F. Bevalot

Gas chromatography–tandem mass spectrometry assay for the quantification of four benzodiazepines and citalopram in eleven postmortem rabbit fluids and tissues, with application to animal and human samples

Pharmacokinetic studies and postmortem toxicological investigations require a validated analytical technique to quantify drugs on a large number of matrices. Three-step liquid/liquid extraction with online derivatization (silylation) ahead of analysis by gas chromatography-tandem mass spectrometry was developed and validated on rabbit specimens in order to quantify citalopram and 4 benzodiazepines (diazepam, nordazepam, oxazepam and temazepam) in 11 biological matrices (blood, urine, bile, vitreous humor, liver, kidney, skeletal muscle, brain, adipose tissue, bone marrow (BM) and lung). Since the 11 biological matrices came from the same animal species, full validation was performed on 1 matrix, bone marrow (considered the most complex), while the other 10 underwent partial validation. Due to non-negligible matrix effects, calibration curves were performed on each matrix. Within-day and between-day precision (less than 12.0% and 12.6%, respectively) and accuracy (from 88.9% to 106.4%) were acceptable on BM at both low and high concentrations. Assessment on the other matrices confirmed accuracy and within-day precision (less than 12%, and generally between 85.1% and 114.5%, respectively). The lower limit of quantification of the method was 1ng/g for nordazepam, 5ng/g for citalopram and 10ng/g for oxazepam, diazepam and temazepam. The combination of 3-step extraction and MS/MS detection provided good selectivity in all matrices, including the most lipid-rich. Application to real-case samples showed that the method was sensitive enough to describe distribution patterns in an animal experiment, and specific enough to detect molecules in highly putrefied samples from human postmortem cases.

Fatal intoxication with milnacipran

The antidepressant milnacipran is a double serotonin/noradrenalin reuptake inhibitor. The low reported incidence of intoxication indicates excellent tolerance in comparison with tricyclic and second generation antidepressants. We report a fatal intoxication associating milnacipran, at blood levels (femoral=21.5 mg/l, cardiac=20 mg/l) 40-fold higher than the usual treatment concentration, and six other molecules (fluoxetine, norfluoxetine, sertraline, cyamemazine, nordazepam and oxazepam) at therapeutic levels. To the best of our knowledge, this is the first reported fatal intoxication involving milnacipran.

Toxicologic aspects of deaths due to falls from height

A prospective study of 161 victims of falls from height is reported. The aim was to determine the interest of systematic qualitative and quantitative toxicological analysis in such fatalities. The primary cause of death was suicide (84.5%), followed by accidents (7%) and homicide (1%). In the remaining 7.5%, cause of death was undetermined. In the suicides, there was evidence of psychotropic medicines in 57% of the observations, with a much higher proportion of benzodiazepines and antidepressants in women than in men. Quantitative toxicologic analysis showed overdosing on medication in 16 suicide victims, with toxic levels in 11 of these. Systematic qualitative and quantitative toxicologic analysis made a significant contribution to the diagnosis of suicide by revealing either an unknown psychiatric treatment or a toxic level.

Interpretation of drug concentrations in an alternative matrix: the case of meprobamate in bile

Investigating toxicological causes of death may require alternative matrices when the usual ones are lacking. Whereas forensic toxicology uses bile almost only for xenobiotic screening, a diagnostic test interpreting postmortem bile concentrations of meprobamate is reported. Based on 128 sets of autopsy data, its intrinsic qualities were good, with 0.95 sensitivity and 0.93 specificity. In a French forensic population, the positive and negative predictive factors were 0.90 and 0.97, respectively. It is a useful means of revealing overdoses where blood samples are not available or of confirming blood tests when postmortem redistribution is suspected.

Postmortem Measurement of Human Chorionic Gonadotropin in Vitreous Humor and Bile

Abstract:u2002 Postmortem human chorionic gonadotrophin (HCG) blood assay can confirm postmortem diagnosis of pregnancy or document situations in which HCG levels are elevated. In some cases, however, blood sampling is not possible at autopsy. In this study, HCG was quantified by enzyme‐linked fluorescent assay (ELFA) in the bile (nu2003=u20035), vitreous humor (nu2003=u20034), and postmortem blood (nu2003=u20034) of five pregnant women. There were no false negatives in the pregnant subjects (nu2003=u20035) or false positives in controls (nu2003=u200334), enabling this test to be recommended for routine use in forensic contexts in which the detection of elevated HCG levels could be of interest.

Interpretation of drug concentrations in an alternative matrix: the case of meprobamate in vitreous humor

The use of vitreous humor (VH) as an alternative matrix to blood in the field of forensic toxicology has been described for numerous drugs. Interpretation of drug concentrations measured in VH, as in other matrices, requires statistical analysis of a data set obtained on a significant series. In the present study, two diagnostic tests interpreting postmortem VH concentrations of meprobamate in 117 sets of autopsy data are reported. (1) A VH meprobamate concentration threshold of 28xa0mg/l was statistically equivalent to that of blood meprobamate concentration threshold of 50xa0mg/l distinguishing overdose from therapeutic use in blood. The intrinsic qualities of the test were good, with sensitivity of 0.95 and absolute specificity of 1. (2) A novel interpretation tool is proposed, allowing blood concentration range to be evaluated, with a known probability, from VH concentration.

P48: Death by 4-methylethcathinone (4-MEC) overdose: A case report

Objective 4-methylethcathinone (4-MEC) is a designer cathinone classified as an illegal narcotic since July 2012 in France. This report presents the first fatality related to consumption of 4-MEC in France. Case description A 36 year-old man, in psychotherapy for drug addiction and suicide attempts, was found dead in his apartment. Three empty syringes were found near the corpse. His boyfriend told the police that the deceased was an intravenous user of drugs purchased on the Internet. No obvious cause of death was identified at autopsy. Several marks from needle and a rectal foreign body were observed. Blood, urine, vitreous humor, bile, gastric contents and hair samples were collected. Methods Immunological screening for amphetamine, methamphetamine, opiates, cannabinoids, cocaine, buprenorphine and MDMA was performed on urine. Identification and quantification of major amphetamine derivatives (amphetamine, methamphetamine, MDMA, MDA, MDEA and MBDB) was performed on urine and blood, following the SFTA GC-MS consensus protocol. General unknown screening (GUS) was performed on peripheral blood and gastric contents by GC-MS (spectral libraries: Nist11, Pfleger2011, Wiley, SWDrug) and on central blood by LC/DAD after liquid-liquid extraction. 4-MEC was quantified in blood by GC-MS/MS, using mephedrone-d3 as internal standard. The assay was validated for linearity, selectivity, limits of detection, precision and accuracy at 3 concentration levels. Analysis of ethanol and other volatile compounds was performed using headspace gas chromatography coupled to flame ionization detector. Results Immunological screening was positive for amphetamine, methamphetamine and MDMA. Confirmation analysis for major amphetamine derivatives was negative. Hydroxyzine was quantified at therapeutic concentration (160ng/ml) in blood. 4-MEC was detected on GUS by GC-MS using the SWDrug mass spectra library. The concentration of 4-MEC was 14,600ng/mL in peripheral blood. Quantification in other matrices is ongoing. No other drugs/toxins were found, and screening for ethanol was negative. Analysis of syringes, performed in a different laboratory, detected 4-MEC. Conclusion Reports of use of 4-MEC have mainly described nonfatal intoxications, with blood concentrations about 100-fold lower than in the present case; only one fatal case has been reported in the literature (Rojek S., Meeting of IAFT, Japan, 2012), in which 4-MEC blood concentration was 1,267ng/ml. In the present case, the circumstances of death, autopsy findings and toxicology results, with a 4-MEC concentration of 14,600ng/ml, were consistent with 4-MEC overdose as cause of death.