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Osimertinib (AZD9291) is a widely used tyrosine kinase inhibitor for the treatment of non-small cell lung cancer patients with activating EGFR mutations. However, the correlation between dose and efficacy has been debated for several years. For this reason, there is a need for standardized methods for routine analysis, clinical studies on pharmacokinetics and dose-response relationships, and greater understanding of preanalytical conditions, such as sample storage stability. The objective of this study was to develop and validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of osimertinib and its two metabolites, AZ7550 and AZ5104, in human plasma and to investigate long-term storage stability of the analytes. Samples were prepared by protein precipitation and separated on a Kinetex EVO C18 column (2.1 × 150 mm, 2.6 µm). Electrospray ionization in positive mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1.25 to 3000 ng/mL. Interassay precisions and accuracies were all ≤ 15 %. Linearity, dilution integrity, and carry-over were also examined and satisfied the validation criteria. Stability was examined under different conditions, and the analytes were found to be stable for more than 3 years at -80°C (< 15 % decline). Finally, the analytical method was successfully applied in a clinical setting on plasma samples from 30 patients with non-small cell lung cancer in treatment with osimertinib, demonstrating its suitability for use in clinical studies and its potential for therapeutic drug monitoring., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Cyclin D-dependent kinase 4/6 inhibitors palbociclib, ribociclib and abemaciclib, in combination with aromatase inhibitors anastrozole and letrozole or oestrogen receptor degrader fulvestrant, are being assessed as candidates for therapeutic drug monitoring. An ideal bioanalytical method for their determination in patient plasma samples is therefore of high interest, as there is no routine reference method yet available in the clinical practice. In this work, three sample preparation approaches - dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), and newly developed phospholipid removal (PLR) for LC-MS determination of these six drugs are comprehensively assessed. The methods are validated in the clinically relevant linear ranges with remarkable precision (RSD ≤6.9 %) and accuracy (bias -13.6 - 11.8 %). To compare the procedures in a real-world setting, they are applied on 38 samples from breast cancer patients. The differences between paired results are below 20 % for more than 92 % of the repeats and the RSD is ≤13.1 % between the corresponding results. Statistical comparison of the results reveals excellent overall agreement between the methods (Lin's concordance correlation coefficient ≥0.9969, maximal Bland-Altman bias 6.3 %). DLLME proved to be the most ecologically acceptable method due to the high degree of miniaturisation (AGREEprep score 0.44), PLR enabled very high sample throughput and cost-effectiveness (BAGI 72.5), while SPE showed the best analytical performance (redness score 100). All three methods are suitable for their designated purpose, and the choice of the ideal method can be made based on the scope of application, available funds and equipment and desired ecological footprint., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tajana Silovski reports a relationship with Novartis Pharmaceuticals that includes: speaking and lecture fees. Tajana Silovski reports a relationship with Pfizer Inc that includes: speaking and lecture fees. Tajana Silovski reports a relationship with Elly Lili that includes: speaking and lecture fees. Other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2 % formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 - 1000 nM abemaciclib, 0.35 - 1000 nM M2 and M18, 0.5 - 1000 nM M20, and 0.75 - 1000 nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13 % and the accuracy was ± 12 %, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11 % and the accuracy was ± 12 % for low, mid, and high and < 19 % at LLOQ. The recovery in human plasma was determined to be between 92 % and 102 % for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers., Competing Interests: Declaration of Competing Interest Kathleen Moore: Consultation or advisory fees from Astra Zeneca, Aravive, Aadi, Blueprint, Clovis, Caris, Duality, Eisai, GSK, Genentech/Roche, Immunogen, Mersana, Merck, Myriad, Novartis, Janssen, Regeneron, VBL Theraeputics, Verastem, and Zentalis. Other activities that could influence the work include GOG Foundation BOD, and ASCO BOD., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lumateperone is a novel agent approved by FDA for treatment of schizophrenia in adults. To elucidate the species differences in the of biotransformation of lumateperone and its pharmacokinetic (PK) characteristics in rats, the metabolite identification of lumateperone was carried out in rat, dog and human liver microsomes, and rat plasma after oral administration using UPLC-Q Exactive Orbitrap high-resolution mass spectrometry HRMS. Furtherly, the PK characteristics of lumateperone and its N-demethylated metabolite (M3) in rat plasma were investigated using a validated LC-MS/MS method following intravenous and oral administration. Fourteen phase I metabolites were found in liver microsomes and ten of them were observed in rat plasma. N-demethylation, carbonylation, dehydrogenation, and piperazine ring cleavage were main metabolic pathway of lumateperone. No unique metabolites were formed in human liver microsomes. After rapid absorption in rats, lumateperone was quickly metabolized and eliminated with bioavailability of less than 5%. The exposure level of M3 was about 1.5-fold higher than that of lumateperone in rat plasma. Lumatperone underwent extensive metabolism and was absorbed rapidly in rats. Metabolite M3 had equivalent or slightly higher exposure levels than lumateperone. This study provides essential PK information to facilitate further pharmacodynamic researches of lumateperone., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Within the family of serotonin (5-HT) receptors, the 5-HT 1A subtype is particularly interesting as it may be involved in various physiological processes or psychological disorders. The p-[ 18 F]MPPF, a highly selective 5-HT 1A antagonist, is used for in vivo studies in human or animal by means of positron emission tomography (PET) [1]. In order to selectively extract p-[ 18 F]MPPF and its main metabolites from plasma, molecularly imprinted polymer (MIP) was prepared against these compounds by using the p-MPPF as template. For the control of the selectivity, non-imprinted polymer (NIP) was also synthesized without template. The MIP sorbent, packed in disposable extraction cartridges (DECs), was then evaluated as molecularly imprinted solid-phase extraction (MISPE) prior to the LC determination. The conditions of extraction were evaluated in order to obtain the highest selective retention of the p-[ 18 F]MPPF and its metabolites on this MIP. The MIP selectivity was exploited in the loading and washing steps by adjusting the pH of plasma samples at a suitable value and by selecting mixtures for the washing step to limit the contribution of non-specific interactions. Other important parameters involved in the conditioning and elution steps were also studied. Finally, a pre-validation was carried out with optimal extraction conditions to demonstrate the performance of this MISPE-LC method as a generic method in the context of evaluation of new MISPE for p-[ 18 F]MPPF and its potential for metabolites extraction from human plasma., Competing Interests: Declaration of Competing Interest Not applicable, (Copyright © 2019 Elsevier B.V. All rights reserved.)

A new method for the quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of five tyrosine kinase inhibitors (afatinib, crizotinib, osimertinib, erlotinib and nintedanib) used in the treatment of non-small cell lung cancer (NSCLC) was developed and validated in human plasma. Separation was performed on an Accucore ® C18 (2.1 × 50 mm; 2.6 μm) column using a gradient elution of water acidified with 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B) at a flow rate of 500 μL/min. The analytes were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with heated electrospray interface. After addition of three isotopically labeled internal standards, plasma pretreatment consisted in a simple protein precipitation. This method presented satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-assay coefficient of variation from 2.6% to 10.6%), accuracy (from 96.1% to 108.5%), recovery and matrix effects. The lower limit of quantification and the linearity of these five tyrosine kinases inhibitors are suitable with the expected concentrations in clinical practice. This new bioanalytical method can be used in daily clinical practice for therapeutic drug monitoring of these tyrosine kinase inhibitors in NSCLC patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro-aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole-D8, has been used as the internal standard (IS) for both analytes. Only 200 μl of human plasma was needed for analyte extraction, using effective phospholipids-eliminating three-step microelution-solid-phase extraction (SPE, Oasis PRiME HLB 96-well μElution Plate). An ACE C18-PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2-μm on-line filter. A combination of ammonium formate (5 mM)-acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re-equilibration time of 3 min, to give a total run time of 8 min. Five μl of the sample was injected into the chromatographic system. Aripiprazole, dehydro-aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18-110 ng/ml and 0.35-100 ng/ml for aripiprazole and dehydro-aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry-over. Our microelution-SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of metronidazole, meropenem, ciprofloxacin, linezolid and piperacillin in human plasma and applied to patients suffering from hospital acquired pneumonia (HAP). The method uses an air assisted dispersive liquid-liquid microextraction for sample preparation. All parameters in the extraction step, including selection of extractant, amount of extractant, ionic strength, pH, and extraction cycles, were investigated and optimized. Chromatography was carried out using a Poroshell 120 SB C 18 (50 × 2.1 mm I.D. 2.6 μm particle size) column and a gradient mobile phase consisting of ammonium acetate buffer (10 mM, pH 4.0) (eluent A); and a mixture of acetonitrile-methanol in a ratio (80/20)(eluent B). Ulifloxacin was used as internal standard. The method demonstrated good linearity with correlation coefficients, r 2  > 0.9995 for the drugs, as well as high precision (RSD% ≤ 9.87%), accuracy ranged from -8.14% to +8.98. The enrichment factor (EF) obtained ranged within 87 and 121. During the validation, the concentrations of the analytes were found to be stable after 3 freeze-thaw cycles and for at least 24 h after extraction. Subsequently, this method was used to quantify the drugs in patients with HAP in order to establish if the dosage regimen given was sufficient to eradicate the infection at the target site., (Copyright © 2017. Published by Elsevier B.V.)

To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of vortioxetine, carvedilol and its metabolite 4-hydroxyphenyl carvedilol in rat plasma. The analytes and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1mm×50mm, 1.7μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 299.2→150.1 for vortioxetine, m/z 407.2→100.3 for carvedilol, m/z 423.2→100.1 for 4-hydroxyphenyl carvedilol and m/z 285.2→193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 0.5-100ng/mL for vortioxetine, 0.5-1000ng/mL for carvedilol and 0.1-50ng/mL for 4-hydroxyphenyl carvedilol. Total time for each chromatograph was 3.0min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD)<11.6% and the accuracy values ranged from -12.2% to 11.3%. The analytical method was successfully applied to a pharmacokinetic interaction study of vortioxetine and carvedilol after oral administration vortioxetine and carvedilol in rats. Results suggested that the co-administration of vortioxetine and carvedilol results in a significant drug interaction in rats., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A procedure based on solid-phase extraction (SPE) followed by high performance liquid chromatography (HPLC) with PDA detection has been developed for the analysis of multiple drugs in rat plasma. The analytes evaluated were ulifloxacin, fenbufen and felbinac. Eight different solid phase extraction cartridges were tested to evaluate their applicability for the isolation of drugs from rat plasma. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by HPLC using a Kinetex C18 EVO column and acetonitrile-10mM ammonium acetate-methanol as the mobile phase under gradient elution conditions. SPE combined with HPLC-PDA allowed the determination of drugs over a linear range of 0.05-15 μg/mL for ulifloxacin while 0.5-50 μg/mL for felbinac and fenbufen, with limit of detection at 0.05 for ulifloxacin and 0.5 for felbinac and fenbufen. Bond Elut Plexa sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 93.54% with relative standard deviation (RSD) <10%. The method was applied with good accuracy and precision in the determination of ulifloxacin, fenbufen and felbinac in rat plasma obtained from rats treated with selected drugs. This method permits its application to pharmacokinetic and pharmacodynamic studies of these analytes and will facilitate detailed investigations on the interactions between new fluoroquinolones and fenbufen., (Copyright © 2016. Published by Elsevier B.V.)

A rapid and sensitive LC-MS/MS method was developed for the quantification of vilazodone in rat plasma using escitalopram as internal standard. After extracted with organic solvent, post-treatment samples were chromatographed on an Agela C18 column. An isocratic mobile phase of acetonitrile: 5mM ammonium acetate: formic acid (35:65:0.1, v/v/v) was applied at a flow rate of 0.25mL/min. Detection was performed using multiple reaction-monitoring (MRM) modes at m/z 442.4→155.3 for vilazodone and m/z 325.1→109.0 for escitalopram. The method was linear in the concentration range of 1.0-100ng/mL with a correlation coefficient ≥0.993. The intra- and inter-assay precision (%RSD) values were within 13.4%, and intra- and inter-day accuracy (%RE) ranged from -9.8 to 6.9%. The total analysis time was 2.2min. The LC-MS/MS method was fully validated for its sensitivity, selectivity, stability, matrix effect and recovery. The data indicated that the developed method was rapid, specific and sensitive. This method was further and successfully applied in the pharmacokinetics study of vilazodone in rat., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A simultaneous, selective, sensitive, and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of bosentan, ambrisentan, sildenafil, and tadalafil in 50μL of human blood plasma. Diluted plasma samples were extracted using a solid-phase extraction procedure with 2% formic acid and methanol. The four drugs were separated by high-performance liquid chromatography using a C18 column and an isocratic mobile phase running at a flow rate of 0.2mL/min for 5min. The drugs were detected by a tandem mass spectrometer with electrospray ionization using deuterated compounds as internal standards. Calibration curves were generated over the linear concentration range of 2-1000ng/mL in plasma with a lower limit of quantification of 2ng/mL for all compounds. Finally, this validated method was applied to a clinical pharmacokinetic study in pediatric patients with pulmonary arterial hypertension (PAH) following the oral administration of PAH drugs. These results indicate that this method is suitable for assessing the risk/benefit of combination therapy in the pediatric population and useful for therapeutic drug monitoring for PAH treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation to the significantly less active CGP74588. Since deuterium-carbon bonds are stronger than hydrogen-carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties, and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl analogue would maintain target inhibition and show a reduced propensity for N-demethylation in in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased exposure of the N-trideuteromethyl analogue following intravenous administration of the compound to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from either parent drug., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This work proposes a liquid chromatography-electrospray ionization ion trap mass spectrometry (LC-ESI-ITMS) method, for the quantification of sildenafil (SDF), tadalafil (TDF) and vardenafil (VDF) and their metabolites N-desmethylSDF, O-desethylSDF and N-desethylVDF, preceded by a sample preparation step based on protein and phospholipid elimination. A C8 column (150 mm × 4.6 mm, 5 μm) with ammonium formate (20mM) and acetonitrile as the mobile phase components have been used. This method has been validated, obtaining limits of quantification ranged from 1 to 2.5 ng/mL and 2 to 5 ng/g in serum and brain tissue respectively, while limits of detection ranged from 0.3 to 0.9 ng/mL in serum and 0.6 to 1.9 ng/g in brain tissue. Assay recoveries for low level QC samples were higher than 83% and the matrix effect ranged between 91% and 108% in serum and between 98% and 107% in brain tissue. The method has been applied to the quantification of these compounds in the serum and brain tissue of rats treated intraperitoneally with 10 mg/kg of SDF, TDF or VDF., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background and Objectives: Current liquid chromatographic tandem mass spectrometry (LC-MS/MS) methods to measure serum levels of aripiprazole (Ar) and dehydroaripiprazole (DHAr) are sensitive, but difficult to use in a hospital context. We aimed to develop a rapid LC-MS/MS method allowing reliable level measurement in the presence of co-administered drugs, withdrawing samples from 22 patients with acute agitation receiving 9.75 mg aripiprazole IM injection., Method: We developed a sensitive and selective HPLC-MS/MS method to measure serum Ar and DHAr levels in a hospital laboratory, requiring minimal sample preparation and inferior sample volume compared to previous LC-MS/MS methods. Analytes were separated on a reversed-phase HPLC (run-time, 10 min). A triple quadrupole tandem mass spectrometer was used for quantitative analysis in positive mode by a multiple reaction monitoring. Samples were drawn 2, 4, 6, and 24h post-injection., Results: Calibration curves (2-1000 ng/mL for Ar and 3.5-500 ng/mL for DHAr) were linear, with mean correlation coefficient >0.9998. Within- and between-day precision and accuracy were within 10%. Mean recovery was 95.2 ± 4.5% for Ar and 97.6 ± 7.2% for DHAr. Ar and DHAr peaks were not affected by other co-administered psychotropic drugs., Conclusion: Our method measured Ar and DHAr concentrations reliably, simply and rapidly without employing many reagents, as currently existing methods., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of PBMC concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple PBMC isolation and extraction procedure were applied on 10-14 mL of blood aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water+formic acid 0.05%) on a C18 reverse phase analytical column with 25 min of analytical run, at flow rate of 0.25 mL/min. Mean intra- and inter-day precision for all compounds were 8.76 and 12.20%; mean accuracy was -3.86%; extraction recovery ranged within 79 and 91%. Calibration curves ranged from 50.0 to 0.25 ng. The limit of quantification was set at 0.25 ng for all the analyzed drugs. This novel developed methodology allows a specific, sensitive and reliable simultaneous intracellular determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in PBMC of patients affected by chronic myeloid leukemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A novel T-type calcium channel blocker, 4-amino-1-{4-[(4-chloro-phenyl)-phenyl-methyl]-piperazin-1-yl}-butan-1-one (HYP-10) has been synthesized, and the compound has shown promise as both a nociceptive and inflammatory pain reliever as well as an analgesic in a rat neuropathic pain model. A quantification method was developed for the determination of HYP-10 in rat plasma. After simple protein precipitation with methanol, HYP-10 and the internal standard, methaqualone were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma HYP-10 concentration after a single intravenous administration of the compound in rats., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Traditional chiral chromatographic separation method development is time consuming even for an experienced chromatographer. This paper describes the application of computer software ACD Lab to facilitate the development of chiral separation for the quantitation of eszopiclone using LC-MS/MS technology. Assisted by ACD/Chrom Manager and LC Simulator software, the optimal chiral chromatographic development was completed within hours. The baseline chiral separation was achieved with a total cycle time of 3 min. For sample extraction method development, a Waters Oasis Sorbent Selection Plate containing four different sorbents was utilized. Optimal conditions were determined using a single plate under various load, wash and elution conditions. This was followed by a GLP validation which demonstrated excellent intra- and inter-day accuracy and precision for the quantitation of eszopiclone in human plasma at 1.00-100 ng/mL range using LC/MS/MS technology. This method was utilized to support multiple clinic bioequivalence studies., (Copyright (c) 2010. Published by Elsevier B.V.)

A sensitive and precise LC-ESI-MS/MS method for determination of nutlin-3a in murine plasma using ketoconazole as an internal standard was developed and validated. Plasma nutlin-3a samples were prepared by either a simple protein precipitation (PP) for the high concentration range (10-20,000ng/mL) or by liquid-liquid extraction (LLE) for the low concentration range (0.25-300ng/mL). Nutlin-3a and ketoconazole were separated on a modified C18 analytical column (4microm, 75mmx2mm) with an isocratic mobile phase (acetonitrile/5mM HCOONH(4)=70/30, v/v). The retention times of nutlin-3a and ketoconazole were 1.14 and 1.45min. Detection was achieved by a tandem MS system, monitoring m/z 582/99 and m/z 532/82 for nutlin-3a and ketoconazole, respectively. The PP method was linear in a range of 10-20,000ng/mL (R(2)>or=0.993) and the LLE method was linear in a range of 0.25-300ng/mL (R(2)>or=0.992). The mean recoveries for PP and LLE were 24% and 78%, respectively. Within-day and between-day precisions were

A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C(18) column (150mmx4.6mm i.d., 5microm particle size, Zhonghuida) protected by a ODS guard column (10mmx4.6mm i.d., 5microm particle size), using acetonitrile-methanol-phosphate buffer (pH 3.0, 30mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0mL/min, and wavelength of the UV detector was set at 281nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05-20microg/mL (r>0.99). The lower limit of quantification was evaluated to be 50ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively.

The purpose of the present study was to determine sildenafil and a novel PDE-5 inhibitor, mirodenafil in the plasma and corpus cavernosum tissue of rats to compare their pharmacokinetic properties. The concentrations of mirodenafil and sildenafil in the rat plasma and corpus cavernosum tissue samples were analyzed using LC-MS/MS after a single oral administration at a dose of 40mg/kg to rats. Although the T(max), Tlambda(1/2) and MRT were not different between mirodenafil and sildenafil, the C(max) and AUC of mirodenafil were significantly higher than those of sildenafil in the plasma and corpus cavernosum tissue. Consequently mirodenafil remained longer than sildenafil in the plasma and tissue. This may provide pharmacokinetic evidence for assessment of the in vivo efficacy of mirodenafil and sildenafil.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of ranolazine in human plasma. The analytical method consists in the precipitation of plasma sample with methanol, followed by the determination of ranolazine by an LC-MS/MS. The analyte was separated on a Peerless Cyano column (33 mm x 4.6 mm, 3 microm) an isocratic mobile phase of methanol-water containing formic acid (1.0%, v/v) (65:35, v/v) at a flow rate of 1.0 ml/min. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard (IS). The MS/MS detection was made by monitoring the fragmentation of m/z 428.20-->279.50 for ranolazine and m/z 448.30-->285.20 for internal standard on a triple quadrupole mass spectrometer. The method was validated over the concentration range of 5-2000 ng/ml for ranolazine in human plasma with correlation coefficient of 0.9937 (S.D.: +/-0.00367, range: 0.9895-0.9963). The accuracy and precision values obtained from six different sets of quality control samples analyzed in separate occasions ranged from 94.53 to 117.86 and 0.14% to 4.56%, respectively. Mean extraction recovery was 82.36-94.25% for three quality control (QC) samples and 88.37% for IS. Plasma samples were stable for three freeze-thaw cycles, or 24h ambient storage, or 1 and 3 months storage at -20 degrees C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 degrees C). The developed method was successfully applied for analyzing ranolazine in plasma samples for a bioequivalence study with 12 healthy volunteers.

The present paper deals with the development of a rapid and feasible high-performance liquid chromatographic method for the determination of trazodone and its main active metabolite 3-(1-clorophenyl)piperazine (m-CPP) in human plasma. Trazodone is a second-generation antidepressant with serotonin antagonist activity. The metabolite seems to be involved in the onset of some side effects of trazodone therapy, thus its determination is very important during therapeutic drug monitoring. Separation was achieved using a C8 reversed-phase column and a mobile phase composed of aqueous phosphate buffer (70%), containing triethylamine, at pH 3.5 and acetonitrile (30%). The UV detector was set at 255 nm and loxapine was used as the internal standard. An original pre-treatment procedure of plasma samples was developed, based on solid-phase extraction with C8 reversed phase cartridges (50mg, 1 mL). The obtained extraction yields values were higher than 90% and precision, expressed as R.S.D., was lower than 5.6%. The method was successfully applied to plasma samples from depressed patients undergoing therapy with trazodone; accuracy results were satisfactory (recovery >91%). Thus, the method seems to be suitable for the therapeutic drug monitoring of trazodone and its main active metabolite in depressed patients' plasma.

A simple, sensitive and specific liquid chromatography/tandem mass spectrometry method for the quantitation of aildenafil, a new phosphodiesterase V inhibitor, in human plasma is presented. The analyte and internal standard, sildenafil, were extracted by a one-step liquid-liquid extraction in alkaline conditions and separated on a C(18) column using ammonia:10mM ammonium acetate buffer:methanol (0.1:15:85, v/v/v) as the mobile phase. The detection by an API 4000 triple quadrupole mass spectrometer in multiple-reaction monitoring mode was completed within 2.5 min. The calibration curve exhibited a linear dynamic range of 0.05-100 ng/ml with a 10 pg/ml limit of detection. The intra- and inter-day precisions measured as relative standard deviation were within 8.04% and 5.72%, respectively. This method has been used in a pharmacokinetic study of aildenafil in healthy male volunteers each given an oral administration of one of the three dosages.

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d7-nefazodone, d7-hydroxynefazodone, d4-BMS-180492 and d4-mCPP as internal standards (IS). The plasma samples were injected into the LC-MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis HLB column (on-line extraction column, 1 mm x 50 mm, 30 microm) and a conventional Luna C8 column (analytical column, 4.6 mm x 50 mm, 5 microm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC-MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.

A method which employed high-performance liquid chromatography coupled with electrochemical detection was developed for the simultaneous determination of sildenafil and its metabolite, N-desmethyl sildenafil, in human plasma has. The method was developed and validated for purposes of its application to a pharmacokinetic study in healthy volunteers after an oral dose of 50mg/tablet under fasting conditions. High precision and accuracy were demonstrated. A one-step liquid-liquid extraction further provides a simple and practical way to process plasma samples containing sildenafil with good quantitative recovery. Sampling lasted for 24h after dosing; consequently a limit of quantitation (LOQ) of 7.858 ng/mL was achieved for sildenafil whereas a LOQ of 8.675 ng/mL was obtained for N-desmethyl sildenafil. The mobile phase consisted of acetonitrile, methanol and phosphate buffer (0.05 M) (18.5:34.5:47.0, v/v/v) pH 7.68. The stationary phase was a C(8) (150 mm x 4.6 mm), 5 microm particle size operated at 27 degrees C. All analytes were stable at the pH of the supernatant, and during the analytical time window. At the applied potential of +1.20 V versus Ag/AgCl, no interferences from endogenous plasma compounds were recorded at the retention times of sildenafil, N-desmethyl sildenafil. High resolution was obtained between the analytes and the employed internal standards.

A simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of LASSBio-579 in plasma rat, using fluconazole as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water (80:20, v/v), containing 0.4mM ammonium hydroxide and 0.2 mM acetic acid at a flow rate of 1.0 ml/min (split ratio 1:5). A Micromass triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the positive mode. Plasma samples were deproteinized with acetonitrile (1:2) and 50 microl of the supernatant were injected into the system. The retention times of LASSBio-579 and IS were approximately 4.7 and 2.4 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 30-2000 ng/ml with determination coefficient >0.98. The lower limit of quantification was 30 ng/ml. The accuracy of method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 13.5% and 6.4%, respectively. The applicability of the LC-MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after intraperitoneal administration of LASSBio-579 to male Wistar rats. No interference from endogenous substances was observed, showing the specificity of the method developed. The reported method can provide the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of LASSBio-579 in pre-clinical pharmacokinetic studies.

Ucb-35440-3 is a new drug entity under investigation at UCB S.A. Due to its physicochemical characteristics, the drug, a poorly water-soluble weak base, shows poor solubility and dissolution characteristics. In rat, the low oral bioavailability (F < 10%) is largely due to poor absorption. In order to enhance the solubility and dissolution characteristics, formulation of ucb-35440-3 as nanocrystals has been achieved in this study. Nanoparticles were prepared using high pressure homogenization and were characterized in terms of size and morphology. In vitro dissolution characteristics were investigated and compared to the un-milled drug in order to verify the theoretical hypothesis on the benefit of increased surface area. In vivo pharmacokinetic evaluation of ucb-35440-3 nanoparticles was also carried out on rats. Crystalline state evaluation before and following particle size reduction was conducted through polarized light microscopy and PXRD to denote any possible transformation to an amorphous state during the homogenization process. Drug chemical stability was also assessed following homogenization. The dissolution rate increased significantly at pH 3.0, 5.0 and 6.5 for ucb-35440-3 nanoparticles. However, the pharmacokinetic profile obtained yielded lower systemic exposure than the un-milled compound (in fed state), this although being thought to be the consequence of the drug and formulation characteristics.

Effects of ranolazine on isosorbide dinitrate- and on sildenafil-induced changes in mean arterial pressure and heart rate were assessed in conscious dogs. Dogs (n = 7) were chronically instrumented for measurements of mean arterial pressure and heart rate. Bolus intravenous injections of either isosorbide dinitrate (0.2 mg/kg) or sildenafil (0.5 mg/kg) caused biphasic changes in mean arterial pressure and heart rate: a transient (approximately 20 s) decrease in mean arterial pressure and an increase in heart rate, followed by prolonged (10-15 min) decreases in mean arterial pressure by 11 +/- 1.6 and 11 +/- 2.2 mm Hg, respectively. Infusion of ranolazine alone (plasma concentrations = 4 or 8 microM) for 10 min did not significantly affect mean arterial pressure and heart rate. The transient hypotension and tachycardia caused by isosorbide dinitrate were not altered by ranolazine. The sildenafil-induced transient tachycardia (Delta change: 114 +/- 10 beats/min) was significantly (P < 0.05) blunted by either 4 (Delta change: 71 +/- 8 beats/min) or 8 (Delta change: 66 +/- 9 beats/min) microM ranolazine. However, the sildenafil-induced transient decrease in mean arterial pressure was not altered by ranolazine. During ranolazine infusion (4-5 or 8-10 microM), isosorbide dinitrate and sildenafil caused prolonged decreases in mean arterial pressure. These results indicate that except for a blunting of the transient tachycardia caused by sildenafil, ranolazine at concentrations up to 10 microM does not alter changes in mean arterial pressure and heart rate induced by either isosorbide dinitrate or sildenafil in conscious dogs.

HPLC method for the determination of sildenafil and its metabolite (N-demethylsildenafil) in rat serum has been developed. The technique included a solid phase extraction of the serum samples on a [poly(divinylbenzene-co-N-vinylpyrrolidone)] solid phase extraction sorbent. After conditioning, the cartridge was loaded with 0.5 mL of buffered serum containing internal standard. Elution was made with 1 mL of acetonitrile. After evaporation of the eluates to dryness and reconstitution with methanol, the samples were analyzed on Kromasil C18 column phase with phosphate buffer 0.05 M/acetonitrile: 54/46, pH 8. Detection was carried out using a photodiode array detector. For sildenafil and demethylsildenafil, full validation of the proposed method was provided (linearity range, calibration curves, average extraction efficiency; average intra-day and interday variabilities, limit of detection, limit of quantification, specificity). The proposed method was successfully utilised to quantify sildenafil and N-demethylsidenafil in rat serum for a pharmacokinetic study.

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C18 column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25-8.0 microg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 microg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 microg/ml, 7.44 to 13.81% at 1.5 microg/ml and 6.10 to 13.94% at 6.0 microg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 microg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.

Objectives: To determine the minimal time to successful intercourse after taking sildenafil citrate for erectile dysfunction (ED)., Methods: Male patients with ED (mean age 60 years; mean ED duration 7.0 years) who were successfully treated with sildenafil (100 mg) for 2 months or longer were randomized to sildenafil (n = 115) or placebo (n = 113) for 4 weeks of double-blind treatment. Using a stopwatch, patients recorded the time needed to obtain an erection hard enough for sexual intercourse after taking the study drug at least 2 hours after eating., Results: Within 14 and 20 minutes of sildenafil dosing, 35% and 51% of sildenafil-treated patients, respectively, versus 22% and 30% of placebo-treated patients, respectively, had an erection that led to successful intercourse (P <0.05 for both). The median time to erection leading to successful intercourse after sildenafil dosing was 36 minutes compared with 141 minutes for placebo., Conclusions: In this study, slightly more than one half of a population of prior sildenafil responders achieved an erection that led to successful sexual intercourse within 20 minutes of sildenafil administration, suggesting that the onset of action of sildenafil can be less than 30 minutes in men with ED.

A liquid chromatography-tandem mass spectrometric method for the simultaneous determination of sildenafil and its active N-demethylated metabolite, UK-103,320 in human plasma was developed. Sildenafil, UK-103,320 and the internal standard (DA-8159) were extracted from human plasma with dichloromethane at basic pH. A reverse-phase LC separation was performed on Luna phenylhexyl column with the mixture of acetonitrile-ammonium formate (10 mM, pH 6.0) (60:40, v/v) as mobile phase. The detection of analytes was performed using an electrospray ionization tandem mass spectrometry in the multiple reaction-monitoring mode. The lower limits of quantification for sildenafil and UK-103,320 were 2.0 ng/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.

Signal Transduction Inhibitor 571 (STI571, formerly known as CGP 57148B) or Gleevec received fast track approval by the US Food and Drug Administration (FDA) for treatment of chronic myeloid leukemia (CML). STI571 (Gleevec) is a revolutionary and promising new oral therapy for CML, which functions at the molecular level with high specificity. The dramatic improvement in efficacy compared with existing treatments prompted an equally profound increase in the pace of development of Gleevec. The duration from first dose in man to completion of the New Drug Application (NDA) filing was less than 3 years. In addition, recently, FDA approved Gleevec for the treatment of gastrointestinal stromal tumor (GIST). In order to support all toxicokinetic (TK) studies with sufficient speed to meet various target dates, a semi-automated procedure using solid phase extraction (SPE) was developed and validated. A Packard Multi-Probe I and a SPE step in a 96-well plate format were utilized. A 3M Empore octyl (C(8))-standard density 96-well plate was used for plasma sample extraction. A Sciex API 3000 triple quadrupole mass spectrometer with an atmospheric pressure chemical ionization (APCI) interface operated in positive ion mode was used for detection. Lower limits of quantification of 1.00 and 2.00 ng/ml were attained for STI571 and its metabolite, CGP 74588, respectively. The method proved to be rugged and allowed the simultaneous quantification of STI571 and CGP 74588 in monkey plasma. Herein, assay development, validation, and representative concentration-time profiles obtained from TK studies are presented.

The current investigation describes the pharmacokinetic-pharmacodynamic correlation of the hypothermic and the corticosterone effect of flesinoxan in the rat simultaneously. A specific objective was to determine the influence of handling the animal. The pharmacokinetic-pharmacodynamic correlation was determined following intravenous administration of 3 and 10 mg/kg flesinoxan in 5 or 15 min. Serial blood samples were obtained for determination of the time course of the flesinoxan and corticosterone concentrations by high performance liquid chromatography. Body temperature was monitored using a telemetric probe. The pharmacokinetics of flesinoxan were described using a three-compartment model. Both the hypothermic and the corticosterone response were successfully described using a physiological indirect response model. It is shown that customizing the animal prior to the experiment has no influence on the pharmacokinetic-pharmacodynamic parameter estimates. Furthermore, the similarity in potency between the hypothermic and corticosterone effects suggests that both are mediated via tissues with a similar receptor-effector coupling efficiency.

Donitriptan is a potent, high efficacy agonist at 5-HT(1B/1D) receptors. We investigated the contractile effects of donitriptan and sumatriptan on human isolated blood vessels of relevance to therapeutic efficacy in migraine (middle meningeal artery) and coronary adverse events (coronary artery). Furthermore, using the concentration-response curves in the middle meningeal artery, we predicted the plasma concentration needed for the therapeutic effect of donitriptan. Both donitriptan and sumatriptan contracted the middle meningeal artery with similar apparent efficacy (E(max): 103+/-8% and 110+/-12%, respectively), but the potency of donitriptan (pEC(50): 9.07+/-0.14) was significantly higher than that of sumatriptan (pEC(50): 7.41+/-0.08). In the coronary artery, the contraction to donitriptan was biphasic with a significantly higher maximal response (E(max): 29+/-6%) than sumatriptan (E(max): 14+/-2%; pEC(50): 5.71+/-0.16), yielding two distinct pEC(50) values (8.25+/-0.16 and 5.60+/-0.24). Incubation with the 5-HT(2) receptor antagonist ketanserin (10 microM) eliminated the low affinity component of the concentration-response curve of donitriptan and the resultant E(max) and pEC(50) were 9+/-2% and 7.33+/-0.21, respectively. Ketanserin was without effect on the sumatriptan-induced contraction. Based on the middle meningeal artery contraction, concentrations (C(max)) of donitriptan that may be expected to have a therapeutic efficacy equivalent to that of 50 and 100 mg sumatriptan are predicted to be around 2.5 and 4.3 nM, respectively. Such concentrations are likely to induce only a small coronary artery contraction of 2.9+/-1.5% and 3.8+/-2.0%, respectively; these are not different from those by C(max) concentrations of sumatriptan (1.7+/-0.4% or 2.2+/-0.4%). The present results suggest that, like sumatriptan, donitriptan exhibits cranioselectivity and would be effective in aborting migraine attacks with a similar coronary side-effect profile as sumatriptan.

CDRI-93/478 is a potent anti-ischemic and anti-hypertensive agent. This compound is in advanced stage of pre-clinical trials. A high-performance liquid chromatographic (HPLC) method was developed for the analysis of CDRI-93/478 in rat serum, a species used for safety evaluation. The HPLC analysis, applicable to 1 ml volumes of serum, involved double extraction of serum samples with diethyl ether at alkaline pH followed by separation on a spheri-5 cyano column and the use of fluorescence detector at excitation wavelength 250 nm and emission wavelength 372 nm. The method was sensitive with a limit of quantitation of 10 ng ml(-1) in rat serum and the recovery was more than 84%. The linearity was satisfactory as indicated by correlation of >0.99, in addition to the visual examination of the calibration curves. The precision and accuracy were acceptable as indicated by relative standard deviation (R.S.D.) ranging from 1.73 to 9.51%, bias values ranging from -7.31 to 8.68%. Moreover, CDRI-93/478 was stable in rat serum after being subjected to three freeze-thaw cycles. In-process stability evaluation showed the stability of the compound in processed samples lasted up to 168 h. The assay was found to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic or toxicokinetic studies.

This manuscript reports development and validation of an assay method in rat serum for the simultaneous estimation of C1, C2, and C3, in-house CDRI molecules, of the class of aryloxy-substituted aryl-piperazinyl derivatives. The assay was applied to determine pharmacokinetic data after simultaneous intravenous administration of these three compounds. A high-performance liquid chromatography assay method using isocratic elution and fluorescence (excitation, 250 nm; emission 350 nm) was developed for simultaneous estimation of all the three compounds in rat serum. Linearity was observed between 12.5 and 400 ng/ml for all the three compounds in serum. Recoveries were highly consistent over the concentration ranges for all the analytes. Variations in the intra- and inter-batch accuracy and precision were within the acceptable limits of +/-20% at the limit of quantitation, whereas at higher concentrations it was +/-15%. A mixture of the three compounds was administered intravenously to rats. Blood samples were collected over a period of 6 h and analyzed to determine serum levels and pharmacokinetics of each compound. The pharmacokinetics of the aforementioned three compounds was also determined after individual administration. The results obtained in the N-in-One dosing correlated well with discrete dosing of compounds. Based on the results obtained, C2 emerges to be the compound with appropriate pharmacokinetic parameters. Thus, the N-in-One method is a useful method for increasing the throughput to obtain the pharmacokinetic information.

The uptake of the antimicrobial quinolone agent, grepafloxacin, both by human circulating blood neutrophils and by those exudated into tissues, was evaluated in vitro by comparing the intracellular drug concentrations. In circulating blood neutrophils, the uptake of grepafloxacin was rapid and saturable at 37 degrees C. The uptake of grepafloxacin into circulating blood neutrophils was reduced by lowering the environmental temperature or by the presence of metabolic inhibitors, suggesting the involvement of an active transport mechanism. Furthermore, the uptake of grepafloxacin by tissue (salivary) neutrophils was also partially temperature-dependent and was significantly greater than that by circulating blood neutrophils, i.e. exudation of neutrophils into tissue results in a markedly enhanced transport mechanism for grepafloxacin. This phenomenon may be related to the higher defense activity against infection seen in exudated tissue neutrophils.

The pharmacodynamics of (S)-4-[3-[4-[1-(4-methylphenyl)-3-[4-(2-methoxyphenyl)piperazine-1-yl]propoxy]benzoyl]indole-1-yl] butyric acid hydrochloride (Z-350), which has alpha(1)-adrenoceptor antagonistic and steroid 5alpha-reductase inhibitory effects, were investigated in rats. The disposition of Z-350 was a function of linear kinetics at doses from 1 to 30 mg/kg; the bioavailability was calculated to be 65.2%. The inhibition of 5alpha-reductase was dependent on the concentration of Z-350 in plasma and in the prostate. Analysis of the relationship between the concentration in the prostate and the inhibition seen after a single oral administration showed that the Hill constant was almost 1.0 and EC(50)(n(H)) was 47.4 ng/g of tissue; these parameters did not change after multiple administration. Z-350 inhibited 5alpha-reductase 1 h after oral administration at a dose of 3 mg/kg; maximum inhibition was observed after 2-4 h, and the inhibition (%) was maintained for 24 h after oral administration.

The pharmacokinetic-pharmacodynamic relationship of S 15535 (1-(benzodioxan-5-yl) 4-(indan-2-yl)piperazine) and its active 5-hydroxy metabolite S 32784 (1-(benzodioxan-5-yl) 4-(5-hydroxyindan-2-yl)piperazine), and buspirone as a reference, were studied in male Wistar rats using a behavioural model of anxiety by determining the reduction in the number of fear-induced ultrasonic vocalisations. S 15535 and buspirone were administered p.o. and i.v. S 32784, present in man but not in rat, was administered i.v. The pharmacokinetics and pharmacokinetic-pharmacodynamic relationships were described using non-linear mixed effects modelling. The no-drug effect was constant and all compounds were active in the model, reducing ultrasonic vocalisations immediately after administration. The sigmoid E(max) model was used to describe the pharmacokinetic-pharmacodynamic relationships, with E(max) values of a 90% decrease in baseline ultrasonic vocalisations. Corrected for plasma protein binding, all compounds showed similar potency. The study shows that ultrasonic vocalisations can be considered a suitable endpoint for the anxiolytic effect when used in conjunction with non-linear mixed effects modelling to overcome the limited sampling and effect measurements.

RGH-1756 (1-(2-methoxy-phenyl)-4-(4-[4-(6-imidazo[2,1-b]-thiazolyl)-phenoxy]-butyl)-piperazine dimethansulphonate) is a novel atypical antipsychotic candidate of Gedeon Richter Ltd. A new HPLC method has been developed and validated for the quantitative determination of RGH-1756 in dog and rat plasma. The compound and the internal standard are extracted from the biological samples by a simple and fast liquid--liquid extraction method, using 1-chlorobutane. The recovery for RGH-1756 is about 90%. The extracts are analyzed by reversed phase HPLC (column: Supelcosil-LC-18-DB 250*4.6 mm, 5 microm, eluent:acetonitrile:methanol:0.2 molar ammonium-acetate 40:25:35, lambda=254 nm). The assay is specific for RGH-1756. The standard curves are linear in the range between 10 and 2000 ng ml(-1). The overall precision (expressed as CV%) and accuracy (expressed as bias%) of quality controls and calibration standards are within 15%. The validated lower limit of quantification is 10 ng/ml. No indications have been found for possible instabilities of RGH-1756 in plasma, in the extraction solvent, or after repeated thawing-freezing cycles. The method has been succesfully applied for the bioavailability studies of RGH-1756 in the two animal species. In these studies results of the inprocess method validation have shown the reliability of the method, too. CV% of quality controls in the rat study has been found between 7.4 and 10.0%, in the dog study between 4.1 and 12.5%. The bias has ranged from 0.4 to 3.8% and from -4.5 to 1.2% in the rat and dog study, respectively.

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.

A simple reversed-phase high-performance liquid chromatography (HPLC) method with UV detection was developed and validated for the quantitation of SYN-2869, a novel triazole antifungal agent and its analogs in rat plasma. The method involved a simple precipitation of plasma protein with acetonitrile (1:10 ratio). The reconstituted sample after evaporation to dryness was injected onto a HPLC column. SYN-2869 and its analogs were separated from the matrix components on a symmetry C18 column using an aqueous mobile phase of acetonitrile and water with a flow rate of 1 ml min(-1). A step gradient of 40-80% acetonitrile eluted all four compounds. The run time was 30 min. The linear range was 0.5 10 microg ml(-1)(r2 > 0.999). The limit of quantitation was 0.5 microg ml(-1). The inter-day precision and accuracy for SYN-2869 standard concentration were from 1.9 to 8.5% and from 1.4 to +/- 4.40%, respectively. The precision and accuracy of intra-day quality control samples were from 4.6 to 5.2% and from 4.6 to 12%, respectively.

A stereoselective method based on high-performance liquid chromatography (HPLC) and ultraviolet detection at 235 nm for the separation of the four possible stereoisomers of compound 1 ((S-(R*,S*))-2-(4-((4-methylpiperazin-1-yl)carbonyl)phenoxy)-3,3-d iethyl-N-(1-(3,4-(methylenedioxy)phenyl)butyl)-4-oxo-1-azetidinecarbo xamide, L-694,458), a potent, selective, and orally active human leukocyte elastase (HLE) inhibitor, in human and monkey plasma has been developed. The molecule of 1 contains two chiral centers and is being developed as a single stereoisomer with the absolute configuration S and R in positions 'a' and 'b', respectively. Although the baseline separation of each of the two pairs of enantiomers (SS/RR and SR/RS) was achieved on a single chiral column (Chiralcel OD-H) using hexane methyl-t-butyl ether (MTBE)-methanol 80/10/10, (v/v/v) as a mobile phase (alpha(RS,SR) = 2.03, alpha(RR,SS) = 4.97), only partial separation of RS from RR was observed under these conditions (k'RS = 3.32, k'RR = 3.08). Baseline separation of all four stereoisomers from each other and from endogenous plasma components required the initial chromatography of the two diastereomeric racemates (SS/RR and SR/RS) on the achiral silica column (50 x 4.6 mm, 5 microm), followed by column switching and further separation of the stereoisomers on a Chiralcel OD-H column (250 x 4.6 mm, 5 microm) using isopropanol (IPA)-hexane diethylamine (DEA), 65/35/0.3, (v/v/v) on both columns as a mobile phase. The drug was extracted from basified (pH 11) plasma (1 ml) using liquid liquid extraction with MTBE. After evaporation of the extract to dryness, the residue was reconstituted in the mobile phase (200 microl) and part of the extract (125 microl) was injected into the HPLC system. Using this method, it was demonstrated that after oral dosing of monkeys at 40 mg kg(-1) with 1 the only stereoisomer detected in the post-dose plasma samples was the starting material 1, and no inversion of the configuration at positions 'a' and 'b' of 1 had occurred in vivo. Based on this observation, a non-chiral assay for 1 in human plasma was also developed. The method was validated in the concentration range 10-500 ng ml(-1) with the assay precision (expressed as the coefficient of variation, CV) better than 9% and assay accuracy in the range of 95-107% of the nominal concentrations at all concentrations within the standard curve range. The total run time in the non-chiral assay was 12 min. The details of both chiral and non-chiral methods are provided.

AD-5423, 2-(4-ethyl-1-piperazinyl)-4-(4-fluorophenyl)-5,6,7,8,9,10-hexahydrocy cloocta [b]pyridine, is a novel anti-psychotic agent. In order to investigate the pharmacokinetics of AD-5423, a simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of AD-5423 and its two N-oxidized metabolites (N-desethyl AD-5423 and AD-5423 N-oxide) in plasma. After pretreatment of a plasma sample by solid-phase extraction, AD-5423 and its metabolites were analyzed on a HPLC with fluorescence detection (335/410 nm). Chromatography was performed on two C18 reversed-phase columns connected by a switching system, with a mobile phase of acetonitrile-methanol-25 mM sodium phosphate buffer (pH 2.5) containing 25 mM sodium 1-heptanesulfonate (36:26:38 v/v/v). The method gives satisfactory accuracy and precision for the determination of AD-5423 and its metabolites. In human plasma, accurate determination are possible over the concentration ranges of 0.04-5 ng ml-1 for AD-5423 and 0.1-5 ng ml-1 for N-oxidized metabolites. The intra- and inter-day assay precision (R.S.D.) of AD-5423 (0.5 ng ml-1) were 3.6 and 7.2%, respectively. In plasma of experimental animals, the validated quantitative range are 0.1-100 ng ml-1 for both AD-5423 and its metabolites.

Objectives: To develop an HPLC method for the analysis of trazodone and its metabolite, 1-m-chlorophenyl-piperazine (m-CPP), in human plasma and red blood cells., Design and Methods: The analytes were extracted by heptane containing 1.5% isoamyl alcohol, back extracted into phosphoric acid and analyzed by reverse phase HPLC with UV detection., Results: In seven randomly selected, male, human subjects plasma concentrations (nmol/l) were 380-5841 for trazodone and 14-237 for m-CPP while those in packed red blood cells were 98-634 for trazodone and 15-155 for m-CPP. Plasma trazodone concentrations were 4-11-fold higher than those in red blood cells while those of m-CPP were about equal. This may be the first report on concentrations of trazodone and m-CPP in human red blood cells., Conclusions: This sensitive method can be used for monitoring trazodone and m-CPP in human plasma and red blood cells.

Inhibiting the action of platelet-activating factor (PAF) is a new therapeutic approach for the treatment of allergic disorders. SCH 44643 is a new orally-active antagonist of response to both PAF and histamine. This study was done to determine plasma drug concentrations using a GC method and to compare them to ex-vivo anti-PAF activity in the plasma of cynomolgus monkeys following a single oral (12.5 mg kg-1) administration. The GC method involved organic solvent extraction of monkey plasma followed by a GC analysis in a RTX-1 capillary column with a nitrogen/phosphorus detector. The method showed good precision (RSD < 8%) and accuracy (bias < 9%) with a limit of quantitation of 20 ng ml-1. The plasma profiles of SCH 44643 concentration and anti-PAF activity were very similar in each of the six monkeys. There was an excellent correlation (r = 0.9003) between anti-PAF activity and plasma concentration of SCH 44643, suggesting that the anti-PAF activity in cynomolgus monkeys was primarily due to unchanged SCH 44643, rather than its potential metabolite(s).

A new labelling approach of haptenic drugs with digoxigenin for the development of competitive enzyme immunoassay (EIA) is reported. It consists of the covalent linking of the hapten to the preactivated digoxigenin derivative and revealing the immune complexes with anti-digoxigenin Fab fragments coupled to alkaline phosphatase. This approach has been applied to the development of an EIA for the pharmacokinetic study of lesopitron (E-4424), a new anxiolytic agent. The assay involves a solid-phase immobilization of IgG purified from polyclonal antiserum developed against the butylamino derivative of lesopitron covalently linked to bovine serum albumin. The tracer consists of the covalent linking of the same butylamino derivative to digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The calibration curve for E-4424 from 12.5 to 6400 pg per well displays an ED50 of 34.5 pg per well, a slope factor of 0.86 and a minimum detectable dose of 4.1 pg per well. The accuracy and the precision of the assay assessed at three different concentrations of E-4424 (500, 1000 and 2000 pg ml-1) give a recovery higher than 95% and intra- and inter-assay RSDs lower than 5 and 10%, respectively. The specificity of the assay was demonstrated by a good correlation of the samples analysed by both HPLC and EIA. A kinetic profile of E-4424 in rats following an oral dose of 50 mg kg-1 has also been established.