LC–MS/MS method for the simultaneous determination of ethyl gallate and its major metabolite in rat plasma

A simple, rapid and sensitive liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed and validated for the determination of ethyl gallate, a pharmacologically active constituent isolated from Lagerstroemia speciosa (Linn.) Pers. This method was used to examine the pharmacokinetics of ethyl gallate and its major metabolite gallic acid in rat plasma using propyl gallate as an internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a Zorbax SB‐C₁₈ column (3.5 μm, 2.1 × 50 mm) with an isocratic mobile phase consisted of methanol–acetonitrile–10 mM ammonium acetate (10 : 25 : 65, v/v/v) containing 0.1% formic acid at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode using the electrospray ionization technique in negative mode. The lower limits of quantification of gallic acid and ethyl gallate of the method were 0.5 and 1.0 ng/mL. The intra‐day and inter‐day accuracy and precision of the assay were less than 8.0%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of ethyl gallate to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for simultaneous determination of R‐bambuterol and its active metabolite R‐terbutaline in human plasma and urine with application to a clinical pharmacokinetic study

A sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for simultaneous determination of R‐bambuterol and its active metabolite R‐terbutaline in human plasma and urine was established. The inhibition for the biotransformation of R‐bambuterol in plasma was fully investigated. Plasma samples were prepared on ice and neostigmine metilsulfate added as a cholinesterase inhibitor immediately after sample collection. All samples were extracted with ethyl acetate and separated on a C₁₈ column under gradient elution with a mobile phase consisting of methanol and water containing 5 mm ammonium acetate at a flow rate of 0.6 mL/min. The analytes were detected by an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte in plasma. In urine samples, the LLOQs were 20.00 and 500.0 pg/mL for R‐bambuterol and R‐terbutaline, respectively. The intra‐ and inter‐day precisions were <12.7 and <8.6% for plasma and urine, respectively. The analytical runtime within 6.0 min per sample made this method suitable for high‐throughput determination. The validated method has been successfully applied to the human pharmacokinetic study of R‐bambuterol involving 10 healthy volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

A sensitive LC‐MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC‐MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at ?80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC‐MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.     

A sensitive LC–MS/MS method for simultaneous determination of cabozantinib and its metabolite cabozantinib N‐oxide in rat plasma and its application in a pharmacokinetic study

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N‐oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC–MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C₁₈ column (2.1 × 150 mm, 3 μm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500–5000 ng/mL for CBZ and 0.525–2100 ng/mL for CBN. The extraction recoveries were constant and the intra‐ and inter‐batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague–Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose‐proportional increases in exposure.     

Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC‐MS/MS using supported liquid extraction

A rapid, sensitive and selective bioanalytical method was developed for the simultaneous determination of fluoxetine and its primary metabolite norfluoxetine in human plasma. Sample preparation was based on supported liquid extraction (SLE) using methyl tert‐butyl ether to extract the analytes from human plasma. Chromatography was performed on a Synergi 4 μ polar‐RP column using a fast gradient. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, m/z 310 → 44 for fluoxetine, m/z 296 → 134 for norfluoxetine and m/z 315 → 44 for fluoxetine‐d₅ (internal standard). The method is linear over the range of 0.05–20 ng/mL (using a human plasma sample volume of 0.1 mL) with a coefficient determination of greater than 0.999. The method is accurate and precise with intra‐batch and inter‐batch accuracy (%bias) of <±15% and precision (%CV) of <15% for both analytes. A run time of 4 min means a high throughput of samples can be achieved. To our knowledge, this method appears to be the most sensitive one reported so far for the quantitation of fluoxetine and norfluoxetine and can be used for routine therapeutic drug monitoring or pharmacokinetic studies. Copyright © 2011 John Wiley & Sons, Ltd.     

An LC–MS/MS method for rapid and sensitive high‐throughput simultaneous determination of various protein kinase inhibitors in human plasma

A reliable, high‐throughput and sensitive LC–MS/MS procedure was developed and validated for the determination of five tyrosine kinase inhibitors in human plasma. Following their extraction from human plasma, samples were eluted on a RP Luna®‐PFP 100 Å column using a mobile phase system composed of acetonitrile and 0.01 m ammonium formate in water (pH ~4.1) with a ratio of (50:50, v /v) flowing at 0.3 mL min⁻¹. The mass spectrometer was operating with electrospray ionization in the positive ion multiple reaction monitoring mode. The proposed methodology resulted in linear calibration plots with correlation coefficients values of r ² = 0.9995–0.9999 from concentration ranges of 2.5–100 ng mL⁻¹ for imatinib, 5.0–100 ng mL⁻¹ for sorafenib, tofacitinib and afatinib, and 1.0–100 ng mL⁻¹ for cabozantinib. The procedure was validated in terms of its specificity, limit of detection (0.32–1.71 ng mL⁻¹), lower limit of quantification (0.97–5.07 ng mL⁻¹), intra‐ and inter assay accuracy (−3.83 to +2.40%) and precision (<3.37%), matrix effect and recovery and stability. Our results demonstrated that the proposed method is highly reliable for routine quantification of the investigated tyrosine kinase inhibitors in human plasma and can be efficiently applied in the rapid and sensitive analysis of their clinical samples.     

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.     

A highly sensitive LC–MS/MS method for simultaneous determination of glycyrrhizin and its active metabolite glycyrrhetinic acid: Application to a human pharmacokinetic study after oral administration

A highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of glycyrrhizin (GL) and its active metabolite, glycyrrhetinic acid (GA), from human plasma was validated and applied to a human pharmacokinetic study. The analytes were extracted from human plasma using an Oasis MAX cartridge and chromatographic separation was performed on an Inertsil ODS‐3 column. The detection was performed using an API 4000 mass spectrometer operating in the positive electrospray ionization mode. Selected ion monitoring transitions of m /z 823 → 453 for GL and m /z 471 → 149 for GA were obtained. The response was a linear function of concentration over the ranges of 0.5–200 ng/mL for GL and 2–800 ng/mL for GA (both R ² > 0.998). Using this method, the pharmacokinetics of GL after single oral administration of a clinical dose (75 mg) to six healthy male Japanese volunteers were evaluated. GL was detected in the plasma of all subjects and the average peak concentration was 24.8 ± 12.0 ng/mL. In contrast, peak concentration of GA was 200.3 ± 60.3 ng/mL, i.e. ~8‐fold higher than that of GL. This is the first report clarifying pharmacokinetic profiles of GL and GA simultaneously at a therapeutic oral dose of a GL preparation.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

LC‐MS/MS assay for the determination of lurasidone and its active metabolite,ID‐14283 in human plasma and its application to a clinical pharmacokinetic study

The authors proposed a sensitive, selective and rapid liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay procedure for the quantification of lurasidone and its active metabolite, i.e. ID‐14283 in human plasma simultaneously using corresponding isotope labeled compounds as internal standards as per regulatory guidelines. After liquid–liquid extraction with tert‐butyl methyl ether, the analytes were chromatographed on a C₁₈ column using an optimized mobile phase composed of 5 mm ammonium acetate (pH 5.0) and acetonitrile (15:85, v/v) and delivered at a flow rate of 1.00 mL/min. The assay exhibits excellent linearity in the concentration ranges of 0.25–100 and 0.10–14.1 ng/mL for lurasidone and ID‐14283, respectively. The precision and accuracy results over five concentration levels in four different batches were well within the acceptance limits. Lurasidone and ID‐14283 were found to be stable in battery of stability studies. The method was rapid with the chromatographic run time 2.5 min, which made it possible to analyze 300 samples in a single day. Additionally, this method was successfully used to estimate the in vivo plasma concentrations of lurasidone and ID‐14283 obtained from a pharmacokinetic study in south Indian male subjects and the results were authenticated by conducting incurred samples reanalysis. Copyright © 2015 John Wiley & Sons, Ltd.     

A simple and sensitive LC‐MS/MS method for the determination of sotalol in rat plasma

A sensitive, rapid and robust HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the quantification of sotalol in rat plasma. Plasma samples were precipitated with acetonitrile before analysis. The chromatographic separation was performed on an Atlantis hydrophilic interaction liquid chromatography Silica column (50 × 2.1 mm, 3 µm) with a gradient mobile phase of 10 mm NH₄COOH (containing 0.2% of formic acid) as buffer A and acetonitrile as mobile phase B. Sotalol (m/z 273.2 → 255.1) and atenolol (the internal standard, IS, m/z 267.2 → 190.1) were monitored under positive ionization mode with 5500 QTRAP. Retention time of sotalol and the IS were 2.69 and 3.43 min, respectively. The linear range was 5–500 nm based on the analysis of 0.1 mL of plasma. The intrabatch precision ranged from 1.2 to 6.1%, and the inter‐batch precision was from 3.3 to 6.5%. The coefficient of variation of IS‐normalized matrix factor was 7.6%. Experiments for stability were performed and the analyte was sufficiently stable. A run time of 6 min for each injection made it possible to analyze a high throughput of plasma samples. The assay was successfully applied to the determination of sotalol in rat plasma after a micro‐dose oral administration. Copyright © 2014 John Wiley & Sons, Ltd.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of piperaquine and its N‐oxidated metabolite in rat plasma using LC‐MS/MS

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N ‐oxidated metabolite (PQ‐M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C₁₈ column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple‐quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0–400.0 ng/mL for PQ and 1.0–50.0 ng/mL for PQ‐M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ‐M, respectively, using 40 μL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co‐administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ‐M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).     

A sensitive LC–MS/MS method for simultaneous quantification of geniposide and its active metabolite genipin in rat plasma and its application to a pharmacokinetic study

Genipin (GP), an active metabolite of geniposide (GE), exhibits more potent pharmacological effects than its parent compound. In this paper, a sensitive LC‐MS/MS method was developed and fully validated for the simultaneous determination of GE and GP in rat plasma. We found that GP degraded rapidly in rat plasma at room temperature as a result of irreversible binding with the endogenous nucleophiles in plasma. GP was stable when the sample's pH was ≤4.0. The degradation of GP in rat plasma was well prevented by immediate addition of 5% glacial acetic acid to the freshly collected plasma. The detection was performed on a tandem mass spectrometer coupled with electrospray ionization source in negative mode. Quantification was conducted by multiple reaction monitoring of the transitions [M + CH₃COO]⁻ m/z 447.3 → 225.3 for GE and [M − H]⁻ m/z 225.2 → 123.1 for GP. The method exhibited high sensitivity (LLOQ 1 ng/mL for GE and 0.2 ng/mL for GP) by selecting the acetate adduct ions as the precursor ions for GE. The robust developed method was successfully applied to a pharmacokinetic study in rats after oral administration of GE.     

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.     

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.     

An APCI LC‐MS/MS method for routine determination of capecitabine and its metabolites in human plasma

The anticancer drug capecitabine and its metabolites [including the active metabolite 5‐fluorouracil (5‐FU)] display high pharmacokinetic inter‐patient variability. Such variability, which may lead to treatment failure or toxicity, could need drug concentration measurement to individualize dosing regimen. However, usual assay methods are often long and fastidious. A simultaneous and cost‐effective method was thus developed for the determination of the concentrations of these compounds in human plasma. Compounds were extracted via a classic liquid–liquid extraction. Chromatographic analysis was performed on a C18 reverse phase column with detection by atmosphere pressure chemical ionization LC‐MS/MS. Our method allows a good chromatographic separation of the compounds and was fully validated following Food and Drug Administration (FDA) recommendations (good selectivity, no carry‐over, linearity of the calibration curves without weighting, deviations from nominal concentrations of standard samples lower than 15%, intra‐ and inter‐assay precision and accuracy lower than 15%). Recovery and stability were also acceptable following the FDA guidelines. A matrix effect impairing the determination of 5‐FU was avoided by using a stable isotopic derivative of 5‐FU as internal standard. Interestingly, this method allows detection of TetraHydroUridine, an inhibitor of ex vivo degradation of metabolites, which is essential for the stability, the adequate conditioning of blood samples and for good laboratory practice, essential in routine determination. This method seems usable to routinely determine concentrations of capecitabine and its metabolites in blood and may be helpful in further studies aiming at performing therapeutic drug monitoring. Copyright © 2010 John Wiley & Sons, Ltd.     

Sensitive LC‐MS/MS‐ESI method for simultaneous determination of nifedipine and atenolol in human plasma and its application to a human pharmacokinetic study

A rapid, simple, sensitive and selective LC‐MS/MS method has been developed and validated for quantification of nifedipine (NF) and atenolol (AT) in human plasma (250 μL). The analytical procedure involves a one‐step liquid–liquid extraction method using carbamazepine as an internal standard (IS). The chromatographic resolution was achieved on a Hypurity Advance C₁₈ column using an isocratic mobile phase consisting of 5 mm ammonium acetate–acetonitrile (15:85, v/v) at flow rate of 1.0 mL/min. The LC‐MS/MS was operated under the multiple‐reaction monitoring mode using electrospray ionization. The total run time of analysis was 2 min and elution of NF, AT and IS occurred at 0.79, 1.04 and 0.76 min, respectively. A detailed method validation was performed as per the FDA guidelines and the standard curves found to be linear in the range of 1.02–101 ng/mL for NF and 5.05–503 ng/mL for AT, with a coefficient of correlation of ≥0.99 for both the drugs. NF and AT were found to be stable in a battery of stability studies, viz. bench‐top, auto‐sampler and repeated freeze–thaw cycles. The validated assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2012 John Wiley & Sons, Ltd.     

Systematic evaluation of matrix effect and cross‐talk‐free method for simultaneous determination of zolmitriptan and N‐desmethyl zolmitriptan in human plasma: a sensitive LC‐MS/MS method validation and its application to a clinical pharmacokinetic study

The objective of the present work was to carry out systematic evaluation to eliminate matrix effect owing to plasma phospholipids as observed during sample preparation and to develop a cross‐talk‐free sensitive, selective and rapid bioanalytical method for the simultaneous determination of zolmitriptan (ZT) and N‐desmethyl zolmitriptan (DZT) in human plasma by liquid chromatography–tandem mass spectrometry using naratriptan as internal standard (IS). The analytes and IS were quantitatively extracted from 200 μL human plasma by solid phase extraction. No cross‐talk was found between ZT and DZT having identical product ions. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The total chromatographic run time was 2.5 min. The method was fully validated for sensitivity, selectivity, specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity and stability studies. The method was validated over a dynamic concentration range of 0.1–15 ng/mL for ZT and DZT. The method was successfully applied to a bioequivalence study of 2.5 mg ZT tablet formulation in 18 healthy Indian male subjects under fasting conditions. Assay reproducibility was assessed by reanalysis of 62 incurred samples. Copyright © 2015 John Wiley & Sons, Ltd.