Determination of N-methyl-4-isoleucine-cyclosporin (NIM811) in human whole blood by high performance liquid chromatography-tandem mass spectrometry

A liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of N-methyl-4-isoleucine-cyclosporin (NIM811) was developed and validated over the concentration range 1-2500 ng/mL in human whole blood using a 0.05 mL sample volume. NIM811 and the internal standard, d(12)-cyclosporin A (d(12)-CsA), were extracted from blood using MTBE via liquid-liquid extraction. After evaporation of the organic solvent and reconstitution, a 10 microL aliquot of the resulting extract was injected onto the LC-MS/MS system. Chromatographic separation of NIM811 and internal standard was performed using a Waters Symmetry RP-8 (50 x 4.6 mm, 3 microm particle size) column. The mobile phase consists of 10 mm ammonium acetate in water (A) and acetonitrile (B), with 45% B from 0 to 0.2 min, 45 to 85% B from 0.2 to 0.8 min and 85% B from 0.8 to 2.2 min. The total run time was 3.5 min with a flow rate of 0.8 mL/min. The method was validated for sensitivity, linearity, reproducibility, stability, dilution integrity and recovery. The precision and accuracy of quality control samples at low (2.00 ng/mL), medium (20.0 and 400 ng/mL) and high (2000 ng/mL) concentrations were in the range 1.1-4.3% relative standard deviation (RSD) and -2.5-10.0% (bias), respectively, from three validation runs. The method has been used to measure the exposure of NIM811 in human subjects.     

A highly sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma

A highly sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of midazolam and its major metabolites 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma was developed and validated. Stable isotope-labeled midazolam-D(4) and 1'-hydroxymidazolam-D(4) were used as internal standards. Compounds were extracted from 0.5 mL plasma by liquid-liquid extraction with ethyl acetate-heptane (1:4). Chromatography was achieved using a Sunfire C(18) column. The mobile phase was a gradient with 10 m m formic acid in Milli-Q water and methanol at a flow rate of 0.3 mL/min. Total run time was 10 min. Detection was performed using a tandem mass spectrometer with positive electrospray ionization. Calibration curves were linear over the range of 0.10-50.0 ng/mL for midazolam and 0.025-25.0 ng/mL for both metabolites. For all compounds the lower limit of quantification was 0.10 ng/mL. Imprecision was assessed according to the NCCLS EP5-T guideline and was below 10% for all compounds. Mean recoveries were between 94 and 109% for midazolam and its metabolites. The validated method was successfully applied in a pharmacokinetic study investigating in vivo CYP3A-activity in a large cohort of renal allograft recipients using sub-therapeutic doses of midazolam as a drug-probe.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the estimation of nicorandil in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of nicorandil in human plasma. Nicorandil was extracted from human plasma using solid-phase extraction technique. Imipramine was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated for a linear range of 1.0-500.0 ng/mL with a correlation coefficient of > or =0.9993. The intra-run and inter-run precision and accuracy was within 10.0%. The overall recovery for nicorandil was 63.81%. The total run time was just 3.0 min.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of amlodipine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of amlodipine in human plasma. Amlodipine was extracted from human plasma by using a solid-phase extraction technique. Imipramine was used as the internal standard. A Hypersil BDS C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.1-10.0 ng/mL with correlation coefficient >or=0.9990. The intrarun and interrun precision and accuracy were within 10.0%. The overall recovery for amlodipine was 63.67%. Total run time was 3.2 min only.     

A rapid and highly sensitive method for the determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a pre-clinical pharmacokinetic study

A sensitive and specific liquid chromatography-positive electrospray ionization-tandem mass spectrometry method has been developed and validated for the determination of glimepiride (GPD) in human plasma. GPD and the internal standard (IS, glibenclamide) were extracted from a small aliquot of human plasma (200 microL) by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. The compounds were separated on a YMC Propack, C18, 4.6x50 mm column using a mixture of ammonium acetate buffer, acetonitrile and methanol (30:60:10, v/v) as mobile phase at 0.5 mL/min on an API 4000 Sciex mass spectrometer connected to an Agilent HPLC system. Method validation and pre-clinical sample analysis was performed as per FDA guidelines and the results met the acceptance criteria. GPD and IS were detected without any interference from human plasma matrix. The method was proved to be accurate and precise at linearity range of 0.02-100.00 ng/mL with a correlation coefficient of 0.999. The method was robust with a lower limit of quantitation of 0.02 ng/mL. Intra- and inter-day accuracies for GPD were 88.60-113.50 and 96.82-103.93%, respectively. The inter-day precision was better than 12.21%. This method enabled faster and reliable determination of GPD in a pre-clinical study.     

Development and validation of a highly sensitive method for the determination of abiraterone in rat and human plasma by LC-MS/MS-ESI: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of abiraterone (ART) in rat and human plasma with liquid chromatography coupled to tandem mass spectrometry and electrospray ionization in the positive-ion mode. The assay procedure involves extraction of ART and phenacetin (internal standard, IS) from rat and human plasma with a simple protein precipitation extraction process. Chromatographic separation was achieved using an isocratic mobile (10 mm ammonium acetate:acetonitrile, 10:90, v/v) at a flow-rate of 0.70 mL/min on an Atlantis dC(18) column maintained at 40 °C with a total run time of 3.5 min. The MS/MS ion transitions monitored were 350.3 → 156.0 for ART and 180.2 → 110.1 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.20 ng/mL and the linearity range extended from 0.20 to 201 ng/mL. The intra- and inter-day precisions were in the ranges 2.39-10.4 and 4.84-9.53% in rat plasma and 3.82-10.8 and 6.97-8.94% in human plasma.     

Determination of plasma topiramate concentration using LC-MS/MS for pharmacokinetic and bioequivalence studies in healthy Korean volunteers

A rapid, simple and validated liquid chromatography coupled to tandem mass spectrometric method (LC-MS/MS) for topiramate analysis in human plasma has been applied to pharmacokinetic and bioequivalence studies in 24 healthy male Korean volunteers. The procedure involves a simple liquid extraction of topiramate and prednisone (internal standard) with acetonitrile and separation by HPLC equipped with a Capcell Pak C18 column using acetonitrile-0.1% triethylamine (80:20, v/v) as a mobile phase. Detection was carried out on an API 2000 MS system by multiple reactions monitoring mode. The ionization was optimized using ESI(-) and selectivity was achieved by MS/MS analysis, m/z 338.0 --> 77.5 and m/z 357.1 --> 327.2 for topiramate and prednisone, respectively. The method had a total run time of 2.5 min and showed good linearity over a working range of 20-5000 ng/mL in human plasma with a lower limit of quantification of 20 ng/mL. No metabolic compounds were found to interfere with the analysis. The inter-day and intra-day accuracy were in the ranges of 99.24-116.63 and 93.45-108.68%, respectively, and inter-day and intra-day precisions were below 6.24 and 5.25%, respectively. This method was successfully applied for pharmacokinetic and bioequivalence studies by analysis of blood samples taken up to 96 h after an oral administration of 100 mg of topiramate in 24 healthy Korean volunteers.     

Sensitive and rapid liquid chromatography/tandem mass spectrometry assay for the quantification of amlodipine in human plasma

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of amlodipine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase C(18) column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 50-10,000 pg/mL for amlodipine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of amlodipine and the IS from spiked plasma samples were 74.7 +/- 4.6 and 72.1 +/- 2.0%, respectively. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The observed maximum plasma concentration (Cmax) of amlodipine (2.5 mg oral dose) was 1425 pg/mL, time to observed maximum plasma concentration (Tmax) was 8.1 h and elimination half-life (T(1/2)) was 50.1 h.     

Highly sensitive method for the determination of omeprazole in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of omeprazole (OPZ) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves alkalinization of plasma followed by simple liquid-liquid extraction of OPZ and lansoprazole (internal standard, IS) from human plasma with acetonitrile. Chromatographic separation was achieved with 0.01 M ammonium acetate:acetonitrile (40:60, v/v) at a flow rate of 0.25 mL/min on an Inertsil ODS 3 column with a total run time 2.5 min. The MS/MS ion transitions monitored were 346.1 --> 198.1 for OPZ and 370.1 --> 252.1 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 0.05 ng/mL and the linearity was observed from 0.05 to 10.0 ng/mL. The intra-day and inter-day precisions were in the ranges 2.09-8.56 and 5.29-8.19%, respectively. This novel method has been applied to a pharmacokinetic study of OPZ in humans.     

Determination of huperzine A in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers

A simple, sensitive and selective LC-MS-MS method has been developed for the quantification of huperzine A in human plasma. Huperzine A and pseudoephedrine hydrochloride (internal standard) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a C(18) column with a mobile phase consisting of 0.2% formic acid-methanol (15:85, v/v) and detected using a tandem mass spectrometer with an electrospray ionization interface. The lower limit of quantification was 0.0508 ng/mL, and the assay exhibited a linear range of 0.0508-5.08 ng/mL (r = 0.9998). The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test vs reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting means of major pharmacokinetic parameters such as AUC(0-24), AUC(0-infinity), C(max), T(max) and t(1/2) of huperzine A were 16.35 +/- 3.42 vs 16.38 +/- 3.61 ng h/mL, 17.53 +/- 3.80 vs 17.70 +/- 3.97 ng h/mL, 2.47 +/- 0.49 vs 2.51 +/- 0.51 ng/mL, 1.3 +/- 0.4 vs 1.2 +/- 0.3 h and 5.92 +/- 0.75 vs 6.18 +/- 0.66 h, respectively, indicating that these two kinds of tablets were bioequivalent.     

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of zafirlukast, a selective leukotriene antagonist in human plasma: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of zafirlukast (ZFK) with 500 microL human plasma using valdecoxib as an internal standard (IS). The API-4,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of ZFK and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with 10 mm ammonium acetate (pH 6.4):acetonitrile (20:80, v/v) on a Hypersil BDS C(18) column. The total chromatographic run time was 2.0 min and the elution of ZFK and IS occurred at approximately 1.11 and 1.58 min, respectively. The MS/MS ion transitions monitored were 574.2 --> 462.1 for ZFK and 313.3 --> 118.1 for IS. The method was proved to be accurate and precise at a linearity range of 0.15-600 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 0.15 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 20 mg ZFK tablet.     

Rapid, simple and highly sensitive LC-ESI-MS/MS method for the quantification of tamsulosin in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of tamsulosin (I), a highly selective alpha1-adrenoceptor antagonist used for the treatment of patients with symptomatic benign prostatic hyperplasia. The analyte and internal standard, mosapride (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reverse phase Waters symmetry C18 column with a mobile phase of 0.03% formic acid-acetonitrile (30:70, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 409.1 solidus in circle 228.1 and m/z 422.3 solidus in circle 198.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.1-50.0 ng/mL for tamsulosin in human plasma. The lower limit of quantitation was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Simultaneous quantification of atorvastatin and active metabolites in human plasma by liquid chromatography-tandem mass spectrometry using rosuvastatin as internal standard

A simple, sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of atorvastatin and its active metabolites ortho-hydroxyatorvastatin and para-hydroxyatorvastatin in human plasma using rosuvastatin as internal standard (IS). Following simple liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 559/440 for atorvastatin, m/z 575/466 for ortho-hydroxyatorvastatin, m/z 575/440 for para-hydroxyatorvastatin and m/z 482/258 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for atorvastatin and its two metabolites in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin and the IS from spiked plasma samples were 54.2 +/- 3.2, 50.1 +/- 3.8, 65.2 +/- 3.6 and 71.7 +/- 2.7%, respectively. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Determination of a novel epothilone D analog (AV-EPO-106) in human plasma using ultra-performance liquid chromatography-tandem mass spectrometry

A novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been established for the determination of a newly synthesized epothilone D analog (AV-EPO-106) in human plasma. The plasma samples were prepared by liquid-liquid extraction with cold tert-butyl methyl ether. The chromatographic separation was achieved within 5 min on a C(18) column with water-methanol (10:90, v/v) as mobile phase at a flow-rate of 0.8 mL/min. Mass transition of m/z 568.2 to 386.1 was measured for AV-EPO-106 in positive atmospheric pressure chemical ionization mode. A detailed validation of the method was performed as per the USFDA guidelines. For AV-EPO-106 at the concentrations of 1.0, 5.0 and 10.0 microg/mL in human plasma, the absolute extraction recoveries were 86.17, 85.24 and 85.69%, respectively. The linear quantification range of the method was 0.10-20.0 microg/mL in human plasma with linear correlation coefficients greater than 0.999. The intra-day and inter-day accuracy for AV-EPO-106 at the levels of 1.0, 5.0 and 10.0 microg/mL in human plasma fell in the ranges of 98.25-100.47 and 94.19-97.25%, and the intra- and inter-day precision were in the ranges of 4.75-6.30% and 8.89-10.45%, respectively. The method was successfully applied to quantify AV-EPO-106 in human plasma to determine the half-life of this compound in human plasma.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.     

Determination of paeoniflorin in rat plasma by a liquid chromatography-tandem mass spectrometry method coupled with solid-phase extraction

A rapid, sensitive and selective liquid chromatography-tandem spectrometry method was developed and validated for determination of paeoniflorin in rat plasma using geniposide as the internal standard. The samples were pretreated with solid-phase extraction using Extract-Clean cartridges. Separation of paeoniflorin and IS was achieved on a reversed-phase C18 column (50x4.6 mm i.d.) with a mobile phase made up of acetonitrile and 0.05% formic acid (25:75, v/v) at a flow rate of 0.5 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring and an electrospray ionization source was employed as the ionization source. The lower limit of quantification obtained was 4 ng/mL (n=6) using 200 microL plasma with an accuracy of -3.67% (relative error) and a precision of 4.13% (relative standard deviation). A good linearity was found in the range of 4-1000 ng/mL. The intra- and inter-day relative standard deviations in the measurement of quality control samples 10, 150 and 800 ng/mL ranged from 3.73 to 4.94% and from 4.31 to 6.56%, respectively. The accuracy was from -3.93 to -1.11% in terms of relative error. The analyte and IS were stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of paeoniflorin after a single oral administration of 53.36 mg/kg paeoniflorin to rats.     

Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma

An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.     

Liquid chromatographic-electrospray tandem mass spectrometric determination of clarithromycin in human plasma

A rapid, sensitive and specific LC-MS-MS method has been developed for the determination of clarithromycin (CLA) in human plasma using roxithromycin (ROX) as the internal standard. Samples were prepared via liquid-liquid extraction with methyl tert-butyl ether (MTBE) and chromatographed on a Supelco RP(18) (4.6 x 50 mm, 3 microm particle size) column with a mobile phase consisting of acetonitrile:methanol:60 mM (pH 3.5) ammonium acetate buffer (32.5:32.5:35) at a constant flow rate of 0.8 mL/min. The run time was 3 min with retention times of approximately 1.65 and 1.70 min for CLA and ROX, respectively. Detection was performed on a PE Sciex API 365 mass spectrometer equipped with a turboionspray ionization source in multiple reaction monitoring (MRM) mode. The MRM pairs were m/z 748.5 --> m/z 158.2 for CLA and m/z 837.7 --> m/z 679.3 for ROX, respectively, with dwell times of 200 ms for each transition. The validated calibration curve range was 5.00-5000 ng/mL, based on 0.100 mL plasma sample volume with signal-to-noise ratio (S/N) greater than 60 for CLA at the lower limit of quantification level (5.00 ng/mL). The correlation coefficients (r(2)) of the calibration curves were better than or equal to 0.996. The inter-day (n = 18) precision and accuracy of the quality control (QC) samples were less than 3.58% RSD (relative standard deviation) and -10.8% bias, respectively. The intra-day (n = 6) precision and accuracy of the quality control samples were less than 5.0 and 12.6%, respectively. There was no significant deviation from the nominal values after a 10-fold dilution of high concentration QC samples using blank matrix. The QC samples were stable when left on the bench for 24 h or after three freeze-thaw cycles. The processed samples were also stable in HPLC autosampler at 10C for over 72 h. No matrix ionization suppression was observed when extracted blank matrix or reconstitution solvent was injected onto the system with post-column infusion of clarithromycin and roxithromycin. No carryover was observed when an extracted blank plasma sample was injected immediately after a 5000 ng/mL ULOQ (the upper limit of quantification) standard. The mean recovery was 81.5 and 78.3%, respectively, for clarithromycin and internal standard.     

High‐performance liquid chromatography tandem mass spectrometry method for quantification of endothelin receptor antagonist drug,ambrisentan, in human plasma and its application in a pharmacokinetic study

A simple and sensitive liquid chromatography tandem mass spectrometry method has been developed for the quantification of ambrisentan (AMB) in human plasma using midazolam (MID) as an internal standard (IS). Chromatographic separation was performed using a Beta Basic‐8 (50 × 4.6 mm, 5 µm) column with an isocratic mobile phase. AMB and MID were detected with proton adducts at m/z 379.09 → 303.12 and 326.15 → 291.14 in multiple reaction monitoring‐positive mode, respectively. A solid‐phase extraction method was used for extraction of the analyte and IS from the plasma samples. The method was shown to be reproducible and reliable with within‐run precision <11%, between‐run precision <14% and linear concentration range from 10.0 to 2000.2 ng/mL, with a correlation coefficient (r²) of >0.995. The method was successfully applied to a pharmacokinetic study of oral administration of AMB (10 mg) in 24 healthy Indian male human volunteers under fasting conditions. Copyright © 2014 John Wiley & Sons, Ltd.