Completed suicides of citalopram users-the role of CYP genotypes and adverse drug interactions.

Depression is known to be a risk factor for suicide. Currently, the most used antidepressants are selective serotonin reuptake inhibitors (SSRIs). Not all users, however, benefit from them. In such cases, treatment failure can be explained in part by genetic differences. In this study, we investigated the role of pharmacogenetic factors in citalopram-positive completed suicides (n = 349). Since citalopram is metabolized by CYP2C19 and CYP2D6 enzymes, the study population was genotyped for clinically relevant CYP2C19 and CYP2D6 polymorphisms and CYP2D6 copy number variation. To assess genetic differences between suicide cases and Finns in general, Finnish population samples (n = 855) were used as controls. Also, the role of drug interactions among suicide cases was evaluated. We found enrichment of a combined group of genetically predicted poor and ultrarapid metabolizer phenotypes (gMPs) of CYP2C19 among suicide victims compared to controls 0.356 [0.31-0.41] vs. 0.265 [0.24-0.30] (p = 0.0065). In CYP2D6 gMPs, there was no difference between cases and controls when the study population was analyzed as a whole. However, there were significantly more poor metabolizers among females who committed suicide by poisoning compared to female controls. In 8% of all drug poisoning deaths, lifetime drug-drug interaction was evaluated having a contribution to the fatal outcome. From clinical perspective, pharmacogenetic testing prior to initiation of SSRI drug could be beneficial. It may also be useful in medico-legal settings as it may elucidate obscure poisoning cases. Also, the possibility of unintentional drug interactions should be taken into account in drug poisoning deaths.

Post-mortem analysis of lactate concentration in diabetics and metformin poisonings.

Lactate is produced in carbohydrate metabolism under anaerobic conditions. Lactic acidosis occurs when the production of lactate exceeds its removal. In post-mortem (PM) context, the lactic acidosis is difficult to interpret due to unknown pathophysiological factors prior to death and PM changes that may affect the lactate levels. We evaluated 1865 medico-legal autopsy cases where the quantitation of glucose, lactate, and ketone bodies was performed as a part of the cause of death (CoD) investigation. Lactate was shown to ascend in a logarithmic manner as the PM interval increased until a plateau was achieved approximately after 8-10 days PM, and the elevation was caused mainly by PM changes. The lactate level was higher than the mean in cases where the CoD was diabetes mellitus type 2 (DM2) or metformin poisoning. Although there was a correlation between metformin and lactate levels, our findings suggest the DM2 and its complications were the cause for elevated lactate levels rather than metformin, since the lactate levels were similar in DM2-associated deaths where no metformin was detected. Elevated lactate levels in PM samples rather referred to metabolic disturbances often caused by DM2. An assay to detect D-lactate in PM samples was described.

Fatal poisoning in drug addicts in the Nordic countries in 2017.

This study is the seventh report on fatal poisonings among drug addicts in the Nordic countries. In this report, we analyse data from the five Nordic countries: Denmark, Finland, Iceland, Norway and Sweden. Data on gender, number of deaths, places of deaths, age, main intoxicants and substances detected in blood were recorded to obtain national and comparable Nordic data, and to allow comparison with earlier studies conducted in 1984, 1991, 1997, 2002, 2007 and 2012. The death rate (number of deaths per 100,000 inhabitants) was highest in Iceland (6.58) followed closely by Sweden (6.46) and then lowest in Denmark (4.29). The death rate increased in Finland (5.84), Iceland and Sweden and decreased in Denmark compared to earlier studies. The death rate in Norway, which has decreased since 2002, has stabilised around 5.7 as of 2017. Women accounted for 7-23% of the fatal poisonings. The percentage was lowest in Iceland and highest in Finland and Norway. The age range was 14-70 years. The median age (41 years) was highest in Denmark and Norway. The other countries had a median age between 33 and 35 years. Opioids were the main cause of death. Methadone remained the main intoxicant in Denmark, while heroin/morphine was still the main intoxicant in Norway, as was buprenorphine in Finland. However, the picture has changed in Sweden compared to 2012, where heroin/morphine caused most deaths in 2017. Sweden also experienced the highest number of deaths from fentanyl analogues (67 deaths) and buprenorphine (61 deaths). Deaths from fentanyl analogues also occurred in Denmark, Finland and Norway, but to a smaller extent. Over the years, the proportion of opioid deaths has decreased in all countries except Sweden, which has experienced an increase. This decline has been replaced by deaths from CNS stimulants like cocaine, amphetamine and methylenedioxymethamphetamine (MDMA). Cocaine deaths have occurred in all countries but most frequently in Denmark. MDMA deaths have increased in all countries but mostly in Finland. Poly-drug use was widespread, as seen in the earlier studies. The median number of detected drugs per case varied from 4-6. Heroin/morphine, methadone, buprenorphine, cocaine, amphetamine, methamphetamine, MDMA, tetrahydrocannabinol (THC) and benzodiazepines were frequently detected. Pregabalin and gabapentin were detected in all countries, especially pregabalin, which was detected in 42% of the Finnish cases. New psychoactive substances (NPS) occurred in all countries except Iceland.

Enzymatic assays for detecting lactose and sucrose in urine to reveal intravenous drug abuse with emphasis on buprenorphine.

Buprenorphine and methadone are commonly used medications for opioid maintenance treatment (OMT), using sublingual and oral administration, respectively. Although beneficial for OMT, these drugs can also be abused by intravenous administration. In intravenous abuse cases, the adjuvants lactose and sucrose are excreted in urine without hydrolysis to monosaccharides, since there are no disaccharidases in the blood. We validated enzymatic methods for the analysis of lactose and sucrose in urine. The analytical performance of both assays was considered appropriate for detecting intravenous drug abuse. The principle was proven by analyzing 93 postmortem (PM) urine samples for lactose, following comprehensive toxicological drug screening. In addition, 32 clinical urine samples from potential drug abusers were analyzed to assess the effect of PM changes on the assay. The mean level of lactose was low in clinical samples and relatively low in PM samples in which no drugs were found. Markedly elevated levels were seen in many of the buprenorphine positive samples, suggesting intravenous administration. Enzymatic methods could provide a simple and cost effective way to assess the intravenous administration of OMT drugs or drugs of abuse. Very high levels of glucose in urine may interfere with the assays. Furthermore, other causes for elevated urine disaccharides, such as hypolactasia and increased intestinal permeability, need to be considered in the interpretation of the results. Copyright © 2016 John Wiley & Sons, Ltd.

Relation of postmortem blood alcohol and drug concentrations in fatal poisonings involving amitriptyline, propoxyphene and promazine.

Drugs and alcohol often occur together in fatal poisonings, complicating the process of determining the cause of death. Especially when found in concentrations generally regarded as toxic but not lethal, the question arises whether the combination of sublethal amounts was the likely cause of death. In this study, we examined poisoning deaths involving amitriptyline, propoxyphene and promazine, which are, after benzodiazepines, the most frequently occurring drugs in Finnish alcohol-related poisonings. From the forensic toxicology database, covering the years 1995-2002, we extracted 332 fatal poisonings, calculated median blood alcohol and drug concentrations, constructed concentration-concentration and concentration-response curves and evaluated the significance of the presence of therapeutic amounts of benzodiazepines. Median amitriptyline and propoxyphene concentrations were lower in alcohol-related cases than in clean drug poisonings. Correspondingly, the median blood alcohol concentrations in all drug-related poisonings were 1.5-2.2 mg/g lower than that found in clean alcohol poisonings. Alcohol concentration proved to be a more sensitive indicator of alcohol-drug interaction than drug concentration. This result suggests that when alcohol is present, relatively small overdoses of the studied drugs may result in fatal poisoning. In this context, fatal drug and alcohol concentrations and the issue of determining the most important agent in fatal drug-alcohol intoxications are discussed.

Fatal poisoning in drug addicts in the Nordic countries in 2012.

This report is a follow-up to a study on fatal poisoning in drug addicts conducted in 2012 by a Nordic working group. Here we analyse data from the five Nordic countries: Denmark, Finland, Iceland, Norway and Sweden. Data on sex, number of deaths, places of death, age, main intoxicants and other drugs detected in the blood were recorded. National data are presented and compared between the Nordic countries and with data from similar studies conducted in 1991, 1997, 2002 and 2007. The death rates (number of deaths per 100,000 inhabitants) increased in drug addicts in Finland, Iceland and Sweden but decreased in Norway compared to the rates in earlier studies. The death rate was stable in Denmark from 1991 to 2012. The death rate remained highest in Norway (5.79) followed by Denmark (5.19) and Iceland (5.16). The differences between the countries diminished compared to earlier studies, with death rates in Finland (4.61) and Sweden (4.17) approaching the levels in the other countries. Women accounted for 15-27% of the fatal poisonings. The median age of the deceased drug addicts was still highest in Denmark, and deaths of addicts >45 years old increased in all countries. Opioids remained the main cause of death, but medicinal opioids like methadone, buprenorphine, fentanyl and tramadol mainly replaced heroin. Methadone was the main intoxicant in Denmark and Sweden, whereas heroin/morphine caused the most deaths in Norway. Finland differed from the other Nordic countries in that buprenorphine was the main intoxicant with only a few heroin/morphine and methadone deaths. Deaths from methadone, buprenorphine and fentanyl increased immensely in Sweden compared to 2007. Poly-drug use was widespread in all countries. The median number of drugs per case varied from 4 to 5. Heroin/morphine, medicinal opioids, cocaine, amphetamines, benzodiazepines and alcohol were the main abused drugs. However, less widely used drugs, like gamma-hydroxybutyric acid (GHB), methylphenidate, fentanyl and pregabalin, appeared in all countries. New psychotropic substances emerged in all countries, with the largest selection, including MDPV, alpha-PVP and 5-IT, seen in Finland and Sweden.

Automated liquid chromatographic/tandem mass spectrometric method for screening beta-blocking drugs in urine.

An automated liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method is presented for the screening and confirmation of 16 beta-blocking drugs in clinical and autopsy urine samples. The described method involved C(18) solid phase extraction, LC separation and MS analysis on a triple-stage quadrupole mass analyser. Samples were initially pre-screened for the presence of any beta-blocking drugs using LC/MS with selected ion monitoring. Any compounds tentatively identified as beta-blocking drugs on the basis of their LC retention time and protonated molecular ion were then automatedly subjected to a second analysis in which the relevant MS/MS product ion mass spectra were acquired. These product ion mass spectra were then automatically searched against a 400-substance mass spectral library containing previously acquired beta-blocking drugs. The results demonstrated that library search of beta-blocking drugs in urine with MS/MS product ion mass spectra was more reliable and produced fewer false negatives than library searching with mass spectra derived from single-stage quadrupole MS. The limits of identification in the MS/MS product ion scan ranged from 0.02 mg l(-1) for carvedilol to 1.2 mg l(-1) for pindolol, the majority of the values being below 0.2 mg l(-1).

Identification of drugs in autopsy liver samples by instrumental qualitative thin-layer chromatography.

An instrumental thin-layer chromatographic (TLC) procedure is described for the identification of drugs with use of corrected RF values (hRcF) and in situ ultraviolet spectra. One hundred and eleven successive autopsy liver samples received from medical examiners were investigated by this technique and the results were compared to those obtained with a reference method. From the nineteen findings by the reference method, sixteen (84%) were correctly identified by the instrumental TLC method, using an hRcF pre-search with a window size of +/-7 units followed by a correlation search. In addition, one drug was identified correctly by correlation search only. There was no serious interference from endogenous substances, and a correlation value of about 0.9 is suggested as a limit to cut the hit list of candidates.

Comparison of four homologous retention index standard series for qualitative gas chromatography of nitrogenous acidic and neutral drugs.

Four homologous retention index standard series, the alkylmethylhydantoins, alkylhydantoins, alkylmaleimides and alkylbis(trifluoromethyl)phosphine sulphides, were evaluated for the screening of blood samples for acidic and neutral drugs on Ultra 2 and HP-1701 capillary columns over a six-month period. An index series consisting of actual drug substances was used as a standard of comparison. All the series produced high precision, and the precision differences between the series were rather small. Considering the limitations of the other series, the alkylmethylhydantoins and alkylhydantoins turned out to be the most feasible internal retention index standard series in the present dual column setting.

Simultaneous screening for 238 drugs in blood by liquid chromatography-ion spray tandem mass spectrometry with multiple-reaction monitoring.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the qualitative screening for 238 drugs in blood samples, which is considerably more than in previous methods. After a two-step liquid-liquid extraction and C(18) chromatography, the compounds were introduced into a triple quadrupole mass spectrometer equipped with a turbo ion spray ion source operating in the positive ionization mode. Identification was based on the compound's absolute retention time, protonated molecular ion, and one representative fragment ion obtained by multiple reaction monitoring (MRM) at an individually selected collision energy of 20, 35, or 50 eV. The limit of detection (LOD) for the majority of the compounds (80%) was < or = 0.05 mg/l, ranging from 0.002 mg/l (e.g., antihistamines) to 5 mg/l (acidic compounds), and for malathion it was 10 mg/l. The LOD values were sufficiently low to allow the majority of compounds to be detected at therapeutic concentrations in the blood.

Precise gas chromatography with retention time locking in comprehensive toxicological screening for drugs in blood.

The long-term precision of three retention parameters, the absolute retention time (RT), the relative retention time related to dibenzepin (RRT), and the internal retention index based on the alkylfluoroaniline series (RI), were studied with 14 basic drugs on HP-5 and DB-17 columns with and without the use of the retention time locking option (RTL). Using the constant flow mode in all experiments, the RTL method was found to produce superior precision with all three retention parameters compared to the non-RTL method on each column. The results showed that RTL offers a significant advantage within a single instrument method, not only between methods, with CV<0.1% by RRT. Consequently, a dual-column gas chromatographic procedure with nitrogen-phosphorus detection was described for comprehensive screening for basic drugs in 1-ml whole blood samples. The method consisted of one-step liquid-liquid extraction with butyl acetate, identification using RRT in the RTL mode, and quantification based on single point calibration. The method allowed reliable screening and quantification of 124 basic drugs at therapeutic and toxic concentration levels in autopsy blood.

Analytical toxicology of fluorinated inhalation anaesthetics.

The chemical and pharmacological properties of the current fluorinated inhalation anaesthetics, halothane, enflurane, isoflurane, sevoflurane and desflurane, are surveyed with implications to toxicity. Analytical methods, especially gas chromatography with head space, purge and trap, or pulse heating extraction, are reviewed in forensic toxicological and occupational/therapeutic monitoring contexts.

Identification of volatile organic compounds in blood by purge and trap PLOT-capillary gas chromatography coupled with Fourier transform infrared spectroscopy.

A purge and trap concentrator with a Tenax trap was coupled to gas chromatography-Fourier transform infrared spectrometry for the identification of volatile organic compounds in blood samples. A styrene-divinyl benzene porous layer capillary column allowed the separation of compounds such as household and medical gases, solvents and alcohol congeners. The identification limits in blood, measured by comparison to an in-house vapour phase spectrum library, generally ranged from 0.05 to 10 mg/l, depending on the analyte structure. Low molecular weight alcohols had identification limits up to 100 mg/l. Six actual casework examples were collected during a 1-year period of routine use to demonstrate the feasibility of the method.

Benzodiazepine findings in blood and urine by gas chromatography and immunoassay.

Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.

Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is presented for the simultaneous screening and quantitation of 18 antihistamine drugs in blood samples. Sample pretreatment involved liquid-liquid extraction of the basic antihistamines followed by a second extraction of the acidic antihistamines. The recoveries were 43-113% for basic drugs and 23-66% for acidic drugs. The combined extracts were run by LC on C(18) reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The mass spectrometric analysis was performed with a triple stage quadrupole mass analyzer. Screening was performed using multiple reaction monitoring (MRM) and any compounds tentatively identified as antihistamine drugs were then automatedly verified by their Product Ion Spectra in a subsequent MS/MS run. Quantitation was based on the MRM data from the screening step. In validation tests, the method showed good linearity at the relevant concentrations. The attained limits of quantitation varied between 0.0005 and 0.01mg/l in blood and were lower than the therapeutic concentrations (C(max)). The limits for identification by Product Ion Spectra were also lower than C(max), except for clemastine, which has exceptionally low concentrations in blood. The intra-assay relative standard deviations were better than 10% and the inaccuracy varied between 39% for levocabastine and 5% for cyclizine, the majority of the values being <20%.

Fast black K salt: a visualization reagent for thin-layer chromatography of beta-adrenergic blocking drugs.

A sensitive visual method for the thin-layer chromatography of beta-adrenergic blocking drugs is described. The Rf values and the detection limits of eleven beta-blockers extracted from urine and as pure drugs are given. Other commonly used cardiovascular drugs are shown not to interfere with the method. The procedure is applicable to both urine and liver extracts.

Automated quantitative screening for acidic and neutral drugs in whole blood by dual-column capillary gas chromatography.

A method consisting of single-step extraction, dual-channel capillary gas chromatography, nitrogen-selective detection, and automated data processing is evaluated for the quantitative screening of acidic and neutral drugs in postmortem blood. The drugs are identified by their cubic spline retention indices using multidetector retention index standards. The data processing is performed with Micman software, which offers several advantages in the maintenance of identification reliability. The precision of the retention indices during a three-month period with a routine loading of 14 samples per day is presented for eight different drugs at therapeutic and toxic concentrations: The CV ranges from 0.11% to 1.12% for NB-54 and from 0.05% to 0.21% for NB-1701. The within-day CV is less than 0.05% in each case. The status of these results and the means of achieving further precision are discussed. The long-term precision of quantification, normally under 10%, is considered adequate for most forensic toxicological purposes.

Quantitative screening for benzodiazepines in blood by dual-column gas chromatography and comparison of the results with urine immunoassay.

A dual-column retention index method is described for quantitative gas chromatographic (GC) screening of 26 benzodiazepine drugs and metabolites in the blood using DB-5 and DB-17 capillary columns and electron capture detection. The method involves a one-step, small-scale liquid-liquid extraction with ethyl acetate and derivatization with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide with 1% tert-butyldimethylsilyl chloride. The results from the GC screening of 514 postmortem blood samples were compared to those obtained from urine immunoassay (Syva ETSplus with a 200-ng/mL cutoff). Both methods gave a negative result in 284 cases and a positive result in 149 cases. In 48 cases, urine was negative by immunoassay but blood was positive by GC. The opposite situation (blood negative, urine positive) was detected only in four cases. In 29 cases, an invalid result was obtained by urine by immunoassay: 26 blood samples of those cases were negative and three samples positive by GC. In postmortem forensic toxicology, the present GC method seems to be a good alternative to the common combination of urinary immunoassay followed by quantitative analysis of blood by chromatography.

Identification limits for volatile organic compounds in the blood by purge-and-trap GC-FTIR.

An analysis method for volatile organic compounds in blood based on purge-and-trap extraction coupled with gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR) was developed. The sample volume was 5 mL, and the internal standard was diethyl ketone. The chromatographic separation was carried out on a PoraPLOT Q capillary column, and the effluent was first directed to the FTIR and then to a flame ionization detector (FID). FTIR identification limits were measured for 27 volatile organic compounds; the criteria for the limit were that the first hit-list position should be obtained against the Sadtler library, which contains 3240 spectra, and that the correlation value should exceed 0.5. It was required that the peak be seen by FID but not necessarily by a Gram-Schmidt chromatogram. The FTIR identification limits, ranging from 0.01 mg/L for ethyl acetate, methylethyl ketone, and sevoflurane to 24 mg/L for methanol, generally allowed the detection of volatile-substance exposure at a lower level than is acutely toxic. Quantitative calibration data were presented for selected substances, based on the FID response, which shows that the method is also amenable to quantitative analysis. The throughput of the method without additional automation is five samples per day, the purge-and-trap stage being the limiting factor.

Peripheral zonal hepatic necrosis caused by accidental ingestion of methyl ethyl ketone peroxide.

Fatal massive peripheral zonal hepatic necrosis developed in a 47-year-old man who accidentally ingested a solution of methyl ethyl ketone peroxide (MEKP) in dimethyl phthalate. Such solutions contain about 10% active oxygen. The clinical course was characterized by temporary cardiac arrest, abdominal burns, severe metabolic acidosis, rapid hepatic failure, rhabdomyolysis and respiratory insufficiency. A fatal outcome resulted 4 d afterwards from hepatic coma associated with blood coagulation disorders. Microscopical examination revealed massive periportal hepatic necrosis accompanied by atypical pseudoductular proliferation. The proliferating cells were probably of bile duct origin and exhibited atypia and mitoses. The pathogenetic mechanism may involve lipid peroxidation caused by free oxygen radicals derived from MEKP.