Rapid and sensitive determination of acetylsalicylic acid and salicylic acid in plasma using liquid chromatography–tandem mass spectrometry: application to pharmacokinetic study

A simple and sensitive analytical method using liquid chromatography–tandem mass spectrometry (LC/MS/MS) for determination of acetylsalicylic acid (aspirin, ASA) and its major metabolite, salicylic acid (SA), in animal plasma has been developed and validated. Both ASA and SA in plasma samples containing potassium fluoride were extracted using acetonitrile (protein precipitation) with 0.1% formic acid in it. 6‐Methoxysalicylic acid was used as the internal standard (IS). The compounds were separated on a reversed‐phase column. The multiple reaction monitoring mode was used with ion transitions of m/z 178.9 → 136.8, 137.0 → 93.0 and 167.0 → 123.0 for ASA, SA and IS, respectively. The lower limits of quantification for ASA and SA were 3 and 30 ng/mL, respectively. The developed method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after p.o. and i.v. administration of 1 mg/kg to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Quantification of lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 μm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002–1 μg/mL. The intra‐ and inter‐assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 μL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of doxofylline in human serum: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of doxofylline (DFL) with 300 microL human serum using imipramine as the internal standard (IS). The API-3,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved direct precipitation of DFL and IS from human serum with acetonitrile. The resolution of peaks was achieved with formic acid (pH 2.5): acetonitrile (10:90, v/v) on an Amazon C(18) column. The total chromatographic run time was 3.0 min and the elution of DFL and IS occurred at approximately 1.46 and 2.15 min, respectively. The MS/MS ion transitions monitored were 267.5 --> 181.1 for DFL and 281.1 --> 86.2 for IS. The method was proved to be accurate and precise at linearity range of 1.00-5,000 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 1.00 ng/mL as lower limit of quantitation. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of DFL tablet.     

Simultaneous quantitative analysis of dextromethorphan,dextrorphan and chlorphenamine in human plasma by liquid chromatography–electrospray tandem mass spectrometry

A sensitive and accurate HPLC‐MS/MS method was developed for the simultaneous determination of dextromethorphan, dextrorphan and chlorphenamine in human plasma. Three analytes were extracted from plasma by liquid–liquid extraction using ethyl acetate and separated on a Kromasil 60‐5CN column (3 µm, 2.1 × 150 mm) with mobile phase of acetonitrile–water (containing 0.1% formic acid; 50:50, v/v) at a flow rate of 0.2 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The calibration curve was linear over the range of 0.01–5 ng/mL for dextromethorphan, 0.02–5 ng/mL for dextrorphan and 0.025–20 ng/mL for chlorphenamine. The lower limits of quantification for dextromethorphan, dextrorphan and chlorphenamine were 0.01, 0.02 and 0.025 ng/mL, respectively. The intra‐ and inter‐day precisions were within 11% and accuracies were in the range of 92.9–102.5%. All analytes were proved to be stable during sample storage, preparation and analytic procedures. This method was first applied to the pharmacokinetic study in healthy Chinese volunteers after a single oral dose of the formulation containing dextromethorphan hydrobromide (18 mg) and chlorpheniramine malaeate (8 mg). Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel LC-MS/MS method for simultaneous quantification of tenofovir and lamivudine in human plasma and its application to a pharmacokinetic study

A new, rapid, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous quantification of tenofovir and lamivudine in human plasma using abacavir as an internal standard. An API‐4000 LC‐MS/MS with electrospray ionization was operated in multiple‐reaction monitoring mode for the analysis. The analytes were extracted from plasma by solid‐phase extraction technique using an Oasis HLB cartridge. The reconstituted samples were chromatographed on a Chromolith ROD speed C₁₈ column using a mixture of 0.1% formic acid in water and acetonitrile (90:10 v/v) at a flow‐rate of 1 mL/min. The method was validated as per the FDA guidelines. The calibration curves were found to be linear in the range of 5–600 ng/mL for tenofovir and 25– 4000 ng/mL for lamivudine. The intra‐ and inter‐day precision and accuracy results were well within the acceptable limits. A run time of 2.8 min consumed for each sample made it possible to analyze more samples per day. The proposed assay method was found to be applicable to a pharmacokinetic study in human male volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

LC–MS/MS method for the simultaneous determination of ethyl gallate and its major metabolite in rat plasma

A simple, rapid and sensitive liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed and validated for the determination of ethyl gallate, a pharmacologically active constituent isolated from Lagerstroemia speciosa (Linn.) Pers. This method was used to examine the pharmacokinetics of ethyl gallate and its major metabolite gallic acid in rat plasma using propyl gallate as an internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a Zorbax SB‐C₁₈ column (3.5 μm, 2.1 × 50 mm) with an isocratic mobile phase consisted of methanol–acetonitrile–10 mM ammonium acetate (10 : 25 : 65, v/v/v) containing 0.1% formic acid at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode using the electrospray ionization technique in negative mode. The lower limits of quantification of gallic acid and ethyl gallate of the method were 0.5 and 1.0 ng/mL. The intra‐day and inter‐day accuracy and precision of the assay were less than 8.0%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of ethyl gallate to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Highly sensitive determination of Schisandrin and Schisandrin B in plasma of rats after administration of Wurenchun (Fructus Schisandrae Chinensis Extracts) preparations by LC‐ESI‐MS/MS

A sensitive and specific method based on liquid chromatography‐tandem mass spectrometry using electrospray ionization (LC‐ESI‐MS/MS) has been developed for the determination of Schisandrin and Schisandrin B in rat plasma. A 100 μL plasma sample was extracted by methyl tert‐butyl ether after spiking the samples with nimodipine (internal standard) and performed on an XTerra®MS‐C₁₈ column (150 mm × 2.1 mm, 3.5 μm) with the mobile phase of acetonitrile–water–formic acid (80:20:0.2, v/v) at a flow rate of 0.2 mL/min in a run time of 8.5 min. The lower limit of quantification of the method was 40 ng/mL for Schisandrin and 20 ng/mL for Schisandrin B. The method showed reproducibility with intra‐day and inter‐day precision of less than 13.8% RSD, as well as accuracy, with inter‐ and intra‐assay accuracies between 93.5 and 107.2%. Finally, the LC‐ESI‐MS/MS method was successfully applied to study the pharmacokinetics of Schisandrin and Schisandrin B in rats after administration of Wurenchun commercial formulations to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Enantioselective determination of carboxyl acid amide fungicide mandipropamid in vegetables and fruits by chiral LC coupled with MS/MS

An efficient enantioselective method for the determination of mandipropamid in vegetables and fruits was presented by LC coupled with MS/MS. The mandipropamid residues in samples (potato, pepper, grape, and watermelon) were extracted with acetonitrile containing 1% acetic acid. An aliquot was cleaned up with primary and secondary amine and C₁₈ sorbent. Complete enantioseparation of mandipropamid enantiomers in <4 min was obtained on a Lux Cellulose‐2 column at 25°C using methanol with 0.1% formic acid/0.1% aqueous formic acid solution (85:15, v/v) as mobile phase. Good linearity was obtained over the concentration range of 0.5–250 μg/L for each enantiomer in the standard solution and sample matrix calibration curves. Quantification was achieved using matrix‐matched standard calibration curves. The interday mean recoveries, intraday repeatability, and inter‐day reproducibility varied from 76.4 to 97.1%, 3.4 to 9.4%, and 3.5 to 11.4%, respectively. The limits of quantification for mandipropamid enantiomers in vegetables and fruits were both 1 μg/kg. Moreover, the absolute configuration of mandipropamid enantiomers was determined by the combination of experimental and predicted electronic circular dichroism spectra, and the first eluted enantiomer was confirmed as (R)‐mandipropamid on five chiral columns.     

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.     

Development and validation of a high-sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with chemical derivatization for the determination of ethinyl estradiol in human plasma

An ultra-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of oral contraceptive ethinyl estradiol (EE) was developed and validated over the curve range of 2.5-500 pg/mL using 1 mL of human plasma sample. Ethinyl estradiol and the internal standard, ethinyl estradiol tetra-deuterated (EE-d4), were extracted from the plasma matrix with methyl t-butyl ether, derivatized with dansyl chloride and then back-extracted into hexane. The hexane phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatographic separation was achieved on a Luna C(18) column (50 x 2 mm, 5 micro m) with an isocratic mobile phase of 20:80 (v/v) water:acetonitrile with 1% formic acid. The offline derivatization procedure introduced the easily ionizable tertiary amine function group to EE. This greatly improved analyte sensitivity in electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 2.5 pg/mL. This high sensitivity method can be used for therapeutic drug monitoring or supporting bio-equivalence and drug-drug interaction studies in human subjects.     

Toxicokinetic and bioavailability studies on retrorsine in mice,and ketoconazole-induced alteration in toxicokinetic properties

Retrorsine (RTS) is a toxic retronecine-type pyrrolizidine alkaloid, which is widely distributed. The purpose of this study was to develop a high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for serum RTS determination in mice. Serum samples were deproteinated by acetonitrile, separated on a C₁₈-PFP column and delivered at 0.8 ml/min with an eluting system composed of water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid as mobile phases. RTS and the internal standard S-hexylglutathione (H-GSH) were quantitatively monitored with precursor-to-product transitions of m/z 352.1 → 120.1 and m/z 392.2 → 246.3, respectively. The method showed excellent linearity over the concentration range 0.05–50 μg/ml, with correlation coefficient r² = 0.9992. The extraction recovery was >86.34%, and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <4.99%. The validated LC–MS/MS method was successfully applied to study the toxicokinetic profiles of serum RTS in mice after intravenous, oral administration and co-treated with ketoconazole, which showed that RTS displayed a long half-life (~11.05 h) and good bioavailability (81.80%). Co-administration of ketoconazole (KTZ) increased the peak serum concentration and area under the concentration–time curve and decreased the clearance and mean residence time. Summing up, a new standardized method was established for quantitative determination of RTS in sera.     

Simultaneous determination of atorvastatin,amlodipine, ramipril and benazepril in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A rapid, simple, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin (ATO), amlodipine (AML), ramipril (RAM) and benazepril (BEN) using nevirapine as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple‐reaction monitoring mode using electrospray ionization. Analytes and IS were extracted from plasma by simple liquid–liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on C₁₈ column by pumping 0.1% formic acid–acetonitrile (15:85, v/v) at a flow rate of 1 mL/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26–210 ng/mL for ATO; 0.05–20.5 ng/mL for AML; 0.25–208 ng/mL for RAM and 0.74–607 ng/mL for BEN with mean correlation coefficient of ≥0.99 for each analyte. The intra‐day and inter‐day precision and accuracy results were well with in the acceptable limits. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of YM155, a novel small-molecule survivin suppressant, in dog plasma

This paper describes a sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for determination of the novel survivin suppressant YM155, 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium, which is developed for the treatment of solid tumors. This method uses a liquid-liquid extraction from 0.25 mL of dog plasma. LC separation was carried out on a Genesis Silica column (50 mm x 3.0 mm i.d.) at a flow-rate of 0.5 mL/min. Compounds were eluted using a mobile phase of 5 mm ammonium acetate and 0.1% formic acid in water-0.1% formic acid in acetonitrile, 17:83 (v/v). MS/MS detection was carried out with an MDS-Sciex API3000 triple quadrupole mass spectrometer in positive electrospray ionization mode. The standard curve was linear from 0.05 to 50 ng/mL (r > or = 0.9968). The lower limit of quantitation was 0.05 ng/mL. Good intra- and inter-day assay precision (within 7.4% RSD) and accuracy (within +/-12.3%) were obtained. The extraction recovery was 66.2%. The method was successfully applied to preclinical pharmacokinetic studies in dogs.     

LC–MS/MS method for the simultaneous determination of tenofovir,emtricitabine, elvitegravir and rilpivirine in dried blood spots

A simple, short, and rugged LC–MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine was developed and validated. Dried blood spots were prepared with 25 μL of spiked whole blood. A 3 mm punch was extracted with methanol containing labeled internal standards. Ten microliters was injected into the LC–MS/MS using isocratic mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (45: 55 v/v) at a flow rate of 0.25 mL/min. The method was validated in the range of 10–2000 ng/mL for all four analytes. The intra‐assay accuracy (RE) of the method was −4.73–4.78, 1.35–2.89, −8.89 to −0.49 and − 1.40–1.81 for tenofovir, emtricitabine, elvitegravir and rilpivirine, respectively. The inter‐assay accuracy was within ±15% of nominal and precision (CV) was <15%. The hematocrit effect on quantification was nonsignificant at the tested hematocrit levels (35–70%). The dried blood spot method showed good agreement with the plasma method, and hence can be used as an alternative to plasma method.     

Assessment of pharmacokinetics,bioavailability and protein binding of anacetrapib in rats by a simple high‐performance liquid chromatography–tandem mass spectrometry method

Anacetrapib is a potent and selective CETP inhibitor and is undergoing phase III clinical trials for the treatment of dyslipidemia. A simple and sensitive high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the quantification of anacetrapib in rat plasma was developed and validated using an easily purchasable compound, chlorpropamide, as an internal standard (IS). A minimal volume of rat plasma sample (20 μL) was prepared by a single‐step deproteinization procedure with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed using selected reaction monitoring modes at the mass/charge transitions m/z 638 → 283 for anacetrapib and m/z 277 → 175 for IS. The assay was validated to demonstrate the selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. The lower limit of quantification was 5 ng/mL. This LC‐MS/MS assay was successfully applied in the rat plasma protein binding and pharmacokinetic studies of anacetrapib. The fraction of unbound anacetrapib was determined to be low (ranging from 5.66 to 12.3%), and the absolute oral bioavailability of anacetrapib was 32.7%.     

Quantitation of unbound sunitinib and its metabolite N-desethyl sunitinib (SU12662) in human plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, selective, and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of unbound sunitinib and its active metabolite N‐desethyl sunitinib in plasma. Plasma and post‐dialysis buffer samples were extracted using a liquid–liquid extraction procedure with acetonitrile–n‐butylchloride (1:4, v/v). Chromatographic separation was achieved on a Waters X‐Terra® MS RP₁₈ column with a mobile phase consisting of acetonitrile and water (60:40, v/v) containing formic acid (0.1%, v/v) using an isocratic run, at a flow‐rate of 0.2 mL/min. Analytes were detected by electrospray tandem mass spectrometry in the selective reaction monitoring mode. Linear calibration curves were generated over the ranges 0.1–100 and 0.02–5 ng/mL for sunitinib and 0.2–200 and 0.04–10 ng/mL for N‐desethyl sunitinib in plasma and in phosphate‐buffered solution, respectively. The values for both within‐day and between‐day precision and accuracy were well within the generally accepted criteria for analytical methods. The analytical range was sufficient to determine the unbound and total concentrations of both analytes. The method was applied for measurement unbound concentrations in addition to total concentrations of sunitinib and its metabolite in plasma of a cancer patient receiving 50 mg daily dose. Copyright © 2012 John Wiley & Sons, Ltd.