da UNIVERSIDADE DA BEIRA INTERIOR ANALYSIS OF ORGANOPHOSPHOROUS PESTICIDES IN POSTMORTEM BIOLOGICAL FLUIDS RAQUEL HELENA CARVALHO SILVA RAPOSO Covilhã, 2009 I UNIVERSIDADE DA BEIRA INTERIOR ANALYSIS OF ORGANOPHOSPHOROUS PESTICIDES IN POSTMORTEM BIOLOGICAL FLUIDS Dissertação apresentada à Universidade da Beira Interior para a obtenção do Grau de Mestre em Bioquímica RAQUEL HELENA CARVALHO SILVA RAPOSO Covilhã, 2009 II Trabalho elaborado sob a supervisão e orientação científica do Mestre Mário João Dias, Director do Serviço de Toxicolgia Forense da Delegação Sul do Instituto Nacional de Medicina Legal e da Prof. Doutora María Eugenia Gallardo Alba, Faculdade de Ciências da Saúde da Universidade da Beira Interior III TABLE OF CONTENTS List of Figures ........................................................................................................................................... VI List of Tables .......................................................................................................................................... VIII Abbreviations ............................................................................................................................................ X Abstract................................................................................................................................................... - 1 - Resumo ................................................................................................................................................... - 3 - Justification and Objectives ................................................................................................................. - 5 - I - Literature Review ............................................................................................................................. - 6 - 1. Introduction ................................................................................................................................... - 7 - 2. Classification and Categorization ............................................................................................... - 8 - 3. Organophosphorous Pesticides .................................................................................................. - 9 - 3.1. Structure ................................................................................................................................. - 9 - 3.2. Physical-Chemical Properties ............................................................................................ - 10 - 3.3. Mechanism of Toxic Action .................................................................................................... 13 3.4. Toxicokinetics ........................................................................................................................... 15 3.4.1. Absorption ........................................................................................................................ 15 3.4.2. Distribution, Metabolism and Excretion ....................................................................... 16 3.5. Toxic Doses and Symptoms .................................................................................................... 17 3.6. Toxic Doses and Treatment .................................................................................................... 18 3.7. Etiology of Pesticide Intoxications ......................................................................................... 19 3.8. Intoxication casuistic in the south region of Portugal ........................................................ 20 4. Legal Background of Pesticide Usage and Management in Portugal ...................................... 21 4.1. European Legislation ............................................................................................................... 22 4.2. Portuguese Legislation ............................................................................................................ 23 4.2.1. Market Introduction ........................................................................................................ 23 4.2.2. Maximum Residue Levels ............................................................................................... 27 4.2.3. Classification, Labeling and Packaging ........................................................................ 28 4.2.4. Seeds treated with plant protection products. ............................................................. 28 4.2.5. Conditions of commercialization and application of plant protection products. ... 29 IV 5. Methodology for the determination of pesticides ....................................................................... 30 II– Experimental ....................................................................................................................................... 49 1. Instrumentation ................................................................................................................................ 50 1.1. Extraction system ..................................................................................................................... 50 1.2. Chromatographic and Detection Systems ............................................................................ 50 2. Material .............................................................................................................................................. 51 2.1. Reagents and Solvents ............................................................................................................. 51 2.2. Standards ................................................................................................................................... 51 2.3. Biological Samples ................................................................................................................... 51 2.4. Working Solutions ................................................................................................................... 52 2.5. Buffer Solutions ........................................................................................................................ 52 3. Chromatographic and detection conditions ................................................................................. 53 4. Extraction procedure ....................................................................................................................... 54 5. Results and Discussion .................................................................................................................... 55 5.1. Identification of Compounds ................................................................................................. 55 5.2. Optimization of the Extraction procedure ............................................................................ 56 6. Validation .......................................................................................................................................... 60 6.1. Selectivity .................................................................................................................................. 60 6.2. Linearity .................................................................................................................................... 65 6.3. Calibration Curves ................................................................................................................... 74 6.4. Limits of detection and quantification .................................................................................. 75 6.5. Intermediate Precision ............................................................................................................. 77 6.6. Repeatability or Intraday Precision ....................................................................................... 78 III – Conclusions ....................................................................................................................................... 80 IV – References ......................................................................................................................................... 83 V LIST OF FIGURES Figure 1 – Structural formula of organophosphorous pesticides. (Casarett, et al, 2001) ........................... - 10 - Figure 2 - Location and Function of Cholinergic Receptors in the Nervous System. (Purves et al, 2004) .... 13 Figure 3 – Symptoms of organophophorus pescitides toxicity. (Abou-Donia, 1992) ...................................... 18 Figure 4 - INML statistics of pesticide intoxications between 2003 and 2006. ................................................. 20 Figure 5 - Detailed statistics of organophosphorous casuistic ........................................................................... 21 Figure 6 – Chromatographic conditions. ............................................................................................................... 54 Figure 7 - Selectivity omethoate (blank sample). ................................................................................................. 62 Figure 8 - Selectivity omethoate (spiked sample). ................................................................................................ 62 Figure 9 - Selectivity diazinon (blank sample)...................................................................................................... 62 Figure 10- Selectivity diazinon (spiked sample). .................................................................................................. 62 Figure 11