Simultaneous determination of metoprolol and its metabolites, α‐hydroxymetoprolol and O‐desmethylmetoprolol,in human plasma by liquid chromatography with tandem mass spectrometry: Application to the pharmacokinetics of metoprolol associated with CYP2D6 genotypes

A rapid and simple LC with MS/MS method for the simultaneous determination of metoprolol and its two CYP2D6‐derived metabolites, α‐hydroxy‐ and O‐desmethylmetoprolol, in human plasma was established. Metoprolol (MET), its two metabolites, and the internal standard chlorpropamide were extracted from plasma (50 μL) using ethyl acetate. Chromatographic separation was performed on a Luna CN column with an isocratic mobile phase consisting of distilled water and methanol containing 0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The total run time was 3.0 min per sample. Mass spectrometric detection was conducted by ESI in positive ion selected‐reaction monitoring mode. The linear ranges of concentration for MET, α‐hydroxymetoprolol, and O‐desmethylmetoprolol were 2–1000, 2–500, and 2–500 ng/mL, respectively, with a lower limit of quantification of 2 ng/mL for all analytes. The coefficient of variation for the assay's precision was ≤ 13.2%, and the accuracy was 89.1–110%. All analytes were stable under various storage and handling conditions and no relevant cross‐talk and matrix effect were observed. Finally, this method was successfully applied to assess the influence of CYP2D6 genotypes on the pharmacokinetics of MET after oral administration of 100 mg to healthy Korean volunteers.     

Development and validation of a highly sensitive LC‐MS/MS‐ESI method for the determination of bicalutamide in mouse plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of bicalutamide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves extraction of bicalutamide and tolbutamide (internal standard, IS) from mouse plasma with a simple protein precipitation method. Chromatographic separation was achieved using an isocratic mobile phase (0.2% formic acid:acetonitrile, 35:65, v/v) at a flow rate of 0.5 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1°C) with a total run time of 3.0 min. The MS/MS ion transitions monitored were m/z 428.9 → 254.7 for bicalutamide and m/z 269.0 → 169.6 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 1.04 ng/mL and the linearity range extended from 1.04 to 1877 ng/mL. The intra‐ and inter‐day precisions were in the ranges of 0.49–4.68 and 2.62–4.15, respectively. Copyright © 2012 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.     

A rapid and efficient analytical method for the quantification of a novel anticholinergic compound,R‐phencynonate,by stable isotope‐dilution LC–MS/MS and its application to bioavailability and dose proportionality studies

A rapid, specific and high‐throughput stable isotope‐dilution LC–MS/MS method was developed and validated with high sensitivity for the quantification of R‐ phencynonate (a eutomer of phencynonate racemate) in rat and dog plasma. Plasma samples were deproteinized using acetonitrile and then separated on a C₈ column with an isocratic mobile phase containing acetonitrile–water–formic acid mixture (60:40:0.1, v /v/v) at a flow rate of 0.2 mL/min. Each sample had a total run time of 3 min. Quantification was performed using triple quadrupole mass spectrometry in selected reaction monitoring mode with positive electrospray ionization. The method was shown to be highly linear (r ² > 0.99) and to have a wide dynamic range (0.1–100 ng/mL) with favourable accuracy and precision. No matrix effects were observed. The detailed pharmacokinetic profiles of R‐ phencynonate at therapeutic doses in rats and dogs were characterized by rapid oral absorption, quick clearance, high volume of distribution and poor absolute bioavailability. R‐ Phencynonate lacked dose proportionality over the oral dose range, based on the power model. However, the area under concentration–time curve and the maximum plasma concentration increased linearly in a dose‐dependent manner in both animal models. The absolute bioavailability of R‐ phencynonate was 16.6 ± 2.75 and 4.78 ± 1.26% in dogs and rats, respectively.     

Simultaneous determination of bupropion,metroprolol, midazolam,phenacetin, omeprazole and tolbutamide in rat plasma by UPLC‐MS/MS and its application to cytochrome P450 activity study in rats

A specific ultra‐performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma with diazepam as internal standard, which are the six probe drugs of the six cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9. Plasma samples were protein precipitated with acetonitrile. The chromatographic separation was achieved using a UPLC® BEH C₁₈ column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with gradient elution. The triple quadrupole mass spectrometric detection was operated by multiple reaction monitoring in positive electrospray ionization. The precisions were <13%, and the accuracy ranged from 93.3 to 110.4%. The extraction efficiency was >90.5%, and the matrix effects ranged from 84.3 to 114.2%. The calibration curves in plasma were linear in the range of 2–2000 ng/mL, with correlation coefficient (r²) >0.995. The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the effects of erlotinib on the activities of CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 in rats. Erlotinib may inhibit the activity of CYP2B6 and CYP3A4, and may induce CYP2C9 of rats. Copyright © 2015 John Wiley & Sons, Ltd.     

High‐performance liquid chromatography–tandem mass spectrometry for simultaneous determination of raltegravir,dolutegravir and elvitegravir concentrations in human plasma and cerebrospinal fluid samples

A simple sample treatment procedure and sensitive liquid chromatography–tandem mass spectrometry method were developed for the simultaneous quantification of the concentrations of human immunodeficiency virus‐1 integrase strand transfer inhibitors – raltegravir, dolutegravir and elvitegravir – in human plasma and cerebrospinal fluid (CSF). Plasma and CSF samples (20 μL each) were deproteinized with acetonitrile. Raltegravir‐d₃ was used as the internal standard. Chromatographic separation was achieved on an XBridge C₁₈ column (50 × 2.1 mm i.d., particle size 3.5 μm) using acetonitrile–water (7:3, v/v) containing 0.1% formic acid as the mobile phase at a flow rate of 0.2 mL/min. The run time was 5 min. Calibration curves for all three drugs were linear in the range 5–1500 ng/mL for plasma and 1–200 ng/mL for CSF. The intra‐ and inter‐day precision and accuracy of all three drugs in plasma were coefficient of variation (CV) <12.9% and 100.0 ± 12.2%, respectively, while those in CSF were CV <12.3% and 100.0 ± 7.9%, respectively. Successful validation under the same LC–MS/MS conditions for both plasma and CSF indicates this analytical method is useful for monitoring the levels of these integrase strand transfer inhibitors in the management of treatment of HIV‐1 carriers.     

Development and validation of a UPLC–MS method for the determination of galantamine in guinea pig plasma and its application to a pre‐clinical bioavailability study of novel galantamine formulations

To evaluate the bioavailability and pharmacokinetic profiles of two novel galantamine formulations as medical countermeasure products, an ultra‐performance liquid chromatography–single quadrupole mass spectrometry (UPLC–MS) method was developed and validated for quantifying galantamine in guinea pig plasma using solid‐phase extraction with a mixed mode strong cation exchange reversed‐phase cartridge. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C₁₈ column maintained at 40°C. The mobile phases were solution A, acetonitrile–water, 5:95 (v/v) and solution B, acetonitrile–water 90:10 (v/v), both containing 2 mM ammonium formate and 0.2% formic acid. The mobile phase was delivered utilizing a 3 min gradient program start with 95%A–5%B at a flow rate of 0.6 mL/min. The analyte and internal standard, galantamine‐d3, were detected by selected ion monitoring mode on a Waters 3100 single quadrupole mass spectrometer with positive electrospray ionization. The method was validated according to the US Food and Drug Administration bioanalytical guidance. The method was selective and was linear over the analytical range of 2–2000 ng/mL. Accuracy and precision were acceptable with intra‐ and inter‐day accuracies between 96.8 and 101% and precisions (RSD) <4.88%. The method was successfully implemented to measure galantamine plasma levels in a series of pre‐clinical bioavailability studies for the evaluation of novel galantamine formulations.     

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

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

Development and validation of a highly sensitive and robust LC‐ESI‐MS/MS method for simultaneous quantitation of simvastatin acid,amlodipine and valsartan in human plasma: application to a clinical pharmacokinetic study

A high‐throughput, simple, highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of simvastatin acid (SA), amlodipine (AD) and valsartan (VS) with 500 µL of human plasma using deuterated simvastatin acid as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using electrospray ionization. The assay procedure involved precipitation of SA, AD, VS and IS from plasma with acetonitrile. The total run time was 2.8 min and the elution of SA, AD, VS and IS occurred at 1.81, 1.12, 1.14 and 1.81 min, respectively; this was achieved with a mobile phase consisting of 0.02 m ammonium formate (pH 4.5):acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X‐Terra C₁₈ column. A linear response function was established for the range of concentrations 0.5–50 ng/mL (r > 0.994) for VS and 0.2–50 ng/mL (r > 0.996) for SA and AD. The method validation parameters for all three analytes met the acceptance as per FDA guidelines. This novel method has been applied to human pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a novel HPLC‐MS/MS method for the determination of aconitine and its application to in vitro and rat microdialysis samples

A sensitive and selective LC‐MS/MS method was developed and validated for the determination of aconitine in microdialysate and rat plasma. Extraction of plasma sample was conducted by use of 1% trichloracetic acid and acetonitrile solution with 10 ng/mL internal standard (propafenone) spiked. Microdialysates were analyzed without sample purification. After sample preparation, 2 µL were injected and separated with an isocratic mobile phase consisting of acetonitrile:0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. Overall, the assay exhibited good precision and accuracy. The diffusion properties of aconitine investigated in in vitro microdialysis experiments revealed unfavourable concentration dependence avertable by keeping a constant pH 5.77 using isotonic phosphate buffer solution as perfusate. The mean relative recoveries were 48.23% [coefficient of variation (CV 4.47%)] and 55.38% (CV 2.89%) for retrodialysis and recovery experiments, respectively. The in vivo recovery of aconitine was 34.48% (CV 3.05%) and was stable over the 6 h study period. Following characterization of aconitine both in vitro and in vivo microdialysis, the developed setting is suitable for application in pharmacokinetics and pharmacodynamics studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a UHPLC‐MS/MS bioanalytical method to quantify in plasma the analgesic candidate PT‐31 following a preliminary pharmacokinetic study in rats

A selective and sensitive UHPLC‐MS/MS bioanalytical method to determine PT‐31, an analgesic drug candidate, in rat plasma was developed and validated. Analyses were performed using a UHPLC‐MS/MS system equipped with an electrospray ionization interface operating in the positive ionization mode using a C₁₈ reversed‐phase column with a mobile phase of water:acetonitrile (68:31, v/v) containing 0.1% acetic acid eluting in a gradient mode with a flow rate of 0.3 mL/min. Plasma samples were deproteinized with cold acetonitrile containing 0.01% TFA (1:2, v/v) and 50 μL of the supernatant were injected into the system. PT‐31 and phenytoin (internal standard) retention times were roughly 1.0 and 1.5 min, respectively. Linear standard curves were plotted for the 0.01–10 µg/mL concentration range, with a coefficient of determination > 0.99. The method's precision was over 88%. Maximum intra‐ and inter‐day relative standard deviations were 14.6% and 11.6%, respectively. Interfering substances were not detected in the chromatogram, indicating that the method was specific. PT‐31 stability was assessed under different temperature and storage settings. The method was used to characterize PT‐31 plasma pharmacokinetics following administration of 5 mg/kg i.v. to Wistar rats. Therefore, the method described is sensitive, linear, precise and specific enough to determine PT‐31 in preclinical pharmacokinetic investigations. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study

Bullatine A is a diterpenoid alkaloid of Xue‐Shang‐Yi‐Zhi‐Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the treatment of rheumatism and pain. The plasma levels of bullatine A were measured by a rapid and sensitive LC‐MS/MS method. Samples were prepared using acetonitrile precipitation and the separation of bullatine A was achieved on a Capcell Pak MG‐C₁₈ column by isocratic elution using acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) as the mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple‐reaction monitoring of the transitions at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the internal standard. The linearity was found to be within the concentration range of 1.32–440 ng/mL with a lower limit of quantification of 1.32 ng/mL. Only 1.3 min was needed for an each analytical run. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of β‐hydroxy‐β‐methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water–acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C₈ (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30–4600 ng/mL (r > 0.998) for HMB. The intra‐ and inter‐day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench‐top, autosampler freeze–thaw cycles and long‐term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

A highly sensitive LC‐MS/MS method for the determination of S‐citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP₁₈ column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometric assay for aliskiren,a novel renin inhibitor in micro‐volumes of human plasma: a pharmacokinetic application in healthy South Indian male subjects

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of aliskiren in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from 100 μL of human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a C₁₈ column using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.10–1013 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 2.2 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

A high‐throughput screening method for determination of multi‐antibiotics in animal feed

A high‐throughput method based on ultrasonic‐assisted extraction, 96‐well plate thin‐film microextraction was established to determinate 18 antibiotics in animal feed. In this method, the extraction was implemented by ultrasonic‐assisted extraction for 30 min with disodium ethylenediaminetetraacetic acid‐McIlvaine buffer (pH 5) containing 6% sodium chloride w/v, purified by thin‐film microextraction and combined with 96‐well plate system to improve the efficiency. Optimization of thin‐film microextraction conditions was performed by methods of single factor and response surface, and finalized as: condition time: 20 min; adsorption time: 55 min; washing time: 5 s with water; desorption time: 30 min with acetonitrile/water (8:2, v/v) containing 0.1% formic acid v/v. Evaluation of different extractive phases showed that polystyrene‐divinylbenzene‐polyacrylonitrile was the optimum coating. The analysis was performed by ultra‐high performance liquid chromatography with tandem mass spectrometry. Recovery, inter‐ and intraday precision, linearity, limit of detection, and quantitation were evaluated. The average recoveries of 18 antibiotics were 66.6–93.5% at three spiked levels, intraday precision was 1–8.4%, and interday precision was 3.0–16.4%. The linearity was good for r² > 0.99. Limits of detection and quantification were found in the range of 1–14 and 4–48 µg/kg, respectively.     

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.