Quantification of 31 illicit and medicinal drugs and metabolites in whole blood by fully automated solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry.

An efficient method for analyzing illegal and medicinal drugs in whole blood using fully automated sample preparation and short ultra-high-performance liquid chromatography-tandem mass spectrometry (MS/MS) run time is presented. A selection of 31 drugs, including amphetamines, cocaine, opioids, and benzodiazepines, was used. In order to increase the efficiency of routine analysis, a robotic system based on automated liquid handling and capable of handling all unit operation for sample preparation was built on a Freedom Evo 200 platform with several add-ons from Tecan and third-party vendors. Solid-phase extraction was performed using Strata X-C plates. Extraction time for 96 samples was less than 3 h. Chromatography was performed using an ACQUITY UPLC system (Waters Corporation, Milford, USA). Analytes were separated on a 100 mm × 2.1 mm, 1.7 µm Acquity UPLC CSH C(18) column using a 6.5 min 0.1 % ammonia (25 %) in water/0.1 % ammonia (25 %) in methanol gradient and quantified by MS/MS (Waters Quattro Premier XE) in multiple-reaction monitoring mode. Full validation, including linearity, precision and trueness, matrix effect, ion suppression/enhancement of co-eluting analytes, recovery, and specificity, was performed. The method was employed successfully in the laboratory and used for routine analysis of forensic material. In combination with tetrahydrocannabinol analysis, the method covered 96 % of cases involving driving under the influence of drugs. The manual labor involved in preparing blood samples, solvents, etc., was reduced to a half an hour per batch. The automated sample preparation setup also minimized human exposure to hazardous materials, provided highly improved ergonomics, and eliminated manual pipetting.

Drug-related death in Denmark in 2007.

INTRODUCTION: We investigated fatal poisonings among drug addicts in 2007. The cause of death, abuse pattern and geographic differences are presented. MATERIAL AND METHODS: All drug-related deaths examined at the three forensic medicine institutes in Denmark in 2007 were evaluated. RESULTS: The number of drug-related deaths in 2007 was 226. Methadone deaths had increased since 1997 while heroin/morphine deaths decreased. In earlier studies, very few deaths from central stimulants like cocaine and amphetamines occurred (1-1.5%), but in 2007 6% of the deaths were caused by these drugs. Multiple drug use was common. Heroin/morphine, cocaine, amphetamine, cannabis, methadone, benzodiazepines and alcohol were included in the poly-drug use. CONCLUSION: This investigation shows stabilization in the number of fatal poisonings in drug addicts. Geographic differences were observed. Methadone was the most frequent cause of fatal poisoning and there was a continuous decrease in heroin/morphine deaths. Fatal deaths from cocaine and amphetamine have increased considerably. Multiple drug use was common. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

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.

Driving under the influence of alcohol and drugs in the eastern part of Denmark in 2015 and 2016: Abuse patterns and trends.

OBJECTIVES: The objective of this study was to examine the frequency of psychoactive drugs and alcohol in drivers under suspicion of driving under the influence of drugs and alcohol in 2015 and 2016 in the eastern part of Denmark. The trends in the number of traffic cases sent for drug analysis since 2000 and alcohol analysis since 2011 are also discussed. METHODS: Blood samples from drivers suspected of being under the influence of alcohol and/or medication and/or illicit drugs in 2015 and 2016 were investigated as requested by the police. The blood samples were screened for alcohol and/or tetrahydrocannabinol (THC) alone, for other drugs (covering all drugs, except THC, listed in the Danish list of narcotic drugs), or for THC and other drugs. Age and gender were also recorded. The number of drug traffic cases since 2000 and the number of alcohol cases since 2011 were extracted from our Laboratory Information Management System (LIMS). RESULTS: In total, 11,493 traffic cases were investigated. Alcohol and/or drugs exceeded the legal limit in 9,657 (84%) cases. Men constituted 95% of the drivers investigated for drugs and 88% of the alcohol cases. The drivers investigated for drugs consisted primarily of young men, whereas drivers investigated for alcohol were older. The frequency was higher for positive alcohol cases above the legal limit (87%) than for drug cases (76%) above the fixed concentration limit. THC (67-69%) was the most frequently detected drug above the legal limit, followed by cocaine (27-28.5%), amphetamine (17%), and clonazepam (6-7%) in both years. Morphine (5.4%), included among the 5 most frequent drugs in 2015, was replaced by methadone (4.6%) in 2016. Few new psychoactive drugs (NPS) were detected. The number of traffic cases sent for drug analysis has increased more than 30-fold since 2000-2006, and the number of traffic cases submitted in 2016 for drug analysis was higher than the number for alcohol analysis; the latter has decreased since 2011. CONCLUSION: Overall, alcohol was the most frequent compound detected above the legal limit in both years, followed by the well-known illicit drugs THC, cocaine, and amphetamine. NPS were seldom seen. One consequence of the increased focus on drugs in traffic has been an immense increase in drug traffic cases sent for analysis since 2006 in the eastern part of Denmark. Although this survey revealed only minimal changes compared to earlier investigations, surveys like this are invaluable for monitoring abuse patterns and trends in drugged and drunken driving.

Fatal poisoning among patients with drug addiction.

INTRODUCTION: Fatal poisonings among drug addicts in Denmark in 2012 were examined. Cause of death, abuse pattern and geographic differences are discussed and data are compared with previous studies. METHODS: All fatal poisonings examined at the three institutes of forensic medicine in Denmark in 2012 were included in the study. RESULTS: A total of 188 fatal intoxications were recorded. The median age increased from 37.5 in 2007 to 41.5 in 2012. The majority were men (77%). Methadone (59%) was the main intoxicant. The decrease in the frequency of heroin/morphine deaths since 1997 (71%) continued, declining to 44% in 2002, 33% in 2007 and finally to 27% in 2012. Few deaths from central stimulants (amphetamine and cocaine) occurred. Multiple drug use was common and consisted mainly of opioids, cocaine, amphetamine, cannabis, benzodiazepines and alcohol. Heroin/morphine use was most frequent on Funen and in South Jutland. Cocaine was most frequently detected in East Denmark, while amphetamine was more frequent in West Denmark. CONCLUSIONS: The number of fatal poisonings among drug addicts has stabilised around 200. The increase in methadone deaths continued and, as in 2007, methadone was the main intoxicant. The increase in methadone deaths seems to be associated with use of methadone in substitution treatment. Nevertheless, methadone treatment also seems to save lives, as indicated by the increasing median age. Use of antidepressants and antipsychotics increased to a high level compared with 2007, indicating that a considerable number of drug addicts also have psychiatric illness. FUNDING: none. TRIAL REGISTRATION: not relevant.

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.

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.

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.

A validated method for simultaneous screening and quantification of twenty-three benzodiazepines and metabolites plus zopiclone and zaleplone in whole blood by liquid-liquid extraction and ultra-performance liquid chromatography- tandem mass spectrometry.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for detection of 23 benzodiazepines and related compounds in whole blood was developed and validated. The method is used for screening and quantitation of benzodiazepines in whole blood received from autopsy cases and living persons. The detected compounds were alprazolam, bromazepam, brotizolam, chlordiazepoxide, demoxepam, clobazam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, estazolam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, midazolam, nitrazepam, 7-aminonitrazepam, oxazepam, temazepam, triazolam, zaleplon, and zopiclone. Whole blood from drug-free volunteers was used for all experiments. Blood samples (0.200 g) were extracted with ethyl acetate at pH 9. Target drugs were quantified using a Waters ACQUITY UPLC system coupled to a Waters Quattro Premier XE triple quadrupole in positive electrospray ionization, multiple reaction monitoring mode. The use of deuterated internal standards for most compounds verified that the accuracy of the method was not influenced by matrix effects. Extraction recoveries were 73-108% for all analytes. Lower limits of quantification ranged from 0.002 to 0.005 mg/kg. Long-term imprecision (CV%) ranged from 6.0 to 18.7%. We present a fully validated UPLC-MS-MS method for 23 benzodiazepines in whole blood with a run-time of only 5 min and using only 0.200 g of whole blood.

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.

Screening and quantitative determination of twelve acidic and neutral pharmaceuticals in whole blood by liquid-liquid extraction and liquid chromatography-tandem mass spectrometry.

We describe a multi-method for simultaneous identification and quantification of 12 acidic and neutral compounds in whole blood. The method involves a simple liquid-liquid extraction, and the identification and quantification are performed using liquid chromatography-tandem mass spectrometry. The method was fully validated for salicylic acid, paracetamol, phenobarbital, carisoprodol, meprobamate, topiramate, etodolac, chlorzoxazone, furosemide, ibuprofen, warfarin, and salicylamide. The method also tentatively includes thiopental, theophylline, piroxicam, naproxen, diclophenac, and modafinil, but these drugs were not included in the full validation program and are not described in detail here. Limit of quantitation was 1 mg/kg for the compounds with coefficients of variation of < 20%, except for furosemide, which had a coefficient of variation of 32% at limit of quantitation. The measuring interval was wide for most components. Extraction efficiencies were high, reflecting the high-yield capacity of the method.

[Fatal cases of poisoning in eastern Denmark during a five-year period (1998-2002)]. / Forgiftningsdødsfald over en femårig periode (1998-2002) i Østdanmark.

INTRODUCTION: The goal of this investigation was to determine the pattern of fatal poisonings in eastern Denmark from 1998 to 2002 and compare it with similar investigations from 1979 to 1996. MATERIALS AND METHODS: The material included 2,996 autopsies from eastern Denmark in which extensive forensic chemical investigations were performed. RESULTS: Of the 2,996 autopsies, 694 cases were drug addicts, in whom 497 fatal overdoses were detected, while in the remaining 2,302 cases of nonaddicts, 443 fatal poisonings were determined. Morphine (heroine) and methadone were the main causes of death among the fatal poisonings of the drug addicts, accounting for 90% of the cases. The fatal poisonings among the nonaddicts were due mainly to medicine (73% of the cases); 13% were due to carbon monoxide and/or cyanide poisoning, and 12% were due to acute ethanol poisoning. The medicine was a wide range of strong and weak analgesics, antidepressants and antipsychotic drugs of older and newer origins. Comparison with earlier investigations from 1979 to 1996 showed that the poisoning pattern had changed, but similarities were also seen. The most frequently occurring drugs involved in fatal poisonings in eastern Denmark from 1979 to 2002 were morphine, methadone, ethanol and carbon monoxide/cyanide, of which methadone increased in occurrence over the 23-year period. CONCLUSION: The investigation of fatal poisonings is an important element in monitoring changes in drug abuse and poisoning patterns and levels.