Can an early diagnostic procedure of metformin-associated lactic acidosis in an emergency unit reduce mortality?

OBJECTIVE: Metformin-associated lactic acidosis (MALA) is a rare but serious adverse reaction with a mortality rate of up to 50%. Unfortunately, diagnosis and care management are often delayed. The objective was to assess the impact on the mortality rate and length of hospital stay of a MALA early diagnosis procedure in diabetic patients with metformin at emergency department (ED) admission. METHOD: From 1/7/2012, a new MALA diagnosis procedure (pH, lactate, metformin) was implemented in all diabetic patients with metformin just after their admission to the ED. The pharmacovigilance staff confirmed the MALA cases (defined as pH≤7.35, lactate concentration>5mmol/L) in patients exposed to metformin and after a causality assessment to eliminate other common causes of lactic acidosis. To assess the impact of this new diagnosis procedure, a before-after study was conducted between two groups: a series of cases with intervention (IG; 1/7/2012-30/6/2013) and a control series of past cases without intervention (CG; 1/1/2011-30/6/2012). The main outcome was the relative reduction of mortality rate and length of hospital stay between the two groups. RESULTS: Thirty-four MALA cases were confirmed in 745 subjects admitted with lactic acidosis, (IG: 12; CG: 22). A higher illness severity score in the IG vs. CG was observed: respectively arterial lactate (14.2±6.9 vs. 8.8±5.8mmol/L, P<0.05), arterial bicarbonate (7.8±4.3 vs. 14.3±6.3mmol/L, P<0.05). The median time up to MALA diagnosis was 20.5 (Q1-Q3: 11.3-38.5) minutes for IG and 55.0 (Q1-Q3: 33.0-132.0) minutes for CG. After procedure implementation, the mortality relative risk reduction was 26.7% (95% CI: -84.3%, 70.8%), and especially 54.2% (95% CI: -265.2%, 94.2%) in the ED. There was no difference in the hospital stay duration between the two groups. CONCLUSION: While the results were not significant, the study suggests that the implementation of a MALA early diagnosis procedure in all patients with metformin admitted to an ED tends to decrease mortality, especially for serious MALA cases detected earlier.

Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology.

Despite a non-invasive sampling, hair samples are generally collected in limited amounts for an obvious esthetic reason. In order to reduce the required quantity of samples, a multianalytes method allowing simultaneous identification and quantification of 35 psychoactive drugs was developed. After incubation of 50 mg of hair in a phosphate buffer pH 5 for one night at room temperature, the substances of interest were extracted by a simple liquid-liquid extraction step, with a dichloromethane/ether mixture (70:30, v/v). After evaporation under a gentle stream of nitrogen and reconstitution in formate buffer (2 mM, pH 3)/acetonitrile (90:10, v/v), twenty microliter were injected into the LC-MS/MS system for a chromatographic run of 29 min using an Atlantis T3 column (150 × 2.1 mm, 3 µm) (Waters Corp, Milford, USA) and a gradient mixture of 2 mM, pH 3.0 ammonium formate, and 2 mM, pH 3.0 ammonium formate/acetonitrile. The data acquisition was performed in scheduled MRM mode. Intra- and inter-day precisions, estimated using the coefficient of variation and relative bias, were lower than 20 % for all concentration levels, except for two compounds. The limits of detection and quantification ranged from 0.5 to 10 pg/mg. After complete validation, this method has been successfully used in several forensic cases, three of which are reported.

Improved liquid chromatography-tandem mass spectrometric method for the determination of ethyl glucuronide concentrations in hair: applications to forensic cases.

Ethyl glucuronide (EtG) is a direct marker of ethanol consumption, and its assay in hair is an efficient tool for chronic alcoholism diagnosis. In 2012, the Society of Hair Testing proposed a new consensus for hair concentrations interpretation, strongly advising the use of analytical methods providing a limit of quantification of less than 3 pg/mg. The present work describes the optimization and validation of a previously developed liquid chromatography-tandem mass spectrometric method in order to comply with this recommendation. The concentration range of this improved method is from 3 to 1,000 pg/mg. Some cases are then described to illustrate the usefulness of hair EtG: a forensic post-mortem case and two cases of suspension of driving licences. Finally, hair samples of some teetotallers (n = 10) have been analyzed, which allowed neither to quantitate nor to detect any trace of EtG.

Determination of dodine in fruit samples by liquid chromatography electrospray ionization-tandem mass spectrometry.

A rapid and sensitive LC/electrospray ionization-MS/MS method has been developed for the determination of dodine in fruit samples. Based on a liquid-liquid extraction of 10 g solid fruit homogenate using an acetone-dichloromethane-hexane mixture and acetate ammonium buffer (pH 4.5), this LC/MS/MS procedure was characterized by recoveries above 50%, with good intra-assay precision (RSD < 13%) and interassay precision (RSD < 18%) for seven different matrixes (apple, apricot, cherry, peach, pear, plum, and quince). This method was validated from 5 to 500 microg/kg according to standard guidelines. Its LOD (1 microg/kg) and LOQ (5 microg/kg) were in accordance with recommendations of the European legislation defined for infant food [maximum residue level (MRL) = 10 microg/kg]. The whole procedure was finally tested on 1022 fruit samples intended for commercialization, both infant food samples and samples not intended in particular for babies. In this study, dodine was detected in 27 samples; none exhibited a concentration higher than the MRL.

Stability of ribavirin concentrations depending on the type of blood collection tube and preanalytical conditions.

Ribavirin pharmacokinetic and exposure effect trials based on either plasma or serum concentrations have yielded diverging results. This study aimed to compare ribavirin concentrations in serum and plasma and to investigate the influence of blood collection and preanalytical conditions on ribavirin concentration stability. Blood samples from patients with hepatitis virus C and receiving ribavirin were collected in plain (dry) tubes, in tubes containing ethylenediaminetetra-acetic acid or lithium-heparinate, in Type II Serum Separating Tubes with clot activator, or Type II lithium heparinate Plasma Separating Tubes. Different time and temperature conditions were tested before and after blood centrifugation. Ribavirin was determined using liquid chromatography-dual mass spectroscopy. Multiple-way analysis of variance was used for statistical analyses. Ribavirin concentrations showed a higher interlaboratory variability in serum than in plasma. Results were fairly stable over 2 hours in whole blood collected in dry or ethylenediaminetetra-acetic acid tubes and very stable up to 24 hours in serum or plasma kept in gel-containing tubes after immediate centrifugation. When Plasma Separating II gel tubes were kept at +4 degrees C or at ambient temperature for up to 24 hours before centrifugation, ribavirin concentrations decreased by 1% to 8% and 12% to 18%, respectively. These results suggest that blood samples should be collected in gel-containing tubes and centrifuged immediately, after which the tubes can be kept at ambient temperature for the next 24 hours. In case of clinical constraints, Plasma Separating II gel tubes can be kept at +4 degrees C for a maximum of 2 hours before centrifugation with limited impact on the measured concentrations.

Screening of pesticides in blood with liquid chromatography-linear ion trap mass spectrometry.

In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS(2) and MS(3) scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS(2) and MS(3)), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 microg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10-100-fold by scanning MS(2) in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS(3) scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.

Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction.

The detection of ethyl-beta-D-6-glucuronide (EtG), a stable phase II metabolite of ethanol, is of interest in both clinical and forensic contexts with the aim of monitoring alcohol abuse. We present a liquid chromatography-electrospray ionisation-tandem mass spectrometry method for the detection and quantification of EtG in hair. Thirty milligrams of washed and cut hair were cleaned up using solid-phase extraction graphite cartridges. Separation was then performed using an Uptisphere-3SI column, and the detection was operated in the negative mode. After validation, the method was applied to hair samples taken from four fatalities (F) with documented excessive drinking habits, 12 heavy drinkers (HD) and seven social drinkers (SD). The method exhibits limits of detection and quantification of 4 and 10 pg/mg, respectively. Intra- and inter-assay standard deviation and relative bias were less than 20% over the calibrating range (10 to 3,000 pg/mg). EtG hair concentrations in SD were <10 pg/mg and >50 pg/mg for F and HD (range, 54 to 497 pg/mg). The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption (EtG hair concentration >50 pg/mg).

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: applications and constraints.

LC coupled to single (LC-MS) and tandem (LC-MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC-MS/MS. Analyses were performed using a previously published LC-MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT-tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their "enhanced" product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.

Pitfalls and prevention strategies for liquid chromatography-tandem mass spectrometry in the selected reaction-monitoring mode for drug analysis.

BACKGROUND: We observed cases of false-positive results with the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Different LC-MS/MS techniques that use the selected reaction-monitoring mode, routinely employed for the analysis and quantification of drugs and toxic compounds in biological matrices, were involved in the false-positive and potentially false-positive results obtained. We sought to analyze the causes of and solutions to this problem. METHODS: We used a previously reported LC-MS/MS general unknown screening method, as well as manual spectral investigation in 1 case, to perform verification and identification of interfering compounds. RESULTS: We observed that false-positive results involved: a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, benzoylecgonine mistaken for atropine, and clomipramine and 3 phenothiazines that share several common ion transitions. CONCLUSIONS: To prevent problems such as those we experienced, we recommend the use of stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, with an experience-based tolerance of +/-15% for transitions with a relative abundance >10% and with an extension to +/-25% for transitions <10% when the concentration is at the limit of quantification. A powerful general unknown screening procedure can help to confirm suspected interferences. Our results indicate that the specificity of screening procedures is questionable for LC-MS/MS analyses performed in the selected reaction-monitoring mode and involving a large number of compounds with only 1 transition per compound.

A fatal case of bupropion (Zyban) overdose.

A sensitive and reproducible method for the identification and the quantitative determination of bupropion (BUP) and its major metabolites, hydroxybupropion (OH-BUP) and threohydrobupropion (T-BUP), was developed in blood and urine. The three compounds were extracted with a solid-phase extraction procedure followed by LC-ESI-MS-MS separation and quantification using decadeuterated lidocaine as internal standard. BUP and its metabolites were satisfactorily identified by multiple reactions monitoring detection. The limits of detection and quantification were determined at 5 and 10 microg/L, respectively, for each analyte. The intraday and interday coefficients of variability were lower than 11.9% for BUP and its metabolites. This method was applied to the forensic case of a 35-year-old male who died after a suspected ingestion of 30 slow-release tablets of Zyban. As samplings were performed at least 72 h after the drug intake, BUP had disappeared from blood, but OH-BUP and T-BUP were present at the concentrations of 5.8 and 30.4 mg/L, respectively. In urine, concentrations ranged from 42.9 mg/L for BUP to 617 mg/L for T-BUP. These results agree with the hypothesis of a successful suicide attempt.

Identification of acepromazine in hair: an illustration of the difficulties encountered in investigating drug-facilitated crimes.

After a drug-facilitated sexual assault (DFSA), a woman was found in a drowsy state at home. She remembered having drunk an unknown beverage by the accused. Blood samples (collected 8 hours after the DFSA), two glasses, and a teaspoon seized by the police were analyzed. Acepromazine, a phenothiazine tranquilizer used in human and veterinary medicine, was detected in the residue of one of the glasses. In spite of acepromazine absence in the victim's blood, the possible use of acepromazine in the DFSA was reported to the police. Two weeks later, a suspect admitted having orally administered acepromazine to the victim. Using a liquid chromatography-tandem mass spectrometry method, this compound was subsequently detected (31 pg/mg) in a sample of the victim's hair collected a month and a half after the DFSA. A potential short elimination half-life in humans and/or the well-known in vitro degradation of acepromazine could explain the negative blood result. DFSA toxicological investigations are challenging and can be complicated when a rather unusual substance is concerned. In particular, special care should be taken when interpreting the results, taking into account elimination and/or instability data, when available.

LC-MS/MS systematic toxicological analysis: comparison of MS/MS spectra obtained with different instruments and settings.

OBJECTIVES: To compare the mass spectra obtained using a linear-ion-trap (LIT) tandem mass spectrometer (QTRAP) operated in the "enhanced product ion scan" (EPI) mode with those obtained in the classical triple-quadrupole product ion scan (PIS) mode run on the same as well as on two other instruments (TSQ-Quantum and Quattro-Micro). DESIGN AND METHODS: After tentative standardization of ion fragmentation and transmission in both polarities using a reference compound (glafenine) on the three instruments, eight test compounds detected in the positive mode and five in the negative mode were systematically infused in different ionization sources and spectral acquisition performed over approximately 5 s. The relative intensity of the ions present in the resulting spectra was quantitatively and statistically compared. Also, the intra-day and inter-day variabilities of these relative intensities, as well as the effect of increasing compound concentration, were studied using QTRAP operated in EPI mode. RESULTS: The EPI and PIS modes operated on a single LIT MS/MS instrument resulted in significant differences in relative ion intensities in both polarities, and so did the other two instruments despite prior standardization with glafenine. Some fragments could be absent in certain spectra, but no unexpected or unique fragments showed up. Intra-day variability was smaller in the LIT EPI than in the regular PIS mode and in the positive than in the negative polarity. In EPI mode, both intra- and inter-day variabilities increased when the relative intensity decreased. The effect of increasing concentration on the relative intensity of major and minor ions was small but significant in both polarities. Finally, contamination and cleansing of the ionization source also had noticeable effects on MS/MS spectra, though the cause is unclear. CONCLUSIONS: MS/MS spectra do not offer the expected inter-instrument reproducibility despite an attempt at standardizing the fragmentation conditions using a reference compound. However, although the inter-instrument differences in ion relative intensity were significant, the spectra obtained looked almost similar. This suggests that in library searching algorithms, higher weight should be assigned for the m/z ratios than for their relative intensity in the spectra.

Bayesian estimation of methotrexate pharmacokinetic parameters and area under the curve in children and young adults with localised osteosarcoma.

BACKGROUND: Methotrexate is the most efficient anticancer drug in osteosarcoma. It requires individual exposure monitoring because of the high doses used, its wide interpatient pharmacokinetic variability and the existence of demonstrated relationships between efficacy, toxicity and serum drug concentrations. OBJECTIVE: To develop a maximum a posteriori (MAP) Bayesian estimator able to predict individual pharmacokinetic parameters and exposure indices such as area under the curve (AUC) for methotrexate from a few blood samples, in order to prevent toxicity and facilitate further studies of the relationships between efficacy and exposure. METHODS: Methotrexate population pharmacokinetics were estimated by a retrospective analysis of concentration data from 40 children and young adults by using the nonparametric expectation maximisation method NPEM. A linear two-compartment model with elimination from the central compartment was assumed. Individual pharmacokinetic parameters and AUC were subsequently estimated in 30 other young patients, using MAP Bayesian estimation as implemented in two programs, ADAPT II and an inhouse program Winphar((R)). RESULTS: The pharmacokinetic parameters used in the model were the volume of the central compartment (V(1)) and the transfer constants (k(10), k(12) and k(21)). The mean values (with percentage coefficient of variation) obtained were: 18.24L (54.1%) and 0.41 (42.3%), 0.0168 (68.7%), and 0.1069 (61.3%) h(-1), respectively. Bayesian forecasting enabled nonbiased estimation of AUC and systemic clearance using a schedule with two sampling times (6 and 24 hours after the beginning of the infusion) and either program. Collection of a third sample at 4 hours improved the precision. CONCLUSION: The Bayesian adaptive method developed herein allows accurate estimation of individual exposure to methotrexate and can easily be used in clinical practice.

Maximum a posteriori Bayesian estimation of oral cyclosporin pharmacokinetics in patients with stable renal transplants.

OBJECTIVE: To develop a maximum a posteriori probability (MAP) Bayesian estimator for the pharmacokinetics of oral cyclosporin, based on only three timepoints, and evaluate its performance with respect to a full-profile nonlinear regression approach. PATIENTS: 20 adult patients with stable renal transplants given orally administered microemulsified cyclosporin and mycophenolate. METHODS: Cyclosporin was assayed by liquid chromatography-mass spectrometry. Nonlinear regression and MAP Bayesian estimation were performed using a home-made program and a previously designed pharmacokinetic model including an S-shaped absorption profile described by a gamma distribution. OUTCOME MEASURES AND RESULTS: MAP Bayesian estimation using the best limited sampling strategy (before administration, and 1 and 3 hours after administration) was compared with nonlinear regression (taken as the reference method) for the prediction of the different pharmacokinetic parameters and exposure indices. Median relative prediction error was -0.49 and -3.42% for area under the concentration-time curve over the administration interval of 12 hours (AUC12) and estimated peak drug concentration (Cmax), respectively (nonsignificant). Relative precision was 2.00 and 4.32%, and correlation coefficient (r) was 0.985 and 0.955, for AUC12 and Cmax, respectively. CONCLUSION: This paper reports preliminary results in a stable renal transplant patient population, showing that MAP Bayesian estimation can allow accurate prediction of AUC12 and Cmax with only three samples (0, 1 and 3 hours). Although these results require confirmation by further studies in other clinical settings, using other drug combinations, other analytical methods and commercially available pharmacokinetic software, the method seems promising as a tool for the therapeutic drug monitoring of cyclosporin in clinical practice or for exposure-controlled studies.

Evaluation of an improved general unknown screening procedure using liquid chromatography-electrospray-mass spectrometry by comparison with gas chromatography and high-performance liquid-chromatography--diode array detection.

This paper presents an improved, comprehensive liquid chromatography-electrospray-mass spectrometry (LC-ES-MS) general unknown screening (GUS) procedure for drugs and toxic compounds and its comparison with conventional techniques in routine laboratory conditions. Chromatographic separation involved an X-TERRA MS C18, 3.5 microm (100 mm x 1 mm i.d.) column together with a 25-min long gradient of acetonitrile in pH 3, 2 mM ammonium formate delivered at a 50 microl/min flow rate. Two different in-source collision-induced dissociation voltages were alternated, both in the positive and in the negative ion modes. Reconstructed spectra were then obtained in both polarities by adding up spectra obtained with low and high energy, resulting in spectra presenting a sufficient number of specific fragment ions for unambiguous and fast identification of compounds. Two large mass spectral libraries of drugs and toxic compounds were built and an efficient automated signal processing, library searching and report editing algorithm developed. Using a common, efficient solid-phase extraction procedure, this LC-ES-MS technique was compared to GC-MS and HPLC-DAD GUS procedures for the identification of a priori unknown compounds in 51 serum samples consecutively sent to the laboratory for GUS. The present LC-MS method identified 75% of the compounds contained in these samples (versus 66% for GC-MS and 71% for HPLC-DAD), including 8% that the other two techniques failed to identify (versus 8% for GC-MS and 9.5% for HPLC-DAD). Therefore, it is complementary to GC-MS and/or HPLC-DAD and helps enlarge the range of drugs detected in clinical toxicology. It could be useful as well in forensic toxicology to confirm a positive result, as 38% of all the compounds were detected by the three techniques and 36% by two of them.

Determination of three beta-blockers in biofluids and solid tissues by liquid chromatography-electrospray-mass spectrometry.

A LC-MS method using clenbuterol as internal standard was developed and validated for three b-blockers (BB) (atenolol, metoprolol, and propranolol) in rabbit postmortem matrices: heart, lung, kidney, liver, brain, blood, vitreous humor, gastric liquid, and urine. The BB were extracted from 2.0 mL of biofluids or 200 mg of solid tissues (after grinding and homogenization) by liquid-liquid extraction using Extrelut columns. Chromatographic separation involved a Nucleosil C18 (150 mm x 1-mm i.d., 5 microm) column together with a gradient of acetonitrile in 2mM, pH 3 ammonium formate. The compounds were ionized in the ionspray source of the atmospheric pressure mass spectrometer and fragmented by in-source collisions. The fragment ions were detected in the positive selected ion monitoring mode, targeting one quantitation and two confirmation ions per compound. The extraction recovery ranged between 10 and 40%, depending on the matrices. The limits of quantitation were 50 ng/g in tissues, 50 microg/L in blood and urine, and 10 microg/L in vitreous humor. Indeed, as preliminary results in one rabbit administered 5 mg/kg of each BB showed that BBs were more concentrated in some postmortem organs, validation was performed in the relevant concentration area in these particular tissues. The technique was found to be linear between 50 ng/g and 5000 ng/g for heart and liver, between 50 microg/L and 5000 microg/L for urine extracts, between 1000 ng/g and 50 000 ng/g for lung and kidney, and between 500 microg/L and 5000 microg/L for gastric content. A quadratic equation best fitted the calibration curve in blood between 50 microg/L and 5000 microg/L, as well as in brain between 50 ng/g and 40,000 ng/g. The correlation coefficients were all higher than 0.997. Intra- and interassay precision and accuracy fulfilled the international requirements. This simple and sensitive assay was applied to the determination of three BB in the biofluids and tissues of a rabbit as part of a preliminary step of a postmortem redistribution study and is also suitable for the routine determination of BB in forensic investigations.

Identification and quantitation of six non-depolarizing neuromuscular blocking agents by LC-MS in biological fluids.

A liquid chromatography-electrospray-mass spectrometry technique for the screening and determination of five non-depolarizing neuromuscular blocking agents (NDBAs), atracurium and its product of degradation/metabolite laudanosine, rocuronium, pancuronium, vecuronium, and mivacurium has been developed using ambenonium as the internal standard (I.S.). Samples were acidified upon reception by adding 20 micro L 0.5M H(2)SO(4) to 500 micro L of biofluid. Sample preparation consisted of simple blood purification and/or protein precipitation using 1 mL I.S. in acetonitrile. Chromatographic separation was carried out on an X-TERRA trade mark column along with a gradient of acetonitrile in 2mM ammonium formate (pH 3). Detection was carried out in the positive selected ion monitoring mode, targeting one quantitation ion and one confirmation ion per compound. The limit of quantitation was 2.5 micro g/L for mivacurium and laudanosine, 5 micro g/L for rocuronium and pancuronium and 10 micro g/L for atracurium and vecuronium in serum (i.e., in the range of, or less than, therapeutic levels). The technique was found to be linear between the respective LOQs and 2000 micro g/L, with correlation coefficients higher than 0.999 in all matrices. Intra- and interday precision and accuracy in serum fulfilled the international criteria. This method was employed for the investigation of a case of suicide by infusion of drugs. Laudanosine, the metabolite or degradation product of both atracurium and cisatracurium, and rocuronium were found in urine and whole blood, at supratherapeutic concentrations in the latter (rocuronium: 1.53 and 2.18 mg/L, laudanosine: 8.86 and 0.31 mg/L, respectively), and even therapeutic concentrations would have been lethal in the absence of respiratory assistance.

Analytical aspects of volatile substance abuse (VSA).

Through a case report, the authors illustrate the volatile substance abuse (VSA) toxicological investigation difficulties mainly due to evaporation of the compounds from postmortem samples and to the lack of reference data for interpretation. A 17-year-old man, student in a chemistry institute, was found dead with a plastic bag placed over his head. Several chemical substances were found in his belongings. Autopsy findings included serious pulmonary lesions and hemorrhagic digestive ulcerations. A large screening of drugs and toxic compounds and selective analyses for several classes of drugs of abuse were carried out in the autopsy samples. In particular, a headspace (HS), -gas chromatography/-mass spectrometry (GC/MS) technique was used to screen for volatile substances and metabolites in the biological samples and for residues of volatile substances on the surface of the plastic bag and in the chemicals found on the scene. The main analytical finding was the presence of alkanes (heptane, methyl-2-pentane, methyl-3-hexane, methylcyclohexane) in the gastric content. The literature data, VSA practices, long time-delay between death and autopsy, preservation conditions of the biological samples before analysis, and in-lab experiments on evaporation of volatile substances were considered to interpret this result. The present fatality was attributed to VSA with a gasoline-based stain remover like "eau écarlate," associated with a hypoxic recreation practice using a plastic bag.

Analytical findings of an acute intoxication after inhalation of methoxetamine.

Methoxetamine (MXE) is increasingly used and abused, as it is frequently presented as being safer than ketamine, and legal. Cases of only MXE consumption being associated with the occurrence of seizures are rarely reported. A single MXE intoxication case by inhalation is described concerning a 21-year-old man, not known to be epileptic, who was found collapsed in his bedroom, supposedly after an epileptic seizure. He was transferred to the Emergency Department of the Henri Mondor Hospital, Aurillac, France. He was conscious, but with a sinus bradycardia (48/min) and an ST-segment elevation on the electrocardiogram, and a slightly increased creatine kinase level (270 U/L) and hyponatremia (127 mmol/L). New seizure activity occurred during hospitalization, but the clinical course in the intensive care unit was favorable. Quantitation of MXE in serum and urine using gas chromatography coupled to mass spectrometry (GC-MS) was developed, as well as a liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) method for the determination of MXE in hair. Limits of detection and quantification were, respectively, 2 and 10 µg/L for the GC-MS method and both 0.5 pg/mg for the LC-MS-MS method. Concentrations of 30 and 408 µg/L were, respectively, measured in serum and urine. Concentrations of 135 and 145 pg/mg were detected in two 2.5 cm hair strands, consistent with one or several consumptions during the 2 ½ months prior to sampling. A sample of the powder consumed was available and also analyzed. This case illustrates the dangers of this drug, which justify its classification as a narcotic in France since August 2013.