TOWARDS FORENSIC APPLICATION OF SOLID-PHASE MICROEXTRACTION Development and validation of an SPME approach to study pharmaceuticals and illicit drugs Hester Peltenburg colofon Towards forensic application of solid-phase microextraction. Development and validation of an SPME approach to study pharmaceuticals and illicit drugs. Copyright © 2016 Hester Peltenburg ISBN/EAN: 978-90-9029292-2 Cover design, layout and printing: proefschrift-aio.nl The research described in this thesis was performed at the Institute for Risk Assessment Sciences, faculty of Veterinary Medicine, Utrecht University. The work was carried out in cooperation with the Netherlands Forensic Institute, department of Forensic Medicine. The prototype C18/SCX fibers used in the research in this thesis were kindly provided by dr. Robert Shirey from Supelco, Sigma Aldrich (Bellafonte, PA, USA). TOWARDS FORENSIC APPLICATION OF SOLID-PHASE MICROEXTRACTION Development and validation of an SPME approach to study pharmaceuticals and illicit drugs “Solid-phase microextraction” als toekomstige bemonsteringstechniek in forensisch onderzoek (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 7 juni 2016 des middags te 12.45 uur door Hester Peltenburg geboren op 12 september 1987 te Utrecht Promotoren: Prof. dr. M. van den Berg Prof. dr. J.L.M. Hermens Copromotoren: Dr. I.J. Bosman Dr. S.T.J. Droge TABLE OF CONTENT 1 7 5 147 Introduction Sensitive determination of plasma protein binding - of cationic drugs using mixed-mode solid-phase microextraction 2 39 - Elucidating the sorption mechanism of “mixed-mode” 187 SPME using the basic drug 6 amphetamine as a model compound Direct tissue sampling of diazepam and amitriptyline - using mixed-mode spme fibers: a feasability study 69 3 - Sorption of amitriptyline and 207 amphetamine to mixed-mode 7 spme in different test conditions General discussion - - 111 4 245 8 Sorption of structurally different pharmaceutical and illicit Summaries drugs to a mixed-mode coated microsampler - - 6 Chapter 1 1 INTRODUCTION 7 BACKGROUND Essential in forensic toxicology is the detection and evaluation of postmortem drug concentrations to determine whether a compound can be identified as the cause of death or a contributor to the cause of death. However, postmortem drug concentrations do not necessarily reflect the concentration at the time of death and vary according to the sampling site and time interval between death and sample collection. A major problem in chemical analysis in forensics studies is that the amount of biological sample is often limited. Moreover, it is not possible to perform repeated sampling after death without disturbance of the biological system. Roughly half of the 57 drugs of abuse that are screened routinely at the Netherlands Forensic Institute are predominantly ionized at physiological pH. Whereas the partition behavior and sampling methodology for neutral toxicants is reasonably well documented, understanding the distribution processes of ionizable compounds and interpreting sampling data poses various scientific challenges. The major aim of this thesis is to develop new analytical techniques that can be applied in postmortem redistribution studies of drugs and pharmaceuticals. To accomplish this, a sampling method using solid-phase microextraction (SPME) was investigated and optimized for a selected number of compounds, specifically focusing on ionizable drugs of abuse. In this chapter, forensic toxicology is introduced. Special attention is given to the uptake, distribution, metabolism and elimination of compounds, and in particular ionizable compounds. These processes are also discussed in the light of a postmortem situation, which leads to the introduction of the phenomenon of postmortem redistribution. Current analytical tools in forensic studies are highlighted, with special attention for solid-phase microextraction. FORENSIC TOXICOLOGY Forensic toxicology is a discipline in toxicology that aids in the legal investigation of death, poisoning or drug use. Most important in this field is the collection of data and the interpretation of the results. At the Netherlands Forensic Institute, most toxicological cases are concerned with driving under the influence of either alcohol or drugs. In 2014, more than 3000 cases were reviewed by the forensic toxicology department (figure 1). While only a small 8 Introduction Chapter 1 Figure 1. Overview of forensic toxicology cases in the Netherlands, as recorded by the Netherlands Forensic Institute. Report year: 2014 Figure 2. Diffusion of charged chemicals is possible across phospholipid membranes (left arrow), epithelium (middle arrow), and basement membrane (right arrow). The numbers indicate the fold increase in permeability of the neutral versus the charged species of an ionizable compound (Avdeef, 2012). Also, the uptake of charged chemicals can be altered by the presence of a pH microclimate and differences in intracellular pH. 9 portion of the total number of cases concerned forensic autopsies and their outcome, the complexity of postmortem toxicology makes it a large part of the work of a forensic toxicologist. Postmortem toxicology The interpretation of toxicological results is mostly based on pharmacokinetic and pharmacodynamic data. In clinical toxicology, or antemortem, this data on concentration-effect relations is readily available. For pharmaceuticals, the deposition of the compound within the body is well studied. Deposition is defined by a number of processes, including absorption, distribution, metabolism and elimination (ADME in short). In a postmortem situation, changes can occur in these processes. The following paragraphs describe the ADME processes, with potential postmortem changes. Absorption Absorption describes the uptake of the compound into the bloodstream and, together with metabolism, determines the bioavailability. The route of administration is an important consideration in absorption. Most pharmaceuticals are administered orally, and need to be taken up from the gastrointestinal tract. The uptake is mainly driven by passive diffusion, and requires ionizable compounds to be in their neutral form. This is also known as the pH partitioning hypothesis (Avdeef 2012). The uptake of ionizable compounds is therefore dependent on the pH inside the gastrointestinal tract. Due to the large pH gradient in the gastrointestinal tract the uptake of ionizable chemicals is highly location-specific. Uptake of anionic compounds mostly occurs in the stomach, whereas cationic compounds are taken up in the colon (Avdeef 2012). However, there are some exceptions to the pH partitioning hypothesis, which allow for the diffusion of the charged form of a compound (figure 2). First of all, passive diffusion is not solely reserved to the neutral form of a chemical. The charged species of a compound is capable of passive diffusion, albeit at much lower rates than for the neutral species. Figure 2 shows the fold increase in permeability of the neutral form of a chemical over the charged form over different intestinal barriers Avdeef( 2012). The permeability of the neutral form of a chemical is 108 greater than for the charged form for the transport across a phospholipid bilayer. For the epithelium itself, this factor is 105, in part due to paracellular transport and ion-selectivity in the tight 10 Introduction junctions. For the basement membrane, ion-selectivity reduces the difference Chapter 1 between neutral and ionized permeability to a factor 10. Secondly, the pH close to the epithelium might be different than the pH in the lumen itself. The microclimate pH in the intestine can be up to two log units lower than the luminal pH, most likely created by the amphoteric nature of the mucus layer (Shiau et al. 1985). In the stomach, reported microclimate pH is 8.0, whereas the normal pH of the stomach is 1.7 in fasted condition (Rechkemmer 1991). Here, the secretion of HCO3- from the gastric epithelium greatly influences the microclimate pH (Rechkemmer 1991). Lastly, intracellular pH also affects absorption. Within a cell, pH can be as low as 4.5 in lysosomes and as high as 8.0 in mitochondria (Asokan & Cho 2002). This can have a dramatic effect on charge and physicochemical properties of weak acids and bases. Specifically, ionizable compounds can enter the cell as a neutral compound, but become charged inside the cell due to the difference in intracellular pH. In its charged form, the compound cannot diffuse as effectively out of the cell, leading to accumulation inside the cell. This is known as ion trapping. In forensic toxicology, one should be aware that illicit drugs usually have a different or unconventional route of administration compared to pharmaceutical drugs. Examples of these types of administration routes include smoking, snorting and self-injecting drugs. The route of administration can affect uptake, blood concentration and rate of metabolism, thereby affecting total body disposition Drummer( 2007). Postmortem, passive diffusion continues, and so does absorption. An example of this is after an overdose of an orally ingested drug. Death might occur before absorption is complete, leading to higher concentrations in blood and tissue near the stomach than elsewhere in the body, due to ongoing absorption (Ferner 2008). Other factors