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  37th TIAFT Triennial Meeting Cracow

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Opening Lectures

Interpretation of Analytical Results

Drugs of Abuse and Doping

Validation of Analytical Procedures

Analysis of Alternative Materials

Driving under the Influence

Free Topics


  Drugs of Abuse and Doping

The Canadian Centre for Ethics in Sport and Doping Control in Canada

uges.jpg Albert D. Fraser
Toxicology Laboratory, Queen Elizabeth II Health Sciences Centre and Dalhousie University, Halifax, Nova Scotia, Canada B3H 2Y9

The Canadian Centre for Ethics in Sport (CCES) is an organization funded by the federal government working toward prevention of doping in amateur sport. The CCES maintains a comprehensive approach involving research, education, advocacy, detection and deterrence of doping in sport. The objective of this presentation is to present the role of the CCES and the activities of the doping control review panel of the CCES.

Doping control involves education of athletes and coaches, urine specimen collection, drug testing, result management, provision of a protest and appeal process, provision of penalties for doping infractions such as sanctions, and a re-instatement mechanism, etc.

All athlete drug testing is performed at the IOC accredited INRS Santé laboratory in Montréal, Quebec. The volume of urine specimens analyzed ranged from 750 in 1987 to 2,500 in 1993. The 1997 test volume was 1,900 specimens. From 1991 to 1997, the percent of unannounced (random) test collections increased from 45 to 78%. Over the past 10 years, anabolic steroid detection accounted for 74% of all doping infractions followed by refusal to submit a urine specimen (13%), stimulant detection (9%) and diuretic use (4%).

The doping control review panel is an advisory body (composed of 2 physicians and 1 toxicologist) that reviews contentious doping control issues. The panels' responsibilities include review of positive test results and refusals to be tested, protests by athletes concerning specimen ownership and/or integrity, selection of unannounced "target" testing in specific cases, and administrative investigations.

In 1997-98, the doping control review panel investigated 9 cases of refusal to comply with doping control procedures and 22 requests for permission to use an IOC banned substance. Positive test findings included detection of b HCG, methyltesterone, dehydroepiandrosterone (DHEA) and pindolol in 1997-98. Other panel activities included investigation of 3-specimen collection and/or ownership cases, follow-up of 2 testosterone/epitestosterone ratio cases, target testing of 2 athletes and media release reports in 3 cases. The panel also recommended an administrative case review of steroid use on a football team.

The CCES and expert advisory committees within the organization provide a body that works effectively toward prevention of drug use in sport in Canada independent from national sport organizations.

Keywords: Doping Control, Drug Testing, Interpretation.



Heroin-Related fatalities in Victoria-Australia 1997-1998: A study of 434 cases

Jim Gerostamoulos*, Voula Staikos, and Olaf H. Drummer
Victorian Institute of Forensic Medicine & Department of Forensic Medicine - Monash University, 57-83 Kavanagh Street, Southbank, 3006 Melbourne, Australia

The number of deaths attributed to the intravenous use of heroin has increased dramatically in Victoria in the past five years. Since 1991, the prevalence of deaths attributed to heroin toxicity has risen from 49 to 268 in 1998. This represents a five-fold increase in seven years. The increase has been particularly dramatic over the last 12 months (an increase in over 60%). In 1998 deaths from intravenous use of heroin each year constituted 47% of all drug deaths reported to the coroner (Annual Report, 1997/98, Victorian Institute of Forensic Medicine).

The heroin death is typified by a median age of 30 years (both male and female), although the age range extends from children as young as 15 to adults in their fifth decade of life. Over 85% of cases are using other central nervous system depressants, with benzodiazepines (45%) and alcohol (36%) being the most common. Approximately 60% of deaths occur indoors at a private residence, the remaining deaths occur in public places and other locations. A similar number (60%) die alone. Disturbingly, the heroin problem is not restricted to the known "hot-spots" in Melbourne; rather most suburban areas appear to be affected. The mean (+/- SD) blood concentration of total morphine was 0.52 +/- 0.53 mg/l. Concentrations ranged from 0.01 - 3.4 mg/l. The median concentration was 0.4 mg/l. A summary of the toxicological findings from 434 heroin-related deaths will be presented.

Keywords: Heroin, Morphine and Toxicology.



Hair Analysis of bAdrenergic Compounds in Doping Control

Veronique Dumestre-Toulet*1, Vincent Cirimele2, J.P. Goulle3, Pascal Kintz2
1 Laboratoire Ruffië & Associés, Bordeaux, France
2Institut de Médecine Légale, Strasbourg, France
3Laboratoire de biochimie et pharmacocinétique - CHU, Le Havre, France

b-adrenergic compounds are sometimes used by sportsmen who wish to improve their performance: b agonists such as salbutamol to increase the respiratory capacity or clenbuterol to increase the muscle mass and b blockers to improve psychomotor coordination.

For doping control, the International Olympic Committee mandates the use of a urine specimen, but urinalysis provides only short term information on an individual's drug use, and cannot distinguish between chronic and single/therapeutic use and can be adulterated.

Hair analysis offers an interesting alternative to solve these problems.

An original procedure was developed to simultaneously test 14 b-adrenergics in hair. After decontamination with methylene chloride, a 100 mg hair strand is pulverized in a ball mill and incubated overnight in 2 ml 0.1 N HCl at 56°C, in the presence of an internal standard. After neutralization of the acid phase with 0.1 N NaOH, 2 ml bicarbonate buffer (pH 8.6) are added to the preparation, which is then purified by solid-phase extraction with Isolute C18 columns. Drugs are derivatized using a mixture of trimethylboroxine-ethyl acetate for 15 min at 80°C, to form methaneboronate derivatives. Drugs are detected using GC/MS on an HP 6890-5973 system. A 4 µl portion of the derivatized extract is injected using a pulsed mode in a 5% phenyl 95% methylsiloxane capillary column. Linearity was observed for all compounds in the range 25 pg/mg to 10 ng/mg. Limits of detection were in the range 2-10 pg/mg. At 1 ng/mg, recoveries were in the range 37 to 100%, with a within-run precision of 5.9 to 14.1% (n=8).

The results of a proficiency testing in which several French laboratories participated will be presented.

Several applications of the method are described showing confirmation of salbutamol use by a 24-year old swimmer (71 pg/mg), who tested positive in urine, doping with metoprolol in a sharpshooter (8.41 ng/mg) and with sotalol in an archer (261 pg/mg), clenbuterol exposure in calves, used to increase the weight of the animals (30 and 48 pg/mg).

Keywords: b-adrenergic Compounds, Hair, Doping.



Tragic Flying Attempt under the Influence of "Magic Mushrooms"

Christian Giroud*, Annick Ménétrey, Marc Augsburger, Thierry Buclin1 and Patrice Mangin
Laboratoire de toxicologie analytique, Institut universitaire de Médecine Légale, CH-1005 Lausanne
1Division de Pharmacologie clinique, CHUV, CH-1011 Lausanne, Switzerland

Whatever its THC concentration, hemp can be legally cultivated in Switzerland provided it is not used for narcotic production. Five g of a commercially available hemp tea containing 35 mg total THC (0.7%) were infused either in 2 dl of hot water or milk for a half an hour, filtered and administered to 2 groups of 6 healthy volunteers. Half of them drank the water infusion while the 3 others ingested the milk hemp tea. Two weeks later, the experiment was repeated with the same volunteers, but this time the type of hemp decoction which was administered was changed. Before administration and during the whole study, biological samples were drawn periodically. Physiological measures included heart rate and conjunctival injection. Subjective effects (rating "high" and sedation) were assessed with a Visual Analog Scale (VAS). Cannabinoids were determined by immunoassays (RIA, EIA) and GC/MS after solid-phase extraction. The presence of cannabinoids in the sweat was tested by immunoassay (Drugwipe®).

Because of its lipophilic character, much higher levels of THC were extracted from hemp into milk (21 mg THC) compared to water (1.7 mg) during the tea-making process. Peak levels of cannabinoids determined in urine by RIA following ingestion of the hemp milk decoction (1400-10,700 ng/ml) were much higher than those measured after drinking the water infusion (104-265 ng/ml). A similar picture was obtained when carboxy-THC was quantified by GC/MS: maximum carboxy-THC concentrations were in the range of 346-1586 ng/ml following milk tea intake and between 16 and and 85 ng/ml urine after drinking the water infusion. GC-MS determinations of THC, 11-OH-THC and carboxy-THC revealed the presence in the whole blood of only very low levels of carboxy-THC (less than 5 ng/ml) after drinking the water infusion while significant concentrations of all these 3 cannabinoids were determined after intake of the milk infusion. The highest mean concentrations of THC, 11-OH-THC and carboxy-THC were 4.0, 3.4 and 24.5 ng/ml, respectively. Trace amounts of cannabinoids could only be found in the saliva collected a short while after ingestion. In contrast to blood, urine and saliva, no cannabinoids could be detected in the sweat collected on the forehead of the volunteers. Blood levels measured at different time-intervals were used to calculate the time of hemp tea administration with the mathematical models developed by Huestis & al. The quality of fit of the models to the data will be discussed.

Keywords: Hemp Tea, THC Intoxication, Elimination Study.



Stability of Drugs in Postmortem Femoral Blood Stored for One Year

Per Holmgren*, Johan Ahlner and Henrik Druid
National Board of Forensic Medicine, Department of Forensic Chemistry, University Hospital, S-581 85 Linköping, Sweden

In order to correctly interpret forensic toxicological results it is important to know the stability of certain compounds. Normally there is a delay of some days between sampling and analysis and in some cases a new question may arise whether a drug was present or not several weeks or months later. The aim of the present study was to evaluate the consequence of long-lasting storage of blood samples with special attention to the stability of various drugs.

Material and methods: An autopsy femoral blood and vitreous humor were collected and forwarded to the laboratory of forensic chemistry. The vitreous humor was divided into two portions and potassium fluoride was added as a preservative to one of the samples as well as to the femoral blood samples. Analysis for drugs in the femoral blood was made within a week or less from the arrival of the sample. The vitreous humor was kept at +4°C for about a month until stored at -20°C. After analysis the blood samples were stored at -20°C. For most drugs, analyses were performed by gas-chromatography and nitrogen sensitive detector. The first analyses were normally performed within two days after the arrival of the sample and the sample was reanalyzed one year later using the same analytical method. The samples from the vitreous humor were only analyzed after one year of storage.

Results: In the present study, we have examined the stability of different kind of drugs including benzodiazepines; analgesics like propoxyphene and acetaminophen; antidepressants like citalopram and amitriptyline; antiepileptics; and hypnotics. In femoral blood, no major changes were seen for most of the drugs studied except for 7-amino-nitrazepam where a significant loss was detected after one year of storage. In the vitreous humor no major differences were noted between the samples with and without preservative. This implies that addition of preservative to vitreous samples does not seem to be necessary. The use of preservative-free samples will also allow analysis for electrolytes e.g. potassium and sodium, which is sometimes important. Generally, the concentration was lower in vitreous compared to femoral blood. However, the concentrations of citalopram and its metabolite desmethylcitalopram were fairly equal in vitreous and the blood.

Conclusions: Storage of blood samples, with a preservative like potassium fluoride added, for one year at -20°C does not affect the concentration except for some drugs like nitro-benzodiazepines. Knowledge of the stability of drugs in stored samples is of great importance when interpreting toxicological results. Vitreous humor could be an alternative medium for performing toxicological analyses, provided the ratio of the drug concentration in the blood to that in vitreous humor is known.



Statistical Estimation of High Alcohol Concentrations Determined in Breath and in Blood

Dariusz Zuba*1, Wojciech Gubala2, Jerzy Labedz2
1 Faculty of Chemistry, Jagiellonian University, Cracow, Poland
2 Institute of Forensic Research, Cracow, Poland

In the study an attempt was made to compare the blood alcohol concentrations obtained from the analysis of breath and from direct blood investigation.

Blood samples were taken from those patients of the Sobering Chamber in Cracow in whom blood alcohol concentration determined by the analysis of breath exceeded 3o/oo. 56 results of simultaneous determinations of alcohol in blood samples and in breath were taken for the comparison.

Breath alcohol concentrations were determined using Alcomat V5, which utilises selective absorption in the IR region. Blood alcohol investigations were carried out by means of gas chromatography with the use of headspace technique.

The results of breath-alcohol analysis differed from -0.83o/oo to 1.37o/oo in comparison with the results of direct blood investigation. The relative difference between these two variables ranged from 18.6% to 54.8% for particular patients.

The correlation between the results of breath- and blood-alcohol analyses were evaluated. The correlation coefficient amounted to 0.242 and was statistically insignificant (p>0.05). Furthermore, the slope and the intercept of the straight line adjusted to the experimental data were significantly different from the expected values (tb0=11.35, tb1=11.16, whereas t0.05,54=1.98). This might indicate a very low precision of the results of breath analyses at high alcohol concentrations.

Moreover, the blood/breath ratios were calculated for each patient. The mean value of this coefficient for the investigated group amounted to 2063:1 +/- 230 (1 standard deviation), and the coefficient of variation amounted to 11.2%. Although the mean value of the blood/breath ratio is nearly the same as the one used in Alcomat, it varies depending on the patient from 1357:1 to 2580:1.

The results of the study show that there is no justification for continuation of the current practice which consists in treating alcohol blood concentrations obtained by the analysis of breath as equivalent to alcohol concentrations determined in the direct analysis of blood samples.

Keywords: Ethanol, Breath Analysis, Blood, Correlation.



The Accuracy of Sequences of Steps in Analytical Procedures in Forensic Toxicology. Its Significance in the Interpretation of the Final Results of Individual Cases of Poisoning

Wlodzimierz J. Gut
An independent expert in toxicology. Os. Piastow 1A/12, 31-623 Cracow, Poland (private address)

The level of accuracy/uncertainty of the determination of a xenobiotic in postmortem tissue will depend on the least precise stage of the analytical procedure. This commonly known factor affects the final results of an analysis in individual cases of poisoning, and hence their interpretation.

In the sequence of analytical operations the final steps of the toxicological analysis of purified and homogenous samples are highly precise. Modern physico-chemical methods allow a high degree of repeatability. The step of isolating the substance is characterised by a much wider variability. In certain cases estimates may be made. But in the process of preparing expert opinions, the significance of this variability is often overlooked. The reasons might include the time-consuming nature and high cost of an accurate determination of repeatability in every single case at this stage of analysis. The biological variability encompasses a wide range and blurred boundary values of concentration. This has made it difficult to categorise the result to a precise interval, which will affect its interpretative evaluation.

This will be made even worse by an inaccurate definition of samples at the initial stage and by the credibility of the samples - associated with the distribution of the xenobiotic within the organ. This issue is not highlighted in the opinions provided by forensic experts. This is probably caused by an intuitive feeling of the inexpedience of trying to make the process of isolation highly precise, with the well-justified assumption that it will disappear later. This inconsistency leads to unreliability in the accuracy of the final result, which does not reflect the total uncertainty.

One should emphasise the need for a holistic approach to the process of forensic toxicological analysis and for attributing various levels of variability to its subsequent stages. The fierce competition in the field of the most modern methods is not justified without paying attention to the whole process. One issue should be particularly highlighted: at which stages will improvement in variability depend on the expertise of the expert, and at which stages is the level of our knowledge the limiting factor?

Keywords: Uncertainty of Results, Interpretation, Blurred Boundary of a Lethal Concentration.