Development and validation of an LC‐MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study

Bullatine A is a diterpenoid alkaloid of Xue‐Shang‐Yi‐Zhi‐Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the treatment of rheumatism and pain. The plasma levels of bullatine A were measured by a rapid and sensitive LC‐MS/MS method. Samples were prepared using acetonitrile precipitation and the separation of bullatine A was achieved on a Capcell Pak MG‐C₁₈ column by isocratic elution using acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) as the mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple‐reaction monitoring of the transitions at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the internal standard. The linearity was found to be within the concentration range of 1.32–440 ng/mL with a lower limit of quantification of 1.32 ng/mL. Only 1.3 min was needed for an each analytical run. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection. Copyright © 2015 John Wiley & Sons, Ltd.     

Novel LC‐ ESI‐MS/MS method for desvenlafaxine estimation human plasma: application to pharmacokinetic study

A simple, sensitive and specific liquid chromatography tandem mass spectrometry (LC‐ESI‐MS/MS) method was developed for the quantification of desvenlafaxine in human plasma using desvenlafaxine d6 as an internal standard (IS). Chromatographic separation was performed using a Thermo‐BDS hypersil C₈ column (50 × 4.6 mm, 3 µm) with an isocratic mobile phase composed of 5 mM ammonium acetate buffer: methanol (20:80, v/v), at a flow rate of 0.80 mL/min. Desvenlafaxine and desvenlafaxine d6 were detected with proton adducts at m/z 264.2/58.1 and 270.2/ 64.1 in multiple reaction monitoring positive mode, respectively. Liquid–liquid extraction was used to extract the drug and the IS. The method was linear over the concentration range 1.001–400.352 ng/mL with a correlation coefficient of ≥0.9994. This method demonstrated intra and inter‐day precision within 0.7–5.5 and 1.9–6.8%, and accuracy within 95.3–107.4 and 93.4–99.5%. Desvenlafaxine was found to be stable throughout the freeze–thaw cycles, bench‐top and long‐term matrix stability studies. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of desvenlafaxine in pharmaceutical dosage forms. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐ESI‐MS/MS method for the determination of hyperoside in beagle dog plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8 µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.0 min. The MS/MS ion transitions monitored were 464.4 → 463.4 for hyperoside and 947.12 → 969.60 for IS. Linear responses were obtained for hyperoside ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were <5.38 and 3.39% and the extraction recovery ranged from 94.39 to 100.78% with an RSD <3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of hyperoside. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of mesalazine in beagle dog plasma and its application to a pharmacokinetic study

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the determination of mesalazine in beagle dog plasma. The plasma samples were prepared by protein precipitation, then the separation of the analyte was achieved on a Waters Spherisorb C₆ column (150 × 4.6 mm, 5 µm) with a mobile phase consisting of 0.2% formic acid in water–methanol (20:80, v/v). The flow rate was set at 1.0 mL/min with a split ratio of 3:2. Mass spectrometric detection was achieved by a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor–product ion transitions at m/z 154 → m/z 108 for mesalazine and m/z 285 → m/z 193 for diazepam (internal standard). The linear calibration curve of mesalazine was obtained over the concentration range 50–30,000 ng/mL. The matrix effect of mesalazine was within ±9.8%. The intra‐ and inter‐day precisions were <7.9% and the accuracy (relative error) was within ±3.5%. The validated method was successfully applied to investigate the pharmacokinetics of mesalazine in healthy beagle dogs after rectal administration of mesalazine suppository. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of an LC‐APCI‐MS/MS method for the determination of phenethyl isothiocyanate in human plasma

Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent present in cruciferous vegetables. This paper describes the development of a method for the determination of PEITC in human plasma by liquid chromatography/tandem mass spectrometry (LC‐MS/MS). Atmospheric‐pressure chemical ionization was found more suitable for ionization of PEITC than electrospray ionization. Because of the lability of PEITC, a combination of low temperature and acidification was applied to minimize the degradation during the sample collection and preparation procedure. A simple protein precipitation with acetonitrile was used for the preparation of plasma samples. The analyte and 1‐phenylpropyl isothiocyanate as internal standard (IS) were subjected to chromatographic analysis on a C₁₈ column (50 × 2.1 mm, 5 µm) using 85% methanol as mobile phase at a flow rate of 0.3 mL/min. The total analysis time for each chromatograph was 3 min and the results were linear over the studied range (5.00–250 ng/mL). The intra‐ and inter‐day precision values were acceptable as per US Food and Drug Administration guidelines. This method was successfully applied in the determination of PEITC concentrations in plasma samples from healthy chinese Volunteers. Copyright © 2014 John Wiley & Sons, Ltd.     

LC‐ESI‐MS/MS analysis and pharmacokinetics of jolkinolide B,a potential antitumor active component isolated from Euphorbia fischeriana,in rat plasma

A simple, specific and reproducible liquid chromatography–electrospray ionization mass spectrometry was developed and validated for the determination of jolkinolide B, a potential antitumor active component isolated from Euphorbia fischeriana, in rat plasma. Chromatographic separation was achieved on a Venusil MP‐C₁₈ column using an isocratic elution. Jolkinolide B and osthole (internal standard) were monitored by positive electrospray ionization in the selected reaction monitoring mode. Good linearity (r² > 0.996) was achieved by a weighted (1/x²) linear least‐squares regression over a concentration range of 6.50–2600 ng/mL. The accuracy and precision of the assay were satisfactory and the method proved to be applicable to pharmacokinetics following a single intravenous bolus injection of jolkinolide B to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Validated LC‐ESI‐MS/MS method for simultaneous quantitation of felodipine and metoprolol in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and specific LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of felodipine (FDP) and metoprolol (MPL) in rat plasma (50 μL) using phenacetin as an internal standard (IS) as per the FDA guidelines. Liquid–liquid extraction method was used to extract the analytes and IS from rat plasma. The chromatographic resolution of FDP, MPL and IS was achieved with a mobile phase consisting of 0.2% formic acid in water–acetonitrile (25:75, v/v) with a time program flow gradient on a C₁₈ column. The total chromatographic run time was 4.0 min and the elution of FDP, MPL and IS occurred at 1.05, 2.59 and 1.65 min, respectively. A linear response function was established for the range of concentrations 0.59–1148 and 0.53–991 ng/mL for FDP and MPL, respectively, in rat plasma. The intra‐ and inter‐day accuracy and precision values for FDP and MPL met the acceptance as per FDA guidelines. FDP and MPL were stable in battery of stability studies viz., bench‐top, auto‐sampler and freeze–thaw cycles. The validated assay was applied to a pharmacokinetic study in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study

A sensitive, specific and simple LC‐MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under multiple‐reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C₁₈ 100 × 2.1 mm column with a mobile phase of water–methanol–0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple‐reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20–10,000 µg/L (r > 0.998) for bivalirudin. The intra‐ and inter‐day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench‐top, freeze–thaw cycles and long‐term stability. The developed assay method was applied to an intravenous administration study in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

Stability‐indicating validation of acitretin and isoacitretin in human plasma by LC‐ESI‐MS/MS bioanalytical method and its application to pharmacokinetic analysis

LC‐ ESI‐ MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin‐d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid–liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin‐d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis‐RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025–753.217 ng/mL for acitretin and 0.394–289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra‐day and inter‐day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of complanatoside A in rat plasma using LC‐MS/MS and its application to a pharmacokinetic study

Complanatoside A is a flavonol glycoside isolated from Astragalus complanatus, and currently it is used as a quality control index for A. complanatus in the 2010 edition of the Chinese Pharmacopoeia. For the first time, a simple and sensitive LC‐MS/MS method was developed for the determination of complanatoside A in rat plasma over the range of 2.3–575 ng/mL. Complanatoside A was extracted from plasma by a protein precipitation procedure, separated by LC and detected by MS/MS in positive electrospray ionization mode. The method was validated for selectivity, carryover, sensitivity, linearity, extraction recovery, matrix effect, accuracy, precision and stability studies. The lower limit of quantification was established at 2.3 ng/mL. Intra‐ and inter‐day precisions (LLOQ, low‐QC, med‐QC and high‐QC) were <7.9%, and accuracies were between 94.0 and 105.1%. Matrix effect was acceptable (97.9–103.0%) and extraction recovery was reproducible (88.5–94.4%). Complanatoside A was stable in the investigated conditions. The method was applied to the pharmacokinetics of complanatoside A in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of piperaquine and its N‐oxidated metabolite in rat plasma using LC‐MS/MS

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N ‐oxidated metabolite (PQ‐M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C₁₈ column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple‐quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0–400.0 ng/mL for PQ and 1.0–50.0 ng/mL for PQ‐M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ‐M, respectively, using 40 μL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co‐administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ‐M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).     

Development of a fast LC‐MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics

A fast and accurate liquid chromatography/tandem mass spectrometric (LC‐MS/MS) assay was first developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 μL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion‐RP 80A column at 30 °C. The mobile phase consisted of methanol and 0.2% formic acid containing 0.2 mM EDTA (60:40, v/v). The flow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor‐to‐parent ion transitions m/z 140.1 → 53.1 for deferiprone and m/z 143.1 → 98.1 for internal standard. A linear range was established from 0.1 to 20 µg/mL. The limit of detection was determined as 0.05 µg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy. Intraday and interday precisions were 4.3–5.5 and 4.6–7.3%, respectively. The recovery of deferiprone was in the range of 80.1–86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of pogostone in rat plasma and its application in pharmacokinetic studies

Pogostone is an important constituent of Pogostemon cablin (Blanco) Benth., and possesses various known bioactivities. A rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the analysis of pogostone in rat plasma using chrysophanol as internal standard (IS). The analytes were extracted with methanol and separated using a reversed‐phase YMC‐UltraHT Pro C₁₈ column. Elution was achieved with a mobile phase consisting of methanol–water (75:25, v/v) for 5 min at a flow rate of 400 μL/min. The precursor/product transitions (m/z) under MS/MS detection with negative electrospray ionization (ESI) were 223.0 → 139.0 and 253.1 → 224.9 for pogostone and IS, respectively. The calibration curve was linear over the concentration range 0.05–160 µg/mL (r = 0.9996). The intra‐ and inter‐day accuracy and precision were within ±10%. The validated method was successfully applied to the preclinical pharmacokinetic investigation of pogostone in rats after intravenous (5, 10 and 20 mg/kg) and oral administration (5, 10 and 20 mg/kg). Finally, the oral absolute bioavailability of pogostone in rats was calculated to be 70.39, 78.18 and 83.99% for 5, 10 and 20 mg/kg, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive LC‐MS/MS method for the determination of osthole in rat plasma: application to pharmacokinetic study

Osthole, a major component isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used in traditional Chinese medicine. We developed and validated a rapid and sensitive LC‐MS/MS method for the quantification of osthole in rat plasma. Sample preparation involved simple liquid–liquid extraction by ethyl acetate after addition of imperatorin as internal standard (IS). The analyte was separated using a C₁₈ column with the mobile phase of methanol–0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The elutes were detected under positive electrospray ionization in multiple reaction monitoring mode. The method was sensitive with 0.5 ng/mL as the lower limit of detection. Good linearity was obtained over the range of 1.0–500.0 ng/mL. The intra and inter‐batch accuracy for osthole in rat plasma samples ranged from 99.5 to 108.1% and the variation was <8.9%. The stability, extraction efficiency and matrix effect were also acceptable. This method was successfully applied to the pharmacokinetic study of osthole in rat after intravenous and oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10‐hydroxynortriptyline (OH‐NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract NTP, OH‐NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH‐NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C₁₈ column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH‐NTP. A linear response function was established for the range of concentrations 1.09–30.0 ng/mL (r > 0.998) for both NTP and OH‐NTP. The intra‐ and inter‐day precision values for NTP and OH‐NTP met the acceptance as per FDA guidelines. NTP and OH‐NTP were stable in a battery of stability studies, i.e. bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly sensitive method for the determination of ropinirole with a lower limit of quantitation of 3.45 pg/mL in human plasma by LC‐ESI‐MS/MS: application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of ropinirole (RPR) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. A solid‐phase process was used to extract RPR and citalopram (internal standard, IS) from human plasma. Chromatographic separation was operated with 0.2% ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Hypurity C₁₈ column with a total run time of 3.2 min. The MS/MS ion transitions monitored were 261.2 → 114.2 for RPR and 325.1 → 209.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 3.45 pg/mL and the linearity was observed from 3.45 to 1200 pg/mL. The intra‐day and inter‐day precisions were in the range of 4.71–7.98 and 6.56–8.31%, respectively. This novel method has been applied to a pharmacokinetic study of RPR in humans. Copyright © 2008 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive and robust LC‐ESI‐MS/MS method for simultaneous quantitation of simvastatin acid,amlodipine and valsartan in human plasma: application to a clinical pharmacokinetic study

A high‐throughput, simple, highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of simvastatin acid (SA), amlodipine (AD) and valsartan (VS) with 500 µL of human plasma using deuterated simvastatin acid as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using electrospray ionization. The assay procedure involved precipitation of SA, AD, VS and IS from plasma with acetonitrile. The total run time was 2.8 min and the elution of SA, AD, VS and IS occurred at 1.81, 1.12, 1.14 and 1.81 min, respectively; this was achieved with a mobile phase consisting of 0.02 m ammonium formate (pH 4.5):acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X‐Terra C₁₈ column. A linear response function was established for the range of concentrations 0.5–50 ng/mL (r > 0.994) for VS and 0.2–50 ng/mL (r > 0.996) for SA and AD. The method validation parameters for all three analytes met the acceptance as per FDA guidelines. This novel method has been applied to human pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of chidamide in rat plasma by LC‐MS and its application to pharmacokinetics study

A sensitive and selective liquid chromatography mass spectrometry method for determination of chidamide in rat plasma was developed. After addition of linezolid as internal standard, protein precipitation by acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB‐C₁₈ (2.1 × 150 mm, 5 µm) column with acetonitrile–0.1% formic acid as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification using target fragment ions m/z 391.5 for chidamide and m/z 338.5 for the IS. Calibration plots were linear over the range of 10–2000 ng/mL for chidamide in rat plasma. The lower limit of quantification for chidamide was 10 ng/mL. The mean recovery of chidamide in plasma was in the range of 86.6–92.1%. The coefficients of variation of intra‐day and inter‐day precision were both <12%. This method is simple and sensitive and was applied successfully in a pharmacokinetic study of chidamide to rats. Copyright © 2013 John Wiley & Sons, Ltd.