Validation of a chiral LC‐MS/MS‐ESI method for the simultaneous quantification of darolutamide diastereomers in mouse plasma and its application to a stereoselective pharmacokinetic study in mice

A simple, selective and reliable LC‐MS/MS method was validated for simultaneous quantitation of darolutamide diastereomers in 50 μL mouse plasma using warfarin as an internal standard (IS) as per regulatory guidelines. Plasma samples were extracted by liquid–liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mm ammonium acetate–absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done in multiple reaction monitoring mode following the transitions m/z 397 → 202 and 307 → 250 for darolutamide diastereomers and the IS, respectively, in the negative ionization mode. The linearity range was 100–2400 ng/mL for each diastereomer. The intra‐ and inter‐day precisions were in the ranges of 1.78–4.20 and 4.34–14.6, and 3.63–4.74 and 4.78–5.15 for diastereomer‐1 and diastereomer‐2, respectively. Both diastereomers were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice. Following oral administration of darolutamide at 10 mg/kg, maximum concentration in plasma was 4189 and 726 ng/mL for diastereomer‐1 and diastereomer‐2, respectively. The terminal half‐life was found to be ~0.50 h for both the diastereomers. The AUC_(0–t) was found to be 18,961 ng*h/mL for diastereomer‐1 and 1340 ng*h/mL diastereomer‐2.     

Bioanalytical method development,validation and quantification of dorsomorphin in rat plasma by LC‐MS/MS

The present investigation describes the development and validation of a sensitive liquid chromatography–mass spectrometry/mass spectrometry (LC‐MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC‐MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C₁₈ column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r² ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter‐day and intra‐day precision and accuracy were found to be within 85–115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats. Copyright © 2013 John Wiley & Sons, Ltd.     

A sensitive and selective method for the quantitative analysis of miglitol in rat plasma using unique solid‐phase extraction coupled with liquid chromatography–tandem mass spectrometry

A sensitive, selective and robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of miglitol in rat plasma. The sample preparation procedures involved protein precipitation and unique solid‐phase extraction, which efficiently removed sources of ion suppression and column degradation interference present in the plasma. Chromatographic separation was achieved on an amide column using 10 mmol/L CH₃COONH₄ and CH₃CN:CH₃OH (90:10, v/v) as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in positive ion mode.The selected reaction monitoring transitions for miglitol and a stable isotope‐labeled internal standard were m/z 208 → m/z 146 and m/z 212 → m/z 176, respectively. The correlation coefficients of the calibration curves ranged from 0.9984 to 0.9993 over a concentration range of 0.5–100 ng/mL plasma. The quantification limit of the proposed method was more than 10 times lower than those of previously reported LC‐MS/MS methods. The novel method was successfully validated and applied to a pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 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.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

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

A highly sensitive and rapid assay method has been developed and validated for the estimation of S‐(−)‐raclopride (S‐RCP) 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 technique for extraction of S‐RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C₁₈ column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S‐RCP and 180.1 → 110.1 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 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra‐day and inter‐day precisions were 0.23–10.5 and 3.74–7.29%, respectively. This novel method was applied to a pharmacokinetic study of S‐RCP in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of leonurine, a novel potential cardiovascular agent, in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study in rats

Leonurine (SCM‐198), an alkaloid from Herba Leonuri, has been suggested as a novel cardiovascular agent by pharmacology studies in preclinical stage. In present study, we report a simple, rapid and sensitive high‐performance liquid chromatography–tandem mass spectrometry method (HPLC‐MS/MS) for determination of leonurine in rat plasma. Leonurine and its internal standard (IS) n‐benzoyl‐l ‐arginine ethyl ester (BAEE) were extracted from plasma samples by one‐step protein precipitation with perchloric acid. Chromatographic separation was performed on an Agilent Zorbax SB‐C₁₈ column (150 × 2.1 mm, 5 µm) using an isocratic elution with acetonitrile–ammonium acetate buffer (10 mm , pH 4.0; 25:75, v/v) as mobile phase at a flow rate of 0.2 mL/min. Analytes were detected by tandem mass spectrometry in positive electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) with the transitions of m/z 312.3 → 181.1 for leonurine and m/z 307.2 → 104.6 for IS. The calibration curves were linear over the range of 4–256 ng/mL with a lower limit of quantitation (LLOQ) of 4 ng/mL. The intra‐ and inter‐day assay precision (as relative standard deviation) were <15%, except which at LLOQ were <20%, with accuracy in the range 98.73‐105.42%. The validated HPLC‐MS/MS method was successfully applied to the pharmacokinetic study in rats following oral administration of leonurine. Copyright © 2011 John Wiley & Sons, Ltd.     

An LC–MS/MS method for quantitation of cyanidin‐3‐O‐glucoside in rat plasma: Application to a comparative pharmacokinetic study in normal and streptozotocin‐induced diabetic rats

A sensitive and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to determine cyanidin‐3‐O‐glucoside (Cy‐3G) in normal and streptozotocin‐induced diabetic rat plasma. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ (50 × 4.6 mm, 5 μm) column and mass spectrometric analysis was performed using a Thermo Finnigan TSQ Quantum Ultra triple‐quadrupole mass spectrometer coupled with an ESI source in the negative ion mode. Selected reaction monitoring mode was applied for quantification using target fragment ions m/z 447.3 → 285.2 for Cy‐3G and m/z 463.0 → 300.1 for quercetin‐3‐O‐glucoside (internal standard). The calibration curve was linear over the range 3.00–2700 ng/mL (r² ≥ 0.99) with the lower limit of quantitation at 3.00 ng/mL. Intra‐ and inter‐day precision was <14.5% and mean accuracy was from −11.5 to 13.6%. Stability testing showed that Cy‐3G remained stable during the whole analytical procedure. After validation, the assay was successfully used to support a preclinical pharmacokinetic comparison of Cy‐3G between normal and diabetic rats. Results indicated that diabetes mellitus significantly altered the in vivo pharmacokinetic characteristics of Cy‐3G after oral administration in rats.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of gymnemagenin in rat plasma using high‐performance liquid chromatography–tandem mass spectrometry: application to pharmacokinetics after oral administration of Gymnema sylvestre extract

A sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method has been developed and validated for the determination of gymnemagenin (GMG), a triterpene sapogenin from Gymnema sylvestre, in rat plasma using withaferin A as the internal standard (IS). Plasma samples were simply extracted using liquid–liquid extraction with tetra‐butyl methyl ether. Chromatographic separation was performed on Luna C₁₈ column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia) at a flow rate of 0.8 mL/min. GMG and IS were eluted at 4.64 and 4.36 min, ionized in negative and positive mode, respectively, and quantitatively estimated using multiple reaction monitoring (MRM) mode. Two MRM transitions were selected at m/z 505.70 → 455.5 and m/z 471.50 → 281.3 for GMG and IS, respectively. The assay was linear over the concentration range of 5.280–300.920 ng/mL. The mean plasma extraction recoveries for GMG and IS were found to be 80.92 ± 8.70 and 55.63 ± 0.76%, respectively. The method was successfully applied for the determination of pharmacokinetic parameters of GMG after oral administration of G. sylvestre extract. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetic study of dendrobine in rat plasma by ultra‐performance liquid chromatography tandem mass spectrometry

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C₁₈ (2.1 ×100 mm, 1.7 µm) column with acetonitrile and 0.1% formic acid 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 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2–1000 ng/mL for dendrobine in rat plasma. The RSDs of intra‐day and inter‐day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright © 2015 John Wiley & Sons, Ltd.     

Method development for quantification of quizartinib in rat plasma by liquid chromatography/tandem mass spectrometry for pharmacokinetic application

Quizartinib is a highly potent inhibitor of the fms‐like tyrosine kinase receptor, which is one of the most commonly mutated genes in acute myeloid leukemia. Quizartinib has shown a significant antileukemic clinical influence among relapsed/refractory acute myeloid leukemia patients. This study aimed at developing and validating an analytical method for the measurement of quizartinib in rat plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method was validated according to US Food and Drug Administration guidelines, and the results obtained in this work met the set criteria. Liquid–liquid extraction was used and chromatographic separation was achieved on a BEHTM C₁₈ column. Detection of quizartinib was achieved in multiple reaction monitoring mode using positive‐ion mode electrospray ionization. The MS/MS ion transitions at mass‐to‐charge ratios (m/z) of 561.129/114.09 and 441.16/84.03 were monitored for quizartinib and ibrutinib, respectively. The linear detection range was 2–1000 ng/mL (r > 0.998), with intra‐ and inter‐day assay precisions ≤13.07 and 13.17%, respectively. This rapid, simple and sensitive method was validated and successfully applied to the pharmacokinetic study of quizartinib in rat samples.     

UPLC‐MS/MS determination of voriconazole in human plasma and its application to a pharmacokinetic study

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was developed to determine voriconazole in human plasma. Sample preparation was accomplished through a simple one‐step protein precipitation with methanol. Chromatographic separation was carried out on an Acquity UPLC BEH C₁₈ column using an isocratic mobile phase system composed of acetonitrile and water containing 1% formic acid (45:55, v/v) at a flow rate of 0.50 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electrospray ionization source in the positive ion mode. The multiple reaction monitoring transitions of m/z 351.0 → 281.5 and m/z 237.1 → 194.2 were used to quantify voriconazole and carbamazepine (internal standard), respectively. The linearity of this method was found to be within the concentration range of 2.0–1000 ng/mL with a lower limit of quantification of 2.0 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 200 mg voriconazole to 20 Chinese healthy male volunteers. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of bencycloquidium bromide in dog plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining bencycloquidium bromide (BCQB) in beagle dog plasma. The plasma sample was deproteinized with methanol which contained l‐ethyl‐bencycloquidium bromide as internal standard, and supernantant was assayed by LC‐MS/MS. The chromatographic separation was performed on a Phenomenex C₁₈ column (100 × 2.0 mm, i.d., 3.0 μm) with a gradient programme mobile phase consisting of methanol and ammonium acetate (5 mm) containing 0.15% acetic acid and at a flow rate of 0.3 mL/min. Electrospray ionization in positive ion mode and selective reaction monitoring was used for the quantification of BCQB with a monitored transitions m/z 330.2 → 142.1 for BCQB and m/z 344.2 → 126.2 for IS. Validation results indicated that the lower limit of quantification was 0.05 ng/mL and the assay exhibited a linear range of 0.05–10.0 ng/mL and gave a correlation coefficient of 0.9998. The intra‐ and inter‐run precisions of the assay were 1.7–4.6 and 3.2–15.6%, respectively, and the intra‐ and inter‐day accuracies were ?8.8 to 1.1 and ?5.0 to 4.6%, respectively. The developed method was applied for the pharmacokinetic study of BCQB in beagle dogs following a single intranasal dose. Copyright © 2009 John Wiley & Sons, Ltd.     

An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and its active metabolites, 1‐caffeoyl‐5‐feruoylquinic acid and 1,5‐O‐diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid–liquid extraction with ethyl acetate, separated on a C₁₈ reversed‐phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1–200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC‐MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5‐DCQA in patients. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a sensitive UHPLC–MS/MS method for quantitative analysis of farrerol in rat plasma: Application to pharmacokinetic and bioavailability studies

Farrerol is a 2,3‐dihydro‐flavonoid isolated from rhododendron. In this study, a sensitive and selective ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the determination of farrerol in rat plasma. Liquid–liquid extraction by ethyl ether was used for sample preparation. Chromatographic separation was achieved on an Agilent UHPLC XDB‐C₁₈ column (2.1 × 100 mm, 1.8 μm) with water and methanol (30:70, v /v) as the mobile phase. An electrospray source was applied and operated in negative ion mode; selection reaction monitoring was used for quantification using target fragment ions m/z 299 → 179 for farrerol and m/z 267 → 252 for internal standard. Calibration plots were linear in the range of 2.88–1440 ng/mL for farrerol in rat plasma. Intra‐ and inter‐day precisions were <11.6%, and the accuracy ranged from −13.9 to 11.9%. The UHPLC–MS/MS method was successfully applied in pharmacokinetics and bioavailability studies of farrerol in rats.     

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.