Benha University Faculty of Science Physics department MASS SPECTROMETRIC STUDY OF SOME FLUOROQUINOLONE DRUGS USING ELECTRON IONIZATION AND CHEMICAL IONIZATION TECHNIQUES IN COMBINATION WITH SEMI-EMPIRICAL CALCULATIONS.
BY
MAMOUN SARHAN MAHMOUD ABD EL KAREEM (M.Sc. PHYSICS) Atomic Energy Authority, Cairo, Egypt
THESIS
SUBMITTED FOR THE Ph.D. DEGREE (EXPERIMENTAL PHYSICS)
From
FACULITY OF SCIENCE BENHA UNIVERSITY
Supervisors Prof. Dr. M. I. El-Zaiki Prof. Dr. Ezzat T.M.Selim Prof. of Nuclear Physics Atomic & Molecular Physics Division Physics Department Experimental Nuclear Physics Department Faculty of Science, (Benha University) Atomic Energy Authority. (Egypt)
Prof. Dr. M.A.Rabbih Prof. Dr. A.M.Hassan Rezk Atomic & Molecular Physics Division National Center for Radiation Research Experimental Nuclear Physics Department and Technology Atomic Energy Authority. (Egypt) Atomic Energy Authority. (Egypt)
2013
Abbreviations and Acronyms
EI Electron Ionization
CI Chemical Ionization
MO Molecular Orbital
MNDO Modified Neglect of Diatomic Overlap
TFC–MS/MS Turbulent Flow Chromatography/Tandem Mass
Spectrometry
SPE Solid-Phase Extraction
FQ Fluoroquinolone
SPME Solid-Phase Microextraction
LC/MS/MS Liquid Chromatography–Tandem Mass Spectrometry
ΔHf Heats of Formation
PA Proton Affinity
AE Appearance Energy
IP Ionization Potential
GC/MS Gas Chromatograph/Mass Spectrometer
RI Relative Intensity m/z Mass to Charge Ratio
ACKNOWLEDGMENT
This work was performed in the Molecular Physics Division of the Experimental Nuclear Physics Department, in cooperation with Radiation Chemistry Department, National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.
I wish to express my deepest thanks to Head of Experimental Nuclear Physics Department of the Egyptian Atomic Energy Authority and Head of Physics Department Faculty of Science, Benha University, for their encouragement interest.
Special thanks to Professor Dr.M.E. El–Zeiki Faculty of Science, Benha University for continuous support and for encouragement interest during the course of this work.
I would like to express my deepest thanks to Professor Dr. Ezzat T.M.Selim, Experimental Nuclear Physics Department of the Egyptian Atomic Energy Authority, for his great efforts in illuminating the discussion.
I would like to express my gratitude and appreciation to Professor Dr. M.A.Rabbih Experimental Nuclear Physics Department of the Egyptian Atomic Energy Authority, for his great efforts for suggesting and supervising this work and also for his valuable guidance and illuminating discussions.
I would like to express my gratitude and appreciation to Professor Dr. A.M.Hassan Rezk, Radiation Chemistry Department, National Center for Radiation Research and Technology, Atomic Energy Authority, for his great efforts in the mass spectrometric measurements and supervising this work.
Abstract
A mass spectrometer of the type QMS (SSQ710) is used to record the electron ionization mass spectra of some 6-fluoroquinolones molecules, namely: Norfloxacin, Pefloxacin, Ciprofloxacin and Levofloxacin.While the chemical ionization mass spectra of these compounds are recorded using Thermo Finnigan TRACE DSQ GC/MS system.
In EI mass spectra, the relative intensities for the molecular ions [M]+• of the studied compounds and the prominent fragment ions are reported and discussed. Furthermore, fragmentation patterns for the four compounds have been suggested and discussed and +• + the most important fragmentation processes such as [M-CO2] , [M-C2H4N] and [M- + CO2-C2H4N] are investigated.
On the other hand, the chemical ionization (CI) mass spectra of the compounds have been recorded using methane as the reagent gas. These spectra are discussed in terms of the structure of the compounds, with particular reference to their conventional electron ionization mass spectra. The protonated molecules [M+H]+ are more relatively intense than [M]+• ions in the recorded EI mass spectra indicating higher stability in the case of [M+H]+. Also, fragmentation patterns for the four compounds have been suggested and discussed (using chemical ionization technique) and the most important fragmentation processes +• + + such as [MH-CO2] , [MH-C2H4N] and [MH-H2O] are investigated.
Using MNDO semi-empirical method for computation together with the experimental results gave valuable information about the heats of formation and ionization energies of the molecules. The effect of substituents on the geometry of the neutral and ionized molecules are reflected in the values of the ionization energy and heats of formation of +∙ neutral ∆Hf(M) and ionized ∆Hf(M) molecules. The calculated values for ionization energies of Norfloxacin, Pefloxacin, Ciprofloxacin and Levofloxacin are 8.1, 8, 8.8 and 8.3 eV, respectively. The calculated charge distributions at N1 and O12 in the qinolone ring of the studied molecules as well as the presence of a lone pair electrons at N1 and O12 atoms indicate that the ionization processes occur at these two atoms. The appearance +• + (AE) and activation energies of the fragment ions [M-CO2] and [M-C2H4N] are also calculated and discussed. It is noteworthy that all the presently calculated values of ionization, appearance and activation energies are not yet published. The MNDO method is also used to probe the protonation of the studied compounds. The calculated proton + affinities (PA's) together with ∆Hf [M+H] values at nitrogen (N1) and at oxygen (O12) atoms are calculated. These results give interesting features for the protonation sites. The protonation at oxygen (O12) site is more favored than that at nitrogen (N1) site. Furthermore, the calculated values of the heats of formation of neutral [M], ionized [M]+•, + protonated molecule [M+H] and PA's values are reported for the first time CONTENTS
Page ABSTRACT і
CHAPTER 1. INTRODUCTION AND AIM OF THE WORK
1.1 Introduction 1 1.2 Aim of the Work 5
CHAPTER 2 . THEORETICAL CONSIDERATIONS
2.1. Processes of Ionization and Dissociation by Electron Ionization 6 2.2. Frank - Condon Principle 9 2.3. Ionization Probability Near Thershold 9 2.4. Determination of Thermochemical Data 10 2.5. Stevenson's Rule 11 2.6. Characteristics of Mass Spectra 12 2.7. Simple Bond Cleavage Processes 12 2.8. Rearrangements Processes 13 2.9. Processes of Ionization and Dissociation by Chemical Ionization 13 2.10. Proton Affinity 14 2.11. Semiempirical quantum chemical methods and the predicting mass spectrometric fragmentations 15
CHAPTER 3. APPARATUS AND EXPERIMENTAL CONDITIONS
3.1. Apparatus 16 3.2. Materials 16 3.3. Experimental Conditions 17
CHAPTER 4 . RESULTS AND DISCUSSION
4.1. Results 18 4.1.1. Experimental Measurements 18 4.1.2. Computational Results 19 4.2. Discussion 20 4.2.1. Mass spectra of Norfloxacin using EI technique 25 4.2.2. Ionization processes of Norfloxacin using EI technique 29 4.2.3. Fragmentation of Norfloxacin using EI technique 31 4.2.4. Mass spectrum of Norfloxacin using CI technique 37 4.2.5. Chemical ionization, proton transfer 40 4.2.6. Fragmentation of Norfloxacin using CI technique 41 4.2.7. The proton affinity (PA) , heat of formation(∆Hf) and the charge distributions of Norfloxacin 43 4.3.1. Mass spectra of Pefloxacin under EI technique 45 4.3.2. Ionization Process of Pefloxacin using EI technique 49 4.3.3. Fragmentation of Pefloxacin using EI technique 51 4.3.4. Fragmentation of Pefloxacin using CI technique 55 4.3.5. The proton affinity (PA), heat of formation (∆Hf) and charge distributions of Pefloxacin 58 4.4.1. Mass spectra of Ciprofloxacin using EI technique 59 4.4.2.Ionization process of Ciprofloxacin using EI technique 63 4.4.3. Fragmentation of Ciprofloxacin using EI technique 65 4.4.4. Fragmentation of Ciprofloxacin using CI technique 70 4.4.5. The proton affinity (PA),heat of formation (∆Hf) and charge distributions of Ciprofloxacin 73 4.5.1. Mass spectra of Levofloxacin using EI technique 74 4.5.2. Ionization process of Levofloxacin using EI technique 78 4.5.3. Fragmentation of Levofloxacin using EI technique 80 4.5.4. Fragmentation of Levofloxacin using CI technique 83 4.5.5. The proton affinity (PA), heat of formation (∆Hf) and charge distributions of Levofloxacin 86
CHAPTER 5. CONCLUSIONS 87
REFRENCES 88
ARABIC SUMMARY
List of Figures and Schemes
figure Name Page
Figure 1 Potential energy curves for a molecule M ionized to either a 7 nondissociative M+• or dissociative state F+ + N• . Path (a) represents the adiabatic transition while path (v) represents the vertical transition.
Figure 2 Structure of fluoroquinolones 20
Figure 3 The structures of Norfloxacin , Pefloxacin , Ciprofloxacin 22 and Levofloxacin
Figure 4 The numbering system of the 6-fluoroquinolone compounds 23 used in this study.
Figure 5 The protonation sites at oxygen(O12) and nitrogen(N1) atoms 24 for 6-fluoroquinolone compounds
Figure 6 The EI mass spectrum of Norfloxacin at 70 eV 26
Figure 7 The EI mass spectrum of Norfloxacin at 15 eV 27
Figure 8 The CI mass spectrum of Norfloxacin 38
Figure 9 The EI mass spectrum of Pefloxacin at 70 eV 46
Figure 10 The EI mass spectrum of Pefloxacin at 15 eV 47
Figure 11 The CI mass spectrum of Pefloxacin 57
Figure 12 The EI mass spectrum of Ciprofloxacin at 70 eV 60
Figure 13 The EI mass spectrum of Ciprofloxacin at 15 eV 61
Figure 14 The CI mass spectrum of Ciprofloxacin 72
Figure 15 The EI mass spectrum of Levofloxacin at 70 eV 75
Figure 16 The EI mass spectrum of Levofloxacin at 15 eV 76
Figure 17 The CI mass spectrum of Levofloxacin 85
Schemes
Scheme 1 Main fragmentation pathways of Norfloxacin at 70 eV 33
Scheme 2 Main fragmentation pathways of Norfloxacin under CI 42 technique
Scheme 3 Main fragmentation pathways of Pefloxacin at 70 eV 52
Scheme 4 Main fragmentation pathways of Pefloxacin under CI 56 technique
Scheme 5 Main fragmentation pathways of Ciprofloxacin at 70 eV 67
Scheme 6 Main fragmentation pathways of Ciprofloxacin under CI 71 technique
Scheme 7. Main fragmentation pathways of Levofloxacin at 70 eV 81
Scheme 8 Main fragmentation pathways of Levofloxacin under CI mode 84
List of Tables
Table Name Page
Table 1 The different functional groups of 6-fluoroquinolones 20
+• Table 2 The molecular ion (M) and the main fragment ions [m/z] with their 28 relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Norfloxacin.
Table 3 Calculated charge distribution of neutral and charged Norfloxacin 30 molecule using MNDO method together with the charge difference (∆).
Table 4 Calculated bond lengths of neutral and charged Norfloxacin using 34 MNDO method together with the bond length difference (∆ L).
+• Table 5 Calculated ∆Hf(M),∆Hf(M) and IE values for the four 6- 35 fluoroquinolone molecules using MNDO method.
+ Table 6 Protonated molecules [M+H] and major fragment Ions [m/z] with their 39 relative intensity [%] for chemical ionization mass spectra of Norfloxacin, Pefloxacin, Ciprofloxacin and Levofloxacin at 70 e V.
Table 7 Calculated heat of formation values for the protonated molecules 44 + ∆Hf(M+H) and proton affinities (PA) at O12 and N1 sites for 6- fluoroquinolone drugs using MNDO method.
+• Table 8 The molecular ion (M) and the main fragment ions [m/z] with their 48 relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Pefloxacin.
Table 9 Calculated charge distribution of neutral and charged Pefloxacin 50 molecule using MNDO method together with the charge difference (∆).
Table 10 Calculated bond lengths of neutral and ionized Pefloxacin using 53 MNDO method together with the bond length difference (∆ L) .
+• Table 11 The molecular ion (M) and the main fragment ions [m/z] with their 62 relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Ciprofloxacin.
Table 12 Calculated charge distribution of neutral and ionized Ciprofloxacin 64 molecule using MNDO method together with the charge difference (∆).
Table 13 Calculated bond lengths of neutral and ionized Ciprofloxacin using 68 MNDO method together with the bond length difference (∆ L).
Table 14 The molecular ion (M)+• and the main fragment ions [m/z] with their 77 relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Levofloxacin.
Table 15 Calculated charge distribution of neutral and ionized Levofloxacin 79 molecule using MNDO method together with the charge difference (∆).
Table 16 Calculated bond lengths of neutral and ionized Levofloxacin using 82 MNDO method together with the bond length difference (∆ L).
CHAPTER (1)
INTRODUCTION AND AIM OF THE WORK
1.1 Introduction:
Mass spectrometer is a microanalytical technique that can be used selectively to detect and determine the amount of a given analyte. Mass spectrometer is also used to determine the elemental composition and some aspects of the molecular structure of an analyte. These tasks are accomplished through the experimental measurement of the mass of gas- phase ions produced from molecules of an analyte. Unique features of mass spectrometer include its capacity for direct determination of the nominal mass (and in some cases, the molar mass) of an analyte, and to produce and detect fragments of the molecule that correspond to discrete groups of atoms of different elements that reveal structural features. Mass spectrometer has the capacity to generate more structural information per unit quantity of an analyte than can be determined by any other analytical technique.(1)
The production of positive ions by electron ionization is a widely employed technique, as it can be utilized for the analysis of nearly all gases, volatile compounds, and metallic vapours. The ion beam current can be accurately controlled because the ionizing electron beam is generally change limited. The energy of the electron beam can also be varied precisely. Thus, ionized species of the complex molecules can be produced both with or without fragmentation so as to reveal details relating to the molecular structure.(2)
As the energy of the electron beam is further increased the probability of ionization increases and the parent ion is formed with the excess energy in its vibrational and electronic degree of freedom. When the excess energy possessed by the molecular ion over the ground state become equal to the dissociation energy in one of the degrees of freedom, fragmentation takes place. If the energy supplied to the molecular ion is again increased, more and more fragmentation occurs and the spectrum become complex. In organic mass spectrometry, generally the mass spectrum is run at 70 eV to get reproducible spectra.(3)
The electron ionization (EI) method together with other techniques such as chemical ionization (CI) mass spectrometry has proved to be a valuable tool in structural characterization of positive ions and for determining fragmentation mechanism.(4) The mass spectrum of each compound is unique and can be used as a chemical " fingerprint " to characterize the compound and the molecular ion peak appears at m/z value equal to the molecular weight of the compound.(4)
However, one of the problems with the conventional electron ionization mode is that molecular ions are often produced too excited that no peak representing the molecular weight of the intact molecule is observed in the spectra. In addition, the spectra tend to be complex and, therefore, difficult to interpret.
On the other hand, in CI mass spectrometry the characteristic ionization of the materials in question is produced by ionic reactions than electron ionization. CI mass spectra are generally quite different and often more useful. The technique gives ions of low internal energy and is generally characterized by a lower abundance of the fragment ions than electron ionization technique. This is an important advantage since one can focus on the molecular weight. The technique has also been used to investigate the relationship between the mass spectra of EI and of CI technique
On the other hand, quantum chemical methods for the calculations of thermochemical data have developed beyond the level of just reproducing experimental data and can now make accurate predictions where the experimental data are unknown or uncertain(5).The semi-emiperical molecular orbital (MO) methods of quantum chemistry (6-17) are widely used in computational studies of large molecules, particularly in organic chemistry and biochemistry. In their implementation, they neglect many of the less important integrals that occur in the ab initio MO formalism. These severe simplifications call for the need to represent the remaining integrals by suitable parametric reference data. This strategy can only be successful if the semi-empirical model retains the essential physics to describe the properties of interest.(18)
Different semi-empirical methods are available to study different molecular properties, both in the ground state and electronically excited states. The present work will focus on the semi-empirical calculation-using MNDO method- of the thermochemical properties for ground state, charged and protonated molecules in the gas phase.(18)
Inspection of the published works done using electron ionization, chemical ionization techniques and semi-empirical calculations for the structural identification of the present compounds show that:
(1) A fast and sensitive method has been developed for the determination of five fluoroquinolones namely: Enrofloxacin, Ciprofloxacin, Difloxacin, Sarafloxacin and Ofloxacin in commercial bovine milk after simple extraction method and LC-MS by Ruiz-Viceo et al.(19)
(2) A simple and rapid method for the determination of residues of Enrofloxacin and Ciprofloxacin in tissues of farm animals using turbulent flow chromatograph/tandem mass spectrometer (TFC–MS/MS)is described by Ralph et al. (20)
(3) A solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry method was developed by Lee et al.(21) for the determination of selected fluoroquinolone (FQ) drugs including ofloxacin, norfloxacin, and ciprofloxacin in wastewater samples.
(4) Kurie et al. (22) developed a sensitive and useful method for the determination of five FQs namely: Enoxacin, Ofloxacin, Ciprofloxacin, Norfloxacin, and Lomefloxacin in environmental waters, using solid-phase microextraction (SPME) coupled with liquid chromatograph–tandem mass spectrometer(LC/MS/MS).
(5) A group of Chinese(23) investigate the fragmentation mechanism of fluroquinolones,six compounds of fluroquinolones were analyzed using electrospray ion trap mass spectrometer by collision induced dissociation in a multi-stage MS full scan postive mode. The mass spectra and structures of the six fluroquinolones were compared with each other and it was observed that fluroquinolones gave characteristic fragment ions by the neutral loss of CO2, HF and CO, corresponding to the carboxy, fluorine and 4-carbonyl group in their structures. These characteristics used by the authors for future structure elucidation in studies of fluroquinolones and analogue compounds.
(6) Time of Flight Mass Spectrometer (TOF MS), with different electron energy for EI and different gas pressure for CI, of Levofloxacin lactate (LL) were studied by R.Q.Li and H.Yin.(24) The authors found a prominent fragmentation rout of Levofloxacin was an elimination of CO 2 from molecular ion at m/z 361, forming cation A at m/z 317, followed by the cleavage of piperizine ring creating caion B at m/z 246 and C at m/z 71. Further fragmentation pathway was the formation of cation D at m/z 231 from B. (7) The ∆Hf(M), ∆Hf [M+H] and local proton affinities (PAs) as well as the charge distributions for the two highly electronegative hetero atoms (O4 and N1) in six quinolone derivatives namely: Quinolone, 1-methyl quinolone, 1-ethyl quinolone, 1- cyclopropopyl quinolone, 3- carboxylic- quinolone, and 6- fluoroquinolone have been calculated using MNDO method by M.A.Rabbih et al.(25)
1.2 Aim of the Work:
The aim of the present thesis is to use the electron ionization and chemical ionization techniques together with semi-empirical calculations (MNDO method) to investigate the following compounds: Norfloxacin, Pefloxacin, Ciprofloxacin, and Levofloxacin from the following point of view:
(1) To record the mass spectra of the studied compounds using electron ionization at 15 as well as 70 eV (2) To record the mass spectra of the studied compounds using chemical ionization technique. (3) To correlate the data obtained from the two techniques. (4) To study the structural-reactivity relationship. (5) To suggest the primary fragmentation and subsequent fragmentation mechanisms for the four compounds. (6) To determine the stability of the product ions using both ionization techniques. (7) To use semi-emiperical calculations using MNDO method to calculate the geometries features and the thermochemical properties for the studied compounds. This include: a. Ionization energies (IE’s) of these molecules b. Heats of formation of neutral (ΔHf (M)) and ionized molecules +• (ΔHf (M) ). + c. Heats of formation of the protonated molecules (ΔHf [M+H] ) d. Proton affinities (PA’s) of the molecules. f. Bond length and charge distribution of the compounds under investigation.
CHAPTER (2)
THEORETICAL CONSIDERATIONS
2.1. Processes Of Ionization and Dissociation By Electron Ionization:
Electron ionization is a familiar method for creating ions from volatile gas-phase molecules [M]. By using fast moving electrons (or photons) to remove an electron from the neutral molecule to create the odd-electron molecular ion M+•.
If an electron is removed from the highest occupied orbital of the molecule [M], the minimum energy necessary for this process, in which the molecular ion [M]+• is formed, is termed the ionization energy, (IE) as in process (1).
e- [M] [M] +• + 2 e- (ionization process) (1)
When the electron energy is increased, the molecular ion [M]+• can dissociate to form the fragment ion [F]+ and a neutral fragment [N]•, as a simple bond cleavage process (2). The minimum energy to do this is called the appearance energy (AE) of the fragment ion [F] + .
[M]+• [F]+ + [N]• (simple bond cleavage process) (2)
Also, [M]+• could dissociate to produce a smaller mass fragment [F]+• and neutral molecule N as a rearrangement process (3) .
[M]+• [F]+•+[N] ( rearrangement process) (3)
The ionization process(26) can be understood as represented in Figure 1. The vibrational energies of both the molecule [M] and the molecular ion [M]+• may represented by potential energy curves as shown in Figure 1.
Io ni za ti on E ne rg y: (27)
Th e ion izat ion ene rgy (IE ), so met ime s call ed (les s cor rect ly) the ion izat ion potential (usually designated by IP, or, in the older literature, I), is the energy required to remove an electron from a molecule or atom: + − M → M + e ΔHrxn= IEa
Ionization energies are characterized as adiabatic or vertical values.
Appearance Energy: (27)
Since IE,s are often determined in experiments in which the ionizing electron or photon energy is varied until the appearance of a fragment ion is observed ("threshold measurements"), IE,s have been called appearance energies.
+ − AB → A + B + e Δ Hrxn = AP
2.2. Franck-Condon Principle:
In electron ionization, the impacting electron pass the molecule in a fraction of the vibrational period, no change occur during the course of the electronic transition in the position and velocities of the nuclei. This means that the nuclear configuration of the system does not change during the transition. This is the well known Franck-Condon principle.(28-29)
2.3. Ionization Probability Near Threshold:
In the ionization of atomic system by electron ionization, one can explain the increase in the cross section qualitatively as follows: when the energy of the electron increases, above critical energy of ionization, an increase in the ionization cross-section will also occur. The theoretical treatment of the ionization probability near the threshold is difficult, but several attempts have been made. Wigner(30) and then Geltman(31) showed theoretically that, the cross-section behavior in the threshold region is given by a power law of the form:
n-1 σ (E) = C (E-Eo) (4)
Where E is the actual electron energy, Eo is the threshold energy, C is a constant and n is the total number of outgoing electrons for the ionization process.
2.4. Determination Of Thermochemical Data:
Mass spectrometer technique allows the determination of many thermochemical quantities. Heats of formation of the different molecular species, ionization and appearance energies of the molecular and fragment ions and electron affinities of ions and radicals can be measured and used to obtain bond dissociation energies.
For the molecule [M] the ionization energy of the molecular ion [M]+• which is formed by the reaction :
M+e- M +• + 2e - (5)
Can be calculated from the relationship:
+• +• IE[M] = ∆Hf[M] - ∆Hf[M] (6)
+• Where ∆Hf[M] and ∆Hf[M] are the heats of formation of the molecular ion and neutral molecule respectively. These values can be calculated using a semi-empirical methods such as MNDO, AM1and PM3.
In fragmentation process such as
[M]+• [F]+ + [N]• (7)
The AE of the fragment ion [F]+ can be calculated from equation (8)
+ AE[F] = IE [F] +D[F-N] + Eexc. (8)
Where IE [F]+ is the ionization energy of the fragment F,D [F-N] represents the dissociation energy and Eexc. is the excess energy.
The (∆Eth) is the calculated thermodynamic threshold value for the formation of an ion by certain process and calculated from the following equation :-
+ (∆Eth) = ∆Hf[F] + ∆Hf[N] - ∆Hf[M] (9)
and Eexc. is calculated according to:-
Eexc.= AE(exp) - ∆Eth (10)
+ The ∆Hf[F] used for ∆Eth calculation must be free from any excess energy and is obtained from the ionization energy of the free radical [F] by the equation :
+ ∆Hf[F] = IE[F] +∆Hf[F] (11)
2.5. Stevenson's Rule
A useful rule regarding the location of the positive charge was first formulated by Stevenson.(32)He noted that for hydrocarbons(for example) the positive charge tend to remain on the fragment with the lower ionization energy. Since the idea arose from the work of Audier,(33) one may call it the Steven-Audier rule.
2.6. Characteristics of Mass Spectra:
There are some observations characterizing the mass spectra:- (34)
(a) The molecular processes leading to the formation of mass spectra consist of a series of competing, and consecutive unimolecular decomposition reactions of excited molecular ion.
(b) The effects of source temperature are far more pronounced in polyatomic mass spectra.
(c) Metastable transitions are observed i.e.unimolecular decomposition reactions occurring with rate of 10-6 sec. These transitions corresponding to either molecular or fragment ions forming other ions by spontaneous decomposition.
(d) Some mass spectra, particularly of oxygen compounds, show the presence of negative ions, they are generally in smaller abundance than the positive ions.
2.7. Simple Bond Cleavage Processes:
Simple bond cleavage occurs when the electron pair of a covalent bond is transferred to two different centers. The site of radical within the molecular ion [A-B]+• is defined by the following equations. Odd-electron ions dissociate by homolytic bond cleavage to an even-electron fragment ion and a radical (equations 12,13)
[A – B] +• A+ + B• (12) odd even odd
[A – B] +• A• + B+ (13) odd odd even
The fragment ion A+ or B+ with the greatest tendency to support an unpaired electron will have a higher appearance energy.
2.8. Rearrangements Processes:
Fragment ions can also be formed by processes in which the initial bond connections in the molecular ion are reordered or rearranged. Fortunately, many of these rearrangement processes have been characterized for organic molecules and therefore can be predicated based on an ion’s structure. Rearrangement reactions occur with the movement of two or more sets of electron pairs.
2.9. Processes of Ionization and Dissociation by Chemical Ionization:
One of the problems with the conventional electron ionization mode is that molecular ions are often produced which are so excited that no peak representing the molecular weight of the compound or the intact molecule is observed in the spectra. This lack of the molecular ion creates a problem in the sample identification because one must depend on the detection of structure from fragment ions alone. In addition, the electron ionization spectra tend to be complex and, therefore, difficult to interpret.(35)
On the other hand, in chemical ionization (CI) mass spectrometry the characteristic ionization of the materials in question is produced by ionic reactions rather than electron ionization. Chemical ionization mass spectra are generally quite different and oftentimes more useful than electron ionization spectra. This technique requires a reaction gas which can produce a set of ions which are either non reactive or only very slightly reactive with the reaction gas itself, but which can react with other materials. The method is certainly applicable for reaction gases: methane, isobutane, ammonia acetaldehyde, di- methylether and iodomethane.
For the methane as the reaction gas, one introduces into the source of the mass spectrometer a mixture of the methane and the added material (analyte) whose spectrum is to be obtained. Under this condition, practically all of the electrons passing through the gas within the source will ionize methane and ionization of the additive (analyte) by electron ionization will be negligible.
All of the primary ions of methane as in equation (14) react rapidly with methane (at virtually every collision) to give product ions by reactions which are well established.
- +• + + + CH4+ e CH4 , CH3 ,CH2 , CH ,...... + 2e (14)
+• + • CH4 + CH4 CH5 + CH3 (15)
+ + CH3 + CH4 C2H5 + H2 (16)
+ + The major fragment ions of methane produced by 70 eV are CH4 and CH3 , + + consequently the major ions are CH5 and C2H5 . The reactions of these ions with the sample produce the major part of chemical ionization spectrum.
2.10. Proton Affinity:
One may define the proton affinity (PA) (36)of a molecule M as the energy required to effect the forward reaction in equation (17), or as the negative enthalpy change associated with the reaction (19):-
[BH]+ + M B+[M+H]+ (17) Where [BH]+ is the protonated molecule
+ The ion CH5 is an efficient proton donor, so that a sample molecule M can be ionized according to equation (18):-
+ + M + CH5 [M+H] + CH4 (18)
M + H+ MH+ (19)
+ + ∆Hº = ∆Hf [M+H] - ∆Hf [M] - ∆Hf [H ] = - PA
... PA = - ∆Hº
2.11. Semi-empirical quantum chemical methods and the predicting mass spectrometric fragmentations:
An impressive number of gas phase chemical studies of ions have emerged during the last fifty years. Most of these studies were experimental, and a wide range of instrumentation methods, mostly mass spectrometric ones, have been used. More recently, these studies have been complemented by high level quantum chemical and other model calculations, providing firm connection between experiment and theory(37).
The semi-empirical molecular orbital (MO) methods of quantum chemistry are widely used in compoutionaal studies of large molecules, particulaary in organic chemistry and biochemistry.Different semi-empirical methods are available to study different molecular properities both in the ground state and electronically excited states(38).
Semi-empirical molecular orbital (MO) methods of quantum chemistry are based on the Hartree–Fock formalism, but make many approximations and obtain some parameters from empirical data. They are very important in computational chemistry for treating large molecules where the full Hartree–Fock method without the approximations is too expensive because the use of empirical parameters appears to allow some inclusion of electron correlation effects into the methods.(39)
Semi-empirical Methods are simplified versions of Hartree-Fock theory using empirical (= derived from experimental data) corrections in order to improve performance. These methods are usually referred to through acronyms encoding some of the underlying theoretical assumptions. The most frequently used methods (MNDO, AM1, PM3) are all based on the Neglect of Differential Diatomic Overlap (NDDO) integral approximation, while older methods use simpler integral schemes such as CNDO and INDO.(40)
The author used the MNDO (Modified Neglected of Diatomic Overlap) is a semi- empirical method for the present study. The method has been developed by Prof. W.Thiel's group at Max-Planck-Institut für Kohlenforschung in Germany.(41)
The basic advantages of the usage of MNDO method can be outlined as: (42)
1- The method enables the computation of structural, electronic and many physical properties of large systems.
2- The method is suitable for studies in chemicals materials and pharmaceutical industrial segments.
3- The method can be used for its fast candidate, structure or transition state identification and perform high accuracy computations with full quantum treatment methods based software such as HyperChem.
4- MNDO is a well tested semi-emperical molecular orbital (MO) method and its accuracy for predicting ΔHf (see for example Ref. 16) values seems to be sufficient for the present study.(25)
5-The data reported for a large number of compounds prove that the MNDO method achieves better agreement with experimental than MINDO-3.(25)
6- The results of many authors(25) for positive ions indicate that using MNDO and AM1 methods lead to geometries and energies close to that obtained at high level of ab initio theory with of course minute faction of the computation effort.
CHAPTER (3)
APPARATUS AND EXPERIMENTAL CONDITIONS
The electron ionization (EI) mass spectra of the four compounds investigated in this work were obtained using the Finnigan Matt SSQ710 Gas Chromatograph/Mass Spectrometer (GC/MS) system. This system is controlled by the Dec 5000 Data Handling System. On the other hand, the chemical ionization (CI) mass spectra were obtained using The Thermo Finnigan TRACE DSQ GC/MS system with Xcalibur ver. 1.4 software.
3.1 Apparatus:
The main features of the the Finnigan Matt SSQ710 mass spectrometer are: a high vacuum system, a confined EI/CI ion source, a single quadrupole mass analyzer and a positive ion / negative ion electron multiplier. The electron multiplier was operated in the positive ion mode.
The main features of the Thermo Finnigan TRACE DSQ mass spectrometer are almost the same as the Finnigan Matt SSQ710 mass spectrometer. However,the former spectrometer is provided with a prefilter between the EI/CI ion source and the single stage quadrupole analyzer.
Samples are introduced into the EI/CI ion source, in both systems, using a direct insertion(solids) probe. The solids probe is supplied with a probe holder that contains a liquid cooling system for the probe. The solids probe was operated in the temperature – programmed mode where it is programmed to heat at a specific rate which is selected according to the nature of the sample being analyzed.
3.2 Materials:
The compounds under investigation in the present work ( Norfloxacin, Pefloxacin, Ciprofloxacin and Levofloxacin) were obtained from El-Obour Modern Pharmaceutical Company, first industrial zone, El-Obour City, Cairo, Egypt. These pure compounds were used as received without any further purification. Methane (purity 99.89% – Air products, England) was used as the chemical ionization reagent gas.
3.3 Experimental Conditions:
In the EI measurements the ion source temperature was maintained at 150 °C. The EI mass spectra were recorded at two electron energies, namely, 15 and 70 eV.
The probe temperature program for the EI measurements was as follows: 30 °C. for 0.1 minute. 250 °C. for 5.1 minutes. 250 °C. for 15.1 minutes.
In the CI mode of operation the ion source temperature was maintained at 130 °C. The CI mass spectra were recorded at 70 eV electron energy
The probe temperature program for the CI measurements was as follows:
Initial temperature was 70 °C. for 60 sec. Ramp rate 50 °C./min. Final temperature 250 °C. for 100 sec.
CHAPTER (4)
RESULTS AND DISCUSSION
4.1 Results:
4.1.1. Experimental measurements:
The experimental results are obtained in the form of mass spectra i.e relative intensity (RI) against mass to charge ratio (m/z) for all the studied compounds using electron ionization (EI) and chemical ionization (CI) techniques.
In this study the ionization of four members of 6-fluoroquinolones molecules, namely: Norfloxacin, Pefloxacin, Ciprofloxacin and Levofloxacin have been investigated using EI and CI techniques. The measurements include the EI mass spectra of the studied molecules at two different electron energy values (70 and 15 eV) while the CI mass spectra have been obtained using methane as a reagent gas at 70 eV only. In the study, one can discuss the mass spectral behaviors of these compounds using the two techniques.
Figures 6,7,9,10,12,13,15 and 16 show the directly measured mass spectra of the studied compounds using EI technique at 70 and 15 eV while Figures 8,11,14 and 17 show the mass spectra of the compounds using CI technique at 70 eV only.
The relative intensities (relative to the base peak) of the molecular ions and different fragment ions at energies 70 and 15 eV of the compounds are listed in Tables 2,8,11 and 14 while the CI mass spectra of the compounds under investigation are listed in Table 6.
The fragmentation pathways of the main fragment ions formed from molecular ions at 70 eV are rationalized in schemes 1,3,5 and 7 while the fragmentation of the protonated molecules of the compounds using CI mode are rationalized in schemes 2,4,6 and 8. 4.1.2. Computational Results:
Theoretical calculations are used for the physical properties of the molecules and the gas phase basicity. The calculations are performed using semi-emperical molecular orbital procedure. The program used in these computations is namely HyperChem™(43) in the modified neglect of diatomic overlap (MNDO) method. These calculations give useful information about the structure of the molecules, which actually used to support the experimental evidences. The most important parameters calculated using MNDO calculations include geometries, bond lengths, charge distribution and heats of formation +• + of the neutral ∆Hf(M),charged ∆Hf(M) and protonated molecule ∆Hf[M+H] . The ionization energy (IE) and proton affinity (PA) values for the studied molecules are calculated using equations 6 and 19(chapter 2).The protonation processes at different sites namely: N1 and O12 of the 6- fluoroquinolones in the gas phase are also calculated.
The bond lengths of neutral and ionized 6-fluoroquinolones molecules are given in Tables 4,10,13 and 16. Also, the charge distribution of the individual oxygen O12 and nitrogen N1 atoms for neutral and charged molecules are reported in Tables 3,9,12 and 15.
+ On the other hand, the calculated values of IE, ∆Hf(M) and ∆Hf(M) for all compounds are given in Table 5 while the values of the heats of formation of the + protonated molecules ∆Hf[M+H] together with the proton affinity of the molecules at oxygen O12 and nitrogen N1 sites are given in Table 7. It is interesting that the calculated IE energy values for the studied molecules have not been previously reported in the literature.
4.2. Discussion:
6-fluoroquinolones are an essential class of antibacterial compounds widely used in clinical application.(44) In 1986, the fluoroquinolones were introduced and they were modified from the class of antibiotics known as quinolones in early 1960. Initially, 6- fluoroquinolones were administrated orally for the treatment of infection caused by gram- negative organisms and pseudomonas species. Several 6-fluoroquinolones play a vital role for the treatment of community acquired pneumonia and intra-abdominal infections. Quinolones consist mainly of bicyclic ring structure and the different functional groups are substituted at position N1 (R1) such as ethyl, cyclopropyl and at C7 (R2) such as piperazyinyl as shown in Figure 2 and Table 1.
Figure 2. Structure of fluoroquinolones
Table 1. The different functional groups of 6-fluoroquinolones
Molecule R1 R2
Norfloxacin ethyl piperazinyl
Pefloxacin ethyl 4-methyl-piprazinyl
Ciprofloxacin cyclopropyl piperazinyl
Levofloxacin 1,8 heterocyclic ring 4-methyl-piprazinyl
The presence of different substituent groups is influenced not only the microbiological and physical properties but also the geometry of the neutral molecule which effect the thermochemical properties. The presence of different functional groups (45) at N1 or C7 positions influence both microbiological and pharmacokinetic properties. This may lead one to investigate and study the mass spectra, the fragmentation pathways, the heats of formation, the ionization energies and the appearance energies of some + + important fragment ions such as [M-CO2] and [M-C2H4N] for the four studied compounds.
The structures of the four compounds are shown in Figure 3 while the numbering system is shown in Figures 4. Figure 5 shows the possible protonation sites (oxygen O12 and nitrogen N1) of 6-fluoroquinolones.
O O
F OH
N N
N H3C CH3
PEFLOXACIN
O O
O O F
OH F OH N N
N N N O H3C CH3 HN
LEVOFLOXACIN CIPROFLOXACIN
Figure 3. The structures of Norfloxacin , Pefloxacin , Ciprofloxacin and Levofloxacin
Figure 4. The numbering system of 6- fluoroquinolone compounds used in the study.
Figure 5. The protonation sites at oxygen atoms and nitrogen (N1) for 6-fluoroquinolone compounds
4.2.1. Mass spectra of Norfloxacin using EI technique:
Electron ionization technique is the oldest and best characterized of all the ionization methods. In this technique, a beam of electrons passes through the gas-phase of the sample. An electron that collides with a neutral analyte (M) molecule can knock off an electron, resulting in a positively charged ion.(46)The ionization process can produce a molecular ion (M)+• which will has the same molecular weight and elemental composition of the starting analyte, and it can produce a fragment ion(s) which corresponds to a smaller piece of the analyte molecule as described in equations 1and 3( chapter 2).
Most mass spectrometers use electrons with energy of 70 electron volts (eV) for recording mass spectra. Decreasing the electron energy can reduce the fragmentation processes, but it also reduces the number of ions formed.(46) In this study, 70 and 15 eV electron energies were used to study the fragmentation processes. The mass spectra of Norfloxacin at these two energies are shown in Figures 6-7 in the range from m/z 56 to m/z 320
Norfloxacin C16H18FN3O3 (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)-3- quinoline carboxylic acid) is a synthetic 6-fluoroquinolone antibiotic which is structurally (47) related to nalidixic acid. The addition of a fluorine atom at C6 and a piperazine ring at (48) C7 has increased its potency in contrast to other fluoroquinolones.
Table 2. The molecular ion (M)+• and the main fragment ions [m/z] with their relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Norfloxacin.
m/z 70 e V 15 e V
320 12 11 319 [M]+· 70 60 278 9 7 + 277 [M-C2H4N] 59 52 276 16 14 + 275 [M-CO2] 100 100 245 9 5 234 13 9 + 233[M-CO2-C2H4N] 88 68 219 5 2 218 9 4 217 4 7 204 11 8 203 13 7 190 6 2 189 6 2 176 6 2 175 6 2 161 7 - 95 6 - 85 6 - 83 7 - 81 6 - 73 6 - 71 8 - 69 7 - 57 14 - 56 15 8
4.2.2. Ionization processes of Norfloxacin using EI technique:
Ionization processes depend on the chemical environment of the atom from which the electron is removed. These have been found to correlate with such chemical parameters as atomic charge, electronegativity, reactivity, effect of substituent parameters, proton affinities and with the predictions of a wide range of semi-empirical parameters used in electronic structure calculations.(49)
The removal of an electron from an organic molecule often leads to change in its relative thermodynamic stability. The energy necessary to produce [M]+• depends on the energy of the electron expelled. The process of ionization occurs mainly in molecules which contain a highly electronegative atoms or groups.
Norfloxacin molecule has a highly electronegative atoms O12 (-0.298 e) and N1 (- 0.309 e) as shown in Table 3. From the calculated charge distributions at N1 and O12 in the qinolone ring of Norfloxacin and the presence of a lone pair electrons at these atoms, one can suggest that the ionization processes may occur at these atoms. The calculated value of the ionization energy of Norfloxacin is equal to 8.10 eV (782 kJ.mol-1) (using equation 6, chapter 2).
Table 3. Calculated charge distribution of neutral and charged Norfloxacin molecule using MNDO method together with the charge difference (∆). Atom Neutral molecule Charged molecule ∆ (e) (e) N1 -0.309 -0.287 0.022 C2 0.199 0.175 -0.024 C3 -0.264 -0.202 0.062 C4 0.338 0.314 -0.024 C4a -0.119 -0.078 0.041 C5 -0.015 0.000 0.015 C6 0.102 0.137 0.035 C7 0.082 0.173 0.091 C8 -0.050 -0.145 -0.095 C8a 0.096 0.190 0.094 4.2.3. C20 0.160 0.149 -0.011 C21 .002 -0.004 -0.006 Fragm C9 0.417 0.399 -0.018 entatio O10 -0.295 -0.291 0.004 n of O11 -0.367 -0.324 0.043 O12 -0.298 -0.230 0.068 Norflo F13 -0.169 -0.128 0.041 xacin N14 -0.395 -0.168 0.227 using C15 0.127 0.082 -0.045 C16 0.095 0.103 0.008 EI N17 -0.320 -0.339 -0.019 techniq C18 0.095 0.105 0.010 ue: C 0.127 0.078 -0.049 19
Most previous research focused on the detection of Norfloxacin in biological samples (21,48,50-51) where other research focused on crystal investigation of Norfloxacin(52-56) However, the present study is interested in the ionization and fragmentation of the compound under electron ionization. Therefore, the main fragmentation pathways at 70 eV for Norfloxacin have been reported in scheme 1 and examined in order to understand the principle of their electron-induced cleavage.
The EI mass spectra of Norfloxacin are recorded at both 15 and 70 eV electron energies. Table 2 contains the relative intensities of the molecular ion and the main fragment ions from m/z 56 up to m/z 319. The molecular ion of Norfloxacin at m/z 319 is observed in the spectra at both 70 eV and 15 eV with relative intensities 70% and 60%, respectively.
The first characteristic fragmentation pathway for Norfloxacin is the formation of the +• [M-CO2] ion at m/z 275 (RI =100% i.e. the base peak in the mass spectrum) . This ion is formed by the elimination of CO2 from the carboxyl group attached to carbon C3 atom of the molecular ion following the migration of hydrogen atom from the OH group to C3 (Scheme 1). This is confirmed by the calculated bond length for C3─C9 in the charged molecular ion which is greater than that of the neutral molecule by 0.0078Å as reported in +• Table 4. The relatively high intensity of [M-CO2] ion indicates the high stability of the +• ion. The positive charge on the oxygen O12 stabilized the [M-CO2] fragment (odd- electron fragment ion). It is worth noting that the fragmentation of the molecular ion to +• produce the fragment [M-CO2] ion is the most favorable process. The fragment [M- +• CO2] ion undergoes further fragmentation resulting in the formation of the fragment [M- + • CO2- C2H4N] ion at m/z 233(RI=88%) by loss of [C2H4N] radical.
The second characteristic fragmentation pathway for Norfloxacin is due to the + formation of the fragment [M-C2H4N] ion at m/z 277 with RI= 59 % by the loss of • C2H4N radical from piperazinyl group. This is confirmed by the calculated bond length for C15─C16 and C18─C19 in charged molecular ion which are greater than that of the neutral + molecule by 0.0098Å and 0.0044Å, respectively, (Table 4).The fragment [M-C2H4N] ion undergoes further fragmentation resulting in the formation of the fragment [M- C2H4N- + + CO2] ion at m/z 233 with RI=88% by the loss of CO2 from [M-C2H4N] ion through simple cleavage of C3-C9 bond and rearrangement of hydrogen atom from O10-H to C3 (Scheme 1).
The peak observed at m/z 218 (RI= 9%) in the mass spectrum of Norfloxacin might be + due to the loss of CH3 radical from the fragment [M-CO2- C2H4N] ion. Also, the fragment + ion at m/z 218 can be formed directly from [M-CO2] by loss of the neutral fragment C3H7N (Scheme 1).
+• It is interesting to calculate thermochemical quantities such as ∆Hf(M) and ∆Hf(M) for Norfloxacin using the MNDO method. Hence, one can calculate the IE value for +• Norfloxacin (using equation 6, chapter 2) as the difference between ∆Hf(M) and ∆Hf(M) leading to calculated value of IE (Norfloxacin) equal to 8.10 eV (782 kJ.mol-1) (Table 5). The ionization of Norfloxacin probably occurs as a result of a removal of one of the lone pair electrons of N1or O12 which may be confirmed by the calculated values for charge difference (∆) at N1and O12 (0.022e and 0.068 e, respectively) as listed in Table +• 3.To the best of knowledge, no experimental or theoretical values for ∆Hf(M) and ∆Hf(M) for Norfloxacin were reported in the literature.
+ +
O O O H F F O H - - CO2 C N N 3 H N HN N 7 N HN rearrangement +
O
M F H m/z = 319 m/z = 275 R.I = 70 % R.I = 100 % N N s
C i
m C
2
p H 2
H
l
e 4
N 4 c m/z N = 218
l
e
a R.I = 9%
v
a
O O g e O F - CO2 O H F H rearrangement N N N N
m/z = 233 m/z = 277 R.I = 88% R.I = 59%
Scheme 1. Main fragmentation pathways of Norfloxacin at 70 eV
Table 4. Calculated bond lengths of neutral and charged Norfloxacin using MNDO method together with the bond length difference (∆ L).
Neutral molecule Charged molecular ion
Bond Bond Length(Å) Bond Bond Length(Å) ∆ L
N1-C2 1.4007 N1-C2 1.4167 0.0160 C2-C3 1.3700 C2-C3 1.3650 -0.0050 C3-C4 1.4881 C3-C4 1.4863 -0.0018 C4-C4a 1.5012 C4-C4a 1.5080 0.0068 C4a-C5 1.4185 C4a-C5 1.4118 -0.0067 Table C5-C6 1.4294 C5-C6 1.4299 0.0005 5. C -C 1.4433 C -C 1.4807 0.0374 6 7 6 7 Calcul C -C 1.4203 C -C 1.4391 0.0188 7 8 7 8 ated C8-C8a 1.4304 C8-C8a 1.4142 -0.0162 ∆Hf(M C8a-N1 1.4204 C8a-N1 1.3980 -0.0224 ),∆Hf( N1-C20 1.4830 N1-C20 1.4934 0.0104 M)+•an C20-C21 1.5379 C20-C21 1.5374 -0.0005 C3-C9 1.4950 C3-C9 1.5028 0.0078 d IE C9-O10 1.3571 C9-O10 1.3527 -0.0044 values C9-O11 1.2304 C9-O11 1.2268 -0.0036 for the C4-O12 1.2275 C4-O12 1.2237 -0.0038 four C6-F13 1.3223 C6-F13 1.3126 -0.0097 6- C7-N14 1.4306 C7-N14 1.3740 -0.0566 fluoro N14-C15 1.4706 N14-C15 1.4962 0.0256 quinol C15-C16 1.5517 C15-C16 1.5615 0.0098 one C16-N17 1.4676 C16-N17 1.4567 -0.0109 molec N17-C18 1.4677 N17-C18 1.4571 -0.0106 ules C18-C19 1.5514 C18-C19 1.5558 0.0044 using C -N 1.4706 C -N 1.4935 19 14 19 14 0.0229 MND O method.
+• Molecule ∆Hf(M) ∆Hf(M) IE
-1 -1 k J. mol k J. mol eV k J. mol-1
Norfloxacin -485 305 8.1 782
Pefloxacin -464 314 8 772
Ciprofloxacin -368 485 8.8 849
Levofloxacin -631 142 8.3 801
+• The appearance energy (AE) of the fragment [M-CO2] ion at m/z= 275 is calculated +• (using the heats of formation of ∆Hf (M) molecule , ∆Hf (CO2) and ∆Hf [M-CO2] ) and is found to be equal 8.30 eV(801 kJ.mol-1). The difference between the value for AE [M- +• -1 CO2] and IENorfloxacin=8.10 eV (782 kJ.mol ) gives the activation energy to produce the +• -1 fragment [M-CO2] ion as equal to 19 kJ.mol (0.20eV) . This indicates that the process +• forming the fragment [M-CO2] ion is the first fragmentation process as discussed in section 4.2.3 and illustrated in scheme 1 by the author.
+ Similarly, the appearance energy of the [M-C2H4N] ion at m/z=277 is calculated (using the heats of formation of the neutral molecule ∆Hf (M), neutral fragment ∆Hf + -1 (C2H4N) and ∆Hf (M-C2H4N) ) and is found to be equal to 9.4 eV (907 kJ.mol ). The + -1 difference between the value for AE [M-C2H4N] and IENorfloxacin=8.10 eV (782 kJ.mol ) + gives the activation energy to produce the fragment [M-C2H4N] ion as equal to 125 -1 + kJ.mol (1.30 eV) which is larger than the activation energy for forming [M-CO2] ion by 106 kJ.mol-1(1.10 eV). This indicates that the process forming the fragment [M- + C2H4N] ion is the second fragmentation process as discussed in section 4.2.3 and illustrated in scheme 1 by the author.
4.2.4. Mass spectrum of Norfloxacin using CI technique:
The chemical ionization technique was introduced as ionization method by Munson and Field in 1966(57) by allowing a reagent gas into an EI source. The pressure in this technique is typically 1 torr . The chemical ionization technique is usually defined as a soft-ionization method. This means that the energy deposition into the molecule is thought to be less than that present in the electron ionization mode. This is reflected on the occurrence and / or on the yield of ions formed by fragmentation processes, which will be less than the fragments in EI method. Hence, the mass spectra of CI are much less complex than the EI spectra and few fragmentations are observed. An ion at m/z [M+H]+ is the base peak in more spectra of these studied molecules. This is an important advantage since this allows one to focus on the molecular ion. In the case of the chemical ionization mass spectrum of Norfloxacin (Figure 8) methane was used as the reagent gas in the ionization chamber.
Table 6. Protonated molecules [M+H]+ and major fragment Ions [m/z] with their relative intensity [%] for chemical ionization mass spectra of Norfloxacin, Pefloxacin, Ciprofloxacin and Levofloxacin at 70 e V.
Norfloxacin Pefloxacin Ciprofloxacin Levofloxacin m/z RI[%] m/z RI[%] m/z RI[%] m/z RI[%]
97 17 130 7 163 8 318 14 218 15 319 9 236 32 320 31 237 6 321 7 250 6 344 5 252 5 346 6 258 7 348 7 264 9 360 9 270 8 361 9 272 5 362[M+H]+ 100 274 6 363 20 + 275 9 376[M+CH3] 5 + 276 60 276 10 390[M+C2H5] 19 277 23 287 8 278 55 288 52 279 13 289 6 289 14 290 10 290 31 290 17 292 9 291 9 302 5 302 8 292 12 304 5 304 20 316 10 314 6 306 12 317 5 316 18 316 5 318 27 317 9 318 10 319 6 318 38 320[M+H]+ 100 332 6 319 9 321 18 333 6 320 14 + + 334[M+CH3] 14 334[M+H] 100 330 5 + + 348[M+C2H5] 20 335 19 332[M+H] 100 346 6 333 18 + 348[M+CH3] 5 334 6 + + 362[M+C2H5] 21 346[M+CH3] 17 + 360[M+C2H5] 23 361 6
4.2.5. Chemical ionization, proton transfer:
Gas-phase proton transfer reactions have been the subject of quantitative studies for more than twenty years and the two fundamental aspects (their thermochemistry and kinetics) are still under active investigation.(58)
Among the wide variety of possible ionization reactions, the most common is proton transfer. Indeed, when analyte molecules M are introduced in the ionization plasma, the + product CH5 ions of the methane reagent gas can transfer a proton to the molecules M producing the protonated molecular ions [M+H]+. This chemical ionization reaction can be described as an acid–base reaction. The tendency for a reagent ion to protonate a particular analyte molecule M may be assessed from its proton affinity values. The observation of protonated molecular ions[M+H]+ implies that the analyte molecule M has a proton affinity much higher than that of the reagent gas (PA(M)>PA(CH4)). If the reagent gas has a proton affinity much higher than that of an analyte (PA(CH4)>PA(M)), + (59) proton transfer from CH5 to M will be energetically unfavorable.
4.2.6. Fragmentation of Norfloxacin using CI technique:
The fragmentation processes in CI spectrum of Norfloxacin are easily distinguished. The protonated molecule [M+H]+ at m/z 320 of Norfloxacin is formed under CI condition with a strong molecular cationic species (RI=100%) (Figure 8). A number of authors( 21,48,60) have detected Norfloxacin in biological samples using different mass spectrometric techniques and detected the protonated molecule at m/z = 320 by CI technique.
The protonated molecule [M+H]+ of Norfloxacin undergoes fragmentation along two different fragmentation pathways. The first fragmentation pathway is the formation of the + fragment [MH- CO2] ion at m/z 276 (RI=60%) which is formed by loss of CO2 as shown in scheme 2.
The second fragmentation pathway in the CI mass spectrum of Norfloxacin is the formation of the fragment ion at m/z 278 (RI=55%) which probably formed by loss of • + neutral radical C2H4N from the protonated molecule to form the fragment [MH- C2H4N] ion.
+ Two peaks observed at m/z 334 and 348 are probably formed due to the methyl (CH3) + + and the ethyl (C2H5 ) cations transfer processes to form methylated [M+CH3] and + ethylated [M+C2H5] molecules with relative intensities 14 and 20%, respectively. Further, the peak observed at m/z 302(RI=8%) is formed by elimination of H2O from the protonated molecule. At the same time the protonation process may occurs at O11 or O10 but the distance between the attached proton and – OH of the carboxylic group is larger (61) than 1.8 Å indicating that the probability of the loss of H2O is low.
+ H+ H O O O H F F O H - CO2 N N N HN N rearrangment HN
[M+H]+ = 320 m/z = 276 - H R.I = 100 % 2 O R.I = 60%
C
2
H
4
N
H+ H+ O O O O F O H F
N N N HN N
m/z = 278 m/z = 302 R.I = 55% R.I = 8 %
Scheme 2. Main fragmentation pathways of Norfloxacin using CI technique
4.2.7. The proton affinity (PA) , heat of formation(∆Hf) and the charge distributions of Norfloxacin:
Proton affinities and heats of formation are important thermodynamic quantities that can be derived from a variety of experimental measurements. Modern computational methods provide the means to estimate reliably the same quantities with an accuracy that often rivals that of experiment.(62) In addition, these methods can provide information to complement results obtained experimentally and to examine problems that are not easily approached directly, such as site-specific proton affinities.(62) A case in point is the protonation of the molecules, which often can take place at more than one position. Computational studies can provide a reliable estimate of the proton affinity of molecules as well as a measure of the thermodynamic difference between the various possible points of attachment of the proton.(62)
One of the computational studies, is the calculated charge distributions of the quinolone ring (pyridinyl) in neutral and charged Norfloxacin molecule (Table 3).The charges on the atoms N1,and O12 (-0.309e and -0.298e, respectively) indicate high electronegativity values. So, these atoms (sites) have a higher affinity to attach the proton than the other atoms in the ring. The calculated values of proton affinities (using equation -1 19 chapter 2 ) for O12 and N1 ( 904 and 749 kJ.mol respectively) together with the heats + of formation of the protonated Norfloxacin molecule, ∆Hf [M+H] at the site N1-H (297 -1 -1 kJ.mol ) and at the site O12-H (142 kJ.mol ) indicate that the protonated Norfloxacin -1 molecule at O12 is more stable than at N1by 155 kJ.mol . One may suggest that the electrostatic interaction between the proton and oxygen O12 stabilized the protonated species.
+ Table 7. Calculated heat of formation values for the protonated molecules ∆Hf(M+H) and proton affinities (PA) at O12 and N1 sites for 6-fluoroquinolone drugs using MNDO method.
+ Molecule ∆Hf(M) Protonated Site ∆Hf(M+H) PA (M) kcal.mol-1 kJ.mol-1 kJ.mol-1
O ─H 142 904 12 Norfloxacin -116 N1─H 297 749
O12─H 151 916 Pefloxacin -111 N1─H 314 753
O12─H 255 904 -88 N1─H 418 745 Ciprofloxacin 4.3.1. Mass
O12─H 2 908 spectr Levofloxacin -149 N1─H 192 715 a of Peflox
acin under EI technique:
Pefloxacin C17H20FN3O3 (1-ethyl-6-fluoro-1-4-dihydro-4-oxo-7(4-methyl-1- piperazinyl) quinolone-3-carboxylic acid) is a second-generation 6-fluoroquinolone antibacterial agent.(63)Most previous research is focused on the detection of Pefloxacin in environmental samples.(53) However, this work is interested in the structure, thermodynamic properties and fragmentation processes of Pefloxacin compound under electron ionization (EI) at two electron energies. The EI mass spectra of Pefloxacin are reported at 70 and 15 eV electron energies and are shown in Figures 9-10 while the relative intensities of the molecular ion and different fragment ions in the mass spectrum relative to the base peak (m/z 289) in the range from m/z 56 up to m/z 333 are listed in Table 8.
Table 8. The molecular ion (M)+• and the main fragment ions [m/z] with their relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Pefloxacin.
m/z 70 eV 15 eV
334 12 10
333 [M]+· 61 55
291 3 3
290 18 21
+ 289 [M-CO2] 100 100
245 4 4
219 6 6
+ 218 [M-CO2-C4H9N] 14 16
+ 203 [M-CO2-C4H9N-CH3] 11 10
96 7 10
81 4 6
79 6 10
+• 71 [C4H9N] 20 30
70 15 26
57 6 7
56 4 4
4.3.2.Ionization Process of Pefloxacin using EI technique:
Pefloxacin molecule has a highly electronegative atoms O12 (-0.300 e) and N1(- 0.307e) as listed in Table 9. From these charge distributions at N1 and O12 in the qinolone ring (pyridinyl) of Pefloxacin and the presence of a lone pair electrons at N1 and O12, one can suggest that the ionization process occur at these atoms.
On the other hand, the calculated ionization energy of Pefloxacin is found to be equal to 8.00 eV (772 kJ mole-1) (using equation 6, chapter 2). This value is nearly within experimental error equal to the ionization energy (8.10 eV) of Norfloxacin (Table 5).
Table 9. Calculated charge distribution of neutral and charged Pefloxacin molecule using MNDO method together with the charge difference (∆).
Atom Neutral molecule Charged molecule ∆ (e) (e)
N1 -0.307 -0.297 0.010 C2 0.198 0.178 -0.020 C3 -0.262 -0.206 0.056 C4 0.337 0.318 -0.019 C4a -0.119 -0.082 0.037 C5 -0.016 0.000 0.016 C6 0.103 0.124 0.021 C7 0.081 0.218 0.137 C8 -0.050 -0.171 -0.121 C8a 0.095 0.190 0.095 C21 0.162 0.151 -0.011 C22 -0.002 -0.003 -0.001 C9 0.4 5 0.401 -0.014 O10 -0.301 -0.293 0.008 O11 -0.357 -0.328 0.029 O12 -0.300 -0.228 0.072 F13 -0.169 -0.124 0.045 N14 -0.391 -0.164 0.227 C15 0.128 0.114 -0.014 C16 0.143 0.124 -0.019 N17 -0.428 -0.418 0.010 C19 0.144 0.126 -0.018 C20 0.128 0.114 -0.014 C 0.193 0.183 -0.010 18
4.3.3. Fragmentation of Pefloxacin using EI technique:
The molecular ion of Pefloxacin at m/z 333 is observed in the EI mass spectrum with relative intensities 61 % and 55 % at 70 and 15 eV, respectively.
The first characteristic fragmentation pathway for Pefloxacin is the formation of the fragment ion at m/z 289 (RI = 100%) which represents the base peak in the mass +• spectrum and is certainly due to the formation of the fragment [M-CO2] ion by the loss of CO2 from the carboxyl group attached to carbon C3 atom of the molecular ion as shown in scheme 3.This is confirmed by the calculated bond length for C3─C9 in charged Pefloxacin molecular ion which is greater than that of neutral molecule by + 0.0055Å (Table 10).The fragment [M-CO2] ion undergoes further fragmentation +• resulting in the formation of the fragment [M-CO2- C4H9N] ion at m/z 218 with relative intensities 14% and 16% at 70 and 15 eV, respectively. The latter fragment [M- +• • CO2- C4H9N] ion fragments to produce the ion at m/z 203 (by loss of CH3 radical) with relative intensities 11% and 10 % at 70 and 15 eV, respectively.
The second characteristic fragmentation process is due to the cleavage of the piperiazinyl group at C15-C16 and C20-N14 bonds to produce the fragment ion at m/z 71 +• [C4H9N] ion directly from the molecular ion with relative intensities 20 % at 70 e V and • 30 % at 15 eV together with neutral fragment [M-C4H9N] (Scheme 3). On the other + hand, the presence of –CH3 group at N17 lead to produce the fragment [M-C2H4N] ion at +• m/z = 291, RI=3% and also to produce the fragment C4H9N at m/z=71, RI=20 % in compariso + + O O O H n with F F O H Norfloxaci - CO2 n, the N N N N N N H3C absence of M –CH3
m/z =333 m/z =289 group lead R.I = 61% R.I = 100 % to produce the
C
4 H fragment
9 N [M- + + C2H4N] O F H ion at m/z = 277, H C 2 N N RI=59%. H2 C m/z = 218 H3C N CH2 R.I = 14%
C -
H2 C H
3 t
n
e
m
g
n O
a r a
e F H r Scheme 3. H H C + 2 2 N N Main C H N CH2 fragmentat CH2 m/z =203 ion H2C R.I = 11% C pathways H 2 of + C4H9N Pefloxacin m/z = 71 R.I=20% at 70 eV at 70 eV R.I=30% at 15 eV
O O F O H H C 2 N N Table 10. Calculated bond lengths of neutral and ionized Pefloxacin using MNDO method together with the bond length difference (∆ L) .
Neutral molecule Molecular ion ∆ L Bond Bond Length(Å) Bond Bond Length(Å)
N1-C2 1.4016 N1-C2 1.4180 0.0164
C2-C3 1.3689 C2-C3 1.3665 -0.0024 The C3-C4 1.4885 C3-C4 1.4877 -0.0008 appearance C4-C4a 1.5009 C4-C4a 1.5072 0.0063 energy (AE) C4a-C5 1.4182 C4a-C5 1.4101 -0.0081 of the C5-C6 1.4295 C5-C6 1.4324 0.0029 C -C 1.4435 C -C 1.4928 0.0493 fragment 6 7 6 7 +• C7-C8 1.4208 C7-C8 1.4547 0.0339 [M-CO2] C8-C8a 1.4299 C8-C8a 1.4088 -0.0211 ion at C8a-N1 1.4220 C8a-N1 1.3994 -0.0226 m/z=289 is N1-C21 1.4819 N1-C21 1.4922 0.0103 calculated C21-C22 1.5379 C21-C22 1.5372 -0.0007 (using the C3-C9 1.4953 C3-C9 1.5008 0.0055 heats of C9-O10 1.3581 C9-O10 1.3534 -0.0047 formation of C9-O11 1.2293 C9-O11 1.2273 -0.0020 ∆Hf(M) C4-O12 1.2274 C4-O12 1.2226 -0.0048 molecule, C6-F13 1.3223 C6-F13 1.3114 -0.0109 ∆Hf (CO2) C7-N14 1.4312 C7-N14 1.3624 -0.0688 and ∆Hf [M- +• N14-C15 1.4704 N14-C15 1.5031 0.0327 CO2] )and C15-C16 1.5521 C15-C16 1.5508 -0.0013 is found to C16-N17 1.4676 C16-N17 1.4609 -0.0067 be equal to N17-C19 1.4677 N17-C19 1.4600 -0.0077 8.30 eV(801 -1 C19-C20 1.5526 C19-C20 1.5504 -0.0022 kJ.mol ). 0.0321 C20-N14 1.4699 C20-N14 1.5020 The 0.0099 N -C 1.4640 N17-C18 1.4739 17 18 difference between the
value for +• -1 AE [M-CO2] and IEPefloxacin= 8.00 eV (772 kJ.mol ) gives the activation energy to +• -1 produce the fragment [M-CO2] ion as equal to 29 kJ.mol (0.30 eV). This indicates that +• the process forming the fragment [M-CO2] ion is the first fragmentation process as discussed in section 4.3.3 and illustrated in scheme 3 by the author.
+ The appearance energy of the [M-C2H4N] ion is calculated (using the heats of formation of the neutral ∆Hf (M) molecule , neutral fragment ∆Hf (C2H4N) and ∆Hf (M- + -1 C2H4N) ) and is found to equal 8.70 eV(840 kJ.mol ). + The difference between the value for AE [M-C2H4N] and IEPefloxacin= 8.00 eV (772 -1 + kJ.mol ) gives the activation energy to produce the fragment [M-C2H4N] ion as equal to 68 kJ.mol-1 (0.7 eV. This indicates that the formation process of the fragment [M- + C2H4N] ion is the second fragmentation process as discussed in section 4.3.3 and illustrated in scheme 3 by the author.
4.3.4. Fragmentation of Pefloxacin using CI technique:
The CI mass spectrum of Pefloaxcin is recorded (Figure 11) and the protonated molecule [M+H]+ at m/z 334 is observed in the spectrum (represents the base peak, RI 100%).
The protonated molecule of Pefloxacin undergoes fragmentation along two different characteristic pathways. The first fragmentation process formed by the loss of CO2 + yielding the fragment [MH-CO2] ion at m/z 290 (RI=31%) while the second + fragmentation process is the formation of the fragment [MH- C2H4N] m/z 292 (RI = • 12%) by loss of C2H4N radical. Two peaks observed at m/z 348 and 362 in the CI + + spectrum of Pefloxacin are probably formed due to methyl (CH3) and ethyl (C2H5) + + cations transfer processes to form methylated [M+ CH3] and ethylated [M+ C2H5] molecules with relative intensities 5% and 21%, respectively. Furthermore, the peak observed at m/z 316 (RI=10%) is formed by elimination of H2O from the protonated molecule.
The peak observed at m/z 318 (RI=27%) may be formed by loss of CO2 molecule from ethylated molecule after migration of hydrogen atom to C3 atom forming the fragment +• [M+C2H5- CO2] ion.
+ + H O O H O H F F O H - CO2 N N N N N N
[M+H]+ m/z =290 m/z =334 R.I = 100% R.I = 31 % - C 2 H
4 N
-
H 2
O
+ O O H
+ F O H O H F C N N O N N N H3C
m/z = 316 m/z = 292 R.I = 10% R.I = 12%
Scheme 4. Main fragmentation pathways of Pefloxacin using CI technique
4.3.5. The proton affinity (PA), heat of formation (∆Hf) and charge distributions of Pefloxacin:
The study of the behavior of molecules in the gas phase has always been a relatively important area of research in mass spectrometry since it produces information about the properties of molecules such as PA,s and heats of formation.(64)The calculated charge distribution of the quinolone ring of the neutral and charged Pefloxacin are calculated and listed in Table 9. The charges on the atoms N1, O12 in the qinolone ring (pyridinyl) of Pefloxacin are -0.307 e and -0.300 e, respectively, indicating high electronegativity values. Consequently, these atoms have higher affinity to attach the proton than the other atoms in the qinolone ring (pyridinyl) of Pefloxacin molecule.
From the thermochemical calculated data (Table 7) of Pefloxacin, one can note that the calculated values of proton affinities at the site N1-H and at the site O12-H (753 and 916 kJ.mol-1 , respectively) together with the calculated heats of formation of the protonated + - Pefloxacin molecule ΔHf[M+H] at these two atoms (sites) (314 and 151 kJ.mol 1 ,respectively ) indicate that the protonated Pefloxacin molecule at O12 is more stable -1 than at N1 by value 163 kJ.mol .
4.4.1. Mass spectra of Ciprofloxacin using EI technique:
Ciprofloxacin C17H18FN3O3 (4-oxo-7-(1-piperazinyl)-6-fluor-1-cyclopropyl-1,4 dihydroquinolin-3-carbonic acid) is one of fluoroquinolone medications and is applied as an effective synthetic antibiotic for treating a wide range of infectious diseases.(65)It is one of nine fluoroquinolones approved in the Russian Federation.(65)
The EI mass spectra of Ciprofloxacin are reported at 70 and 15 eV electron energies and are shown in Figures 12-13. While the relative intensities of the molecular ion and different fragment ions in the mass spectrum relative to the base peak (m/z 287) in the range from m/z 56 up to m/z 332 are listed in Table 11.
Table 11. The molecular ion (M)+• and the main fragment ions [m/z] with their relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Ciprofloxacin.
m/z 70 eV 15 eV
332 14 11
331 [M]+ 74 62
290 13 11
+ 289 [M-C2H4N] 71 67
288 19 18
+ 287 [M-CO2] 100 100
246 11 11
+ 245 [M-CO2-C2H4N] 69 68
231 5 5
230 8 7
229 9 9
218 4 4
215 5 4
204 5 5
202 5 6
201 6 6
57 9 8
56 20 19
4.4.2.Ionization process of Ciprofloxacin using EI technique:
Ciprofloxacin molecule has a highly electronegative atoms s O12(-0.295 e) and N1 (- 0.277 e) as listed in Table 12. From the calculated charge distributions at N1 and O12 in the qinolone ring (pyridinyl) of Ciprofloxacin and the presence of a lone pair electrons at these two atoms, one can suggest that the ionization process may occur at these atoms.
+• It is interesting to calculate thermochemical quantities such as ∆Hf(M) and ∆Hf(M) for Ciprofloxacin using the MNDO method and consequently, one can calculate the IE +• value for Ciprofloxacin (using equation 6, chapter 2) as the difference between ∆Hf(M) and ∆Hf(M). The calculated value for IE of Ciprofloxacin is found to be equal to 8.80 eV(849 kJ.mol-1) (Table 5).To the best of knowledge, no experimental or theoretical +• values for ∆Hf[M] and ∆Hf[M] for Ciprofloxacin were reported in the literature.
Table 12. Calculated charge distribution of neutral and ionized Ciprofloxacin molecule using MNDO method together with the charge difference (∆).
Atom Neutral molecule Charged molecule ∆ (e) (e) (e) N1 -0.277 -0.125 0.152 C2 0.206 0.233 0.027 C3 -0.266 -0.179 0.087 C4 0.339 0.206 -0.133 C4a -0.117 -0.085 0.032 C5 -0.021 -0.029 -0.008 C6 0.133 0.147 0.014 C7 0.076 0.157 0.081 C8 -0.075 -0.059 0.016 C8a 0.100 0.116 0.016 C20 0.035 -0.042 -0.077 C21 -0.053 -0.026 0.027 C22 -0.057 -0.034 0.023 O10 -0.295 -0.285 0.010 C9 0.418 0.380 -0.038 O11 -0.370 -0.297 0.073 O12 -0.295 0.064 0.359 F13 -0.159 -0.137 0.022 N14 -0.387 -0.419 -0.032 C15 0.128 0.133 0.005 C16 0.098 0.099 0.001 N17 -0.312 -0.324 -0.012 C18 0.096 0.100 0.004 C 0.127 0.138 19 0.011
4.4.3. Fragmentation of Ciprofloxacin using EI technique:
Ciprofloxacin is a broad spectrum antibiotic belonging to the second generation quinolones. It is distributed widely and absorbed very well in different body tissues and fluids. It is widely used in various types of infections: urinary track, respiratory track, gastrointestinal track and also for skin and soft tissues infections as recommended by the Food and Drug Administration (FDA).(66)
The EI mass spectrum of Ciprofloxacin has three major fragment ions at m/z 289,287 and 245 beside the molecular ion at m/z 331.The main fragment ions and its relative intensities at 15 and 70 eV are listed in Table 11 while the mass spectra are shown in Figures 12-13 .The molecular ion observed at m/z 331with RI= 74 %.It is worth noting that Nakata et al.(67) had recorded the molecular ion peak of Ciprofloxacin with reasonable intensity.
The molecular ion [M]+• undergoes fragmentation by two different pathways. The first +• fragmentation pathway is the formation of the fragment [M-CO2] at m/z 287 with RI=100% which represents the base peak in the mass spectra at both 70 and 15 eV by the loss of CO2 through a C3-C9 simple bond cleavage and hydrogen migration from the carboxylic group to C3 atom as shown in scheme 5.This is confirmed by the calculated bond length for C3─C9 in charged molecular ion which are greater than that of neutral +• molecule by 0.013Å (Table 13). The relatively high intensity of [M-CO2] ion indicates +• the high stability of the ion. The fragment [M-CO2] ion undergoes further fragmentation by the cleavage of C15-C16 and C18-C19 bonds to produce the fragment ion + [M-CO2-C2H4N] at m/z 245 ( Scheme 5) with relative intensities 69% and 68% at 70 and 15 eV, respectively.
The second fragmentation pathway in the mass spectrum of Ciprofloxacin is the + formation of the fragment [M-C2H4N] ion at m/z 289 with relative intensities 71 and 67 % • at 70 and 15 e V, respectively. This fragment ion is due to the loss of C2H4N radical from +• the molecular ion [M] by C15-C16 and C18-C19 bond cleavage and hydrogen migration from piperazinyl group to C8 as shown in scheme 5. This is confirmed by the calculated bond lengths for C15─C16 and C18─C19 bonds in the charged molecular ion which are greater than that of neutral molecule by 0.0004Å and 0.0005Å, respectively, (Table 13). + The fragment [M-C2H4N] ion undergoes fragmentation by loss of CO2 to produce the + fragment ion [M-C2H4N-CO2] at m/z 245 (Scheme 5) with relative intensities 69% and 68% at 70 and 15 eV, respectively.
+ An ion at m/z 56 (C3H6N) with RI= 20%, 19 % at 70 and 15 eV, respectively is also reported in the spectrum of Ciprofloxacin and is formed by the cleavage of C15-C16 and C18-N17 bonds and hydrogen migration to N14 in piperazinyl group.
+ + O O O H F F O H - CO2 N H rearengment N N N N N H H H re a re n g m M = 287 e m/z n t H2 m/z = 331 R.I = 100 % C R.I = 74%
C CH2
2 C N H
2 4
H N C
4 N H2 O O O - CO m/z 56 2 F H R.I = 20% F O H N N N N
m/z = 245
m/z = 289 R.I = 69% R.I = 71%
Scheme 5. Main fragmentation pathways of Ciprofloxacin at 70 eV
Table 13. Calculated bond lengths of neutral and ionized Ciprofloxacin using MNDO method together with the bond length difference (∆ L).
Neutral molecule Molecular ion ∆ L Bond Bond Length(Å) Bond Bond Length(Å) N1-C2 1.4017 N1-C2 1.3703 -0.0314 C2-C3 1.371 C2-C3 1.4123 0.0413 C3-C4 1.4908 C3-C4 1.4229 -0.0679 C4-C4a 1.5019 C4-C4a 1.4585 -0.0434 C4a-C5 1.4169 C4a-C5 1.4303 0.0134 C -C 1.431 C -C 1.4214 5 6 5 6 -0.0096 C6-C7 1.4441 C6-C7 1.4567 0.0126
C7-C8 1.4194 C7-C8 1.4189 -0.0005 C8-C8a 1.4281 C8-C8a 1.4311 0.0030 N1-C20 1.4534 N1-C20 1.4762 0.0228 C20-C21 1.5421 C20-C21 1.5436 0.0015 C21-C22 1.5186 C21-C22 1.5157 -0.0029 C22-C20 1.5420 C22-C20 1.5449 0.0029 C3-C9 1.4939 C3-C9 1.5069 0.0130 C9-O10 1.3571 C9-O10 1.3502 -0.0069 C9-O11 1.2308 C9-O11 1.2250 -0.0058 C4-O12 1.2269 C4-O12 1.3096 0.0827 C6-F13 1.3203 C6-F13 1.3167 -0.0036 C7-N14 1.4310 C7-N14 1.4102 -0.0208 N14-C15 1.4700 N14-C15 1.4754 0.0054 C15-C16 1.5530 C15-C16 1.5534 0.0004 C16-N17 1.4670 C16-N17 1.4645 -0.0025 N17-C18 1.4661 N17-C18 1.4636 -0.0025 C18-C19 1.5528 C18-C19 1.5533 0.0005 C -N 1.4701 C -N 1.4746 19 14 19 14 0.0045
+• The appearance energy of [M-CO2] ion( m/z = 287) is calculated (using the heats of formation of the neutral ∆Hf (M) molecule , neutral fragment ∆Hf (CO2) and ∆Hf (M- +• -1 CO2) ) and is found to be equal to 9.30eV(897 kJ.mol ).The difference between the +• -1 value for AE [M-CO2] and IECiprofloxacin= 8.80 eV (849 kJ.mol ) gives the activation +• -1 energy to produce the fragment [M-CO2] ion as equal to 48 kJ.mol (0.50 eV).This +• indicates that the process forming the fragment [M-CO2] ion is the first fragmentation process as discussed in section 4.4.3 and illustrated in scheme 5 by the author.
+ The appearance energy of [M-C2H4N] ion is calculated (using the heats of formation + of the neutral ∆Hf (M) molecule , neutral fragment ∆Hf (C2H4N) and ∆Hf [M- C2H4N] ) and is found to be equal to 10 eV(965 kJ.mol-1).The difference between the value for AE + -1 [M-C2H4N] and IECiproflxacin= 8.80 eV (849 kJ.mol ) gives the activation energy to + -1 produce the fragment [M-C2H4N] ion as equal to 115 kJ.mol (1.20 eV). This indicates + that the process of forming the fragment [M-C2H4N] ion is the second fragmentation process as discussed in section 4.4.3 and illustrated in scheme 5 by the author. .
4.4.4. Fragmentation of Ciprofloxacin using CI technique:
As mentioned before the molecular ion of Ciprofloxacin using EI technique was observed at m/z 331.Similary,the protonated molecule[M+H]+ was formed at m/z 332 using CI technique with RI= 100% which represents the base peak in the CI mass spectrum of Ciprofloxacin (Figure 14).
The protonated molecule [M+H]+ of Ciprofloxacin undergoes fragmentation along two different fragmentation pathways. The first fragmentation pathway is formed by the loss + of CO2 to form the fragment [MH-CO2] ion m/z 288 (RI=52%). The second + fragmentation pathway is the formation of the fragment [MH-C2H4N ] m/z • 290(RI=17%) by the loss of C2H4N radical as shown in scheme 6. On the other hand, a peak observed at m /z 314 (RI=6%) is formed by elimination of H2O from the protonated + molecule to form the fragment [MH-H2O] ion
Two peaks observed at m/z 346 and 360 in the CI mass spectrum of Ciprofloxacin are + + probably formed due to the methyl (CH3) and the ethyl (C2H5 ) cations transfer + + processes to form methylated [M+CH3] and ethylated [M+C2H5] molecules with relative intensities 17 and 23%, respectively.
H+ O O + O H H F F O H - CO2
N N N N N N H H
[M+H]+ = 332 m/z = 288 R.I = 100% R.I = 52%
C - 2 H H 2 O 4 N H+ O O F O H N N O O H+
F
N N N H m/z= 290 R.I = 17% m/z = 314 R.I = 6% Scheme 6. Main fragmentation pathways of Ciprofloxacin using CI technique
4.4.5. The proton
affinity (PA),heat of formation (∆Hf) and charge distributions of Ciprofloxacin:
The proton affinity of a molecule is one of its fundamental properties as its heats of formation and ionization energy.(68)
The calculated charge distributions of the quinolone ring of neutral and charged Ciprofloxacin are listed in Table 12. The charge on the atoms N1 and O12 are -0.277 e and -0.295 e, respectively indicating high electronegativity values. So, these atoms (sites) have higher affinity to attach the proton than the other atoms in the quinolone ring (pyridinyl) of Ciprofloxacin molecule.
From the thermochemical calculated data (Table 7) of Ciprofloxacin, one can observe that the calculated values of proton affinities of the sites N1-H and O12-H are 745kJ.mol-1 and 904 kJ.mol-1 respectively, together with the calculated heats of + formation of protonated molecule ∆Hf(M+H) at the sites N1-H and O12-H (418 and 255 -1 kJ.mol respectively,) indicating that the protonted molecule of Ciprofloxacin at O12 site -1 is more stable than that at N1 site by 163 kJ.mol .
4.5.1. Mass spectra of Levofloxacin using EI technique:
Levofloxacin C18H20FN3O4 (6-fluoro-2-methyl-6-(4-methylpiperazin-1-yl)-10-oxo-4 oxa-1-azatricyclo-7-trideca-5,7,11-tetraene,11-carboxylic acid) is a synthetic fluoroquinolone antibacterial agent.(69) Levofloxacin is the active levo-isomer of ofloxacin, and has a wide spectrum of antibacterial activity against both Gram-positive and Gram-negative bacteria, as well as a typical pathogens such as Mycoplasma, Chlamydia and Legionella.(70)
The EI mass spectra of Levofloxacin are recorded at both 70 and 15 eV electron energies as shown in Figures15-16 while the relative intensities of the molecular ion and the main fragment ions in the mass spectrum (relative to the base peak at m/z 361) in the range from m/z 56 up to m/z 362 are listed in Table14.
Table 14. The molecular ion (M)+• and the main fragment ions [m/z] with their relative intensities [%] at 70 and 15 eV electron energies in the mass spectra of Levofloxacin.
m/z 70 e V 15 e V
362 17 16
361[M]+• 100 100
+• 317[M-CO2] 17 14
276 5 5
273 7 5
254 6 5
247 12 9
+• 246[M-CO2-C4H9N] 22 19
232 11 8
231 10 7
+• 71(C4H9N) 29 34
56 4 -
4.5.2.Ionization process of Levofloxacin using EI technique:
Levofloxacin molecule has a highly electronegative atoms O12(-0.297 e) and N1(- 0.299 e) as listed in Table 15. From the charge distributions at N1 and O12 in the quinolone ring (pyridinyl) of Levofloxacin and the presence of a lone pair electrons at these two atoms, one suggest that the ionization processes occur at these atoms.
+• It is interesting to calculate thermochemical quantities such as ∆Hf(M) and ∆Hf(M) for Levofloxacin using the MNDO method and consequently, one can calculate the IE +• value for Levofloxacin (using equation 6, chapter 2) as the difference between ∆Hf(M) and ∆Hf(M). The calculated value for IE of Levofloxacin is found to be equal to 8.30 eV(801 kJ.mol-1) (Table 5).To the best of knowledge, no experimental or theoretical +• values for ∆Hf[M] and ∆Hf[M] for Levofloxacin were reported in the literature.
Table 15. Calculated charge distribution of neutral and ionized Levofloxacin molecule using MNDO method together with the charge difference (∆).
Atom Neutral molecule Charged molecule ∆ (e) (e)
N1 -0.299 -0.292 0.007 C2 0.201 0.181 -0.02 C3 -0.261 -0.209 0.052 C4 0.338 0.322 -0.016 C4a -0.099 -0.062 0.037 C5 -0.028 -0.005 0.023 C6 0.112 0.14 0.028 C7 0.092 0.081 -0.011 C8 0.079 0.104 0.025 C8a 0.051 0.057 0.006 O21 -0.227 -0.252 -0.025 C22 0.15 0.155 0.005 C24 0.091 0.088 -0.003 C23 0.016 0.011 -0.005 C9 0.416 0.402 -0.014 O10 -0.295 -0.291 0.004 O11 -0.366 -0.332 0.034 O12 -0.297 -0.244 0.053 F13 -0.167 -0.141 0.026 N14 -0.395 -0.081 0.314 C15 0.142 0.1 -0.042 C16 0.144 0.133 -0.011 N17 -0.429 -0.434 -0.005 C19 0.143 0.135 -0.008 C20 0.142 0.108 -0.034 C 0.194 0.188 -0.006 18
4.5.3. Fragmentation of Levofloxacin using EI technique:
The molecular ion peak of Levofloxacin at m/z 361 (represent the base peak ,RI=100 %) is observed in the mass spectra at 70 and 15 eV indicating the stability of the molecular ion of Levofloxacin due to the presence of heterocyclic substituent at N1 and C8 atoms .The molecular ion undergoes fragmentation by the loss of CO2 from the + carboxyl group to form the fragment [M-CO2] ion at m/z 317(RI=17%). This is confirmed by the calculated bond length for C3─C9 in the molecular ion which is greater + than that of neutral molecule by 0.0057Å. The fragment ion [M-CO2] undergoes further + fragmentation resulting in the formation of the fragment [M-CO2-C4H9N] ion at m/z 246 • ( RI=20% ) by loss of the neutral (C4H9N) (Scheme 7).
+ An ion at m/z 71 (C4H9N) is also found in the mass spectrum of Levofloxacin (RI=29 % and 34%) at 70 and 15 eV, respectively. This ion is formed by the cleavage of C15-C16 and C20-N14 bonds in 4-methyl piperizinyl with consecutive rearrangement process.
+• The appearance energy of the [M-CO2] ion m/z (317) is calculated (using the heats of formation of the neutral ∆Hf (M) molecule , neutral molecule ∆Hf (CO2) and fragment +• -1 ion ∆Hf (M-CO2) ) and is found to be equal = 8.50eV(820 kJ.mol ). The difference +• -1 between the value for AE [M-CO2] and IELevofloxacin= 8.30 eV (801 kJ.mol ) gives the +• -1 activation energy to produce the fragment [M-CO2] ion as equal to 19 kJ.mol (0.20 eV).
+ + H O O O F F H O CO2 N N N N H-Rearangment N O N O H C CH H3C CH3 3 3 M + m/z = 361 m/z = 317 R.I= 100% R.I= 17%
C4H9N
+ CH H 3 O F H N H2C N N N H C 2 CH2 H2C CH2 O CH3 C C H2 H2 m/z = 71 m/z = 246 Scheme 7. Main fragmentation R.I = 29% R.I = 20% pathways of Levofloxacin at 70 eV
Table 16. Calculated bond lengths of neutral and ionized Levofloxacin using MNDO method together with the bond length difference (∆ L).
Neutral molecule Molecular ion ∆ L Bond Bond Le ngth(Å) Bond Bond Length(Å)
N1-C2 1.3975 N1-C2 1.4088 0.0113 C2-C3 1.3719 C2-C3 1.3706 -0.0013 C3-C4 1.4898 C3-C4 1.4890 -0.0008 C4-C4a 1.5017 C4-C4a 1.5094 0.0077 C4a-C5 1.4187 C4a-C5 1.4207 0.0020 C5-C6 1.4314 C5-C6 1.4253 -0.0061 C6-C7 1.4450 C6-C7 1.4713 0.0263 C7-C8 1.4368 C7-C8 1.4520 0.0152 C8-C8a 1.4446 C8-C8a 1.4459 0.0013 C8a-N1 1.4153 C8a-N1 1.4031 -0.0122 C8-O21 1.3588 C8-O21 1.3472 -0.0116 O21-C22 1.4028 O21-C22 1.4140 0.0112 C22-C24 1.5634 C22-C24 1.5611 -0.0023 C24-N1 1.4823 C24-N1 1.4852 0.0029 C24-C23 1.5475 C24-C23 1.5482 0.0007 C3-C9 1.4961 C3-C9 1.5018 0.0057 C9-O10 1.3569 C9-O10 1.3532 -0.0037 C9-O11 1.2301 C9-O11 1.2275 -0.0026 C4-O12 1.2276 C4-O12 1.2239 -0.0037 C6-F13 1.3223 C6-F13 1.3164 -0.0059 C7-N14 1.4236 C7-N14 1.3877 -0.0359 N14-C15 1.4675 N14-C15 1.4978 0.0303 C15-C16 1.5526 C15-C16 1.5561 0.0035 C16-N17 1.4676 C16-N17 1.4620 -0.0056 N17-C19 1.4678 N17-C19 1.4630 -0.0048 C19-C20 1.5524 C19-C20 1.5521 -0.0003 C19-N14 1.4680 C19-N14 1.4955 0.0275 N -C 1.4632 N -C 1.4730 17 18 17 18 0.0098
4.5.4. Fragmentation of Levofloxacin using CI technique:
The CI mass spectrum of Levofloxacin is recorded and investigated. The resulting protonated molecule [M+H]+ at m/z 362 (RI= 100% ) represents the base peak (Figure 17).This protonated molecule [M+H]+ undergoes fragmentation along two different fragmentation pathways. The first fragmentation process is the formation of the fragment + [MH-CO2] ion with m/z 318 (RI= 14%) by loss of CO2. The second fragmentation + pathway leads to the formation of the fragment ion [MH-C2H4N] with m/z 320 (RI= • 31%) by loss of C2H4N from the protonated molecule. Another peak has been observed at m/z 344 (RI=5%) is formed by elimination of H2O from the protonated molecule to + form the fragment [MH-H2O] ion.
Two peaks observed at m/z 376 and 390 in the CI mass spectrum of Levofloxacin are + + formed due to the methyl (CH3) and the ethyl (C2H5 ) cations transfer processes to form + + methylated [M+CH3] and ethylated [M+C2H5] molecules with relative intensities 5 and 19%, respectively.
+• One can note that, the relative intensities of the [M+CH3] ions which are produced in all mass spectra of the studied molecules have low intensities in comparison with the +• + relative intensities of the [M+C2H5] ions indicating the presence of C2H5 ion with + large intensity than that of CH3 in the source of chemical ionization.
H+ H H+ O O O F F H O CO2 N N N N N O N O H C CH H3C CH3 3 3
[M+H]+ m/z = 318 m/z = 362 R.I= 14% R.I = 100% - C 2 H
- 4 N
H
2 O
+ + H H H H O O O O F F O O N N N N O N O CH3 H3C CH3
CH3 m/z= 320 m/z = 344 R.I = 31% R.I = 5%
Scheme 8. Main fragmentation pathways of Levofloxacin using CI technique
4.5.5. The proton affinity (PA), heat of formation (∆Hf) and charge distributions of Levofloxacin:
The calculated charge distributions of the quinolone ring(pyridinyl) of neutral and charged Levofloxacin molecule are calculated and listed in Table 7. The charges on the atoms N1 and O12 are -0.299e and -0.297 e respectively, indicating high electronegativity values. So, these atoms (sites) have higher affinity to attach the proton than the other atoms of the ring.
Using the thermochemical calculated data (Table 7) of Levoloxacin, one observes that the calculated values of the proton affinities at the sites N1-H and O12-H are 715 and 908 kJ.mol-1 , respectively, together with the calculated heat of formation of the protonated Levofloxacin molecule at these two atoms (sites ) N1 and O12 ( 192 and 2 -1 kJ.mol , respectively) indicate that the protonated Levofloxacin molecule at the site O12 is more stable than that at the site N1.
CHAPTER (5)
CONCLUSIONS
The present EI mass spectra of four 6- fluoroquinolones shows that the molecular ions [M]+• of Norfloxacin, Pefloxacin,Ciprofloxacin and Levofloxacin have relative intensities 70,61,74 and 100 %, respectively. The relatively high intensity of [M]+• for Levofloxacin indicates its higher stability due to the presence of heterocyclic substituent at N1 and C8 atoms. In comparison, the CI mass spectra of the four compounds show that the protonated molecules [M+H]+ are more relatively intense than [M]+• ions in the EI mode indicating higher stability in the case of [M+H]+ .
The important primary fragmentation pathways of the studied compounds in the EI mass spectra indicated:
+• (a) The fragment [M-CO2] ions produced from Norfloxacin, Pefloxacin and Ciprofloxacin have relative intensity 100% (representing the base peak) while Levofloxacin have only 17% relative intensity. + (b) The fragment [M-C2H4N] ions produced from Norfloxacin and Pefloxacin have relative intensities 59 and 71 %, respectively, while not appeared in the mass spectra of Pefloxacin and Levofloxacin.
On the other hand, semi-empirical MNDO calculations gave useful information which help in explaining the experimental events. The appearance and activation energies for the fragment ions in EI mass spectra are calculated for the first time. Of most important, the calculated results show that the protonation at O12 site is more favored than that at N1 site for the compounds under investigation.
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جبهعخ ثٌِب كليخ العلْم قسن النيزيبء
دراسة الطيف الكتىل ملركبات الفلوروكينولني ابس تخدام طريقىت الت أين ا أللكرتوىن والكميياىئ مصحوبة ابحلساابت الش به وضعية
هقدهت هي
هأهىى سسحبى هحوىد عبد الكسين هدزض هسبعد – قسن الطبيعت النىويت التجسيبيت هيئت الطبقت الرزيت-هصس
للحصىل على دزجت الدكتىزاة فى فلسفت العلىم-فيصيبء تجسيبيت
األستبذ الدكتىز / عصث طه هحود سلين أستبذ الفيصيبءالرزيت والجصيئيت –قسن الطبيعت النىويت األستبذ الدكتىز / هصطفى ﺇبساهين التجسيبيت هسكص البحىث النىويت الصعيقى هيئت الطبقت الرزيت-هصس أستبذ الفيصيبء النىويت –قسن الفيصيبء – كليت العلىم األستبذ الدكتىز / عبد الىهبة حسي جبهعت بنهب زشق السبع األستبذ الدكتىز / هحود عبد الفتبح زبيع أستبذ الفيصيبء – الوسكص القىهى لبحىث وتكنىلىجيب االشعبع أستبذ الفيصيبءالرزيت والجصيئيت –قسن الطبيعت النىويت التجسيبيت هيئت الطبقت الرزيت-هصس هسكص البحىث النىويت هيئت الطبقت الرزيت-هصس
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الملخص العربى
رممن ﺇسممزامام هايممبك ال زلممخ هممي مم ا SSQ 710 الوممزّد ثوحلممر ثممبعٔ االقاممبة ّف مممخ للزممريي ثا ي ممخ الزممريي اإلل ز ًّمممٔ لزسمممطير الايمممل ال زلمممٔ لمممجعو ه كجمممبد النلْ ّكيٌمممْليي ُّمممٔ الٌْ ملْكسبسممميي ّالجينلْكسبسممميي ّالسج ّملْكسبسيي ّالينْملْكسبسيي مٔ حبلخ الزريي االل ز ًّٔ. ّأهب الايل ال زلٔ لِذح الو كجمبد ممٔ حبلمخ الزمريي ال يويبئٔ م م رن رسطيلخ ثبسزامام هايبك ال زلخ هي ا .Thermo Finnigan TRACE DSQ GC/MS.
ثبلٌسجَ للزريي اإلل ز ًّٔ رن رسطير الايل ال زلٔ للو كجبد األ ثعخ عٌمم مبقزيي هازلنزميي ُّومب 72ّ15 ﺇل زم ّى مْلذ ( ّرن رسطير األيًْبد األسبسيخ للطزيئبد. كذلك رن ٳقز اح ّهٌبقشخ عوليبد الز سي الوازلنخ للو كجبد األ ثعخ +• + الوسزامهخ مٔ الم اسخ ّّجم أى أحم عوليمبد الز سمي رشمور االيًْمبد: -M-CO2] , [M-C2H4N] and [M] + CO2-C2H4N]
ثبلٌسجخ للزريي ال يويبئٔ رن ﺇسزامام ي خ الزريي ال يويبئٔ الوزّدح ثغب الويثبى الال م لعوليخ الزريي مٔ رسطير الايل ال زلٔ للو كجبد األ ثعخ. ّ هٌبقشخ الايل ال زلٔ للو كجبد األ ثعخ الٌبرطخ عي ي خ الزمريي ال يويمبئٔ ﺇعزومبدا علمٔ ر كيممت ُممذٍ الو كجممبد الٌممبري عممي الايممل ال زلممٔ لِممب مممٔ حبلممخ الزممريي اإلل ز ًّممٔ كو جممل لِممب . ّّجممم أى األيًْممبد األسبسيخ الزمٔ جمذثذ ه ٍ الِيمم ّجيي +[M+H] ممٔ الو كجمبد األ ثعمخ هاد بجمبد ّامماد عبليمخ%100 ثبلو ب ًمخ ثثجبد ّاماد األيًْبد االسبسيخ•+[M] مٔ حبلخ الزريي اإلل ز ًّٔ .
رن ايضب ٳقز اح ّهٌبقشخ عوليبد الز سي الوازلنخ للو كجبد األ ثعخ الوسزامهخ مٔ الم اسخ مٔ حبلخ الزريي ال يويمبئٔ ّّجم أى أحم عوليبد الز سي أدد الٔ ر ْيي االيًْبد: +• + + [MH-CO2] , [MH-C2H4N] and [MH-H2O]
رن الحصْل علٔ ال ثي هي الوعلْهبد عي الو كجبد األ ثعخ رحمذ الم اسمخ همي خمالل الايمل ال زلمٔ لِمب ممٔ حمبلزٔ الزريي اإلل ز ًّٔ ّال يويبئٔ ّهي ُذٍ الوعلْهبد : 1- ه الز سي الوازلنَ للو كجبد األ ثعخ. 2- بجبد األيًْبد الوز ًْخ . ل م أه ي أيضب الحصْل علٔ ال ثيم همي الوعلْهمبد عمي حم ا ح الز مْيي سمْاء الو كمت أّ أيمْى الو كمت ّ بقمبد الزريي للو كجبد األ ثعخ ثإسزامام الزاجي بد الوازلنخ لوي بًي ب ال ن ال يويبئيخ الوزوثلخ مٔ ي مخ ال-MNDO(semi (emiprical method ه ز ًخ ثبلٌزبئي الوعوليخ .
روذ هٌبقشخ الزربي الٌبري هي ّضل الزجميالد الٌِمسيخ مٔ اا لب الو كجبد األ ثعخ ّالمذٓ ظِم همي خمالل ﺇخمزالك ال ين الٌبرطخ ممٔ حسمبثبد حم ا ح الز مْيي ّ بقمبد الزمريي لِمب سمْاء ممٔ الحبلمخ الوزعبدلمخ اّ الحبلمخ األيًْيمخ . ّكمذلك هي بًي يخ الز سي لجعو ال ّاثط ال يويبئيخ هي خالل ال ين الوازلنخ لاْل ال ّاثط.
رمممن اسمممزامام ي مممخ MNDOممممٔ حسمممبة بقمممبد الزمممريي للو كجمممبد اال ثعمممخ الٌْ ملْكسبسممميي ّالجينلْكسبسممميي ّالسج ّملْكسبسيي ّالينْملْكسبسيي( ّالزٔ رسمبّٓ ال مين 8.1 8ّ ّ 8.8 8.3ّ ﺇل زم ّى مْلمذ علمٔ الز ريمت. ّقمم أه ي رنسي عوليخ الزريي ًزيطخ لٌزع أحم اإلل ز ًّبد األقر روبس ب مٔ ه رٔ الٌيز ّجييN1 اّ ه ح االّكسمطيي O12 الوْجْدح مٔ حل خ ال يٌْلْيي. جويل ال ين السبث خ لابقبد الزريي الزٔ رن حسبثِب ألّل ه ح ممٔ ُمذٍ الم اسمخ ّلمن رٌشم هي قجر .
أيضب رن حسبة ح ا ح الز ْيي الوازلنخ للو كجبد األ ثعخ مٔ الحبلخ الوزعبدلخ ّ الحبلخ األيًْيخ.
اسزامهذ ًنس الا ي خ ممٔ حسمبة عوليمخ ﺇضمبمخ الِيمم ّجيي للو كجمبد األ ثعمخ . ّأيضمب رمن حسمبة قبثليمخ ﺇسمز جبل الِيم ّجيي للو دكجب األ ثعمخ (PA's) عٌمم هْضمل ه رمٔ الٌزيزم ّجييN1 ّ عٌمم هْضمل ه ح االّكسمطيي O12 ّهلممك ل جمم الشممحٌخ السممبلجخ لِممذٍ الممذ اد مممٔ الو كجممبد األ ثعممخ . ّقممم أه ممي رنسممي الٌزممبئي علممٔ اى عوليممبد ﺇسممز جبل الِيم ّجيي مٔ الو كجبد األ ثعخ عٌم هْضل ه ح األّكسطييO12 أمضر همي ﺇسمز جبل الِيمم ّجيي عٌمم هْضمل ه ح الٌيزممممم ّجيي N1 . أيضمممممب رمممممن حسمممممبة حممممم ا ح الز مممممْيي لايًْمممممبد األسبسممممميخ للو كجمممممبد األ ثعمممممخ الوسمممممز جلخ + للِيم ّجيي [M+H] عٌم الوْاقل O12ّ N1 . جويمل ال مين السمبث خ الزمٔ رمن حسمبثِب لحم ا ح الز مْيي الحبلمخ الوزعبدلمخ + +• [Hf [M∆ ّ ممٔ الحبلمخ األيًْيمخ [Hf [M∆ ّأيضمب ممٔ حبلمخ الو كجمبد الوسمز جلخ للِيمم ّجيي [Hf [M+H∆ ّقبثليخ جذة الِيم ّجيي(PA's) رن حسبثِب ألّل ه ح ّلن رٌش سبث ب حيث رعزج ﺇضبمخ هِوخ مٔ الميٌبهي ب الح ا يخ لزلك الو كجبد.