Development and validation of a method for quantification of human insulin and its synthetic analogues in plasma and post-mortem sera by LC-MS/HRMS.

Analysis of human insulin and its synthetic analogues is increasingly requested for clinical monitoring, for anti-doping purposes, but also for forensic cases. Indeed, insulin analogues may be abused for suicide or homicide - whence their forensic interest. Collection and storage conditions, as well as the phenomenon of degradation make post-mortem serum samples analytically challenging and consequently, the rate of exogenous insulin administration as cause of death is undoubtedly underestimated. However, with recent technological advances and the development of new extraction techniques particularly for anti-doping analyses, detection of insulins in post-mortem samples seems to be achievable. This study describes the first validated quantitative method for analysis human insulin and its six analogues (lispro, aspart, glulisine, glargine, detemir and degludec) in plasma and post-mortem sera. Various extraction processes, namely precipitation + solid phase extraction (SPE), filtration + SPE, precipitation + SPE + immunopurification, and filtration + immunopurification, were assessed to evaluate the lowest limit of detection for all target analogues. The selected sample preparation consists of filtration step followed by immunopurification extraction with an anti-body precoated ELISA plate for plasma. For post-mortem sera, the first step of precipitation was added to remove matrix interferences. The extracts were analyzed by ultra-high-performance liquid chromatography-high resolution mass spectrometry (LC-HRMS), interfaced by electrospray (ESI). The method was validated with respect linearity, precision, accuracy, recovery, matrix effect, dilution and carryover. The limit of quantification (LOQ) in plasma was 0.5 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. Two types of post-mortem sera were studied based on the post-mortem interval (PMI): inferior or superior to 48 h. The obtained LOQ were the same for each analogue, independent from the PMI: 1.0 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. At the LOQ level, for all insulins and all samples, accuracy was between 70 and 130% and precision inferior to 30%. The validated method was applied to five subjects participating in therapeutic monitoring of insulin and to seven post-mortem cases.

Helium poisoning: new procedure for sampling and analysis.

An increasing number of suicidal asphyxiation with a plastic bag with inert gases, and in particular helium (He), have been reported from numerous countries over the last decade. These cases are differently managed and lead to different and variable interpretations. Based on the 12 last cases analysed in the laboratory and on the review of the most recent literature about this topic, updated autopsy guidelines for sampling have been proposed regarding to the samples choice and analytical challenges required by the gaseous state of this substance. Biological samples from airways (lungs lobe) followed by brain and cardiac blood are the best matrices to take during the autopsy to diagnose He exposure. Gaseous samples from trachea, pulmonary bronchi, gastric and cardiac areas are also recommended as alternative samples. The anatomical site of sampling must be carefully detailed, and to this end, forensic imaging constitutes a beneficial tool. Even if He detection is sufficient to conclude to He exposure, He concentrations in samples may be related to He exposure conditions (duration, breathing rate, etc.). A quantification in biological samples could be helpful to document more precisely the case. He concentrations in gaseous samples are reported up to 6.0 µmol/mL (tracheal gas), 2.4 µmol/mL (pulmonary gas), 0.64 µmol/mL (cardiac gas) and 12 µmol/mL (gastric gas). He concentrations in solid/liquid samples are reported up to 28 µmol/g (lungs) and 0.03 µmol/g (cardiac blood). The other matrices usually sampled during autopsy such as urine, peripheral blood, liver, fat matter and kidney appear as not relevant.

State of the art in bile analysis in forensic toxicology.

In forensic toxicology, alternative matrices to blood are useful in case of limited, unavailable or unusable blood sample, suspected postmortem redistribution or long drug intake-to-sampling interval. The present article provides an update on the state of knowledge for the use of bile in forensic toxicology, through a review of the Medline literature from 1970 to May 2015. Bile physiology and technical aspects of analysis (sampling, storage, sample preparation and analytical methods) are reported, to highlight specificities and consequences from an analytical and interpretative point of view. A table summarizes cause of death and quantification in bile and blood of 133 compounds from more than 200 case reports, providing a useful tool for forensic physicians and toxicologists involved in interpreting bile analysis. Qualitative and quantitative interpretation is discussed. As bile/blood concentration ratios are high for numerous molecules or metabolites, bile is a matrix of choice for screening when blood concentrations are low or non-detectable: e.g., cases of weak exposure or long intake-to-death interval. Quantitative applications have been little investigated, but small molecules with low bile/blood concentration ratios seem to be good candidates for quantitative bile-based interpretation. Further experimental data on the mechanism and properties of biliary extraction of xenobiotics of forensic interest are required to improve quantitative interpretation.

Using bone marrow matrix to analyze meprobamate for forensic toxicological purposes.

Bone marrow (BM) analysis is of forensic interest for postmortem toxicological investigations where blood samples are unavailable or unusable. Due to the lack of studies, it remains difficult to interpret concentrations of xenobiotics measured in this matrix. Based on a statistical approach published previously to interpret meprobamate concentrations in bile and vitreous humor, we propose here a diagnostic test for interpretation of BM meprobamate concentrations from analysis of 99 sets of autopsy data. The mean age was 48 years (range 18-80 years, one unknown) for males and 50 years (range 19-80 years, one unknown) for females, with a male/female ratio at 0.768. A BM concentration threshold of 11.3 µg/g was found to be statistically equivalent to that of a blood meprobamate concentration threshold of 50 µg/ml in distinguishing overdose from therapeutic use. The intrinsic qualities of this diagnostic test were good with sensitivity of 0.82 and specificity of 0.92. Compared to previous tests published with the same objective on vitreous humor and bile, this study shows that BM is a useful alternative matrix to reveal meprobamate overdose when blood, vitreous humor, and bile are not available or unusable.

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.

Postmortem measurement of caffeine in bone marrow: influence of sample location and correlation with blood concentration.

Bone marrow (BM) analysis is of forensic interest in postmortem toxicological investigation in case of limited, unavailable or unusable blood samples. However, it remains difficult to determine whether a drug BM concentration is therapeutic or represents overdose, due to the lack of studies on this alternative matrix. Given the variations in BM composition in the body, sample location was suggested to be a relevant factor in assessing BM concentration. The aim of the present study was to compare postmortem caffeine concentrations in various BM sample locations and secondly to consider the correlation between BM and blood concentrations. Six BM samples (right and left side: proximal and medial femur and 5th rib) and a blood sample were collected from 21 forensic autopsies. Gas chromatography coupled to tandem mass spectrometry was performed. Blood caffeine concentrations ranged from 60 to 7591ng/mL. Femoral and rib BM concentrations ranged from 51 to 6171ng/g and 66 to 7280ng/g, respectively. Blood concentrations were always higher than BM concentrations. As a good correlation was demonstrated between blood and rib BM and between blood and the average of the four femoral BM concentrations, blood caffeine concentrations could be correctly extrapolated from BM concentrations. BM caffeine concentration was found to depend on sample location. Rib BM caffeine concentrations appeared to be systematically greater than averaged femur values and concentrations were much more variable between the 4 femur BM samples than between the 2 ribs. From a practical point of view, for caffeine analysis, rib BM appeared more relevant than femoral BM, which requires multisampling to overcome the concentration variability problem.

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.

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.

A bioassay using the human hepatoblastoma cell line HepG2 for detecting phototoxicity of furocoumarins.

We successfully evaluated the human hepatoblastoma cell line HepG2 as a model to assess phototoxicity of coumarins. Five natural furocoumarins were tested and their phototoxic activities, obtained by measuring cell viability in the presence of UV using the MTT test, were as follows: xanthotoxin (8-MOP) >> heraclenol = trichoclin = imperatorin >> peucedanin, both in growing and confluent cell cultures. This easy-to-perform, miniaturised, quantitative and sensitive method could therefore be used as a primary screening test for phototoxicity of a large number of compounds and plant extracts.

Analysis of corticosteroids in hair by liquid chromatography-electrospray ionization mass spectrometry.

The present study describes a confirmatory method for the quantitative determination in hair of the most common corticosteroids illegaly used as doping agents by athletes. Corticosteroids are extracted from 50 mg of powdered hairs by methanolic extraction follows by a solid-phase extraction on C18 cartridge. After extraction, the dried residue is reconstituted with 50 microl acetonitrile and injected in a liquid chromatograph. Liquid chromatography separation is performed on a reversed-phase C18 column with a binary gradient of formiate buffer pH 3-acetonitrile as mobile phase. Detection is performed with an electrospray ionization mass spectrometer in negative ion and selected-ion monitoring mode. The limits of sensitivity achieved is 0.1 ng/mg in hair. Application to hair sample collected during an antidoping control and comparison to results obtain on urines, collected on the same athletes at the same time, shows the interest and the complementarity of both matrices. Hair analysis could allow the detection of corticosteroids on a large period preceding the control, and the detection of natural corticosteroids administered as pro-drug, like hydrocortisone acetate.

Efficient synthesis of octandrenolone and related dipyranoacetophenones.

Octandrenolone (1) was prepared in high yield by condensation of 2', 4',6'-trihydroxyacetophenone with 3-chloro-3-methylbut-1-yne in the presence of a catalytic amount of copper(I) iodide. Methylation of 1 afforded O-methyloctandrenolone (2). Oxidation of 2 with m-chloroperoxybenzoic acid followed by hydrolysis gave the racemic trans-(+)-1-(9,10-dihydro-9,10-dihydroxy-5-methoxy-2,2,8, 8-tetramethyl-2H,8H-benzo[1,2-b:3,4-b']dipyran-6-yl)ethanone (3), which confirmed the structure of the natural product previously isolated from Melicope erromangensis.

Polyoxygenated flavones from the leaves of comptonella microcarpa

The leaves of Comptonella microcarpa have yielded one alkaloid, dictamnine, and four known polyoxygenated flavonoids, meliternatin, 3,5,8-trimethoxy-6,7-3',4'-dimethylenedioxyflavone, 7-(3-methylbut-2-enyloxy)-3,5,8-trimethoxy-3', 4'-methylenedioxyflavone (3), 7-hydroxy-3,5,8-trimethoxy-3', 4'methylenedioxyflavone. In addition, two new flavonoids were found whose structures were established on the basis of their spectral data as 7-hydroxy-3,5,6,8-tetramethoxy-3',4'-methylenedioxyflavone (1) and 7-(3-methylbut-2-enyloxy)-3,5,6,8-tetramethoxy-3', 4'-methylenedioxyflavone (2).

[Rutacea from New Caledonia. Chemical composition of stem bark of Citrus macroptera Montr]. / Rutacées de Nouvelle-Calédonie. Composition chimique des écorces de Citrus macroptera Montr.

A preliminary screening showed the occurrence of alkaloids only in stem bark of Citrus macroptera Montr. In a first work on this Citrus [only one alkaloid was identified = edulinine]. We report in this paper the isolation of two other alkaloids: (+/-) Ribalinine and Isoplatydesmine and of five aromatic compounds: two derived from cinnamic acid, three identified as syringaldehyde, vanillin and methyl/vanillinate.

[Antifungal activity in vitro of Scutellaria baicalensis Georgi upon cutaneous and ungual pathogenic fungi]. / Activité antifongique in vitro de Scutellaria baicalensis Georgi vis-à-vis de champignons pathogènes cutanés et unguéaux.

The root of Scutellaria baicalensis Georgi (Lamiaceae) is one of the traditional drugs commonly used in the Far East. The extracts obtained by the successive exhaustion in chloroform and in ethyl acetat present clear fungistatic activities in vitro against to some cutaneous and ungual pathogenic fungi, and particularly upon strains of Candida albicans, Cryptococcus neoformans and Pityrosporum ovale. For each of these, its minimum inhibitory concentration is determined. The ethyl acetat extract appears the most interesting because of its efficiency and of its stability. An antifungal substance is found out as the baicalein.

Alkaloids from Esenbeckia pilocarpoides.

A preliminary screening showed the occurrence of alkaloids only in root bark and roots of ESENBECKIA PILOCARPOIDES H. B. K., (Rutaceae). Six alkaloids have been isolated and identified from root bark: one acridone, 1-hydroxy-3-methoxy- N-methyl-acridone; four furoquinolines, maculine, flindersiamine, kokusaginine, kokusagine; the sixth, isomaculine, a furo-4-quinolone, known as a synthetic product, has been isolated for the first time from a natural source.