Forensic toxicology of MJ’s death

Forensic toxicology of Michael Jackson’s death

Eugene Tan, Zhao Zhenyu, Fan Jiahuan

M12605

1. Introduction

2. Detailed cause of MJ’s death

2.1 Confession by doctor

2.2 Physical evidence

3. Toxicology

3.1 Clinical toxicology

3.2  Forensic toxicology

4. Analysis of drugs in forensic toxicology

4.1 Blood and urine samples

4.1.1 Toxicology screen--Antibody-mediated toxicology tests

4.1.2 Blood and Urine sample preparation

4.1.3 Gas Chromatography-Mass Spectrometry

4.1.3.1 Why GC-MS is used?

4.1.3.2 GC-MS process

4.1.3.3 Some applications of GC-MS

4.2 Liver, Stomach and vitreous humor samples

4.2.1 Preparation of samples:

4.3.1.1 Liver

4.3.2.2 Vitreous humor

4.3.3.3 Stomach

5. Conclusion

6. Reference

1. Introduction:

Michael (Joseph) Jackson (August 29, 1958 – June 25, 2009) was an American recording artist, entertainer and businessman. Often referred to as the King of Pop, or by his initials MJ. On June 25, 2009, Jackson died while in his bed. His sudden death was a huge shock to the public. While investigating his startling death, an autopsy report stated that his hips, thighs and shoulders were riddled with needle wounds — believed to be the result of injections of narcotic painkillers, given three times a day for years.
 

Pathologists found his stomach empty aside from partially-dissolved pills he took before the painkiller injection which stopped his heart. Samples were sent for toxicology tests. It was later found out that his death was caused by acute propofol intoxication (an overdose of the anesthetic propofol), with the drug lorazepam listed as an additional primary cause. Several other drugs were also found in Jackson's system, including midazolam, diazepam, lidocaine and nordiazepam.

And in this article, detailed cause of death will be presented, knowledge of toxicology will be introduced and the specific procedures of drug testing will also be analyzed.

2. Detailed cause of MJ’s death:

Drugs found in victim’s body and their effects are shown in table 1:

Drug/location	Blood	Urine	Liver	Vitreous humor (part of the eye)	Stomach
propofol	present	present	present	present	present
lorazepam	present	-	-	-	-
midazolam	present	present	-	-	-
lidocaine	present	present	present	-	present
diazepam	present	-	-	-	-
nordiazepam	present	-	-	-	-

Table 1: presence of drugs in different samples

Propofol is a fast-acting drug that reduces anxiety and tension, and promotes relaxation and sleep or loss of consciousness. Propofol is injected into a vein and ideally should be given by trained anaesthesia professionals in a controlled environment. Side-effects of propofol can include respiratory depression, difficulties in breathing. Other drugs such as diazepam may interfere with the effects of propofol if both are taken simultaneously.

Lorazepam (Ativan) is used for treating anxiety and is in the same benzodiazepines group as diazepam (Valium). It is also effective for insomnia and panic attacks. Michael Jackson had taken eight 2mg tablets on the day he died. Side-effects include a feeling of depression, loss of orientation, headache and sleep disturbance.

Midazolam (Versed) is usually injected and is used before operations or procedures to increase sleepiness and decrease your memory of the event. Rapid intravenous (IV) use of midazolam can cause severe breathing problems which could cause brain damage or be fatal if left untreated.

Lidocaine is an anaesthetic sometimes used to treat skin inflammations. It is usually used by dentists to anaesthetise patients' gums.

Diazepam (Valium) is a drug taken in tablet form for the treatment of anxiety disorders. The most frequent side-effects of diazepam are drowsiness, fatigue, and loss of balance. Confusion, depression, speech problems, and double vision are also rare side-effects.

Nordiazepam otherwise known as desoxydemoxepam, nordazepam or desmethyldiazepam, is a 1,4-benzodiazepine derivative. It is an anaesthetic and has sedative and muscle relaxant properties. Therefore it is primarily used for treating patients with a case of anxiety. The side-effects of this drug cause the patient to feel sleepy and sleep for an abnormally long period of time.

2.1 Confession by doctor:

Michael Jackson had problems sleeping even after multiple sedatives has been administered into his body. It was when Jackson pleaded with the doctor that he needed some propofol in order to sleep, that was when the tragedy happened. Jackson had taken three powerful benzodiazepines on his final morning on earth. These were diazepam (also known as Valium), lorazepam (Ativan) and midazolam (Hypnovel). In Britain Hypnovel, which when injected intravenously causes very deep sedation, is almost exclusively used for heavy sedation in intensive care and induction of anaesthesia. It is not used as a sleeping medication in this country, as a rule. These drugs are not normally lethal when used on their own, but when mixed with other CNS (central nervous system) depressants like alcohol or something like propofol, they can be. Propofol changes the body's state very rapidly so that the patient will go unconscious and stop breathing. It can affect the breathing even before unconsciousness. Since the confession of the doctor states that Michael Jackson was still alive (there is still a faint pulse and his body was still warm) and he was unconscious on his bed and not breathing, it is very likely caused by the effects of overdose from anaesthetic propofol (and possibly with the other narcotics the victim took before being administered with propofol).

2.2 Physical evidence:

Propofol and lidocaine were detected (in approximately 0.19 g of white fluid) from a 10cc syringe barrel with plunger. There were also anaesthetic drugs (all drugs that were also found in Michael jackson’s system) found around his room.

  

3. Toxicology

Michael Jackson’s cause of death was determined by a series of toxicology tests. Toxicology is the study of toxic substances and drugs. Those include natural toxins from plants and animals, poisonous chemicals found in the nature such as heavy metals, as well as drugs taken for medical uses or for illegal uses. Toxicology tests are performed to investigate cases of suspected drug overdose or deaths caused by drugs. They can by very time-consuming and tedious because many types of sample have to be analyzed. There are two types of toxicology tests, they are clinical toxicology and forensic toxicology.

3.1 Clinical toxicology

Clinical toxicology is performed for patients who are sent to the emergency department in hospitals and are suspected to be suffering from drug intoxication. In this test, a screening of drugs of abuse is first performed using immunoassay on blood or urine samples of the patients. After the initial screening, confirmation tests are also done using alternative methods sometimes to verify the identity of the drugs. Those confirmation tests make use of methods like chromatography or mass spectrometry, and sometimes both are used.

3.2 Forensic toxicology

Forensic toxicology makes use of toxicology and also some other disciplines such as analytical and clinical chemistry or pharmacology to investigate causes of death by drugs or poison, for legal purposes. This type of test is much more complicated than clinical toxicology, because for most postmortem cases, the amount of drugs present in the body is much lower than that of a patient with a drug overdose or intoxication. Therefore, the analytical instrumentations used in this type of test is a lot more sophisticated, because they have to be able to detect drugs and toxins at very low concentrations. Besides, the range of samples used for the test is also much wider, and they include hair, brain, liver, kidneys, and oral fluid from the body and so on.
 

After the various tests are done, the team of forensic toxicologists, often made up of clinical toxicologists, pathologists and analytical chemists, will interpret the results, and determine the identity and quantity of drugs or toxins present in the body as well as their effects, and finally, the cause of death. This process will often take weeks or sometimes even months because there are a lot of samples to be screened and tested. Besides, the confirmation tests mentioned previously also need time. And finally, sometimes it is difficult for the forensic toxicologists to interpret the test results.

For Michael Jackson’s case, a forensic toxicology was performed to investigate his death, for it was suspected to be caused by a drug overdose.  Blood, urine, liver, vitreous humor and stomach samples were taken for toxicology.

 4. Analysis of drugs in forensic toxicology

As mentioned before, blood, urine, liver, vitreous humor and stomach samples were taken from Michael Jackson’s body for testing of possible drugs existed. Toxicology was carried out, but no specific procedures were described in the Autopsy Report. After research, we constructed the following procedures and this could only be one of the possible ways of testing.

4.1 Blood and urine samples

In blood and urine samples, a toxicology screen is normally conducted first to narrow down the range of the drugs in the sample and followed by Gas Chromatography-Mass Spectrometry (GC-MS in short) to confirm the results previously. GC-MS also helps to further test for existence of more possible drugs which could have not been discovered under antibody-mediated toxicology tests.

4.1.1 Toxicology screen--Antibody-mediated toxicology tests

Immunoassays are the most common drug screening techniques.  Various tests such as Enzymatic Immunoassay (EIA), Enzyme Mediated Immunoassay Technique (EMIT), Enzyme Linked Immunosorbent Assay (ELISA), and Cloned Enzyme Donor Immuno-Assay (CEDIA) undergo the same basic process: urine or blood sample is added with an anti-drug antibody, the antibody will bind to the drug if the specific drug is present, and this enables a measurable indicator reaction to occur that is reported as “positive”.
 

All these immunoassays use an anti-morphine antibody to detect opiates; and when it comes to Benzodiazepam immunoassays, an anti-diazepam antibody is used Because these antibodies are specific to morphine and diazepam, there will be both false-negative and false-positive results with non-morphine opioids (ex., fentanyl) and non-diazepam benzodiazepines (ex., lorazepam), which may confuse the clinical and forensic picture and suggest diversion. (Sample with non-diazepam benzodiazepines (ex. Lorazepam) has to produce a significant number of negative benzodiazepine immunoassays due to lower reactivity of clonazepam with the immunoassays ’diazepam antibody. Diazepam is positive in immunoassays). 
 

Although immunoassays are inexpensive, reproducible, rapid, and easy to perform, a disadvantage of immunoassays testing is that only the class of drug is identified (ex. benzodiazepine) and not the specific drug (ex. diazepam); further testing such as Gas Spectroscopy- Mass Spectrometry are often needed to make a specific identification.

4.1.2 Blood and Urine sample preparation

The preparation of GC-MS samples was as follows.

1. Urine samples are first hydrolyzed (2-mL sample, 100 mL b-glucuronidase, 50 mL internal standard, and 2 mL of pH 6.8 phosphate buffer (0.1M)).
2. Then the sample is heated for 2 h at 37^oC prior to extraction.
3. Blood or hydrolyzed urine (2 mL), internal standard solution (diazepam-d₅, 200 mL of a 10 g/mL solution in methanol), and pH 7.0 phosphate buffer (1 mL) are mixed.
4. Then extracted with n-butyl chloride (6mL) and rotated slowly for 15 min. The organic layer is transferred to a conical tube and evaporated to dryness.
5. Then reconstituted with acetonitrile (200 mL), and vortex mixed. Heptane (500 mL) is added as a wash, vortex mixed, and the heptane discarded.
6. The acetonitrile layer is evaporated to dryness then reconstituted and derivatized using acetonitrile/MTBSTFA+ 1% TBDMCS (3:1) (75 mL).
7. Samples are allowed to derivatize in a 70^oC oven for 30 min, then transferred to an auto-sampler vial for GC-MS analysis.

4.1.3 Gas Chromatography-Mass Spectrometry

4.1.3.1 Why GC-MS is used?

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. Gas Chromatography (GC) separates different components which would travel in the column in a different rate; thus providing information about various compounds existing in the sample. Mass spectrometry (MS) provides information on the molecular weight and elemental composition from the molecular ion and then likely structural aspects of the molecule from the fragmentation patterns of the ions.
 

However, due to various limitations of the two components, it is so far impossible to conduct an accurate determination of the specific compounds existing in a sample with any of the two along. As a result, these two components are used together to conduct a much finer degree of substance identification. MS process normally requires a very pure sample and GC helps to separate the molecule with high purity before going into the mass spectrometer. While with a traditional detector (e.g. Flame ionization detector) in GC, it cannot differentiate between multiple molecules that happen to take the same amount of time to travel through the column, which are the result of two or more molecules that co-elute. A similar fragmentation pattern can also be observed for two different molecules in MS. Combining the two processes reduces the possibility of error, as it is extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore, whenever both MS and GC patterns are matched in a test, it indicates the results that the analysis of interest is in the sample.
 

And as its high certainty of testing, the GC-MS result is regarded as the “gold standard” for forensic substances identification.

4.1.3.2 Gas Chromatography-Mass Spectrometry process:

            a.  Gas Chromatography (GC):
 

1.The sample is first being introduced into the machine by auto-sample vial.
 

2.Samples are vaporised and then introduced to the head of the chromatographic column (normally packed column or open tubular column (capillary)).
 

3.A carrier gas (normally nitrogen gas/inert gases so that they do not react with the analyte in the process) then transports the sample in the column towards the end of the column where the detector is.
 

4.Molecules that have more interaction with the stationary phase would stay in the column for a longer time and to be eluted slower. As a result, separation occurs.
 

5.Once the molecule reaches the top, an analyzer would record concentrations as separate peaks on a graph. Each peak is then sent through MS, where ideally each pea should only have only 1 compound so that when it enters the mass spectrometer, it will be pure.
 

          b.  Mass spectrometry:
 

6.Vaporised molecules from GC have flowed into the Mass Spectrometer and then an electron beam is introduced into these molecules, causing the electron from the molecules to be ejected, forming a cation.
 

7.The ions are then accelerated down a chamber by an accelerating plate and focused into a beam.
 

8.As the beam travels to the magnetic sector, deflection of the ion beams is caused by the magnetic field present. Smaller ions are deflected more as compared to a larger one.
 

9.The ions will then reach a quadrupole filter. There are four rods then arranged parallel to the ion beam. Direct current and radio frequency is applied to the rods, causing the rods generate an oscillating electrostatic field. Ions then obtain an oscillation based on their mass to charge ratio. Ions with incorrect mass to charge ratio for the specific electrostatic filter applied under go into an unstable oscillation and do not make it through the mass filter to the detector. Only ions with the correct charge to mass ratio will be able to pass the filter and reach the detector (usually in increasing order of mass to charge ratio).
 

10.The detector will detect the ions and produce a current proportional to the number of ions striking it. Thus recording the abundance of a particular ion as seen on the mass spectrometer data.
 

These results are then compared with the computer database of GC-MS and finally the specific component is identified. Samples are quantitated using a selected ion monitoring (SIM) mode and confirmed using a full scan mode.

4.1.3.3 Possible applications of GC-MS:

1. Monitoring the pollution level in our environment: GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The cost of GC-MS equipment has decreased significantly, and the reliability has increased at the same time, which has contributed to its increased adoption in environmental studies. There are some compounds for which GC-MS is not sufficiently sensitive, including certain pesticides and herbicides, but for most organic analysis of environmental samples, including many major classes of pesticides, it is very sensitive and effective.
2.  Astrochemistry: bringing back samples from space to detect the actual composition of the compounds so that we can have a better understanding of the universe
3. Security: at airports, there are explosive detectors, to prevent the likelihood of terrorism

4.2 Liver, Stomach and vitreous humor samples

Being different from the test of urine and blood samples, for stomach, liver and vitreous humor samples, GC-MS is used right away after preparation of samples.

4.2.1 Preparation of samples:

4.2.1.1 Liver

1. About 10 grams of liver is firstly grinded to pulp and homogenized for 15 minutes in 10 ml of distilled water.
2.  The pH of the homogenate is then buffered to 10 using sodium hydroxide solution and digested for 2 hours at 55^oC  using 10 mg of subtilisin.
3.  After that, its pH is buffered back to 7. To prepare the urine sample, 0.25 ml of urine is first mixed with 0.25 ml of sodium acetate buffer with a pH value of 5.1.
4.    The mixture is then hydrolyzed using 5000 units of β-glucuronidase by incubating at 37 ^oC for 2 hours.

4.2.1.2 Vitreous humor

1. 2.5 ml of fluid firstly will be extracted from each eye using a needled syringe.
2.  Hyaluronidase will then be added to the fluid to lower its viscosity.
3.  Also, the fluid will be heated at 100 ^oC for 5 minutes and then cooled to room temperature.
4. This will also make the fluid less viscous thus improve accuracy of the analysis.
5. After that, 2% sodium fluoride preservative will be added to the fluid to preserve it.
   

4.2.1.3 Stomach

Different from how the druges were found in other parts of Jackson's body, MJ’s stomach was empty at the time of death except for a few half dissolved pills he had before going to the bed. So the pill was then analyzed. However, due to the limitation of the source, we could not figure out how the sample was prepared for GC-MS, but we believe that dissolving of pills followed by extraction and dilution may be used.
 

After all preparations were done, the samples were sent for GC-MS analysis, and results are again compared with the database.

5. Conclusion

Reason of MJ’s death has been discussed in detail, toxicology has been introduced and forensic toxicology was used in this case.  The detailed process of forensic toxicology has been suggested upon the research we have down. And details of Antibody-mediated toxicology tests, Gas spectroscopy-Mass spectrometry have been introduced. Also, the preparation of GC-MS sample has been discussed.

6. References

1. Hamett-Stabler C, Webster L. Clinical guide to urine drug testing monograph. Newark, NJ: University of Medicine and Dentistry of New Jersey, Center for Continuing and Outreach Education, 2008.
2. Schneider RS, Lindquist P, Tong-inWong E, Rubenstein E, Ullman EF. Homogeneous enzyme immunoassay for opiates in urine. Clin Chem 1973; 19:821–5.
3. Webster LR, Dove B. Avoiding opioid abuse while managing pain: a guide for ractitiones. North Branch, MN: Sunrise Press, 2007.
4. Jones A.W, Holmgren P. , Uncertainty in estimating blood ethanol concentrations by analysis of vitreous humour, Journal of Clinical Pathology, 2001;54:699-702
5. Drummer, O. H., A Fatality Due to Propofol Poisoning,  Journal of Forensic Sciences, JFSCA, July 1992, Vol. 37, No. 4, 1186-1189.
6. Kim A. C. , “Postmortem Vitreous Analyses”,  MedScape reference, [Online] 2011 (Adapted from: http://emedicine.medscape.com/article/1966150-overview#aw2aab6b3)
7. Peter L. Tenore, Advanced Urine Toxicology Testing, Journal of Addictive Diseases, 2010; 436-448
8. Jayne E. Clarkson, Ann Marie Gordon, Lorazepam and Driving Impairment, Journal of Analytical Toxicology, September 2004; Vol. 28, 475-480
9. Christopher R, autopsy report of Jackson,  no-2009-04415, August 2009

Contribution of members:

	Eugene	Zhenyu	Jiahuan
Contribution	-Introduction to the topic chosen -types of drugs found and where they are found -how mass spectroscopy works -alternative evidence apart from chemical evidence (e.g. confession by doctor) -editing after final compilation	-explanation on what toxicology is -how  samples are prepared from certain tissues for toxicology testing (Liver +vitreous humor )	-Compiling the information gathered and organised it into a journal -how gas chromatography works - how  samples are prepared from certain tissues for toxicology testing (urine+blood) - Antibody-mediated toxicology tests