Induced in-source fragmentation pattern of certain novel (1Z,2E)-N-(aryl)propanehydrazonoyl chlorides by electrospray mass spectrometry (ESI-MS/MS)
© Abdelhameed et al; licensee Chemistry Central Ltd. 2013
Received: 11 November 2012
Accepted: 23 January 2013
Published: 25 January 2013
Collision 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.
The 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.
This 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.
KeywordsIn-source fragmentation Hydrazones Fragmentor voltage Electrospray ionization
Several types of ionization methods are available for use in mass spectrometry studies, such as electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), or electron impact (EI). Electrospray ionization mass spectrometry (ESI-MS), as a powerful tool for the analysis, has been widely applied to the analysis of glycoproteins , oligonucleotides [2, 3], oligosaccharides [4, 5], drugs and drug metabolites , environmental contaminants [7, 8], and numerous other types of compounds . In atmospheric pressure ion sources, e.g. APCI or electrospray ionisation (ESI), fragmentation of ions can occur in the ion source (in-source fragmentation) before ions reach the analyzer. In spite of the fact that this method of in source fragmentation is less selective than tandem mass spectrometry, as all ions in the source will be fragmented simultaneously, it has been used by several research groups [10–19]. On the other hand, collision 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 (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. Another drawback to the IT is the low mass cut-off in product ion scans. This arises from the fact that the precursor ion and the lowest m/z fragment ion must be stable simultaneously in the IT [20, 21]. In the present study, we combine the usage of in-source fragmentation with product ion scan (MS2) to obtain structural information of certain novel (1Z 2E)-N-(aryl)propanehydrazonoyl chlorides using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The use of this approach was designed to improve the structure elucidation power of tandem mass spectrometry to simulate the MS3 of ion trap overcoming the aforementioned drawbacks of the latter.
Results and discussion
General procedure for the synthesis of (1Z,2E)-2- (benzoyl/benzothiazol-2-oyl)-N-arylpropanehydrazonoyl chlorides 3a–e. A mixture of benzoyl hydrazine 1a or benzothiazole-2-carbohydrazide 1b (10 mmol) and 2-oxo-N-arylpropanehydrazonoyl chloride 2a–d (10 mmol) in absolute ethanol (50 mL) was refluxed for 9 h. Then left to cool, the formed solid was filtered off, washed with ethanol, and recrystallized from EtOH/DMF to afford the corresponding hydrazonoyl chlorides 3a–e, respectively. The target compounds were synthesized via reacting 4-methylbenzohydrazide (1) with 2-oxo-N-arylpropanehydrazonoyl chlorides 2a-e in refluxing ethanol. The latter reaction produced, in each case, a single yellow product was identified as (1Z,2E)-2-(2-(4-methylbenzoyl)hydrazono)-N’-arylpropanehydrazonoyl chloride 3a-e.
Multistage MS data of compounds 3a-e by ESI-MS/MS
Product ions of protonated compounds 3a-e [M+H]+(m/z)
General method for synthesis of (1Z,2E)-2-(2-(4-methylbenzoyl)hydrazono)-N’-(aryl)propanehydrazonoyl chloride 3a-e
Reagents and solvents
HPLC grade water was obtained by Milli-Q connected to Elix Millipore water purification system (Millipore, USA). Acetonitrile (ACN) HPLC grade was purchased from BDH laboratory supplies (Poole-UK).
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 thermostatted column compartment. A connector is used instead of the column to allow direct injection of samples. Mobile phase composed of two solvents: A is HPLC grade water, and B is acetonitrile mixed in the ratio 1:1. Compounds 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). Flow rate is 0.4 ml/min, run time was 3 minutes. 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. For optimization of the ionization conditions and of fragment ion spectra, analytes concentrations of 20 to 50 μg.mL-1-depending on the ions intensities- were employed. For screening of mass signals of the different compounds and to search for parent ions for MS/MS experiments, MS2 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 selectivity problem of in-source fragmentation, a product ion scan was performed prior to the in-source fragmentation to determine each compound’s related fragment ions. The 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.
Mass spectrometry has shown to be an effective tool for the structural characterization of the synthesized compounds 3a-e. This work demonstrated a comprehensive fragmentation mechanism study of compounds 3a-e using the combination of induced in-source fragmentation with product ion scan (MS2). The highly sensitive product ion spectra of compounds 3a-e were obtained from a single-stage MS2 scan with abundant product ions and no low mass cut-off. The detailed fragmentation pathways of all ions observed in the in-source fragmentation spectrum of compounds 3a-e were elucidated by further dissociation of each of these fragment ions using the product ion (MS2) scan mode. The substructures of all fragment ions were unambiguously assigned. The approach described here is simple, sensitive, rapid and powerful for the identification of unknown compounds. Additionally, it can be used to identify trace amounts of metabolites and can be potentially applied to the analysis of impurities and degradation products in complex systems.
Collision induced dissociation
Triple quadrupole mass spectrometer system
Ion trap mass spectrometer system
Multiple stages of fragmentation
Matrix assisted laser desorption ionization
Atmospheric pressure chemical ionization
Electrospray ionization mass spectrometry
High-performance liquid chromatography.
Authors would like to thank the Deanship of Scientific Research and the Research Center of the College of Pharmacy, King Saud University for supporting this study.
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