Determination of Response Factors for Analytes Detected during Migration Studies, Strategy and Internal Standard Selection for Risk Minimization.

Migration studies are one of the few domains of pharmaceutical analysis employing wide-scope screening methodologies. The studies involve the detection of contaminants within pharmaceutical products that arise from the interaction between the formulation and materials. Requiring both qualitative and quantitative data, the studies are conducted using Liquid Chromatography or Gas Chromatography coupled to a mass spectrometer (LC-MS and GC-MS). While mass spectrometry allows wide-scope analyte detection and identification at the very low Analytical Evaluation Threshold (AET) levels used in these studies, MS detectors are far from "universal response" detectors. Regulation brings the application of uncertainty factors into the picture to limit the risk of potential analytes detected escaping report and further evaluation; however, whether the application of a default value can cover any or all relevant applications is still debatable. The current study evaluated the response of species usually detected in migration studies, generating a suitable representative sample, analyzing said species, and creating a strategy and evaluation mechanism for acceptable classification of the detected species. Incorporating novel methodologies, i.e., Design of Experiments (DoE) for Design Space generation, the LC-MS-based methodology is also evaluated for its robustness in changes performed.

Advantages of automation in plasma sample preparation prior to HPLC/MS/MS quantification: application to the determination of cilazapril and cilazaprilat in a bioequivalence study.

An HPLC/MS/MS method characterized by complete automation and high throughput was developed for the determination of cilazapril and its active metabolite cilazaprilat in human plasma. All sample preparation and analysis steps were performed by using 2.2 mL 96 deep-well plates, while robotic liquid handling workstations were utilized for all liquid transfer steps, including liquid-liquid extraction. The whole procedure was very fast compared to a manual procedure with vials and no automation. The method also had a very short chromatographic run time of 1.5 min. Sample analysis was performed by RP-HPLC/MS/MS with positive electrospray ionization using multiple reaction monitoring. The calibration curve was linear in the range of 0.500-300 and 0.250-150 ng/mL for cilazapril and cilazaprilat, respectively. The proposed method was fully validated and proved to be selective, accurate, precise, reproducible, and suitable for the determination of cilazapril and cilazaprilat in human plasma. Therefore, it was applied to a bioequivalence study after per os administration of 2.5 mg tablet formulations of cilazapril.

Validation of a novel, fully automated high throughput high-performance liquid chromatographic/tandem mass Spectrometric method for quantification of pantoprazole in human plasma.

An automated high-throughput HPLC/MS/MS method was developed for the quantitative determination of pantoprazole in human plasma. Only 100 microL plasma was placed in 2.2 mL 96 deep-well plates, and both pantoprazole and omeprazole (IS) were extracted from human plasma by liquid-liquid extraction, using diethyl ether-dichloromethane (70:30, v/v) as the organic solvent. Robotic liquid-handling workstations were used 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. Sample analysis was performed by utilizing the combination of RP-HPLC/MS/MS, with positive-ion electrospray ionization and multiple reaction monitoring detection. The chromatographic run time was set at 1.8 min with a flow rate of 0.6 mL/min on a Nucleosil octylsilyl (C8) analytical column. The method was proven to be sensitive, specific, accurate, and precise for the determination of pantoprazole in human plasma. The method was applied to a bioequivalence study after per os administration of a 40 mg pantoprazole gastric retentive tablet.

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 microL), 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 1mg tablet within a 4-day period of time.

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 C(18) 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.

An improved and fully validated LC-MS/MS method for the simultaneous quantification of simvastatin and simvastatin acid in human plasma.

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of simvastatin (SV) and simvastatin acid (SVA) in human plasma. Plasma samples were treated by acetonitrile (ACN) addition for protein precipitation (PP) and subsequent two-step liquid-liquid extraction (LLE) in 96-deepwell plates, using methyl t-butyl ether (MTBE) as the organic solvent. ACN addition step was proven to enhance method sensitivity, as well as producing cleaner samples for injection. Lovastatin (LV) and lovastatin acid (LVA) were used as internal standards (IS) for SV and SVA quantification respectively. A relatively small plasma volume (300 microL) was employed and all procedure liquid transfer steps were performed automatically, by the use of robotic liquid handling workstations. Both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources were applied and compared for LC-MS/MS sample analysis, with ESI proven to be more sensitive for the specific analytes. Polarity switch (from negative to positive ionization mode) was performed during the same analytical run, so as for the simultaneous SV and SVA determination to be possible. The method had a short sample preparation time, as well as a chromatographic run time of just 1.9 min, the shortest so far reported for SV determination. It was validated and fulfilled all preset criteria for sensitivity, specificity, linearity (0.100-40.0 ng/mL), inter- and intra-accuracy and precision for both molecules. The proposed method was applied to the rapid and reliable simultaneous determination of SV and SVA in a bioequivalence study, after per os administration of a SV tablet (80 mg).

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 2.2 mL 96-deep-well plates. Roxithromycin and the internal standard clarithromycin were extracted from 100 microL 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 50.0-20.0x10(3) ng mL(-1). 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.

An improved high-throughput liquid chromatographic/tandem mass spectrometric method for terbinafine quantification in human plasma, using automated liquid-liquid extraction based on 96-well format plates.

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for terbinafine quantification in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2 mL 96-deepwell plates. Terbinafine and the internal standard (IS) N-methyl-1-naphthalenemethylamine were extracted from human plasma by LLE, using a mixture of methyl t-butyl ether (MTBE)-hexane (70:30, v/v) as the organic solvent. All liquid transfer steps, including preparation of calibration standards and quality control samples, as well as the addition of the IS, were performed automatically by using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of a reconstitution solution. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The method had a very short sample preparation time and a chromatographic run time of 2.2 min. It was proved to have excellent sensitivity, specificity, accuracy as well as inter- and intraday precision for the quantification of terbinafine in human plasma. The calibration curve was linear for the range of concentrations 5.0-2000.0 ng/mL. The proposed method was applied to the rapid and reliable determination of terbinafine in a bioequivalence study after per os administration of 250 mg tablet formulations of terbinafine.

Application of a semi-automated 96-well format solid-phase extraction, column-switching, fluorescence detection protocol for the determination of alendronate in human urine samples obtained from a bioequivalence study.

In the current study, a semi-automated, 96-well format, solid-phase extraction (SPE), analytical column-switching method for alendronate determination in human urine is developed, validated and applied to a bioequivalence study. The current protocol was a substantial improvement of an existing classical method. A robotic liquid handling system was employed to simplify and reduce the time of sample preparation procedure. Automated SPE was carried out using a 96-well cartridge plate and a vacuum control system. Urine samples were determined by applying a column-switching protocol with fluorescence detection. Analysis time, due to the column-switching procedure, was about half of the conventional LC approach (11.5 min instead of 21 min). The method application required the determination of alendronate in urine samples obtained from 96 healthy volunteers as part of a bioequivalence study of two 70 mg alendronate sodium tablets. All major pharmacokinetic parameters of the bioequivalence study were estimated and reported.

Simultaneous determination of losartan, EXP-3174 and hydrochlorothiazide in plasma via fully automated 96-well-format-based solid-phase extraction and liquid chromatography-negative electrospray tandem mass spectrometry.

An automated, sensitive and high-throughput liquid chromatographic/electrospray tandem mass spectrometric (LC-MS/MS) assay was developed for the simultaneous determination of losartan (LOS), its major circulating metabolite EXP-3174 and hydrochlorothiazide (HCTZ) in human plasma. LOS and HCTZ coexist in the same drug formulation, and this is the first method that enables the simultaneous determination of both drugs along with the active metabolite of LOS. Since these drugs have different physicochemical properties, the employment of a liquid-liquid extraction (LLE) protocol was precluded. A fully automated solid-phase extraction (SPE) protocol, based on 96-well format plates, was used to isolate these compounds and furosemide (internal standard, IS) from plasma. Washing and elution steps were amended accordingly in order to minimize any matrix effect from components of the plasma without reducing the elution of the molecules of interest. The compounds were eluted from a C18 column and detected with an API 3000 triple-quadrupole mass spectrometer using negative electrospray ionization and multiple reaction monitoring (MRM). The assay was linear over the range 1.00-400 ng/mL for LOS and EXP-3174 and 0.500-200 ng/mL for HCTZ, respectively, when 200 microl of plasma was used in the extraction. The overall intra- and interassay variations were within acceptance limits. The analysis time for each sample was 4 min, and more than 300 samples could be analyzed in one day by running the system overnight. The assay was simple, highly sensitive, selective, precise, fast, and it enables the reliable determination of LOS, EXP-3174 and HCTZ in pharmacokinetic or bioequivalence studies after per os administration of a single tablet containing both drugs.

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 MicroL 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 5mg donepezil tablet.

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 2mg tablet of risperidone and allowed the completion of the study (>1400 samples) in only 4 days time.

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.

Development of a high-throughput method for the determination of itraconazole and its hydroxy metabolite in human plasma, employing automated liquid-liquid extraction based on 96-well format plates and LC/MS/MS.

A semi-automated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of the antifungal drug itraconazole (ITZ) and its coactive metabolite hydroxyitraconazole (OH-ITZ) in human plasma. The plasma samples underwent liquid-liquid extraction (LLE) in 2.2 mL 96 deepwell plates. ITZ, OH-ITZ and the internal standard (IS) R51012 were extracted from plasma, using a mixture of acetonitrile (ACN) and methyl t-butyl ether (MTBE) as the organic solvent. This specific mixture, due to its composition, had a significant impact on the performance of the assay. All liquid transfer steps, including preparation of calibration standards and quality control samples as well as the addition of the IS, were performed automatically using robotic liquid handling workstations for parallel sample processing. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analytes and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by combined reversed phase LC/MS/MS, with positive ion electrospray ionization and a TurboIonSpray interface, using multiple reactions monitoring (MRM). The method was shown to be sensitive and specific to both ITZ and OH-ITZ, it revealed excellent linearity for the range of concentrations 2-500 ng mL(-1) for ITZ and 4-1000 ng mL(-1) for OH-ITZ, it was very accurate and it gave very good inter- and intra-day precisions. The proposed high-throughput method was employed in a bioequivalence study after per os administration of two 100 mg tablets of ITZ, and it allowed this study to be completed in under four days.

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.