A two-sample approach to retrograde extrapolation of blood THC concentrations - Is it feasible?

BACKGROUND: Retrograde extrapolation of drug concentrations in blood can be relevant in cases of drug-impaired driving and is regularly used in forensic toxicology in Norway. Δ9-tetrahydrocannabinol (THC) has complex, multi-compartmental pharmacokinetics, which makes retrograde extrapolation of blood THC concentrations problematic. In the present study, we evaluated an approach to retrograde extrapolation in which momentary rates of decrease of THC were estimated from two consecutive blood samples in apprehended drivers. MATERIAL AND METHODS: Data were collected from apprehended drivers in Norway 2000-2020. We included 548 cases in which THC was detected in two consecutive blood samples collected ≥ 20 min apart. THC concentrations were measured by GC-MS and UHPLC-MS/MS. In each case, THC concentrations and the time between the two sampling points (Δt) were used to estimate the rate constant k. The relationship between THC concentration and k was modelled by linear regression. RESULTS: The median Δt was 31 min (interquartile range, IQR = 9). The median blood THC concentration was 2.4 µg/L (IQR = 3.4) at the first sampling point and 2.3 µg/L (IQR =3.1) at the second. The concentration decreased in 62% and increased in 38% of all cases. However, considering measurement uncertainty, the changes were not statistically significant in 87% of cases. The mean of k was 0.12 h-1, corresponding to an apparent t1/2 of 6.0 h. The t1/2 predicted from linear regression of k against THC concentration ranged from 0.93 to 13 h for the highest and lowest concentrations observed (36 and 0.63 µg/L, respectively). The time from driving to blood collection had a median of 1.7 h (IQR = 1.5), and did not correlate with k. CONCLUSIONS: The apparent t1/2 of THC calculated from the mean of k was 6.0 h, which is shorter than the terminal elimination t1/2 suggested in previous population studies. This indicates that blood samples were often taken during the late distribution phase of THC. Because Δt was short relative to the rates of decrease expected in the late distribution and elimination phases, the underlying true concentration changes related to in vivo pharmacokinetics were small and masked by the relatively larger "false" changes introduced by random analytical and pre-analytical error. Therefore, individual values of k calculated from only two blood samples taken a short time apart are unreliable, and a two-sample approach to retrograde extrapolation of THC cannot be recommended.

Rate of elimination of γ-hydroxybutyrate from blood determined by analysis of two consecutive samples from apprehended drivers in Norway.

AIM: Gamma-hydroxybutyrate (GHB) is a common drug of abuse with an elimination half-life of 20-45 min. However, there is some evidence that GHB might exhibit saturation kinetics after ingesting high recreational doses. The aim of this study was to investigate the elimination kinetics of GHB from blood in people apprehended by the police for impaired driving and secondary to describe concentrations in all GHB-positive drivers. METHODS: Two consecutive blood samples were taken about 30-40 min apart from N = 16 apprehended drivers in Norway. GHB was determined in blood by an Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method. The changes in GHB between the two consecutive blood samples allowed estimating GHB's elimination half-life, assuming first-order and zero-order elimination kinetics. GHB concentrations are also reported for N = 1276 apprehended drivers with GHB in blood. RESULTS: The median time interval between collecting the two blood samples was 36 min (range 20-56 min). The median concentration of GHB in the first blood sample was 56.5 mg/L (range 14.1-142 mg/L) compared with 47.8 mg/L in the second sample (range 9.75-113 mg/L). The median elimination half-life was 103 min (range 21-187 min), and GHB's median zero-order elimination rate constant was 21.0 mg/L/h (range 6.71-45.4 mg/L/h). Back-calculation to the time of driving resulted in GHB concentrations up to 820 mg/L assuming first-order kinetics and up to 242 mg/L assuming zero-order kinetics. In all drivers (N = 1276), the median GHB concentration was 73.7 mg/L and highest was 484 mg/L. CONCLUSION: The elimination half-life of GHB in blood samples from apprehended drivers was longer than expected compared with results of controlled dosing studies. Zero-order kinetics seems a more appropriate model for GHB when concentrations are back-calculated, and the median elimination rate was 21 mg/L/h.

Detection Time of Oxazepam and Zopiclone in Urine and Oral Fluid after Experimental Oral Dosing.

Data from previous experimental studies on the detection time of oxazepam and zopiclone in biological matrices are limited. The aim of this study was to examine the detection time in urine and oral fluid after single oral doses of oxazepam and zopiclone. Ten healthy volunteers received 25 mg of oxazepam in the evening of Day 1 and 7.5 mg of zopiclone in the evening of Day 3. Urine and oral fluid samples were collected twice daily for 9 days, with an additional sampling the day after ingestion of zopiclone. A total of 19 samples of both urine and oral fluid from each participant were analyzed using fully validated chromatographic methods. The median detection time for oxazepam was 91 h (range 73-108) in urine and 67 h (range 50-98) in oral fluid. The median detection time for zopiclone in urine was 49 h (range 25-98) and 59 h (range 48-146) in oral fluid. The metabolite zopiclone N-oxide showed a detection time of 36 h (range 25-84) in urine. The area under the concentration-time curve (AUCTotal) in urine corrected for creatinine was 150 µmol/L/mmol/L*h (range 105-216) for oxazepam and 1.60 µmol/L/mmol/L*h (range 0.79-4.53) for zopiclone. In oral fluid, the AUCtotal was 673 nmol/L*h (range 339-1,316) for oxazepam and 2,150 nmol/L*h (range 493-4,240) for zopiclone. In conclusion, oxazepam can be detected longer in urine than in oral fluid, while zopiclone can be detected longer in oral fluid than in urine. The high AUCTotal for zopiclone in oral fluid shows that the transfer into oral fluid is significant. In certain individuals the detection time of zopiclone in oral fluid is long. These results can be helpful when interpreting drug testing analyzes.

BioAlder: a tool for assessing chronological age based on two radiological methods.

We have created the tool BioAlder as an age prediction model based on the systems Greulich and Pyle (hand) and the Demirjian's grading of the third molar tooth. The model compiles information from studies representing a total of 17,151 individuals from several parts of the world. The model offers a solution where issues as group-wise data format and age mimicry bias are bypassed. The model also provides a solution for combining the two grading systems, hand and tooth, to one combined age prediction result assuming independency. We have tested our model of age prediction and the independency assumption on a separate data set from Lebanon with 254 young individuals. The prediction intervals of BioAlder covered most of the data points; however, we observed some outliers. Our analyses indicate at least a weak dependency between the two methods.

An Experimental Study of Diazepam and Alprazolam Kinetics in Urine and Oral Fluid Following Single Oral Doses.

Benzodiazepines are commonly seen in samples submitted for drug testing of patients, people involved in child welfare cases, work-place drug testing, as well as in drug-facilitated assaults. Limited previous experimental studies are available regarding the excretion of benzodiazepines in urine and oral fluid. The aim of this study was to investigate the concentrations of diazepam and alprazolam in oral fluid and urine for up to 2 weeks after ingestion of a single oral dose in healthy volunteers. A total of 11 healthy volunteers ingested 10 mg diazepam at the start of the study and 0.5 mg alprazolam on Day 3 of the study. A total number of 10 oral fluid samples and 17 urine samples were collected from each participant. The samples were analyzed by liquid chromatography with tandem mass spectroscopy and ultra-high performance liquid chromatography tandem mass spectrometry methods. The median detection time was 252 h for the longest detected diazepam metabolite in urine (oxazepam, range 203-322) and 132 h in oral fluid (N-desmethyldiazepam, range 109-136). For alprazolam, the median detection time was 36 h (metabolite α-OH-alprazolam, range 26-61) in urine and 26 h (alprazolam, range 4-37) in oral fluid. These results show that detection times are only 36 h for alprazolam in urine after intake of a single therapeutic oral dose. For diazepam in urine, detection times were 11 days. Detection times were generally shorter in oral fluid compared to urine. The results could be helpful in the interpretation of diazepam or alprazolam findings in drug testing cases involving urine or oral fluid.

THC and CBD in blood samples and seizures in Norway: Does CBD affect THC-induced impairment in apprehended subjects?

BACKGROUND AND AIMS: Several publications have suggested increasing cannabis potency over the last decade, which, together with lower amounts of cannabidiol (CBD), could contribute to an increase in adverse effects after cannabis smoking. Naturalistic studies on tetrahydrocannabinol (THC) and CBD in blood samples are, however, missing. This study aimed to investigate the relationship between THC- and CBD concentrations in blood samples among cannabis users, and to compare cannabinoid concentrations with the outcome of a clinical test of impairment (CTI) and between traffic accidents and non-accident driving under the influence of drugs (DUID)-cases. Assessment of THC- and CBD contents in cannabis seizures was also included. METHODS: THC- and CBD concentrations in blood samples from subjects apprehended in Norway from April 2013-April 2015 were included (n=6134). A CTI result was compared with analytical findings in cases where only THC and/or CBD were detected (n=705). THC- and CBD content was measured in 41 cannabis seizures. RESULTS: Among THC-positive blood samples, 76% also tested positive for CBD. There was a strong correlation between THC- and CBD concentrations in blood samples (Pearson's r=0.714, p<0.0005). Subjects judged as impaired by a CTI had significantly higher THC- (p<0.001) and CBD (p=0.008) concentrations compared with not impaired subjects, but after multivariate analyses, impairment could only be related to THC concentration (p=0.004). Analyzing seizures revealed THC/CBD ratios of 2:1 for hashish and 200:1 for marijuana. CONCLUSIONS: More than ¾ of the blood samples testing positive for THC, among subjects apprehended in Norway, also tested positive for CBD, suggesting frequent consumption of high CBD cannabis products. The simultaneous presence of CBD in blood does, however, not appear to affect THC-induced impairment on a CTI. Seizure sample analysis did not reveal high potency cannabis products, and while CBD content appeared high in hashish, it was almost absent in marijuana.

Urinary Kinetics of Heroin Metabolites in Pigs Shortly After Intake.

In previous experimental studies on heroin metabolites excretion in urine, the first sample was often collected a few hours after intake. In forensic cases, it is sometimes questioned if a positive urine result is expected e.g., 30 min after intake. The aim of this study was to investigate urinary excretion of heroin metabolites (morphine, 6-monoacetylmorphine (6-MAM) and morphine-3-glucuronide (M3G)) every 30 min until 330 min after injection of a 20 mg heroin dose in six pigs. Samples were analyzed using a previously published, fully validated liquid chromatography-tandem mass spectrometry method. All metabolites were detected after 30 min in all pigs. The time to maximum concentration (Tmax) median (range) for 6-MAM and morphine was 30 min (first sample) (30-120), and 90 min (30-330) for M3G. In four of the six pigs, the Tmax of 6-MAM and morphine was reached within 30 min. All analytes were still detectable at the end of study. This study showed that positive results in urine are expected to be seen shortly after use of heroin in pigs. Detection times were longer than previously indicated, especially for 6-MAM, but previous studies used lower doses. As the physiology of these animals resembles that of the humans, transferability to man is expected.

Impairment due to alcohol, tetrahydrocannabinol, and benzodiazepines in impaired drivers compared to experimental studies.

OBJECTIVE: In some countries, per se laws for other drugs than alcohol are used to judge drunk and drugged drivers. These blood concentration limits are often derived from experimental studies on traffic relevant behavior of healthy volunteers. Knowledge about how results from experimental studies could be transferred to a real-life setting is missing. The aim of this study was to compare impairment seen in experimental studies to the impairment seen at equivalent concentrations in apprehended drunk and drugged drivers. METHODS: Results from previously performed meta-analyses of experimental studies regarding impairment from alcohol, tetrahydrocannabinol (THC), and benzodiazepines were compared to impairment in apprehended drunk and drugged drivers as judged by a clinical test of impairment. Both experimental studies and real-life cases were divided into 4 groups according to increasing blood drug concentration intervals. The percentage of impaired test results in experimental studies was compared to the percentage of impaired subjects among drivers within the same blood drug concentration window. RESULTS: For ethanol, the percentage of impaired drivers (n = 1,223) increased from 59% in the lowest drug concentration group to 95% in the highest drug concentration group, compared to 7 and 72% in the respective groups in experimental studies. For THC, the percentage of impaired drivers (n = 950) increased from 42 to 58%, the corresponding numbers being 11 and 42% for experimental studies. For benzodiazepines, the percentage of impaired drivers (n = 245) increased from 46 to 76%, the corresponding numbers being 16 and 60% for experimental studies. The increased odds ratio for impairment between 2 concentration groups was comparable for experimental studies and impaired drivers. CONCLUSIONS: Fewer test results indicated impairment in experimental studies compared to impaired drivers in real life when influenced by similar blood concentrations of either ethanol, THC, or benzodiazepines. In addition, a comparable relationship between drug concentration and impairment was seen for both experimental studies and real-life cases. We believe that the present study strengthens the background for using experimental studies to establish fixed concentration limits for drunk and drugged drivers, but experimental studies in an impaired driver population could further expand our knowledge.

Pharmacokinetics of heroin and its metabolites in vitreous humor and blood in a living pig model.

Vitreous humor (VH) is an alternative matrix for drug analysis in forensic toxicology. However, little is known about the distribution of xenobiotics, such as opioids, into VH in living organisms. The aim of this study was to simultaneously measure heroin and metabolite concentrations in blood and VH after injection of heroin in a living pig model. Six pigs were under non-opioid anesthesia during the surgical operation and experiment. Ocular microdialysis was used to acquire dialysate from VH, and a venous catheter was used for blood sampling. Twenty milligrams of heroin was injected intravenously with subsequent sampling of blood and dialysate for 6 h. The samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. Heroin was not detected in VH; 6-monoacetylmorphine (6-MAM) and morphine were first detected in VH after 60 min. The morphine concentration in VH thereafter increased throughout the experimental period. For 6-MAM, Cmax was reached after 230 min in VH. In blood, 6-MAM reached Cmax after 0.5 min, with a subsequent biphasic elimination phase. The blood and VH 6-MAM concentrations reached equilibrium after 2 h. In blood, morphine reached Cmax after 4.3 min, with a subsequent slower elimination than 6-MAM. The blood and VH morphine concentrations were in equilibrium about 6 h after injection of heroin. In conclusion, both 6-MAM and morphine showed slow transport into VH; detection of 6-MAM in VH did not necessarily reflect a recent intake of heroin. Because postmortem changes are expected to be small in VH, these experimental results could assist the interpretation of heroin deaths.

Influence of alcohol and other substances of abuse at the time of injury among patients in a Norwegian emergency department.

BACKGROUND: The presence of alcohol or other substances of abuse in blood or urine from injured patients is often used as a proxy for substance influence at the time of injury. The aim of this study was to obtain an estimate of substance influence at the time of injury based on blood concentrations of alcohol and other substances of abuse, and to explore the relationship between the substance prevalence at the time of admittance to the hospital and the actual influence at the time of the injury. METHODS: The study included all adult patients admitted to the emergency department of a university hospital during 1 year (n = 996). Quantification in blood was done by an enzymatic method for alcohol, and by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry for 28 other substances of abuse. Concentrations of alcohol and other substances in blood at the time of injury were calculated. The degree of influence was assessed on the basis of the calculated blood concentrations, with a threshold of influence set at a blood alcohol concentration (BAC) of 0.05 %, or a substance concentration leading to an influence similar to that of a BAC of 0.05 %. RESULTS: A total of 324 patients (32.5 %) were determined to be under the influence at the time of injury. In comparison, 394 patients (39.6 %) had one or more substances above the cut-off limit in blood at the time of admittance to the hospital. Alcohol was the most prevalent substance causing influence at 25.9 %. Among patients with violence-related injuries, almost 75 % were under the influence of alcohol and/or substances. Patients under the influence were younger, and men were more often under the influence than women. More patients were under the influence at nighttime and during weekends than at daytime and on weekdays. CONCLUSIONS: About one third of the injured patients were determined to be under the influence at the time of injury, with alcohol being the most prevalent substance causing influence. Approximately 98 % of the patients with alcohol detected in blood at the time of admittance to the hospital were under the influence of alcohol at the time of injury.

Drugged driving arrests in Norway before and after the implementation of per se law.

Norway introduced legislative limits for driving under the influence of drugs (DUID) February 1st, 2012, to harmonize with the legislation on driving under the influence of alcohol. Per se limits corresponding to blood alcohol concentrations (BACs) of 0.02% were established for 20 drugs and concentration limits for graded sanctions corresponding to BACs of 0.05% and 0.12% were established for 13 of these drugs as well. The new system is not applied to individuals with valid prescriptions for medicinal drugs. The aim of this study was to investigate if the implementation of legislative limits for drugs affected the number of blood samples taken from suspected drugged drivers, drug findings and the number of expert witness statement requests. The number of blood samples taken in suspected DUID cases increased by 20% after introduction of legislative limits (3320 cases in 2010 and 3970 in 2013). The number of samples with at least one drug above the per se limit corresponding to BAC of 0.02% increased by 17% (from 2646 in 2010 to 3090 in 2013), whereas the number of expert witness statements was reduced by the half (from 63.4% in 2010 and 28.7% in 2013).

Impairment due to amphetamines and benzodiazepines, alone and in combination.

INTRODUCTION: The impairing effects of combined use of amphetamines and benzodiazepines among recreational drug users are not well described, but knowledge about this is important in the risk assessment of such combined drug use. The aim of this study was to compare the impairment, among apprehended drivers, as judged by a clinical test of impairment (CTI), in cases where a combination of amphetamines and benzodiazepines was detected, in blood, with cases where only one of the two drug groups was detected. METHODS: The results of CTI judgments were compared to toxicological drug tests of blood samples that were obtained at the time of CTI screening in cases containing amphetamines only, cases containing different benzodiazepines only, and cases containing a combination of amphetamines and benzodiazepines. RESULTS: There were significantly more drivers being judged as impaired in the combined group (n = 777), compared both with amphetamines alone (n = 267, χ(2) = 47.8, p < 0.001) and benzodiazepines alone (n = 153, χ(2) = 7.0, p = 0.008). This was also seen when only including the lowest concentrations of benzodiazepines (χ(2) = 4.3, p = 0.038). The concentrations of the drugs were higher in the single drug groups, compared with the combined group. CONCLUSION: This study indicates that during real-life driving, those influenced by both amphetamines and benzodiazepines are more impaired, as judged by the CTI, compared with those influenced by either drug alone, although the combined group showed lower drug concentrations.

Codeine to morphine concentration ratios in samples from living subjects and autopsy cases after incubation.

The codeine to morphine concentration ratio is used in forensic toxicology to assess if codeine has been ingested alone or if morphine and/or heroin have been ingested in addition. In our experience, this interpretation is more difficult in autopsy cases compared with samples from living persons, since high morphine concentrations are observed in cases where only codeine is assumed to have been ingested. We have investigated if codeine and morphine glucuronides are subject to cleavage to the same extent in living and autopsy cases in vitro. We included whole blood samples from eight living subjects and nine forensic autopsy cases, where only codeine ingestion was suspected. All samples were incubated for 2 weeks at 37°C and analyzed for codeine and six codeine metabolites using liquid chromatography tandem mass spectrometry. A reduction in the codeine to morphine concentration ratio was found, both in samples from living subjects (mean 33%, range 22-50%) and autopsy cases (mean 37%, range 13-54%). The increase in the morphine concentrations was greater in the autopsy cases (mean 85%, max 200%) compared with that of the living cases (mean 51%, max 87%). No changes were seen for codeine or codeine-6-glucuronide concentrations. The altered ratios might mislead the forensic toxicologist to suspect morphine or heroin consumption in cases where only codeine has been ingested.

Suicide due to cyclizine overdose.

Cyclizine is an antihistamine with sedative effect used to treat motion sickness. A few studies have reported on cyclizine abuse among teenagers, and cyclizine abuse has been reported among opioid dependants receiving methadone, with the combination having been reported to produce strong psychoactive effects. Few reports exist on the possible toxic effects of cyclizine, and it is regarded as a safe drug most often sold as a non-prescription/over-the-counter drug. Very few cases of fatalities resulting from cyclizine overdose can be found in the literature. We present a case where a 22-year-old female was found unconscious and intoxication with drugs and alcohol was suspected. Whole blood from the femoral vein, urine and stomach content were collected during autopsy and screened for drugs of abuse and medicinal drugs. GC-MS screening of the stomach contents revealed presence of cyclizine and meclozine. Cyclizine and meclozine concentrations in blood were determined using a UPLC-MS-MS method. Quantification of femoral blood revealed a high concentration of cyclizine (16 mg/L), a low concentration of meclozine (0.2 mg/L) and ethanol 0.16 g/dL. No other medicinal drugs or drugs of abuse were detected. We report on a case of suicide where cyclizine was found to be the principal drug and question the safety of this drug.

Analyses of beverage remains in drug rape cases revealing drug residues--the possibility of contamination from drug concentrated oral fluid or oral cavity contained tablets.

In drug-facilitated sexual assault (DFSA) cases, drug residues may be detected in beverage remains found in cups or glasses known to have been used by the victims. In this small naturalistic study, the possibility of beverages being contaminated, either by drug concentrated oral fluid or by oral cavity contained tablets, was investigated. Analysis of residues from cups containing soft drinks was performed by immunoassay and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Beverage with both added tablets and spiked oral fluid was investigated, as well as simulation of swallowing tablets. Only the residues from added tablets were positive with immunoassay, while drugs were detectable in all cups using more sensitive UPLC-MS/MS. In conclusion, the possibility of detecting drug residues in beverages due to a contamination, from either drug concentrated oral fluid or oral cavity contained tablets at a time of consumption, should be kept in mind when performing sensitive analysis.

[Intoxation with paramethoxymethamphetamine]. / Forgiftning med parametoksymetamfetamin.

BACKGROUND: Since the summer of 2010, there has been an epidemic of deaths related to paramethoxymethamphetamine (PMMA) in Norway. We present a review of the pharmacology and toxicology of the substance. MATERIAL AND METHOD: The review is based on a literature search in the databases PubMed, Ovid and MEDLINE. A discretionary selection was made of relevant articles. RESULTS: Paramethoxymethamphetamine and paramethoxyamphetamine (PMA) are two so-called designer amphetamines which appear from time to time on the illegal narcotics market in many countries. They are frequently sold as ecstasy or amphetamine, often mixed with amphetamine or methamphetamine. The substances, known on the street as «Death¼, have potent serotonergic effects and are associated with significant toxicity. Many deaths have been reported worldwide, even after intake of an «ordinary user dose¼. The narcotic effect is not very pronounced and the onset is slow, which may lead to unintentional overdosing. INTERPRETATION: In cases of severe intoxation that are apparently related to intake of amphetamine or ecstasy, PMMA/PMA intoxation should be suspected.

[Interactions with methadone and buprenorphine]. / Interaksjoner med metadon og buprenorfin.

In Norway, about 5000 patients receive opioid maintenance treatment; 60 % receive methadone and 40 % buprenorphine. An increasing number of regular general practitioners and hospital doctors are in contact with this group of patients. This article presents a short overview of drug interactions with methadone and buprenorphine, as an aid to medical doctors in contact with these patients.