neurotoxicity and mechanisms of induced hyperexcitability

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ORIGINAL ARTICLE ORIGINALNI NAUČNI RAD ORIGINAL ARTICLE ORIGINALNI NAUČNI RADSTRUCTURAL INVESTIGATION OF DEXTROMETHORPHANUSING MASS SPECTROMETRY AND THERMALANALYSES COMBINEDWITH MOCALCULATIONSM.A. Zayed *1 , M.A. Fahmey 2 , M.F. Hawash 2 , S.A.M. Abdallah 31Chemistry Department, Faculty of Science, Cairo University, 12316 Giza, Egypt.2Nuclear Physics Dept., N.R.C., Atomic Energy Authority 13759, Cairo, Egypt.3University of Workers, 10 Aswan, A.R. EgyptReceived / Primljen: 10. 03. 2011. Accepted / Prihvaćen: 07. 04. 2011.ABSTRACT(C 18H 25NO, mole mass = 271) was investigated usingthermal analyses (TA) measurements (TG/DTG and DSC)compared with EI mass spectral (MS) electron impact fragmentationat 70 eV of electron energy. Semi-empirical MOcalculations using the PM-3 procedure were performed withdextromethorphan (DMP) as a neutral molecule and the correspondingpositively charged ion. These included moleculargeometry (bond length), bond order, bond strain, charge distributionon different atoms, heat of formation, and ionisationenergy. The mass spectral fragmentation pathways and thermalanalyses decomposition were proposed and comparedwith each other to select the most suitable scheme. In electronionisation (EI) mass spectral fragmentation, the initial rup-ture is due to C 3H 7N (bridge) loss followed by C 9H 11O. In TA,the primary loss is due to loss of C 3H 7N Bridge + HBr (afterH 2O loss of crystallisation). TA revealed a high response ofthe drug to the temperature variation with very fast rate. Itdecomposed in several sequential steps in temperature rangefrom 100ºC to 600ºC. The initial thermal decomposition wassimilar to that obtained using MS. So, it is possible to selectthe proper pathway using both techniques. This comparisonwas successfully confirmed by MO calculation. The structuralreactivityeffect on metabolism and the biological effect comparedwith codeine are discussed.Keywords: Dextromethorphan structure, Mass Spectrometry,Thermal analyses, MO calculationINTRODUCTIONDextromethorphan (DMP, methoxy-methyl morphinan)is an antitussive agent used in many nonprescriptioncough and cold medications [1]. DM has an IUPACname (+)-3-methoxy-17-methyl-(9α, 13α, 14α)- morphinan(C 18H 25NO, m/z=271) and is a synthetic product thatis chemically related to codeine [2]. The drug is a safe, oralantitussive that is widely available [3 – 6]. The structural formulaand numbering system of the drug is given in Fig. 1.A number of methods have been reported to measureDM in biological fluids, including high-performance liquidchromatography (HPLC) [7, 8], gas chromatography (GC)[9, 10] and gas chromatography–mass spectrometry (GC–MS) [11-14, 1]. The latter technique is highly sensitive [5].Rapid advances in biological sciences have led to anincreased demand for chemical and structural informationfrom biological systems. Mass spectrometry plays apivotal role in the structural characterisation of biologicalmolecules [15]. The technique is important because it providesa substantial amount of structural information usinga small amount of sample. Moreover, the techniques offercomparative advantages in speed and productivity forpharmaceutical analysis [16]. In contrast, thermal analysisdelivers extremely sensitive measurements of heat change,which can be applied on a broad scale with pharmaceuticaldevelopment. These methods provide unique informationrelating to thermodynamic data of the system studied[17]. The increasing use of the combined techniques in TAcould provide more specific information and thus facilitiesa more rapid interpretation of the curves obtained [17].In the electron ionisation (EI) mass spectra, the fragmentationconsists of competitive and consecutive unimolecularfragmentation [18]. The fragmentation of the ionisedmolecule depends mainly on their internal energy [19]. Thethermo-gravimetric TG/DTG analysis provides quantitativeinformation on weight losses due to decomposition and/orevaporation of low molecular weight fragments as a functionof time and temperature. In conjunction with mass spectrometricanalysis [20-22], the nature of the released fragmentsUDK 615.233.074 / Ser J Exp Clin Res 2011; 12 (1): 21-28Correspondence to: mazayed429@yahoo.com, Tel: 00202-22728437, 002-010577665.21
EXPERIMENTALMass spectrometry (MS)Dextromethorphan drug (DMP)Figure 1. Structure and numbering system of DMP for, C, N and O skelton.may be deduced, thus greatly facilitating the interpretation ofthermal degradation processes. In contrast, the computationalquantum chemistry could provide additional informationabout the atoms and bonds, which could be used successfullyin an interpretation of experimental results [23]. The applicationof the computational quantum chemistry in addition toexperimental results (MS and TA) gives valuable informationabout the atoms and bonds, which helps in the descriptionand prediction of the primary fragmentation site of cleavageand subsequent ones [24-28].Although the literature is wealthy in information relatedto the biological activities of DMP and its metabolites in vivoand in vitro [3-6], there seems to be a lack of any correlationbetween chemical behaviour and its electronic structure.The main aim of this work was to perform an experimentaland theoretical investigation of DM using thermalanalyses (TA) and EI mass spectral (MS) fragmentation at70 eV. In addition, MO calculations were performed usingthe PM3 procedure on the neutral molecule and chargedmolecule to investigate geometrical parameters (bondlength, bond order, bond strain, heats of formation ionisationenergy) and charge distribution.The calculations correlated the experimental results[TA and MS] to obtained information regarding the stabilityof the drug and the prediction of the site of primaryfragmentation and subsequent ones. This study could behelpful in establishing a quantitative and qualitative structure–activityrelationship for the drug, which is the mainobjective of this work. It is worth mentioning that there isscant literature concerning such a comparative study.Moreover, the authors tried to correlate and discuss thestructure reactivity and relationship between DM and Codeine[26], which are chemically related to each other [2]and which affect the O and OH on MS and TA and MOparameters. In addition, they attempted to find the metabolitestructures in the mass spectrum and correlated thebiological behaviour of the two drugs.The electron ionisation (EI) mass spectrum of DMPwas obtained using a Shimadzu GC-MS-Qp 1000 PX quadruplemass spectrometer with an electron multiplier detectorequipped with the GC-MS data system. The directprobe for solid material was used in this study. The samplewas put into a glass sample micro vial by a needle (˜ 1 μgmax). The vial was installed on the tip of the DP containingthe heating cable and was inserted into the evacuatedion source. The sample was ionised by an electron beamemitted from the filament with the generated ions beingeffectively introduced into the analyser by the focusing andextractor lens systems. The MS was continuously scanned,and the spectra obtained were stored. The electron ionisationmass spectra were obtained at an ionising energyvalue of 70 eV, ionisation current of 60 μA and a vacuumgreater than 10 -6 tor r.Thermal analysis (TA)The thermal analyses of DMP were made using a conventionalthermal analyser (Shimadzu system of DTA-50and 30 series TG-50). The mass loss for 5 mg of sample andthe heat response to changes in the sample were measuredfrom room temperature up to 600ºC. The heating rate in aninert argon atmosphere was 10ºC min -1 . These instrumentswere calibrated using indium metal as a thermal stable material.The reproducibility of the instrument reading was determinedby repeating each experiment more than twice.Quantum chemical calculationsThe MO calculations were performed using a semiempiricalmolecular orbital calculation. The method usedin these computations was the parametric PM-3 methoddescribed by Stewart [29]. The geometry of all stable speciesstudied was completely optimised with respect to allgeometrical variables using the Eigen vector following (EF)routine [30]. The program was run under the molecularorbital calculation package MOPAC2000 by Stewart [31]for microcomputers.RESULTS AND DISCUSSIONThe chemistry and reactivity of pharmaceutical drugsare of great interest because of their importance in treatingvarious diseases. Knowledge of the thermal decompositionmechanism of the drug is very important to understandthe chemical processes in biological systems. It is difficultto establish the exact major fragmentation pathway in EIusing conventional MS. However, the combination of experimentaltechniques (TA and MS) and MO calculation isvery important to understand the following topics:22
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