Dissertation committee:
Ειρήνη Παντερή, Καθηγήτρια, Τμήμα Φαρμακευτικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ
Νικόλαος Θωμαΐδης, Καθηγητής, Τμήμα Χημείας, Σχολή Θετικών Επιστημών, ΕΚΠΑ
Βικτωρία Σαμανίδου, Καθηγήτρια, Τμήμα Χημείας, Σχολή Θετικών Επιστημών, ΑΠΘ
Ιωάννης Λουκάς, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ
Ιωάννης Ντότσικας, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, Σχολή Επιστημών Υγείας, ΕΚΠΑ
Αναστάσιος Οικονόμου, Καθηγητής, Τμήμα Χημείας, Σχολή Θετικών Επιστημών, ΕΚΠΑ
Ευάγγελος Γκίκας, Καθηγητής, Τμήμα Χημείας, Σχολή Θετικών Επιστημών, ΕΚΠΑ
Summary:
In this dissertation, new LC-MS/MS analytical methods were developed and validated for identification and quantitation of psychoactive substances in antemortem and postmortem whole blood, urine and selected "alternative" biological materials such as bile, spleen, liver, brain, skeletal muscle, and vitreous fluid. The developed and validated methods can be used for systemic toxicological analysis.
In the theoretical part of this thesis, chapter 1 provides the definition of narcotics and a detailed description of Greek and American legislation regarding the control of these substances. The importance of quantifying psychoactive substances is also described. Chapter 2 analyzes the chemical, pharmacological and pharmacokinetic properties of the studied substances. An introduction is also made to the derivatives of phenethylamine and for each of the substances studied, a description is presented of their chemical characteristics, as well as of their pharmacological and pharmacokinetic properties. Chapter 3 describes the "traditional" and "alternative" biological samples used in systemic toxicological analysis, the UHPLC-MS/MS system, and the process of systemic toxicological analysis. Finally, a detailed description is made of the guidelines applied in this dissertation for the acceptance of mass spectra data as well as the validation of the methods. Specifically, reference is made to the guidelines of the Scientific Working Group for Forensic Toxicology (SWGTOX), the guidelines of the Organization of Scientific Area Committees of Forensic Science (OSAC), as well as the European Directive 2002/657/EC.
In the experimental part of this thesis, chapter 4 describes the laboratory equipment, reagents, buffer solutions, and reference standards which were used. In chapter 5, a method was developed and validated for the screening and the identification of amphetamine, methamphetamine, phenylpropanolamine, ephedrine, pseudoephedrine, MDA, MDMA, MDEA, phentermine, methylphenidate, bupropion, hydroxy bupropion, alpha-PVP, ethylone, MDPV, mephedrone, methcathinone, methedrone and of methylone in samples of antemortem and postmortem whole blood, using LC-MS/MS and electrospray ionization. The method was evaluated according to the SWGTOX guideline for the main quality characteristics, such as specificity, selectivity, carry-over, matrix effect, LOD and recovery in both antemortem and postmortem whole blood. Comparing the results of antemortem with those of postmortem whole blood, no significant differences were found between them. The method has been applied successfully in Proficiency Testing Scheme and real blood samples. In chapter 6, a method was developed and validated for the quantitative determination of 12 analtytes in samples of antemortem and postmortem whole blood, using LC-MS/MS and electrospray ionization. The method was evaluated according to the SWGTOX guideline for the main quality characteristics, such as linearity, accuracy, repeatability, specificity, selectivity, carry-over, both in antemortem and postmortem whole blood. Comparing the results of antemortem with those of postmortem whole blood, no significant differences were found between them. The method has been applied successfully in Proficiency Testing Scheme and real blood samples and it has already been included in the routine testing protocol of the toxicological laboratory. In chapter 7, the effect of urine and selected alternative biological samples in ion suppression/enhancement as well as the % recovery of the 19 analytes using the LC-MS/MS method, were studied. The urine and the selected alternative biological samples (liver, brain, bile, spleen, skeletal muscle, gastric content, and vitreous fluid) were used after first being tested for the presence or absence of the above substances. For most of the analytes, the nature of biological matrix did not significantly affect the suppression/enhancement of their signal, it was also observed that the matrix was an important factor affecting the results for most of the analytes. The % recovery of many analytes was affected by the nature of the matrix with a representative example of phenylpropanolamine for which the recovery ranged from 39% in bile samples to 99% in vitreous fluid.
Keywords:
Psychoactive substances, whole blood, alternative biological samples, chromatography, mass spectrometry, solid phase extraction, pharmaceutical analysis, toxicological analysis
