Lim Zhan Feng, Teri Danielle Yeoh, Le Vu INTRODUCTION Forensic toxicology is the eld of study that entails the

elucidation of chemicals for medical and/or legal purposes. This falls under three categories, namely, postmortem examinations, human performance assessments and drug testing. Postmortem forensic toxicology is widely used in fatalities that are suspected to be due to drug intoxication, homicides and accidental deaths, as the cause of death often cannot be conclusively determined through an autopsy (surgical procedure) alone. These postmortem forensic toxicology tests involve a laboratory analysis that will identify and quantify the substances present in the body to ascertain if the contributed to the death. It employs techniques fromanalytical chemistry, pharmacology and clinical chemistry to determine information such as types of drugs present, concentration of these drugs and considering their effects on the human system. The main obstacle a forensic toxicologist faces is to gure out what substances to test for. There is no machine which can directly tell the identity of substances present; rather, one has to make a guess. Often, guesses are made from the various medicines present at deceaseds bedside or home. Similarly, the deceaseds doctor(s) can also provide information on what medication and drugs the deceased is consuming. Tests are then conducted on these hypothesized candidates. In the event of negative test results, one will have to continue guessing. Given the millions of known substances to chemistry (there are about 21 million registered compounds), this is task is akin to searching for the proverbial needle in a haystack. Bodily processes further complicate things as certain substances are metabolized more quickly than others, and travel to different parts of the body or becomes infused with body tissue. Postmortem redistribution is yet another complication. Drugs may be redistributed into the blood from solid organs such as the lungs, liver and heart. As such, drug concentrations may be inuenced by the sampling site and time interval between death and sample collection.Thus, forensic toxicology requires the understanding of these metabolic pathways and transport systems in the body to effectively analyze the original circumstances involved in the victim's death. In this essay, we will rst discuss methods on extraction of these evidence, proceeded by the methods employed to analyze them. We will conclude with an analysis of the circumstances surrounding Michael Jackson's death (Propofol toxicology test).

SAMPLE COLLECTION Forensic toxicology tests require the collection of samples from the human body for testing. These include urine, blood, hair and vitreous humour from the eyes among many others. Blood samples are collected from twice, once at the heart and the other at another site, often the femoral vein. The femoral vein is selected due to its relative distance from solid

organs (lungs, heart, etc.) as this reduces the inuence of the phenomenon of postmortem redistribution. Vitreous humor is collected due to its stability and anatomical isolation. It is often used to determine bodily concentrations of ethanol and is analyzed in conjunction with blood ethanol levels. Urine is also collected. Drugs often remain in urine for a long period after administration and is thus, benecial to forensic toxicology tests. Urine samples are also amenable to several color and immunoassay tests, such that no pre-treatment is required. Bile samples may be collected in the absence of urine. Liver samples are crucial as the liver is the site for drug metabolism. As such, drugs and their respective metabolites are often found in higher concentrations at the liver than in the blood. ANALYSIS The rst analytical step is analyte separation. In most cases, analytes need to be isolated from the biological matrix. Thic can be achieved through heating of the sample, wherein the gaseous phase containing volatile substances will be studied. Protein precipitation via inorganic acids (trichloroacetic acid) or organic solvents (methanol) is another method. Liquid-liquid extractions are frequently conducted too. Analyte separation ensures that subsequent tests such as HPLC will be more accurate. Upon completion of separation, samples are subject to further tests- spectrophotometry, immunoassays, and chromatography. Spectrophotometry Color tests are a simple example of visible spectrophotometry. Easy to conduct, they can be carried out directly on a protein-free sample. Color tests are used to screen for compounds such as cyanide, salicylate and acetaminophen. Ultraviolet spectrophotometry is used to screen for certain drug classes such as benzodiazepines (of relevance to the Jackson case), antidepressants and barbiturates. However, the technique is poor at determining concentrations as well as distinguishing between drugs and their metabolites. Doing so is especially important in the case of prodrugs, which are administered in an inactive form, but whose metabolites are active and have signicantly higher pharmacological activity. Immunoassay Immunoassays are biochemical tests which are advantageous in that they can be conducted directly on urine samples immediately. Highly sensitive to a specic drug, the method depends on the fact that the analyte (antigen: drug in sample) undergoes a unique immune reaction with a second substance (antibody). Immunoassays involve competition between a labeled drug (eg. radioactive elements as labels) and the drug in the sample for sites on the antibody. Chromatography

Gas chromatography is a major component of forensic toxicology testing. Highly modiable, changes can be made to improve resolution, sensitivity as well as specicity. By altering the stationary/mobile phase and/or ow rate, a more desirable resolution for a specic substance can be obtained. Tweaks to temperature allow for the identication of compounds with different volatilities within a single test. Detectors can also be modied. Flame ionization detectors are used for tests involving carbon and hydrogen atoms. Dectectors with rubidium beads are used for nitrogen atoms. Electron capture detectors are utilized for compounds with halogens (eg. benzodiazepines). High performance liquid chromatography is favored for polar and heat-sensitive compounds. This technique can be modied by altering stationary/mobile phase compositions. Detection is carried out in various ways: uorescence, ultraviolet, electrochemical, etc. Today, the main method employed is some form of chromatography, equipped with mass spectrometry. Either chromatography is used as an analytical technique with the mass spectrometer as the detector, or mass spectrometry is the analytical technique that uses chromatography to get the samples in. Toxicologists dissolve tissue in acid or alkaline solution followed by HPLC or GC. Tests can be directly performed on the outputs, rather than having to go through the arduous process of extraction. The gas chromatograph with mass spectrometry is the most useful technique working at a crime scene. Many samples are complex organic mixtures and this method is able to separate it into its pure constituents. A small amount of the suspect substance is dissolved in a solvent and injected by needle into a hollow tube. A ow of inert gas propels the heated mixture through the coiled glass tube, where a highly sensitive detector identies the separate elements at the other end. Each element moves at a different speed, so they can be identied and have their concentration detected when they cross the nish line. Control substances are put in to the GC for comparison as well to identify suspicious substances against 'normal' ones MICHAEL JACKSONS DEATH Based on toxicology ndings, the cause of his death was determined to be acute propofol intoxication with a contributory benzodiazepine effect. Propofol is a widely used intravenous agent for induction and maintenance of anesthesia and for sedation in intensive care patients, but it is also associated with abuse and dependency due to the euphoric feelings and relaxing effects of propofol, some of which are fatal. Drugs found in Jackson's body included lorazepam (Ativan), midazolam (Versed), diazepam (Valium), mordiazepam (metabolite of diazepam). Ephedrine, lidocaine and propofol (Diprivan). Of utmost relevance to the case are the lorazepam and Propofol results. Diazepam and its metabolite, nordiazepam, lorazepam and midazolam are all in the family of drugs known as benzodiazepines. Benzodiazepines have a depressant effect on the central nervous system. However, while making one extremely sleepy or unconscious in overdose, it very rarely results in apnea. Nevertheless, it amplies the effects of propofol, thereby lowering the body tolerance of propofol before death.

A very small amount of midazolam was found in the blood and urine. Therapeutic blood levels of midazolam is 0.08-0.25 mcg/ml. 0.0046 mcg/ml was found in blood plasma. From a scientic point of view, this means that midazolam was administered 24-48 hours before his death and it played no part in his death, although his doctor claims otherwise; that he did administer to him that morning. Lorazepam, a benzodiazepine, was available in Michael Jackson's house in tablet form readily, as well as in intravenous form. The defense (his doctor) claims that Jackson consumed 8 2mg tablets while out of the room. This would mean that either pill fragments will be found in the stomach, with very low level in blood, or no pill fragments, but a high level of it in the blood. However, tests shows that there were no fragments and the level of lorazepam was instead a therapeutic low, one consistent with the intravenous dosage,0.169 mcg/ml. For someone whose body was addicted to it due to regular administration, its minute presence should have little part to play. Textbook studies on 5 fatal cases of acute propofol poisoning had postmortem blood propofol levels ranged from 0.5 5.3 mcg/ml. After a typical 2.5 mg/kg anesthesia induction dose of propofol, the accepted therapeutic dosage is 1.3 6.8 mcg/ml. These past cases thus show that it is possible to die of propofol intoxication at blood levels below that of those needed to anesthetize a patient, halting breathing. Blood levels in the heart blood were found to be 3.2 mcg/ml; in the hospital blood a concentration of 4.1 mcg/ml and in the femoral blood, a concentration of 2.6 mcg/ml. This put the Jacksons propofol blood concentration the therapeutic range for someone having a general anesthetic. As there was no intention to anesthetize Jackson to the point of halting breathing entirely, the level of propofol was clearly far too high, and thus, ultimately proved fatal. We can objectively deduce that propofol had been injected intravenously as the amount of propofol found in Jackson's stomach was inadequate. 150mg-200mg of propofol must be consumed in order to attain the concentrations of propofol in Jacksons body. This is in stark contrast to the mere 0.13mg found in his stomach. ANALYTICAL PROCEDURE IN DETERMINING PROPOFOL CONCENTRATIONS Determining the amount of propofol in human tissues A group of researchers from the Graduate School of Medical Sciences, Kyushu University have developed a simple and reliable method for the quantitative determination of low nanogram-per-gram concentrations of propofol in solid tissues by GCMS. They analysed samples from a forensic autopsy case in which a suspected propofol misinjection was made eight days before the death of a victim. Experiment Reagents and standard solution

Stocks of pure propofol, thymol were purchased beforehand. Other necessary chemicals are of analytical reagent grade. Separate 5mL stock solutions of propofol and thymol of concentration 1.0 mg/mL were prepared. Working stock solutions, 0.1 and 0.01 mg/mL of each, were made from the stock solution of each substance. Preparation of biological samples for quantication Human tissue samples (blood, liver, brain and adipose) obtained from autopsy were stored at -200C. Propofol- and thymol-free tissue samples were used for making control samples and calibrators; they were supplemented with propofol at the concentrations of 0, 10, 50, 100, 500, and 1000 ng/mL or ng/g for the preparation of the calibration curve. Extraction Liver, brain, and adipose tissue samples (0.2 g each) were nely sliced and mixed with 1 mL of KCl/NaOH buffer (pH 12.4) in a round-bottom centrifuge tube (10 mL volume). The samples were then homogenized. Blood sample (0.2 mL) was mixed with 1 mL of the same buffer. The mixture was transferred to a polypropylene tube (2-mL volume), and 50ng of thymol as internal standard (IS). 100 L of heptane were added. The solution was thoroughly mixed on a vortex mixer for 10 s and centrifuged at 1500 x g for 5 min to separate the phases. The mixture was kept at 20C for 20 min. The organic layer obtained was then transferred to an injection vial, and a 2-L aliquot of the solution was injected into the GCMS. Human tissue control samples were also extracted similarly and submitted to GCMS. Determination of propofol With appropriate conditions and instrumentation, GC-MS was carried out for the tissue samples. The calibration curves were obtained by plotting the peak area ratio of propofol to thymol versus the amount of propofol. The limit of detection, absolute recovery and precision was then measured, followed my determination of the amount of propofol using the constructed calibration curve.

Prior to this research, several methods have been published previously for the extraction of propofol in human solid tissues. Iwansen-Bergmann used the supernatant of tissue homogenate to extract 7.627g/g of propofol in brain and liver samples with cyclohexane for GCMS analysis. Another published approach was mixing the digested solution of tissue homogenate with acetonitrile, then submitting it to HPLC for the analysis of propofol in the order of microgram-per-gram. However, these methods were not

suitable for the quantication of propofol in order of nanogram-per-gram in tissue samples. Another simple method was introduced by Klausz but it involved an evaporating process of the organic layer, which the authors believe signicantly increased the risk of substance loss when quantifying a volatile substance in the order of nanogram-per-gram. Headspace GC analysis was used by Chao et al. for the detection of propofol in liver, kidney, and brain in the order of microgram-per-gram, but its sensitivity was not suitable for the detection of the substance in the order of nanogram-per-gram. Articulated below is the method proposed by Iwansen-Bergman, which can be used for higher microgram-per-gram or microgram-per-ml degree of concentrations of propofol in solid tissues. Experiment Reagents

Pure propofol and thymol was obtained before the experiment. All other chemicals and reagents were of analytical grade Extraction

Solid tissue samples were minced and 1g was homogenized with two parts of water and ultrasonicated for 30min, then centrifuged. Blood samples were only centrifuged. Urine was analyzed untreated and acidic hydrolyzed at 1000C for 30min. The supernatants of the tissue samples, blood, and (neutralised) urine samples were spiked with thymol and subsequently diluted with one volume of KH2PO4 buffer (1.5mol/l, pH 6.8). The samples were extracted twice with 3ml cyclohexane. Ethanolic NaOH (100l, 0.1mol/l in ethanol) was added to the organic phase and the extract was dried at 400C under a slow stream of nitrogen. The samples were reconstituted with 50l ethanol and 1l was injected into GC-MS. Hair samples were taken by cutting the hair as close to the scalp as possible. They were cut into segments of 2cm, washed three times (DI water, acetone, CH2Cl2) and dried. Afterwards the segments were separately pulverized using a ball mill. 2ml methanol and 200ng of the internal standard methaqualon were added to 50mg of the pulverized hair. This mixture was incubated in an ultrasonic bath for 4h. After centrifugation the supernatant was transferred to a clean vessel and 2ml methanol was added to the residue and again incubated for 4 hours. After centrifugation the two supernatants were combined, 100l ethanolic NaOH was added and evaporated to dryness under a stream of nitrogen at 400C. The reconstituted samples were examined by GC-MS. Determination of propofol

With appropriate conditions and instrumentation, GC-MS was carried out for the tissue samples. Standard 6-point calibration curves were obtained using 0.0110g propofol/ml

serum blank and for hair using 0.01g - 0.5g propofol/50mg hair blank. The limit of detection, absolute recovery and precision was then measured, followed the determination of the amount of propofol using the constructed calibration curve.

References
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