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Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure-toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95-106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22-55%),with high recovery rates (81-93%) and minimal matrix effects (ME 0.9-1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

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.)

Background: Therapeutic drug monitoring (TDM) using cyclin-dependent kinase inhibitors (CDK4/6is) is a novel approach for optimizing treatment outcomes. Currently, palbociclib, ribociclib, and abemaciclib are the available CDK4/6is and are primarily coadministered with letrozole. This study aimed to develop and validate an LC-MS/MS method for the simultaneous analysis of CDK4/6is, 2 active metabolites of abemaciclib (M2 and M20), and letrozole in human plasma for use in TDM studies., Methods: Sample pretreatment comprised protein precipitation with methanol and dilution of the supernatant with an aqueous mobile phase. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column (2.5 μm, 3.0 × 75 mm XP), with methanol serving as the organic mobile phase and pyrrolidine-pyrrolidinium formate (0.005:0.005 mol/L) buffer (pH 11.3) as the aqueous mobile phase. A triple quadrupole mass spectrometer was used for the detection, with the ESI source switched from negative to positive ionization mode and the acquisition performed in multiple reaction monitoring mode., Results: The complete validation procedure was successfully performed in accordance with the latest regulatory guidelines. The following analytical ranges (ng/mL) were established for the tested compounds: 6-300, palbociclib and letrozole; 120-6000, ribociclib; 40-800, abemaciclib; and 20-400, M2 and M20. All results met the acceptance criteria for linearity, accuracy, precision, selectivity, sensitivity, matrix effects, and carryover. A total of 85 patient samples were analyzed, and all measured concentrations were within the validated ranges. The percent difference for the reanalyzed samples ranged from -11.2% to 7.0%., Conclusions: A simple and robust LC-MS/MS method was successfully validated for the simultaneous quantification of CDK4/6is, M2, M20, and letrozole in human plasma. The assay was found to be suitable for measuring steady-state trough concentrations of the analytes in patient samples., Competing Interests: F. Puglisi has received honoraria from Amgen, AstraZeneca, Daiichi Sankyo, Celgene, Eisai, Eli Lilly, Exact Sciences, Gilead, Ipsen, Menarini, MSD, Novartis, Pierre Fabre, Pfizer, Roche, Seagen, Takeda, and Viatris. F. Puglisi is currently receiving research grants from AstraZeneca, EISAI, and Roche. L. Gerratana has received honoraria from AstraZeneca, Daiichi Sankyo, Eli Lilly, GlaxoSmithKline, Incyte, Novartis, Pfizer, Menarini Stemmline, and AbbVie. L. Gerratana is currently receiving research grants from Menarini Silicon Biosystems. S. Corsetti has received a travel grant from AstraZeneca. The remaining authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.)

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05-50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5-107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sharon A. Riddler reports a relationship with Gilead Sciences Inc that includes: funding grants. Sharon A. Riddler reports a relationship with Merck & Co Inc that includes: funding grants. If there are 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 Elsevier B.V. All rights reserved.)

Therapeutic drug monitoring (TDM) is strongly suggested to define the proper drug dosage to overcome inter- and intra-patient variability in drug exposure, which is typically observed with oral anticancer agents, such as palbociclib (PALBO), ribociclib (RIBO) and letrozole (LETRO), all approved for the treatment of HR+, HER2- locally advanced or metastatic breast cancer (BC). Optimal TDM implementation requires a blood sampling organization that can be hampered by limited availability of health and laboratory personnel. Dried Blood Spot (DBS) sampling is proposed to overcome such limitations. The aim of this work was the development of a new LC-MS/MS method to analyze DBS samples containing PALBO, RIBO, and LETRO. Analytes extraction from DBS was performed by adding a methanolic solution containing the corresponding internal standards. LC-MS/MS analysis was performed using a LC Nexera (Shimadzu) system coupled with an API 4000 QTrap (SCIEX) mass spectrometer. The chromatographic separation was performed on a Luna Omega Polar C18 column (Phenomenex). The method was applied to 38 clinical samples collected by finger prick. The influence of hematocrit and spot size, sample homogeneity, stability, and correlation between finger prick and venous DBS measurement were assessed. The analytical validation was performed according to EMA and FDA guidelines. The analytical range of the method was 1 to 250 ng/mL for PALBO, 40 to 10000 ng/mL for RIBO, and 2 to 500 ng/mL for LETRO, where linearity was assessed, obtaining mean coefficients of determination (R 2 ) of 0.9979 for PALBO, 0.9980 for RIBO, and 0.9987 for LETRO). The LC-MS/MS method runtime was 6.6 min. Incurred sample reanalysis demonstrated reproducibility, as the percentage difference between the two quantifications was lower than 20% for 100% of PALBO, 81.8% of RIBO, and 90.9% of LETRO paired samples. Intra- and inter-day precision (CV (%)) was lower than 11.4% and intra- and inter-day accuracy was between 90.0 and 106.5%. DBS sample stability at room temperature was confirmed for 2.5 months. A positive correlation was observed between DBS and plasma concentrations for the 3 drugs, Lin's concordance correlation coefficients obtained by DBS normalization applying a selected strategy were 0.958 for PALBO, 0.957 for RIBO, and 0.963 for LETRO. In conclusion, a fast, easy, and reproducible DBS LC-MS/MS method for the simultaneous quantification of palbociclib; ribociclib and letrozole was developed to be used in clinical practice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7-111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

AMG 510 is the first-in-class KRAS G12C inhibitor, currently in phase 2 clinical trials as an orphan drug to treat non-small cell lung cancer patients. We developed and validated a sensitive, selective, and high-throughput HPLC-MS/MS method for the quantitation of AMG 510 in mouse plasma per the regulatory guideline of the US Food and Drug and Administration. AMG 510 and the IS (MRTX-1257) were extracted from mouse plasma using tert-butyl methyl ether and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 25:75, v/v) at a flow rate of 0.65 mL/min on an Atlantis dC 18 column. AMG 510 and the IS eluted at ~0.95 and 0.73 min, respectively. AMG 510 and the IS were detected by positive electrospray ionization in multiple reaction monitoring using transition pair (Q1 → Q3) m/z 561.1 → 134.1 and m/z 566.5 → 98.2, respectively. Excellent linearity was achieved in the concentration range of 1.08-5040 ng/mL (r > 0.0996). No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. AMG 510 was demonstrated to be stable under the tested storage conditions. This novel method has been applied to a pharmacokinetic study in mice., (© 2020 John Wiley & Sons, Ltd.)

Therapeutic drug monitoring (TDM) is used in certain clinically selected cases and in research settings to optimize the response to antiretroviral therapy. Plasma of blood is commonly used for TDM, but blood sampling is invasive and at risk for transmission of infectious agents. On the other hand, saliva sampling is noninvasive, safe, cheap, and easily performed compared to blood. Dolutegravir (DTG) is now widely prescribed as a key component of antiretroviral therapy for HIV infection. In this study, we examined the relationship between DTG concentrations in plasma and saliva of treated patients to explore the possibility of using saliva as an alternative body fluid of TDM. A total of 17 pairs of blood and saliva samples were obtained from 15 consented HIV-1-infected subjects treated with DTG containing regimens for more than one month. Both blood and saliva samples were collected within 1 h of each other. Drug concentrations were determined by liquid chromatography-tandem mass spectrometry using DTG-d5 as an internal standard. The LLOQ was 0.5 ng/mL. The calibration curves were prepared with pooled plasma or saliva containing DTG in a range of 0.5-100 ng/mL with precision of <14.4% and accuracy within ±14.7%. The DTG concentrations in the plasma and saliva were significantly correlated (Pearson's correlation coefficient r = 0.76, p < 0.001). The median ratio of the drug concentration in saliva to those in plasma was 0.0056, which is close to the rate of non-protein-bound DTG in plasma (0.70%), suggesting that only free DTG in plasma is transported to the salivary glands and secreted into saliva. The present study demonstrates that DTG concentration in saliva reflects the pharmacologically active drug concentration in plasma and may provide an easily accessible alternative for monitoring effective antiretroviral treatment., Competing Interests: The authors have read the journal’s policy and declare the following competing interests: KS and SK are employees of Hanah Meditech Co. Ltd. However, this company was established after this study concluded, and KS and SK were employees of Keio University, Tokyo, Japan at the time the study was conducted. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometric method was developed to quantify ipatasertib in dog plasma. The dog plasma sample was deproteinated by using acetonitrile with ulixertinib as an internal standard followed by separation on a Spursil C 18 -EP column with a gradient mobile phase comprising 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile. Positive ion electrospray was used, and multiple reaction monitoring transitions were m/z 458.2 > 387.2 for ipatasertib and m/z 433.1 > 262.1 for the internal standard. The developed method was validated with a linear range of 0.3-1500 ng/mL, and with correlation coefficient greater than 0.9989. The lower limit of quantification was 0.3 ng/mL. The intra- and inter-day precision ranged from 3.58 to 14.32%, whereas the intra- and inter-day accuracy was in the range of -2.50-13.25%. No carry-over and matrix effects were observed under the current conditions. The extraction recovery was demonstrated to be greater than 85.43%. Ipatasertib was stable during the storage, processing, and determination. The validated assay was further successfully applied to a pharmacokinetic study of ipatasertib in dogs after oral and intravenous administrations. The bioavailability of ipatasertib was determined to be 19.3%., (© 2020 John Wiley & Sons, Ltd.)

A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 μL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992-0.9983) over the concentration ranges of 0.3-250 ng/mL for palbociclib, 10-10000 ng/mL for ribociclib and 0.5-500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Dr. Fabio Puglisi reports grants from Astrazeneca and from Roche, outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometry method for the detection of tandospirone (TDS) and its active metabolite 1-[2-pyrimidyl]-piperazine (1-PP) in Sprague-Dawley rat plasma is described. It was employed in a pharmacokinetic study. These analytes and the internal standards were extracted from plasma using protein precipitation with acetonitrile, then separated on a CAPCELL PAK ADME C 18 column using a mobile phase of acetonitrile and 5 mm ammonium formate acidified with formic acid (0.1%, v/v) at a total flow rate of 0.4 mL/min. The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. The method was validated to quantify the concentration ranges of 1.000-500.0 ng/mL for TDS and 10.00-500.0 ng/mL for 1-PP. Total time for each chromatograph was 3.0 min. The intra-day precision was between 1.42 and 6.69% and the accuracy ranged from 95.74 to 110.18% for all analytes. Inter-day precision and accuracy ranged from 2.47 to 6.02% and from 98.37 to 105.62%, respectively. The lower limits of quantification were 1.000 ng/mL for TDS and 10.00 ng/mL for 1-PP. This method provided a fast, sensitive and selective analytical tool for quantification of tandospirone and its metabolite 1-PP in plasma necessary for the pharmacokinetic investigation., (© 2019 John Wiley & Sons, Ltd.)

Mefunidone (MFD) is a promising anti-fibrotic candidate molecule with greater anti-fibrotic activity than pirfenidone (PFD). However, there has been no report on the methodology used for the quantification of MFD or on any investigation of its pharmacokinetics. In this study, an efficient and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to assay MFD in monkey plasma. This assay method was validated and applied to a pharmacokinetics study in monkeys. The lower limit of quantification of this assay was 0.1 μg·mL -1 , and the linear calibration curve was acquired with R 2  > 0.99 between 0.1 and 60 μg·mL -1 . The intra-day and inter-day precision were evaluated with coefficient of variations of 1.5%-5.8%, whereas the mean accuracy ranged from 91.7% to 106.9%. A negligible matrix effect and good recovery were obtained using this assay, with average extraction recoveries of MFD and the internal standard (IS) in the range of 85.5%-124.8% and 84.1%-94.0%, respectively. The precision of the absolute matrix effect of MFD and the IS was 1.2-3.0% and 1.2-7.3%, respectively. The samples were stable under all experimental conditions. Linear pharmacokinetics were observed for MFD in monkeys, where the exposures of MFD increased proportionally with increasing MFD doses at the range of 10-90 mg·kg -1 . Moderate elimination of MFD from the body was observed, with t 1/2 of 5-7 h, and the elimination rate of MFD was stable during multiple dosing. In conclusion, this method provides an reliable analytical approach for quantification of MFD in plasma and was successfully applied to a pharmacokinetics study in monkeys., (© 2019 John Wiley & Sons, Ltd.)

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 μm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169., Competing Interests: The authors have declared that no competing interests exist.

Osimertinib (AZD9291) is a highly selective irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) designed to treat nonsmall-cell lung cancer patients with EGFR active and T790 M resistant mutations. A rapid and sensitive method for quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of osimertinib and its metabolite AZ5104 in human plasma was developed and validated. The samples were prepared by protein precipitation and separated on a BEH C 18 column (2.1 × 50 mm, 1.7 μm) by gradient elution with 0.1% (v/v) formic acid and 10 mm ammonium acetate in water and acetonitrile as the mobile phase. Electrospray ionization in positive ion mode and multiple reaction monitoring were used to monitor the ion transitions at m/z 500.4 → 385.3 and 486.3 → 371.1. The results indicated that the method had excellent sensitivity and specificity. The validation was performed in a range from 0.5 to 100 ng/mL. Intra-day and inter-day precisions (in terms of RSD) were all <15%, and the accuracies (in terms of RE) were within ±15%. The lower limit of quantification, matrix effect, extraction recovery, stability and dilution integrity were also validated and satisfied the validation criteria. Finally, this method was successfully applied in a retrospective analysis, and the predose samples of 52 nonsmall-cell lung cancer patients who were enrolled in a novel third EGFR TKI clinical trial were analyzed for screening regardless of whether they had previously received osimertinib treatments., (© 2018 John Wiley & Sons, Ltd.)

A fast and easy-to-use liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of a novel antifungal drug, olorofim (F901318), a member of the novel class of orotomides, in human plasma and serum was developed and validated. Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. An isotope-labeled analogue of F901318 was employed as an internal standard. Chromatographic separation was achieved using a 50-mm by 2.1-mm, 1.9-μm, polar Hypersil Gold C 18 column and isocratic mobile phase consisting of 0.1% formic acid-acetonitrile (60%-40%, vol/vol) at a flow rate of 330 μl/min. The analyte was detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode with positive heated electrospray ionization (HESI + ) within a single runtime of 2.00 min. The present LC-MS/MS method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the U.S. Food and Drug Administration (FDA). Linearity of F901318 concentration ranges was verified by the Mandel test. The calibration curve was tested linear across the range and fitted using least-squares regression with a weighting factor of the reciprocal concentration. The limit of detection was 0.0011 mg/liter, and the lower limit of quantitation was 0.0033 mg/liter. Intraday and interday precisions ranged from 1.17% to 3.23% for F901318, and intraday and interday accuracies (percent bias) ranged from 0.75% to 5.01%. In conclusion, a method was established for the rapid quantitation of F901318 concentrations in serum and plasma samples in patient trials, and it optimizes therapeutic drug monitoring in applying an easy-to-use single method., (Copyright © 2018 American Society for Microbiology.)

Background: A simple, rapid, and sensitive liquid chromatography coupled with tandem mass spectrometry method has been developed and validated for the quantification of ruxolitinib, olaparib, vismodegib, and pazopanib in human plasma., Methods: After a simple protein precipitation of plasma samples, the chromatographic separation was performed using an ultraperformance liquid chromatography system coupled with mass tandem spectrometry in a positive ionization mode. The mobile phase consisted of a gradient elution of 10-mmol/L formate ammonium buffer containing 0.1% (vol/vol) formic acid (phase A) and acetonitrile with 0.1% (vol/vol) formic acid (phase B) at a flow rate at 300 µL/min., Results: Analysis time was 5.0 minutes per run, and all analytes and internal standards eluted within 1.5-1.73 minutes. The calibration curves were linear over the range from 10 to 2500 ng/mL for ruxolitinib and from 100 to 100,000 ng/mL for olaparib, vismodegib, and pazopanib with coefficients of correlation above 0.99 for all analytes. The intraday and interday coefficients of variation were below 14.26% and 14.81%, respectively, for lower concentration and below 9.94% and 6.37%, respectively, for higher concentration., Conclusions: Using liquid chromatography coupled with tandem mass spectrometry, we have developed and validated a simple and rapid assay for the simultaneous quantification of olaparib, vismodegib, pazopanib, and ruxolitinib in human plasma. This method is now part of our therapeutic drug monitoring service provision and is currently used clinically to manage patients prescribed these drugs.

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.)

As a new hypnotic, zopiclone is widely used in clinical treatment. There are many methods for determination of zopiclone, including spectrophotometry, chromatography and chromatography mass spectrum, etc. Present paper reviews different kinds of biological samples associated with zopiclone, extraction and purification methods, and determination and analysis methods, which aims to provide references for the relevant research and practice., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Osimertinib is a new-generation epidermal growth factor inhibitor for the treatment of non-small cell lung cancer. In the present study, a rapid and sensitive LC with tandem MS method was developed and validated for the determination of osimertinib in rat plasma. Chromatographic separation was carried out on a C18 column using acetonitrile and water containing 0.1% formic acid. The assay was validated over a concentration range of 1.0-1000 ng/mL for osimertinib, with a lower LOQ of 1.0 ng/mL. The intra- and interday accuracy values for osimertinib ranged from 92.66 to 101.50% and from 97.08 to 99.15%, respectively, and the intra- and interday precision values for osimertinib ranged from 6.25 to 10.34% and from 3.43 to 10.44%, respectively. The method was successfully applied in a pharmacokinetic study of osimertinib after oral administration of osimertinib (4.5 mg/kg) to rats.

Paper spray (PS) is an ambient ionization technique applicable to ionizing analytes from untreated dried biofluid samples. In-field sample analysis could benefit from the capability to use a finger prick of blood to measure drugs in whole blood at low cost and in a short time. Some studies may require specialized blood collection devices that can be used in remote areas. In this study, four different dried blood spot (DBS) devices are used with PS sources and tested for rapid quantification of imatinib and N-desmethyl-imatinib. A triple quadrupole mass spectrometer allows analyte detection with high sensitivity. Analytical figures of merit for the four devices are compared, and it is concluded that several of the novel devices successfully deploy DBS with PS and yield similar results to traditional manual PS methods. Clinical samples collected in a remote location were analyzed as a proof of concept for in-field blood collection and subsequent rapid laboratory analysis. Graphical abstract Dried blood spot analyis by paper spray ionization MS/MS for in field sample collection.

A high-throughput method was developed for the detection of 31 benzodiazepine drugs and tandospirone in human plasma by on-line column-switching ultra-fast liquid chromatography-tandem mass spectrometry. Plasma samples (100μl) spiked with the 32 drugs and oxazepam-d 5 (internal standard) were diluted with 300μl of 13.3mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced into an analytical column (SUMIPAX ODS D-Swifter column, 30mm×3.0mm i.d.; particle size 2μm) by column switching. Quantification was performed by multiple reaction monitoring with positive-ion electrospray ionization. Distinct peaks appeared for each drug and the internal standard on each channel within 7min, including the extraction time. All drugs spiked into plasma showed recoveries of 83-95%. The regression equations for the 32 drugs showed excellent linearities in the range of 50-2000pg/ml of plasma and the limits of detection ranged from 20 to 50pg/ml. The lower and upper limits of quantitation were 50-100ng/ml and 2000pg/ml, respectively. Intra- and interday coefficients of variation for none of the drugs were greater than 13.6%. The accuracies of quantitation were 87-112%. The multiple reaction monitoring information-dependent acquisition of enhanced product ions method enabled the quantification and confirmation of diazepam, triazolam, and lorazepam obtained from actual plasma., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A cost effective, sensitive, simple, and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the prasugrel metabolite in human plasma. Following solid phase extraction, the analyte (prasugrel active metabolite; R-138727) and internal standard (emtricitabine) were separated using a mobile phase in an isocratic elution mode on a reverse phase C18 column and were analyzed by an LC-MS/MS in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 498.3-206.0 for R-138727 and m/z 248.2-130.1 for the internal standard. The assay exhibited a linear dynamic range of 0.2-120 ng/mL. The lower limit of quantification was 0.2 ng/mL with a relative standard deviation of 5.0%. This LC-MS/MS method was validated with intra-batch and inter-batch precision and accuracy. Results for precision and accuracy are in range of 3.9-9.6% and 95.2-102.2% respectively. This validated method is simple and repeatable to use in bioequivalence/pharmacokinetic studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, a simple, sensitive and fast ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitative determination of vortioxetine in rat plasma. Plasma samples were processed with a protein precipitation. The separation was achieved by an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7μm) column with a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The validated method had an excellent linearity in the range of 0.05-20ng/mL (R(2)>0.997) with a lower limit of quantification (0.05ng/mL). The extraction recovery was in the range of 78.3-88.4% for vortioxetine and 80.3% for carbamazepine (internal standard, IS). The intra- and inter-day precision was below 8.5% and accuracy was from -11.2% to 9.5%. No notable matrix effect and astaticism was observed for vortioxetine. The method has been successfully applied to a pharmacokinetic study of vortioxetine in rats for the first time, which provides the basis for the further development and application of vortioxetine., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vilazodone hydrochloride (CAS 163521-12-8) is polymorphic and has 15 crystal forms, referred to as I-XI and XIII-XVI. In the study, we prepared and performed structural identification of a new crystal form named XVII. To investigate this in vivo, a rapid and sensitive method based on liquid-liquid extraction, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the determination of vilazodone hydrochloride in dog plasma. This HPLC-MS/MS method was successfully applied to a bioavailability comparison of two crystal forms of vilazodone hydrochloride (IV and XVII) in six healthy beagles using a single-dose, two-way crossover design. The maximum plasma concentration (C(max)), the time taken to reach C(max), and the area under the concentration-time curve were determined following oral administration of 10 mg vilazodone hydrochloride (IV or XVII) to beagles. These analyses revealed no significant bioavailability differences between vilazodone hydrochloride forms IV and XVII in dogs., (Copyright © 2014 John Wiley & Sons, Ltd.)

An ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of zopiclone and zolpidem in whole blood, for use in cases with suspected driving under influence of drugs (DUID) and autopsy cases. Sample preparation was performed with liquid-liquid extraction (LLE) using ethyl acetate/n-heptane (80:20, v/v) and 0.1mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7μm), with a flow rate of 0.5mL/min, 1μL injected and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode. The run time of the method was 4.5min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R(2) values above 0.999 for both compounds. Intermediate precision and accuracies (bias) were 2.4-12.9% RSD and from -5.9 to 6.8%, respectively. Recoveries of the compounds were ≥70%. The lower limit of quantification (LLOQ) for zopiclone was 0.50nmol/L (0.19ng/mL) or 0.05pg injected on column, and 3.5nmol/mL (1.10ng/mL) for zolpidem, or 0.27pg injected on column. The limit of detection (LOD) was 0.2nmol/L (0.08ng/mL) for zopiclone and 0.3nmol/L (0.09ng/mL) for zolpidem. Matrix effects (ME) were between 108 and 115% when calculated against IS. A comparison with former confirmation LC-MS method at the Norwegian Institute of Public Health, Division of Forensic Medicine (NIPH) was performed during method validation. Good correlation was seen for both compounds. The method has been running on a routine basis for two years, and has proven to be very robust and reliable with satisfactory long term precision and bias and with results for external quality samples corresponding well to consensus mean or median. Zopiclone and zolpidem concentrations in post mortem and ante mortem cases were reported. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs introduced in the Norwegian Road Traffic Act Law from 2012., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC-MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse-phase C18 column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra- and inter-day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats.

A specific and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric method was developed for the quantification of imatinib and its primary metabolite N-desmethyl imatinib in human plasma. Protein precipitation with methanol was used for sample preparation. High-performance liquid chromatographic separation was performed on a Thermo BDS Hypersil C18 column (4.6 × 100 mm, 2.4 µm) with methanol-water (55:45, v/v) containing 0.1% formic acid and 0.2% ammonium acetate as the mobile phase, using isocratic elution at a flow rate of 0.7 mL/min. Detection was conducted with positive electrospray ionization multiple reaction monitoring of the ion transitions at m/z 494 → 394 for imatinib, 480 → 394 for N-desmethyl imatinib and 297 → 110 for the internal standard (palonosetron). The assay was validated in the concentration ranges of 8-5,000 ng/mL for imatinib and 3-700 ng/mL for N-desmethyl imatinib. The quantification limits for imatinib and N-desmethyl imatinib were 8 and 3 ng/mL, respectively. The intra-day and inter-day precision values of the assay (expressed as percentage relative standard deviation) were less than 15% at all concentration levels within the tested range, and the accuracy values were between 85 and 115%. The established method was successfully applied to the pharmacokinetic study of imatinib mesylate capsules in 24 healthy Chinese volunteers.

A rapid, sensitive and selective high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method (HPLC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of lomerizine in human plasma. Protein precipitation process was used to extract lomerizine from human plasma. Plasma samples were separated by HPLC on an Agela Venusil XBP Phenyl column (100 mm × 2.1 mm, 5 μm) using a mobile phase consisting of methanol-2mM ammonium acetate-formic acid (70:30:0.1, v/v/v) and the flow rate was set at 0.35 mL/min. The total run time was 4.0 min and the elution of lomerizine was at 1.9 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective [M+H](+) ions m/z 469.2→181.0 for lomerizine and m/z 405.2→202.9 for the I.S. The calibration curve was linear over the range of 0.1-25 ng/mL (r(2)>0.99) with a limit of quantitation (LOQ) of 0.1 ng/mL. The intra- and inter-day precision (relative standard deviation, RSD) values were below 9.65% and the mean accuracy was from 99.00 to 103.00% at four quality control levels. Lomerizine was stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of lomerizine after single oral doses of 10 mg lomerizine to 18 healthy volunteers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Sildenafil is used for the treatment of pediatric pulmonary hypertension. A dried blood spot (DBS)-based LC-MS method for sildenafil quantitation was developed and applied to a group of patients., Results: DBS showed high portability and stability of samples, and the method was selective and linear for quantitation of sildenafil (5-3,000 ng/ml) and N-desmethyl-sildenafil (3-1,500 ng/ml). After a single oral dose of sildenafil (1 mg/kg), method evidenced poor metabolism in these patients., Conclusion: The method was successfully applied in peripheral blood and can be used for both pharmacokinetics and therapeutic drug monitoring. DBS proved to have advantages during sample translation and preservation of analytes. Data suggest that hazardous blood sildenafil levels may be reached in this population.

Olaparib is an inhibitor of poly ADP ribose polymerase 1 (PARP-1). Phase I and II trials showed promising results of olaparib against tumours in BRCA mutation carriers. Currently an increasing number of clinical trials with olaparib in combination with other compounds or radiotherapy are conducted. To support these clinical trials an LC-MS/MS method was developed and validated for the quantification of olaparib in human plasma. Human plasma samples were collected in the clinic and stored at nominally -20°C. Olaparib was isolated from plasma by liquid-liquid extraction, separated on a C18 column with gradient elution and analyzed with triple quadrupole mass spectrometry in positive ion mode. A deuterated isotope was used as internal standard for the quantification. The assay, ranging from 10 to 5000ng/mL, was linear with correlation coefficients (r(2)) of 0.9994 or better. The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±7.6% of nominal and inter-assay and intra-assay precision ≤9.3% at the lower limit of quantification and ≤5.7% at the other concentration levels tested. All results were within the acceptance criteria of the US FDA and the latest EMA guidelines for method validation. A quantitative method was developed and validated for the quantification of olaparib in human plasma. The method could successfully be applied for the pharmacokinetic quantification of olaparib in cancer patients treated with olaparib., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A sensitive and rapid method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the simultaneous determination of blonanserin, its major active metabolite (N-deethyl form) and other three metabolites (N-oxide form, Ethylenediamine form and Carboxylate form) in human plasma. Plasma samples were pre-purified by solid-phase extraction (SPE) and analyzed using a gradient chromatographic separation over an Acquity UPLC CSH C18 column. The mobile phase consisted of acetonitrile-water containing 5mM ammonium formate and 0.1% formic acid at a flow rate of 0.5mL/min. Positive electrospray ionization was employed as the ionization source in the multiple reaction monitoring (MRM) mode. The analysis time was about 3.5min. The method was fully validated over the concentration range of 0.01-1ng/mL for all analytes. The lower limit of quantification (LLOQ) was 0.01ng/mL. Inter- and intra-batch precision was less than 15% and the accuracy was within 85-115%. The mean extraction recoveries of all analytes at two concentration levels were consistent. Selectivity, matrix effect and stability were also validated. The method was applied to the pharmacokinetic study of blonanserin in Chinese healthy subjects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Methamphetamine abuse continues as a major problem in the USA owing to its powerful psychological addictive properties. AZ66, 3-[4-(4-cyclohexylpiperazine-1-yl)pentyl]-6-fluorobenzo[d]thiazole-2(3H)-one, an optimized sigma receptor ligand, is a promising therapeutic agent against methamphetamine. To study the in vivo pharmacokinetics of this novel sigma receptor ligand in rats, a sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed in rat plasma and validated. The developed method requires a small volume of plasma (100 μL) and a simple liquid-liquid extraction. The chromatographic separations were achieved in 3.3 min using an Acquity UPLC BEH Shield RP18 column. The mass spectrophotometric detection was carried out using a Waters Micromass Quattro MicroTM triple-quadrupole system. Multiple reaction monitoring was used for the quantitation with transitions m/z 406 → m/z 181 for AZ66 and m/z 448 → m/z 285 for aripiprazole. The method was validated over a concentration range of 1-3500 ng/mL and the lower limit of quantitation was determined to be 1 ng/mL. Validation of the assay demonstrated that the developed UPLC/MS/MS method was sensitive, accurate and selective for the determination of AZ66 in rat plasma. The present method has been successfully applied to an i.v. pharmacokinetic study in Sprague-Dawley rats., (Copyright © 2013 John Wiley & Sons, Ltd.)

The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra(®)), tadalafil (Cialis(®)) and vardenafil hydrochloride (Levitra(®)) has increased dramatically over the past 2 years. These substances are prescription drugs and must be used under medical supervision. However, they can easily be obtained over the internet from illegal sites, being a potential for a threat to public health. The development of an electrospray ionisation (ESI) ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) procedure for the simultaneous identification and quantification of three PDE5 inhibitors in blood samples was desired. Samples were prepared using Oasis(®) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC(®) HSS T3 (100 × 2.1 mm i.d., 1.8 μm particles) column with a gradient mobile phase of 0.1% formic acid and acetonitrile at a 0.5 mL/min flow rate. Quantification was achieved by multiple reaction monitoring (MRM) of two transitions per compound: m/z 475.1 > 58 e m/z 475.1 > 311.1 for sildenafil; m/z 389.9 > 267.9 e m/z 389.9 > 134.8 for tadalafil and m/z 489 > 71.9 e m/z 489 > 150.9 for vardenafil. Zolpidem-d6 (m/z 314.5 > 235.3) was used as the internal standard. Calibration curves were linear over the concentration range of 5-1000 ng/mL, with a coefficient of determination better than 0.997. The lower limits of detection and quantification for these substances were ≤ 3 ng/mL and ≤ 8 ng/mL, respectively. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. A rapid, selective and sensitive UPLC-MS/MS method using solid-phase extraction was developed for the simultaneous determination and quantification of sildenafil, vardenafil and tadalafil in blood samples., (Copyright © 2013 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of urapidil and aripiprazole in human plasma. A simple liquid-liquid extraction with ethyl acetate was used for the sample preparation. Chromatographic separation was achieved on a Phenomenex C18 (4.6 × 50 mm, 5 µm) column with 0.1% formic acid-acetonitrile (10:90, v/v) as the mobile phase with flow rate of 0.6 mL/min. The quantitation of the target compounds was determined in a positive ion multiple reaction monitoring mode. Calibration plots were linear over the range of 2.0-2503.95 ng/mL for urapidil and 1.0-500.19 ng/mL for aripiprazole. The lower limit of quantitation for urapidil and aripiprazole was 2.0 and 1.0 ng/mL, respectively. Mean recovery was in the range of 69.94-75.62% for both analytes and internal standards. Intra-day and inter-day precisions of the assay at three concentrations were 2.56-5.89% with accuracy of 92.31-97.83% for urapidil, and 3.14-6.84% with accuracy of 91.38-94.42% for aripiprazole. The method was successfully applied to human pharmacokinetic study of urapidil and aripiprazole in healthy human male volunteers., (Copyright © 2013 John Wiley & Sons, Ltd.)

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, performed by electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been developed and validated for the simultaneous determination of bortezomib (BORT), dasatinib (DASA), imatinib (IMAT), nilotinib (NILO), erlotinib (ERLO), lapatinib (LAPA), sorafenib (SORA), sunitinib (SUNI) and vandetanib (VAND) in human plasma. Separation is achieved on an Hypersil Gold(®) PFP column using a gradient elution of 10mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3 mL/min. After addition of the internal standard and protein precipitation, the supernatant is diluted 2-fold in a mixture A and B (50/50, v/v). Two selected reaction monitoring transitions are used for each analyte: one is used for quantitation, the second one is used for confirmation. The standard curves are ranged from 2 ng/mL to 250 ng/mL for BORT, DASA and SUNI and from 50 ng/mL to 3500 ng/mL for the others and were fitted to a 1/x weighted linear regression model. The lowest limits of quantification were 2 ng/mL for BORT, DASA and SUNI and 50 ng/mL for the other TKIs. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day RSD from 3.7% to 13.8%), accuracy (from 86.8% to 113.5%), recovery as well as stability of the analytes under various conditions. The method also may contribute to better understand the relationship between pharmacokinetics and pharmacodynamics of TKIs in hematological malignancies and solid tumors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An improved and rugged UPLC-MS/MS method has been developed and validated for sensitive and rapid determination of aripiprazole in human plasma using aripiprazole-d8 as the internal standard (IS). The analyte and IS were extracted from 100 μL of human plasma by solid-phase extraction using Phenomenex Strata-X (30 mg, 1 cc) cartridges. Chromatography was achieved on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column using methanol: 10mM ammonium formate (85:15, v/v) as the mobile phase with isocratic elution. Quantitation was done using multiple reaction monitoring in the positive ionization mode. The linearity of the method was established in the concentration range 0.05-80 ng/mL. The mean extraction recovery was greater than 96% across QC levels, while intra- and inter batch accuracy and precision (% CV) values ranged from 97.4 to 101.9% and from 1.20 to 3.72% respectively. The relative matrix effect in eight different lots of plasma samples, expressed as % CV for the calculated slopes of calibration curves was 1.08%. The stability of aripiprazole was studied under different storage conditions. The validated method was used to support a bioequivalence study of 10mg aripiprazole formulation in 36 healthy Indian subjects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We have developed a method of liquid chromatography in tandem with mass spectrometry to monitor therapeutic levels of imatinib in plasma, a selective inhibitor of protein tyrosine kinase. After solid-phase extraction of plasma samples, imatinib and its internal standard, imatinib-D8, were eluted with Zorbax SB-C18 at 60 °C, under isocratic conditions through a mobile phase consisting of 4 mm ammonium formate, pH: 3.2 (solution A) and acetonitrile solution B. The flow rate was 0.8 mL/min with 55% solution A + 45% solution B. Imatinib was detected and quantified by mass spectrometry with electrospray ionization operating in selected-reaction monitoring mode. The calibration curve was linear in the range 10-5000 ng/mL, the lower limit of quantitation being 10 ng/mL. The method was validated according to the recommendations of the Food and Drug Administration, including tests of matrix effect (bias < 10%) and recovery efficiency (>80 and <120%). The method is precise (coefficient of variance intra-day <2% and inter-day <7%), accurate (95-108%), sensitive and specific. It is a simple method with very fast recording time (1.2 min) that is applicable to clinical practice. This will permit improvement of the pharmacological treatment of patients., (Copyright © 2012 John Wiley & Sons, Ltd.)

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.)

Imatinib is a small-molecule tyrosine kinase inhibitor with large inter-individual but low intra-individual pharmacokinetic variability with consistent concentration-efficacy and concentration-toxicity relationships. For these reasons imatinib therapeutic drug monitoring is based on total plasma concentrations. However, since a significant impact of unbound imatinib concentrations on clinical response and/or toxicity evaluation has been suggested, the quantification of free fraction of imatinib and its active metabolite are of interest for therapeutic monitoring. Hence a reliable method for both separation and assay of the free fraction is needed. Using plasma samples spiked with imatinib (from 1000 to 7500 ng/mL) and its metabolite (from 1000 to 2500 ng/mL), an ultrafiltration procedure and an UPLC assay which give reproductive values for unbound fractions of imatinib (mean 3.0±1.0%) and metabolite N-desmethyl imatinib (3.6±1.8%) have been developed. The validation of the analytical UPLC-MS/MS method associated to ultrafiltration for quantification of imatinib and N-desmethyl imatinib was reported. The LOQ was set at 10 ng/mL for imatinib and 20 ng/mL for N-desmethyl imatinib, intraday CV (%) ranged from 2.7 to 4.8% for imatinib and from 5.4 to 12.4% for N-desmethyl imatinib and interday CV (%) ranged from 5.6 to 6.5% for imatinib and from 5.4 to 16.1% for N-desmethyl imatinib. Methodological modifications were attempted to overcome non specific binding (NSB) on the ultrafiltration device. Two types of devices previously used for unbound determination of drugs were tested. Our results clearly showed that the methodology and the features of devices used for ultrafiltration could totally compromise the determination of unbound concentrations of a drug., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Imatinib is a first-line treatment for chronic myelogenous leukaemia (CML). The pharmacokinetics of imatinib in patients with CML are characterised by large interpatient variability. Concentration monitoring of imatinib and its active metabolite N-desmethyl imatinib (DMI) is considered necessary to enhance the safe and effective use of imatinib. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry assay was developed for the simultaneous determination of imatinib and its metabolite DMI in human plasma. After proteins were precipitated with acetonitrile, imatinib, DMI and the internal standard D8-imatinib were resolved on a Gemini-NX 3 μm C18 column using gradient elution of 0.05 % formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves of imatinib and DMI were adequately fitted by quadratic equations (r > 0.999) over the concentration range of 10 to 2,000 ng/mL which encompasses clinical concentrations. Bias was ≤±8.3 %, intra- and inter-day coefficients of variation (imprecision) were ≤8.0 % and the limit of quantification was 10 ng/mL for both imatinib and DMI. The assay is being used successfully in clinical practice to enhance the safe and effective use of imatinib.

4-Methyl-piperazine-1-carbodithioc acid 3-cyano-3, 3-diphenylpropyl ester hydrochloride (TM208), a newly synthesized anticancer compound, was quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the first time. A simple, rapid and sensitive assay method using propranolol as internal standard (IS) after one-step precipitation with acetonitrile was developed and validated to determine TM208 in rat plasma. Separation was achieved on a reverse-phase C(18) column with a mobile phase composed of methanol-water (pH4.0) containing 5 m m ammonium acetate in gradient elution mode. A triple quadrupole tandem mass spectrometer with electrospray ionization source was used as detector and operated by multiple reaction monitoring in the positive ion mode. Calibration curves were linear (r > 0.99) between 0.2 and 500 ng/mL. The quantitative limit was 0.2 ng/mL; reliable precision and accuracy were validated by relative standard deviation values in the range 3.44-13.15% and relative error values between -0.58 and -9.78%. The method was successfully applied to preclinical pharmacokinetic studies of TM208., (Copyright © 2011 John Wiley & Sons, Ltd.)

This paper reports an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method to quantitate 21 benzodiazepines, zolpidem and zopiclone in serum and plasma. After liquid-liquid extraction, an Acquity UPLC with a TQ Detector and BEH C18 column was used (Waters, Milford, MA). The injection-to-injection run time was 7.5 min. Forty-eight authentic serum and plasma patient specimens were analyzed and results compared to those obtained using a previously published method. Average r(2) values for linearity (1 to 1,000 ng/mL over five days) were all above 0.995, except α-hydroxytriazolam (0.993). Intra-day and inter-day relative standard deviation values were within ± 15% and the percent deviation from the expected concentrations were within ± 11%. Recovery ranged from 62 to 89%. Matrix effects ranged from -28% to +6%. The limits of detection were 1 ng/mL, except for lorazepam, nordiazepam, oxazepam and temazepam (5 ng/mL). Ion ratios were ± 15% for all analytes. For authentic patient specimens (n = 48, 76 positive results), there was excellent correlation between the UPLC-MS-MS results and the previous method. The best least-squares fit had an equation of y = 1.0708x + 1.6521, r(2) = 0.9822. This UPLC-MS-MS method is suitable for the quantification of benzodiazepines and hypnotics in serum and plasma, and offers fast, reliable and sensitive results.

Blonanserin is a novel atypical antipsychotic with highly selective receptor antagonist activity to dopamine D₂ and 5-HT(2A). N-desethyl blonanserin (blonanserin C) is its major active metabolite in human plasma. Herein we report a new highly sensitive, selective, and rapid liquid chromatography-tandem mass spectrometry method to determine blonanserin and blonanserin C simultaneously in human plasma and urine, with N-desethyl-chlor-blonanserin (blonanserin D) as internal standard (IS). Blonanserin and blonanserin C were extracted from aliquots of plasma and urine with ethyl acetate and dichloromethane (4:1) as the solvent and chromatographic separation was performed using an Agilent Eclipse Plus C₁₈ column. The mobile phase was composed of: acetonitrile and ammonium formate-formic acid buffer containing 5mM ammonium formate and 0.1% formic acid (87:13, v/v). To quantify blonanserin, blonanserin C, and blonanserin D, respectively, multiple reaction monitoring (MRM) transition of m/z 368.2→297.2, m/z 340.2→297.1, and m/z 356.2→313.3 was performed in positive mode. The analysis time was about 5.5 min. The calibration curve was linear in the concentration range of 0.01-2 ng/ml. The lower limit of quantification reached 0.01 ng/ml. The intra and inter-day precision and relative errors were less than 8.0% and 6.6% for three QC levels in plasma and urine. The current LC-MS/MS method was validated as simple, sensitive, and accurate and has been successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A simple, sensitive and high-throughput ultra high-performance liquid chromatography electrospray ionization mass spectrometry (U-HPLC-ESI-MS/MS) method has been developed and validated for the determination of ranolazine in human plasma. Propafenone was employed as the internal standard (I.S.). The analytes were chromatographically separated on a BEH C(18) column (50 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of acetonitrile and aqueous ammonium acetate solution (0.06% formic acid, 7.5 mmol L(-1) ammonium acetate, 40:60, v/v). Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 428.3→279.3 for ranolazine and m/z 342.4→115.9 for propafenone. The assay was linear over the concentration range 1-3000 ng mL(-1), with correlation coefficients ≥0.997. The intra- and inter-day coefficients of variation were less than 8.9% in terms of relative standard deviation and accuracy ranged from 93.0 to 108.9% at all quality control levels. The validated method was a simple sample preparation procedure and short run-time (<2.0 min) method, which was successfully applied to a phase I pharmacokinetic study of ranolazine in Chinese healthy volunteers., (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 rapid, sensitive and reliable LC-MS/MS method was developed and validated for the simultaneous determination of ranolazine and its three metabolites, CVT-2514, CVT-2738, and CVT-4786, in human plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on a Gemini C(18) column (50 mm × 2.0 mm, 5 μm) using methanol: 5 mM ammonium acetate as the mobile phase with gradient elution. Mass detection was carried out by electrospray ionization in both positive and negative ion multiple reaction monitoring (MRM) modes. The calibration curves were linear over a concentration range of 4-2000 ng/mL for ranolazine, 4-1000 ng/mL for CVT-2514 and CVT-2738 and 8-1000 ng/mL for CVT-4786. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the simultaneous estimation of ranolazine and its three metabolites in human plasma from a clinical pharmacokinetics study., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of G-856 in human plasma to support clinical development. The method consisted of a solid phase extraction for sample preparation and LC-MS/MS analysis in the positive ion mode using TurboIonSpray for analysis. d₈-G-856 was used as the internal standard. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 5.00-2000 pg/mL for G-856. There were no significant endogenous interference components in the multiple lots of blank human plasma tested. The accuracy (%Acc) at the lower limit of quantitation (LLOQ) was 98.2% with a precision (%CV) of 5.38%. For quality control samples at 15.0, 800, and 1600 pg/mL, the inter-day %CV was ≤ 5.03%. Inter-day %Acc ranged from 96.9 to 99.3%. G-856 was stable in human plasma for 184 days at -20 °C and -70 °C storage. G-856 was stable in human plasma at room temperature for up to 16 h and through four freeze/thaw cycles. This validated LC-MS/MS method for determination of G-856 was used to support Phase 1 clinical studies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid-liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1-500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C(max); 616 ± 73 ng/mL) was achieved at 0.5 h (T(max)) and area under curve (AUC(0-24)) was 1841 ± 308 ng h/mL. The half-life (t(1/2)) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials., (Copyright © 2011 John Wiley & Sons, Ltd.)

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 μm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple-reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002-1 μg/mL. The intra- and inter-assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 μL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats., (Copyright © 2011 John Wiley & Sons, Ltd.)