Development and validation of an LC‐APCI‐MS/MS method for the determination of phenethyl isothiocyanate in human plasma

Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent present in cruciferous vegetables. This paper describes the development of a method for the determination of PEITC in human plasma by liquid chromatography/tandem mass spectrometry (LC‐MS/MS). Atmospheric‐pressure chemical ionization was found more suitable for ionization of PEITC than electrospray ionization. Because of the lability of PEITC, a combination of low temperature and acidification was applied to minimize the degradation during the sample collection and preparation procedure. A simple protein precipitation with acetonitrile was used for the preparation of plasma samples. The analyte and 1‐phenylpropyl isothiocyanate as internal standard (IS) were subjected to chromatographic analysis on a C₁₈ column (50 × 2.1 mm, 5 µm) using 85% methanol as mobile phase at a flow rate of 0.3 mL/min. The total analysis time for each chromatograph was 3 min and the results were linear over the studied range (5.00–250 ng/mL). The intra‐ and inter‐day precision values were acceptable as per US Food and Drug Administration guidelines. This method was successfully applied in the determination of PEITC concentrations in plasma samples from healthy chinese Volunteers. Copyright © 2014 John Wiley & Sons, Ltd.     

UHPLC‐Q‐TOF‐MS/MS‐based screening and characterization of metabolites of cnidilin in human liver microsomes

Cnidilin is an active natural furocoumarin ingredient originating from well‐known traditional Chinese medicine Radix Angelicae Dahuricae . In the present study, an efficient approach was developed for the screening and identification of cnidilin metabolites using ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. In this approach, an on‐line data acquisition method multiple mass defect filter combined with dynamic background subtraction was developed to trace all probable metabolites. Based on this analytical strategy, a total of 24 metabolites of cnidilin were detected in human liver microsomal incubation samples and the metabolic pathways were proposed. The results indicated that oxidation was the main biotransformation route for cnidilin in human liver microsomes. In addition, the specific cytochrome P450 (CYP) enzymes involved in the metabolism of cnidilin were identified using chemical inhibition and CYP recombinant enzymes. The results showed that CYP1A2 and CYP3A4 might be the major enzymes involved in the metabolism of cnidilin in human liver microsomes. The relationship between cnidilin and the CYP450 enzymes could provide us a theoretical basis of the pharmacological mechanism.     

Development and validation of automated SPE‐LC‐MS/MS method for determination of indapamide in human whole blood and its application to real study samples

A fast and simple liquid chromatography–electrospray ionization tandem mass spectrometry method for determination of indapamide in human whole blood was developed and validated. The sample extraction of indapamide from human whole blood was achieved using automated solid‐phase extraction. Chromatographic separation was performed on Kinetex C₁₈ column (100 × 2.1 mm, 1.7 µm particle size) using acetonitrile and 2 mm ammonium formate in ratio 90:10 (v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode using positive electrospray ionization for indapamide and the internal standard (zolpidem tartarate). The total run time was 2.5 min. The present method was found to be linear in the concentration range of 1–50 ng/mL with the coefficient of determination 0.9987. The absolute recoveries of indapamide were 90.51–93.90%. The method was validated according the recommendations for validation of bioanalytical methods of European Medicines Agency guideline and was successfully used to analyze human whole blood samples for application in a pharmacokinetic study. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study

A sensitive, specific and simple LC‐MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under multiple‐reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C₁₈ 100 × 2.1 mm column with a mobile phase of water–methanol–0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple‐reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20–10,000 µg/L (r > 0.998) for bivalirudin. The intra‐ and inter‐day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench‐top, freeze–thaw cycles and long‐term stability. The developed assay method was applied to an intravenous administration study in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10‐hydroxynortriptyline (OH‐NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). 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 NTP, OH‐NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH‐NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C₁₈ column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH‐NTP. A linear response function was established for the range of concentrations 1.09–30.0 ng/mL (r > 0.998) for both NTP and OH‐NTP. The intra‐ and inter‐day precision values for NTP and OH‐NTP met the acceptance as per FDA guidelines. NTP and OH‐NTP were stable in a battery of stability studies, i.e. bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of 3‐α‐hydroxytibolone in human plasma by LC‐MS/MS: application for a pharmacokinetic study after administration of a tibolone formulation

A new method was developed for the quantitation of 3‐α‐hydroxy tibolone, in human plasma, after oral administration of a tablet formulation containing tibolone (2.5 mg). 3‐α‐Hydroxy tibolone was extracted by a liquid–liquid procedure, using cyproterone acetate as internal standard and chlorobutane as extraction solvent. After extraction, samples were submitted to a derivatization step with p‐toluenesulfonyl isocyanate. A mobile phase consisting of acetonitrile and water (72:28 v/v) was used and chromatographic separation was achieved using Agilent XDB C₁₈ column (100 × 4.6 mm i.d.; 5 µm particle size), at 40°C. Mass spectrometric detection was performed using atmospheric pressure chemical ionization in negative mode for 3‐α‐hydroxy tibolone and in positive mode for cyproterone acetate. The fragmentation transitions were m/z 510.2 → m/z 170.1 and m/z 417.0 → m/z 357.1 for 3‐α‐hydroxy tibolone and cyproterone acetate, respectively. Calibration curves were constructed over the range 100–30,000 pg/mL and the method was shown to be specific, precise and accurate, with a mean recovery rate of 94.2% for 3‐α‐hydroxy tibolone. No matrix effect or carry‐over was detected in the samples. The validated method was applied in a pharmacokinetic study with a tibolone formulation in healthy female volunteers. Copyright © 2013 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.     

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.     

The development and validation of a high‐throughput LC–MS/MS method for the analysis of endogenous β‐hydroxy‐β‐methylbutyrate in human plasma

A high‐throughput, sensitive, and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid quantitation of β ‐hydroxy‐β ‐methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 μL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC–MS/MS technique under negative mode electrospray ionization conditions. A ¹³C–labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze–thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. Inter‐day accuracies and coefficients of variation (CV) were 91.2–98.1 and 3.7–7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers.     

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.     

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.     

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.     

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 an LC‐ESI‐MS/MS quantification method for a potential γ‐aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays

Binding assays for the γ‐aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry‐based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC‐ESI‐MS/MS quantification method for DDPM‐1007 {(RS)‐1‐[4,4,4‐Tris(4‐methoxyphenyl)but‐2‐en‐1‐yl]piperidine‐3‐carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C₈ column in combination with a mobile phase composed of 10 mm ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 μL/min DDPM‐1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM‐1007 [(²H₉)DDPM‐1007] was synthesized and employed as internal standard. This way DDPM‐1007 could be quantified in a range from 100 pm to10 nm in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra‐ and inter‐batch accuracy. Based on this LC‐ESI‐MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM‐1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM‐1007 at GAT3 could be unambiguously detected. Additionally, the established LC‐MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM‐1007, as exemplified for its logD determination. Copyright © 2012 John Wiley & Sons, Ltd.     

Novel LC‐ ESI‐MS/MS method for desvenlafaxine estimation human plasma: application to pharmacokinetic study

A simple, sensitive and specific liquid chromatography tandem mass spectrometry (LC‐ESI‐MS/MS) method was developed for the quantification of desvenlafaxine in human plasma using desvenlafaxine d6 as an internal standard (IS). Chromatographic separation was performed using a Thermo‐BDS hypersil C₈ column (50 × 4.6 mm, 3 µm) with an isocratic mobile phase composed of 5 mM ammonium acetate buffer: methanol (20:80, v/v), at a flow rate of 0.80 mL/min. Desvenlafaxine and desvenlafaxine d6 were detected with proton adducts at m/z 264.2/58.1 and 270.2/ 64.1 in multiple reaction monitoring positive mode, respectively. Liquid–liquid extraction was used to extract the drug and the IS. The method was linear over the concentration range 1.001–400.352 ng/mL with a correlation coefficient of ≥0.9994. This method demonstrated intra and inter‐day precision within 0.7–5.5 and 1.9–6.8%, and accuracy within 95.3–107.4 and 93.4–99.5%. Desvenlafaxine was found to be stable throughout the freeze–thaw cycles, bench‐top and long‐term matrix stability studies. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of desvenlafaxine in pharmaceutical dosage forms. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a simple,sensitive and accurate LC‐MS/MS method for the determination of guanfacine,a selective α2A‐adrenergicreceptor agonist,in plasma and its application to a pharmacokinetic study

A simple, practical, accurate and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and fully validated for the quantitation of guanfacine in beagle dog plasma. After protein precipitation by acetonitrile, the analytes were separated on a C₁₈ chromatographic column by methanol and water containing 0.1% (v/v) formic acid with a gradient elution. The subsequent detection utilized a mass spectrometry under positive ion mode with multiple reaction monitoring of guanfacine and enalaprilat (internal standard) at m/z 246.2 → 159.0 and m/z 349.2 → 205.9, respectively. Good linearity was obtained over the concentration range of 0.1–20 ng/mL for guanfacine in dog plasma and the lower limit of quantification of this method was 0.1 ng/mL. The intra‐ and inter‐day precisions were <10.8% relative standard deviation with an accuracy of 92.9–108.4%. The matrix effects ranged from 89.4 to 100.7% and extraction recoveries were >90%. Stability studies showed that both analytes were stable during sample preparation and analysis. The established method was successfully applied to an in vivo pharmacokinetic study in beagle dogs after a single oral dose of 4 mg guanfacine extended‐release tablets. Copyright © 2013 John Wiley & Sons, Ltd.     

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

A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8 µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.0 min. The MS/MS ion transitions monitored were 464.4 → 463.4 for hyperoside and 947.12 → 969.60 for IS. Linear responses were obtained for hyperoside ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were <5.38 and 3.39% and the extraction recovery ranged from 94.39 to 100.78% with an RSD <3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of hyperoside. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for the determination of dexamethasone in nude mice plasma and its application to a pharmacokinetic study

In the current study, a simple, sensitive and rapid analytical method for the determination of dexamethasone was developed and applied to a pharmacokinetic study in nude mice. Using testosterone as an internal standard, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) approach after one‐step precipitation with acetonitrile was validated and used to determine the concentrations of dexamethasone in nude mice plasma. The method utilized a simple isocratic reverse phase separation over a Dionex C₁₈ column with a mobile phase composed of acetonitrile–water (40:60, v/v). The analyte was detected by a triple quadrupole tandem mass spectrometer via electrospray and multiple reaction monitoring was employed to select both dexamethasone at m/z 393.0/147.1 and testosterone at m/z 289.5/97.3 in the positive ion mode. The calibration curves were linear (r >0.99) ranging from 2.5 to 500 ng/mL with a lower limit of quantitation of 2.5 ng/mL. The relative standard deviation ranged from 1.69 to 9.22% while the relative error ranged from ?1.92 to ?8.46%. This method was successfully applied to a preclinical pharmacokinetic study of dexamethasone and its pharmacokinetics was characterized by a two‐compartment model with first‐order absorption in female nude mice. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of mesalazine in beagle dog plasma and its application to a pharmacokinetic study

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the determination of mesalazine in beagle dog plasma. The plasma samples were prepared by protein precipitation, then the separation of the analyte was achieved on a Waters Spherisorb C₆ column (150 × 4.6 mm, 5 µm) with a mobile phase consisting of 0.2% formic acid in water–methanol (20:80, v/v). The flow rate was set at 1.0 mL/min with a split ratio of 3:2. Mass spectrometric detection was achieved by a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor–product ion transitions at m/z 154 → m/z 108 for mesalazine and m/z 285 → m/z 193 for diazepam (internal standard). The linear calibration curve of mesalazine was obtained over the concentration range 50–30,000 ng/mL. The matrix effect of mesalazine was within ±9.8%. The intra‐ and inter‐day precisions were <7.9% and the accuracy (relative error) was within ±3.5%. The validated method was successfully applied to investigate the pharmacokinetics of mesalazine in healthy beagle dogs after rectal administration of mesalazine suppository. Copyright © 2014 John Wiley & Sons, Ltd.