Ultra‐performance LC MS/MS method for quantification of clopidogrel active metabolite

A sensible ultra‐performance LC–MS method was developed and validated for the quantification of clopidogrel active metabolite in human plasma, with clopidogrel D4 as internal standard. Plasma pretreatment involved a one‐step protein precipitation with acetonitrile. The separation was performed by reverse‐phase chromatography on a C8 column. The method was linear over the concentration range of 1–150 ng/mL. The intra‐ and inter‐day precision values were below 17% and accuracy was from 1.7 to 7.5% at all quality control levels. The lower LOQ was 0.8 ng/mL. Sample analysis time was reduced to 5 min including sample preparation (limited to protein precipitation). The present method was successfully applied to a clopidogrel active metabolite pharmacokinetic study following oral administration to healthy volunteers.     

Stereoselective determination of ginsenosides Rg3 and Rh2 epimers in rat plasma by LC‐MS/MS: Application to a pharmacokinetic study

We developed and validated an accurate and sensitive LC–MS/MS method for the simultaneous quantitation of ginsenoside Rg3 and Rh2 epimers (R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2) in rat plasma. Analytes were extracted from 0.1 mL aliquots of rat plasma by liquid–liquid extraction, using 2 mL of ethyl acetate. In this assay, dioscin (500 ng/mL) was used as an internal standard. Chromatographic separation was conducted using an Acclaim RSLC C₁₈ column (150 × 2.1 mm, 2.2 μm) at 40°C, with a gradient mobile phase consisting of 0.1% formic acid in distilled water and in acetonitrile, a flow rate of 0.35 mL/min, and a total run time of 20 min. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with negative electrospray ionization at m/z 783.4 → 161.1 for R‐Rg3 and S‐Rg3, m/z 621.3 → 161.1 for R‐Rh2 and S‐Rh2, and m/z 867.2 → 761.5 for the internal standard. For R‐Rg3 and S‐Rg3, the lower limit of quantification was 5 ng/mL, with a linear range up to 500 ng/mL; for R‐Rh2 and S‐Rh2, the lower limit of quantification was 150 ng/mL, with a linear range up to 6000 ng/mL. The coefficient of variation for assay precision was less than 10.5%, with an accuracy of 86.4–112%. No relevant cross‐talk or matrix effect was observed. The method was successfully applied to a pharmacokinetic study after oral administration of 400 mg/kg and 2000 mg/kg of BST204, a fermented ginseng extract, to rats. We found that the S epimers exhibited significantly higher plasma concentrations and area under curve values for both Rg3 and Rh2. This is the first report on the separation and simultaneous quantification of R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2 in rat plasma by LC‐MS/MS. The method should be useful in the clinical use of ginseng or its derivatives.     

Quantitative determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood by liquid chromatography–tandem mass spectrometry with pre‐column derivation and its pharmacokinetic application

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood. DASK was pretreated using pre‐column derivatization with 2‐mercaptoethanol followed by liquid–liquid extraction with cyclohexane. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring mode via electrospray ionization source. The linear range for the determination of DASK spiked in rat whole blood (0.25 mL) was 3–3000 ng/mL. The accuracy was within 9%. Intra‐ and inter‐day precisions were no more than 16.1 and 13.3%, respectively. The validated LC‐MS/MS method was successfully applied to the preliminary pharmacokinetic study in rats. After DASK administration (60 mg/kg, p.o.) in rats, pharmacokinetic parameters were obtained, where the area under the drug concentration–time curve was 2393.7 ± 224.4 ng h/mL and the elimination half‐life was 27.6 ± 5.3 h. Copyright © 2008 John Wiley & Sons, Ltd.     

Pharmacokinetic properties of isradipine after single‐dose and multiple‐dose oral administration in Chinese volunteers: a randomized,open‐label,parallel‐group phase I study

Isradipine could be used for the treatment of high blood pressure or Parkinsonism, yet the study on pharmacokinetics (PK) of isradipine is lacking in the Chinese population. The current study aims to assess the dose proportionality, pharmacokinetics and gender effect of isradipine following oral single and multiple doses in Chinese subjects. A randomized, open‐label, parallel‐group trial was conducted in 30 healthy Chinese volunteers. Subjects randomly received a single dose of 2.5, 5 or 10 mg, and multiple doses (2.5 mg) of isradipine. Blood samples were collected pre‐dose (0 h) and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36 and 48 h post‐dose. Isradipine was rapidly absorbed with the time to maximum concentration <1.27 h for all dosage groups. The maximum concentrations were 2.46, 5.34, 10.93 and 3.32 ng/mL and area under the concentration–time curve from time zero to the last time point (AUC_last) were 7.05, 12.58, 24.68 and 5.31 ng/ml·h for the 2.5, 5 and 10 mg single‐dose and 2.5 mg multiple‐dose groups, respectively. The half‐life ranged from 5.76 to 7.94 h. The maximum concentration and AUC were found to increase linearly and dose‐dependently for isradipine. No statistical gender differences were found. These findings indicated that the pharmacokinetic parameters of isradipine in Chinese population were dose‐proportional and predictable over a range of 2.5–10 mg isradipine oral doses. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Validation of LC‐MS/MS method applied to evaluation of free tissue concentrations of vildagliptin in diabetic rats by microdialysis

A novel LC‐MS/MS method was developed for the quantification of vildagliptin in an aqueous matrix. The method was successfully validated, meeting all the requisites of US Food and Drug Administration guide for a bioanalytical method. The developed method presented a limit of quantification of 10 ng/mL and the range of concentration achieved was 10–1875 ng/mL. The injection volume necessary was only 10 μL, and retention time was 4.60 min. The mobile phase employed was methanol–ammonium acetate 5 mm (95:5). The stability of the drug was evaluated in the different conditions through which the samples passed. A pharmacokinetic experiment was conducted with diabetic male Wistar rats, and the concentration of drug in liver was evaluated through a microdialysis technique. The perfusion fluid employed was ultrapure water. The dose administrated was 50 mg/kg and the method allowed the quantification of vildagliptin for more than three half lives, successfully characterizing the pharmacokinetic profile when the developed method was applied. This is the first report on the tissue pharmacokinetics of a DPP‐4 inhibitor and could contribute to drug dosage optimization in the future. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a robust LC‐MS‐MS with atmospheric pressure chemical ionization for quantitation of carbazochrome sodium sulfonate in human plasma: application to a pharmacokinetic study

A highly selective and sensitive liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC‐APCI‐MS‐MS) was developed and validated for the quantitation and pharmacokinetic study of carbazochrome sodium sulfonate in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and amiloride hydrochloride was employed as an internal standard. The analytes were separated on a Hypersil ODS‐2 column by a multiple‐step linear gradient elution with a mobile phase consisting of 0.2% formic acid solution and methanol pumped at a flow rate of 1.0 mL/min. The determination was optimized and carried out with positive atmospheric pressure chemical ionization by selective reaction monitoring of the ion of m/z 148, the protonated thermodegraded fragment of the free acidic form of carbazochrome sodium sulfonate selected as the parent, and the ion of m/z 107 as the optimum collision induced dissociation (CID) product. The method was fully validated over a concentration range of 0.5–50 ng/mL, with the lower limit of quantitation of 0.5 ng/mL. The application of the LC‐MS‐MS method was demonstrated for the specific and quantitative analysis of carbazochrome sodium sulfonate in human plasma from a pharmacokinetic study in 24 healthy male Chinese volunteers after a single oral administration of 90 mg carbazochrome sodium sulfonate capsules. Copyright © 2010 John Wiley & Sons, Ltd.     

A rapid and sensitive LC-MS/MS method for quantification of donepezil and its active metabolite, 6-o-desmethyl donepezil in human plasma and its pharmacokinetic application

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and fully validated for simultaneous quantification of donepezil and its active metabolite, 6‐o‐desmethyl donepezil in human plasma. Analytes and the internal standard were extracted from human plasma by liquid–liquid extraction technique using a 30:70 v/v mixture of ethyl acetate and n‐hexane. The reconstituted samples were chromatographed on a C₁₈ column by using a 70:30 v/v mixture of acetonitrile and ammonium formate (5 mm , pH 5.0) as the mobile phase at a flow rate of 0.6 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.09–24.2 ng/mL for donepezil and 0.03–8.13 ng/mL for 6‐o‐desmethyl donepezil. The results of the intra‐day and inter‐day precision and accuracy studies were well within the acceptable limits. The proposed method was successfully applied for the estimation of the drug in real time plasma samples for pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.     

An LC-MS/MS method for simultaneous determination of vincristine and verapamil in rat plasma after oral administration of a dual agent formulation

A rapid and sensitive method for simultaneous determination of vincristine and verapamil in rat plasma was first developed and validated, using high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) in multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). The method, which required a small sample volume (25 µL) of plasma, was linear in the concentration range of 0.5–500 ng/mL for vincristine and 0.1–100.0 ng/mL for verapamil. Finally, the method was successfully employed in a pharmacokinetic study of vincristine and verapamil in rats after an oral administration of a dual‐agent formulation containing vincristine and verapamil. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of automated SPE-HPLC-MS/MS methods for the quantification of asenapine, a new antipsychotic agent, and its two major metabolites in human urine

To support the evaluation of the pharmacokinetic parameters of asenapine (ASE) in urine, we developed and validated online solid‐phase extraction high‐performance liquid chromatography methods with tandem mass spectrometry detection (SPE‐LC‐MS/MS) for the quantification of ASE and two of its major metabolites, N‐desmethylasenapine (DMA) and asenapine‐N⁺‐glucuronide (ASG). The linearity in human urine was found acceptable for quantification in a concentration range of 0.500–100 ng/mL for ASE and DMA and 10.0–3000 ng/mL for ASG, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

LC–MS/MS method for simultaneous determination of ramelteon and its metabolite M‐II in human plasma: Application to a clinical pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M‐II in human plasma. After extraction from 200 μL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS‐2 (5 μm, 150 × 2.1 mm) column with a mobile phase consisted of methanol–0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500–30.0 ng/mL for ramelteon and 1.00–250 ng/mL for M‐II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (C_max), the time to the C_max and the elimination half‐life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M‐II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.     

Development and validation of an LC-MS-MS method for determination of methyl kulonate in rat plasma

A sensitive, rapid and specific LC‐MS‐MS method was established and validated for determination of methyl kulonate, a major bioactive constituent isolated from Meliae Cortex, in rat plasma. Plasma samples were treated by precipitating protein with methanol and were chromatographed using a Capcell Pak C₁₈ column (100 × 4.6 mm, 5 µm) with the mobile phase comprising a mixture of methanol, 10 m m ammonium formate and formic acid (95:5:0.1, v/v/v). Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive atmospheric ionization at m/z 467 → 311 for methyl kulonate, and m/z 469 → 451 for dubione B (internal standard), respectively. A good linear response was observed over the concentration range 1.00–500 ng/mL with the lower limit of quantification 1.00 ng/mL in rat plasma. The method also afforded satisfactory results base on sensitivity, specificity, precision, accuracy, recovery, freeze–thaw and long‐time stability. The validated method was successfully applied to determine the pharmacokinetic properties of methyl kulonate in rats after oral administration at dose of 100 mg/kg. This pharmacokinetic study of methyl kulonate is reported here for the first time. Copyright © 2011 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 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.     

Pharmacokinetics of luteolin and tetra‐acetyl‐luteolin assayed by HPLC in rats after oral administration

Accurate and reproducible HPLC methods were developed and validated for the determination of concentrations of luteolin (LT) and tetra‐acetyl‐luteolin (TALT) in rat plasma. HPLC analyses were performed on an Agilent TC‐C₁₈ column protected by a guard Agilent Zorbax Eclipse Plus. The mobile phase for LT was a binary mixture of acetonitrile–water (40:60, v/v) containing 0.5% phosphoric acid at a flow rate of 1.0 mL/min, and that for TALT was a binary mixture of methanol–water (70 : 30, v/v) containing 0.5% glacial acetic acid at the same flow rate. The UV detection wavelength for both analytes was set at 350 nm. The calibration curve was linear over the range of 40–1800 ng/mL, the lower limit of quantitation was 40 ng/mL and the lower limit of detection was 20 ng/mL for both LT and TALT. The intra‐ and inter‐day precision (RSD) values for all samples were within 7.9%. The concentration–time curves of LT and TALT after oral administration (30 mg/kg) were both fitted to a two‐compartment model. The pharmacokinetic characteristics of TALT were better than that of LT in the maximum plasma concentration (C_max) and the area under the concentration–time curve (AUC). Copyright © 2010 John Wiley & Sons, Ltd.     

Development of an LC/MS/MS method in order to determine arctigenin in rat plasma: its application to a pharmacokinetic study

In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2–500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and ?80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half‐life and area under the concentration–time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

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

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

A high‐performance liquid chromatography–tandem mass spectrometry method coupled with protein precipitation for determination of granisetron in human plasma and its application to a comparative pharmacokinetic study

A rapid, simple and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) has been developed for the determination of granisetron in human plasma. Plasma samples were pre‐purified by protein precipitation procedure. The chromatographic separation was achieved with Synergi Polar‐RP (75 × 2 mm, 4 µm) column using a mixture of 5 mm pH4.0 ammonium formate and methanol (300:316, v/v) under isocratic conditions at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The analysis time was about 2.5 min. The method was fully validated over the concentration range 0.1–10 ng/mL. The lower limit of quantification was 0.1 ng/mL. Inter‐ and intra‐batch precision was <6.1% and the accuracy was within 95.6–100.0%. The mean extraction recovery was 96.3%. Selectivity, matrix effect and stability were also validated. The method was applied to the comparative pharmacokinetic study of granisetron in Chinese healthy subjects. Copyright © 2014 John Wiley & Sons, Ltd.