Enantioselective LC‐ESI‐MS/MS determination of dropropizine enantiomers in rat plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective and reliable LC–ESI‐MS/MS method for direct quantitation of dropropizine enantiomers namely levodropropizine (LDP) and dextrodropropizine (DDP) in rat plasma without the need for derivatization as per regulatory guidelines. Dropropizine enantiomers and carbamazepine (internal standard) were extracted from 50 μL rat plasma using ethyl acetate. LDP and DDP resolved with good baseline separation (R_s = 4.45) on a Chiralpak IG‐3 column. The mobile phase consisted of methanol with 0.05% diethylamine pumped at a flow rate of 0.5 mL/min. Detection and quantitation were done in multiple reaction monitoring mode following the transitions m/z 237 → 160 and 237 → 194 for dropropizine enantiomers and the internal standard, respectively, in the positive ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 3.23–2022 ng/mL for each enantiomer. The intra‐ and inter‐day precisions were in the ranges of 3.38–13.6 and 5.11–13.8 for LDP and 4.19–11.8 and 8.89–10.1 for DDP. Both LDP and DDP were found to be stable under different stability conditions. The method was successfully used in a stereoselective pharmacokinetic study of dropropizine enantiomers in rats following oral administration of racemate dropropizine at 100 mg/kg. The pharmacokinetic results indicate that the disposition of dropropizine enantiomers is not stereoselective and chiral inversion does not occur in rats.     

LC–ESI–MS/MS determination of copanlisib,a novel PI3K inhibitor,in mouse plasma and its application to a pharmacokinetic study in mice

A sensitive, selective and rapid LC–ESI–MS/MS method has been developed and validated for the quantification of copanlisib in mouse plasma using enasidenib as an internal standard (IS) as per regulatory guideline. Copanlisib and the IS were extracted from mouse plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid–acetonitrile; 25:75, v/v) on a HyPURITY C₁₈ column. Copanlisib and the IS eluted at ~0.95 and 2.00 min, respectively. The MS/MS ion transitions monitored were m/z 481.1 → 360.1 and m/z 474.0 → 456.0 for copanlisib and the IS, respectively. The calibration range was 3.59–3588 ng/mL. The intra‐ and inter‐batch accuracy and precision (RE and RSD) across quality controls met the acceptance criteria. Stability studies showed that copanlisib was stable in mouse plasma for one month. This novel method has been applied to a pharmacokinetic study in mice.     

Validated LC–ESI–MS/MS method for the determination of tunicamycin in rat plasma: Application to a pharmacokinetic study

A liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X‐Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC–MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A–D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r²) was >0.99 for all homologs with accuracy 90.7–107.4% and precision 0.74–15.1%. The recovery of homologs was 78.6–90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench‐top for 6 h, for up to three freeze–thaw cycles, in the injector for 24 h and for 1 month at ?80 ° C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.     

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.     

Determination of epacadostat,a novel IDO1 inhibitor in mouse plasma by LC–MS/MS and its application to a pharmacokinetic study in mice

A sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of epacadostat in mouse plasma using tolbutamide as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation. Chromatographic separation was performed on an Atlantis dC₁₈ column using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.90 mL/min. Elution of epacadostat and IS occurred at ~2.41 and 2.51 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 1.07–533 ng/mL. The intra‐ and inter‐day accuracy and precision were in the ranges of 1.81–12.9 and 3.80–11.1%, respectively. This novel method has been applied to a pharmacokinetic study in mice.     

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.     

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 an LC–MS/MS method for the determination of hinokiflavone in rat plasma and its application to a pharmacokinetic study

Hinokiflavone has drawn a lot of attention for its multiple biological activities. In this study, a sensitive and selective method for determination of hinokiflavone in rat plasma was developed for the first time, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Amentoflavone was used as an internal standard. Separation was achieved on a Hypersil Gold C₁₈ column with isocratic elution using methanol–water (65:35, v /v) as mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the negative electrospray mode with selected reaction monitoring was used to detect the transitions of m/z 537 → 284 for hinokiflavone and m/z 537 → 375 for IS. The LOQ was 0.9 ng/mL with a linear range of 0.9–1000 ng/mL. The intra‐ and inter‐day accuracy (RE%) ranged from −3.75 to 6.91% and from −9.20 to 2.51% and the intra‐ and inter‐day precision (RSD) was between 0.32–14.11 and 2.85–10.04%. The validated assay was successfully applied to a pharmacokinetic study of hinokiflavone in rats. The half‐life of drug elimination at the terminal phase was 6.10 ± 1.86 h, and the area under the plasma concentration‐time curve from time zero to the time of last measurable concentration and to infinity values obtained were 2394.42 ± 466.86 and 2541.93 ± 529.85 h ng/mL, respectively.     

Simultaneous determination of evodiamine and its four metabolites in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography tandem mass spectrometry method was validated for simultaneous quantification of evodiamine and its metabolites 10‐hydroxyevodiamine (M1), 18‐hydroxyevodiamine (M2), 10‐hydroxyevodiamine‐glucuronide (M3) and 18‐hydroxy‐ evodiamine‐glucuronide (M4) in rat plasma for the first time. The analytes were extracted with acetonitrile and separated on a C₁₈ column within 3 min. The detection was achieved in positive selected reaction monitoring mode with precursor‐to‐product transitions at m/z 304.1 → 161.1 for evodiamine, m/z 320.1 → 134.1 for M1, m/z 320.1 → 150.1 for M2, m/z 496.2 → 134.1 for M3, m/z 496.2 → 171.1 for M4 and m/z 349.2 → 305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients >0.9991. The lower limits of quantification for evodiamine, M1, M2, M3 and M4 were 0.1, 0.1, 0.1, 0.25 and 0.25 ng mL⁻¹, respectively. Extraction recoveries and matrix effects of the analytes were within the ranges of 84.51–97.21 and 90.13–103.30%, respectively. The accuracy (relative error) ranged from −8.14 to 7.23% while the intra‐ and inter‐day precisions (relative standard deviation) were < 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study will be helpful in understanding the in vivo disposition of evodiamine.     

A sensitive LC–MS/MS method for simultaneous determination of cabozantinib and its metabolite cabozantinib N‐oxide in rat plasma and its application in a pharmacokinetic study

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N‐oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC–MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C₁₈ column (2.1 × 150 mm, 3 μm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500–5000 ng/mL for CBZ and 0.525–2100 ng/mL for CBN. The extraction recoveries were constant and the intra‐ and inter‐batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague–Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose‐proportional increases in exposure.     

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.     

Sensitive method for the determination of rocilinostat in small volume mouse plasma by LC‐MS/MS and its application to a pharmacokinetic study in mice

A highly sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of rocilinostat in small volume mouse plasma (20 μL) using vorinostat as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation procedure with acetonitrile. Chromatography was achieved on Prodigy ODS‐2 column using a binary gradient using mobile phase A (0.2% formic acid in water) and B (acetonitrile) at a flow rate of 0.38 mL/min. The total chromatographic run time was 4.1 min and the elution of rocilinostat and IS occurred at ~3.2 and 2.9 min, respectively. A linear response function was established in the concentration range of 0.28–1193 ng/mL in mouse plasma. The intra‐ and inter‐day accuracy and precisions were in the ranges of 3.12–8.93 and 6.41–11.6%, respectively. This novel method has been applied to a pharmacokinetic study in mice. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative determination of euphol in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C₁₈ short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was <10.83%. This LC‐MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%. Copyright © 2014 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.     

Simultaneous determination of tandospirone and its active metabolite, 1‐[2‐pyrimidyl]‐piperazine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone (TDS) and its active metabolite 1‐[2‐pyrimidyl]‐piperazine (1‐PP) in Sprague–Dawley rat plasma is described. It was employed in a pharmacokinetic study. These analytes and the internal standards were extracted from plasma using protein precipitation with acetonitrile, then separated on a CAPCELL PAK ADME C₁₈ column using a mobile phase of acetonitrile and 5 mm ammonium formate acidified with formic acid (0.1%, v/v) at a total flow rate of 0.4 mL/min. The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. The method was validated to quantify the concentration ranges of 1.000–500.0 ng/mL for TDS and 10.00–500.0 ng/mL for 1‐PP. Total time for each chromatograph was 3.0 min. The intra‐day precision was between 1.42 and 6.69% and the accuracy ranged from 95.74 to 110.18% for all analytes. Inter‐day precision and accuracy ranged from 2.47 to 6.02% and from 98.37 to 105.62%, respectively. The lower limits of quantification were 1.000 ng/mL for TDS and 10.00 ng/mL for 1‐PP. This method provided a fast, sensitive and selective analytical tool for quantification of tandospirone and its metabolite 1‐PP in plasma necessary for the pharmacokinetic investigation.     

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.     

Quantification of β‐eudesmol in rat plasma using LC–MS/MS and its application to a pharmacokinetic study

A sensitive and specific LC–MS/MS assay for determination of β ‐eudesmol in rat plasma was developed and validated. After liquid–liquid extraction with ethyl ether , the analyte and IS were separated on a Capcell Pak C₁₈ column (50 × 2.0 mm, 5 μm) by isocratic elution with acetonitrile—water–formic acid (77.5:22.5:0.1, v /v/v) as the mobile phase at a flow rate of 0.4 mL/min. An ESI source was applied and operated in positive ion mode; a selected reaction monitoring scan was used for quantification by monitoring the precursor–product ion transitions of m/z 245.1 → 163.1 for β ‐eudesmol and m/z 273.4 → 81.2 for IS. Good linearity was observed in the concentration range of 3–900 ng/mL for β ‐eudesmol in rat plasma. Intra‐ and inter‐day precision and accuracy were both within ±14.3%. This method was applied for pharmacokinetic studies after intravenous bolus of 2.0 mg/kg or intragastric administration of 50 mg/kg β ‐eudesmol in rats.     

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.     

LC‐MS/MS assay for the determination of lurasidone and its active metabolite,ID‐14283 in human plasma and its application to a clinical pharmacokinetic study

The authors proposed a sensitive, selective and rapid liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay procedure for the quantification of lurasidone and its active metabolite, i.e. ID‐14283 in human plasma simultaneously using corresponding isotope labeled compounds as internal standards as per regulatory guidelines. After liquid–liquid extraction with tert‐butyl methyl ether, the analytes were chromatographed on a C₁₈ column using an optimized mobile phase composed of 5 mm ammonium acetate (pH 5.0) and acetonitrile (15:85, v/v) and delivered at a flow rate of 1.00 mL/min. The assay exhibits excellent linearity in the concentration ranges of 0.25–100 and 0.10–14.1 ng/mL for lurasidone and ID‐14283, respectively. The precision and accuracy results over five concentration levels in four different batches were well within the acceptance limits. Lurasidone and ID‐14283 were found to be stable in battery of stability studies. The method was rapid with the chromatographic run time 2.5 min, which made it possible to analyze 300 samples in a single day. Additionally, this method was successfully used to estimate the in vivo plasma concentrations of lurasidone and ID‐14283 obtained from a pharmacokinetic study in south Indian male subjects and the results were authenticated by conducting incurred samples reanalysis. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly sensitive LC‐MS/MS method for the determination of S‐citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP₁₈ column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.