A high‐throughput LC–MS/MS method for determination of flunarizine in human plasma: Pharmacokinetic study with different doses

A high‐throughput and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of flunarizine in human plasma. Liquid–liquid extraction under acidic conditions was used to extract flunarizine and flunarizine‐d8 from 100 μL human plasma. The mean extraction recovery obtained for flunarizine was 98.85% without compromising the sensitivity of the method. The chromatographic separation was performed on Hypersil Gold C₁₈ (50 × 2.1 mm, 3 μm) column using methanol–10 mm ammonium formate, pH 3.0 (90:10, v/v) as the mobile phase. A tandem mass spectrometer (API‐5500) equipped with an electrospray ionization source in the positive ion mode was used for detection of flunarizine. Multiple reaction monitoring was selected for quantitation using the transitions, m/z 405.2 → 203.2 for flunarizine and m/z 413.1 → 203.2 for flunarizine‐d8. The validated concentration range was established from 0.10 to 100 ng/mL. The accuracy (96.1–103.1%), intra‐batch and inter‐batch precision (CV ≤ 5.2%) were satisfactory and the drug was stable in human plasma under all tested conditions. The method was used to evaluate the pharmacokinetics of 5 and 10 mg flunarizine tablet formulation in 24 healthy subjects. The pharmacokinetic parameters C_max and AUC were dose‐proportional.     

Simultaneous determination of evobrutinib and its metabolite evobrutinib‐diol in dog plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography hyphenated with electrospray ionization tandem mass spectrometric method (LC–ESI–MS/MS) was developed and validated for simultaneous determination of evobrutinib and evobrutinib‐diol in dog plasma. The plasma sample was processed using acetonitrile and chromatographic separation was carried out on a Waters Acquity BEH C₁₈ column (50 × 2.1 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid and acetonitrile, with an optimized gradient elution at a flow rate of 0.4 mL/min. Detection was accomplished in selective reaction monitoring mode via electrospray ionization interface operated in positive ion mode. The precursor‐to‐product transitions for quantification were m/z 430.2 → 98.1 for evobrutinib, m/z 464.2 → 98.1 for evobrutinib‐diol and m/z 441.2 → 138.1 for ibrutinib (internal standard). The developed assay was linear over the tested concentration ranges with correlation coefficient >0.995. The LLOQ was 0.1 ng/mL for both analytes. The inter‐ and intra‐day precisions were <9.65% and the accuracy ranged from ?3.94 to 6.37%. The extraction recovery was >85.41% and no significant matrix effect was observed. The developed assay was successfully applied to the pharmacokinetic study of evobrutinib and evobrutinib‐diol in dogs after oral administration of evobrutinib at a single dose of 5 mg/kg.     

Overcoming interference of plasma phospholipids using HybridSPE for the determination of trimetazidine by UPLC–MS/MS

An improved, precise and reliable ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the quantification of trimetazidine, using trimetazidine‐d8 as the internal standard (IS). Interference owing to plasma phospholipids during sample preparation was overcome using a hybrid solid‐phase extraction–phospholipid ultra cartridge. The mean extraction recovery of trimetazidine (98.66%) and trimetazidine‐d8 (97.63%) from spiked plasma was consistent and reproducible. Chromatographic analysis was performed on a UPLC Ethylene Bridged Hybrid (BEH) C18 (50 × 2.1 mm, 1.7 μm) column with isocratic elution using acetonitrile–5 mm ammonium formate, pH 3.5 (40:60, v/v) as the mobile phase. The parent → product ion transitions for trimetazidine (m/z 267.1 → 181.1) and trimetazidine‐d8 (m/z 275.2 → 181.1) were monitored on a triple quadrupole mass spectrometer with electrospray ionization functioning in the positive multiple reaction monitoring mode. The linearity of the method was established in the concentration range of 0.05–100 ng/mL for trimetazidine. The intra‐batch and inter‐batch accuracy and precision (CV) were 97.3–103.1 and 1.7–5.3%, respectively. Qualitative and quantitative assessment of matrix effect showed no interference of endogenous/exogenous components. The developed method was used to measure plasma trimetazidine concentration for a bioequivalence study with 12 healthy subjects.     

A validated LC–MS/MS method for the simultaneous determination of thalidomide and its two metabolites in human plasma: Application to a pharmacokinetic assay

An accurate and sensitive LC–MS/MS method for determining thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in human plasma was developed and validated using umbelliferone as an internal standard. The analytes were extracted from plasma (100 μL) by liquid–liquid extraction with ethyl acetate and then separated on a BETASIL C₁₈ column (4.6 × 150 mm, 5 μm) with mobile phase composed of methanol–water containing 0.1% formic acid (70:30, v/v) in isocratic mode at a flow rate of 0.5 mL/min. The detection was performed using an API triple quadrupole mass spectrometer in atmospheric pressure chemical ionization mode. The precursor‐to‐product ion transitions m/z 259.1 → 186.1 for thalidomide, m/z 273.2 → 161.3 for 5‐hydroxy thalidomide, m/z 273.2 → 146.1 for 5′‐hydroxy thalidomide and m/z 163.1 → 107.1 for umbelliferone (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentrations of 10.0–2000.0 ng/mL for thalidomide, 0.2–50.0 ng/mL for 5‐hydroxy thalidomide and 1.0–200.0 ng/mL for 5′‐hydroxy thalidomide. The method was validated with respect to linear, within‐ and between‐batch precision and accuracy, extraction recovery, matrix effect and stability. Then it was successfully applied to estimate the concentration of thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in plasma samples collected from Crohn's disease patients after a single oral administration of thalidomide 100 mg.     

An LC–MS/MS method for quantification of demethylzeylasteral,a novel immunosuppressive agent in rat plasma and the application to a pharmacokinetic study

In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of demethylzeylasteral in rat plasma. Electrospray ionization was operated in the negative ion mode while demethylzeylasteral and oleanolic acid (internal standard) were measured by selected reaction monitoring (demethylzeylasteral: m/z 479.2 → 436.0; oleanolic acid: m/z 454.9 → 407.2). This LC–MS/MS method had good selectivity, sensitivity, accuracy and precision. The pharmacokinetic profiles of demethylzeylasteral were subsequently examined in Wistar rats after oral or intravenous administration.     

Preclinical pharmacokinetic analysis of armillarisin succinate ester in mouse plasma and tissues by LC–MS/MS

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated to determine the concentration of armillarisin succinate ester in mouse plasma and tissues, used for preclinical evaluation. Bavachin was employed as the internal standard. Separation was performed on a 3.5 µm Zorbax SB‐C₁₈ column (30 × 2.1 mm), with a mobile phase consisting of methanol and aqueous 20 mm ammonium acetate. Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via selected ion monitoring in negative scanning mode. Quantification was performed using the transitions m/z 333 → 233 and 323 → 221 for armillarisin succinate ester and internal standard, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic and tissue distribution study after intravenous injection of ASE in mouse in a dose of 10 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma by liquid chromatography–tandem mass spectrometry

A simple and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma (mRCC). After simple one‐step protein precipitation with methanol–acetonitrile (1:1, v/v), all three analytes were separated on an Agilent Zorbax SB‐C₁₈ column using a gradient mobile phase consisting of water (0.1% formic acid)–acetonitrile (0.1% formic acid) at a flow rate of 0.50 mL/min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 399.0 → 326.2, m/z 371.0 → 283.1, m/z 343.0 → 283.1 and m/z 386.3 → 122.2 for sunitinib, M1, M2 and buspirone, respectively. The method was linear over the range of 0.10–100 ng/mL for all three analytes using only 50 μL of plasma and the lower limit of quantifications for the three analytes were all 0.10 ng/mL. The intra‐day and inter‐day precisions were all less than 15% and the accuracies were within the range of ±15%; recoveries were between 85.0 and 115%. The validated method was successfully applied to an explorative pharmacokinetic study of sunitinib in Chinese patients with mRCC following multi‐dose oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU‐48 in rat plasma and its application to a pharmacokinetic study

A specific, sensitive and stable high‐performance liquid chromatographic–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantitative determination of methyl 3‐amino‐6‐methoxythieno [2,3‐b]quinoline‐2‐carboxylate (PU‐48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU‐48 was achieved with a reversed‐phase C₁₈ column (100 × 2.1 mm, 3 μm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple‐quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU‐48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass‐to‐charge ratio (m/z) 289.1 → 229.2 for PU‐48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU‐48 was linear over the concentration range of 0.1–1000 ng/mL (r² > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU‐48 in rats.     

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.     

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS)

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolic profiles and pharmacokinetics of goitrin in rats through liquid chromatography combined with electrospray ionization–tandem mass spectrometry

Several chemical and biological studies have revealed R,S‐goitrin as the main bioactive constituent of Isatis indigotica Fort., responsible for antiviral antiendotoxin activity; however, few pharmacokinetic studies have been conducted. To comprehend the kinetics of R,S‐goitrin and promote its curative application, a rapid and sensitive UHPLC–MS/MS method was developed. The selected reaction monitoring transitions were m/z 130.0 → 70.0 for R,S‐goitrin and m/z 181.1 → 124.0 for the internal standard in a positive‐ion mode. The established UHPLC–MS/MS method achieved good linearity for R,S‐goitrin at 10–2000 ng/mL. The intra‐ and interday accuracy levels were within ±9.7%, whereas the intraday and interday precision levels were <11.3%. The extraction recovery, stability and matrix effect were within acceptable limits. The validated method was successfully applied for the pharmacokinetic analysis of R,S‐goitrin in rats after oral administration. Moreover, a total of six metabolites were structurally identified through UHPLC–Q/TOF–MS. The proposed metabolic pathways of R,S‐goitrin in rats involve demethylation, acetylation, glutathionylation and oxygenation.     

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.     

High‐throughput salting‐out‐assisted liquid–liquid extraction for the simultaneous determination of atorvastatin,ortho‐hydroxyatorvastatin,and para‐hydroxyatorvastatin in human plasma using ultrafast liquid chromatography with tandem mass spectrometry

A high‐throughput, specific, and rapid liquid chromatography with tandem mass spectrometry method was established and validated for the simultaneous determination of atorvastatin and its two major metabolites, ortho‐hydroxyatorvastatin and para‐hydroxyatorvastatin, in human plasma. A simple salting‐out‐assisted liquid–liquid extraction using acetonitrile and a mass‐spectrometry‐friendly salt, ammonium acetate, was employed to extract the analytes from human plasma. A recovery of more than 81% for all analytes was achieved in 1 min extraction time. Chromatographic separation was performed on a Kinetex XB C₁₈ column utilizing a gradient elution starting with a 60% of water solution (1% formic acid), followed by increasing percentages of acetonitrile. Analytes were detected on a tandem mass spectrometer equipped with an electrospray ionization source that was operated in the positive mode, using the transitions of m/z 559.3 → m/z 440.2 for atorvastatin, and m/z 575.3 → m/z 440.2 for both ortho‐ and para‐hydroxyatorvastatin. Deuterium‐labeled compounds were used as the internal standards. The method was validated over the concentration ranges of 0.0200–15.0 ng/mL for atorvastatin and ortho‐hydroxyatorvastatin, and 0.0100–2.00 ng/mL for para‐hydroxyatorvastatin with acceptable accuracy and precision. It was then successfully applied in a bioequivalence study of atorvastatin.     

Determination of tigecycline in human plasma by LC–MS/MS and its application to population pharmacokinetics study in Chinese patients with hospital‐acquired pneumonia

A selective, sensitive and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of tigecycline (TGC) in human plasma, using tigecycline‐d₉ as an internal standard (IS). Analytical samples were prepared using a protein precipitation method coupled with a concentration process. The analyte and IS were separated on a reversed‐phase Waters Acquity UPLC^® BEH‐C₁₈ column (2.1 × 50 mm i.d., 1.7 μm) with a flow rate of 0.25 mL/min. The mobile phase consisted of water, containing 0.2% formic acid (v/v) with 10 mm ammonium formate (A) and acetonitrile (B). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 586.2 → 513.1 and m/z 595.1 → 514.0 for TGC and IS, respectively. The linearity of the method was in the range of 10–5000 ng/mL. Intra‐ and inter‐batch precision (CV) for TGC was <9.27%, and the accuracy ranged from 90.06 to 107.13%. This method was successfully applied to the analysis of samples from hospital‐acquired pneumonia patients treated with TGC, and a validated population pharmacokinetic model was established. This developed method could be useful to predict pharmacokinetics parameters and valuable for further pharmacokinetics/pharmacodynamics studies.     

An LC–MS/MS method for quantitation of cyanidin‐3‐O‐glucoside in rat plasma: Application to a comparative pharmacokinetic study in normal and streptozotocin‐induced diabetic rats

A sensitive and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to determine cyanidin‐3‐O‐glucoside (Cy‐3G) in normal and streptozotocin‐induced diabetic rat plasma. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ (50 × 4.6 mm, 5 μm) column and mass spectrometric analysis was performed using a Thermo Finnigan TSQ Quantum Ultra triple‐quadrupole mass spectrometer coupled with an ESI source in the negative ion mode. Selected reaction monitoring mode was applied for quantification using target fragment ions m/z 447.3 → 285.2 for Cy‐3G and m/z 463.0 → 300.1 for quercetin‐3‐O‐glucoside (internal standard). The calibration curve was linear over the range 3.00–2700 ng/mL (r² ≥ 0.99) with the lower limit of quantitation at 3.00 ng/mL. Intra‐ and inter‐day precision was <14.5% and mean accuracy was from −11.5 to 13.6%. Stability testing showed that Cy‐3G remained stable during the whole analytical procedure. After validation, the assay was successfully used to support a preclinical pharmacokinetic comparison of Cy‐3G between normal and diabetic rats. Results indicated that diabetes mellitus significantly altered the in vivo pharmacokinetic characteristics of Cy‐3G after oral administration in rats.     

Simultaneous determination of three dipeptides (JBP485, Gly–Sar and JBP923) in the cell lysates by liquid chromatography‐tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell

A simple and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of JBP485, Gly–Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly–Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C₁₈ HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water–methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00?5000 nm with coefficient of 0.9968 for Gly–Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra‐ and inter‐day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC‐MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly–Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1. Copyright © 2014 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.     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

A quantitative LC–MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue

Compound 27 {1, 12‐bis[4‐(4‐amino‐6,7‐dimethoxyquinazolin‐2‐yl)piperazin‐1‐yl]dodecane‐1,12‐dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC–MS/MS) has been developed for the quantification of this compound. Liquid–liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse‐phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2–200 ng/mL with the intra‐ and inter‐day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.