Anni Steentoft

Simultaneous Screening and Quantification of 29 Drugs of Abuse in Oral Fluid by Solid-Phase Extraction and Ultraperformance LC-MS/MS

BACKGROUND The European DRUID (Driving under the Influence of Drugs, Alcohol And Medicines) project calls for analysis of oral fluid (OF) samples, collected randomly and anonymously at the roadside from drivers in Denmark throughout 2008-2009. To analyze these samples we developed an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detection of 29 drugs and illicit compounds in OF. The drugs detected were opioids, amphetamines, cocaine, benzodiazepines, and Delta-9-tetrahydrocannabinol. METHOD Solid-phase extraction was performed with a Gilson ASPEC XL4 system equipped with Bond Elut Certify sample cartridges. OF samples (200 mg) diluted with 5 mL of ammonium acetate/methanol (vol/vol 90:10) buffer were applied to the columns and eluted with 3 mL of acetonitrile with aqueous ammonium hydroxide. Target drugs were quantified by use of a Waters ACQUITY UPLC system coupled to a Waters Quattro Premier XE triple quadrupole (positive electrospray ionization mode, multiple reaction monitoring mode). RESULTS Extraction recoveries were 36%-114% for all analytes, including Delta-9-tetrahydrocannabinol and benzoylecgonine. The lower limit of quantification was 0.5 mug/kg for all analytes. Total imprecision (CV) was 5.9%-19.4%. With the use of deuterated internal standards for most compounds, the performance of the method was not influenced by matrix effects. A preliminary account of OF samples collected at the roadside showed the presence of amphetamine, cocaine, codeine, Delta-9-tetrahydrocannabinol, tramadol, and zopiclone. CONCLUSIONS The UPLC-MS/MS method makes it possible to detect all 29 analytes in 1 chromatographic run (15 min), including Delta-9-tetrahydrocannabinol and benzoylecgonine, which previously have been difficult to incorporate into multicomponent methods.

Drugged driving in the Nordic countries--a comparative study between five countries

The purpose of this study was to compare whether the high incidence of drugged driving in Norway was different to that in the other Nordic countries. All blood samples received by Nordic forensic institutes during one week in 1996, from drivers suspected by the police of driving under the influence (Denmark: n = 255, Finland: n = 270, Iceland: n = 40, Sweden: n = 86, Norway: n = 149), were analysed for alcohol and drugs (benzodiazepines, cannabinoids, amphetamines, cocaine, opiates and a number of antidepressant drugs) independent of the primary suspicion, and using the same analytical cut-off levels at the different institutes. The primary suspicion was directed towards drugs in more than 40% of the Norwegian cases, drugs were detected in more than 70% of these samples. In only 0-3% of the cases from Denmark, Finland and Iceland, were drugs suspected, while the corresponding frequency for Sweden was 17%. However, evidential breath analyses were used for about three-quarters of the Swedish drivers suspected to be influenced by alcohol. Blood alcohol concentrations (BACs) below the legal limits were found in 32, 18 and 2% of the Norwegian, Icelandic and Finnish cases, respectively (BAC < 0.05%), in 10% of the Danish cases (BAC < 0.08%) and in 20% of the Swedish cases (BAC < 0.02%). Drugs were most frequently found in the Norwegian and Swedish cases with no alcohol (80-83%). Similar frequencies of drugs in samples with BACs above the legal limits (19-22%), were obtained for all countries. Benzodiazepines, tetrahydrocannabinol and amphetamine represented the most commonly detected drugs. Our results show that differences between Norway and other Nordic countries with regard to drugs and driving, are connected to the selection criteria made by the police and with more focus on drugged driving in Norway.

Medicinal and illegal drugs among Danish car drivers

The objective of this study was to get an insight into the prevalence of medicinal and illegal drugs among car drivers in a Danish rural area. The police randomly stopped about 1000 car drivers and asked them to deliver a saliva sample and gave them a questionnaire to fill in at home. Laboratory analyses by specific methods of samples, which a screening found positive, confirmed that 2% were positive for benzodiazepines or illegal drugs (amphetamine, cannabis, cocaine or opiates): 1.3% were positive for illegal drugs and 0.7% for benzodiazepines. Questionnaire statements from some of the drivers confirm that occasionally some of these drive despite a suspicion to be under the influence of an illegal drug (2.8%), an illegal drug including alcohol (4%), a hazardous medicinal drug including alcohol (8.5%), or alcohol alone above the legal limit (24.5%). These results are considered reliable for the survey area and may not reflect national conditions. The overall results indicate that in this study driving under the influence of illegal drugs or alcohol seems to be associated to especially men, aged 22-44 years. Driving under the influence of hazardous medicinal drugs seems to be associated to middle-aged/elderly drivers, both men and women.

Drugs related to motor vehicle crashes in northern European countries: A study of fatally injured drivers

The aim of this study was to find which drugs and drug combinations were most common in drivers who died, in particular, in single vehicle crashes where the responsibility for the crash would be referred to the driver killed. The study included all available blood samples from drivers, who died within 24h of the accident, in the years 2001 and 2002 in the five Nordic countries (total population about 24 million inhabitants). The samples were analysed for more than 200 different drugs in addition to alcohol, using a similar analytical programme and cut-off limits in all countries. In three countries (Finland, Norway and Sweden) blood samples were available for more than 70% of the drivers, allowing representative prevalence data to be collected. 60% of the drivers in single vehicle crashes had alcohol and/or drug in their blood samples, compared with 30% of drivers killed in collisions with other vehicles. In single vehicle accidents, 66% of the drivers under 30 years of age had alcohol and/or drugs in their blood (alcohol only - 40%; drugs only - 12%; alcohol and drugs - 14%). The drugs found were mostly illicit drugs and psychoactive medicinal drugs with warning labels (in 57% and 58% respectively of the drivers under 30 with drugs present). Similar findings were obtained for drivers 30-49 years of age (63% with alcohol and/or drugs). In drivers aged 50 years and above, killed in single vehicle crashes (48% with alcohol and/or drugs) illicit drugs were found in only one case, and psychoactive medicinal drugs were detected less frequently than in younger age groups. In 75% of single vehicle crashes, the driver was under 50 years. Thus, the majority of accidents where the drivers must be considered responsible, occurred with drivers who had recently used alcohol, or drugs, alone or in combination. The drugs involved were often illicit and/or psychoactive drugs with warning labels. Therefore a large proportion of single vehicle accidents appear to be preventable, if more effective measures against driving after intake of alcohol and drugs can be implemented.

Methadone and drug addicts

SummaryDrug addicts who had died in Copenhagen City and County in 1981 and 1989 were analysed for methadone. In 1981, 94 cases were analysed of which 16% were found positive for methadone, and in 1989, 70 cases were analysed of which 37% were positive. Methadone alone was found to be the cause of death in 50% more cases in 1989 than in 1981. Only half of the drug addicts who were found positive for methadone had been under methadone treatment. Morphine and benzodiazepines were the most frequently occurring other substances in both 1981 and 1989. Alcohol was found in only about 30% of the methadone-positive cases. The median whole blood concentrations of methadone found in addicts where methadone was the cause of death was 0.3 mg/kg where no alcohol was present and 0.2 mg/kg where alcohol was present. In living persons using methadone, the median was 0.1 mg methadone/kg whole blood with or without alcohol present.ZusammenfassungEs wurden Drogenabhängige, die im Bereich der Stadt und des Kreises Kopenhagen in den Jahren 1981 and 1989 verstarben, hinsichtlich einer Methadoneinnahme untersucht. 1981 wurden 94 derartige Fälle untersucht, von denen 16% einen positiven Methadonbefund erbrachten. 1989 wurden 70 Fälle untersucht, von denen 37 positiv waren. Eine Methadonintoxikation als alleinige Todesursache wurde 1989 50% häufiger diagnostiziert als 1981. Nur die Hälfte der Drogenabhängigen, die Methadonpositiv waren, befanden sich in einer therapeutischen Methadonbehandlung. Morphin und Benzodiazepine waren sowohl 1981 als auch 1989 die am häufigsten nachzuweisenden weiteren Substanzen. Alkohol konnte nur in 30% der Methadon-positiven Fälle detektiert werden. Der Median der Vollblutkonzentration von Methadon betrug in jenen Fällen, in denen eine Methadonintoxikation die Todesursache war und keine zusätzliche Alkoholbeeinflußung vorlag, 0,3 mg/kg, hingegen bei einer zusätzlichen Alkoholbeeinflußung nur 0,2 mg/kg. Bei lebenden Personen, welche Methadon einnahmen, betrug der Median jeweils 0,1 mg Methadon/kg Vollblut unabhängig von der Alkoholbeeinflussung.

Deaths among drug addicts in Denmark in 1987-1991.

In the period 1987-1991 a total of 739 fatalities among drug addicts was investigated at the three University Institutes of Forensic Medicine in Denmark. The annual number rose from 130-140 in the first 4 years to 192 in 1991, and 80% were males. The mean and median age for both males and females increased by 1 year in the period. The main drug of abuse was heroin, in most cases supplemented by various other drugs, and in almost all cases taken intravenously. In about one-third of the cases each year there was information of abuse of alcohol in addition. In the poisoning cases, the main drug of poisoning was morphine/heroin, constituting 35-55% of the cases each year. As regards methadone-poisoning cases, the number increased significantly in 1991 compared to the first 4 years. Furthermore, the number and proportion of addicts dying while in methadone treatment increased during the 5-year period. In about half of the methadone poisoning cases, there was information of methadone treatment at the time of death. The other half obviously obtained the methadone completely illegally. Ketobemidone was the third most frequent drug of poisoning, while propoxyphene and barbituric acid only were found in a very few cases each. The results are compared to those from an earlier investigation concerning drug deaths in Denmark in 1968-1986. The importance of registering drug deaths is emphasized.

Risk of severe driver injury by driving with psychoactive substances

Driving with alcohol and other psychoactive substances imposes an increased risk of severe injury accidents. In a population-based case-control design, the relative risks of severe driver injury (MAIS≥2) by driving with ten substance groups were approximated by odds ratios (alcohol, amphetamines, benzoylecgonine, cocaine, cannabis, illicit opiates, benzodiazepines and Z-drugs, i.e. zolpidem and zopiclone, medicinal opioids, alcohol-drug combinations and drug-drug combinations). Data from six countries were included in the study: Belgium, Denmark, Finland, Italy, Lithuania and the Netherlands. Case samples (N=2490) were collected from severely injured drivers of passenger cars or vans in selected hospitals in various regions of the countries. Control samples (N=15,832) were sampled in a uniform sampling scheme stratified according to country, time, road type and season. Relative risks were approximated by odds ratios and calculated by logistic regression. The estimates were adjusted for age, gender and country. The highest risk of the driver being severely injured was associated with driving positive for high concentrations of alcohol (≥0.8 g/L), alone or in combination with other psychoactive substances. For alcohol, risk increased exponentially with blood alcohol concentration (BAC). The second most risky category contained various drug-drug combinations, amphetamines and medicinal opioids. Medium increased risk was associated with medium sized BACs (at or above 0.5 g/L, below 0.8 g/L) and benzoylecgonine. The least risky drug seemed to be cannabis and benzodiazepines and Z-drugs. For male drivers, the risk of being severely injured by driving with any of the psychoactive substances was about 65% of that of female drivers. For each of the substance groups there was a decrease in the risk of severe driver injury with increasing age. It is concluded that among psychoactive substances alcohol still poses the largest problem in terms of driver risk of getting injured.

DRUGS IN BLOOD SAMPLES FROM UNCONSCIOUS DRUG ADDICTS AFTER THE INTAKE OF AN OVERDOSE

This investigation includes whole blood samples from 53 drug addicts found unconscious in the Copenhagen area with evidence of a heroin overdose. Heroin/morphine was detected in 85% of the patients and other opioids in 11%. One or more benzodiazepines, most often diazepam, were detected in 75% of the patients. A blood alcohol concentration higher than 1.00 mg/g was detected in 57% of the patients. Methadone was detected in seven patients, ketobemidone in four, amphetamine in five and cocaine in one. This investigation showed widespread multi-drug abuse and heroin/morphine alone was detected in only one patient. As indicators of heroin intake, 6-monoacetylmorphine (MAM) and morphine were detected in this investigation.

Morphine concentrations in autopsy material from fatal cases after intake of morphine and/or heroin

Abstract This paper presents 245 fatal cases due to morphine and/or heroin over an 8-year period from East Denmark. All but 13 of the cases were from narcotics addicts. In 40% of the cases, only morphine was detected; in 32%, morphine and ethanol; in 19%, morphine and other drugs; and in 9%, morphine, ethanol and other drugs. Morphine concentrations in blood and liver for these four groups, and the age and sex distribution are shown. Morphine levels in blood and liver were also compared for: cases with a short survival time after the injection of morphine and/or heroin; cases from non-addicts; and cases where the drug used was obviously not the cause of death. The blood values in these groups were compared with blood concentrations found in living persons. Concentrations of morphine in stomach contents after intravenous injection of morphine and/or heroin are shown to be of the same level as in blood and liver. Ten fatal morphine cases are presented in which no morphine was detected in the urine after hydrolysis of the urine.

Muscle as an Autopsy Material for Evaluation of Fatal Cases of Drug Overdose

Abstract Striated muscle constitutes a large portion of the body weight and is nearly always available even in burned, putrefied, mummified or exhumed bodies. Drug concentrations have been estimated in extremity muscle, blood and liver in 142 fatal cases where drug overdose was implicated. The results suggest that muscle should be included as a standard autopsy material in examinations for drug poisoning.