Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a high‐performance liquid chromatographic method for the determination of stemoninine in rat plasma after administration of Stemona tuberosa extracts

For the first time, an HPLC method was developed and validated for the determination of stemoninine in plasma after oral and intravenous administration of the extract of the roots of Stemona tuberosa to rats. Plasma samples were analyzed on a Waters reversed‐phase C₁₈ column using a gradient mobile‐phase of eluent A (water containing 0.1% formic acid and 0.2% triethylamine, pH 3.68) and eluent B (acetonitrile–water, 50:50, v/v). The flow rate was 1.0 mL/min and the detector wavelength was 210 nm. The Waters Oasis solid‐phase extraction cartridge was applied for the preparation of plasma samples with high recovery. A good linear relationship was obtained in the concentration range of 1.55–124 µg/mL (r = 0.9995). The limits of quantification and detection were 1.55 and 0.42 µg/mL, respectively. The average recoveries ranged from 91.11 to 96.43% in plasma at stemoninine concentrations of 3.10, 62.0 and 99.2 µg/mL. Intra‐ and inter‐batch coefficient of variations were 3.27–5.37% and 2.49–3.92%, respectively. This method was successfully applied to pharmacokinetic studies after oral and intravenous administration of Stemona tuberosa extract in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

High‐performance liquid chromatography method for determination of carnosic acid in rat plasma and its application to pharmacokinetic study

A sensitive and reproducible high‐performance liquid chromatography method with ultraviolet detection (UV) was developed for the determination of carnosic acid (CA) in rat plasma. After simple acidification and liquid–liquid extraction of plasma samples using gemfibrozil as an internal standard, the supernatant was evaporated to dryness under a gentle stream of nitrogen. The residue was reconstituted in 200 µL before being injected into the chromatographic system. The analysis was performed on a C₁₈ column protected by an ODS guard column using acetonitrile–0.1% phosphoric acid (55:45, v/v) as mobile phase, and the wavelength of the UV detector was set at 210 nm. The calibration curve was linear over the range of 0.265–265.0 µg/mL with a correlation coefficient of 0.9997. The recovery for plasma samples of 0.530, 13.25, 132.5 and 265.0 µg/mL was 72.2, 87.9, 90.4 and 94.7%, respectively. The intra‐day and inter‐day relative standard deviations for the measurements of quality control samples were less than 3.1%. The stability of the plasma samples was also validated. This method was successfully used to study the pharmacokinetics and bioavailability of CA in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous estimation of levodopa,carbidopa and 3‐oxymethyldopa in rat plasma using HPLC‐ECD

The aim of study was to develop a suitable analytical method for simultaneous estimation of levodopa, carbidopa and 3‐O‐methyl dopa in rat plasma. Chromatographic separation of plasma samples was achieved using a reverse‐phase C₁₈ column. The mobile phase used consisted of a mixture of methanol and phosphate buffer (10 mM , pH 3.50) in the ratio of 90:10 v/v. All analytes were estimated by electrochemical detection at +800 mV. The developed method has been validated as per the standard guidelines. Precision study results were found to be satisfactory, with percentage relative standard deviation for repeatability and intermediate precision <3.96 and 6.56%, respectively, for all analytes detected in rat plasma. The developed method in rat plasma was found to be simple, rapid, accurate, precise and specific. The proposed method has been successfully applied for analysis of rat plasma samples obtained during an oral pharmacokinetic study of sustained release pellets of levodopa and carbidopa in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Simultaneous determination of four trace estrogens in feces,leachate, tap and groundwater using solid–liquid extraction/auto solid‐phase extraction and high‐performance liquid chromatography with fluorescence detection

A simple and selective high‐performance liquid chromatography method coupled with fluorescence detection was developed for the simultaneous measurement of trace levels of four estrogens (estrone, estradiol, estriol and 17α‐ethynyl estradiol) in environmental matrices. For feces samples, solid–liquid extraction was applied with a 1:1 v/v mixture of acetonitrile and ethyl acetate as the extraction solvent. For liquid samples (e.g., leachate and groundwater), hydrophobic/lipophilic balanced automated solid‐phase extraction disks were selected due to their high recoveries compared to conventional C₁₈ disks. Chromatographic separations were performed on a reversed‐phase C₁₈ column gradient‐eluted with a 45:55 v/v mixture of acetonitrile and water. The detection limits were down to 1.1 × 10^?2 (estrone), 4.11 × 10^?4 (estradiol), 5.2 × 10^?3 (estriol) and 7.18 × 10^?3 μg/L (17α‐ethynyl estradiol) at excitation/emission wavelengths of 288/310 nm, with recoveries in the range of 96.9 ± 3.2–105.4 ± 3.2% (n = 3). The method was successfully applied to determine estrogens in feces and water samples collected at livestock farms and a major river in Northeast China. We observed relatively high abundance and widespread distribution of all four estrogens in our sample collections, implying the urgency for a comprehensive and intricate investigation of estrogenic fate and contamination in our researched area.     

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of fenofibric acid in rat plasma

A rapid and sensitive LC‐MS/MS method for the quantification of fenofibric acid in rat plasma was developed and validated. Plasma samples were prepared by liquid–liquid extraction with a mixture of N‐hexane–dichloromethane–isopropanol (100:50:5, v/v/v). Isocratic chromatographic separation was performed on a reversed‐phase Discovery C₁₈ column (2.1 × 50 mm, 5 µm). The mobile phase was methanol–water–formic (75:25:0.25, v/v/v). Detection of fenofibric acid and the internal standard (IS) diclofenac acid was achieved by ESI MS/MS in the negative ion mode using m/z 317 → m/z 213 and m/z 294 → m/z 250 transitions, respectively. The method was linear from 0.005 to 1.250 µg/mL when 100 μL plasma was analyzed. The lower limit of quantification was 0.005 µg/mL. The intra‐ and inter‐day precision values were below 8.2%, and accuracy ranged from ?0.9 to 2.1% in all quality control samples. The recovery was 90.3–94.7% and 83.3% for fenofibric acid and IS, respectively. Total run time for each sample analysis was 2.5 min. The validated method was successfully applied to a pharmacokinetic study in six rats after oral administration of fenofibrate, the ester prodrug of fenofibric acid (equivalent to fenofibric acid 5 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid fenofibric acid determination. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantification of berberine and lysergol by HPLC-UV: evidence that lysergol enhances the oral bioavailability of berberine in rats

A sensitive and simple HPLC method was developed for the simultaneous quantification of berberine and lysergol in rat plasma. The chromatographic separation was achieved on a C₁₈ column using isocratic elution with methanol–acetonitrile–0.1% ortho‐phosphoric acid (25:20:55, v/v/v), pH adjusted to 6.5 with triethylamine and detected at a UV wavelength of 230 nm. The extraction of the berberine and lysergol from the rat plasma with methylene chloride resulted in their high recoveries (82.62 and 90.17%). HPLC calibration curves for both berberine and lysergol based on the extracts from the rat plasma were linear over a broad concentration range of 50–1000 ng/mL. The limit of quantification was 50 ng/mL. Intra‐ and inter‐day precisions were <15% and accuracy was 87.12–92.55% for berberine and 87.01–92.26% for lysergol. Stability studies showed that berberine and lysergol were stable in rat plasma for short‐ and long‐term period for sample preparation and analysis. The described method was successfully applied to study the pharmacokinetics of berberine as well as lysergol following oral administration in Sprague–Dawley rats. The results of the study inferred that lysergol improved the oral bioavailability of berberine. Copyright © 2011 John Wiley & Sons, Ltd.     

A simple and rapid high‐performance liquid chromatography method for determination of alendronate sodium in beagle dog plasma with application to preclinical pharmacokinetic study

A simple and rapid high performance liquid chromatographic (HPLC) method for quantifying alendronate in beagle dog plasma was developed, validated and applied to a pharmacokinetic study. The sample preparation involved coprecipitation with CaCl₂ and derivatization with o‐phthalaldehyde. Chromatographic separation was achieved on a Diamonsil? C₁₈ (250 × 4.6 mm, 5 µm) using acetonitrile–0.4% EDTA‐Na₂ (16:84, v/v) containing 0.034% of NaOH as mobile phase. The fluorimetric detector was operated at 339 nm (excitation) and 447 nm (emission). The linearity over the concentration range of 5.00–600 ng/mL for alendronate was obtained and the lower limit of quantification was 5.00 ng/mL. For each level of quality control samples, inter‐day and intra‐day precisions were less than 8.52 and 7.42% and accuracies were less than 9.07%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70 mg alendronate sodium to beagle dogs, the maximum plasma concentration (C_max) and elimination half‐life were 152 ± 27.3 and 1.75 ± 0.267 h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of a hydrophilic paclitaxel derivative, 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma by LC–MS/MS

A LC‐MS/MS method for the determination of a hydrophilic paclitaxel derivative 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma was developed to evaluate the pharmacokinetics of 7‐xylosyl‐10‐deacetylpaclitaxel in the rats. 7‐Xylosyl‐10‐deacetylpaclitaxel and docetaxel (IS for 7‐xylosyl‐10‐deacetylpaclitaxel) were extracted from rat plasma with acetic ether and analyzed on a Hypersil C₁₈ column (4.6 × 150 mm i.d., particle size 5 µm) with the mobile phase of ACN/0.05% formic acid (50:50, v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring mode. The standard curves for 7‐xylosyl‐10‐deacetylpaclitaxel in plasma were linear (r >0.999) over the concentration range of 2.0–1000 ng/mL with a weighting of 1/concentration². The method showed a satisfactory sensitivity (2.0 ng/mL using 50 µL plasma), precision (CV ≤ 10.1%), accuracy (relative error ?12.4 to 12.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma after intravenous administration of 7‐xylosyl‐10‐deacetylpaclitaxel to female Wistar rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of a novel diarylheptanoid (Juglanin B) from green walnut husks (Juglans regia L.) in rat plasma by high‐performance liquid chromatography

A simple and reliable analytical method based on high‐performance liquid chromatography (HPLC) coupled with a diode array detector (DAD) was developed for the determination of a novel diarylheptanoid (Juglanin B) from green walnut husks (Juglans regia L.) in rat plasma using rhoiptelol as an internal standard. Chromatographic separation was carried out on a Sinochrom ODS‐AP C₁₈ column (250 × 4.6 μm i.d., 5 mm) with acetonitrile–10 mM postassium dihydrogen phosphate (pH = 3; 55:45, v/v) as mobile phase, and the detection wavelength was set at 214 nm. The plasma samples were prepared using methanol as protein precipitator. The extraction recovery of Juglanin B ranged from 70.26 to 78.59%, and the calibration curve had a good linearity in the range 0.08–50 μg/mL (r² = 0.9932). The RSDs of intra‐ and inter‐day precision ranged from 1.19 to 4.92% and 4.35 to 4.54%, respectively. The HPLC‐DAD method described is a simple, rapid and reliable method for the determination of Juglanin B level and for use in studies involving pharmacokinetics. Copyright © 2009 John Wiley & Sons, Ltd.     

RP-HPLC Method for Determination of Oseltamivir Phosphate in Capsules and Spiked Plasma

A new, sensitive RP-HPLC method was developed for the determination of oseltamivir phosphate in capsules and plasma. The method was based on the reaction of the drug with 4-chloro-7-nitrobenzofurazan in borate buffer solution of pH 8.50. Isocratic chromatography was performed on a C₁₈ column with acetonitrile–10 mM nitric acid (pH 3, 60 + 40, v/v) as the mobile phase with fluorescence detection (λex: 470 nm, λem: 541 nm). Mexiletine hydrochloride was used as an internal standard. Analytical parameters were evaluated. The calibration range was linear from 50.0–750.0 ng/ml. The mean percentage recovery in capsules and plasma were 99.95% and 95.42%, respectively.     

A rapid and sensitive LC‐MS/MS method for quantification of 3,29‐dibenzoyl rarounitriol in rat plasma: application to a pharmacokinetic study

A rapid, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry was established and validated to assay the concentrations of 3,29‐dibenzoyl rarounitriol in rat plasma. Plasma samples were processed by liquid–liquid extraction with ethyl acetate and separated on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 3 µm) at an isocratic flow rate of 0.5 mL/min using methanol–10 mm ammonium acetate–formic acid (90:10:0.1, v/v/v) as mobile phase. The total run time was 5 min for each sample. MS/MS detection was accomplished in selected reaction monitoring mode with positive electrospray ionization. The calibration curve was linear over the concentration range of 0.125–50 ng/mL with lower limit of quantification of 0.125 ng/mL. The intra‐ and inter‐day precisions were <10.1% in terms of coefficient of variation, and the accuracy was within ±11.7% in terms of relative error. The developed method was successfully applied to a pharmacokinetic study of 3,29‐dibenzoyl rarounitriol following intragastric administration of 3.65 mg/kg to Wistar rats. Copyright © 2015 John Wiley & Sons, Ltd.     

HPLC with fluorescence detection assay of perampanel,a novel AMPA receptor antagonist,in human plasma for clinical pharmacokinetic studies

Perampanel (Fycompa®), a novel α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial‐onset seizures. To support therapeutic drug monitoring, a simple high‐performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C₁₈ column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile–water–acetic acid–sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0–500 ng/mL) and this could be extended to 25 µg/mL by 50‐fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug‐free blank human plasma from six individuals and no interference was observed with co‐medications tested. Intra‐ and inter‐batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel. Copyright © 2015 John Wiley & Sons, Ltd.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

Developing a robust LC–MS/MS method to quantify Zn‐DTPA,a zinc chelate in human plasma and urine

Quantitation of Zn‐DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of Zn‐DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 μm; Shiseido, Ginza, Tokyo, Japan) and a C₁₈ guard column. The mobile phase consisted of methanol–2 mm ammonium formate (pH 6.3)–ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn‐DTPA were 1–100 μg/mL in plasma and 10–2000 μg/mL in urine. The intra‐ and inter‐day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn‐DTPA and the accuracies for QC samples were from −4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn‐DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn‐DTPA. The result showed that the concentration of Zn‐DTPA in urine was about 20 times that in plasma, and Zn‐DTPA was completely (94.7%) excreted through urine in human.     

Development and validation of a UPLC‐MS/MS method for the determination of 7‐hydroxymitragynine,a μ‐opioid agonist,in rat plasma and its application to a pharmacokinetic study

A simple, sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated to determine the concentrations of 7‐hydroxymitragynine in rat plasma. Following a single‐step liquid–liquid extraction of plasma samples using chloroform, 7‐hydroxymitragynine and the internal standard (tryptoline) were separated on an Acquity UPLC^TM BEH C₁₈ (1.7 µm, 2.1 × 50 mm) column using an isocratic elution at a flow rate of 0.2 mL/min. The mobile phase consisted of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile (10:90, v/v). The run time was 2.5 min. The analysis was carried out under the multiple reaction‐monitoring mode using positive electrospray ionization. Protonated ions [M + H]⁺ and their respective product ions were monitored at the following transitions: 415 → 190 for 7‐hydroxymitragynine and 173 → 144 for the internal standard. The calibration curve was linear over the range of 10–4000 ng/mL (r² = 0.999) with a lower limit of quantification of 10 ng/mL. The extraction recoveries ranged from 62.0 to 67.3% at concentrations of 20, 600 and 3200 ng/mL). Intra‐ and inter‐day assay precisions (relative standard deviation) were <15% and the accuracy was within 96.5–104.0%. This validated method was successfully applied to quantify 7‐hydroxymitragynine in rat plasma following intravenous administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a rapid LC-MS/MS method for the determination of JCC76, a novel antitumor agent for breast cancer, in rat plasma and its application to a pharmacokinetics study

JCC76 is a novel nimesulide analog that selectively inhibits the human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cell proliferation and tumor progression. To support further pharmacological and toxicological studies of JCC76, a novel and rapid method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated for the quantification of the compound in rat plasma. A C₁₈ column was used for chromatographic separation, and the mobile phase was aqueous ammonium formate (pH 3.7; 5 mm )–methanol (1:9, v/v) with an isocratic elution. With a simple liquid–liquid extraction procedure using the mixture of methyl tert‐butyl ether–hexane (1:2, v/v), the mean extraction efficiency of JCC76 in rat plasma was determined as 89.5–97.3% and no obvious matrix effect was observed. This method demonstrated a linear calibration range from 0.3 to 100 ng/mL for JCC76 in rat plasma and a small volume of sample consumption. The intra‐ and inter‐assay accuracy and precision were within ±10%. The pharmacokinetics of JCC76 was also profiled using this validated method in rats. In conclusion, this rapid and sensitive method has been proven to effectively quantify JCC76 for pharmacokinetics study. Copyright © 2011 John Wiley & Sons, Ltd.