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.     

Determination and toxicokinetics comparison of ketamine and S(+)‐ketamine in dog plasma by HPLC‐MS method

;A simple and reproducible method was developed for the quantification of ketamine and S(+)‐ketamine in dog plasma using a high‐performance liquid chromatography system coupled to a positive ion electrospray mass spectrometric analysis. Solid‐phase extraction was used for extracting analytes from dog plasma samples. The analytes were separated on a Zorbax SB C₁₈ column (100 × 2.1 mm, 3.5 μm) with acetonitrile–formate buffer (10 mM ammonium formate and 0.3% formic acid) (17 : 83, v/v) as mobile phase at a flow‐rate of 0.2 mL/min. Detection was operated under selected ion monitoring mode. [M + H]⁺ at m/z 238 for ketamine and S(+)‐ketamine and [M + H]⁺ at m/z 180 for phenacetin (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration range 51.6–2580 ng/mL. The intra‐ and inter‐day precisions (RSD %) were within 11.3% and the assay accuracies ranged from 80.0 to 101.4%. Their average recoveries were greater than 91.1% at all test concentrations. The analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the toxicokinetics study and comparison of ketamine and S (+)‐ketamine following intravenous administration to dogs. Copyright © 2009 John Wiley & Sons, Ltd.     

Pharmacokinetics of methyl salicylate‐2‐O‐β‐D‐lactoside,a novel salicylic acid analog isolated from Gaultheria yunnanensis,in dogs

Methyl salicylate‐2‐O‐β‐D‐lactoside (MSL), a natural salicylate derivative of Gaultheria yunnanensis (Franch.) Rehder (G. yunnanensis), has been shown to provide a beneficial anti‐inflammatory effect in animal models. Studies on the pharmacokinetics and bioavailability of MSL can provide both a substantial foundation for understanding its mechanism and empirical evidence to support its use in clinical practice. A simple and sensitive high‐performance liquid chromatography (HPLC) method, coupled with ultraviolet analyte detection, was developed for determining the concentration of MSL and its metabolite in beagle plasma. Chromatographic separation was achieved on a Agilent Zorbax SB‐C₁₈ column (5 μm ,4.6 × 250 mm). The mobile phase consisted of aqueous solution containing 0.1% phosphoric acid and acetonitrile (82:90, v/v), at a flow rate of 1 mL/min. Validation of the assay demonstrated that the developed HPLC method was sensitive, accurate and selective for the determination of MSL and its metabolite in dog plasma. After orally administering three doses of MSL, it could no longer be detected in dog plasma and its metabolite, salicylic acid, was detected. Salicylic acid showed a single peak in the plasma concentration–time curves and linear pharmacokinetics following the three oral doses (r² > 0.99). In contrast, only MSL was detected in plasma following intravenous administration. These results will aid in understanding the pharmacological significance of MSL. The developed method was successfully used for evaluation of the oral and intravenous pharmacokinetic profile of MSL in dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive LC–MS/MS method for quantification of quercetin‐3‐O‐β‐d‐glucopyranosyl‐7‐O‐β‐d‐gentiobioside in plasma and its application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method with good accuracy and precision was developed and validated for the pharmacokinetic study of quercetin‐3‐O‐β‐d ‐glucopyranosyl‐7‐O‐β‐d ‐gentiobioside (QGG) in Sprague–Dawley rats. Plasma samples were simply precipitated by methanol and then analyzed by LC–MS/MS. A Venusil® ASB C₁₈ column (2.1 × 50 mm, i.d. 5 μm) was used for separation, with methanol–water (50:50, v/v) as the mobile phase at a flow rate of 300 μL/min. The optimized mass transition ion‐pairs (m/z) for quantitation were 787.3/301.3 for QGG, and 725.3/293.3 for internal standard. The linear range was 7.32–1830 ng/mL with an average correlation coefficient of 0.9992, and the limit of quantification was 7.32 ng/mL. The intra‐ and inter‐day precision and accuracy were less than ±15%. At low, medium and high quality control concentrations, the recovery and matrix effect of the analyte and IS were in the range of 89.06–92.43 and 88.58–97.62%, respectively. The method was applied for the pharmacokinetic study of QGG in Sprague–Dawley rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous estimation of levodopa,carbidopa and 3‐oxymethyldopa in rat plasma using HPLC‐ECD

The aim of study was to develop a suitable analytical method for simultaneous estimation of levodopa, carbidopa and 3‐O‐methyl dopa in rat plasma. Chromatographic separation of plasma samples was achieved using a reverse‐phase C₁₈ column. The mobile phase used consisted of a mixture of methanol and phosphate buffer (10 mM , pH 3.50) in the ratio of 90:10 v/v. All analytes were estimated by electrochemical detection at +800 mV. The developed method has been validated as per the standard guidelines. Precision study results were found to be satisfactory, with percentage relative standard deviation for repeatability and intermediate precision <3.96 and 6.56%, respectively, for all analytes detected in rat plasma. The developed method in rat plasma was found to be simple, rapid, accurate, precise and specific. The proposed method has been successfully applied for analysis of rat plasma samples obtained during an oral pharmacokinetic study of sustained release pellets of levodopa and carbidopa in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4‐O‐deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 µL plasma aliquot was protein precipitated with acetonitrile–methanol (1:1, v/v) containing the internal standard vinorelbine‐d3 and 20 µL volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using isocratic elution with 1 mm ammonium acetate–ammonia buffer pH 10.5–acetonitrile–methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4‐O‐deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 µL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4‐O‐deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre‐)clinical pharmacologic studies with vinorelbine in humans and mice. Copyright © 2009 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 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 UPLC‐MS/MS method for the determination of 7‐hydroxymitragynine,a μ‐opioid agonist,in rat plasma and its application to a pharmacokinetic study

A simple, sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated to determine the concentrations of 7‐hydroxymitragynine in rat plasma. Following a single‐step liquid–liquid extraction of plasma samples using chloroform, 7‐hydroxymitragynine and the internal standard (tryptoline) were separated on an Acquity UPLC^TM BEH C₁₈ (1.7 µm, 2.1 × 50 mm) column using an isocratic elution at a flow rate of 0.2 mL/min. The mobile phase consisted of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile (10:90, v/v). The run time was 2.5 min. The analysis was carried out under the multiple reaction‐monitoring mode using positive electrospray ionization. Protonated ions [M + H]⁺ and their respective product ions were monitored at the following transitions: 415 → 190 for 7‐hydroxymitragynine and 173 → 144 for the internal standard. The calibration curve was linear over the range of 10–4000 ng/mL (r² = 0.999) with a lower limit of quantification of 10 ng/mL. The extraction recoveries ranged from 62.0 to 67.3% at concentrations of 20, 600 and 3200 ng/mL). Intra‐ and inter‐day assay precisions (relative standard deviation) were <15% and the accuracy was within 96.5–104.0%. This validated method was successfully applied to quantify 7‐hydroxymitragynine in rat plasma following intravenous administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Simple HPLC‐UV for the quantification of a new leishmanicidal candidate (E)‐1‐4(trifluoromethyl) benzylidene)‐5‐(2‐4‐dichlorozoyl) carbonylhydrazine (LASSBio‐1736) in rat plasma for pharmacokinetics assessment

In this study, a sensitive HPLC‐UV assay was developed and validated for the determination of LASSBio‐1736 in rat plasma with sodium diclofenac as internal standard (IS). Liquid–liquid extraction using acetonitrile was employed to extract LASSBio‐1736 and IS from 100 μL of plasma previously basified with NaOH 0.1 M. Chromatographic separation was carried on Waters Spherisorb^®S5 ODS2 C₁₈ column (150 × 4.6 mm, 5 μm) using an isocratic mobile phase composed by water with triethylamine 0.3% (pH 4), methanol and acetonitrile grade (45:15:40, v/v/v) at a flow rate of 1 mL/min. Both LASSBio‐1736 and IS were eluted at 4.2 and 5 min, respectively, with a total run time of 8 min only. The lower limit of quantification was 0.2 μg/mL and linearity between 0.2 and 4 μg/mL was obtained, with an R² > 0.99. The accuracy of the method was >90.5%. The relative standard deviations intra and interday were <6.19 and <7.83%, respectively. The method showed the sensitivity, linearity, precision, accuracy and selectivity required to quantify LASSBio‐1736 in preclinical pharmacokinetic studies according to the criteria established by the US Food and Drug Administration and European Medicines Agency. Copyright © 2016 John Wiley & Sons, Ltd.     

LC‐MS/MS method for simultaneous analysis of uracil, 5,6‐dihydrouracil, 5‐fluorouracil and 5‐fluoro‐5,6‐dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5‐fluorouracil (5‐FU) is widely used for treating solid tumors. Response to 5‐FU treatment is variable with 10–30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6‐dihydrouracil (UH₂), and analogously, 5‐FU into 5‐fluoro‐5,6‐dihydrouracil (5‐FUH₂). Combined quantification of U and UH₂ with 5‐FU and 5‐FUH₂ may provide a pre‐therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography–tandem mass spectrometry assay for simultaneous quantification of U, UH₂, 5‐FU and 5‐FUH₂ in human plasma. Samples were prepared by liquid–liquid extraction with 10:1 ethyl acetate‐2‐propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC₁₈ column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01–10 μm for U, 0.1–10 μm for UH₂, 0.1–75 μm for 5‐FU and 0.75–75 μm for 5‐FUH₂, covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5‐FU‐treated colorectal cancer patients. The present method merges the analysis of 5‐FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5‐FU‐based chemotherapy. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of 1‐O‐acetylbritannilactone in rat plasma and its application to a preclinical pharmacokinetic study

A novel, rapid and sensitive LC‐MS/MS method for the determination of 1‐O‐Acetylbritannilactone (ABL), a sesquiterpene lactone abundant in Inula britannica, was developed and validated using heteroclitin D as internal standard. Separation was achieved on a reversed phase Hypersil Gold C₁₈ column (50 × 4.6 mm, i.d., 3.0 µm) using isocratic elution with methanol–5 mM ammonium acetate buffer aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min. Calibration curve was linear (r > 0.99) in a concentration range of 1.60–800 ng/mL with the lower limit of quantification of 1.60 ng/mL. Intra‐ and inter‐day accuracy and precision were validated by relative error (RE) and relative standard deviation (RSD) values, respectively, which were both less than ±15%. The validated method has been successfully applied to a pharmacokinetic study of ABL in rats. The elimination half‐lives were 0.412 ± 0.068, 0.415 ± 0.092 and 0.453 ± 0.071 h after a single intravenous administration of 0.14, 0.42, and 1.26 mg/kg ABL, respectively. The area under the plasma concentration–time curve from time zero to the last quantifiable time point and from time zero to infinity and the plasma concentrations at 2 min were linearly related to the doses tested. Copyright © 2015 John Wiley & Sons, Ltd.     

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

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

Quantitation and pharmacokinetics of 1,4‐diamino‐2,3‐dicyano‐1,4‐bis (2‐aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography‐tandem mass spectrometry

A simple, robust, and rapid LC‐MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 μL) were deproteinized using 200 μL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC‐C₁₈ column (4.6 × 50 mm, 2.7 μm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20–5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra‐ and interday, was less than 10.1% with an accuracy of 90.7–99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3‐h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.     

Accurate determination of a novel vasodilatory β‐blocker TJ0711 using LC‐MS/MS: Resolution of an isobaric metabolite interference in dog plasma

A rapid, robust and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for bioanalysis of TJ0711, a novel vasodilatory β‐blocker in dog plasma. This assay is able to chromatographically separate TJ0711 from its isobaric metabolite as well as glucuronide conjugates. Chromatographic separation was achieved on a Welch Ultimate‐XB C₁₈ column (2.1 × 100 mm, 3 μm). The analyte and internal standard (propranolol) were extracted from plasma by liquid–liquid extraction using ethyl acetate. The mass spectrometric detection was carried out in positive ion multiple reaction monitoring mode. Good linearity was obtained over the concentration range of 0.5–500 ng/mL (r > 0.99) for TJ0711. Moreover, the method had good accuracy (RE ranging from −2.70 to −0.32%) and precision (RSD < 7.55%). TJ0711 was stable in dog plasma for at least 6 h at ambient temperature, for at least 30 days at −20°C and after three freeze–thaw cycles. This method was successfully applied to a preclinical pharmacokinetic study and the results demonstrated linear pharmacokinetics of TJ0711 over a dose range from 0.03 to 0.3 mg/kg. No significant gender differences were observed in TJ0711 plasma pharmacokinetic parameters.     

A new,validated HPLC‐MS/MS method for the simultaneous determination of the anti‐cancer agent capecitabine and its metabolites: 5′‐deoxy‐5‐fluorocytidine, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 5‐fluorodihydrouracil,in human plasma

A rapid and selective liquid chromatography/tandem mass spectrometric method was developed for the simultaneous determination of capecitabine and its metabolites 5′‐deoxy‐5‐fluorocytidine (5′‐DFCR), 5′‐deoxy‐5‐fluorouracil (5′‐DFUR), 5‐fluorouracil (5‐FU) and dihydro‐5‐fluorouracil (FUH₂) in human plasma. A 200 μL human plasma aliquot was spiked with a mixture of internal standards fludarabine and 5‐chlorouracil. A single‐step protein precipitation method was employed using 10% (v/v) trichloroacetic acid in water to separate analytes from bio‐matrices. Volumes of 20 μL of the supernatant were directly injected onto the HPLC system. Separation was achieved on a 30 × 2.1 mm Hypercarb (porous graphitic carbon) column using a gradient by mixing 10 mm ammonium acetate and acetonitrile–2‐propanol–tetrahydrofuran (1 : 3 : 2.25, v/v/v). The detection was performed using a Finnigan TSQ Quantum Ultra equipped with the electrospray ion source operated in positive and negative mode. The assay quantifies a range from 10 to 1000 ng/mL for capecitabine, from 10 to 5000 ng/mL for 5′‐DFCR and 5′‐DFUR, and from 50 to 5000 ng/mL for 5‐FU and FUH₂ using a plasma sample of 200 μL. Correlation coefficients (r²) of the calibration curves in human plasma were better than 0.99 for all compounds. At all concentration levels, deviations of measured concentrations from nominal concentration were between ?4.41 and 3.65% with CV values less than 12.0% for capecitabine, between ?7.00 and 6.59% with CV values less than 13.0 for 5′‐DFUR, between ?3.25 and 4.11% with CV values less than 9.34% for 5′‐DFCR, between ?5.54 and 5.91% with CV values less than 9.69% for 5‐FU and between ?4.26 and 6.86% with CV values less than 14.9% for FUH₂. The described method was successfully applied for the evaluation of the pharmacokinetic profile of capecitabine and its metabolites in plasma of treated cancer patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a single HPLC method for determination of α‐tocopherol in cell culture and in human or mouse biological samples

A straightforward and common analytical method for α‐tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase‐chromatographic method (RP‐HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C₁₈ column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H₂O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter‐ and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Rapid high‐performance liquid chromatography–tandem mass spectrometry method for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma and its application in comparative bioavailability study

A rapid high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma using fluoxetine as an internal standard. In the liquid–liquid extraction method, compounds and internal standard were extracted from plasma using methyl tertiary butyl ether as an extraction solvent. The HPLC separation of the analytes was performed on a Zorbax SB‐C₁₈, 50 × 4.6 mm, 5 µm column, using a isocratic elution program using a mobile phase consisting of HPLC‐grade methanol: 5 mm ammonium acetate (80:20 v/v) at a flow‐rate of 1.0 mL/min with a total runtime of 3.0 min. The proposed method has been validated with a linear range of 4–400 ng/mL for venlafaxine and 5–500 ng/mL for O‐desmethyl venlafaxine. The method was applied for a bio‐equivalence study of 75 mg tablets formulation in 32 Indian male healthy subjects under fasting conditions. Copyright © 2009 John Wiley & Sons, Ltd.