Pseudo-MS3 Approach Using Electrospray Mass Spectrometry (ESI-MS/MS) to Characterize Certain (2E)-2-[3-(1H-Imidazol-1-yl)-1-phenylpropylidene]hydrazinecarboxamide Derivatives

Abdelhameed, Ali S.; Kadi, Adnan A.; Attia, Mohamed I.; Angawi, Rihab F.; Attwa, Mohamed W.; Darwish, Hany W.

doi:https://doi.org/10.1155/2014/386301

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Research Article | Open Access

Volume 2014 | Article ID 386301 | https://doi.org/10.1155/2014/386301

Pseudo-MS³ Approach Using Electrospray Mass Spectrometry (ESI-MS/MS) to Characterize Certain (2E)-2-[3-(1H-Imidazol-1-yl)-1-phenylpropylidene]hydrazinecarboxamide Derivatives

Ali S. Abdelhameed,¹Adnan A. Kadi,¹Mohamed I. Attia,^1,2Rihab F. Angawi,³Mohamed W. Attwa,¹and Hany W. Darwish^1,4

Academic Editor: Irene Panderi

Received23 Oct 2013

Accepted02 Dec 2013

Published05 Jan 2014

Abstract

An approach for the use of in-source fragmentation with electrospray ionization followed by product ion scan in a triple quadrupole mass spectrometer system is described. This approach is based on the elucidation of the various fragmentation pathways by further dissociation of each fragment ion in the ion spectrum. This can be achieved predominately, by combining fragmentor voltage induced dissociation (in-source fragmentation) with subsequent collision-induced dissociation; this process can be referred to as pseudo-MS³ scan mode. This technique permitted unambiguous assignment and provided sufficient sensitivity and specificity. It is advantageous for structure elucidation of unknown compounds. We investigate the possibility of using in-source fragmentation with the diverse novel chemical entities encompassing different substituents. This process was intended to improve the qualitative capability of tandem mass spectrometry simulating the MS³ of ion trap for studying fragmentation mechanisms. The approach is to implement the investigated technique as a well established tool for the characterization of new pharmacologically important chemical entities. The data presented in this paper provided useful information on the effect of different substituents on the ionization/fragmentation processes and can be used in the characterization of (2E)-2-[3-(1H-imidazol-1-yl)-1-phenylpropylidene]-hydrazinecarboxamide derivatives 3a–h.

1. Introduction

It has always been known that the use of a triple quadrupole mass spectrometer system (QqQ) for quantitative analysis is superior to that of any ion trap mass spectrometry systems. This is due to the fact that one can always utilize the collision-induced dissociation (CID) in the QqQ which usually produces more abundant fragment ions than those shown in case of resonance excitation in the ion trap (IT) [1]. However, the opposite has always remained correct with regard to qualitative analysis. On the other hand, electrospray ionization mass spectrometry (ESI-MS)—as a robust technique for quantification studies—has been excessively applied for the analysis of different types of compounds such as oligosaccharides, glycoproteins, oligonucleotides, and drug metabolites [2–7]. In electrospray ionization (ESI), ions fragmentation may take place inside the ion source (in-source fragmentation) prior to reaching the mass analyzer. Although this method of in-source fragmentation has low specificity, as most of the ions in the ion source are fragmented simultaneously, it has been reported by different scientists [8–17]. Elaborate ion spectra can be acquired from a single stage MS² scan using QqQ. By contrast, generating a similar number of fragment ions in IT demands various fragmentation stages () utilizing CID through the in-trap resonance excitation, leaving it to be time consuming and less sensitive. Additionally, the low mass cutoff in product ion scans represents another drawback of IT. This is due to the fact that both precursor ion and the lowest m/z fragment ion have to be stable simultaneously inside the IT [1, 18]. In this study, we use in-source fragmentation (ISF) prior to product ion (MS²) scan to elucidate the structure of certain (2E)-2-[3-(1H-imidazol-1-yl)-1-phenylpropylidene]-hydrazinecarboxamide derivatives 3a–h using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The use of this approach was designed to boost the structure elucidation power of QqQ to mimic the MS³ of ion trap avoiding the IT drawbacks mentioned above.

Compounds 3a–h are considered as hybrid structures incorporating both imidazole moiety, a pharmacophore of alkylimidazole anticonvulsants (nafimidone (A) and denzimol (B), Figure 1) and arylsemicarbazone moiety, a pharmacophore of arylsemicarbazone anticonvulsants with the general structural formula C (Figure 1) [19–22]. The test compounds 3a–h displayed anticonvulsant activity in the preliminary anticonvulsant screening assays [23]. Accordingly, in the light of interesting structural and biological results the situation definitely urges some additional research in their analyses.

2. Materials and Methods

Unless otherwise indicated, all chemicals were purchased from Sigma (St. Louis, MO).

2.1. Chemistry

2.1.1. General Procedure for Preparation of the Ketones 1a–c

The appropriate acetophenone (200 mmol), dimethylamine hydrochloride (270 mmol), and paraformaldehyde (90 mmol) were heated to reflux in absolute ethanol (35 mL) in the presence of catalytical amount of concentrated hydrochloric acid (0.5 mL). Reflux of the reaction mixture was continued under stirring for two hours, the mixture was cooled, and acetone (200 mL) was added. The formed Mannich base hydrochlorides were precipitated, filtered off, and dried. Subsequently, Mannich base hydrochlorides (100 mmol) were dissolved in water (100 mL) and imidazole (200 mmol) was added. The reaction mixture was heated to reflux for five hours, cooled and the precipitated solids were collected by filtration to give ketones 1a–c [24–26] which were pure enough to be used in the next step.

2.1.2. General Procedure for the Synthesis of Arylsemicarbazides 2a–e

A solution of the appropriate aniline derivative 1a–e (20 mmol) in CH₂Cl₂ (20 mL) was added dropwise to a stirred solution of ethyl chloroformate (10 mmol) in CH₂Cl₂ (5 mL). The reaction mixture was stirred at room temperature for 0.5 hr (2a and 2c–e) and 24 hrs (2b). After completion of the reaction, the reaction mixture was filtered and the filtrate was washed with 1 N HCl, dried (Na₂SO₄), and evaporated under reduced pressure to give the respective crude carbamates. A mixture containing the formed carbamates (10 mmol) and hydrazine hydrate (10 mL) was heated to reflux for 1 hr (2e), 2 hrs (2d), and 24 hrs (2a–c). The reaction mixture was cooled and filtered to yield the corresponding crude arylsemicarbazides 2a–e [27]. The crude products were used as such for the next reactions.

2.1.3. General Procedure for the Synthesis of the Test Compounds 3a–g

A solution containing the appropriate arylsemicarbazide 2a–e (10 mmol), appropriate ketone 1a–c (10 mmol), and few drops of glacial acetic acid in ethanol (15 mL) was stirred at room temperature for 18 hrs. The reaction mixture was rotovapped and the residue was crystallized from ethanol to give the title compounds 3a–g [23].

2.1.4. Synthesis of (2E)-2-[1-(4-Bromophenyl)-3-(1H-imidazol-1-yl)propylidene]hydrazinecarboxamide (3h)

A solution containing 1a (4.3 mmol), semicarbazide hydrochloride (4.3 mmol) and anhydrous sodium acetate (4.3 mmol), in absolute ethanol (40 mL) was stirred at room temperature for 18 hrs. The reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The residue was crystallized from ethanol to give the title compound 3h [23].

2.2. Mass Spectrometry

2.2.1. Reagents and Solvents

HPLC water was purified using cartridge system (Milford, Bedford, USA). Ultrapure water of 18 μΩ was obtained from Milli-Q Plus Purification System (Millipore, Bedford, MA, USA). Acetonitrile (ACN) HPLC grade was purchased from BDH Laboratory Supplies (Poole, UK).

2.2.2. LC-MS/MS

An Agilent 6410 triple quadrupole mass spectrometer (Agilent technologies, USA) equipped with an electrospray ionization interface (ESI) coupled to an Agilent 1200 HPLC (Agilent Technologies, USA) was used. Agilent 1200 series system consists of G1311A binary pump, G1322A degasser, G1367B HIP-ALS autosampler, and G1316A thermostated column compartment. A connector is used instead of the column to allow direct injection of samples. Mobile phase consists of water and acetonitrile (ACN) (1 : 1) running for 3 minutes with a flow rate of 0.4 mL/min. Compounds 3a–h were prepared by weighing the solid substances to 1 mg·mL⁻¹ in ACN. Test solutions for MS were prepared by diluting the stock solutions with ACN/H₂O mixture (1 : 1). 10 μL of each sample was injected into the LC-MS/MS. MS parameters were optimized for each compound by varying fragmentor voltage of the ion source for scan mode and collision energy for product ion mode. Analytes concentrations of 20 to 50 μg·mL⁻¹—depending on the ions intensities—were used for optimization of the ionization conditions and fragment ion spectra. For screening of mass signals of the different compounds and identifying of the parent ions for MS/MS experiments, MS/MS scans were performed in the mass range of m/z 100–600. Because of the flow rate dependency of the ESI process, ion source specific parameters were readjusted. The ESI was operated in positive mode. The source temperature was set to 350°C and ion spray voltage was 4.5 kV.

To overcome the reduced specificity issue of ISF, an MS² scan took place prior to ISF to determine each compound’s related fragment ions. Fragmentor voltage was optimized to produce adequate in-source fragmentation; values of 100, 120, 140, 160, 180, and 200 V were tested to obtain the fragments of each compound in the scan spectra. The optimum fragmentor voltage to generate in-source fragments was 200 V. Furthermore, the collision energy used for product ion (MS²) analysis was also optimized by varying collision energy values (4, 6, 8, 10, 12, 14, 16, 18, and 20 eV) and was set to 20 eV to attain the fragment ions.

3. Results and Discussion

3.1. Chemistry

The test compounds 3a–h have been successfully achieved as illustrated in Scheme 1. Thus, the appropriate ketone 1a–c [24–26] was allowed to react with the appropriate semicarbazide 2a–e [27] and/or semicarbazide hydrochloride to produce the corresponding semicarbazones 3a–h [23].

3.2. Mass Spectrometry

An initial MS/MS scan followed by a product ion scan of each compound was performed to distinguish the parent ion peaks and the fragment ions of compounds 3a–h. The data obtained played a guidance role prior to the pseudo-MS³ process for the same compounds. The highly sensitive product ion spectra of compounds 3a–h acquired from a one stage product ion scan with abundant ions and no low mass cutoff are represented in Figures 2 and 3. The ISF step showed numerous fragments including the daughter ion peaks produced by MS/MS scans, which in turn were used as precursor ions for the pseudo-MS³ step.

(a)

(b)

(c)

(d)

(e)

(f)

(a)

(b)

(c)

(d)

(e)

(f)

(g)

A pattern of seven major fragments (I–VII) was observed following in-source fragmentation (ISF) for all compounds 3a–h regardless of the different substituents either in “X” or “R” position of the main nucleus. A common fragment (I) was observed for the substituted compounds 3a–h with [M + H]⁺at m/z (266.21, 300.30, 280,22, 374.10, 364.10397.92, 358.10 and 268.12, resp.) as shown in Scheme 2. These [M + H]⁺ values suggested that this fragment is characterized by the removal of imidazole ring. Upon exposure to further MS/MS fragmentation, fragment (I) showed defined pattern of [M + H]⁺ at m/z (250.83, 203.21, and 173.10 for X = Br, OCH₃, and H, resp.) suggesting the breakage of the amide linkage. The latter (II) was also a common fragment of in-source fragmentation that finally produced a fragment of [M + H]⁺ at m/z 77.10 for a benzene ring. Two other common fragments (III and IV) were seen after ISF and a result of MS/MS of fragment (I). One fragment (III) with [M + H]⁺ at m/z (207.98, 160.20, and 130.12 for X = Br, OCH₃, and H, resp.) was suggested to have lost an imidazole ring and an HN–CO–NH–R. While the other fragment (IV) with [M + H]⁺ at m/z (225.10, 177.21, and 147.12 for X = Br, OCH₃, and H, resp.) was perhaps characterized by the removal of the imidazole ring in addition to an HCO–NH–R moiety. Additionally, fragment (III) was also seen in MS/MS spectrum of fragment (IV). Fragment (V) commonly appeared by ISF of the main nucleus with [M + H]⁺ at m/z (319.10, 271.10, and 241.10 for X = Br, OCH₃, and H, resp.) which assumed a loss of an R–NH moiety followed by removal of imidazole ring and HN–NH–CHO after product ion scan. Moreover, fragments (VI and VII) with [M + H]⁺ at m/z (262.10 and 214.20 for X = Br and OCH₃, resp. for fragment VI) and [M + H]⁺ at m/z 183.10 for fragment VII. Both fragments were identified after ISF and presumed to involve the loss of an HN–NH–CO–NH–R moiety. Alternatively, a fragment appeared only as a result of MS/MS of fragment (VI) in case of X = Br with [M + H]⁺ at m/z 121.20 assuming the loss of the imidazole ring and releasing a CH₂–CH₂moiety. However, in case of fragment VII where X = H, a typical rearrangement to form a tropylium ion was suggested, producing a fragment after MS/MS with [M + H]⁺ at m/z 118.14. All ion peaks together with their corresponding proposed structures obtained from ISF and MS² scans for compounds 3a–h are shown in Scheme 2 and are also summarized in Table 1.

4. Conclusions

ESI-QqQ has proven to be an efficient technique for the structural elucidation of compounds 3a–h. The approach described in this study comprehensively studied the fragmentation pathways of compounds 3a–h utilizing the integration of ISF with the collision-induced dissociation. Those fragmentation pathways of compounds 3a–h were elaborated by further dissociating individual fragment ions in the MS² scan mode. Substructures of all fragment ions were unambiguously identified. Sensitive ion spectra of compounds 3a–h were acquired from one stage product ion scan with abundant ions and absence of the low mass cutoff. The described technique is characterized by its simplicity, sensitivity, and time saving and can be used to identify unknown compounds, metabolites, impurities, and drug degradates.

Conflict of Interests

The authors declare no conflict of interests.

Acknowledgment

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the Research Group Project no. RGP-VPP-322.

References

M.-Y. Zhang, N. Pace, E. H. Kerns, T. Kleintop, N. Kagan, and T. Sakuma, “Hybrid triple quadrupole-linear ion trap mass spectrometry in fragmentation mechanism studies: application to structure elucidation of buspirone and one of its metabolites,” Journal of Mass Spectrometry, vol. 40, no. 8, pp. 1017–1029, 2005.
View at: Publisher Site | Google Scholar
N. Kawasaki, M. Ohta, S. Hyuga, O. Hashimoto, and T. Hayakawa, “Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry,” Analytical Biochemistry, vol. 269, no. 2, pp. 297–303, 1999.
View at: Publisher Site | Google Scholar
M. T. Krahmer, Y. A. Johnson, J. J. Walters, K. F. Fox, A. Fox, and M. Nagpal, “Electrospray quadrupole mass spectrometry analysis of model oligonucleotides and polymerase chain reaction products: determination of base substitutions, nucleotide additions/deletions, and chemical modifications,” Analytical Chemistry, vol. 71, no. 14, pp. 2893–2900, 1999.
View at: Publisher Site | Google Scholar
A. Kapur, J. L. Beck, and M. M. Sheil, “Observation of daunomycin and nogalamycin complexes with duplex DNA using electrospray ionisation mass spectrometry,” Rapid Communications in Mass Spectrometry, vol. 13, no. 24, pp. 2489–2497, 1999.
View at: Google Scholar
W. Mo, H. Sakamoto, A. Nishikawa et al., “Structural characterization of chemically derivatized oligosaccharides by nanoflow electrospray ionization mass spectrometry,” Analytical Chemistry, vol. 71, no. 18, pp. 4100–4106, 1999.
View at: Publisher Site | Google Scholar
A. Risberg, H. Masoud, A. Martin, J. C. Richards, E. R. Moxon, and E. K. H. Schweda, “Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd,” European Journal of Biochemistry, vol. 261, no. 1, pp. 171–180, 1999.
View at: Publisher Site | Google Scholar
G. Lhoëst, T. Zey, R. K. Verbeeck et al., “Isolation from pig liver microsomes, identification by electrospray tandem mass spectrometry and in vitro immunosuppressive activity of a rapamycin tris-epoxide metabolite,” Journal of Mass Spectrometry, vol. 34, no. 1, pp. 28–32, 1999.
View at: Google Scholar
A. Putschew and M. Jekel, “Induced in-source fragmentation for the selective detection of organic bound iodine by liquid chromatography/electrospray mass spectrometry,” Rapid Communications in Mass Spectrometry, vol. 17, no. 20, pp. 2279–2282, 2003.
View at: Publisher Site | Google Scholar
A. Hütteroth, A. Putschew, and M. Jekel, “Selective detection of unknown organic bromine compounds and quantification potentiality by negative-ion electrospray ionization mass spectrometry with induced in-source fragmentation,” International Journal of Environmental Analytical Chemistry, vol. 87, no. 6, pp. 415–424, 2007.
View at: Publisher Site | Google Scholar
J. H. Gil, J. Hong, J. C. Choe, and Y. H. Kim, “Analysis of fatty acyl groups of diacyl galactolipid molecular species by HPLC/ESI-MS with in-source fragmentation,” Bulletin of the Korean Chemical Society, vol. 24, no. 8, pp. 1163–1168, 2003.
View at: Google Scholar
Q. Tian, C. J. G. Duncan, and S. J. Schwartz, “Atmospheric pressure chemical ionization mass spectrometry and in-source fragmentation of lutein esters,” Journal of Mass Spectrometry, vol. 38, no. 9, pp. 990–995, 2003.
View at: Publisher Site | Google Scholar
D. J. Carrier, C. Eckers, and J.-C. Wolff, “‘In-source’ fragmentation of an isobaric impurity of lamotrigine for its measurement by liquid chromatography tandem mass spectrometry after pre-concentration using solid phase extraction,” Journal of Pharmaceutical and Biomedical Analysis, vol. 47, no. 4-5, pp. 731–737, 2008.
View at: Publisher Site | Google Scholar
Z. Yan, G. W. Caldwell, W. J. Jones, and J. A. Masucci, “Cone voltage induced in-source dissociation of glucuronides in electrospray and implications in biological analyses,” Rapid Communications in Mass Spectrometry, vol. 17, no. 13, pp. 1433–1442, 2003.
View at: Publisher Site | Google Scholar
G. J. Van Berkel, S. A. McLuckey, and G. L. Glish, “Electrospray ionization of porphyrins using a quadrupole ion trap for mass analysis,” Analytical Chemistry, vol. 63, no. 11, pp. 1098–1109, 1991.
View at: Publisher Site | Google Scholar
J. A. Loo, H. R. Udseth, and R. D. Smith, “Collisional effects on the charge distribution of ions from large molecules, formed by electrospray-ionization mass spectrometry,” Rapid Communications in Mass Spectrometry, vol. 2, no. 10, pp. 207–210, 1988.
View at: Publisher Site | Google Scholar
C. Buré, W. Gobert, D. Lelièvre, and A. Delmas, “In-source fragmentation of peptide aldehydes and acetals: influence of peptide length and charge state,” Journal of Mass Spectrometry, vol. 36, no. 10, pp. 1149–1155, 2001.
View at: Publisher Site | Google Scholar
W. Weinmann, M. Stoertzel, S. Vogt, and J. Wendt, “Tune compounds for electrospray ionisation/in-source collision-induced dissociation with mass spectral library searching,” Journal of Chromatography A, vol. 926, no. 1, pp. 199–209, 2001.
View at: Publisher Site | Google Scholar
G. Hopfgartner, C. Husser, and M. Zell, “Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer,” Journal of Mass Spectrometry, vol. 38, no. 2, pp. 138–150, 2003.
View at: Publisher Site | Google Scholar
S. Dalkara and A. Karakurt, “Recent progress in anticonvulsant drug research: strategies for anticonvulsant drug development and applications of antiepileptic drugs for non-epileptic central nervous system disorders,” Current Topics in Medicinal Chemistry, vol. 12, no. 9, pp. 1033–1071, 2012.
View at: Publisher Site | Google Scholar
E. Perucca, J. French, and M. Bialer, “Development of new antiepileptic drugs: challenges, incentives, and recent advances,” The Lancet Neurology, vol. 6, no. 9, pp. 793–804, 2007.
View at: Publisher Site | Google Scholar
M. Bialer and B. Yagen, “Valproic acid: second Generation,” Neurotherapeutics, vol. 4, no. 1, pp. 130–137, 2007.
View at: Publisher Site | Google Scholar
H. Nau and W. Löscher, “Pharmacologic evaluation of various metabolites and analogs of valproic acid: teratogenic potencies in mice,” Fundamental and Applied Toxicology, vol. 6, no. 4, pp. 669–676, 1986.
View at: Publisher Site | Google Scholar
M. I. Attia, M. N. Aboul-Enein, A. A. El-Azzouny, Y. A. Maklad, and H. A. Ghabbour, “Anticonvulsant potential of certain new (2E)-2-[1-aryl-3-(1H-imidazol-1-yl)- propylidene]-N-(aryl/H)hydrazinecarboxamides,” The Scientific World Journal, Article ID 357403, 2013.
View at: Publisher Site | Google Scholar
M. N. Aboul-Enein, A. A. E.-S. El-Azzouny, M. I. Attia, O. A. Saleh, and A. L. Kansoh, “Synthesis and anti-candida potential of certain novel 1-[(3-substituted-3- phenyl)propyl]-1H-imidazoles,” Archiv der Pharmazie, vol. 344, no. 12, pp. 794–801, 2011.
View at: Publisher Site | Google Scholar
G. Roman, M. Mares, and V. Nastasa, “A novel antifungal agent with broad spectrum: 1-(4-Biphenylyl)-3-(1H-imidazol-1-yl)-1-propanone,” Archiv der Pharmazie, vol. 346, no. 2, pp. 110–118, 2013.
View at: Publisher Site | Google Scholar
J. Wan, Z.-Z. Peng, X.-M. Li, P.-K. Ouyang, and S.-S. Zhang, “3-(1H-lmidazol-1-yl)-1-(4-methoxyphenyl)-propan-1-one,” Acta Crystallographica E, vol. 61, no. 8, pp. o2585–o2586, 2005.
View at: Publisher Site | Google Scholar
M. N. Aboul-Enein, A. A. El-Azzouny, M. I. Attia et al., “Design and synthesis of novel stiripentol analogues as potential anticonvulsants,” European Journal of Medicinal Chemistry, vol. 47, pp. 360–369, 2012.
View at: Publisher Site | Google Scholar

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Copyright © 2014 Ali S. Abdelhameed et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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