A fatal mono-intoxication with 4-fluoroisobutyrylfentanyl: Case report with postmortem concentrations.

We report on a case of a 35-year-old man who died suddenly and unexpectedly due to a 4-fluoroisobutyrylfentanyl (4-FIBF) mono-intoxication. Pathological, toxicological and chemical investigations were conducted at the Netherlands Forensic Institute. A full three-cavity forensic pathological examination was performed according to international guidelines. Biological samples obtained during autopsy were comprehensively investigated for the presence of toxic substances using headspace gas chromatography (GC) with flame ionization detection, liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS), GC-MS, high-performance LC with diode array detection and LC-tandem MS (LC-MS-MS). The seized crystalline substance found next to the body was investigated using a presumptive color test, GC-MS, Fourier-transform infrared spectroscopy and nuclear magnetic resonance. Pathological investigation identified minor lymphocytic infiltrates in the heart, considered irrelevant for the cause of death. Toxicological analysis of the victims' blood indicated the presence of a fluorobutyrylfentanyl (FBF) isomer, with no other compounds detected. The FBF isomer was identified in the seized crystalline substance as 4-FIBF. 4-FIBF concentrations were quantified in femoral blood (0.030 mg/L), heart blood (0.12 mg/L), vitreous humor (0.067 mg/L), brain tissue (>0.081 mg/kg), liver tissue (0.44 mg/kg) and urine (approximately 0.01 mg/L). Based on the outcomes of the pathological, toxicological and chemical investigations, the cause of death of the deceased was attributed to a fatal 4-FIBF mono-intoxication. The presented case underlines the added value of a combined bioanalytical and chemical investigative approach to identify and subsequently quantify fentanyl isomers in postmortem cases. Furthermore, it demonstrates the importance of investigating the postmortem redistribution of novel fentanyl analogs to establish reference values and to subsequently allow for correct interpretation of cause of death analysis in future casework.

Sorption of structurally different ionized pharmaceutical and illicit drugs to a mixed-mode coated microsampler.

The mixed-mode (C18/strong cation exchange-SCX) solid-phase microextraction (SPME) fiber has recently been shown to have increased sensitivity for ionic compounds compared to more conventional sampler coatings such as polyacrylate and polydimethylsiloxane (PDMS). However, data for structurally diverse compounds to this (prototype) sampler coating are too limited to define its structural limitations. We determined C18/SCX fiber partitioning coefficients of nineteen cationic structures without hydrogen bonding capacity besides the charged group, stretching over a wide hydrophobicity range (including amphetamine, amitriptyline, promazine, chlorpromazine, triflupromazine, difenzoquat), and eight basic pharmaceutical and illicit drugs (pKa>8.86) with additional hydrogen bonding moieties (MDMA, atenolol, alprenolol, metoprolol, morphine, nicotine, tramadol, verapamil). In addition, sorption data for three neutral benzodiazepines (diazepam, temazepam, and oxazepam) and the anionic NSAID diclofenac were collected to determine the efficiency to sample non-basic drugs. All tested compounds showed nonlinear isotherms above 1mmol/L coating, and linear isotherms below 1mmol/L. The affinity for C18/SCX-SPME for tested organic cations without Hbond capacities increased with longer alkyl chains, ranging from logarithmic fiber-water distribution coefficients (log Dfw) of 1.8 (benzylamine) to 5.8 (triflupromazine). Amines smaller than benzylamine may thus have limited detection levels, while cationic surfactants with alkyl chain lengths >12 carbon atoms may sorb too strong to the C18/SCX sampler which hampers calibration of the fiber-water relationship in the linear range. The log Dfw for these simple cation structures closely correlates with the octanol-water partition coefficient of the neutral form (Kow,N), and decreases with increased branching and presence of multiple aromatic rings. Oxygen moieties in organic cations decreased the affinity for C18/SCX-SPME. Log Dfw values of neutral benzodiazepines were an order of magnitude higher than their log Kow,N. Results for anionic diclofenac species (logKow,N 4.5, pKa 4.0, log Dfw 2.9) indicate that the C18-SCX fiber might also be useful for sampling of organic anions. This data supports our theory that C18-based coatings are able to sorb ionized compounds through adsorption and demonstrates the applicability of C18-based SPME in the measurement of freely dissolved concentrations of a wide range of ionizable compounds.

Consequences of Decontamination Procedures in Forensic Hair Analysis Using Metal-Assisted Secondary Ion Mass Spectrometry Analysis.

Today, hair testing is considered to be the standard method for the detection of chronic drug abuse. Nevertheless, the differentiation between systemic exposure and external contamination remains a major challenge in the forensic interpretation of hair analysis. Nowadays, it is still impossible to directly show the difference between external contamination and use-related incorporation. Although the effects of washing procedures on the distribution of (incorporated) drugs in hair remain unknown, these decontamination procedures prior to hair analysis are considered to be indispensable in order to exclude external contamination. However, insights into the effect of decontamination protocols on levels and distribution of drugs incorporated in hair are essential to draw the correct forensic conclusions from hair analysis; we studied the consequences of these procedures on the spatial distribution of cocaine in hair using imaging mass spectrometry. Additionally, using metal-assisted secondary ion mass spectrometry, we are the first to directly show the difference between cocaine-contaminated and user hair without any prior washing procedure.

Sensitive determination of plasma protein binding of cationic drugs using mixed-mode solid-phase microextraction.

Freely dissolved concentrations are considered to be the most relevant concentration in pharmacology and toxicology, as they represent the active concentration available for interaction with its surroundings. Here, a solid-phase microextraction (SPME) coating that combines octadecyl and propylsulfonic acid groups as strong cation exchange sites, known as C18/SCX or "mixed-mode" SPME, is used to measure freely dissolved concentrations of amitriptyline, amphetamine, diazepam and tramadol to different binding matrices, including bovine serum albumin (BSA), human serum albumin (HSA), human plasma and human whole blood. A potential confounding factor in binding studies is that proteins may sorb to the fiber coating leading to incorrect measurement of protein sorption or changes in uptake kinetics to the fiber coating. Sorption of bovine serum albumin (BSA) was observed and quantified using a Lowry assay. BSA binds to the C18/SCX fiber in small amounts, but large changes in uptake kinetics were not observed. All experiments were performed at equilibrium. In addition, however, the effect of depletion and non-equilibrium extraction on the estimation of protein binding affinities was also studied. Binding affinities to BSA and human serum albumin (HSA) were calculated as log KBSA or log KHSA. These values were very similar to reported literature values. Sampling at either equilibrium or non-equilibrium resulted in similar binding affinities. Furthermore, SPME fibers were used to measure freely dissolved concentrations in undiluted human plasma and whole blood. Analysis of SPME extracts could be performed using HPLC-UV or HPLC with fluorescence detection without prior clean-up of the samples. Measured bound fractions in plasma using this SPME approach were comparable to literature reference values. Bound fractions in whole blood were always higher than in plasma, due to red blood cell partitioning. This work shows the potential of SPME as sampling tool for freely dissolved concentrations, especially for highly protein-bound compounds. Conventional SPME coatings such as polyacrylate (PA) or polydimethylsiloxane (PDMS) might be lacking sensitivity when sampling the small neutral fraction of highly protein-bound positively charged compounds, but the C18/SCX fiber is able to sorb the charged species of organic cations, thereby improving sensitivity for these types of compounds.

Sorption of amitriptyline and amphetamine to mixed-mode solid-phase microextraction in different test conditions.

A solid-phase microextraction (SPME) method based on a sampler coating that includes strong cation groups (C18/SCX) is explored as a rapid direct sampling tool to detect and quantify freely dissolved basic drugs. Sampling kinetics, sorption isotherms and competitive effects on extraction yields in mixtures were tested for amphetamine and the relatively large/hydrophobic tricyclic antidepressant amitriptyline. Both compounds are >99% ionized at pH 7.4 but their affinity for the C18/SCX fiber is markedly different with distribution coefficients (Dfw values) of 2.49±0.02 for amphetamine and 4.72±0.10 for amitriptyline. Typical changes in electrolyte homeostasis that may occur in biomedical samples were simulated by altering pH and ionic composition (Na(+) and K(+) concentrations). These changes were shown to affect C18/SCX sorption affinities of the tested drugs with less than 0.2log units. At relatively low fiber loadings (<10mmol/L coating) and at all tested exposure times, linear sorption isotherms were obtained for both compounds but at aqueous concentrations of the individual drugs corresponding to concentrations in blood that are lethal, sorption isotherms became strongly nonlinear. Competition effects within binary mixtures occurred only if combinations of aqueous concentrations resulted in total fiber loadings that were in the nonlinear range of the SPME sorption isotherm for the individual compounds. We also compared sorption to the (prototype) C18/SCX SPME coating with analogue (biocompatible) C18 coated SPME fibers. C18/SCX fibers show increased sorption affinity for cationic compounds compared to C18 fibers, as tested using amitriptyline, amphetamine and trimethoprim. Surprisingly, sorption affinity of these ionized compounds for the C18 SPME fibers were within 1log unit of the C18/SCX SPME fibers. This shows that the strong cation exchange groups within the C18/SCX coating only has a relatively small contribution to the total sorption affinity of cationic compounds. Also the role of negatively charged silanol groups in both the C18 and C18/SCX coating seems small, as anionic diclofenac species sorbed strongly to the C18 fiber. Ionized organic species seem to be substantially adsorbed to the high surface area of C18 in SPME types using porous silica based coatings.

Hydrogen peroxide reactions on cocaine in hair using imaging mass spectrometry.

Today, forensic hair analysis is considered to be a standard method for identifying chronic drug users since information about drug use stored and located in hair can cover several months to even years. When interpreting these results, one should be aware of all kind of pitfalls. External factors such as bleaching might influence the analytical result. Although the effect of hydrogen peroxide on cocaine in a solution was described before, it was never investigated whether the described reaction products (ecgonine methylester, benzoylecgonine, hydroxynorcocaine and dihydroxycocaine) are indeed found on contaminated or user hair. Since it is of great importance in forensic hair analysis to know whether cocaine and/or reaction products are detectable in hair after bleaching, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was used to study the effect of hydrogen peroxide treatment on incorporated cocaine in hairs. Cocaine oxidation products were identified in a solution based on MS/MS spectra and spatial distribution of these products in hair was explored using MALDI TOF-MS. All images were accomplished by spraying α-Cyano-4-hydroxycinnamic acid (CHCA) as a MALDI-matrix. Images revealed a loss of detectability of cocaine and its reaction products in hairs already after a short bleaching period. Since all compounds of interest are found in the hydrogen peroxide and wash solution, these findings indicate that all evidence of cocaine use might be lost after a hair bleaching treatment. Therefore, forensic toxicologists should take into consideration whether hair samples were bleached before making any conclusions from hair analysis results.

Elucidating the sorption mechanism of "mixed-mode" SPME using the basic drug amphetamine as a model compound.

We studied the sorption of amphetamine as a model drug to represent small, polar organic cations to a new SPME coating combining C18 and propylsulfonic acid. This combination of hydrophobic and strong cation exchange (SCX) groups was compared to conventional SPME fibers with polyacrylate (PA) or C18 coating. The affinity of amphetamine at physiological pH (PBS) was 20 to 180 times greater for the new C18/SCX coating than for C18 alone and PA of different coating thickness. As amphetamine is a base and >99% protonated at physiological pH, this enhanced affinity is attributed to the ion-exchange phase in the coating. Tests at pH above the pKa of amphetamine show that, when normalized to the coating volume, neutral amphetamine also has a higher affinity compared to PA. As ion-exchange groups are not unlimitedly present in the coating, amphetamine isotherms level off to a saturation concentration on the C18/SCX fiber at the highest tested aqueous concentrations. Also, other cations (Na(+), K(+), Ca(2+)) compete for the SCX sites and decrease the sorption coefficients, e.g. by 1.7 log units when comparing Milli-Q water with PBS. The C18/SCX fiber provides improved sensitivity over some of the classic SPME fibers. However, care should be taken near the cation exchange capacity of the fiber and the fiber should be calibrated in an appropriate matrix so as to eliminate competition effects.

Toxicological findings in cases of sexual assault in the Netherlands.

Reports on cases of alleged drug-facilitated sexual assault (DFSA) have increased since the mid-1990s. The aim of this study was to identify the extent and types of drugs found in cases of alleged sexual assault (DFSA) in the Netherlands. In total, 135 cases of alleged DFSA were identified. Most of the victims were women (94%), and the mean age of the victims was 25 years. Blood and urine samples were tested for the presence of alcohol, drugs (drugs of abuse and prescription drugs), or both. In 27% of the cases, no alcohol and/or drugs were found. With increasing time delay, more cases were found to be negative. Alcohol is the most commonly found drug followed by nonopiate analgesics, illicit drugs, and benzodiazepines. In some cases, the absence of alcohol and drugs may represent false-negative results owing to the time delay between alleged sexual assault and sampling.

Post-mortem cases involving amphetamine-based drugs in The Netherlands. Comparison with driving under the influence cases.

In this study we reviewed the post-mortem cases in the years 1999-2004 that were presented at the Netherlands Forensic Institute. The concentrations of amphetamine-based drugs in femoral blood from cases of suspected unnatural death were compared with concentrations in whole blood from non-fatal cases of driving under the influence (DUI cases) and with literature. Furthermore, the combinations with other drugs and/or alcohol were investigated. Amphetamine-based drugs were present in 70 post-mortem cases and 467 DUI cases. The most detected amphetamine-based drug was MDMA, followed by amphetamine. The presence of MDA could usually be explained by metabolism of MDMA. Methamphetamine and MDEA were rarely present. Frequently, the amphetamine-based drugs were taken in combination with alcohol and/or other non-amphetamine-based drugs such as cocaine or cannabinoids. The 70 post-mortem cases were divided into 38 amphetamine-based drug caused (i.e. the amphetamine-based drug directly caused or contributed to the death) and 32 amphetamine-based drug related deaths (i.e. death was not directly caused by the amphetamine-based drug). In the latter category, other (poly)drug intoxications and death by violence or drowning were the most frequent causes of death. In 30 cases, MDMA caused death directly. The range in blood concentrations of MDMA in these cases was substantial, i.e. 0.41-84 mg/L with a median concentration of 3.7 mg/L (n=30). MDMA blood concentrations in the MDMA related deaths (n=20) and in the DUI cases (n=360) varied up to 3.7 and 4.0 mg/L, respectively. Seven victims died from the direct effects of amphetamine; the blood concentration of amphetamine ranged from 0.24 to 11.3 mg/L, with a median concentration of 1.7 mg/L (n=7). The median concentrations of amphetamine in the amphetamine related deaths (n=13) and the DUI cases (n=208) were much lower, i.e. 0.28 and 0.22 mg/L, respectively. Amphetamine blood concentrations up to 6.0 and 2.3 mg/L were seen in the drug related deaths and DUI cases, respectively. The most frequently encountered amphetamine-based drugs in the investigated deaths were MDMA and amphetamine. The majority of MDMA- and amphetamine-caused deaths, i.e. 90% of these deaths, occurred with blood concentrations above 1.5 and 0.80 mg/L, respectively. MDMA and amphetamine blood concentrations in drug related deaths and DUI cases, however, overlap the range of fatal concentrations. Therefore, MDMA or amphetamine concentrations should never be used alone to establish the cause of death.

Forensic cases involving the use of GHB in The Netherlands.

In this study, forensic cases involving the use of Gamma Hydroxy Butyric acid (GHB) from the second half of 1999 through the second half of 2001 in The Netherlands (blood >5mg/l and urine >10mg/l) are described. GHB was analysed by GC-MS after lactone formation and using GHB-d6 as internal standard. The results are divided into three groups: cases of chemical submission, cases of driving under the influence and cases of unknown causes of death.GHB was found in six cases of possible chemical submission. In these cases, relatively low concentrations of GHB were found. The results show that in cases of chemical submission, urine should be analyzed, because GHB is present longer in urine than in blood. The police should collect the samples in containers that do not contain citrate as anticoagulant. Especially at low levels of GHB, the formation of GHB in these tubes hampers an interpretation of the results.GHB was found in 13 cases of driving under the influence. In contrast to the cases of chemical submission, high concentrations of GHB were found, corresponding with observations of extreme sleepiness or temporary loss of consciousness.GHB was found in 16 cases of unexplained death: the measured range of GHB concentrations in blood might correspond to effects such as drowsiness, but not to serious toxicity of GHB. In 4 of these 16 cases, the role of GHB could be excluded. In the remaining cases, the role of GHB remains unclear; more research into "background" concentrations of GHB in post-mortem material is required. The incidence of the use of GHB in The Netherlands cannot be derived from these toxicological data. As GHB is not routinely found during systematical toxicological analyses, these data may seriously underestimate the use of GHB. Therefore, information from the police to the forensic institute is essential.

Hair analysis for cannabinoids and amphetamines in a psychosis incidence study.

It remains often uncertain whether the use of illicit substances has contributed to the aetiology of psychosis. Gas chromatography-mass spectrometry can be used to detect them in hair of the head. Given a monthly growth rate between 1.0 and 1.5 cm, one can examine hair segments that originated during the pre-psychotic period. We examined the usefulness of hair analysis to detect the use of cannabinoids or amphetamines during this period. One hundred patients participated in a psychosis incidence study and 64 yielded hair. Refusal was associated with non-Dutch ethnicity, not with a clinical diagnosis of use. A monthly growth rate of 1.5 cm was assumed and 33 specimens were found to be long enough. Cannabinoids or amphetamines were detected in nine specimens. In seven they were not detected, whereas the patients had reported their use. It is likely that their hair grew at a slower rate and that the examined segments belonged to an earlier period of time, during which the substances were not used. Lack of knowledge about the individual hair growth rate is an important limitation to the usefulness of this method.