Turbulent flow and ternary column-switching on-line clean-up system for high-throughput quantification of risperidone and its main metabolite in plasma by LC-MS/MS. Application to a bioequivalence study

A high-throughput LC-MS/MS method was developed for the simultaneous determination of Risperidone and 9-OH-risperidone in human plasma. A semi-automated sample preparation procedure was applied, including protein precipitation after addition of ACN, via a robotic system, and subsequent sub-zero temperature extraction of the latter. Injections of the ACN extractants were performed on a turbulent flow ternary column-switching system, consisted of dual extraction columns in parallel for on-line purification of samples and an analytical column. Toggling with the assistance of two valves provided a run cycle time of 3 min and the whole procedure minimized carry-over effect. On-line clean-up procedure along with sub-zero temperature extraction increased sample purification and extended column life. The analytical range of the method was 0.1-200 ng mL-1 for both analytes with excellent linearity and very good accuracy and precision. The proposed method was employed in a bioequivalence study after per os administration of a 2 mg tablet of risperidone and allowed the completion of the study (>1400 samples) in only 4 days time. © 2007 Elsevier B.V. All rights reserved

LC-Ion Trap-MS Method for the Determination of Fluconazole in Plasma for Bioequivalence Studies of Pharmaceutical Formulations Using Semi-Automated Sample Handling

An LC-ion trap-MS method for the determination of fluconazole in human plasma has been developed to be applied for bioequivalence studies. The human plasma samples were extracted with methyl-tert-butyl ether. The extract was evaporated to dryness, reconstituted with mobile phase and injected into the LC-MS apparatus. Fluconazole d4 was used as the internal standard (IS). A C18 analytical column was used with a mobile phase consisting of 40% formic acid 0.1% and 60% acetonitrile. A robotic liquid handler was used to increase the methods throughput. Fluconazole was detected by monitoring the mass transition of m/z 307.1 to m/z 238.0 (m/z 311.1 to m/z 242.0 for the IS). The method was validated according to the European Medicines Agency (EMEA) regulations and was found to be accurate, precise, and selective throughout the calibration range (0.05-10.0 μg/mL). Compared to the published methods for the determination of fluconazole in its usual therapeutic levels, the method provides the shortest sample runtime. For the first time, it was demonstrated that ion trap mass spectrometry can be used for the determination of fluconazole in plasma. © Copyright 2015 Taylor & Francis Group, LLC

Comparison of hydrophilic interaction and reversed-phase liquid chromatography coupled with tandem mass spectrometric detection for the determination of three pharmaceuticals in human plasma

In the present study, hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) combined with tandem mass spectrometric detection (MS/MS) were evaluated and compared for the determination of donepezil, cetirizine and loratadine in human plasma, in terms of sensitivity and sample preparation procedure. A retention study for the above compounds of various polarities was performed, using both C18 and silica columns, with several aqueous-organic mobile phase ratios, in order to investigate their retention mechanism profile under HILIC and RPLC. Both chromatographic conditions were compared for chromatographic analysis of plasma samples processed with a liquid-liquid extraction (LLE) method for donepezil determination, resulting in significantly higher sensitivity under HILIC. Furthermore, HILIC and RPLC were compared for direct injection, and novel methods including LLE, solid-phase extraction and protein precipitation protocols were developed. Direct injection technique significantly reduced sample preparation time, increasing at the same time method sensitivity. The current study contributes to broadening the range of analyzable compounds by HILIC-MS/MS to molecules of medium polarity. Copyright © 2008 John Wiley & Sons, Ltd

Quantitative determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry employing an automated liquid-liquid extraction based on 96-well format plates. Application to a bioequivalence study

An automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for quantitative determination of donepezil in human plasma. 150 μL of plasma samples were placed in 2.2 mL 96-deepwell plates and both donepezil and loratadine (IS) were extracted from human plasma by liquid-liquid extraction (LLE), using hexane as the organic solvent. Robotic liquid handling workstations were employed for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. The method developed, includes a sample analysis performed by reversed phase LC-MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The chromatographic run time was set for 2.0 min with a flow rate of 0.7 mL/min in a C18 analytical column. The method was significantly sensitive, specific, accurate and precise for the determination of donepezil in human plasma and had the shortest run time. The curve was proven to be linear for the concentration range of 0.1-100 ng/mL. After validation, the method was applied to the rapid and reliable quantitative determination of donepezil in a bioequivalence study after per os administration of a 5 mg donepezil tablet. © 2006 Elsevier B.V. All rights reserved

Development and validation of an improved high-throughput method for the determination of anastrozole in human plasma by LC-MS/MS and atmospheric pressure chemical ionization

In the present study, an automated, 96-well format LC-MS/MS method for the determination of anastrozole in human plasma was developed and fully validated. Within method development procedure, atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) were compared in terms of sensitivity and specificity, with the former proven to be more appropriate and thus being chosen for analyte ionization. In addition, the effect of declustering potential (DP) and collision energy (CE) in sensitivity was, as well, studied and compared between APCI and ESI source employment. Samples were treated with an acetonitrile (ACN) protein precipitation step followed by liquid-liquid extraction (LLE) with methyl t-butyl ether (MTBE) as the organic solvent, using omeprazole as the internal standard (IS). The statistical evaluation for the APCI protocol revealed excellent linearity, accuracy and precision values for the range of concentrations 0.100-100 ng/mL. The method proposed involves the lowest plasma volume so far reported (190 μL), as well as the shortest run time (1.6 min) and along with the employment of two robotic liquid handling systems enabled the rapid and reliable determination of anastrozole in a bioequivalence study (>1000 plasma samples) after per os administration of 1 mg tablet within a 4-day period of time. © 2008 Elsevier B.V. All rights reserved

Development of a rapid liquid chromatography tandem mass spectrometry method for the determination of lisinopril, applicable for a bioequivalence study, employing a 96-well format solid phase extraction protocol

A rapid liquid chromatography/tandem mass spectrometry (LC/MS-MS) method was developed for the determination of lisinopril in human plasma. Lisinopril and the internal standard enalaprilat (IS) were extracted from human plasma by semi-automated solid phase extraction (SPE) using a 96-well format extraction plate. Initially, a 1:1 plasma: acetonitrile (ACN) mixture was prepared and vortexed to achieve protein precipitation. After centrifugation, an aliquot of the supernatant water/ACN solvent mixture was evaporated. The residue was dissolved in a certain volume of a reconstitution solution, which was passed through the extraction plate and the eluent was analyzed by combined reversed phase liquid chromatography tandem mass spectrometry with positive ion electrospray ionization using multiple reactions monitoring (MRM). The method was proved to be sensitive and specific for both drugs and its statistical evaluation revealed excellent linearity for the range of concentrations 2.0-200.0 ng mL-1 and very good accuracy, and inter- and intraday precisions. The proposed method enables the rapid and reliable determination of lisinopril in pharmacokinetic or bioequivalence studies after per os administration of 20 mg tablet formulations of lisinopril and it was applied in such study from our laboratory. © 2005 Elsevier B.V. All rights reserved

Development of a rapid method for the determination of glimepiride in human plasma using liquid-liquid extraction based on 96-well format micro-tubes and liquid chromatography/tandem mass spectrometry

A semi-automated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of glimepiride in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Glimepiride and the internal standard (IS) glibenclamide were extracted from human plasma by LLE, using a mixture of ethyl acetate/diethyl ether 50:50 (v/v) as the organic solvent. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analyte and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by reversed-phase LC/ MS/MS with positive ion electrospray ionization, using multiple reaction monitoring. The method proved to be sensitive and specific for both drugs, and statistical evaluation revealed excellent linearity for the range of concentrations 2.0-500.0 ng/mL with very good accuracy and inter- and intra-day precisions. The proposed method enabled the rapid and reliable determination of glimepiride in pharmacokinetic or bioequivalence studies after per os administration of a 3 or 4 mg tablet of glimepiride. Copyright © 2005 John Wiley & Sons, Ltd

Validation of a fully automated high throughput liquid chromatographic/tandem mass spectrometric method for roxithromycin quantification in human plasma. Application to a bioequivalence study

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of roxithromycin in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2mL 96-deep-well plates. Roxithromycin and the internal standard clarithromycin were extracted from 100 μL of human plasma by LLE, using methyl t-butyl ether as the organic solvent. All liquid transfer steps were performed automatically using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple-reaction monitoring. The method had a very short chromatographic run time of 1.6 min. The calibration curve was linear for the range of concentrations 50.0-20.0 × 103 ng mL-3. The proposed method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of roxithromycin in human plasma. Therefore, it was applied to the rapid and reliable determination of roxithromycin in a bioequivalence study after per os administration of 300 mg tablet formulations of roxithromycin. Copyright © 2007 John Wiley & Sons, Ltd

Development and validation of a rapid 96-well format based liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analysis method for ondansetron in human plasma

A rapid and sensitive LC-MS/MS method for the quantification of ondansetron was developed and validated. The plasma samples were treated by a semi-automated liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Ondansetron and the internal standard (IS) granisetron were analyzed by combined reversed phase LC-MS/MS, with positive ion electrospray ionization, using multiple reactions monitoring (MRM). The statistical evaluation for this method reveals excellent linearity, accuracy and precision values for the range of concentrations 0.25-40.0 ng/mL. The proposed method enabled the reliable determination of ondansetron in bioequivalence studies after per os administration of a 4 or 8 mg tablet. © 2006 Elsevier B.V. All rights reserved