- Research article
- Open Access
Characterization of in vivo metabolites in rat urine following an oral dose of masitinib by liquid chromatography tandem mass spectrometry
© The Author(s) 2018
- Received: 21 August 2017
- Accepted: 4 May 2018
- Published: 15 May 2018
- In vivo metabolism
- Sprague–Dawley rats
- Phase II glucuronide conjugates
Cancer became a major reason of death . More than four millions new cancer cases reported in developed countries [2, 3]. Molecular targeting strategies were used to treat distributed cancer depending on identifying the tumor suppressors and oncogenes involved in the progress of human cancers . Tyrosine kinase inhibitors (TKIs) (e.g. masitinib) are compounds that target tyrosine kinases enzymes, which are responsible for the activation of numerous proteins in a number of cell signaling pathways. They initiate or stop many functions inside living cells . Blocking the selected activation of these proteins has been shown to have therapeutic benefits in cancer diseases and central nervous system disorders mast cells and macrophages [6, 7]. Tyrosine kinase inhibitors (TKIs) are considered a very important class of targeted therapy .
Drug metabolism research is an integral part of the drug discovery process and is very often the factor that determines the success of a given drug to be marketed and clinically used . Drug metabolism research is generally conducted using in vitro and/or in vivo techniques. In vitro techniques involve the incubation of drugs with different types of in vitro preparations (e.g. liver microsomes, hepatocytes) isolated from rats and subsequent sample processing and analysis using spectroscopic techniques [14, 15]. In vivo techniques involve the administration of a single dose of the drug to rat, and the subsequent collection of urine that contain the drugs and their potential metabolites. In this work, we focused in the in vivo phase I metabolites and in vivo phase II MST metabolites identification using LC–MS/MS . All measurements were done using Agilent LC–MS/MS system that consisted of LC (Agilent HPLC 1200) coupled to MS/MS detector (6410 QqQ MS) through an electrospray ionization source (Agilent Technologies, USA) .
MST chemical structure contains cyclic tertiary amine. Phase I metabolism of cyclic tertiary amines produces metabolites of oxidative products including N-dealkylation, ring hydroxylation, α-carbonyl formation, N-oxygenation, and ring opening metabolites that can be formed through iminium ion intermediates [18, 19].
List of materials and chemicals
LC Labs (USA)
Eurostar Scientific Ltd. (UK)
Ammonium formate, HPLC grade acetonitrile (ACN), Dimethyl Sulfoxide (DMSO), Polyethylene glycol 300 (PEG 300) and formic acid
Water (HPLC grade)
Milli-Q plus purification system (USA)
Animal Care Center, College of Pharmacy, King Saud University (Saudi Arabia)
In vivo metabolism of MST in Sprague–Dawley Rats
Rat dosing protocol
So the dose for each rat was 33.3 mg/kg. All rats except one were given a single dose of MST. All MST doses were administered by oral gavage. Urine draining into the special urine compartments fitted to the metabolism cages were collected prior to drug dosing as blank control reference and at 6, 12, 18, 24, 48, 72 and 96 h following MST dosing. Urine samples taken from all metabolism cages were pooled together, labeled, and stored at (− 20 °C).
Urine samples were thawed to room temperature and filtered over 0.45 µm syringe filters. Liquid liquid extraction (LLC) was used to extract MST and its related metabolites. Equal volume of ice cold acetonitrile (ACN) was added to each sample then vigorously shaken by vortexing for 1 min. Phase separation [23, 24] between an aqueous sample and a water-miscible solvent (ACN) into two layers achieved by using ice cold ACN that was added to urine and the mixture was stored at 4 °C overnight . Low temperature leads to phase separation of ACN/urine mixture. The pH of urine and the nature of urine matrix which contains high concentration of salt participated in phase separation . As we did not want to miss any MST-related metabolites, both layers were removed and evaporated to dryness under stream of nitrogen. The dried extracts were reconstituted in 1 mL of mobile phase and transferred to 1.5 mL HPLC vials for LC–MS/MS analysis. Control urine samples obtained from rats prior to drug dosing were prepared in the exact way described for each method of sample purification.
Adjusted parameters of the supposed LC–MS/MS methodology
Parameters of LC
Parameters of MS/MS
Agilent 6410 QQQ
Gradient mobile phase
A: H2O (10 mM Ammonium formate,
Drying gas: N2 gas
Flow rate (12 L/min)
Pressure (55 psi)
Flow rate: 0.2 mL/min
Run time: 45 min
Injection volume: 20 µL
Agilent eclipse plus C18 column
ESI temperature: 350 °C
Capillary voltage: 4000 V
High purity N2
Mass scan and product ion (PI)
MST and its related in vivo phase I and phase II metabolites
Fragmentor voltage: 130 V
Post time (15 min)
Collision energy of 20 eV
Identification of in vivo MST metabolites
MST-related metabolites were concentrated in the ACN layer while endogenous urine components and polar metabolites (e.g. glucuronide conjugates) were found in the aqueous layer. Extracted ion chromatograms for the expected metabolites were used to find metabolites in the total ion chromatogram of both organic and aqueous layers. PI studies were for the suspected compounds and results were interpreted and compared with the PI of MST. Mass scan and PI scan modes of the triple quadrupole mass analyzer were used for detection of in vivo phase I and phase II MST metabolites. PI mass spectra were used to propose the metabolite chemical structure by reconstructing the marker daughter ions.
Identification of in vivo phase I metabolic pathways of MST
In vivo phase I MST metabolites
[M + H]+
In vivo phase I metabolic reaction
Carbonyl group reduction
N-demethylation and Hydroxylation of pyridine ring
N-demethylation and Hydroxylation of N-methyl piperazine
Benzyl oxidation to carboxylic acid
Pyridine ring hydroxylation and N-methyl piperazine oxidation
511,482 399, 247
Oxidation and Hydroxylation of N-methyl piperazine
497.2, 415, 396.8
Pyridine ring hydroxylation
497, 399, 415, 217
Pyridine ring N-oxidation
497, 399, 415, 217
428, 415, 400, 381.3, 98.1,
Piperazine ring N-oxidation
488, 402, 123
Pyridine ring hydroxylation and piperazine ring hydroxylation
415, 381, 123
Piperazine ring hydroxylation and benzyl hydroxylation
Oxidative cleavage of N-methyl piperazine ring to carboxylic acid
N-oxide formation of pyridine and piperazine ring and Benzylic hydroxylation 
Phenyl hydroxylation and oxidative deamination
285, 271, 164, 111
Benzyl hydroxylation and oxidative deamination
MST excretion of in rat urine
M1 in vivo phase I metabolite
The major metabolic pathway for MST is N-demethyalation. M1 was detected at m/z 485 in mass scan spectrum.
M2, M3 and M4 in vivo phase I metabolite
M2, M3 and M4 were detected at m/z 501 at different retention times in mass scan spectrum of organic urine extract. PI scan for the three metabolites gave different daughter ions. In the case of M2, parent ion at m/z 501 was fragmented to one ion at m/z 401. The daughter ion at m/z 401 supposed that there is no change in the methyl piperazine group. The metabolic pathway for M2 metabolite was supposed to be the reduction of the carbonyl group.
MO1 to MO6 in vivo phase I metabolite
Oxidized MST metabolite (M + O) was detected at m/z 515 in mass scan spectrum at different retention times. Fragmentation of parent ions at m/z 515 gave different daughter ions as shown in the Table 3. The structure of each metabolite was supposed The metabolic pathway for MO metabolites was supposed to be either by hydroxylation or N-oxidation of MST .
M5, M6 and M7 in vivo phase I metabolite
M8, M9 and M10 in vivo phase I metabolite
M11 in vivo phase I metabolite
M11 was detected at m/z 547 in mass scan spectrum of the urine organic extract. PI chromatogram of urine organic extract at m/z 547 showed one peak at 30.72 min. PI scan for M11 at m/z 547 gave daughter ions at m/z 511. Metabolic reactions for M11 metabolite were supposed to be hydroxylation of benzylic carbon, oxidation of pyridine nitrogen and oxidation of piperazine nitrogen.
In vivo phase I oxidative deamination metabolic pathway (MA1, MA2 and MA3)
The loss of the piperazine moiety by oxidative deamination and rapid further oxidation of the intermediate aldehyde to a carboxylic acid metabolite were observed for MA1, MA2 and MA3 in the aqueous layer of the urine/ACN mixture. Fragmentation of parent ions at m/z 431 and at m/z 447 gave different daughter ions. The structure of each metabolite was supposed.
Identification of in vivo phase II metabolic pathways of MST
In vivo phase II MST metabolites
Retention time (min)
Phase II metabolic pathway
Direct N-conjugation with glucuronic acid
N-demethylation and direct N-conjugation with glucuronic acid
Glucuronidation of hydroxy MST at N-methyl piperazine ring
Glucuronidation of hydroxy MST at benzyl carbon
MG1 in vivo phase II metabolite
MG2 in vivo phase II metabolite
MG3 and MG4 in vivo Phase II metabolites
AK, SA, HD and MA established the experiment design. Practical work was performed by MA. Data were analyzed by AK, HD, SA and MA. HD and MA wrote the first draft of the manuscript. AK and SA contributed in editing the manuscript. AK, SA and HD supervised the research work. All authors read and approved the final manuscript.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at the King Saud University for funding this work through the Research Group Project No. RGP-322.
The authors declare that they have no competing interests.
All data supporting the results in this article can be found in the manuscript or the Additional file.
Ethics approval and consent to participate
Animal Care Center guidelines of Pharmacy College at King Saud Univesity were applied for Rats’ maintenance. The Local Animal Care and Use Committee at KSU approved these guidelines.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al (2008) Cancer statistics, 2008. CA Cancer J Clin 58(2):71–96View ArticleGoogle Scholar
- Sinha R, El-Bayoumy K (2004) Apoptosis is a critical cellular event in cancer chemoprevention and chemotherapy by selenium compounds. Curr Cancer Drug Targets 4(1):13–28View ArticleGoogle Scholar
- Cozzi P, Mongelli N, Suarato A (2004) Recent anticancer cytotoxic agents. Curr Med Chem Anti Cancer Agents 4(2):93–121View ArticleGoogle Scholar
- Barinaga M (1997) From bench top to bedside. Science 278(5340):1036–1039View ArticleGoogle Scholar
- Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103(2):211–225View ArticleGoogle Scholar
- Özvegy-Laczka C, Cserepes J, Elkind NB, Sarkadi B (2005) Tyrosine kinase inhibitor resistance in cancer: role of ABC multidrug transporters. Drug Resist Updates 8(1):15–26View ArticleGoogle Scholar
- Steeghs N, Nortier JW, Gelderblom H (2007) Small molecule tyrosine kinase inhibitors in the treatment of solid tumors: an update of recent developments. Ann Surg Oncol 14(2):942–953View ArticleGoogle Scholar
- Natoli C, Perrucci B, Perrotti F, Falchi L, Iacobelli S (2010) Tyrosine kinase inhibitors. Curr Cancer Drug Targets 10(5):462–483View ArticleGoogle Scholar
- Daly M, Sheppard S, Cohen N, Nabity M, Moussy A, Hermine O et al (2011) Safety of masitinib mesylate in healthy cats. J Vet Intern Med 25(2):297–302View ArticleGoogle Scholar
- Marech I, Patruno R, Zizzo N, Gadaleta C, Introna M, Zito AF et al (2014) Masitinib (AB1010), from canine tumor model to human clinical development: where we are? Crit Rev Oncol Hematol 91(1):98–111View ArticleGoogle Scholar
- Dubreuil P, Letard S, Ciufolini M, Gros L, Humbert M, Castéran N et al (2009) Masitinib (AB1010), a potent and selective tyrosine kinase inhibitor targeting KIT. PLoS ONE 4(9):e7258View ArticleGoogle Scholar
- Lortholary O, Chandesris MO, Livideanu CB, Paul C, Guillet G, Jassem E et al (2017) Masitinib for treatment of severely symptomatic indolent systemic mastocytosis: a randomised, placebo-controlled, phase 3 study. Lancet 389(10069):612–620View ArticleGoogle Scholar
- Kumar GN, Surapaneni S (2001) Role of drug metabolism in drug discovery and development. Med Res Rev 21(5):397–411View ArticleGoogle Scholar
- Kadi AA, Al-Shakliah NS, Yin W, Rahman AM (2017) In vitro investigation of metabolic profiling of newly developed topoisomerase inhibitors (ethyl fluorescein hydrazones, EtFLHs) in RLMs by LC–MS/MS. J Chromatogr B 1054:93–104View ArticleGoogle Scholar
- Rahman AM, Al-Shakliah NS, Yin W, Kadi AA (2017) In vitro Investigation of Metabolic Profiling of a Potent Topoisomerase Inhibitors Fluorescein Hydrazones (FLHs) in RLMs by LC-MS/MS. J Chromatogr B 1054:27–35View ArticleGoogle Scholar
- Attwa MW, Kadi AA, Darwish HW, Alrabiah H (2018) LC-MS/MS reveals the formation of reactive ortho-quinone and iminium intermediates in saracatinib metabolism: phase I metabolic profiling. Clin Chim Acta 482:84–94View ArticleGoogle Scholar
- Attwa MW, Kadi AA, Darwish HW, Amer SM, Alrabiah H (2018) A reliable and stable method for the determination of foretinib in human plasma by LC-MS/MS: application to metabolic stability investigation and excretion rate. Eur J Mass Spectrom 1469066718768327. https://doi.org/10.1177/1469066718768327
- Masic LP (2011) Role of cyclic tertiary amine bioactivation to reactive iminium species: structure toxicity relationship. Curr Drug Metab 12(1):35–50View ArticleGoogle Scholar
- Kadi AA, Attwa M, Darwish HW (2018) LC-ESI-MS/MS reveals the formation of reactive intermediates in brigatinib metabolism: elucidation of bioactivation pathways. RSC Adv 8(3):1182–1190View ArticleGoogle Scholar
- Shin J-W, Seol I-C, Son C-G (2010) Interpretation of animal dose and human equivalent dose for drug development. J Korean Med 31(3):1–7Google Scholar
- Nair AB, Jacob S (2016) A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 7(2):27–31View ArticleGoogle Scholar
- Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J Off Publ Fed Am Soc Exp Biol 22(3):659–661 (Epub 2007/10/19) Google Scholar
- Varaprath S, Salyers KL, Plotzke KP, Nanavati S (1999) Identification of metabolites of octamethylcyclotetrasiloxane (D(4)) in rat urine. Drug Metab Dispos Biol Fate Chem 27(11):1267–1273Google Scholar
- Bylda C, Thiele R, Kobold U, Volmer DA (2014) Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC–MS/MS. Analyst 139(10):2265–2276View ArticleGoogle Scholar
- Yoshida M, Akane A (1999) Subzero-temperature liquid − liquid extraction of benzodiazepines for high-performance liquid chromatography. Anal Chem 71(9):1918–1921View ArticleGoogle Scholar
- Valente IM, Gonçalves LM, Rodrigues JA (2013) Another glimpse over the salting-out assisted liquid–liquid extraction in acetonitrile/water mixtures. J Chromatogr A 1308:58–62View ArticleGoogle Scholar
- Amer S, Kadi AA, Darwish HW, Attwa MW (2017) Identification and characterization of in vitro phase I and reactive metabolites of masitinib using a LC-MS/MS method: bioactivation pathway elucidation. RSC Adv 7(8):4479–4491View ArticleGoogle Scholar
- Amer SM, Kadi AA, Darwish HW, Attwa MW (2017) LC–MS/MS method for the quantification of masitinib in RLMs matrix and rat urine: application to metabolic stability and excretion rate. Chem Cent J 11(1):136View ArticleGoogle Scholar