Journal of Analytical Toxicology 2014;38:46 –51 doi:10.1093/jat/bkt094 Advance Access publication December 9, 2013
Article
Concentrations of Cocaine and Benzoylecgonine in Femoral Blood from Cocaine-Related Deaths Compared with Venous Blood from Impaired Drivers Alan Wayne Jones1,2* and Anita Holmgren1 1
Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Artillerigatan 12, SE-587 58 Linko¨ping, Sweden, and 2Department of Clinical Pharmacology, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden
*Author to whom correspondence should be addressed. Email: [email protected]
The concentrations of cocaine and its major metabolite benzoylecgonine (BZE) were determined in femoral blood from 132 cocainerelated deaths and compared with venous blood from 988 apprehended drivers. Cocaine and BZE were determined by solidphase extraction and isotope dilution gas chromatography –mass spectrometry with limits of quantitation of 0.02 mg/L for both substances. Significantly more men (95–98%) than women (2–5%) abused cocaine, although their mean age was about the same (29– 30 years). Mean age (+ + SD) of cocaine-related deaths was 29 + 7 years, which was not significantly different from 30 + 8 years in traffic cases (P > 0.05). The median concentration of cocaine in blood in 61 fatalities was 0.10 mg/L compared with 0.06 mg/L in traffic cases (P < 0.001). In drug intoxication deaths, the median concentration of cocaine was 0.13 mg/L (N 5 25), which was not significantly different from 0.09 mg/L (N 5 36) in other causes of death. Cocaine-related deaths mostly involved mixed drug intoxications including co-ingestion of heroin, cannabis, amphetamines as well as legal drugs, such as benzodiazepines and/or ethanol. The concentrations of cocaine in blood from living and deceased persons overlapped, which makes it infeasible to predict toxicity from the analytical toxicology results alone.
Introduction Abuse of cocaine, in the form of its hydrochloride salt and as the free-base (crack), is highly prevalent worldwide and has become a growing problem in some nations (1, 2). Cocaine exerts a powerful reinforcing effect on the individual and the associated psychological dependence and craving means more of the drug and higher doses are taken (3, 4). Indeed, the abuse potential of cocaine was recognized early after it was isolated from coca leaves in 1860 (5) and when introduced into medicine as a local anesthetic for eye surgery in 1874 (6). Compared with other recreational drugs, such as opiates, cocaine-related deaths are relatively infrequent in Sweden and when they do occur they are mostly attributed to multidrug intoxications (7, 8). The combined use of cocaine and heroin is especially dangerous when this mixture is administered intravenously (9). Likewise, the toxicity of cocaine is enhanced when co-ingested with ethanol, owing to the biosynthesis of a pharmacologically active metabolite (cocaethylene), which has a longer elimination half-life from blood compared with cocaine itself (10). The physiological effects of cocaine include increase in heart rate and blood pressure, which in predisposed individuals might result in hemorrhage stroke and death (11). Other causes of death in chronic cocaine users include excited delirium, myocardial infarctions and cardiac arrhythmias, although these are not easy to recognize at autopsy (12). Forensic toxicologists are in general agreement that toxicity of cocaine is unpredictable
and that the concentrations determined in blood from living and deceased persons show a significant overlap, which makes interpretation difficult (13). This paper compares the concentrations of cocaine and its main metabolite benzoylecgonine (BZE) in forensic blood samples from people suspected of driving under the influence of drugs (DUID) with cocaine-related deaths in which femoral blood was analyzed. These medical examiner cases were classified as either drug intoxication deaths or other causes of death, e.g., trauma, traffic or natural disease. The number and types of co-ingested drugs together with cocaine were compared and contrasted in living and deceased persons.
Material and methods Forensic autopsies The number of forensic autopsies made in Sweden (current population 9.3 million) has remained remarkably constant at just over 5000 per year over for the past 10 years. The police authorities order a forensic autopsy in all unnatural or suspicious deaths. Forensic pathologists take specimens of femoral blood for toxicology before evisceration of the body and after addition of a potassium fluoride preservative (2%, w/v), blood, urine and vitreous humor are transported to the toxicology laboratory in refrigerated containers. A national forensic toxicology database (TOXBASE) was searched retrospectively from 2001 to 2010 to find autopsy causes in which cocaine and/or its major metabolite BZE were verified present in femoral blood. Cause and manner of death was assigned by forensic pathologists after consideration of all aspects of the case including anatomical and histological findings, police reports, witness statements, discoveries at the scene, toxicology report, etc. International Classification of Disease Codes (ICD-9) were assigned by medical examiners and used by us to distinguish drug intoxication ( poisoning) deaths from other causes of death. The postmortem interval was not known in individual cases, although none of the bodies showed signs of decomposition. If femoral blood was not obtainable, owing to massive trauma or decomposition of the body, these cases were not used in the present study.
Apprehended drivers Blood and urine specimens from people arrested for DUID in Sweden are sent to the same central toxicology laboratory responsible for analysis of postmortem specimens. All analytical results are entered into a database (TOXBASE). The venous blood samples from DUID suspects were taken using gray-stopper evacuated tubes (10 mL) containing a mixture of sodium fluoride (100 mg)
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and potassium oxalate (25 mg) as preservatives. Whenever possible, a specimen of urine (10 mL) is also collected from traffic offenders in plastic tubes containing sodium fluoride (100 mg) as preservative. Results from 5 years of traffic cases (2001–2005) were searched to find those in which either cocaine and/or BZE were verified positive in blood samples. In routine forensic cases, other metabolites of cocaine, such as norcocaine, ecgonine methylester or cocaethylene, were not analyzed.
Police procedures The first suspicion of DUID arises when the police investigate a moving traffic offence, a road-traffic collision or when a vehicle is stopped in connection with a routine sobriety control. Other road users might call emergency services to report seeing a dangerous driver, which results in a police investigation of alcohol or drug use. The traffic offenders first provide a roadside breathalcohol screening test and a positive result (.0.1 mg/L) leads to an evidential breath-alcohol test done at the nearest police station. Refusal to comply with the roadside breath test or the evidential test will result in a blood sample being taken from the driver by force if necessary. If the roadside breath-alcohol test is negative and the driver shows signs and/or symptoms of drug influence, then the police request a venous blood sample for toxicological analysis. The samples of venous blood are usually taken by a physician or nurse at a police station or if a driver is injured in a crash blood samples are taken at a local hospital. In connection with the introduction of a zero-tolerance law for driving under the influence of drugs other than alcohol (1 July 1999), the police were permitted to examine a driver’s eyes with a flashlight to record reaction to light, and to measure pupil-size and other effects of drugs such as gaze nystagmus as supporting evidence of drug influence. These observations of clinical drug influence are recorded by the police on arrest forms, which are sent to the toxicology laboratory together with the specimens of blood and urine.
Determination of cocaine and BZE in blood The analysis of forensic blood samples starts with a broad screening using immunoassay methods (EMIT and CEDIA), and this is targeted at five classes of drugs, namely opiates, cannabinoids, amphetamine analogs, cocaine metabolite and benzodiazepines. If urine is available, this specimen is used for the immunologic
screening analysis, otherwise blood is used after precipitation of proteins. The urinary cut-off concentration for positive BZE was 0.30 mg/L (EMIT) and 0.05 mg/L, if blood extracts (CEDIA) were analyzed. Positive results from the screening are always verified by more sensitive and specific analytical methods, such as gas chromatography –mass spectrometry (GC–MS) or LC–MS for illicit drugs or GC-NPD for prescription drugs and headspace gas chromatography for volatiles. The method used to determine cocaine and BZE in blood was described in more detail elsewhere and this involved use of solid-phase extraction (Bond Elute Certify columns) followed by GC– MS after adding deuterium (d3) labeled internal standards (14). Verification of cocaine was done by GC–MS using mass fragments m/z 182/185 for quantitation analysis and m/z 198 and m/z 303 as the qualifier ions. Verification of BZE was done after making the pentafluoropropyl derivatives (PFPA and PFPOH) and using mass fragments m/z of 300/303 for quantitative analysis and m/z 316 and m/z 421 as the qualifier ions. Standard curves were prepared by spiking drug-free blood with known concentrations of cocaine and BZE and these were linear over a range from 0.02 to 2.0 mg/L for both analytes. When concentrations in blood exceeded 2.0 mg/L, the samples were reanalyzed after dilution with fresh drug-free blood. The analytical limit of quantitation for reporting positive cocaine and BZE in whole blood was 0.02 mg/L from both substances.
Statistical evaluation of results Mean, median and highest concentrations of cocaine and BZE in blood were used as descriptive statistics. Mean age of traffic offenders and autopsy cases were compared by Student’s t-test. Because frequency distributions of cocaine and BZE in blood were markedly skewed to the right, medians are preferred as an indicator of central tendency and two medians were compared by a Mann –Whitney non-parametric test. Comparison of cocaine and BZE concentrations in blood from living and deceased persons was done with the help of box-and-whisker plots.
Results Demographics of cocaine users The mean age of cocaine-related deaths and traffic cases with cocaine in blood was about the same 29 –30 years (range 15 –70
Table I Subject Demographics and Concentrations of Cocaine and BZE in Blood from Autopsy Cases and in People Arrested for DUID (Traffic) Case type
Drugs present
N
N (%) Male
Autopsy
Traffic
Cocaine and BZE BZE only BZE all cases Cocaine and BZE BZE only BZE all cases
61 71 132 300 688 988
a
58 (95) 67 (95) 125 (95) 293 (98) 656 (95) 949 (96)
Age (years) b
Cocaine (mg/L)
BZE (mg/L)c
Female
mean + SD
Mean (median) max.
Mean (median) max.
3 (5) 4 (5) 7 (5) 7 (2) 32 (5) 39 (4)
30 + 8.1 30 + 7.7 30 + 7.8 29 + 7.1 29 + 7.3 29 + 7.3
0.62 (0.10)d 8.5 ,0.02 mg/L 0.62 (0.10) 8.5 0.08 (0.06) 2.1 ,0.02 mg/L 0.08 (0.06) 2.1
1.80 (0.70)e 22 0.21 (0.11) 1.1 0.95 (0.30) 22 0.90 (0.70)e 4.2 0.19 (0.13) 1.3 0.41 (0.20) 4.2
a
Higher proportion of males to females (P , 0.001). No significant age differences (P . 0.05). c Median concentrations of BZE were not significantly different between autopsy and traffic cases (P . 0.05). d Higher median concentration of cocaine in the autopsy cases (P , 0.001). e Higher median concentration of BZE in blood when cocaine was also present (P , 0.001). b
Blood-Cocaine Concentrations in Living and Deceased Persons 47
years) as shown in Table I (P . 0.05). However, there was a clear predominance of males (95 –98%) that used cocaine in both the living and deceased persons.
Concentrations of cocaine and BZE in blood Table I shows that the median concentration of cocaine in autopsy blood (0.1 mg/L) was significantly higher than in blood from apprehended drivers (0.06 mg/L) (P , 0.001). The concentrations of parent drug (cocaine) and metabolite (BZE) were highly correlated in autopsy cases r ¼ 0.86 (N ¼ 61, P , 0.001) and traffic offenders, r ¼ 0.38 (N ¼ 300, P , 0.001). The median concentration of BZE in blood was higher when cocaine was also present in blood, which suggests more recent use of the stimulant (P , 0.001). The concentration distributions of cocaine and BZE in blood from living and deceased are shown in Figures 1 and 2 as box-and-whisker plots. A logarithm transformation of drug concentrations was used because the distributions were markedly skewed to the right. There was also a considerable overlapping in concentrations of cocaine (Figure 1) and BZE (Figure 2) for samples taken from living and deceased persons. Among autopsy cases, there were 31 cases (51%) with a cocaine concentration in blood .0.1 mg/L and 22 cases (36%) exceeded 0.2 mg/L. This compares with 71 traffic cases (24%) over 0.1 mg/L and 19 cases (6.3%) .0.2 mg/L.
Prevalence of co-ingested drugs Poly-drug use was more common in the medical examiner (autopsy) cases compared with traffic cases and the number of co-ingested substances varied from one to seven. Table II shows that cocaine and BZE were the only drugs identified in 110 traffic offenders (34%) and the median concentration of cocaine was 0.07 mg/L. In another 325 traffic cases, BZE, with or without cocaine, was the only drug identified at a median concentration of 0.20 mg/L. In the motorists, the concentrations of cocaine and BZE did not seem to depend on number of other drugs identified. Highest concentrations of cocaine and BZE were found in four cocaine-related deaths (3%) at a median concentration of 0.59 mg/L (median BZE 0.70 mg/L, N ¼ 12) when no other drugs were present.
Types of drugs co-ingested with cocaine Table III lists the top-10 drugs identified in blood samples together with cocaine and/or BZE. In both DUID suspects and autopsy cases, the top-three drugs were the same, namely ethanol, cannabis and diazepam. Other illicit drugs identified in blood along with cocaine were amphetamine, morphine (heroin), GHB and MDMA. The major class of prescription drugs used with cocaine were benzodiazepines exemplified by alprazolam, flunitrazepam, clonazepam and nitrazepam. The median concentrations of cocaine in blood tended to be higher in the autopsy cases, although these concentrations did not seem to depend on the type of co-ingested substances (Table III), neither in living or deceased persons. 48 Jones and Holmgren
Figure 1. Box-and-whiskers plots comparing concentration distributions of cocaine in cocaine-related deaths (autopsy) and traffic cases (impaired drivers). The boxes depict median and lower and upper quartiles, whereas the whiskers extend to +1.5 times the interquartile range and outliers are shown as separate data points.
Figure 2. Box-and-whiskers plots comparing concentration distributions of BZE in cocaine-related deaths (autopsy) and traffic cases (impaired drivers). The boxes depict median and lower and upper quartiles, whereas the whiskers extend to +1.5 times the interquartile range and outliers are shown as separate data points.
Table II Number of Cases (N) and Median Concentrations of Cocaine and BZE in Blood from Living (Traffic Cases) and Deceased Persons in Relation to the Number of Co-ingested Substances Co-ingested drugs
0 1 2 3 4 –7
Autopsy (N)a
Median conc. (mg/L)
Cocaine
BZE
Cocaine
BZEa
Cocaine
BZE
Cocaine
BZEa
4 17 11 8 17
12 31 23 16 50
0.59 0.09 0.20 0.08 0.10
0.70 0.40 0.30 0.45 0.15
110 104 47 29 10
325 292 201 121 49
0.07 0.05 0.05 0.06 0.09
0.20 0.20 0.17 0.19 0.14
Traffic (N)
Median conc. (mg/L)
N ¼ number of cases. a With or without cocaine also present in blood.
Cocaine concentrations in intoxication deaths Table IV shows the concentrations of cocaine and BZE in blood samples from intoxication deaths (drug overdose) or other
Table III Number of Cases (N) and Median Concentrations of Cocaine and BZE in Blood from Living (traffic cases) and Deceased Persons (Autopsy Cases) in Relation to the Types of Co-ingested Substances Autopsy cases
Ethanol THC Diazepam Morphine (heroin)b Amphetamines Paracetamol Flunitrazepam Alprazolam Clonazepam Nitrazepam
Median conc. (mg/L)
Traffic cases
a
Cocaine (N)
BZE (N)
0.07 (23) 0.08 (13) 0.10 (12) 0.10 (11) 0.12 (9) 0.22 (10) 0.15 (3) 0.06 (6) 0.10 (3) 0.08 (2)
0.30 (40) 0.20 (33) 0.20 (31) 0.16 (42) 0.14 (30) 0.65 (16) 0.16 (14) 0.45 (12) 0.22 (8) 0.23 (8)
Ethanol THC Diazepam Amphetamines Flunitrazepam Morphine (heroin)b MDA/MDMA GHB Alprazolam Clonazepam
Median conc. (mg/L) Cocaine (N)
BZEa (N)
0.05 (61) 0.05 (57) 0.05 (53) 0.05 (43) 0.06 (40) 0.04 (22) 0.03 (6) 0.07 (7) 0.04 (6) 0.03 (5)
0.20 (124) 0.16 (238) 0.20 (211) 0.14 (238) 0.19 (152) 0.18 (110) 0.19 (36) 0.17 (19) 0.12 (25) 0.19 (18)
N ¼ number of cases. a BZE with or without cocaine present in blood samples. b Heroin use confirmed from presence of 6-acetlylmorphine in blood or urine or morphine-to-codeine concentration ratio in blood above unity.
Table IV Comparison of Femoral Blood Concentrations of Cocaine and/or BZE in Autopsy Cases Classified as Drug Intoxication (Poisoning) or Other Causes of Death Cause of death
Drugs in blood
N
Cocaine (mg/L) Mean (median) highest
BZE (mg/L) Mean (median) highest
Drug intoxicationa
Cocaine and BZE BZE only Cocaine and BZE BZE only
25
1.25 (0.13)b 8.5
2.96 (1.3)c 22.0
37 36
,0.02 mg/L 0.18 (0.09) 1.0
0.22 (0.12)b 1.1 0.99 (0.50) 4.1
34
,0.02 mg/L
0.20 (0.10) 0.9
Other causes (trauma, traffic, natural)
a
Mainly mixed-drug overdose deaths involving ethanol, morphine (heroin) and/or amphetamine, diazepam in various combinations. b Median concentration of cocaine in drug intoxication deaths not significantly different from other causes of death (P . 0.05). c Median BZE concentration was higher in the intoxication deaths (P , 0.01).
cause of death (trauma, traffic, etc.). In 25 intoxication deaths (40%), the median concentration of cocaine was 0.13 mg/L, which was not significantly different from a median of 0.09 mg/ L found in 36 other causes of death (P . 0.05). High concentrations of cocaine were determined in three intoxication deaths when no other substances were present; 4.1 mg/L (BZE 0.7 mg/ L), 8.5 mg/L (BZE 22 mg/L) and 6.7 mg/L (BZE 11 mg/L), respectively.
Discussion In this study, most cocaine-related deaths involved co-ingestion of other psychoactive substances, such as ethanol, heroin or amphetamine, and only four cases were mono-intoxications, which is in good agreement with other studies (11, 15, 16). A cocaine-related death implies that the toxicology results were positive for cocaine and/or BZE when femoral blood was analyzed by GC –MS analysis, but the death was not necessarily a drug overdose. Cocaine poisoning deaths are sometimes observed in body-packers after leakage of the concealed drugs or in suicides when massive doses of cocaine are administered intravenously (17). Cocaine-related fatalities might also reflect
adverse cardiovascular events, such as myocardial infarction, cardiac arrhythmia and/or QT syndrome (12, 18). The death certificates issued in chronic cocaine users sometimes incriminate paranoia, delusions, excited delirium and convulsions as important factors in the death (19, 20). The concentrations of drugs in postmortem blood samples, including cocaine and BZE, show temporal and site-dependent changes (21). Standardized methods of sampling postmortem blood for toxicology are important and the specimen tubes should contain sodium or potassium fluoride as a preservative, which stabilizes blood-cocaine concentrations after sampling. Storage and transport of blood samples frozen or refrigerated (þ48C) also improves stability by preventing in vitro degradation of cocaine (22, 23). The concentrations of cocaine and BZE were determined in peripheral blood samples, that is, femoral vein (autopsy) or cubital vein (traffic cases) and a fluoride preservative was always included (23). The biological elimination half-life of cocaine is fairly short (0.5–1.5 h) compared with its metabolite BZE (6–10 h), which explains why many blood samples were positive for BZE but negative for parent drug cocaine (24). When both cocaine and BZE were present together in blood, this speaks towards fairly recent use of the stimulant as found in N ¼ 61 autopsy cases and N ¼ 300 traffic cases. It is not possible to calculate the amount of drug taken or the route of administration or time of last use from the analytical toxicology results alone (25). The median concentration of cocaine in 25 drug intoxication deaths in Melbourne, Australia was 0.1 mg/L, which was not significantly different from people taking cocaine but dying from other causes, such as external injury or natural disease (26). In our study, we found the same overall median concentration in all cocaine-related deaths (0.1 mg/L, N ¼ 61), although the median was higher in 25 drug intoxication deaths (0.13 mg/L) compared with median of 0.09 mg/L in other causes of death (N ¼ 36). These results support the generally accepted notion that cocaine concentrations in blood from living (DUID suspects and people admitted to hospital for treatment) overlap with cocaine concentrations in medical examiner cases. Cocaine is more popular as a recreational drug in the United States and many more cocaine-related deaths provides a large forensic material for scrutiny and evaluation (8, 27 –29). In one recent survey, Molina and Hargrove (30) identified 461 fatalities over a 16-year period in Bexar County in Texas. In 143 cases (31%), cocaine was the only intoxicant found at a median concentration of 0.89 mg/L (BZE 4.0 mg/L). This concentration is appreciably higher than that found in Sweden and other nations, which probably reflects cross-cultural differences in patterns of cocaine abuse, including dose and route of administration. Ethanol was present together with cocaine in 42% of these Bexar County autopsy cases and the next most prevalent drug was heroin followed by psychoactive medicinal drugs (30). The concentrations of cocaine in blood from 48 drug intoxication deaths in San Francisco were considered not significantly different from 51 cocaine-related deaths in the same city when presence of cocaine was deemed to be an incidental finding (31). The mean (+SD) concentration of cocaine in the intoxication deaths (1.12 + 2.71 mg/L) was almost double that of causes of death (0.487 + 0.75 mg/L), but use of a parametric statistical test (Student’s t-test) makes such a comparison of mean values
Blood-Cocaine Concentrations in Living and Deceased Persons 49
suspect. The existence of highly skewed distributions should have been obvious because the standard deviations were double the size of the means, which calls for use of non-parametric tests. The office of the chief medical examiner in Maryland classified cocaine-related deaths as either a result of intoxication (drug overdose) or some other cause (32). The mean (median) concentrations were 0.908 mg/L (0.30 mg/L) in 13 intoxication deaths compared with 0.146 mg/L (0.10 mg/L) in 19 deaths attributed to other causes. A Student’s t-test showed no statistically significant difference in mean cocaine concentration, although use of a non-parametric test would have been more appropriate. The same U.S. study identified opiates, including heroin, as a common co-ingested drug and multidrug deaths were a prominent finding (32). Over a 10-year period, over 50,000 forensic autopsies were performed in Sweden and the vast majority of these include a complete toxicological screening and verification analysis for ethanol and other drugs. Despite the large size of this postmortem material, only 132 cases were verified positive for cocaine and/or BZE. This either suggests an infrequent use of cocaine as a recreational drug in Sweden or a low inherent toxicity of this stimulant. Epidemiological surveys of drug use by drivers in Sweden found that 2 –3% were positive for cocaine at a median concentration of 0.05 mg/L (median BZE 0.7 mg/L) (14). In Switzerland, the prevalence of cocaine use by drivers was much higher 13% (33), although the median concentration of the stimulant in blood was the same as in Sweden (0.05 mg/L). There were 22% of drivers in the Netherlands with cocaine in blood at a median concentration of 0.05 mg/L (34). In a German study, a much higher median concentration of cocaine was reported (0.379 mg/L, N ¼ 734) and 16.4% of all drivers tested had used cocaine (35). A recent study of apprehended drivers in UK found 44 individuals with cocaine in blood and the median concentration was 0.025 mg/L (median BZE 1.0 mg/L) (36). The results from these different countries show that median concentration of cocaine in blood can vary widely, probably depending on the dose taken and the time elapsed after last use of the drug in relation to sampling blood for toxicology. In DUID suspects and autopsy cases, highest concentrations of cocaine in blood were observed when no other drugs were present apart from the BZE metabolite. These mono-drug intoxications were more commonly encountered in the traffic offenders (N ¼ 110) compared with medical examiner cases (N ¼ 4). The present study verifies that concentrations of cocaine and/ or BZE in blood in deaths caused by drug intoxication were not significantly different from other causes of deaths. The results also show that the co-ingestion of ethanol and other drugs is common in such fatalities leading to mixed-drug intoxication deaths. Illicit drugs such as THC, heroin, amphetamine, GHB and/or MDMA were often identified in blood together with cocaine. The size of the present material, which comprised 132 cocaine-related deaths and 988 traffic offenders, makes the results of this study a useful contribution to the forensic toxicology of cocaine.
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Blood-Cocaine Concentrations in Living and Deceased Persons 51
Article
Concentrations of Cocaine and Benzoylecgonine in Femoral Blood from Cocaine-Related Deaths Compared with Venous Blood from Impaired Drivers Alan Wayne Jones1,2* and Anita Holmgren1 1
Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Artillerigatan 12, SE-587 58 Linko¨ping, Sweden, and 2Department of Clinical Pharmacology, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden
*Author to whom correspondence should be addressed. Email: [email protected]
The concentrations of cocaine and its major metabolite benzoylecgonine (BZE) were determined in femoral blood from 132 cocainerelated deaths and compared with venous blood from 988 apprehended drivers. Cocaine and BZE were determined by solidphase extraction and isotope dilution gas chromatography –mass spectrometry with limits of quantitation of 0.02 mg/L for both substances. Significantly more men (95–98%) than women (2–5%) abused cocaine, although their mean age was about the same (29– 30 years). Mean age (+ + SD) of cocaine-related deaths was 29 + 7 years, which was not significantly different from 30 + 8 years in traffic cases (P > 0.05). The median concentration of cocaine in blood in 61 fatalities was 0.10 mg/L compared with 0.06 mg/L in traffic cases (P < 0.001). In drug intoxication deaths, the median concentration of cocaine was 0.13 mg/L (N 5 25), which was not significantly different from 0.09 mg/L (N 5 36) in other causes of death. Cocaine-related deaths mostly involved mixed drug intoxications including co-ingestion of heroin, cannabis, amphetamines as well as legal drugs, such as benzodiazepines and/or ethanol. The concentrations of cocaine in blood from living and deceased persons overlapped, which makes it infeasible to predict toxicity from the analytical toxicology results alone.
Introduction Abuse of cocaine, in the form of its hydrochloride salt and as the free-base (crack), is highly prevalent worldwide and has become a growing problem in some nations (1, 2). Cocaine exerts a powerful reinforcing effect on the individual and the associated psychological dependence and craving means more of the drug and higher doses are taken (3, 4). Indeed, the abuse potential of cocaine was recognized early after it was isolated from coca leaves in 1860 (5) and when introduced into medicine as a local anesthetic for eye surgery in 1874 (6). Compared with other recreational drugs, such as opiates, cocaine-related deaths are relatively infrequent in Sweden and when they do occur they are mostly attributed to multidrug intoxications (7, 8). The combined use of cocaine and heroin is especially dangerous when this mixture is administered intravenously (9). Likewise, the toxicity of cocaine is enhanced when co-ingested with ethanol, owing to the biosynthesis of a pharmacologically active metabolite (cocaethylene), which has a longer elimination half-life from blood compared with cocaine itself (10). The physiological effects of cocaine include increase in heart rate and blood pressure, which in predisposed individuals might result in hemorrhage stroke and death (11). Other causes of death in chronic cocaine users include excited delirium, myocardial infarctions and cardiac arrhythmias, although these are not easy to recognize at autopsy (12). Forensic toxicologists are in general agreement that toxicity of cocaine is unpredictable
and that the concentrations determined in blood from living and deceased persons show a significant overlap, which makes interpretation difficult (13). This paper compares the concentrations of cocaine and its main metabolite benzoylecgonine (BZE) in forensic blood samples from people suspected of driving under the influence of drugs (DUID) with cocaine-related deaths in which femoral blood was analyzed. These medical examiner cases were classified as either drug intoxication deaths or other causes of death, e.g., trauma, traffic or natural disease. The number and types of co-ingested drugs together with cocaine were compared and contrasted in living and deceased persons.
Material and methods Forensic autopsies The number of forensic autopsies made in Sweden (current population 9.3 million) has remained remarkably constant at just over 5000 per year over for the past 10 years. The police authorities order a forensic autopsy in all unnatural or suspicious deaths. Forensic pathologists take specimens of femoral blood for toxicology before evisceration of the body and after addition of a potassium fluoride preservative (2%, w/v), blood, urine and vitreous humor are transported to the toxicology laboratory in refrigerated containers. A national forensic toxicology database (TOXBASE) was searched retrospectively from 2001 to 2010 to find autopsy causes in which cocaine and/or its major metabolite BZE were verified present in femoral blood. Cause and manner of death was assigned by forensic pathologists after consideration of all aspects of the case including anatomical and histological findings, police reports, witness statements, discoveries at the scene, toxicology report, etc. International Classification of Disease Codes (ICD-9) were assigned by medical examiners and used by us to distinguish drug intoxication ( poisoning) deaths from other causes of death. The postmortem interval was not known in individual cases, although none of the bodies showed signs of decomposition. If femoral blood was not obtainable, owing to massive trauma or decomposition of the body, these cases were not used in the present study.
Apprehended drivers Blood and urine specimens from people arrested for DUID in Sweden are sent to the same central toxicology laboratory responsible for analysis of postmortem specimens. All analytical results are entered into a database (TOXBASE). The venous blood samples from DUID suspects were taken using gray-stopper evacuated tubes (10 mL) containing a mixture of sodium fluoride (100 mg)
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and potassium oxalate (25 mg) as preservatives. Whenever possible, a specimen of urine (10 mL) is also collected from traffic offenders in plastic tubes containing sodium fluoride (100 mg) as preservative. Results from 5 years of traffic cases (2001–2005) were searched to find those in which either cocaine and/or BZE were verified positive in blood samples. In routine forensic cases, other metabolites of cocaine, such as norcocaine, ecgonine methylester or cocaethylene, were not analyzed.
Police procedures The first suspicion of DUID arises when the police investigate a moving traffic offence, a road-traffic collision or when a vehicle is stopped in connection with a routine sobriety control. Other road users might call emergency services to report seeing a dangerous driver, which results in a police investigation of alcohol or drug use. The traffic offenders first provide a roadside breathalcohol screening test and a positive result (.0.1 mg/L) leads to an evidential breath-alcohol test done at the nearest police station. Refusal to comply with the roadside breath test or the evidential test will result in a blood sample being taken from the driver by force if necessary. If the roadside breath-alcohol test is negative and the driver shows signs and/or symptoms of drug influence, then the police request a venous blood sample for toxicological analysis. The samples of venous blood are usually taken by a physician or nurse at a police station or if a driver is injured in a crash blood samples are taken at a local hospital. In connection with the introduction of a zero-tolerance law for driving under the influence of drugs other than alcohol (1 July 1999), the police were permitted to examine a driver’s eyes with a flashlight to record reaction to light, and to measure pupil-size and other effects of drugs such as gaze nystagmus as supporting evidence of drug influence. These observations of clinical drug influence are recorded by the police on arrest forms, which are sent to the toxicology laboratory together with the specimens of blood and urine.
Determination of cocaine and BZE in blood The analysis of forensic blood samples starts with a broad screening using immunoassay methods (EMIT and CEDIA), and this is targeted at five classes of drugs, namely opiates, cannabinoids, amphetamine analogs, cocaine metabolite and benzodiazepines. If urine is available, this specimen is used for the immunologic
screening analysis, otherwise blood is used after precipitation of proteins. The urinary cut-off concentration for positive BZE was 0.30 mg/L (EMIT) and 0.05 mg/L, if blood extracts (CEDIA) were analyzed. Positive results from the screening are always verified by more sensitive and specific analytical methods, such as gas chromatography –mass spectrometry (GC–MS) or LC–MS for illicit drugs or GC-NPD for prescription drugs and headspace gas chromatography for volatiles. The method used to determine cocaine and BZE in blood was described in more detail elsewhere and this involved use of solid-phase extraction (Bond Elute Certify columns) followed by GC– MS after adding deuterium (d3) labeled internal standards (14). Verification of cocaine was done by GC–MS using mass fragments m/z 182/185 for quantitation analysis and m/z 198 and m/z 303 as the qualifier ions. Verification of BZE was done after making the pentafluoropropyl derivatives (PFPA and PFPOH) and using mass fragments m/z of 300/303 for quantitative analysis and m/z 316 and m/z 421 as the qualifier ions. Standard curves were prepared by spiking drug-free blood with known concentrations of cocaine and BZE and these were linear over a range from 0.02 to 2.0 mg/L for both analytes. When concentrations in blood exceeded 2.0 mg/L, the samples were reanalyzed after dilution with fresh drug-free blood. The analytical limit of quantitation for reporting positive cocaine and BZE in whole blood was 0.02 mg/L from both substances.
Statistical evaluation of results Mean, median and highest concentrations of cocaine and BZE in blood were used as descriptive statistics. Mean age of traffic offenders and autopsy cases were compared by Student’s t-test. Because frequency distributions of cocaine and BZE in blood were markedly skewed to the right, medians are preferred as an indicator of central tendency and two medians were compared by a Mann –Whitney non-parametric test. Comparison of cocaine and BZE concentrations in blood from living and deceased persons was done with the help of box-and-whisker plots.
Results Demographics of cocaine users The mean age of cocaine-related deaths and traffic cases with cocaine in blood was about the same 29 –30 years (range 15 –70
Table I Subject Demographics and Concentrations of Cocaine and BZE in Blood from Autopsy Cases and in People Arrested for DUID (Traffic) Case type
Drugs present
N
N (%) Male
Autopsy
Traffic
Cocaine and BZE BZE only BZE all cases Cocaine and BZE BZE only BZE all cases
61 71 132 300 688 988
a
58 (95) 67 (95) 125 (95) 293 (98) 656 (95) 949 (96)
Age (years) b
Cocaine (mg/L)
BZE (mg/L)c
Female
mean + SD
Mean (median) max.
Mean (median) max.
3 (5) 4 (5) 7 (5) 7 (2) 32 (5) 39 (4)
30 + 8.1 30 + 7.7 30 + 7.8 29 + 7.1 29 + 7.3 29 + 7.3
0.62 (0.10)d 8.5 ,0.02 mg/L 0.62 (0.10) 8.5 0.08 (0.06) 2.1 ,0.02 mg/L 0.08 (0.06) 2.1
1.80 (0.70)e 22 0.21 (0.11) 1.1 0.95 (0.30) 22 0.90 (0.70)e 4.2 0.19 (0.13) 1.3 0.41 (0.20) 4.2
a
Higher proportion of males to females (P , 0.001). No significant age differences (P . 0.05). c Median concentrations of BZE were not significantly different between autopsy and traffic cases (P . 0.05). d Higher median concentration of cocaine in the autopsy cases (P , 0.001). e Higher median concentration of BZE in blood when cocaine was also present (P , 0.001). b
Blood-Cocaine Concentrations in Living and Deceased Persons 47
years) as shown in Table I (P . 0.05). However, there was a clear predominance of males (95 –98%) that used cocaine in both the living and deceased persons.
Concentrations of cocaine and BZE in blood Table I shows that the median concentration of cocaine in autopsy blood (0.1 mg/L) was significantly higher than in blood from apprehended drivers (0.06 mg/L) (P , 0.001). The concentrations of parent drug (cocaine) and metabolite (BZE) were highly correlated in autopsy cases r ¼ 0.86 (N ¼ 61, P , 0.001) and traffic offenders, r ¼ 0.38 (N ¼ 300, P , 0.001). The median concentration of BZE in blood was higher when cocaine was also present in blood, which suggests more recent use of the stimulant (P , 0.001). The concentration distributions of cocaine and BZE in blood from living and deceased are shown in Figures 1 and 2 as box-and-whisker plots. A logarithm transformation of drug concentrations was used because the distributions were markedly skewed to the right. There was also a considerable overlapping in concentrations of cocaine (Figure 1) and BZE (Figure 2) for samples taken from living and deceased persons. Among autopsy cases, there were 31 cases (51%) with a cocaine concentration in blood .0.1 mg/L and 22 cases (36%) exceeded 0.2 mg/L. This compares with 71 traffic cases (24%) over 0.1 mg/L and 19 cases (6.3%) .0.2 mg/L.
Prevalence of co-ingested drugs Poly-drug use was more common in the medical examiner (autopsy) cases compared with traffic cases and the number of co-ingested substances varied from one to seven. Table II shows that cocaine and BZE were the only drugs identified in 110 traffic offenders (34%) and the median concentration of cocaine was 0.07 mg/L. In another 325 traffic cases, BZE, with or without cocaine, was the only drug identified at a median concentration of 0.20 mg/L. In the motorists, the concentrations of cocaine and BZE did not seem to depend on number of other drugs identified. Highest concentrations of cocaine and BZE were found in four cocaine-related deaths (3%) at a median concentration of 0.59 mg/L (median BZE 0.70 mg/L, N ¼ 12) when no other drugs were present.
Types of drugs co-ingested with cocaine Table III lists the top-10 drugs identified in blood samples together with cocaine and/or BZE. In both DUID suspects and autopsy cases, the top-three drugs were the same, namely ethanol, cannabis and diazepam. Other illicit drugs identified in blood along with cocaine were amphetamine, morphine (heroin), GHB and MDMA. The major class of prescription drugs used with cocaine were benzodiazepines exemplified by alprazolam, flunitrazepam, clonazepam and nitrazepam. The median concentrations of cocaine in blood tended to be higher in the autopsy cases, although these concentrations did not seem to depend on the type of co-ingested substances (Table III), neither in living or deceased persons. 48 Jones and Holmgren
Figure 1. Box-and-whiskers plots comparing concentration distributions of cocaine in cocaine-related deaths (autopsy) and traffic cases (impaired drivers). The boxes depict median and lower and upper quartiles, whereas the whiskers extend to +1.5 times the interquartile range and outliers are shown as separate data points.
Figure 2. Box-and-whiskers plots comparing concentration distributions of BZE in cocaine-related deaths (autopsy) and traffic cases (impaired drivers). The boxes depict median and lower and upper quartiles, whereas the whiskers extend to +1.5 times the interquartile range and outliers are shown as separate data points.
Table II Number of Cases (N) and Median Concentrations of Cocaine and BZE in Blood from Living (Traffic Cases) and Deceased Persons in Relation to the Number of Co-ingested Substances Co-ingested drugs
0 1 2 3 4 –7
Autopsy (N)a
Median conc. (mg/L)
Cocaine
BZE
Cocaine
BZEa
Cocaine
BZE
Cocaine
BZEa
4 17 11 8 17
12 31 23 16 50
0.59 0.09 0.20 0.08 0.10
0.70 0.40 0.30 0.45 0.15
110 104 47 29 10
325 292 201 121 49
0.07 0.05 0.05 0.06 0.09
0.20 0.20 0.17 0.19 0.14
Traffic (N)
Median conc. (mg/L)
N ¼ number of cases. a With or without cocaine also present in blood.
Cocaine concentrations in intoxication deaths Table IV shows the concentrations of cocaine and BZE in blood samples from intoxication deaths (drug overdose) or other
Table III Number of Cases (N) and Median Concentrations of Cocaine and BZE in Blood from Living (traffic cases) and Deceased Persons (Autopsy Cases) in Relation to the Types of Co-ingested Substances Autopsy cases
Ethanol THC Diazepam Morphine (heroin)b Amphetamines Paracetamol Flunitrazepam Alprazolam Clonazepam Nitrazepam
Median conc. (mg/L)
Traffic cases
a
Cocaine (N)
BZE (N)
0.07 (23) 0.08 (13) 0.10 (12) 0.10 (11) 0.12 (9) 0.22 (10) 0.15 (3) 0.06 (6) 0.10 (3) 0.08 (2)
0.30 (40) 0.20 (33) 0.20 (31) 0.16 (42) 0.14 (30) 0.65 (16) 0.16 (14) 0.45 (12) 0.22 (8) 0.23 (8)
Ethanol THC Diazepam Amphetamines Flunitrazepam Morphine (heroin)b MDA/MDMA GHB Alprazolam Clonazepam
Median conc. (mg/L) Cocaine (N)
BZEa (N)
0.05 (61) 0.05 (57) 0.05 (53) 0.05 (43) 0.06 (40) 0.04 (22) 0.03 (6) 0.07 (7) 0.04 (6) 0.03 (5)
0.20 (124) 0.16 (238) 0.20 (211) 0.14 (238) 0.19 (152) 0.18 (110) 0.19 (36) 0.17 (19) 0.12 (25) 0.19 (18)
N ¼ number of cases. a BZE with or without cocaine present in blood samples. b Heroin use confirmed from presence of 6-acetlylmorphine in blood or urine or morphine-to-codeine concentration ratio in blood above unity.
Table IV Comparison of Femoral Blood Concentrations of Cocaine and/or BZE in Autopsy Cases Classified as Drug Intoxication (Poisoning) or Other Causes of Death Cause of death
Drugs in blood
N
Cocaine (mg/L) Mean (median) highest
BZE (mg/L) Mean (median) highest
Drug intoxicationa
Cocaine and BZE BZE only Cocaine and BZE BZE only
25
1.25 (0.13)b 8.5
2.96 (1.3)c 22.0
37 36
,0.02 mg/L 0.18 (0.09) 1.0
0.22 (0.12)b 1.1 0.99 (0.50) 4.1
34
,0.02 mg/L
0.20 (0.10) 0.9
Other causes (trauma, traffic, natural)
a
Mainly mixed-drug overdose deaths involving ethanol, morphine (heroin) and/or amphetamine, diazepam in various combinations. b Median concentration of cocaine in drug intoxication deaths not significantly different from other causes of death (P . 0.05). c Median BZE concentration was higher in the intoxication deaths (P , 0.01).
cause of death (trauma, traffic, etc.). In 25 intoxication deaths (40%), the median concentration of cocaine was 0.13 mg/L, which was not significantly different from a median of 0.09 mg/ L found in 36 other causes of death (P . 0.05). High concentrations of cocaine were determined in three intoxication deaths when no other substances were present; 4.1 mg/L (BZE 0.7 mg/ L), 8.5 mg/L (BZE 22 mg/L) and 6.7 mg/L (BZE 11 mg/L), respectively.
Discussion In this study, most cocaine-related deaths involved co-ingestion of other psychoactive substances, such as ethanol, heroin or amphetamine, and only four cases were mono-intoxications, which is in good agreement with other studies (11, 15, 16). A cocaine-related death implies that the toxicology results were positive for cocaine and/or BZE when femoral blood was analyzed by GC –MS analysis, but the death was not necessarily a drug overdose. Cocaine poisoning deaths are sometimes observed in body-packers after leakage of the concealed drugs or in suicides when massive doses of cocaine are administered intravenously (17). Cocaine-related fatalities might also reflect
adverse cardiovascular events, such as myocardial infarction, cardiac arrhythmia and/or QT syndrome (12, 18). The death certificates issued in chronic cocaine users sometimes incriminate paranoia, delusions, excited delirium and convulsions as important factors in the death (19, 20). The concentrations of drugs in postmortem blood samples, including cocaine and BZE, show temporal and site-dependent changes (21). Standardized methods of sampling postmortem blood for toxicology are important and the specimen tubes should contain sodium or potassium fluoride as a preservative, which stabilizes blood-cocaine concentrations after sampling. Storage and transport of blood samples frozen or refrigerated (þ48C) also improves stability by preventing in vitro degradation of cocaine (22, 23). The concentrations of cocaine and BZE were determined in peripheral blood samples, that is, femoral vein (autopsy) or cubital vein (traffic cases) and a fluoride preservative was always included (23). The biological elimination half-life of cocaine is fairly short (0.5–1.5 h) compared with its metabolite BZE (6–10 h), which explains why many blood samples were positive for BZE but negative for parent drug cocaine (24). When both cocaine and BZE were present together in blood, this speaks towards fairly recent use of the stimulant as found in N ¼ 61 autopsy cases and N ¼ 300 traffic cases. It is not possible to calculate the amount of drug taken or the route of administration or time of last use from the analytical toxicology results alone (25). The median concentration of cocaine in 25 drug intoxication deaths in Melbourne, Australia was 0.1 mg/L, which was not significantly different from people taking cocaine but dying from other causes, such as external injury or natural disease (26). In our study, we found the same overall median concentration in all cocaine-related deaths (0.1 mg/L, N ¼ 61), although the median was higher in 25 drug intoxication deaths (0.13 mg/L) compared with median of 0.09 mg/L in other causes of death (N ¼ 36). These results support the generally accepted notion that cocaine concentrations in blood from living (DUID suspects and people admitted to hospital for treatment) overlap with cocaine concentrations in medical examiner cases. Cocaine is more popular as a recreational drug in the United States and many more cocaine-related deaths provides a large forensic material for scrutiny and evaluation (8, 27 –29). In one recent survey, Molina and Hargrove (30) identified 461 fatalities over a 16-year period in Bexar County in Texas. In 143 cases (31%), cocaine was the only intoxicant found at a median concentration of 0.89 mg/L (BZE 4.0 mg/L). This concentration is appreciably higher than that found in Sweden and other nations, which probably reflects cross-cultural differences in patterns of cocaine abuse, including dose and route of administration. Ethanol was present together with cocaine in 42% of these Bexar County autopsy cases and the next most prevalent drug was heroin followed by psychoactive medicinal drugs (30). The concentrations of cocaine in blood from 48 drug intoxication deaths in San Francisco were considered not significantly different from 51 cocaine-related deaths in the same city when presence of cocaine was deemed to be an incidental finding (31). The mean (+SD) concentration of cocaine in the intoxication deaths (1.12 + 2.71 mg/L) was almost double that of causes of death (0.487 + 0.75 mg/L), but use of a parametric statistical test (Student’s t-test) makes such a comparison of mean values
Blood-Cocaine Concentrations in Living and Deceased Persons 49
suspect. The existence of highly skewed distributions should have been obvious because the standard deviations were double the size of the means, which calls for use of non-parametric tests. The office of the chief medical examiner in Maryland classified cocaine-related deaths as either a result of intoxication (drug overdose) or some other cause (32). The mean (median) concentrations were 0.908 mg/L (0.30 mg/L) in 13 intoxication deaths compared with 0.146 mg/L (0.10 mg/L) in 19 deaths attributed to other causes. A Student’s t-test showed no statistically significant difference in mean cocaine concentration, although use of a non-parametric test would have been more appropriate. The same U.S. study identified opiates, including heroin, as a common co-ingested drug and multidrug deaths were a prominent finding (32). Over a 10-year period, over 50,000 forensic autopsies were performed in Sweden and the vast majority of these include a complete toxicological screening and verification analysis for ethanol and other drugs. Despite the large size of this postmortem material, only 132 cases were verified positive for cocaine and/or BZE. This either suggests an infrequent use of cocaine as a recreational drug in Sweden or a low inherent toxicity of this stimulant. Epidemiological surveys of drug use by drivers in Sweden found that 2 –3% were positive for cocaine at a median concentration of 0.05 mg/L (median BZE 0.7 mg/L) (14). In Switzerland, the prevalence of cocaine use by drivers was much higher 13% (33), although the median concentration of the stimulant in blood was the same as in Sweden (0.05 mg/L). There were 22% of drivers in the Netherlands with cocaine in blood at a median concentration of 0.05 mg/L (34). In a German study, a much higher median concentration of cocaine was reported (0.379 mg/L, N ¼ 734) and 16.4% of all drivers tested had used cocaine (35). A recent study of apprehended drivers in UK found 44 individuals with cocaine in blood and the median concentration was 0.025 mg/L (median BZE 1.0 mg/L) (36). The results from these different countries show that median concentration of cocaine in blood can vary widely, probably depending on the dose taken and the time elapsed after last use of the drug in relation to sampling blood for toxicology. In DUID suspects and autopsy cases, highest concentrations of cocaine in blood were observed when no other drugs were present apart from the BZE metabolite. These mono-drug intoxications were more commonly encountered in the traffic offenders (N ¼ 110) compared with medical examiner cases (N ¼ 4). The present study verifies that concentrations of cocaine and/ or BZE in blood in deaths caused by drug intoxication were not significantly different from other causes of deaths. The results also show that the co-ingestion of ethanol and other drugs is common in such fatalities leading to mixed-drug intoxication deaths. Illicit drugs such as THC, heroin, amphetamine, GHB and/or MDMA were often identified in blood together with cocaine. The size of the present material, which comprised 132 cocaine-related deaths and 988 traffic offenders, makes the results of this study a useful contribution to the forensic toxicology of cocaine.
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50 Jones and Holmgren
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