Determination of limonin in dog plasma by liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A highly sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of limonin in beagle dog plasma using nimodipine as internal standard. The analyte and internal standard (IS) were extracted with ether followed by a rapid isocratic elution with 10 mm ammonium acetate buffer–methanol (26:74, v/v) on a C₁₈ column (150 × 2.1 mm i.d.) and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 469.4 → 229.3 and m/z 417.2 → 122.0 were used to measure the analyte and the IS. The assay was linear over the concentration range of 0.625–100 ng/mL for limonin in dog plasma. The lower limit of quantification was 0.312 ng/mL and the extraction recovery was >90.4% for limonin. The inter‐ and intra‐day precision of the method at three concentrations was less than 9.9%. The method was successfully applied to pharmacokinetic study of limonin in dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid quantification of levosulpiride in human plasma using RP‐HPLC‐MS/MS for pharmacokinetic and bioequivalence study

A rapid and validated method for analysis of levosulpiride in human plasma using liquid chromatography coupled to tandem mass spectrometry was developed. Levosulpiride and tiapride (IS, internal standard) were extracted from alkalized plasma samples with ethylacetate and separation by RP‐HPLC. Detection was performed by positive ion electrospray ionization in multiple‐reaction monitoring mode, monitoring the transitions m/z 342.1 → m/z 112.2 and m/z 329.1 → m/z 213.2, for quantification of levosulpiride and IS, respectively. The standard calibration curves showed good linearity within the range of 2–200 ng/mL (r² ≥ 0.9990). The lower limit of quantitation was 2 ng/mL. The retention times of levosulpiride (0.63 min) and IS (0.66 min) presented a significant time saving benefit of the proposed method. No significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence study of a 25 mg of levosulpiride tablet in 24 healthy Korean volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

A UPLC–MS/MS method for comparative pharmacokinetics study of morusin and morin in normal and diabetic rats

The aim of this study was to establish and validate a rapid, selective and reliable ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for simultaneous quantitations of morin and morusin, and to investigate their pharmacokinetics difference between normal and diabetic rats after oral administration. Plasma samples were pretreated via protein precipitation with acetonitrile. Genkwanin was used as internal standard (IS). Analytes and IS were separated on a Thermo Hypersil Gold C₁₈ column (50 × 4.6 mm, 3 μm) using gradient elution. The mobile phase consisted of acetonitrile and 0.1% formic acid in water at a flow rate of 0.5 mL/min. Mass spectrometry detection was carried out by means of negative electrospray ionization source and multipe‐reaction monitoring mode. The transitions of m/z 300.9 → 151.2 for morin, m/z 419.2 → 297.1 for morusin and m/z 283.1 → 268.2 for IS were chosen for quantification. Calibration curves were linear in the range of 1.01–504.2 ng/mL (r² ≥ 0.99) for morin and 1.02–522.3 ng/mL (r² ≥ 0.99) for morusin. The lower limit of quantification was 1.02 ng/mL for morin and 1.05 ng/mL for morusin. The extraction recovery was >85.1% for each analyte. No obvious matrix effect was observed under the present UPLC–MS/MS conditions during all of the bioanalysis. The stability study demonstrated that morin and morusin remained stable during the whole analytical procedure. The method was successfully applied to support the pharmacokinetic comparisons of morin and morusin between normal and diabetic rats.     

Simultaneous determination of plasma nicotine and cotinine by UHPLC–MS/MS in C57BL/6 mice and its application in a pharmacokinetic study

Plasma concentrations of nicotine and its active metabolite cotinine are highly correlated with its biological effects. A UHPLC–MS/MS method was developed, validated and applied for nicotine and cotinine analysis in mice plasma. Chromatographic separation was achieved on a BEH HILIC column using acetonitrile (0.1% formic acid) and 10 mm ammonium formate as mobile phase. The gradient elution was performed at 0.4 mL/min with a run time of 3.6 min. The quantitative ion transition was m/z 163.1 > 130.0 for nicotine, m/z 177.1 > 80.0 for cotinine and m/z 167.1 > 134.0 for nicotine‐D₄ (internal standard, IS). For both nicotine and cotinine, the calibration range was 5–500 ng/mL with 5 ng/mL as the lower limit of quantitation, and the intra‐ and inter‐day bias and imprecision were ?4.61–12.00% and <11.12%. The IS normalized recovery was 90.62–98.95% for nicotine and 89.18–101.53% for cotinine, and the IS normalized matrix factor was 106.00–116.44% for nicotine and 100.34–109.85% for cotinine. Both nicotine and cotinine were stable under conventional storage conditions. The validated method has been applied to a pharmacokinetic study in mice to calculate the pharmacokinetic parameters for both analytes.     

Development and validation of UHPLC‐MS/MS assay for rapid determination of a carvone Schiff base of isoniazid (CSB‐INH) in rat plasma: application to pharmacokinetic study

In this study, a fast UHPLC‐MS/MS method was developed and validated for the determination of a novel potent carvone Schiff base of isoniazid (CSB‐INH) in rat plasma using carbamazepine as an internal standard (IS). After a single‐step protein precipitation by acetonitrile, CSB‐INH and IS were separated on an Acquity BEH^TM C₁₈ column (50 × 2.1 mm, 1.7 µm) under an isocratic mobile phase, consisting of acetonitrile: 10 mM ammonium acetate (95:5, v/v), at a flow rate of 0.3 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring mode by using positive electrospray ionization source. The precursor to product ion transitions were set at m/z 270.08 → 79.93 for CSB‐INH and m/z 237.00 → 178.97 for IS. The proposed method was validated in compliance with US Food and Drug Administration and European Medicines Agency guidelines for bioanalytical method validation. The method was found to be linear in the range of 0.35–2500 ng/mL (r² ≥ 0.997) with a lower limit of quantification of 0.35 ng/mL. The intra‐ and inter‐day precision values were ≤12.0% whereas accuracy values ranged from 92.3 to 108.7%. In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of CSB‐INH in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

Determination of corypalmine in mouse blood by UPLC–MS/MS and its application to a pharmacokinetic study

In this work, a selective and sensitive ultra‐performance liquid chromatography tandem mass spectrometry method was established and validated for determination of corypalmine in mouse blood after oral or intravenous administration. A UPLC BEH C₁₈ column was used to separate corypalmine and berberrubine (internal standard) at 40°C. The mobile phase was composed of acetonitrile and 10 mmol/L ammonium acetate (containing 0.1% formic acid) at a flow rate of 0.4 mL/min, and the total run time was 4.0 min. Electrospray ionization in positive ion mode was applied; target fragment ions m/z 342.2 → 178.0 for corypalmine and m/z 322.1 → 307.0 for berberrubine were identified with multiple reaction monitoring mode. The linear range was 1–1000 ng/mL (r > 0.995) and the lower limit of quantification for corypalmine in plasma was 1.0 ng/mL. The intra‐ and inter‐day precisions were both <14%. The range of accuracy in this method was 97.5–109.0%. Mean recovery was >69.6%, and the matrix effect was 96.8–107.6%. Based on its high sensitivity, specificity and reliability, this method was successfully applied to study the pharmacokinetic parameters of corypalmine in mouse by oral and intravenous administration, and finally, the bioavailability of corypalmine was identified at 4.6%.     

Determination of tubuloside B by LC‐MS/MS and its application to a pharmacokinetic study in rats

Tubuloside B, a novel neuroprotective phenylethanoid, is a major active constituent of Cistanche tubulosa and Cistanche deserticola. A specific and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the quantification of tubuloside B in rat plasma. Sample preparation was conducted through a protein‐precipitation extraction with methanol using tubuloside A as internal standard (IS). Chromatographic separation was achieved using a Capcell Pak C₁₈ column (2.0 × 50 mm, 5 μm) with a mobile phase of methanol–10 mm ammonium acetate buffer (70:30, v/v) in an isocratic elution. Mass spectrometry analysis was performed in negative ionization mode with selected reaction monitoring transitions at m/z 665.1 → 160.9 for tubuloside B, and m/z 827.1 → 160.9 for IS. Calibration curves were linear over the range of 1.64–1640 ng/mL for plasma samples samples (R² > 0.990). The lower limit of quantification (LLOQ) was 1.64 ng/mL. The intra‐ and inter‐day accuracy was between 92.3 and 113.0% with the RSD <9.23% at all LLOQ and quality control levels. Finally, this method was successfully applied in the pharmacokinetics study of tubuloside B after intravenous administration.     

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.     

An analytical method for cyclosporine using liquid chromatography–mass spectrometry

A liquid chromatographic mass spectrometric (LC‐MS) assay has been developed for cyclosporine A (CyA) in rat plasma using amiodarone as internal standard (IS). Rat plasma (100 µL) containing drug and IS were extracted using liquid–liquid extraction with 4 mL of 95:5 ether:methanol. After evaporation of the organic layer the residue was reconstituted with 500 µL of water. Then the aqueous layer was transferred to LC‐MS sample vials. A 10 µL volume was injected. The analysis was performed on a C₈ column 3.5 µm (2.1 × 50 mm) heated to 60°C with a mobile phase consisting of acetonitrile:methanol:0.2% NH₄OH (60:20:20) at an isocratic flow‐rate of 0.2 mL/min. The ions used for quantitation of CyA and IS were m/z 1202.8 and 645.9, with retention times of 3.35 and 4.72 min, respectively. Linear relationships (r² > 0.99) were achieved between plasma or blood concentration and peak height ratios (drug:IS) over the concentration range 50–5000 ng/mL. The CV% and mean error were <19%. Based on validation data, the lower limit of quantification for the assay was 50 ng/mL. The reported assay method displayed high measures of linearity, sensitivity, reliability and precision, allowing its applicability in pharmacokinetic studies in rat. Copyright © 2009 John Wiley & Sons, Ltd.     

A high performance liquid chromatography-tandem mass spectrometric method for the determination of mefenamic acid in human plasma: application to pharmacokinetic study

In the present study, the development and validation of an LC‐MS/MS method for quantifying mefenamic acid in human plasma is described. The method involves liquid–liquid extraction using diclofenac as an internal standard (IS). Chromatographic separation was achieved on a Thermo Hypurity C₁₈, 50 × 4.6 mm, 5 µm column with a mobile phase consisting of 2 m m ammonium acetate buffer and methanol (pH 4.5 adjusted with glacial acetic acid; 15:85, v/v) at a flow‐rate of 0.75 mL/min and the total run time was 1.75 min. Analyte was introduced to the LC‐MS/MS using an atmospheric pressure ionization source. Both the drug and IS were detected in negative‐ion mode using multiple reaction monitoring m/z 240.0 → 196.3 and m/z 294.0 → 250.2, respectively, with a dwell time of 200 ms for each of the transitions. The standard curve was linear from 20 to 6000 ng/mL. This assay allows quantification of mefenamic acid at a concentration as low as 20 ng/mL in human plasma. The observed mean recovery was 73% for the drug. The applicability of this method for pharmacokinetic studies has been established after successful application during a 12‐subject bioavailibity study. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid and sensitive determination of aucubin in rat plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

A sensitive, accurate, rapid and robust LC‐MS‐MS method for the quantification of aucubin, a major bioactive constituent of Aucuba japonica, Eucommia ulmoides and Plantago asiatica, was established and validated in rat plasma. Plasma samples were simply precipitated by adding methanol and the supernatant was chromatographed by a Diamonsil® C₁₈(2) column with the mobile phase comprising a mixture of 10 mm ammonium acetate in methanol and that in water with the ratio of 50:50 (v/v). Quantification of aucubin was performed by mass spectrometry in the multiple‐reaction monitoring mode with positive atmospheric ionization at m/z 364 → 149 for aucubin, and m/z 380 → 165 for catalpol (IS), respectively. The retention time was 2.47 and 2.44 min for aucubin and the IS, respectively. The calibration curve (10.0–30,000 ng/mL) was linear (r² > 0.99) and the lower limit of quantification was 10.0 ng/mL in the rat plasma sample. The method showed satisfactory results such as sensitivity, specificity, precision, accuracy, recovery, freeze–thaw and long‐term stability. This simple LC‐MS method was successfully applied in a pharmacokinetic study carried out in Sprague–Dawley rats after oral administration of aucubin at a single dose of 50 mg/kg. Herein the pharmacokinetic study of aucubin is reported for the first time. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of cyclobenzaprine in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry and its application in a pharmacokinetic study

A sensitive, rapid and simple liquid chromatography–electrospray ionization mass spectrometry (LC‐ESI‐MS/MS) method was developed for the quantitative determination of cyclobenzaprine in human plasma, to study the pharmacokinetic behavior of cyclobenzaprine capsule in healthy Chinese volunteers. With escitalopram as the internal standard (IS), sample pretreatment involved a one‐step liquid–liquid extraction using saturated sodium carbonate solution and hexane–diethyl ether (3:1, v/v). The separation was performed on an Ultimate XB‐CN column (150 × 2.1 mm, 5 µm). Isocratic elution was applied using acetonitrile–water (40:60, v/v) containing 10 m M ammonium acetate and 0.1% formic acid. The detection was carried out on a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionization. The ion‐pairs including m/z 276.2–216.2 for cyclobenzaprine and m/z 325.2–109.1 for IS were used for monitoring. Linear calibration curves were obtained over the range of 0.049–29.81 ng/mL with the lower limit of quantification at 0.049 ng/mL. The intra‐ and inter‐day precision showed ≤6.5% relative standard deviation. The established method laid the groundwork for follow‐up studies and provided basis for the clinical administration of cyclobenzaprine. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of a liquid chromatography–mass spectrometry method for determination of agrimol B in rat plasma: application to preclinical pharmacokinetics

A sensitive and accurate liquid chromatography coupled with mass spectrometry (LC‐MS) method was developed for the determination of agrimol B, a main active ingredient isolated from Agrimonia pilosa Ledeb., in rat plasma. Chromatographic separation was achieved on a Zorbax CN column (150 × 4.6 mm, 5 µm), with isocratic elution consisting of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Agrimol B and dryocrassin ABBA, an internal standard (IS), were analyzed by selected ion monitoring at m/z transitions of 681.3 and 819.4, respectively. This assay exhibited a good linearity with a correlation coefficient >0.99 and showed no endogenous interference with the analyte and IS. The limit of quantification of agrimol B was 8.025 ng/mL with acceptable precision and accuracy. The method was successfully applied in the pharmacokinetic study of agrimol B in rats after intravenous (1 mg/kg) and oral (2, 5 and 10 mg/kg) doses of agrimol B. The absolute bioavailability of agrimol B was 16.4–18.0% in rat. Our study clarifies the pharmacokinetic behavior of agrimol B in animals. Copyright © 2014 John Wiley & Sons, Ltd.     

LC–MS–MS Assay for Simultaneous Quantification of Fexofenadine and Pseudoephedrine in Human Plasma

A rapid, sensitive, and simple HPLC–MS–MS method, with electro-spray ionization and cetirizine as internal standard (IS), has been developed and validated for simultaneous quantification of fexofenadine and pseudoephedrine in human plasma. The analytes were isolated from plasma by solid-phase extraction (SPE) on Oasis HLB cartridges. The compounds were chromatographed on an RP 18 column with a mixture of ammonium acetate (10 mm, pH 6.4) and methanol as mobile phase. Quantification of the analytes was based on multiple reaction monitoring (MRM) of precursor-to-product ion pairs m/z 502 → 466 for fexofenadine, m/z 166 → 148 for pseudoephedrine, and m/z 389 → 201 for cetirizine. The linear calibration range for both analytes was 2–1,700 ng mL⁻¹ (r = 0.995), based on analysis of 0.1 mL plasma. Extraction recovery was 91.5 and 80.88% for fexofenadine and pseudoephedrine, respectively. The method was suitable for analysis of human plasma samples obtained 72 h after administration of a drug containing both fexofenadine and pseudoephedrine.     

Determination of asperosaponin VI in dog plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pilot pharmacokinetic study

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the determination of asperosaponin VI in beagle dog plasma using glycyrrhizic acid as the internal standard (IS). Plasma samples were simply pretreated with methanol for deproteinization. Chromatographic separation was performed on a Hedera ODS‐2 column using mobile phase of methanol–10 mm ammonium acetate buffer solution containing 0.05% acetic acid (71:29, v/v) at a flow rate of 0.38 mL/min. Asperosaponin VI and the IS were eluted at 2.8 and 1.9 min, respectively, ionized in negative ion mode, and then detected by multiple reaction monitoring. The detection used the transitions of the deprotonated molecules at m/z 927.5 → 603.4 for asperosaponin VI and m/z 821.4 → 645.4 for glycyrrhizic acid (IS). The assay was linear over the concentration range of 0.15–700 ng/mL and was successfully applied to a pilot pharmacokinetic study in beagle dogs. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

A liquid chromatography–tandem mass spectrometric assay for the antihypertensive agent azelnidipine in human plasma with application to clinical pharmacokinetics studies

A robust and sensitive high‐performance liquid chromatographic–tandem mass spectrometric (HPLC‐MS/MS) assay for the high‐throughput quantification of the antihypertensive drug azelnidipine in human plasma was developed and validated following bioanalytical validation guidelines. Azelnidipine and internal standard (IS), telmisartan, were extracted from human plasma by precipitation protein and separated on a C₁₈ column using acetonitrile–methanol–ammonium formate with 0.1% formic acid as mobile phase. Detection was performed on a turbo‐spray ionization source (ESI) and mass spectrometric positive multiple reaction monitoring mode (+MRM) using the respective transitions m/z 583.3 → 167.2 for azelnidipine and m/z 515.3 → 497.2 for IS. The method has a wide analytical measuring range from 0.0125 to 25 ng/mL. For the lowest limit of quantitation, low, medium and high quality controls, intra‐ and interassay precisions (relative standard deviation) were 3.30–7.01% and 1.78–8.09%, respectively. The drug was sufficiently stable under all relevant analytical conditions. The main metabolite of azelnidipine, M‐1 (aromatized form), was monitored semiquantitatively using the typical transition m/z 581.3 → 167.2. Finally, the method was successfully applied to a clinical pharmacokinetic study in human after a single oral administration of azelnidipine 8 mg. The assay meets criteria for the analysis of samples from large research trials. Copyright © 2014 John Wiley & Sons, Ltd.