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.     

Simultaneous quantitative analysis of phosphocreatine,creatine and creatinine in plasma of children by HPLC–MS/MS method: Application to a pharmacokinetic study in children with viral myocarditis

A simple and rapid HPLC–MS/MS method was developed and validated for simultaneous measurement of phosphocreatine and its metabolites creatine and creatinine in children's plasma. A 50 μL aliquot of plasma was prepared by protein precipitation with acetonitrile–water (1000 μL, 1:1, v/v) followed by separation on a Hypersil Gold C₁₈ column (35°C) with gradient mobile phase consisting of 2 mm ammonium acetate aqueous solution (pH 10) and methanol at a flow rate of 0.3 mL/min and analyzed by mass spectrometry in both positive (phosphocreatine) and negative (creatine and creatinine) ion multiple reaction monitoring mode. Good linearity (r > 0.99) was obtained for the three analytes. The intra‐day and inter‐day values of CV were <5.46% (?13.09% ≤ RE ≤ 2.57%). The average recoveries of the three analytes were 70.9–97.5%. No obvious impact was found for the quantitation of three analytes in normal, hemolyzed and hyperlipemic plasma. In the end, this method was successfully applied to a pharmacokinetic study of phosphocreatine in children (six cases) with viral myocarditis of children after intravenous infusion of 2 g of the test drug. The pharmacokinetc parameters of phosphocreatine/creatine were as follows: t_1/2 0.24/0.83 h, T_max 0.49/0.55 h, C_max 47.34/59.29 μg/mL, AUC_last 17.07/59.63 h μg/mL, AUC_inf 17.16/79.01 h μg/mL and MRT 0.29/0.67 h.     

Liquid chromatography–tandem MS/MS method for simultaneous quantification of paracetamol,chlorzoxazone and aceclofenac in human plasma: An application to a clinical pharmacokinetic study

A facile, fast and specific method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous quantitation of paracetamol, chlorzoxazone and aceclofenac in human plasma was developed and validated. Sample preparation was achieved by liquid–liquid extraction. The analysis was performed on a reversed‐phase C₁₈ HPLC column (5 μm, 4.6 × 50 mm) using acetonitrile–10 mM ammonium formate pH 3.0 (65:35, v/v) as the mobile phase where atrovastatin was used as an internal standard. A very small injection volume (3 μL) was applied and the run time was 2.0 min. The detection was carried out by electrospray positive and negative ionization mass spectrometry in the multiple‐reaction monitoring mode. The developed method was capable of determining the analytes over the concentration ranges of 0.03–30.0, 0.015–15.00 and 0.15–15.00 μg/mL for paracetamol, chlorzoxazone and aceclofenac, respectively. Intraday and interday precisions (as coefficient of variation) were found to be ≤12.3% with an accuracy (as relative error) of ±5.0%. The method was successfully applied to a pharmacokinetic study of the three analytes after being orally administered to six healthy volunteers.     

Validated UPLC‐MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study

A simple and sensitive analytical method based on ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood–brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC^TM BEH C₁₈ column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol–water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0–83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra‐ and inter‐day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1–9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood–brain barrier permeability. Copyright © 2013 John Wiley & Sons, Ltd.     

High-throughput protein precipitation method with 96-well plate for determination of doxepin and nordoxepin in human plasma using LC–MS/MS

The aim of this study was to establish a high-throughput and sensitive LC–MS/MS method for the determination of doxepin and its major active metabolite nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of doxepin. Doxepin and nordoxepin were extracted from human plasma samples by protein precipitation with acetonitrile by using protein precipitation 96-well plates. The analyte was separated using a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm) using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 v:v (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 μL. The detection was performed using a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 280.4 → 107.0 and 283.4 → 235.0 for doxepin and doxepin-D3, respectively, and 266.3 → 106.9 and 269.3 → 235.0 for nordoxepin and nordoxepin-D3, respectively, in positive electrospray ionization mode. The total run time was 3.5 min. The method was validated over a concentration range of 50–10,000 pg/mL using a Triple Quad 4500 MS System (Sciex) for both analytes. The developed and validated method can be successfully used to study the bioequivalence/pharmacokinetics of doxepin and nordoxepin.     

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.     

Development and validation of a sensitive and high‐throughput LC‐MS/MS method for the simultaneous determination of esomeprazole and naproxen in human plasma

A sensitive and high‐throughput LC‐MS/MS method has been developed and validated for the combined determination of esomeprazole and naproxen in human plasma with ibuprofen as internal standard. Solid‐phase extraction was used to extract both analytes and internal standard from human plasma. Chromatographic separation was achieved in 4.0 min on XBridge C₁₈ column using acetonitrile–25 mM ammonium formate (70:30, v/v) as mobile phase. Mass detection was achieved by ESI/MS/MS in negative ion mode, monitoring at m/z 344.19 → 194.12, 229.12 → 169.05 and 205.13 → 161.07 for esomeprazole, naproxen and IS, respectively. The calibration curves were linear from 3.00 to 700.02 ng/mL for esomeprazole and 0.50 to 150.08 ng/mL for naproxen. The intra‐ and inter‐batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. The assay is suitable for measuring accurate esomeprazole and naproxen plasma concentrations in human bioequivalence study following combined administration. Copyright © 2013 John Wiley & Sons, Ltd.     

The simultaneous UPLC–MS/MS determination of emerging drug combination; candesartan and chlorthalidone in human plasma and its application

A novel, precise, sensitive and accurate ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the simultaneous determination of a novel drug combination, candesartan (CAN) and chlorthalidone (CHL), in human plasma. Chromatographic separation was achieved on Waters Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm). Mobile phase consisting of 1 mm ammonium acetate in water–acetonitrile (20:80 v /v) was used. The total chromatographic runtime was 1.9 min with retention times for CAN and CHL at 0.7 and 1.1 min respectively. Ionization and detection of analytes and internal standards was performed on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and negative ionization mode. Quantitation was done to monitor protonated precursor → product ion transition of m /z 439.2 → 309.0 for CAN, 337.0 → 189.8 for CHL and 443.2 → 312.1 for candesartan D4 and 341.0 → 189.8 for chlorthalidone D4. The method was validated over a wide dynamic concentration range of 2.0–540.0 ng/mL for candesartan and 1.0–180.0 ng/mL for chlorthalidone. The validated method was successfully applied for the assay of CAN and CHL in healthy volunteers.     

Validated UHPLC–MS/MS assay for quantitative determination of etoposide,gemcitabine, vinorelbine and their metabolites in patients with lung cancer

A fully valid UHPLC–MS/MS method was developed for the determination of etoposide, gemcitabine, vinorelbine and their metabolites (etoposide catechol, 2′,2′‐difluorodeoxyuridine and 4‐O ‐deacetylvinorelbine) in human plasma. The multiple reaction monitoring mode was performed with an electrospray ionization interface operating in both the positive and negative ion modes per compound. The method required only 100 μL plasma with a one‐step simple de‐proteinization procedure, and a short run time of 7.5 min per sample. A Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) provided chromatographic separation of analytes using a binary mobile phase gradient (A, 0.1% formic acid in acetonitrile, v /v; B, 0.1% formic acid in water, v /v). Linear coefficients of correlation were >0.995 for all analytes. The relative deviation of this method was <10% for intra‐ and inter‐day assays and the accuracy ranged between 86.35% and 113.44%. The mean extraction recovery and matrix effect of all the analytes were 62.07–105.46% and 93.67–105.87%, respectively. This method was successfully applied to clinical samples from patients with lung cancer.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitative bioanalysis of bavachalcone in rat plasma by LC–MS/MS and its application in a pharmacokinetics study

This study aims to develop and validate a simple and sensitive liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for investigating the pharmacokinetic characteristics of bavachalcone. Liquid–liquid extraction was used to prepare plasma sample. Chromatographic separation of bavachalcone and IS was achieved using a Venusil ASB C₁₈ (2.1 × 50 mm, 5 μm) column with a mobile phase of methanol (A)–water (B) (70:30, v /v). The detection and quantification of analytes was performed in selected‐reaction monitoring mode using precursor → product ion combinations of m/z 323.1 → 203.2 for bavachalcone, and m/z 373.0 → 179.0 for IS. Linear calibration plots were achieved in the range of 1–1000 ng/mL for bavachalcone (r ² > 0.99) in rat plasma. The recovery of bavachalcone ranged from 84.1 to 87.0%. The method was precise, accurate and reliable. It was fully validated and successfully applied to pharmacokinetic study of bavachalcone.     

A simple and fast LC–MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma

A simple and fast liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C₁₈ column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate–formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d₅ and TNF-d₇ were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25–500 ng/mlfor TAF and 0.300–15.0 ng/ml for TNF with acceptable accuracy and precision.     

UPLC‐MS/MS assay for the simultaneous quantification of carvedilol and its active metabolite 4′‐hydroxyphenyl carvedilol in human plasma to support a bioequivalence study in healthy volunteers

An ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the simultaneous determination of carvedilol and its pharmacologically active metabolite 4′‐hydroxyphenyl carvedilol in human plasma using their deuterated internal standards (IS). Samples were prepared by solid‐phase extraction using 100 μL human plasma. Chromatographic separation of analytes was achieved on UPLC C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile‐4.0 mm ammonium formate, pH 3.0 adjusted with 0.1% formic acid (78:22, v/v) as the mobile phase. The multiple reaction monitoring transitions for both the analytes and IS were monitored in the positive electrospray ionization mode. The method was validated over a concentration range of 0.05–50 ng/mL for carvedilol and 0.01‐10 ng/mL for 4′‐hydroxyphenyl carvedilol. Intra‐ and inter‐batch precision (% CV) and accuracy for the analytes varied from 0.74 to 3.88 and 96.4 to 103.3% respectively. Matrix effect was assessed by post‐column analyte infusion and by calculation of precision values (coefficient of variation) in the measurement of the slope of calibration curves from eight plasma batches. The assay recovery was within 94–99% for both the analytes and IS. The method was successfully applied to support a bioequivalence study of 12.5 mg carvedilol tablets in 34 healthy subjects. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of peimine and peiminine in rat plasma by LC‐ESI‐MS employing solid‐phase extraction

A simple and reliable LC‐ESI‐MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid‐phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C₁₈ column using 10 mM ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)–acetonitrile (85:15, v/v) as mobile phase. The linear range was 1–100 ng/mL for peimine and peiminine. Intra‐ and inter‐day precisiond were less than 10%. Accuracies were within 85–115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method. Copyright © 2009 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.     

A sensitive non‐aqueous capillary electrophoresis‐mass spectrometric method for multiresidue analyses of β‐agonists in pork

Non‐aqueous capillary electrophoresis–mass spectrometry (NACE‐MS) was developed for trace analyses of β‐agonists (i.e. clenbuterol, salbutamol and terbutaline) in pork. The NACE was in 18 mM ammonium acetate in methanol–acetonitrile–glacial acetic acid (66 : 33 : 1, v/v/v) using a voltage of 28 kV. The hyphenation of CE with a time‐of‐flight MS was performed by electrospray ionization interface employing 5 mM ammonium acetate in methanol–water (80 : 20, v/v) as the sheath liquid at a flow rate of 2 μL/min. Method sensitivity was enhanced by a co‐injection technique (combination of hydrodynamic and electrokinetic injection) using a pressure of 50 mbar and a voltage of 10 kV for 12 s. The method was validated in comparison with HPLC–MS‐MS. The NACE‐MS procedure provided excellent detection limits of 0.3 ppb for all analytes. Method linearity was good (r² > 0.999, in a range of 0.8–1000 ppb for all analytes). Precision showed %RSDs of <17.7%. Sample pre‐treatment was carried out by solid‐phase extraction using mixed mode reversed phase/cation exchange cartridges yielding recoveries between 69 and 80%. The NACE‐MS could be successfully used for the analysis of β‐agonists in pork samples and results showed no statistical differences from the values reported by the Ministry of Public Health, Thailand using HPLC‐MS‐MS method. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of silodosin and its active glucuronide metabolite KMD‐3213G in human plasma by LC–MS/MS for a bioequivalence study

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is described for the simultaneous determination of silodosin (SLD) and its active metabolite silodosin β‐d ‐glucuronide (KMD‐3213G) in human plasma. Liquid–liquid extraction of plasma samples was carried out with ethyl acetate and methyl tert‐butyl ether solvent mixture using deuterated analogs as internal standards. The extraction recoveries of SLD and KMD‐3213G were in the ranges 90.8–93.4 and 87.6–89.9%, respectively. The extracts were analyzed on a Symmetry C₁₈ (50 × 4.6 mm, 5 μm) column under gradient conditions using 10 mm ammonium formate in water and methanol–acetonitrile (40:60, v/v), within 6.0 min. For MS/MS measurements, ionization of the analytes was carried out in the positive ionization mode and the transitions monitored were m/z 496.1 → 261.2 for SLD and m/z 670.2 → 494.1 for KMD‐3213G. The method showed good linearity, accuracy, precision and stability in the range 0.10–80.0 ng/mL for SLD and KMD‐3213G. The IS‐normalized matrix factors obtained were highly consistent, ranging from 0.962 to 1.023 for both analytes. The method was used to support a bioequivalence study of SLD and its metabolite in healthy volunteers after oral administration of 8 mg silodosin capsules.     

CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

An LC–MS/MS assay for the quantitative determination of 2‐pyridyl acetic acid,a major metabolite and key surrogate for betahistine,using low‐volume human K2EDTA plasma

Betahistine is widely used for the treatment of vertigo. Owing to first‐pass metabolism, 2‐pyridyl acetic acid (2PAA, major metabolite of betahistine) was considered as surrogate for quantitation. A specific and sensitive LC–MS/MS method was developed and validated for quantitation of 2PAA using turbo‐ion spray in a positive ion mode. A solid‐phase extraction was employed for the extraction of 2PAA and 2PAA d₆ (IS) from human plasma. Chromatographic separation of analytes was achieved using an ACE CN, 5 μm (50 × 4.6 mm) column with a gradient mobile phase comprising acetonitrile–methanol (90:10% v /v) and 0.7% v/v formic acid in 0.5 mm ammonium trifluoroacetate in purified water (100% v/v). The retention times of 1.15 and 1.17 min for 2PAA and internal standard, respectively, were achieved. Quantitation of 2PAA and internal standard was achieved by monitoring multiple reaction monitoring transition pairs (m /z 138.1 to m /z 92.0 and m /z 142.1 to m /z 96.1, respectively). The developed method was validated for various parameters. The calibration curves of 2PAA showed linearity from 5.0 to 1500 ng/mL, with a lower limit of quantitation of 5.0 ng/mL. The bias and precision for inter‐ and intra‐batch assays were <10%. The developed method was used to support clinical sample analysis.     

Determination of sunitinib and its active metabolite,N‐desethyl sunitinib in mouse plasma and tissues by UPLC‐MS/MS: assay development and application to pharmacokinetic and tissue distribution studies

A simple, sensitive and specific method using ultraperformance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) was developed to determine sunitinib and N‐desethyl sunitinib in mouse plasma and tissues. The analytes were separated by an isocratic mobile phase consisting of acetonitrile and buffer solution (water with 0.1% formic acid and 5 m m ammonium acetate; 40: 60, v/v) running at a flow rate of 0.35 mL/min for 2 min. Quantification was performed using a mass spectrometer by multiple reaction monitoring in positive electrospray ionization mode. The transition was monitored at m/z 399 → 283, m/z 371 → 283 and m/z 327 → 270 for sunitinib, N‐desethyl sunitinib and internal standard, respectively. Calibration curves were linear over concentration ranges of 2–500, 0.5–50 and 1–250 ng/mL for plasma, heart and other biosamples. The method was successfully applied to animal experiments. The pharmacokinetic study indicated that sunitinib was eliminated quickly in mice with a half‐life of 1.2 h; tissue distribution data showed more sunitinib and its metabolite in liver, spleen and lung, which provided reference for further study. Copyright © 2014 John Wiley & Sons, Ltd.