A sensitive LC–MS/MS‐based bioanalytical method for quantification of salviaflaside and rosmarinic acid in rat plasma and its application in a pharmacokinetic study

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of salviaflaside and rosmarinic acid in rat plasma. Sample preparation was carried out through liquid–liquid extraction with ethyl acetate using curculigoside as internal standard (IS). The analytes were determined by selected reaction monitoring operated in the positive ESI mode. Chromatographic separation was performed on an Agilent Eclipse Plus C₁₈ column (100 × 4.6 mm, 1.8 μm) with a mobile phase consisting of methanol–water–formic acid (50:50:0.1, v/v/v) at a flow rate of 0.3 mL/min. The run time was 1.9 min per sample and the injection volume was 5 μL. The method had an LLOQ of 1.6 ng/mL for salviaflaside and 0.94 ng/mL for rosmarinic acid in plasma. The linear calibration curves were fitted over the range of 1.6–320 ng/mL for salviaflaside and 0.94–188 ng/mL for rosmarinic acid in plasma with correlation coefficients (r²) >0.99. Intra‐ and inter‐day precisions (relative standard deviation) were < 13.5%, and accuracies (relative error) were between −8.6% and 14.5% for all quality control samples. The method was validated and applied to the pharmacokinetics of salviaflaside and rosmarinic acid in plasma after oral administration of Prunella vulgaris extract to rats.     

Rapid high‐performance liquid chromatography–tandem mass spectrometry method for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma and its application in comparative bioavailability study

A rapid high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma using fluoxetine as an internal standard. In the liquid–liquid extraction method, compounds and internal standard were extracted from plasma using methyl tertiary butyl ether as an extraction solvent. The HPLC separation of the analytes was performed on a Zorbax SB‐C₁₈, 50 × 4.6 mm, 5 µm column, using a isocratic elution program using a mobile phase consisting of HPLC‐grade methanol: 5 mm ammonium acetate (80:20 v/v) at a flow‐rate of 1.0 mL/min with a total runtime of 3.0 min. The proposed method has been validated with a linear range of 4–400 ng/mL for venlafaxine and 5–500 ng/mL for O‐desmethyl venlafaxine. The method was applied for a bio‐equivalence study of 75 mg tablets formulation in 32 Indian male healthy subjects under fasting conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of ambroxol and salbutamol in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A rapid, selective and sensitive liquid chromatography–tandem mass spectrometry assay method was developed for simultaneous determination of ambroxol and salbutamol in human plasma using citalopram hydrobromide as internal standard (IS). The sample was alkalinized with ammonia water (33:67, v/v) and extracted by single liquid–liquid extraction with ethyl acetate. Separation was achieved on Waters Acquity UPLC BEH C₁₈ column using a gradient program at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 378.9 → 263.6 (ambroxol), m/z 240.2 → 147.7 (salbutamol) and m/z 325.0 → 261.7 (IS). The total analytical run time was relatively short (3 min). Calibration curves were linear in the concentration range of 0.5–100.0 ng/mL for ambroxol and 0.2–20.0 ng/mL for salbutamol, with intra‐ and inter‐run precision (relative standard deviation) <15% and accuracy (relative error) ranging from 97.7 to 112.1% for ambroxol and from 94.5 to 104.1% for salbutamol. The method was successfully applied in a clinical pharmacokinetic study of the compound ambroxol and salbutamol tablets.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Bioanalysis of tolvaptan,a novel AVP‐V2 receptor antagonist in human plasma by a novel LC‐ESI‐MS/MS method: a pharmacokinetic application in healthy South Indian male subjects

A simple, rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) assay method is proposed for the determination of tolvaptan in human plasma samples using tolvaptan d₇ as internal standard (IS). Analyte and the IS were extracted from 100 μL of human plasma via simple liquid–liquid extraction. The chromatographic separation was achieved on a C₁₈ column using a mixture of methanol and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.05–501 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The intra‐day and inter‐day precision (coefficient of variation) and accuracy results in three validation batches across five concentration levels were well within the acceptance limits. A run time of 2.0 min for each sample made it possible to analyze more samples in a short time, thus increasing the productivity. The proposed method was successfully applied to a pharmacokinetic study of 15 mg and 60 mg tolvaptan tablet formulation in healthy South Indian male subjects under fasting condition. Copyright © 2013 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.     

A highly sensitive method for the quantification of fludrocortisone in human plasma using ultra‐high‐performance liquid chromatography tandem mass spectrometry and its pharmacokinetic application

A simple and high sensitive ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the determination of fludrocortisone in human plasma was developed and validated as per guidelines. The analyte and internal standard (IS), fludrocortisone‐d₅, were extracted from human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a Chromolith RP_18e column using a mixture of acetonitrile and 2 mm ammonium formate (70:30, v/v) as the mobile phase at a flow rate of 0.7 mL/min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The precursors to product ion transitions monitored for fludrocortisone and IS were m/z 381.2 → 343.2 and 386.2 → 348.4, respectively. The assay was validated with linear range of 40–3000 pg/mL. The intra‐ and inter‐day precisions (relative standard deviation) were within 0.49–7.13 and 0.83–5.87%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometric assay for the determination of tetrabenazine and its active metabolites in human plasma: a pharmacokinetic study

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and fully validated for the simultaneous quantification of tetrabenazine and its active metabolites α‐dihydrotetrabenazine and β‐dihydrotetrabenazine in human plasma. Tetrabenazine d7 was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via solid‐phase extraction using C₁₈ solid‐phase extraction cartridges. The reconstituted samples were chromatographed on a Zorbax SB C₁₈ column using a 60:40 (v/v) mixture of acetonitrile and 5 mm ammonium acetate as the mobile phase at a flow rate of 0.8 mL/min. The API‐4000 LC‐MS/MS in multiple reaction‐monitoring mode was used for detection. The calibration curves obtained were linear (r² ≥ 0.99) over the concentration range of 0.01–5.03 ng/mL for tetrabenazine and 0.50–100 ng/mL for α‐dihydrotetrabenazine and β‐dihydrotetrabenazine. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

Development and validation of UPLC‐MS/MS method for determination of domperidone in human plasma and its pharmacokinetic application

A sensitive, rapid and selective ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC‐MS/MS) method was developed for the determination and pharmacokinetic study of domperidone in human plasma. Diphenhydramine was used as the internal standard. Plasma sample pretreatment involved a one‐step liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (3:2, v/v). The analysis was carried out on an Acquity UPLC^TM BEH C₁₈ column. The mobile phase consisted of methanol–water containing 10 mmol/L ammonium acetate and 0.5% (v/v) formic acid (60:40, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionizationsource with positive mode. Each plasma sample was chromatographed within 2.1 min. The standard curves for domperidone were linear (r² ≥ 0.99) over the concentration range of 0.030–31.5 ng/mL with a lower limit of quantification of 0.030 ng/mL. The intra‐ and inter‐day precision (relative standard deviation) values were not higher than 13% and accuracy (relative error) was from ?7.6 to 1.2% at three quality control levels. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of domperidone in healthy Chinese volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of sitagliptin and simvastatin in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and validated for simultaneous quantification of sitagliptin and simvastatin in human plasma. Carbamazepine was used as an internal standard (IS). The analytes and IS were extracted from the human plasma by liquid–liquid extraction technique. The reconstituted samples were chromatographed on an Alltima HP C₁₈ column using an isocratic solvent mixture [acetonitrile–5 mm ammonium acetate (pH 4.5), 85:15 (v/v)] at a flow rate of 1.0 mL/min. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curves obtained were linear (r² ≥ 0.99) over the concentration range of 0.10–501 and 0.05–105 ng/mL for sitagliptin and simvastatin, respectively. The results of the intra‐ and inter‐day precision and accuracy studies were well within the acceptable limits. Both the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 3.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay was successfully applied to a pharmacokinetic study in human volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetic study

A selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry bioanalytical method has been developed for the estimation of cycloserine in human plasma, employing cytosine as the internal standard. The extraction of the analyte was facilitated by solid‐phase extraction using 100 μL of human plasma. The separation was carried out on a BDS Hypersil C₁₈ (150 × 4.6 mm, 5 μm) column using a mixture of 0.2% formic acid in HPLC‐grade water, methanol and acetonitrile (70:15:15, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was linear over the range of 0.20–20 μg/mL with r² > 0.99. Complete validation of the method was performed as per US Food and Drug Administration guidelines and the results met acceptance criteria. Applying the present method, the clinical pharmacokinetics of cycloserine following oral administration of 250 mg cycloserine was studied under fasting conditions. Assay reproducibility was also verified by incurred sample reanalysis.     

A rapid and sensitive liquid chromatography–tandem mass spectrometric assay for moexipril,an angiotensin‐converting enzyme inhibitor in human plasma

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of angiotensin‐converting enzyme inhibitor, moexipril, in human plasma. Benazepril was used as an internal standard (IS). Analyte and IS were extracted from the human plasma by liquid–liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on a C₁₈ column by using a mixture of methanol and 0.1% formic acid buffer (85:15, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.2–204 ng/mL. The multiple reaction‐monitoring mode was used for quantification of ion transitions at m/z 499.4/234.2 and 425.2/351.1 for moexipril and IS, respectively. The results of the intra‐ and inter‐day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 400 plasma samples per day. The proposed method was found to be applicable to clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a rapid and sensitive SPE-LC-MS/MS method for the simultaneous estimation of fluoxetine and olanzapine in human plasma

A high‐performance liquid chromatographic assay with tandem mass spectrometric detection was developed to simultaneously quantify fluoxetine and olanzapine in human plasma. The analytes and the internal standard (IS) duloxetine were extracted from 500 μL aliquots of human plasma through solid‐phase extraction. Chromatographic separation was achieved in a run time of 4.0 min on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of acetonitrile–water containing 2% formic acid (70:30, v/v), at a flow‐rate of 0.5 mL/min. Detection of analytes and internal standard was performed by electrospray ionization tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions monitored for fluoxetine, olanzapine and IS were m/z 310.01 → 147.69, 313.15 → 256.14 and 298.1 → 153.97, respectively. The method was validated over the concentration range of 1.00–150.20 ng/mL for fluoxetine and 0.12–25.03 ng/mL for olanzapine in human plasma. The intra‐batch and inter‐batch precision (%CV) across four quality control levels was ≤6.28% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate plasma concentration in bioequivalence study and therapeutic drug monitoring as well, following combined administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Application of a rapid and selective method for the simultaneous determination of carebastine and pseudoephedrine in human plasma by liquid chromatography–electrospray mass spectrometry for bioequivalence study in Korean subjects

We describe a simple, rapid and sensitive high‐performance liquid chromatography–electrospray ionization tandem mass spectrometric method that was developed for the simultaneous determination of carebastine and pseudoephedrine in human plasma using cisapride as an internal standard. Acquisition was performed in multiple‐reaction monitoring mode by monitoring the transitions: m/z 500.43 > 167.09 for carebastine and m/z 166.04 > 147.88 for pseudoephedrine. The devised method involves a simple single‐step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a C₁₈ reversed‐phase chromatographic column at 0.2 mL/min by isocratic elution with 10 mM ammonium formate buffer–acetonitrile (30:70, v/v; adjusted to pH 3.3 with formic acid). The devised method was validated over 0.5–100 ng/mL of carebastine and 5–1000 ng/mL of pseudoephedrine with acceptable accuracy and precision, and was successfully applied to a bioequivalence study involving a single oral dose (10 mg of ebastine plus 120 mg of pseudoephedrine complex) to healthy Korean volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

A simple,rapid and sensitive liquid chromatography–tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K₂EDTA plasma using levonorgestrel d₆ as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid–liquid extraction. Chromatographic separation was achieved on a Zorbax XDB‐Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile–5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003–200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra‐ and inter‐day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of terbinafine in human plasma using UPLC–MS/MS: Application to a bioequivalence study in healthy subjects

A high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the determination of terbinafine in human plasma. The method employed liquid–liquid extraction of terbinafine and terbinafine‐d7 (used as internal standard) from 100 μL human plasma with ethyl acetate–n‐hexane (80:20, v/v) solvent mixture. Chromatography was performed on a BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile–8.0 mm ammonium formate, pH 3.5 (85:15, v/v) under isocratic elution. For quantitative analysis, MS/MS ion transitions were monitored at m/z 292.2/141.1 and m/z 299.1/148.2 for terbinafine and terbinafine‐d7, respectively, using electrospray ionization in the positive mode. The method was validated according to regulatory guidance for selectivity, sensitivity, linearity, recovery, matrix effect, stability, dilution reliability and ruggedness with acceptable accuracy and precision. The method shows good linearity over the tested concentration range from 1.00 to 2000 ng/mL (r² ≥ 0.9984). The intra‐batch and inter‐batch precision (CV) was 1.8–3.2 and 2.1–4.5%, respectively. The method was successfully applied to a bioequivalence study with 250 mg terbinafine in 32 healthy subjects. The major advantage of this method includes higher sensitivity, small plasma volume for processing and a short analysis time.     

A rapid and sensitive LC-MS/MS method for quantification of donepezil and its active metabolite, 6-o-desmethyl donepezil in human plasma and its pharmacokinetic application

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and fully validated for simultaneous quantification of donepezil and its active metabolite, 6‐o‐desmethyl donepezil in human plasma. Analytes and the internal standard were extracted from human plasma by liquid–liquid extraction technique using a 30:70 v/v mixture of ethyl acetate and n‐hexane. The reconstituted samples were chromatographed on a C₁₈ column by using a 70:30 v/v mixture of acetonitrile and ammonium formate (5 mm , pH 5.0) as the mobile phase at a flow rate of 0.6 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.09–24.2 ng/mL for donepezil and 0.03–8.13 ng/mL for 6‐o‐desmethyl donepezil. The results of the intra‐day and inter‐day precision and accuracy studies were well within the acceptable limits. The proposed method was successfully applied for the estimation of the drug in real time plasma samples for pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.