Mohamed W. Attwa

Identification and characterization of in vitro phase I and reactive metabolites of masitinib using a LC-MS/MS method: bioactivation pathway elucidation

Masitinib is a selective tyrosine kinase inhibitor (TKI). It is currently registered in Europe for the treatment of mast cell tumors in dogs. The current study reports the identification and characterization of fourteen phase I metabolites of masitinib by reversed phase liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS). Phase I metabolic reactions were reduction, demethylation, hydroxylation, oxidation and N-oxide formation. Structures of the proposed phase I metabolites showed high lability to form reactive metabolites. So incubation was performed in the presence of 1.0 mM GSH or 1.0 mM KCN to check for reactive metabolites. No GSH adduct was found, while eight cyano adduct structures were determined based on full MS scan and MS2 scan data for each metabolite. Interestingly, a literature review showed no previous studies have been made on the in vitro metabolism of masitinib or detailed structural identification of the formed metabolites.

Validated LC-MS/MS Method for the Quantification of Ponatinib in Plasma: Application to Metabolic Stability.

In the current work, a rapid, specific, sensitive and validated liquid chromatography tandem mass-spectrometric method was developed for the quantification of ponatinib (PNT) in human plasma and rat liver microsomes (RLMs) with its application to metabolic stability. Chromatographic separation of PNT and vandetanib (IS) were accomplished on Agilent eclipse plus C18 analytical column (50 mm × 2.1 mm, 1.8 μm particle size) maintained at 21±2°C. Flow rate was 0.25 mLmin-1 with run time of 4 min. Mobile phase consisted of solvent A (10 mM ammonium formate, pH adjusted to 4.1 with formic acid) and solvent B (acetonitrile). Ions were generated by electrospray (ESI) and multiple reaction monitoring (MRM) was used as basis for quantification. The results revealed a linear calibration curve in the range of 5–400 ngmL-1 (r2 ≥ 0.9998) with lower limit of quantification (LOQ) and lower limit of detection (LOD) of 4.66 and 1.53 ngmL-1 in plasma, 4.19 and 1.38 ngmL-1 in RLMs. The intra- and inter-day precision and accuracy in plasma ranged from1.06 to 2.54% and -1.48 to -0.17, respectively. Whereas in RLMs ranged from 0.97 to 2.31% and -1.65 to -0.3%. The developed procedure was applied for quantification of PNT in human plasma and RLMs for study metabolic stability of PNT. PNT disappeared rapidly in the 1st 10 minutes of RLM incubation and the disappearance plateaued out for the rest of the incubation. In vitro half-life (t1/2) was 6.26 min and intrinsic clearance (CLin) was 15.182± 0.477.

Detection and characterization of ponatinib reactive metabolites by liquid chromatography tandem mass spectrometry and elucidation of bioactivation pathways

Ponatinib (PNT), as a multi-targeted tyrosine kinase inhibitor, is active against T315I and other BCR-ABL mutants. PNT is registered in the U.S. and EU under the trade name of Iclusig®. The current study reports the identification and characterization of in vitro metabolites of PNT, which were produced by its incubation with rat liver microsomes (RLMs). PNT and its metabolites were extracted from the incubation mixture by the protein precipitation procedure and the supernatants were injected into high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) equipment. Reversed phase liquid chromatography resolved seven in vitro PNT metabolites. Each metabolite displayed one or more metabolic reaction pathways including N-demethylation, N-oxide formation, oxidation, reduction and hydroxylation. Structures of the PNT metabolites showed high liability to form reactive metabolites. Since bioactivation is often speculated to be responsible for observed idiosyncratic toxicities including hepatotoxicity, incubation of PNT with RLMs was carried out in the presence of 1.0 mM GSH or 1.0 mM KCN to check its reactive metabolites. No GSH adduct was found while four cyano adduct metabolites were determined and their structures were proposed based on the mass scan and product ion data for each metabolite.

Microwave-Assisted Solution-Phase Synthesis and DART-Mass Spectrometric Monitoring of a Combinatorial Library of Indolin-2,3-dione Schiff Bases with Potential Antimycobacterial Activity

A combinatorial library composed of eleven hydrazides A-K and eleven indolin-1,2-dione derivatives 1-11 has been designed to formally generate sublibraries of 22 mixtures, M1-M22 comprising of 121 Schiff bases, A-K(1-11). The designed library has been synthesized by the solution-phase method and microwave-assisted synthetic techniques. The formation of individual compounds of each mixture was confirmed by Direct Analysis in Real Time (DART) as ionization technique connected to an Ion Trap as a mass detector. The synthesized mixtures were evaluated for their antimycobacterial activity against four Mycobacterium strains; M. intercellulari, M. xenopi, M. cheleneoi and M. smegmatis. Variable antimycobacterial activity was revealed with the investigated mixtures and maximum activity was shown by M8, M10, M11, and M15 with MIC values of 1.5, 3.1, 6.2 and 0.09 μg/mL, respectively. Application of the indexed method of analysis on these active mixtures revealed that compounds D8, D10 and D11 may contribute to the activity of the tested mixtures.

An LC‐MS/MS method for rapid and sensitive high‐throughput simultaneous determination of various protein kinase inhibitors in human plasma

A reliable, high-throughput and sensitive LC-MS/MS procedure was developed and validated for the determination of five tyrosine kinase inhibitors in human plasma. Following their extraction from human plasma, samples were eluted on a RP Luna®-PFP 100 Å column using a mobile phase system composed of acetonitrile and 0.01 m ammonium formate in water (pH ~4.1) with a ratio of (50:50, v/v) flowing at 0.3 mL min-1 . The mass spectrometer was operating with electrospray ionization in the positive ion multiple reaction monitoring mode. The proposed methodology resulted in linear calibration plots with correlation coefficients values of r2  = 0.9995-0.9999 from concentration ranges of 2.5-100 ng mL-1 for imatinib, 5.0-100 ng mL-1 for sorafenib, tofacitinib and afatinib, and 1.0-100 ng mL-1 for cabozantinib. The procedure was validated in terms of its specificity, limit of detection (0.32-1.71 ng mL-1 ), lower limit of quantification (0.97-5.07 ng mL-1 ), intra- and inter assay accuracy (-3.83 to +2.40%) and precision (<3.37%), matrix effect and recovery and stability. Our results demonstrated that the proposed method is highly reliable for routine quantification of the investigated tyrosine kinase inhibitors in human plasma and can be efficiently applied in the rapid and sensitive analysis of their clinical samples.

LC-ESI-MS/MS reveals the formation of reactive intermediates in brigatinib metabolism: elucidation of bioactivation pathways

Brigatinib (BGB) is a newly approved anaplastic lymphoma kinase (ALK) inhibitor. On April 28, 2017, BGB was approved by the U.S. FDA for the treatment of metastatic anaplastic lymphoma kinase-positive non-small cell lung cancer. The toxicity profile of BGB includes nausea, fatigue, diarrhea, elevated lipase, dyspnoea, hypertension, hypoxia, pneumonia, elevated amylase, pulmonary embolism, elevated ALT, hyponatraemia and hypophosphatemia. Using LC-MS/MS, we investigated the in vitro phase I metabolism of for BGB in rat liver microsomes (RLMs). In the in vitro metabolism of BGB, iminium reactive intermediates were trapped by potassium cyanide forming a stable complex that can be characterized by LC-MS/MS. Four BGB in vitro phase I metabolites were identified. In vitro phase I metabolic pathways were N-dealkylation, α hydroxylation and α oxidation. Additionally, three iminium reactive metabolites were found and the bioactivation mechanisms were proposed. A piperidine ring was found to be responsible for BGB bioactivation. The presence of these three reactive metabolites may be the main reason for BGB side effects. A literature review showed no previous article reported the in vitro phase I metabolism study of BGB or structural identification of the formed reactive metabolites.

A highly efficient and sensitive LC-MS/MS method for the determination of afatinib in human plasma: application to a metabolic stability study

Afatinib (AFT) is a new tyrosine kinase inhibitor approved for the treatment of nonsmall cell lung cancer. In the present study, a simple, specific, rapid and sensitive liquid chromatography tandem mass-spectrometric method for the quantification of AFT in human plasma, was developed and validated. Chromatographic separation of the analytes was accomplished on a reversed-phase Luna(®) -PFP 100 Å column (50 × 2.0 mm; 3.0 μm) maintained at ambient temperature. Isocratic elution was carried out using acetonitrile-water (40:60, v/v) containing 10 mm ammonium formate buffer (pH 4.5) adjusted with formic acid at a flow rate of 0.4 mL min(-1) . The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring mode. The method yields a linear calibration plot (r(2)  = 0.9997) from a quantification range of 0.5-500 ng mL(-1) with the lower limit of quantification and lower limit of detection of 1.29 and 0.42 ng mL(-1) , respectively. The intra- and inter-day precision and accuracy were estimated and found to be in the ranges of 1.53-4.11% for precision and -2.80-0.38% for accuracy. Finally, quantification of afatinib in a metabolic stability study in rat liver microsomes was achieved through the proposed method. Copyright

Induced in-source fragmentation pattern of certain novel (1Z,2E)-N-(aryl)propanehydrazonoyl chlorides by electrospray mass spectrometry (ESI-MS/MS)

BackgroundCollision induced dissociation (CID) in the triple quadrupole mass spectrometer system (QQQ) typically yields more abundant fragment ions than those produced with resonance excitation in the presence of helium gas in the ion trap mass spectrometer system (IT). Detailed product ion spectra can be obtained from one stage MS2 scan using the QQQ. In contrast, generating the same number of fragment ions in the ion trap requires multiple stages of fragmentation (MSn) using CID via in-trap resonance excitation with the associated time penalties and drop in sensitivity.ResultsThe use of in-source fragmentation with electrospray ionization (ESI) followed by product ion scan (MS2) in a triple quadrupole mass spectrometer system, was demonstrated. This process enhances the qualitative power of tandem mass spectrometry to simulate the MS3 of ion trap for a comprehensive study of fragmentation mechanisms. A five pharmacologically significant (1Z, 2E)-N-arylpropanehydrazonoyl chlorides (3a-e) were chosen as model compounds for this study. In this work, detailed fragmentation pathways were elucidated by further dissociation of each fragment ion in the ion spectrum, essentially, by incorporating fragmentor voltage induced dissociation (in-source fragmentation) and isolation of fragments in a quadrupole cell Q1. Subsequently, CID occurs in cell, Q2, and fragment ions are analyzed in Q3 operated in product ion mode this process can be referred to as pseudo-MS3 scan mode.ConclusionsThis approach allowed unambiguous assignment of all fragment ions using tandem mass spectrometer and provided adequate sensitivity and selectivity. It is beneficial for structure determination of unknown trace components. The data presented in this paper provide useful information on the effect of different substituents on the ionization/fragmentation processes and can be used in the characterization of this important class of compounds.

Investigation of metabolic stability of the novel ALK inhibitor brigatinib by liquid chromatography tandem mass spectrometry

Brigatinib (BGB) belongs to a class of drugs called ALK inhibitor. On April 28, 2017, BGB has been approved by U.S. FDA for use in metastatic ALK-positive NSCLC. A fast, specific, sensitive and validated LC-MS/MS method was developed for the quantification of BGB in human plasma matrix. This method was applied successfully to study metabolic stability of BGB. Reversed phase (C18 column) and isocratic binary mobile phase (55% 0.1% formic acid: 45% ACN) were used for chromatographic separation of BGB and ponatinib (IS). The flow rate, total run time and injection volume were fixed at 0.2 mL/min, 4 min, 5 μL respectively. ESI source was utilized for ions formation, while multiple reaction monitoring (MRM) mode was used for ion analysis. In human plasma matrix, the Linearity range of the calibration curve was 5-500 ng/mL (r2 ≥ 0.9982). LOQ and LOD were found to be 1.89 and 5.72 ng/mL. The precision and accuracy for the intra-day and inter-day were 0.45 to 1.85% and 97.37 to 104.85%. In vitro half-life (t1/2) and intrinsic clearance (CLint) were equal to 12.0 min and 13.1 ± 0.15 mL/min/kg respectively. The quantification of BGB in human plasma or its metabolic stability has not been studied as seen in literature review.

LC-MS/MS reveals the formation of aldehydes and iminium reactive intermediates in foretinib metabolism: phase I metabolic profiling

Foretinib (GSK1363089) is an inhibitor of multiple receptor tyrosine kinases including MET and VEGFR, with the potential for treatment of solid tumors. In this study, we investigated the in vitro metabolic pathways for foretinib in rat liver microsomes using LC-MS/MS. Methoxylamine and potassium cyanide were used as trapping agents for aldehyde and iminium reactive intermediates, respectively, of foretinib to form a stable complex that can be identified by LC-MS/MS. Six foretinib phase I metabolites were characterized. The phase I metabolic pathways were oxidation, defluorination, reduction and hydroxylation. Additionally, four potential reactive metabolites, two aldehydes and two iminium ions, were found and the bioactivation pathways were proposed. Reporting the in vitro and reactive metabolites of foretinib is very crucial in the development stage. A literature review showed that no previous articles have provided an in vitro metabolism study of foretinib or detailed structural identification of the formed reactive metabolites.