Evaluating the hip-flask defence using analytical data from ethanol and ethyl glucuronide. A comparison of two models.

AIM: Claimed intake of alcohol after a traffic incident, called the hip-flask defence, can be objectively assessed by different methods. One of them is the use of two consecutive ethanol concentrations in urine and the ratio between ethanol concentrations in urine and blood. Another one is the concentrations of ethyl glucuronide (EtG) and ethyl sulphate (EtS) in blood and their ratio to ethanol. The experimental basis for both these models is from single dose studies only. The aim of this study was therefore to describe the kinetics of ethanol, EtG and EtS after ingestion of two repeated doses of ethanol and to investigate the usefulness of the different models for the assessment of the hip-flask defence. METHODS: Thirty-five subjects ingested a first dose of 0.51 g of ethanol per kilo body weight, and two hours later a second dose (the hip-flask drink) of 0.25, 0.51 or 0.85 g of ethanol per kilo body weight. Ten urine and 17 blood samples were collected and analysed for ethanol, EtG and EtS using fully validated methods. It was investigated if all subjects fulfilled the criteria for recent drinking, according to the two different models, when using the samples collected 180-240 minutes after start of first dose drinking. According to the first model, increase in urinary ethanol concentrations and a ratio UAC/BAC below 1.3 indicated recent drinking. According to the second model, increase in blood EtG concentrations and a ratio ethanol (g/kg)/EtG (mg/L) above 1 indicated recent drinking. RESULTS: All subjects in the high dose group fulfilled all criteria for recent drinking. One subject in the medium dose group and nine subjects in the low dose group failed to show increasing UAC and/or a UAC/BAC ratio below 1.3. One subject in the low dose group failed to show increasing concentrations of blood EtG, but all subjects showed a ratio ethanol/EtG above 1. CONCLUSIONS: The present study showed, by the use of experimental data, that both two models used to investigate the hip-flask defence can be used, but only when the hip-flask dose is sufficiently high.

Fatal intoxications associated with the designer opioid AH-7921.

AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a designer opioid with ∼80% of morphine's µ-agonist activity. Over a 6-month period, we encountered nine deaths where AH-7921 was involved and detected in blood from the deceased. Shortly after the last death, on August 1 2013, AH-7921 was scheduled as a narcotic and largely disappeared from the illicit market in Sweden. AH-7921 was measured by a selective liquid chromatography-MS-MS method and the concentrations of AH-7921 ranged from 0.03 to 0.99 µg/g blood. Six of our cases had other drugs of abuse on board and most had other medications such as benzodiazepines, antidepressants and analgesics. However, the other medicinal drugs encountered were present in postmortem therapeutic concentrations and unlikely to have contributed to death. In addition to the parent compound, we identified six possible metabolites where two N-demethylated dominated and four mono-hydroxylated were found in trace amounts in the blood. In conclusion, deaths with AH-7921 seem to occur both at low and high concentrations, probably a result of different tolerance to the drug. Hence, it is reasonable to assume that no sharp dividing line exists between lethal and non-lethal concentrations. Further, poly-drug use did not seem to be a major contributing factor for the fatal outcome.

Concentration-time profiles of gamma-hydroxybutyrate in blood after recreational doses are best described by zero-order rather than first-order kinetics.

The recreational drug gamma-hydroxybutyrate (GHB) has a short plasma elimination half-life (t(1/2)) reported to be about 30-50 min. However, this represents a terminal half-life and therefore might not necessarily apply after large (abuse) doses are taken. Clinical studies with sodium oxybate (sodium salt of GHB) suggest that zero-order rather than first-order kinetics are more appropriate to describe post-peak concentration-time (C-T) profiles. We report the case of a 23-year-old male found unconscious by the police and a blood sample contained 100 mg/L GHB and 0.14 g% ethanol. On regaining consciousness the man admitted drinking alcohol about 6 h earlier but claimed that his drink must have been spiked with GHB. The police wanted to know how much GHB had been administered to account for the man's clinical condition. A back-calculation for 6 h, assuming a GHB half-life of 40 min, gives a very high concentration in blood of approximately 900 mg/L, which would probably have proven fatal. Back-calculating using zero-order kinetics and a proposed elimination rate of 18 mg/L per hour leads to a GHB concentration of 208 mg/L, which is much more realistic. Toxicologists should not arbitrarily apply the principles of first-order kinetics after abuse doses of drugs, when zero-order or saturation kinetics (Michaelis-Menten) are more appropriate.

Retention of opioids and their glucuronides on a combined zwitterion and hydrophilic interaction stationary phase.

A stationary phase combining zwitterionic ion chromatography and hydrophilic interaction chromatography (ZIC-HILIC) from SeQuant was evaluated for the chromatography of some opiates and their polar metabolites. The effects of mobile phase constitution on retention and resolution were extensively evaluated. Different aspects of mobile phase constitution such as ion strength and type of buffer, type and amount of organic modifier and pH were examined. The selectivity and retention of the opiates compared to their glucuronides could be substantially altered with small changes of the mobile phase, especially when the type of buffer, i.e., formate or acetate and organic modifier, i.e., acetonitrile or methanol were changed. The retention on the ZIC-HILIC was dominated by hydrophilic interaction chromatography (HILIC) but considerable effects on the selectivity was observed, possibly caused by an ion exchange mechanism due to interactions with the charges on the stationary phase.

Quantitation of seven low-dosage antipsychotic drugs in human postmortem blood using LC-MS-MS.

In forensic toxicology, antipsychotic drugs are of considerable interest because of their abuse potential and their involvement in intoxications and suicides. In recent years, several new drugs dosed at low levels have entered the market and have put further demands on assays used. The aim of this work was to develop a validated liquid chromatography-tandem mass spectrometry assay for the quantitation of the low-dosage antipsychotic drugs buspirone, fluphenazine, flupenthixol, perphenazine, risperidone, ziprasidone, and zuclopenthixol in human postmortem blood. After liquid-liquid extraction using methyl t-butyl ether, compounds were separated on a Zorbax SB-CN column. Calibration curves were linear in the range 0.8-100 microg/L (r > 0.998) for all drugs. Both within- and between-day coefficients of variation were lower than 25% for all drugs at the LOQ, and extraction recoveries ranged between 58 and 112%. The possible presence of matrix effects was closely investigated. Fifty-four authentic samples were analyzed within the routine postmortem investigation, which resulted in the diagnosis of three fatal intoxications. Even though only a few intoxications were identified, the assay may present valuable information on suicidal deaths in psychotic patients where a true negative result implies noncompliance and a higher susceptibility for suicide. Without a sensitive enough method, this conclusion cannot be drawn. Therefore, we believe that antipsychotic drugs must be measured not only in toxic concentrations but also in therapeutic levels in postmortem cases.

Dose-hair concentration relationship and pigmentation effects in patients on low-dose clozapine.

Several hair components have been suggested as possible molecular sites for drug binding and interaction. Of these, keratin and melanin have been investigated in some detail in order to assess the mechanisms by which the binding occurs. Substances that are positively charged at physiological pH may interact by electrostatic forces between their cationic groups and the anionic carboxylic groups on the surface of the melanin polymer. Studies in human subjects with grey hair have shown that various drugs are detectable in both the coloured (melanin rich) and white (melanin free) hair shafts of these individuals. Again this supports the proposition that keratin and hair proteins play an important role in the binding of drugs in hair. However, drugs are often found in significantly higher concentrations in pigmented hair strands than in senile white hair strands. Another interesting question is if the concentration measured in hair reflects the dose taken. Previous reports have both verified and rejected this hypothesis, but most agree that many factors have impact on the incorporation rate, melanin being one. In this study we obtained blood and hair samples from 12 grey haired patients treated with low-dose clozapine as an adjunct medication in their treatment against Parkinson disease. Each patient's hair was divided into a pigmented and a non-pigmented portion and those were analyzed separately. Clozapine and desmethylclozapine were analyzed with LC-MS-MS after extraction of the analytes from hair and plasma. Paired results from the analysis of pigmented and white hair confirmed the preference for binding to pigmented hair for both clozapine and its metabolite. A majority of the incorporated clozapine was found in the pigmented hair but, as drugs could be detected in white hair, binding to hair protein or association with other hair matrix account for a significant part of drug accumulation in hair. High correlations between dose and the measured concentration of analyte were found for both clozapine (r = 0.91) and desmethylclozapine (r = 0.88).

Evaluation of electrospray ionisation liquid chromatography-tandem mass spectrometry for rational determination of a number of neuroleptics and their major metabolites in human body fluids and tissues.

A study of liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS) with positive electrospray ionisation (ESI) for the determination of selected drugs in human tissues and body fluids such as blood, urine and hair is described. The possibility to screen for and quantify the 19 most commonly prescribed neuroleptics on the Swedish market and determine the presence of their major metabolites within a single LC-MS-MS analysis was evaluated on a PE Sciex API2000 instrument. Chromatographic conditions were optimised and the best separation, with individual retention times for most of the analytes, was obtained on a Zorbax SB-CN column within a 9-min gradient run. The MS-MS fragmentation conditions were optimised for each compound in order to obtain both specific fragments and high signal intensity. Since neuroleptics are a heterogeneous group of compounds, a markedly difference in collision energy needed to achieve fragments of the selected parent ions was seen and the number of fragments achieved varied as well. For sensitive quantification the transition of the most intense fragment of the protonated molecular ion (M+1)(+) was selected for multiple reaction monitoring analysis. More than 70 transitions were finally included in the assay. Detection levels down to the lower ng/ml level were achieved for all analytes, but between analytes more than a 10-fold difference in signal response was seen. By evaluation of extracted ion chromatograms from the analysis of authentic human blood, urine and hair sample the proposed concept for rational drug analysis was found to be both selective and sensitive for the neuroleptics included. A great number of metabolites could be determined in blood, urine and hair as well. A full method validation was not performed since the objective was to evaluate the method design rather than to validate a final method set-up.

Characterization of [3H]flunitrazepam binding to melanin.

In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 +/- 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.

Incorporation of selegiline metabolites into hair after oral selegiline intake.

We have previously shown that melanin in human hair has a great impact on the incorporation of codeine into hair. The present study on 10 subjects was performed to investigate whether or not these findings could also be extrapolated to other therapeutic drugs. We chose selegiline because it metabolizes to two commonly abused central stimulants, methamphetamine and amphetamine. The results would therefore also be of interest when studying the intake of such drugs and their incorporation into human hair. Selegiline and metabolites were determined by gas chromatography-mass spectrometry, total melanin by spectrophotometry, and pyrrole-tricarboxylic acid by high-performance liquid chromatography with ultraviolet detection. Our results show strong positive exponential relationships (y = e(x)) between melanin and the metabolites, which for methamphetamine improved by normalizing for plasma area under the curve. We conclude that the major metabolites of selegiline can be detected in hair up to four weeks after a single oral dose and that the incorporation closely relates to the melanin contents.

Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

Codeine concentration in hair after oral administration is dependent on melanin content.

BACKGROUND: Analysis of drugs in hair has been used on a qualitative basis to estimate earlier exposure to drugs. Clinical applications are rare because of the lack of dose-response relationships in the studies performed to date, and questions remain regarding the mechanisms of drug incorporation into hair. Several human studies have shown differences in drug accumulation between pigmented and nonpigmented hair. However, the melanin concentration in hair was not determined and correlated to the amount of drug incorporated. METHODS: Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography-mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography-mass spectrometry, respectively. RESULTS: There was an exponential relationship between codeine and melanin concentrations in hair, (r(2) = 0.95 with total melanin and r(2) = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r(2) = 0.86 for codeine vs total melanin and r(2) = 0.90 vs eumelanin. CONCLUSIONS: Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.

Incidence of opiates, amphetamines, and cocaine in hair and blood in fatal cases of heroin overdose.

The purpose of the present study was to investigate the occurrence in hair, of some drugs of abuse in deaths caused by heroin overdose, in comparison to findings in blood. Blood, urine and hair samples were obtained during routine post mortem examinations. Samples were analysed for amphetamines, opiates, and cocaine. Immunometric drug screening was performed in urine and positive results confirmed with gas chromatography-mass spectrometry (GC-MS) of blood samples. All hair samples were analyzed with GC-MS. Hair samples were either incubated with methanol for determination of opiates and cocaine, or dissolved in sodium hydroxide for determination of amphetamines. All 19 blood samples were positive for morphine (0.04-0.4 microgram g-1) and ten were also positive for 6-acetylmorphine (0.003-0.02 microgram g-1). Thirteen of the hair samples were positive for 6-acetylmorphine and seven of which were positive also for morphine. Concentrations ranged from 0.3-7.4 and 0.3-1.3 (ng mg-), respectively. Amphetamine was found in three blood samples (0.04-1.2 micrograms g-1) and in eleven hair samples (0.4-18.3 ng mg-). Cocaine was determined in one blood sample (0.03 microgram g-1) and two hair samples (0.7-6.5 ng mg-). Out of the nineteen cases studied, eight showed chronic multi drug use on the basis of the results of hair analysis. In six subjects no opiates could be detected in hair, suggesting; "first" or occasional intake of heroin, which could be a contributing factor to the overdose death, because of lack of tolerance. We conclude that analysis of hair can be a useful complement to analysis of more conventional autopsy material, especially when investigating overdose deaths and previous histories of drug use and abuse.

A cluster of fentanyl-related deaths among drug addicts in Sweden.

During a 16-month period, nine fatalities occurred among white male drug-addicts, where fentanyl was detected at postmortem toxicological analysis. The street samples associated with these cases confirmed the presence of fentanyl as an additive in low-concentration amphetamine powders with caffeine, phenazone and sugar as cutting agents. In seven of the cases, an acute intoxication by fentanyl was considered to be the immediate cause of death, and in one case, it was likely, but no analysis of fentanyl was performed in blood, and in another case the death was suicide by hanging. This appears to be the first report of a cluster of fentanyl-related deaths outside the United States, and the occurrence of fentanyl in combination with amphetamine has not previously been reported. In addition, in all cases, femoral blood was collected, and samples were handled and analysed according to standardized, quality-controlled procedures. The previous history, circumstances surrounding the death, autopsy findings, histology and toxicology examination of each case are presented. The gas chromatographic-mass spectrometric method for fentanyl is also described. Fentanyl concentrations ranged from 0.5 to 17 ng g-1 blood, and from 5 to 160 ng ml-1 urine. Other drugs found were amphetamine (8 cases), ethanol (5 cases) and benzodiazepines (5 cases). Morphine was found in only one case. The average age of men was 33.9 years (range 22-44); six were found in their own of friend's apartment, two inside buildings (stairways) and one was found outdoors. We conclude that fentanyl is a dangerous substance that should be considered in drug-addict deaths even outside the United States, particularly when the remaining toxicology is unremarkable, and the cause of death cannot be ascertained

Identification of N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in urine from drug users.

The psychoactive agent N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) was identified in urine samples from ten people suspected of petty drug offences. MBDB is a selective serotonin-releasing agent and the alpha-ethyl homologue of 3, 4-methylenedioxymethamphetamine (MDMA). The presence of MBDB was confirmed by comparing the mass spectra of the urine samples with the mass spectrum of authentic reference substance. Quantitation of MBDB, MDMA, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDE) was performed with gas chromatography-mass spectrometry with trifluoroacetic anhydride as the derivatizing agent. Concentrations of MBDB in urine specimens ranged from 0.1-24 micrograms/mL. The demethylated metabolite of MBDB, 3,4-(methylenedioxyphenyl)-2-butanamine (BDB), was also detected in the urine specimens. In seven of the ten samples, MBDB and BDB were the only ecstasy analogues found, but the samples were positive for several other drugs. This appears to be the first report on MBDB as an abused drug in Sweden.

Determination of Phenmetrazine in urine by gas chromatography-mass spectrometry.

A sensitive and simple gas chromatographic--mass spectrometric method is described for the determination of the central nervous system (CNS) stimulant phenmetrazine in urine. The extraction and derivatization were combined into a single step with isooctane and methyl chloroformate. The limit of quantitation was 0.05 micrograms/mliters urine, and the method was linear up to 100 micrograms/mliters. The coefficients of variation (CV) for within-day runs were 1.2% and 2.4% (n = 5) for two controls containing 1.0 micrograms/mliters and 50 micrograms/mliters, respectively. During a six-month period, the same controls showed CVs of 9.1% and 8.7%, respectively (n = 40), indicating a somewhat lower between-run precision. Phenmetrazine was present in 83 out of 3000 urine samples that were screened for CNS stimulants during this period, and the concentrations ranged from 0.5-370 micrograms/mliters.

A convenient derivatization method for the determination of amphetamine and related drugs in urine.

The most commonly abused CNS stimulant in Sweden is amphetamine followed by phenmetrazine. Methamphetamine and phentermine are rarely seen but still of interest. This paper describes a rapid and sensitive method for the analysis of amphetamine, methamphetamine, phentermine, and phenmetrazine in urine using gas chromatography with nitrogen sensitive detection (GC-NPD). The method also qualitatively determines ephedrine and norephedrine. The derivatization was carried out at room temperature with methyl chloroformate to form the corresponding carbamates. Other chloroformate analogues were also tested. Because methyl chloroformate is relatively stable in the presence of water the extraction and derivatization were combined in one step. A concentration step was not necessary to achieve sufficient sensitivity. The recovery was more than 83% for all analytes. The LOQ was 0.05, 0.03, 0.07 and 0.01 (microgram/mL urine) for amphetamine, methamphetamine, phentermine and phenmetrazine respectively. The cut-off was set at 0.2 microgram/mL. The within-day and between-day relative standard deviation (RSD) for amphetamine were 2.2% (n = 9) and 4.7% (n = 5) respectively. There was a good quantitative correlation (r2 = 0.995) between GC-NPD using chloroformate derivatives and gas chromatography-mass spectrometry (GC-MS) using trifluoroacetic anhydride (TFA) as derivatizing agent for the determination of amphetamine in authentic samples.