Rapid determination of losartan and losartan acid in human plasma by multiplexed LC–MS/MS

A rapid LC–MS/MS method has been developed and validated for the determination of losartan (LOS) and its metabolite losartan acid (LA) (EXP‐3174) in human plasma using multiplexing technique (two HPLC units connected to one MS/MS). LOS and LA were extracted from human plasma by SPE technique using Oasis HLB® cartridge without evaporation and reconstitution steps. Hydroflumethiazide (HFTZ) was used as an internal standard (IS). The analytes were separated on Zorbax SB C‐18 column. The mass transition [M–H] ions used for detection were m/z 421.0 → 127.0 for LOS, m/z 435.0 → 157.0 for LA, and m/z 330.0 → 239.0 for HFTZ. The proposed method was validated over the concentration range of 2.5–2000 ng/mL for LOS and 5.0–3000 ng/mL for LA with correlation coefficient ?0.9993. The overall recoveries for LOS, LA, and IS were 96.53, 99.86, and 94.16%, respectively. Total MS run time was 2.0 min/sample. The validated method has been successfully used to analyze human plasma samples for applications in 100 mg fasted and fed pharmacokinetic studies.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2‐carboxyethyl)phosphine as a reducing agent by LC–MS/MS

A simple, robust, and rapid LC–MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2‐carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide‐bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel‐d₄ as an internal standard (IS). The mixtures were separated on a C₁₈ RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI‐MS. The detector was operated in selected reaction‐monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05–20 and 0.5–200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra‐ and interday, was less than 8.26% with an accuracy of 87.6–106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.     

A rapid and sensitive determination of hypoxic radiosensitizer agent nimorazole in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A highly sensitive, accurate and robust LC‐MS/MS method was developed and validated for determination of nimorazole (NMZ) in rat plasma using metronidazole (MNZ) as internal standard (IS). The analyte and IS were extracted from plasma by precipitating protein with acetonitrile and were chromatographed using an Agilent Poroshell 120, EC‐C₁₈ column. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid (85:15 v/v). The total run time was 1.5 min and injection volume was 5 μL. Multiple reaction monitoring mode using the transitions of m/z 227.1 → m/z 114.0 for MNZ and m/z 172.10 → m/z 128.1 for IS were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The calibration curve was linear in the range of 0.25–200 ng/mL (r² > 0.9996) and the lower limit of quantification was 0.25 ng/mL in the rat plasma samples. Recoveries of NMZ ranged between 88.05 and 95.25%. The precision (intra‐day and inter‐day) and accuracy of the quality control samples were 1.25–8.20% and ?2.50–3.10, respectively. The analyte and IS were found to be stable during all sample storage and analysis procedures. The LC‐MS/MS method described here was validated and successfully applied to pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of swertianolin in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method has been developed and validated for quantifying swertianolin in rat plasma using rutin as an internal standard (IS). Following liquid–liquid extraction with ethyl acetate, chromatographic separation for swertianolin was achieved on a C₁₈ column with a gradient elution using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.3 mL/min. The detection was performed on a tandem mass spectrometer using multiple reaction monitoring via an electrospray ionization source and operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 435.1/272.0 for swertianolin and 609.2/300.1 for IS. The lower limit of quantitation was 0.5 ng/mL within a linear range of 0.5–500 ng/mL. Intra‐day and inter‐day precision was less than 6.8%. The accuracy was in the range of ?13.9 to 12.0%. The mean recovery of swertianolin was >66.7%. The proposed method was successfully applied in evaluating the pharmacokinetics of swertianolin after an oral dose of 50 mg/kg Swertia mussotii extract in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

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 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.     

Development and validation of a robust LC‐MS‐MS with atmospheric pressure chemical ionization for quantitation of carbazochrome sodium sulfonate in human plasma: application to a pharmacokinetic study

A highly selective and sensitive liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC‐APCI‐MS‐MS) was developed and validated for the quantitation and pharmacokinetic study of carbazochrome sodium sulfonate in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and amiloride hydrochloride was employed as an internal standard. The analytes were separated on a Hypersil ODS‐2 column by a multiple‐step linear gradient elution with a mobile phase consisting of 0.2% formic acid solution and methanol pumped at a flow rate of 1.0 mL/min. The determination was optimized and carried out with positive atmospheric pressure chemical ionization by selective reaction monitoring of the ion of m/z 148, the protonated thermodegraded fragment of the free acidic form of carbazochrome sodium sulfonate selected as the parent, and the ion of m/z 107 as the optimum collision induced dissociation (CID) product. The method was fully validated over a concentration range of 0.5–50 ng/mL, with the lower limit of quantitation of 0.5 ng/mL. The application of the LC‐MS‐MS method was demonstrated for the specific and quantitative analysis of carbazochrome sodium sulfonate in human plasma from a pharmacokinetic study in 24 healthy male Chinese volunteers after a single oral administration of 90 mg carbazochrome sodium sulfonate capsules. Copyright © 2010 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.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐ESI‐MS/MS determination of 4‐methylpyrazole in dog plasma and its application to a pharmacokinetic study in dogs

A simple, specific, sensitive and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of 4‐methylpyrazole in dog plasma using N‐methylnicotinamide‐d₄ as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 4‐methylpyrazole and the IS was performed on a monolithic (Chromolith RP_18e) column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min. Elution of 4‐methylpyrazole and the IS occurred at ~1.60 and 1.56 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.96–4955 ng/mL. The intra‐ and inter‐day accuracy and precision were in the ranges 1.81–12.9 and 3.80–11.1%, respectively. This novel method has been applied to a pharmacokinetic study in dogs.     

Quantitative determination of saroglitazar,a predominantly PPAR alpha agonist,in human plasma by a LC–MS/MS method utilizing electrospray ionization in a positive mode

A sensitive LC–MS/MS method was developed and validated for quantitation of saroglitazar using turboion spray interface with positive ion mode. A liquid–liquid extraction, with a mixture of dichloromethane and diethyl ether, was employed for the extraction of saroglitazar and glimepiride (IS) from human plasma. The chromatographic separation was achieved using an ACE‐5, C₁₈ (4.6 × 100 mm) column with a gradient mobile phase comprising acetonitrile and ammonium acetate buffer with trifluoracetic acid in purified water. Both analytes were separated within 10 min with retention times of 4.52 and 2.57 min for saroglitazar and IS, respectively. Saroglitazar quantitation was achieved by the summation of two MRM transition pairs (m/z 440.2 to m/z 366.0 and m/z 440.2 to m/z 183.1), while that of IS was achieved using transition pair m/z 491.3 to m/z 352.0. The calibration standards of saroglitazar showed linearity from 0.2 to 500 ng/mL, with a lower limit of quantitation of 0.2 ng/mL. The biases for inter‐ and intra‐batch assays were ?7.51–1.15% and ?11.21 to ?3.25%, respectively, while the corresponding precisions were 5.04–8.06% and 1.53–7.68%, respectively. The developed method was used to monitor the plasma concentrations of saroglitazar in clinical samples.     

Validation of an enantioselective LC–MS/MS method to quantify enantiomers of (±)‐OTX015 in mice plasma: Lack of in vivo inversion of (−)‐OTX015 to its antipode

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)‐OTX015 and (−)‐OTX015] in mice plasma on LC–MS/MS‐electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)‐OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(−)‐OTX015] and inactive [(+)‐OTX015] enantiomers were resolved on a Chiralpak‐IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v /v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)‐OTX015, (−)‐OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r ² > 0.998) in the concentration range 1.03–1030 ng/mL. The inter‐ and intraday precisions were in the range 2.20–13.3 and 8.03–12.1% and 3.80–14.4 and 8.97–13.6% for (+)‐OTX015 and (−)‐OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (−)‐OTX015 and unequivocally demonstrated that (−)‐OTX015 does not undergo chiral inversion to its antipode in vivo in mice.     

LC–MS/MS method for simultaneous determination of ramelteon and its metabolite M‐II in human plasma: Application to a clinical pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M‐II in human plasma. After extraction from 200 μL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS‐2 (5 μm, 150 × 2.1 mm) column with a mobile phase consisted of methanol–0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500–30.0 ng/mL for ramelteon and 1.00–250 ng/mL for M‐II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (C_max), the time to the C_max and the elimination half‐life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M‐II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.     

Bioanalytical LC‐MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers

A LC‐MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge‐coupled solid‐phase extraction and reverse‐phase chromatographic separation was performed on an Ascentis C₁₈ column. Turbo‐spray negative‐ion mode multiple‐reaction monitoring was selected for mass pair detection at m/z 338.3 → 78.0 and m/z 407.3 → 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half‐life matching for test and reference drug was achieved with 73.43 ± 9.68 and 73.06 ± 14.03 h, respectively, and intra‐subject coefficient of variation achieved within 11% for AUCs and C_max evaluated by non‐compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Measurement of fexofenadine concentration in micro‐sample human plasma by a rapid and sensitive LC‐MS/MS employing protein precipitation: application to a clinical pharmacokinetic study

A simple, rapid and sensitive liquid chromatography/positive ion electro‐spray tandem mass spectrometry method (LC‐MS/MS) was developed and validated for the quantification of fexofenadine with 100 μL human plasma employing glipizide as internal standard (IS). Protein precipitation was used in the sample preparation procedure. Chromatographic separation was achieved on a reversed‐phase C₁₈ column (5 μm, 100 × 2.1 mm) with methanol : buffer (containing 10 mmol/L ammonium acetate and 0.1% formic acid; 70 : 30, v/v) as mobile phase. The total chromatographic runtime was approximately 3.0 min with retention time for fexofenadine and IS at approximately 1.9 and 2.1 min, respectively. Detection of fexofenadine and IS was achieved by LC‐MS/MS in positive ion mode using 502.1 → 466.2 and 446.0 → 321.1 transitions, respectively. The method was proved to be accurate and precise at linearity range of 1–600 ng/mL with a correlation coefficient (r) of ≥0.9976. The validated method was applied to a pharmacokinetic study in human volunteers following oral administration of 60 or 120 mg fexofenadine formulations, successfully. Copyright © 2009 John Wiley & Sons, Ltd.