Kirsten Wiese Simonsen

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.

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.

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.

Alcohol and drugs in seriously injured drivers in six European countries

The objective of this study was to determine the presence of alcohol and drugs in drivers severely injured in traffic crashes in six European countries. Data were collected from 2492 seriously injured drivers of cars and vans in Belgium, Denmark, Finland, Italy, Lithuania, and the Netherlands, between 2007 and 2010. Toxicological analysis was performed with chromatographic techniques on whole blood for 23 substances. The percentage of drivers positive for at least one psychoactive substance ranged between 28% (Lithuania) and 53% (Belgium). Alcohol (≥0.1 g/L) was the most common finding with the highest percentage in Belgium (42.5%). Among the alcohol-positive drivers, 90.5% had a blood alcohol count (BAC) ≥0.5 g/L and 65.7% had a BAC ≥1.3 g/L. Benzodiazepines (0.0-10.2%) and medicinal opioids (0.5-7.8%) were the most prevailing medicinal drugs, but half of the concentrations were lower than therapeutic. Cannabis (0.5-7.6%) was the most prevailing illicit drug. Alcohol was found in combination with drugs in 2.3-13.2% of the drivers. Drug combinations were found in 0.5-4.3% of the drivers. This study confirms the high prevalence of psychoactive substances in injured drivers, but we observed large differences between the participating countries. Alcohol was the most common finding, followed by cannabis and benzodiazepines. Notable are the many drivers having a BAC ≥ 1.3 g/L. The majority of the substances were found in combination with another psychoactive substance, mostly alcohol. The high prevalence of high BACs and combinations (compared to roadside surveys) suggest that those drivers are most at risk and that preventive actions should target them preferentially.

A prospective toxicology analysis in alcoholics

A prospective and comprehensive investigation was done on 73 medico-legal autopsies in alcoholics. The results of the toxicology analyses are described. Alcohol intoxication was the cause of death in 8%, combined alcohol/drug intoxication in 15% and drugs alone in 19%. Alcoholic ketoacidosis was found to be the cause of death in 7%. Altogether toxicology analyses were necessary for determining the cause of death in 51% of the cases. In four cases the cause of death would not have been found, had this investigation not been made. It is concluded that toxicology analyses should be the rule rather than the exception in deaths in alcoholics.

Narcotics at street level in Denmark a prospective investigation from 1995 to 2000

This article describes an investigation of illicit drugs at street level in six selected police districts in different regions of Denmark. The investigation was carried out during a 6-year period from 1995 to 2000. During the period, a total of 1244 samples were examined, as about 200 samples were seized each year. A total of 94% of the seized samples were familiar drugs: heroin base, heroin hydrochloride, cocaine hydrochloride and amphetamine sulphate. Only 2% of the samples contained designer drugs. From having constituted 53% of the samples in 1995, the frequency of heroin base fell during the period to 27% of the samples in 2000. The frequency of heroin hydrochloride was unchanged. In the same period, the frequency of cocaine hydrochloride increased from 10% of the samples in 1995 to about 25% of the samples in 2000. Apart from a few exceptions, cocaine had the same extension in all regions of Denmark after 1996. Amphetamine was more frequent in the west of Denmark, while heroin hydrochloride was more frequent in central Denmark. The purity of heroin base was lower in the period 1997-1999 than in the other years. During the entire period, the purity of cocaine hydrochloride and amphetamine sulphate fell, while the purity of heroin hydrochloride was unchanged. No significant differences between the various regions of Denmark were detected during the period in the purity of heroin hydrochloride, heroin base, cocaine hydrochloride or amphetamine sulphate. MDMA was the most frequent designer drug, but other types of designer drugs such as MDA and MDE and the less common PMA, PMMA and MBDB were also found.

The Frequency of Drugs Among Danish Drivers Before and After the Introduction of Fixed Concentration Limits

Objective: Until July 2007, the driving under the influence of drugs (DUID) legislation in Denmark was based on impairment, evaluated on the basis of a clinical investigation and toxicological analyses, but in 2007 fixed concentration limits were introduced into the Danish traffic legislation. The objective for this study was to investigate the prevalence of medication and illicit drugs among Danish drivers before and after 2007. Methods: Blood samples from drivers suspected of being under the influence of medication and/or illicit drugs were investigated as requested by the police. The results for a 10-year period before and for one year after the introduction of fixed concentration limits are presented. Results: A total of 2340 blood samples were analyzed for the presence of medications and/or illicit drugs for the period 1997–2006. The average number of cases per year was 234 (213–283), and on average 87 percent of the investigated cases were positive for one or more drugs. For 2008 the number of investigated traffic cases was increased to 1176. Seventy-three percent of the cases from 2008 were positive for one or more drugs. Benzodiazepines, cannabis (THC), amphetamine, heroin/morphine, methadone, cocaine, and ecstasy were the most frequently detected drugs for the period 1997–2006 and also in 2008. The number of these cases in which an ethanol level was detected above 0.5 mg/g (the Danish legal limit) was on average 18 percent (9–26%) for the period 1997–2006 and 19 percent for 2008. The average age of the drivers ranged from 31 to 34 years for the period 1997–2006 and was 31 years for 2008. The percentage of females per year ranged from 3 to 20 percent. Conclusion: The number of traffic cases investigated for substances other than ethanol were consistently low, in the range of 200 to 300 per year during the period from 1997 to 2006, but after the introduction of fixed concentration limits in 2007 a 5-fold increase was seen already in 2008.

Quantitative Analysis of 30 Drugs in Whole Blood by SPE and UHPLC-TOF- MS

An Ultra-High Pressure Liquid Chromatography Time-of-Flight Mass Spectrometry (UHPLC-TOF-MS) method for quantitative analysis of 30 drugs in whole blood was developed and validated. The method was used for screening and quantification of common drugs and drugs of abuse in whole blood received from autopsy cases and living persons. The compounds included: alprazolam, amphetamine, benzoylecgonine, bromazepam, cathine, cathinone, chlordiazepoxide, cocaine, codeine, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, flunitrazepam, 7-aminoflunitrazepam, ketamine, ketobemidone, 3,4-Methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, methadone, morphine, 6-monoacetylmorphine, nitrazepam, 7-aminonitrazepam, oxazepam, temazepam, tramadol, O-desmethyltramadol, and zolpidem. Blood samples (200 μL) were subjected to Solid Phase Extraction (SPE). Target drugs were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2 TOF-MS apparatus. Extraction recoveries ranged from 41% (7-aminoclonazepam) to 111% (ketamine) and matrix effects ranged from -13% (temazepam) to 50% (7-aminonitrazepam). For all compounds, a quadratic polynomial was applied for fitting the calibration curves. Lower Limits of Quantification (LOQ) ranged from 0.005 to 0.05 mg/kg. Satisfactory precisions below 15% and accuracies within 85-115% were obtained for all compounds at concentrations exceeding the LOQ. In conclusion, we present a validated UHPLC-TOF-MS method for simultaneous quantification of 30 drugs in whole blood with a run time of 15 min using 200 μL of whole blood.

Random and systematic errors in case-control studies calculating the injury risk of driving under the influence of psychoactive substances

Between 2006 and 2010, six population based case-control studies were conducted as part of the European research-project DRUID (DRiving Under the Influence of Drugs, alcohol and medicines). The aim of these case-control studies was to calculate odds ratios indicating the relative risk of serious injury in car crashes. The calculated odds ratios in these studies showed large variations, despite the use of uniform guidelines for the study designs. The main objective of the present article is to provide insight into the presence of random and systematic errors in the six DRUID case-control studies. Relevant information was gathered from the DRUID-reports for eleven indicators for errors. The results showed that differences between the odds ratios in the DRUID case-control studies may indeed be (partially) explained by random and systematic errors. Selection bias and errors due to small sample sizes and cell counts were the most frequently observed errors in the six DRUID case-control studies. Therefore, it is recommended that epidemiological studies that assess the risk of psychoactive substances in traffic pay specific attention to avoid these potential sources of random and systematic errors. The list of indicators that was identified in this study is useful both as guidance for systematic reviews and meta-analyses and for future epidemiological studies in the field of driving under the influence to minimize sources of errors already at the start of the study.