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.     

A simple,selective and rapid validated method for estimation of anastrazole in human plasma by liquid chromatography–tandem mass spectrometry and its application to bioequivalence study

A rapid, simple and specific method for estimation of anastrazole in human plasma was validated using letrozole as internal standard. The analyte and internal standard were extracted from plasma using simple solid‐phase extraction. The compound were separated on a reverse‐phase column with an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (12 : 88, v/v) and detected by tandem mass spectrometry in positive ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 294.1 → 225.1 for anastrazole and m/z 286.1 → 217.1 for internal standard. Linearity in plasma was observed over the concentration range 0.3–30 ng/mL for anastrazole. The mean recovery for anastrazole was 83.7% with a lower limit of quantification of 0.3 ng/mL. The coefficient of variation of the assay was less than 6.8% and the accuracy was 96.1–102.2%. The validated method was applied to a bioequivalence study of 1 mg anastrazole tablet in healthy human volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Assessment of pharmacokinetics,bioavailability and protein binding of anacetrapib in rats by a simple high‐performance liquid chromatography–tandem mass spectrometry method

Anacetrapib is a potent and selective CETP inhibitor and is undergoing phase III clinical trials for the treatment of dyslipidemia. A simple and sensitive high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the quantification of anacetrapib in rat plasma was developed and validated using an easily purchasable compound, chlorpropamide, as an internal standard (IS). A minimal volume of rat plasma sample (20 μL) was prepared by a single‐step deproteinization procedure with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed using selected reaction monitoring modes at the mass/charge transitions m/z 638 → 283 for anacetrapib and m/z 277 → 175 for IS. The assay was validated to demonstrate the selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. The lower limit of quantification was 5 ng/mL. This LC‐MS/MS assay was successfully applied in the rat plasma protein binding and pharmacokinetic studies of anacetrapib. The fraction of unbound anacetrapib was determined to be low (ranging from 5.66 to 12.3%), and the absolute oral bioavailability of anacetrapib was 32.7%.     

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.     

Sensitive and specific LC‐ESI‐MS/MS method for determination of ZYDPLA1, a novel long‐acting dipeptidyl peptidase 4 inhibitor in rat plasma: An application for toxicokinetic study in rats

A rapid and highly specific assay was developed and validated for the estimation of ZYDPLA1 in rat plasma using liquid chromatography coupled to tandem mass spectrometry with positive electrospray ionization. Method validation comprised of parameters such as specificity, matrix effect, precision, accuracy, recovery, stability, etc. The assay procedure involved a simple protein precipitation of ZYDPLA1 and alprazolam (internal standard) from rat plasma using acetonitrile. Chromatographic separation was achieved with a gradient mobile phase comprising: (A) 0.2% ammonia in purified water; (B) 0.1% formic acid in isopropyl alcohol/methanol (1: 1 v /v); and (C) acetonitrile at a flow rate of 1 mL/min on an ACE‐5, C18 (4.6 × 50 mm) column with a run time of 5.5 min. The quantitation of ZYDPLA1 was achieved by the summation of four multiple reaction mode transitions (m/z 399.7 → 383.0, 399.7 → 276.10, 399.7 → 153.20 and 399.7 → 127.20), while that of the internal standard was by a single multiple reaction mode transition (m/z 309.10 → 281.00). The lower limit of quantitation achieved was 0.01 μg/mL and the method showed linearity from 0.01 to 25 μg/mL. The intra‐ and inter‐day precision (%CV) of the quality control samples was within 8.81% and accuracy was ±10% of nominal values. This novel method was applied for evaluation of toxicokinetics of ZYDLA1 in rats.     

A chiral LC‐MS/MS method for the enantioselective determination of R‐(+)‐ and S‐(–)‐pantoprazole in human plasma and its application to a pharmacokinetic study of S‐(–)‐pantoprazole sodium injection

Pantoprazole, a proton pump inhibitor, is clinically used for the treatment of peptic diseases. An enantioselective LC‐MS/MS method was developed and validated for the simultaneous determination of pantoprazole enantiomers in human plasma. Pantoprazole enantiomers and the internal standard were extracted from plasma using acetonitrile. Chiral separation was carried on a Chiralpak IE column using the mobile phase consisted of 10 mm ammonium acetate solution containing 0.1% acetic acid–acetonitrile (28 : 72, v /v). MS analysis was performed on an API 4000 mass spectrometer. Multiple reactions monitoring transitions of m /z 384.1→200.1 and 390.1→206.0 were used to quantify pantoprazole enantiomers and internal standard, respectively. For each enantiomer, no apparent matrix effect was found, the calibration curve was linear over 5.00–10,000 ng/mL, the intra‐ and inter‐day precisions were below 10.0%, and the accuracy was within the range of –5.6% to 0.6%. This method was applied to the stereoselective pharmacokinetic studies in human after intravenous administration of S ‐(–)‐pantoprazole sodium injections. No chiral inversion was observed during sample storage, preparation procedure and analysis. While R ‐(+)‐pantoprazole was detected in human plasma with a slightly high concentration, which implied that S ‐(–)‐pantoprazole may convert to R ‐(+)‐pantoprazole in some subjects.     

Development and validation of HPLC‐MS/MS method for the determination of lixivaptan in mouse plasma and its application in a pharmacokinetic study

The aim of the present study was to develop a simple, sensitive and accurate liquid chromatography–electrospray ionization tandem mass spectrometry (ESI‐MS/MS) method for the determination of lixivaptan (LIX) in mouse plasma using vildagliptin as the internal standard (IS). A precipitation procedure was used for the extraction of LIX and vildagliptin from mouse plasma. Chromatographic separation of LIX was achieved using a C₁₈ analytical column (50 × 2.1 mm, 1.8 μm) at 25°C. The mobile phase comprised acetonitrile and ammonium formate (10 mm , pH 3.1; 40:60, v /v) pumped at a flow rate of 0.3 mL min⁻¹. A tandem mass spectrometer with an electrospray ionization source was used to perform the assay. Quantification of LIX at m/z 290 → 137 and IS at 154 → 97 was attained through multiple reaction monitoring. The investigated method was authenticated following the bio‐analytical method of validation guidelines of the US Food and Drug Administration. The developed method showed a good linearity over the concentration range from 5 to 500 ng mL⁻¹, and the calibration curve was linear (r = 0.9998). The mean recovery of LIX from mouse plasma was 99.2 ± 0.68%. All validation parameters for LIX were within the levels required for acceptance. The proposed method was effectively used for a pharmacokinetic study of LIX in mouse plasma.     

A sensitive quantitative assay for the determination of propafenone and two metabolites, 5‐hydroxypropafenone and N‐depropylpropafenone,in human K2EDTA plasma using LC–MS/MS with ESI operated in positive mode

Propafenone is a potent antiarrhythmic agent; clinically propafenone has been used for a number of cardiac arrhythmias because it possesses multiple modes of action, via beta adrenergic receptor blockade and calcium antagonistic activity. Propafenone (PPF) exhibits extensive saturable presystemic biotransformation (first‐pass effect) resulting in two active metabolites: 5‐hydroxypropafenone (5‐OH PPF) formed by CYP2D6 and N‐ depropylpropafenone (NDP) formed by both CYP3A4 and CYP1A2 enzymes. A specific and sensitive LC–MS/MS method was developed and validated for quantitation of PPF, 5‐OH PPF and NDP using turboion spray in a positive ion mode. A solid‐phase extraction was employed for the extraction from human plasma. Chromatographic separation of analytes was achieved using an ACE‐5 C₈ (50 × 4.6 mm) column with a gradient mobile phase comprising ammonium acetate containing 0.01% TFA in purified water and acetonitrile. The retention times achieved were 1.36, 1.23, 1.24 min and 1.34 min for PPF, 5‐OH PPF, NDP and IS (carbamazepine), respectively. Quantitation was performed by monitoring multiple reaction monitoring transition pairs of m /z 342.30 to m /z 116.20, m /z 358.30 to m /z 116.20, m /z 300.30 to m /z 74.20 and m /z 237.20 to m /z 194.10, respectively. The developed method was validated for various parameters. The calibration curves of PPF and 5‐OH PPF showed linearity from 1 to 500 ng/mL, with a lower limit of quantitation of 1.0 ng/mL and for NDP linearity from 0.1 to 25 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The bias and precision for intra‐ and‐inter batch assays were <10 and 5%, respectively. The developed assay was used to evaluate pharmacokinetic properties of propafenone and its major metabolites in healthy human subjects.     

Simple and sensitive LC‐ESI‐MS method for the quantitation of sildenafil in plasma samples

Sildenafil is a selective inhibitor of phosphodiesterase type 5 enzyme. A simple and sensitive LC‐MS assay was developed to determine the concentration of sildenafil in human plasma. Sildenafil and omeprazole (internal standard) were extracted from the plasma with diethyl ether. The extract was evaporated under nitrogen and the residue was constituted with ACN and injected onto Novapak C₁₈ column (75×3.9 mm, 4 μm). The mobile phase consisted of 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid and was delivered isocratically at a flow rate of 0.2 mL/min. Sildenafil and omeprazole were monitored using a positive electrospray mode with single‐ion recording set at m/z 475 and 346, respectively, which are consistent with [M+H]⁺ molecular ions, and the run time was less than 5 min. The detection limit of sildenafil was 0.5 ng/mL, and the calibration curve was linear between 0.5 and 2000 ng/mL (R²>0.99). Within‐ and between‐day coefficients of variation were less than 7%. This method has been successfully used to measure sildenafil plasma concentrations in a beagle dog model following an oral administration of the drug.     

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.     

Development of a fast LC‐MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics

A fast and accurate liquid chromatography/tandem mass spectrometric (LC‐MS/MS) assay was first developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 μL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion‐RP 80A column at 30 °C. The mobile phase consisted of methanol and 0.2% formic acid containing 0.2 mM EDTA (60:40, v/v). The flow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor‐to‐parent ion transitions m/z 140.1 → 53.1 for deferiprone and m/z 143.1 → 98.1 for internal standard. A linear range was established from 0.1 to 20 µg/mL. The limit of detection was determined as 0.05 µg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy. Intraday and interday precisions were 4.3–5.5 and 4.6–7.3%, respectively. The recovery of deferiprone was in the range of 80.1–86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated LC‐MS/MS assay for the quantitative determination of vardenafil in human plasma and its application to a pharmacokinetic study

A sensitive high‐performance liquid chromatography–tandem mass spectrometric (HPLC‐MS/MS) assay has been developed for the quantitative analysis of vardenafil in human plasma. Vardenafil and the internal standard, alprazolam, were extracted from 0.2 mL aliquots of alkalinized plasma by a single solvent extraction into hexane : dichloromethane. Reversed‐phase chromatographic separation was affected by gradient elution with mobile phases consisting of 10 mM ammonium formate pH 7.0 (solvent A) and methanol (100%, solvent B), delivered at a flow rate of 0.4 mL/min. The analytes were detected by using an electrospray ion source on a 4000 QTrap triple quadrupole mass spectrometer operating in positive ionization mode. The mass transitions were m/z 489.3 → 312.2 for vardenafil and m/z 309.2 → 281.0 for alprazolam. The assay was linear over the concentration range of 0.2–100 ng/mL, with correlation coefficients ≥0.995. The intra‐ and inter‐day precision was less than 5.4% in terms of relative standard deviation and the accuracy was within 12.7% in terms of relative error. The lower limit of quantitation was set at 0.2 ng/mL. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of plasma samples obtained following the oral administration of vardenafil to healthy male volunteers in a pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

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

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

Pharmacokinetic study of ardisiacrispin A in rat plasma after intravenous administration by UPLC–MS/MS

In this work, a sensitive and selective UPLC‐MS/MS method for determination of ardisiacrispin A in rat plasma was developed. Cyasterone used as an internal standard (IS) and protein precipitation by acetonitrile–methanol (9:1, v /v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m /z 1083.5 → 407.1 for ardisiacrispin A and m /z 521.3 → 485.2 for IS. Calibration plots were linear throughout the range 5–2000 ng/mL for ardisiacrispin A in rat plasma. Mean recoveries of ardisiacrispin A in rat plasma ranged from 80.4 to 92.6%. The values of RSD of intra‐ and inter‐day precision were both <11%. The accuracy of the method was between 97.3 and 105.6%. The method was successfully applied to pharmacokinetic study of ardisiacrispin A after intravenous administration in rats.     

A highly sensitive method for the quantification of fludrocortisone in human plasma using ultra‐high‐performance liquid chromatography tandem mass spectrometry and its pharmacokinetic application

A simple and high sensitive ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the determination of fludrocortisone in human plasma was developed and validated as per guidelines. The analyte and internal standard (IS), fludrocortisone‐d₅, were extracted from human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a Chromolith RP_18e column using a mixture of acetonitrile and 2 mm ammonium formate (70:30, v/v) as the mobile phase at a flow rate of 0.7 mL/min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The precursors to product ion transitions monitored for fludrocortisone and IS were m/z 381.2 → 343.2 and 386.2 → 348.4, respectively. The assay was validated with linear range of 40–3000 pg/mL. The intra‐ and inter‐day precisions (relative standard deviation) were within 0.49–7.13 and 0.83–5.87%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2015 John Wiley & Sons, Ltd.     

Validation of an ultra‐performance liquid chromatography–tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope‐labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra‐ and inter‐batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC‐MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

Rapid determination of corticosterone in mouse plasma by ultra fast liquid chromatography‐tandem mass spectrometry

Major depressive disorder is a severe, life‐threatening and highly prevalent psychiatric disorder. A high percentage of people suffering from depression are characterized by hyperactivity of the hypothalamic–pituitary–adrenal axis, resulting in plasma glucocorticoid (cortisol in human and corticosterone in rodent) elevations. Glucocorticoid is a critical molecule in the onset of pathology of depression. A simple, highly sensitive and specific method based on ultra‐fast liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of corticosterone in mouse plasma for the first time, which provides technical support for the high‐throughput measurement for clinical determination of corticosterone in biological samples. Samples were spiked with methanol to precipitate the protein, and then chromatographed on an Agilent Zorbax Eclipse Plus C₁₈ (100 × 2.1 mm,1.8 µm) column by linear gradient elution with methanol and 0.1% formic acid as the mobile phase within 5 min. The detection of corticosterone was performed on ultra‐fast liquid chromatography–triple quadrupole tandem mass spectrometry in the positive ion. The ions [M + H]⁺ m/z 347.2 → m/z 311.1 for corticosterone and [M + H]⁺ m/z 363.2 → m/z 327.2 for hydrocortisone (internal standard) were used for quantitative determination. The lower quantification limit for corticosterone was 1 ng/mL. The validated method was successfully applied to the quantitation of corticosterone in mouse plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of an LC–MS/MS method for quantification of two pairs of isomeric flavonoid glycosides and other ones in rat plasma: Application to pharmacokinetic studies

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of six flavonoid glycosides – isoorientin ( 1 ), orientin ( 2 ), 2″‐O‐β ‐d ‐xylopyranosyl isoorientin ( 3 ), 2″‐O‐β ‐d ‐xylopyranosyl isovitexin ( 4 ), 6‐C‐l ‐α ‐arabipyranosyl vitexin ( 5 ) and vitexin ( 6 ) – in rat plasma using isoquercitrin as the internal standard (IS). Plasma samples were prepared by a one‐step protein precipitation with acetonitrile. Chromatographic analysis was carried out on a 25 cm C₁₈ column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. Six analytes and IS were detected through electrospray ionization in negative‐ion selection reaction monitoring mode. The mass transitions were as follows: m/z 447.2 → 327.0 for 1 , m/z 447.2 → 327.0 for 2 , m/z 579.3 → 458.9 for 3 , m/z 563.0 → 293.1 for 4 , m/z 563.0 → 353.0 for 5 , m/z 431.1 → 311.1 for 6 , and m/z 463.1 → 300.2 for IS. Calibration curves exhibited good linearity (r ² > 0.9908) over a wide concentration range for all compounds. Intra‐ and inter‐day precision (RSD, %) at four different levels were both <14.2% and the accuracy (RE, %) ranged from −11.9 to 12.0%. The extraction recoveries of the six components ranged from 88.2 to 103.6%. The validated assay was successfully applied to the pharmacokinetic studies of the six components in male rat plasma after intravenous administration of total flavonoids of Scorzonera austriaca Wild.