Mass spectrometry detection of organophosphorus pesticide adducts on butyrylcholinesterase and albumin.

This study established a method to prepare and detect OPs adducts on butyrylcholinesterase (BChE) and human serum albumin (HSA). OPs (methyl paraoxon, ethyl paraoxon, methyl parathion, parathion) were incubated with BChE or HSA in vitro, and the adducts of OPs-BChE or OPs-HSA were prepared and qualitatively analyzed by ultra-performance liquid chromatography data-dependent high-resolution tandem mass spectrometry (UPLC-ddHRMS/MS). The amounts of BChE and HSA in the incubating systems were varied and the resulting amounts of the adducts were determined using linear regression. OPs-BChE in the blood were isolated by immunomagnetic separation (IMS), and then digested into the OPs-nonapeptide adduct by pepsin. The proteins in the remaining blood plasma were precipitated and digested by pronase to OPs-tyrosines(OPs-Tyr), which were quantified by UPLC-ddHRMS/MS. 4 OPs-nonapeptides and 4 OPs-Tyr adducts were obtained through the process above. The relative mass deviation of incubated adducts between the actual and theoretical exact masses was less than 10 ppm, and further confirmed by fragmentation mass spectra analysis. Calibration curves were linear for all adducts with a coefficient of determination value (R2) ≥0.995. The limits of detection (LOD) and limits of quantification (LOQ) for adducts detected by MS ranged from 0.05 to 1.0 ng/mL, and from 0.1 to 2.0 ng/mL, respectively. The recovery percentages for adducts ranged from 76.1 % to 107.1 %, matrix effects ranged from 83.4 % to 102.1 %. The inter-day and intra-day precision were 6.1-10.1 % and 6.9-12.9 % for adducts. This study provides a new reference method for the detection of organophosphorus pesticide poisoning. In addition, two blood samples with organophosphorus poisoning were tested by the designed method, and the corresponding adducts were detected in both samples.

Postmortem Diffusion of Aconitum Alkaloids and Their Metabolites in Rabbits.

OBJECTIVES: To explore the postmortem diffusion rule of Aconitum alkaloids and their metabolites in poisoned rabbits, and to provide a reference for identifying the antemortem poisoning or postmortem poisoning of Aconitum alkaloids. METHODS: Twenty-four rabbits were sacrificed by tracheal clamps. After 1 hour, the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration. Then, they were placed supine and stored at 25 ℃. The biological samples from 3 randomly selected rabbits were collected including heart blood, peripheral blood, urine, heart, liver, spleen, lung and kidney tissues at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h after intragastric administration, respectively. Aconitum alkaloids and their metabolites in the biological samples were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: At 4 h after intragastric administration, Aconitum alkaloids and their metabolites could be detected in heart blood, peripheral blood and major organs, and the contents of them changed dynamically with the preservation time. The contents of Aconitum alkaloids and their metabolites were higher in the spleen, liver and lung, especially in the spleen which was closer to the stomach. The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration. In contrast, the contents of Aconitum alkaloids and their metabolites in kidney were all lower. Aconitum alkaloids and their metabolites were not detected in urine. CONCLUSIONS: Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits, diffusing from high-content organs (stomach) to other major organs and tissues as well as the heart blood. The main mechanism is the dispersion along the concentration gradient, while urine is not affected by postmortem diffusion, which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

PURPOSE: A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). METHOD: Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. RESULTS: The method attended method validation criteria. The limits of detection were equal or lower than 2.0 ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0 ng/mL, from 0.1 to 5.0 ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24 °C for 24 h, all analytes were stable at -20 °C for 14 days and at -80 °C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24 h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. CONCLUSIONS: A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Rapid Screening of 34 Emerging Contaminants in Surface Water by UHPLC-Q-TOF-MS.

OBJECTIVES: To establish a rapid screening method for 34 emerging contaminants in surface water by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS). METHODS: The pretreatment conditions of solid phase extraction (SPE) were optimized by orthogonal experimental design and the surface water samples were concentrated and extracted by Oasis® HLB and Oasis® MCX SPE columns in series. The extracts were separated by Kinetex® EVO C18 column, with gradient elution of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution. Q-TOF-MS 'fullscan' and 'targeted MS/MS' modes were used to detect 34 emerging contaminants and to establish a database with 34 emerging contaminants precursor ion, product ion and retention times. RESULTS: The 34 emerging contaminants exhibited good linearity in the concentration range respectively and the correlation coefficients (r) were higher than 0.97. The limit of detection was 0.2-10 ng/L and the recoveries were 81.2%-119.2%. The intra-day precision was 0.78%-18.70%. The method was applied to analyze multiple surface water samples and 6 emerging contaminants were detected, with a concentration range of 1.93-157.71 ng/L. CONCLUSIONS: The method is simple and rapid for screening various emerging contaminants at the trace level in surface water.

Toxicokinetics of MDMA and Its Metabolite MDA in Rats.

OBJECTIVES: To investigate the toxicokinetic differences of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and its metabolite 4,5-methylene dioxy amphetamine (MDA) in rats after single and continuous administration of MDMA, providing reference data for the forensic identification of MDMA. METHODS: A total of 24 rats in the single administration group were randomly divided into 5, 10 and 20 mg/kg experimental groups and the control group, with 6 rats in each group. The experimental group was given intraperitoneal injection of MDMA, and the control group was given intraperitoneal injection of the same volume of normal saline as the experimental group. The amount of 0.5 mL blood was collected from the medial canthus 5 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h after administration. In the continuous administration group, 24 rats were randomly divided into the experimental group (18 rats) and the control group (6 rats). The experimental group was given MDMA 7 d by continuous intraperitoneal injection in increments of 5, 7, 9, 11, 13, 15, 17 mg/kg per day, respectively, while the control group was given the same volume of normal saline as the experimental group by intraperitoneal injection. On the eighth day, the experimental rats were randomly divided into 5, 10 and 20 mg/kg dose groups, with 6 rats in each group. MDMA was injected intraperitoneally, and the control group was injected intraperitoneally with the same volume of normal saline as the experimental group. On the eighth day, 0.5 mL of blood was taken from the medial canthus 5 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h after administration. Liquid chromatography-triple quadrupole tandem mass spectrometry was used to detect MDMA and MDA levels, and statistical software was employed for data analysis. RESULTS: In the single-administration group, peak concentrations of MDMA and MDA were reached at 5 min and 1 h after administration, respectively, with the largest detection time limit of 12 h. In the continuous administration group, peak concentrations were reached at 30 min and 1.5 h after administration, respectively, with the largest detection time limit of 10 h. Nonlinear fitting equations for the concentration ratio of MDMA and MDA in plasma and administration time in the single-administration group and continuous administration group were as follows: T=10.362C-1.183, R2=0.974 6; T=7.397 3C-0.694, R2=0.961 5 (T: injection time; C: concentration ratio of MDMA to MDA in plasma). CONCLUSIONS: The toxicokinetic data of MDMA and its metabolite MDA in rats, obtained through single and continuous administration, including peak concentration, peak time, detection time limit, and the relationship between concentration ratio and administration time, provide a theoretical and data foundation for relevant forensic identification.

Melatonin attenuates fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice.

Melatonin is a neurohormone synthesized by the pineal gland to regulate the circadian rhythms and has proven to be effective in treating drug addiction and dependence. However, the effects of melatonin to modulate the drug-seeking behavior of fentanyl and its underlying molecular mechanism is elusive. This study was designed to investigate the effects of melatonin on fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice. The accompanying changes in the expression of Brain and Muscle Arnt-Like (BMAL1), tyrosine hydroxylase (TH), and monoamine oxidase A (MAO-A) in relevant brain regions including the suprachiasmatic nucleus (SCN), nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus (Hip) were investigated by western blot assays to dissect the mechanism by which melatonin modulates fentanyl - induced behavioral sensitization and circadian rhythm disorders. The present study suggest that fentanyl (0.05, 0.1 and 0.2 mg/kg) could induce behavioral sensitization and melatonin (30.0 mg/kg) could attenuate the behavioral sensitization and circadian rhythm disorders in mice. Fentanyl treatment reduced the expression of BMAL1 and MAO-A and increased that of TH in relevant brain regions. Furthermore, melatonin treatment could reverse the expression levels of BMAL1, MAO-A, and TH. In conclusion, our study demonstrate for the first time that melatonin has therapeutic potential for fentanyl addiction.

Aptamer-based fluorescent sensor for highly sensitive detection of methamphetamine.

The construction of a fluorescence aptamer sensor was achieved by employing the fundamental principle of fluorescence resonance energy transfer. By employing molecular modeling technologies to identify the binding site, the high-affinity aptamer APT-40nt was derived from the whole sequence and utilized on the graphene oxide (GO) fluorescent platform for the purpose of achieving a highly sensitive detection of methamphetamine (METH). The aptamer tagged with fluorescein (FAM) dye undergoes quenching in the presence of GO due to π-stacking interaction. With the addition of the target, the aptamer that has been tagged was detached from the GO surface, forming a stable complex with METH. This process resulted in fluorescence restoration of the system, and the degree of fluorescence restoration was proportional to METH concentration in the linear range of 1-50 and 50-200 nM. Notably, under optimized conditions, the detection limit of this aptasensor was as low as 0.78 nM, which meets the detection limit requirements of METH detection in saliva and urine in some countries and regions. Moreover, other common illicit drugs and metabolites had minimizing interference with the determination. The established aptasensor, therefore, has been successfully applied to detect METH in saliva and urine samples and exhibited satisfactory recoveries (87%-111%). This aptasensor has the advantages of low detection limit, excellent selectivity, ease of operation, and low cost, providing a promising strategy for on-site detection of METH in saliva and urine.

DNA methylation profiling reveals potential biomarkers of ß-lactams induced fatal anaphylactic shock.

Anaphylaxis is a serious reaction of systemic hypersensitivity with that rapid onset and sudden death. Drug hypersensitivity, particularly induced by ß-lactams, is one of the most frequent causes of anaphylaxis in adults. But identification of anaphylactic shock, in forensic sciences recently, is difficult, because it mainly depends on nonspecific characteristic morphological changes, as well as exclusion and circumstantial evidence. Here, we detected DNA methylation signatures of ß-lactams-induced fatal anaphylactic shock with the Illumina Infinium Human Methylation EPIC BeadChip, to screen potential forensic biomarkers and reveal the molecular mechanisms of drug-induced anaphylaxis with fatal shock and sudden death. Our results indicated that DNA methylation was associated with ß-lactams-induced fatal anaphylactic shock, in which the hypomethylation played a vital role. We found that 1459 differentially methylated positions (DMPs) were mainly involved in ß-lactams-induced fatal anaphylactic shock by regulating MAPK and other signaling pathways. 18 DNA methylation signatures that could separate ß-lactams-induced anaphylactic shock from healthy individuals were identified. The altered methylation of DMPs can affect the transcription of corresponding genes and promote ß-lactams-induced fatal anaphylactic shock. The results suggest that DNA methylation can detect forensic identification markers of drug-induced anaphylaxis with fatal shock and sudden death, and it is an effective method for the forensic diagnosis.

Dynamic Study of Kinetically Trapped Byproducts during DNA Assembly: Case Study on a Pathway-Dependent Assembly.

Despite 40 years of development of DNA nanotechnology, the fundamental knowledge of the process of DNA strand assembly into targeted nanostructures remains unclear. Study of the dynamic process, especially the competing hybridizations in kinetic traps, provides insight into DNA assembly. In this study, a system of middle-domain first assembly (MDFA) was proposed to enable oligonucleotides to assemble into a 2D DNA monolayer in a pathway-dependent approach. This system was an ideal case to study the dynamic interactions between competing hybridizations during oligonucleotide assembly. Dynamic study revealed the coexistence of the kinetically trapped dead-end byproduct and target product at the early stage of annealing, followed by transformation of the byproduct into the target product by reverse disassembly, due to the equilibrium of the competing hybridizations increasingly favoring the target product pathway. This study offered a better understanding of the assembly pathway of DNA nanostructures for future design.

Stability of diazepam's phase II metabolites in dried blood spots on filter paper.

Phase II metabolites play an important role in diazepam-related cases. The study aimed to assess the stability of diazepam's phase II metabolites in dried blood spots on filter paper. METHODS: A piece of filter paper was spotted with 100 µL of whole blood (added 1% sodium fluoride as needed) obtained from participant who received 5 mg diazepam orally, air dried for 2 h at room temperature, and then stored at different conditions. Whole spots were cut at 0.1 cm from the outer edge of blood spots at post-consumption time-points of prior (zero), 5, 16, 35, 61, 120 days and 1, 1.5 years. Analytes were extracted with methanol/water mixture (8:2, v/v) and determined using HPLC-MS/MS. Decomposition rules were analyzed by a statistical software "SPSS". RESULTS: Temazepam glucuronide remained stable (0.5-18.6% loss) at 20 â„ƒ and at 20 â„ƒ with 1% sodium fluoride for 16 days, while it was unstable after 5 days at 4 â„ƒ (21.1-26.2% loss) and - 20 â„ƒ (28.9 - 34.4% loss). After 35 days, temazepam glucuronide concentrations began to fluctuate significantly under all conditions, and an obvious increase (290.4-355.1%) was observed in 1.5 years. Oxazepam glucuronide was always unstable after 5 days, the percentage loss was even 100% when it was stored for 61 days and 1.5 years. CONCLUSIONS: Dried blood spots on ordinary filter paper are recommended to be stored at 20 â„ƒ or 20 â„ƒ with 1% sodium fluoride within 16 days. Samples should be analyzed immediately or stored in sterile and dry media.

How to sample a seizure plant: the role of the visualization spatial distribution analysis of Lophophora williamsii as an example.

Natural compounds in plants are often unevenly distributed, and determining the best sampling locations to obtain the most representative results is technically challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide the basis for formulating sampling guideline. For a succulent plant sample, ensuring the authenticity and in situ nature of the spatial distribution analysis results during MSI analysis also needs to be thoroughly considered. In this study, we developed a well-established and reliable MALDI-MSI method based on preservation methods, slice conditions, auxiliary matrices, and MALDI parameters to detect and visualize the spatial distribution of mescaline in situ in Lophophora williamsii. The MALDI-MSI results were validated using liquid chromatography-tandem mass spectrometry. Low-temperature storage at -80°C and drying of "bookmarks" were the appropriate storage methods for succulent plant samples and their flower samples, and cutting into 40 µm thick sections at -20°C using gelatin as the embedding medium is the appropriate sectioning method. The use of DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile) as an auxiliary matrix and a laser intensity of 45 are favourable MALDI parameter conditions for mescaline analysis. The region of interest semi-quantitative analysis revealed that mescaline is concentrated in the epidermal tissues of L. williamsii as well as in the meristematic tissues of the crown. The study findings not only help to provide a basis for determining the best sampling locations for mescaline in L. williamsii, but they also provide a reference for the optimization of storage and preparation conditions for raw plant organs before MALDI detection. Key Points: An accurate in situ MSI method for fresh water-rich succulent plants was obtained based on multi-parameter comparative experiments.Spatial imaging analysis of mescaline in Lophophora williamsii was performed using the above method.Based on the above results and previous results, a sampling proposal for forensic medicine practice is tentatively proposed.

Predicting the retention time of Synthetic Cannabinoids using a combinatorial QSAR approach.

Background: Abuse of Synthetic Cannabinoids (SCs) has become a serious threat to public health. Due to the various structural and chemical group modified by criminals, their detection is a major challenge in forensic toxicological identification. Therefore, rapid and efficient identification of SCs is important for forensic toxicology and drug bans. The prediction of an analyte's retention time in liquid chromatography is an important index for the qualitative analysis of compounds and can provide informatics solutions for the interpretation of chromatographic data. Methods: In this study, experimental data from high-resolution mass spectrometry (HRMS) are used to construct a regression model for predicting the retention time of SCs using machine learning methods. The prediction ability of the model is improved by adopting a strategy that combines different descriptors in different independent machine-learning methods. Results: The best model was obtained with a method that combined Substructure Fingerprint Count and Finger printer features and the support vector regression (SVR) method, as it exhibited an R2 value of 0.81 for the validation set and 0.83 for the test set. In addition, 4 new SCs were predicted by the optimized model, with a prediction error within 3%. Conclusions: Our study provides a model that can predict the retention time of compounds and it can be used as a filter to reduce false-positive candidates when used in combination with LC-HRMS, especially in the absence of reference standards. This can improve the confidence of identification in non-targeted analysis and the reliability of identifying unknown substances.

Forensic toxicokinetics of difenidol hydrochloride in vivo verified by practical lethal cases.

A gas chromatography-mass spectrometry (GC-MS) method for the determination of difenidol hydrochloride in biological specimens has been developed. The method exhibited excellent recovery (> 90%) and precision (RSD < 10%), and the LOD was 0.05 µg/mL or µg/g, which met the requirements of bioanalytical method. Through the animal model of the forensic toxicokinetics, the dynamic distribution, postmortem redistribution (PMR) and stability in specimen preservation process of difenidol in animals were studied. The experimental results showed that after intragastric administration, the difenidol's concentrations in the heart-blood and various organs increased over time except stomach, and then decreased gradually after reaching the peaks of concentration. The toxicological kinetics equation and toxicokinetic parameters were established by processing the data of the mean drug concentration of difenidol changing with time. In PMR experiment, the concentrations of difenidol in some organs closer to the gastrointestinal tract (heart-blood, heart, liver, lung, kidney, and spleen) changed significantly at different time points. But the concentration of difenidol in brain tissues which were far away from the gastrointestinal tract and muscles with larger overall mass was relatively stable. PMR of difenidol was therefore confirmed. Thus, the effect of PMR on the concentration of difenidol in the specimens should be considered in cases involving difenidol poisoning or death. Furthermore, the stability of difenidol in heart-blood samples from poisoned rats was investigated at various time points and under different preservation conditions (20 °C, 4 °C, - 20 °C and 20 °C (1% NaF)) for a period of two months. Difenidol was stable and did not decompose in the preserved blood. Therefore, this study provided the experimental basis for the forensic identification of the cases of difenidol hydrochloride poisoning (death). PMR has been verified by practical lethal cases.

Combined metabolomics and tandem machine-learning models for wound age estimation: a novel analytical strategy.

Wound age estimation is one of the most challenging and indispensable issues for forensic pathologists. Although many methods based on physical findings and biochemical tests can be used to estimate wound age, an objective and reliable method for inferring the time interval after injury remains difficult. In the present study, endogenous metabolites of contused skeletal muscle were investigated to estimate the time interval after injury. Animal model of skeletal muscle injury was established using Sprague-Dawley rat, and the contused muscles were sampled at 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h postcontusion (n = 9). Then, the samples were analysed using ultraperformance liquid chromatography coupled with high-resolution mass spectrometry. A total of 43 differential metabolites in contused muscle were determined by metabolomics method. They were applied to construct a two-level tandem prediction model for wound age estimation based on multilayer perceptron algorithm. As a result, all muscle samples were eventually divided into the following subgroups: 4, 8, 12, 16-20, 24-32, 36-40, and 44-48 h. The tandem model exhibited a robust performance and achieved a prediction accuracy of 92.6%, which was much higher than that of the single model. In summary, the multilayer perceptron-multilayer perceptron tandem machine-learning model based on metabolomics data can be used as a novel strategy for wound age estimation in future forensic casework. Key Points: The changes of metabolite profile were correlated with the time interval after injury in contused skeletal muscle.A panel of 43 endogenous metabolites screened by ultraperformance liquid chromatography coupled with high-resolution mass spectrometry could distinguish the wound ages.The multilayer perceptron (MLP) algorithm exhibited a robust performance in wound age estimation using metabolites.The combination of matabolomics and MLP-MLP tandem model could improve the accuracy of inferring the time interval after injury.

Exploration and Practice of the "One Combination, Two Highlights, Three Combinations, Four in One" Innovative Talents Training Mode in Forensic Medicine.

Talent is one of the basic and strategic supports for building a modern socialist country in all aspects. Since the 1980s, the establishment of forensic medicine major and the cultivation of innovative talents in forensic medicine have become hot topics in higher education in forensic medicine. Over the past 43 years, the forensic medicine team of Shanxi Medical University has adhered to the joint education of public security and colleges, and made collaborative innovation, forming a training mode of "One Combination, Two Highlights, Three Combinations, Four in One" for innovative talents in forensic medicine. It has carried out "5+3/X" integrated reform, and formed a relatively complete talent training innovation mode and management system in teaching, scientific research, identification, major, discipline, team, platform and cultural construction. It has made a historic contribution to China's higher forensic education, accumulated valuable experience for the construction of first-class major and first-class discipline of forensic medicine, and provided strong support for the construction of the national new forensic talent training system. The popularization of this training mode is conducive to the rapid and sustainable development of forensic science, and provides more excellent forensic talents for national building, regional social development and the discipline construction of forensic science.

Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS.

OBJECTIVES: To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring. RESULTS: The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 µg/mL and 252.14 ng/mL, respectively. CONCLUSIONS: This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

A Target-Triggered Emission Enhancement Strategy Based on a Y-Shaped DNA Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristic for microRNA Imaging in Living Cells.

DNA self-assembled fluorescent nanoprobes have been developed for bio-imaging owing to their high resistance to enzyme degradation and great cellular uptake capacity. In this work, we designed a new Y-shaped DNA fluorescent nanoprobe (YFNP) with aggregation-induced emission (AIE) characteristic for microRNA imaging in living cells. With the modification of the AIE dye, the constructed YFNP had a relatively low background fluorescence. However, the YFNP could emit a strong fluorescence due to the generation of microRNA-triggered AIE effect in the presence of target microRNA. Based on the proposed target-triggered emission enhancement strategy, microRNA-21 was detected sensitively and specifically with a detection limit of 122.8 pM. The designed YFNP showed higher bio-stability and cell uptake than the single-stranded DNA fluorescent probe, which has been successfully applied for microRNA imaging in living cells. More importantly, the microRNA-triggered dendrimer structure could be formed after the recognition of target microRNA, achieving a reliable microRNA imaging with a high spatiotemporal resolution. We expect that the proposed YFNP will become a promising candidate for bio-sensing and bio-imaging.

HPLC-MS/MS determination and the postmortem distribution or postmortem redistribution of paraquat and its metabolites in four fatal intoxication cases.

HPLC-MS/MS analysis and postmortem distribution or postmortem redistribution of paraquat and its two metabolites in poisoning death cases were reported. Paraquat, monoquat, and paraquat monopyridone were extracted from the sample with acetonitrile or methanol, respectively, detected by ZORBAX HILIC Plus (4.6 × 100 mm, 3.5 µm) chromatographic column, with 0.1 % formic acid aqueous solution - 0.1 % formic acid acetonitrile solution (v/v) as mobile phase. Paraquat, monoquat, and paraquat monopyridone had a good linear relationship within the range of 10-1000, 1-400, and 1-1000 ng/mL (or g), the correlation coefficient (r) were all ≥ 0.9996. Their detection limits were lower than 1 ng/mL (or g). The detection accuracy was 91.25∼113.44 %. The intra-day and inter-day precision were 1.51-3.99 % and 1.92-4.93 %, respectively. This method was used to detect and analyze four rare paraquat poisoning cases. The distribution of paraquat, monoquat, and paraquat monopyridone is uneven, which is relatively high in the heart, blood, lung, and kidney. Heart blood/Peripheral blood ratio of paraquat, monoquat, paraquat monopyridone concentration in two poisoned cases were 1.4, 2.0, 1.5 and 1.9, 1.3, 1.2, which showed a location dependent postmortem redistribution. This is the first time that HPLC-MS/MS and the postmortem distribution or postmortem redistribution of paraquat metabolites in poisoned death cases have been reported. This research provides scientific basis for forensic identification of paraquat poisoning cases and extraction of biological specimen.

Simulating dynamic interaction between diazepam and ethanol targeting the GABAA receptor via in silico model.

Combined diazepam-ethanol poisoning is common in forensic toxicology. Both diazepam and ethanol can inhibit the central nervous system via the γ-aminobutyric acid (GABA) ligand gated chloride ion channel, GABAA Receptor (GABAAR). As the common target of diazepam and ethanol, whether GABAAR is the key target of their combined action remains unclear. This study aimed to explore their interaction based on the synergistic mechanisms between diazepam and ethanol targeting the GABAAR. Four models were built in silico based on the crystal structure of GABAAR. Molecular dynamic processes of the four models were simulated by the GPU-accelerated pmemd.cuda program in the Amber18 package. Results showed that ethanol inclined to combine the adjacent GABA or diazepam sites, minimized fluctuations of the root-mean-square deviation (RMSD) in the molecular dynamic process of GABA or diazepam binding the GABAAR, and increased the release of binding energy of GABA or diazepam binding the GABAAR. Results also showed that diazepam had less effect on the RMSD fluctuation or the binding energy release of GABA binding GABAAR. The formation of complex of diazepam and GABAAR could minimize the RMSD fluctuation and increase binding energy release of ethanol binding GABAAR. Thus, ethanol, bridging GABA and diazepam, could strengthen the complex of GABA binding the GABAAR, as well as the complex of diazepam binding the GABAAR. However, whether diazepam binds GABAAR or not, it cannot affect GABA binding the GABAAR; and yet the complex of diazepam and GABAAR can stabilize the complex of ethanol and GABAAR.

The Effects of Drug Addiction and Detoxification on the Human Oral Microbiota.

Drug addiction can powerfully and chronically damage human health. Detoxification contributes to health recovery of the body. It is well established that drug abuse is associated with poor oral health in terms of dental caries and periodontal diseases. We supposed that drug addiction and detoxification might have significant effects on the oral microbiota. To test the hypothesis, we assessed the effects of drug (heroin and methylamphetamine) addiction/detoxification on the oral microbiota based on 16S rRNA gene sequencing by an observational investigation, including 495 saliva samples from participants. The oral microbial compositions differed between non-users, current and former drug users. Lower alpha diversities were observed in current drug users, with no significant differences between non-users and former drug users. Heroin and METH addiction can cause consistent variations in several specific phyla, such as the enrichment of Acidobacteria and depletion of Proteobacteria and Tenericutes. Current drug users had significantly lower relative abundances of Neisseria subflava and Haemophilus parainfluenzae compared to non-users and former drug users. The result of random forest prediction model suggested that the oral microbiota has a powerful classification potential for distinguishing current drug users from non-users and former drug users. A cooccurrence network analysis showed that current drug users had more complex oral microbial networks and lower functional modularity. Overall, our study suggested that drug addiction may damage the balance of the oral microbiota. These results may have benefits for further understanding the effects of addiction-related oral microbiota on the health of drug users and promoting the microbiota to serve as a potential tool for accurate forensic identification. IMPORTANCE Drug addiction has serious negative consequences for human health and public security. The evidence indicates that drug abuse can cause poor oral health. In the current study, we observed that drug addiction caused oral microbial dysbiosis. Detoxication have positive effects on the recovery of oral microbial community structures to some extent. Understanding the effects of drug addiction and detoxification on oral microbial communities will promote a more rational approach for recovering the oral function and health of drug users. Furthermore, specific microbial species might be considered biomarkers that could provide information regarding drug abuse status for saliva left at crime scenes. To the best of our knowledge, this is the first report on the role of the oral microbiota in drug addiction and detoxification. Our findings give new clues to understand the association between drug addiction and oral health.