐ and (?)‐DMN was observed with higher excretion levels of (+)‐DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers. Copyright © 2014 John Wiley & Sons, Ltd.