Strategies for Collection and Analysis of Samples in Simple Asphyxiant Gas Acute Poisoning Death Cases / 法医学杂志

At present, the death cases of simple asphyxiant gas acute poisoning are increasing sharply. Common asphyxiant gases in death cases include nitrogen, helium, carbon dioxide, methane, propane, laughing gas, etc. Simple asphyxiant gas has no affinity for biological matrices and escapes quickly, which puts forward new requirements for autopsy procedures, selection and collection of samples, laboratory analysis and identification. This paper reviews the research and development process of death cases caused by simple asphyxiant gas acute poisoning and put forwards the collection and analysis strategy of the samples in such cases. The most valuable biological samples in such cases should be lung tissues associated with the airways, followed by brain tissue and cardiac blood. Gaseous samples from the esophageal cavity, tracheal cavity, pulmonary bronchi, gastric and cardiac areas are also recommended as valuable samples. In the case of postmortem examination, the gas should be injected into gas sample bag directly. Biological materials such as tissue and blood should be directly sealed in head-space vials and analyzed by using the headspace gas chromatography-mass spectrometry.

Forensic Analysis of 9 Poisoning Death Cases Caused by Oral Administration of Diphenidol / 法医学杂志

OBJECTIVES@#To analyze the characteristics of diphenidol poisoning cases and to provide clues and technical means for the identification of such cases.@*METHODS@#Biological samples of 9 deaths caused by diphenidol poisoning were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the characteristics of these cases were analyzed retrospectively.@*RESULTS@#Most of the deaths caused by diphenidol poisoning were young females. The dosage was between 60 and 300 tablets, and the mass concentration of diphenidol in the postmortem blood ranged from 0.87 to 99.00 μg/mL. There was no correlation between the dosage and the concentration of diphenidol in the blood.@*CONCLUSIONS@#Diphenidol poisoning has the characteristics of high concealment and lethality. More attention should be paid to suicide cases, and diphenidol should be recommended as a routine detection item to avoid missing detection.

Analysis of the Distribution of Total Phosphine and the Characteristics of Phosphine Poisoning in 29 Victims / 法医学杂志

OBJECTIVES@#To study the distribution of total phosphine in phosphine poisoning victims and summarize the characteristics of phosphine poisoning cases.@*METHODS@#The phosphine and its metabolites in the biological samples of 29 victims in 16 phosphine poisoning cases were qualified and quantified by headspace gas chromatography-mass spectrometry.@*RESULTS@#Five victims among 29 were poisoned by ingestion of aluminium phosphide and 24 by inhalation of phosphine gas. Phosphine metabolites were detected in the biological samples of 23 victims, and the concentrations of total phosphine in blood ranged 0.5-34.0 μg/mL. The total concentration of phosphine in liver tissue was up to 71.0 μg/g. Phosphine was not detected in the blood of the other six survived victims, which may be related to the small amount of phosphine exposure and the delay in blood sampling.@*CONCLUSIONS@#The total concentration of phosphine in blood and tissues caused by aluminum phosphine ingestion is higher than that caused by phosphine gas inhalation. The death cases of phosphine inhalation are characterized by long exposure time, repeated exposures and age susceptibility.

Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology / 中国实验方剂学杂志

ObjectiveTo explore the structural characteristics and functional differences of intestinal flora in patients with type 2 diabetes mellitus （T2DM） of dampness heat trapping spleen（DHTS） syndrome and Qi-Yin deficiency（QYD） syndrome. MethodFrom June 2018 to January 2020，62 T2DM patients with DHTS syndrome and 60 with QYD syndrome were selected from Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine. Serum and fecal samples were collected to compare body mass index（BMI），glucose and lipid metabolism，fasting insulin （FINS） and fasting C-peptide （FCP） levels，and homeostasis model assessment of insulin resistance（HOMA-IR） of the two syndrome types. Fecal samples were extracted for DNA database construction，and 16S rDNA high-throughput sequencing was used to analyze and compare the intestinal flora and metabolic pathways. Result① The BMI，fasting plasma glucose（FPG），2-hour postprandial blood glucose （2 h PBG），total cholesterol（TC），triglyceride（TG），low density lipoprotein（LDL），FINS，FCP，and HOMA-IR were higher in patients with DHTS syndrome than in patients with QYD syndrome，and the high density lipoprotein（HDL） of the former was lower than that of the latter，（P<0.05，P<0.01）. ② In terms of species composition and differences，Bacteroidetes， Clostridia and Gammaproteobacteria were dominant at the class level，and the relative abundance of Clostridia，Mollicutes and Verrucomicrobiae in QYD syndrome group was higher than that in DHTS syndrome group. At the order level，Bacteroidales，Clostridiales and Enterobacteriales were mainly found. The relative abundance of Clostridiales，Erysipelotrichales and Verrucomicrobiales in QYD syndrome group was obviously higher than that in DHTS syndrome group，while Aeromonadales in the former was lower than that in the latter （P<0.05）. At the family level，Bacteroidaceae，Prevotellaceae and Ruminococcaceae were predominant. The relative abundance of Ruminococcaceae，Porphyromonadaceae and Erysipelotrichaceae in QYD syndrome group was higher than that in DHTS syndrome group（P<0.05）. At the genus level，Bacteroides，Prevotella and Parabacteroides were mainly found. The relative abundance of Parabacteroides，Butyrivibrio and Ruminiclostridium in QYD syndrome group was higher than that in DHTS syndrome group，while that of Klebsiella and Megasphaera in DHTS syndrome group was higher than that in QYD syndrome group（P<0.05）. ③ Through Venn analysis of operational taxonomic units（OTU），it was found that there were 49 OTUs in patients with DHTS syndrome patients and 47 OTUs in QYD syndrome patients. ④ The results of OTU β diversity and α analysis showed that Shannon and Simpson indexes had statistical differences，while Ace and Chao indexes had no statistical differences. The intestinal microbial diversity of patients with QYD syndrome was higher than that of patients with DHTS syndrome（P<0.05）. The analysis of similarities （ANOSIM） showed that the difference of β diversity between the two groups was significant（P<0.05）. ⑤ Linear discriminant analysis Effect Size（LEfSe） results demonstrated that Klebsiella，Megasphaera and Aeromonadales could be selected as the key biomarkers for DHTS syndrome； 14 bacteria such as Ruminiclostridium，Burkholderiaceae，Lautropia，Butyrivibrio，Erysipelotrichales can be selected as the key biomarkers for QYD syndrome. ⑥Functional annotation and analysis showed that the DHTS syndrome involved 9 metabolic pathways，including arginine and proline metabolism，lipopolysaccharide biosynthesis，nicotinic acid and nicotinamide metabolism，while the QYD syndrome involved 10 metabolic pathways，including acarbose and valinomycin biosynthesis，glucagon signaling pathway and NOD-like receptor signaling pathway. ConclusionThere are obvious differences in intestinal flora and functions in T2DM patients of DHTS syndrome and QYD syndrome，which can be used as reference for traditional Chinese medicine （TCM） syndrome differentiation and the target of TCM treatment.

Metabolism of New Psychoactive Substances 4F-MDMB-BUTINACA in Zebrafish / 法医学杂志

Objective To study the metabolic transformation pathways of 4F-MDMB-BUTINACA in vivo by establishing zebrafish models. Methods Six adult zebrafish were randomly divided into blank control group and experimental group, with three fish in each group. After the zebrafish in the experimental group were exposed to 1 μg/mL 4F-MDMB-BUTINACA for 24 h, they were transferred to clean water and cleaned three times, then pretreated for instrumental analysis. The zebrafish in blank control group were not exposed to 4F-MDMB-BUTINACA. Mass spectrometry and structural analysis of 4F-MDMB-BUTINACA and its metabolites were conducted by liquid chromatography-high resolution mass spectrometry and Mass Frontier software. Results A total of twenty-six metabolites of 4F-MDMB-BUTINACA were identified in zebrafish, including eighteen phase Ⅰ metabolites and eight phase Ⅱ metabolites. The main metabolic pathways of phase Ⅰ metabolites of 4F-MDMB-BUTINACA in zebrafish were ester hydrolysis, N-dealkylation, oxidative defluorination and hydroxylation, while the main metabolic pathway of phase Ⅱ metabolites was glucuronidation. Conclusion Metabolite Md24 （ester hydrolysis） and Md25 （ester hydrolysis combined with dehydrogenation） would be recommended to be potentially good biomarkers for abuse of 4F-MDMB-BUTINACA.

Analysis and Application of Seven Common New Psychoactive Substances of Synthetic Cannabinoids in Hair / 法医学杂志

Objective To establish a detection method for common new psychoactive substances of synthetic cannabinoids in hair with ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. Methods In the 1 mL of internal standard methanol solution, 20 mg hair was added. After cryogenic grinding and ultrasonic extraction, the extract was separated by ACQUITY UPLC HSS T3 column （100 mm×2.1 mm, 1.8 μm）. The mobile phase A was aqueous solution that composed of 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. The mobile phase B was acetonitrile. Electrospray ionization source in positive ion mode was used for data acquisition in multi-reaction monitoring （MRM） mode. Results The seven common new psychoactive substances of synthetic cannabinoids in hair had a good linear relationship within their respective linear ranges （r>0.99）, the limits of detection were 0.5-2 pg/mg, the limits of quantification were 1-5 pg/mg, the intra-day and inter-day precisions were 0.1%-12.6%, the intra-day and inter-day accuracies were 89.2%-110.7%, the recovery rates were 52.3%-93.3%, and the matrix effects were 19.1%-95.2%. Conclusion The established method has a simple sample preparation process and high sensitivity. It is suitable for qualitative and quantitative analysis of common new psychoactive substances of synthetic cannabinoids in hair.

Establishment and Its Application of UPLC-MS/MS Method for 20 Fentanyl-related Substances in Blood / 法医学杂志

Objective To establish an ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） rapid determination method for simultaneous analysis of 20 fentanyl-related substances in blood. Methods With fentanyl-D5 as an internal standard, the blood was extracted by liquid-liquid extraction （LLE）, then separated with an ACQUITY UPLC HSS T3 chromatographic column, and finally 20 fentanyl-related substances were simultaneously analyzed with multiple reaction monitoring （MRM） mode. Results The limits of detection （LOD） of all compounds were 0.02-0.03 ng/mL, and the limits of quantitation （LOQ） were 0.05-0.2 ng/mL. Within the mass concentration range of 0.05-40 ng/mL, 20 fentanyl-related substances had a good linear relationship, and correlation coefficients were larger than 0.99. The accuracy of the method was 87.69%-114.68% and the extraction recovery rate was 85.35%-101.80%, and no significant matrix effect was observed. The established method was successfully applied to the detection of sufentanil in rat blood after sufentanil was injected. Sufentanil could still be detected in blood of rats 10 h after sufentanil injection. Conclusion The established method has the advantages of simple pretreatment, high sensitivity and good selectivity, and can be used for the determination of fentanyl-related substances in forensic toxicology analysis.

Rapid Determination of Cocaine and Its Metabolite Benzoylecgonine in Hair by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a rapid determination method with LC-MS/MS for cocaine and its metabolite benzoylecgonine in hair.@*METHODS@#Deuterated internal standards （cocaine-D₃ and benzoylecgonine-D₈） were added to the decontaminated hair. After the extraction by ultrasonication with methanol, the compounds were separated by the Restek Allure PFP propyl column, and cocaine and benzoylecgonine were simultaneously analysed in multiple reaction monitoring mode.@*RESULTS@#The cocaine and benzoylecgonine in hair showed a good linearity in the range of mass fraction between 0.02 and 10.00 ng/mg with the limits of detection of 0.01 ng/mg.@*CONCLUSIONS@#The developed method is simple and rapid with a good selectivity, which is suitable for the determination of cocaine and its metabolite benzoylecgonine in hair.

Identification of New Designer Benzodiazepine Diclazepam in Drug Facilitated Sexual Assault / 法医学杂志

OBJECTIVES@#To identify the new designer drugs which are totally unknown and not in the routine testing list by the technologies such as high-resolution mass spectrometry in drug facilitated sexual assault, in order to solve the problem in actual cases.@*METHODS@#The milky fluid from an actual case was extracted and analyzed using LC-QE, ¹H-NMR and GC-MS, respectively. The accurate masses and cluster ions isotope patterns of unknown compound were obtained by LC-QE. The molecular formula was confirmed as C₁₆H₁₂C₂N₂O based on the protons number of ¹H-NMR. The isomers diclazepam and 4-chlorodiazepam were separated and detected with GC-MS.@*RESULTS@#The new designer benzodiazepine as diclazepam in the milky fluid was identified. The results provided direct evidence for the investigation and qualitative analysis of such cases.@*CONCLUSIONS@#The combined application of various methods, including LC-QE, ¹H-NMR and GC-MS, can be used to detect unknown new psychoactive substances.

Determination of Oleandrin in Blood and Liver Samples by LC-MS/MS / 法医学杂志

Objective To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method for the determination of oleandrin in blood and liver tissues, which could be applied to the cases of death caused by oleander poisoning.Methods Blood or liver tissues underwent a liquidliquid extraction (LLE) using ethyl acetate, and the extract was separated on an Agilent ZORBAX SB-C18column and eluted with a gradient of acetonitrile and 20 mmol/L ammonium acetate (containing0.1％formic acid).Oleandrin was detected using electrospray positive ionization (ESI+) with multiplereaction monitoring (MRM) mode.Results Oleandrin showed excellent linearity in both blood and liver samples in the corresponding linear range (r＞0.995 0), with detection limits 1 ng/mL and 2 ng/g, respectively, extraction recovery rates greater than 70.50％, both intra-and inter-day precisions less than10.71％, accuracies 98.42％-111.63％, and matrix effects 91.52％-106.39％.The method was successfully applied to a case of suspected oleander poisoning.Oleandrin was detected in the blood, urine, liver tissues, bile, stomach wall tissues and stomach contents of the cadaver, with the content ranging from65.5 to 29 600.0 ng/mL (ng/g).Conclusion The method developed in this study is simple and convenient to operate with good selectivity, and is suitable for the analysis of oleandrin in biological samples such as blood and liver tissues, which can provide technical support for forensic identification and clinical diagnosis and treatment of oleander poisoning.

Determination of Sulfide Ion in Blood from Hydrogen Sulfide Poisoning Cases / 法医学杂志

OBJECTIVES@#To establish a gas chromatography-mass spectrometry （GC-MS） method for the determination of sulfide ion in blood and apply it to the practical cases.@*METHODS@#The 1, 3, 5-tribromobenzene was selected as an internal standard, and 0.2 mL blood sample was collected and analyzed using GC-MS after α-Bromo-2, 3, 4, 5, 6-pentafluorobenzyl bromide derivatization.@*RESULTS@#The mass concentration of sulfide ion in blood had good linearity in the range of 0.2-40 μg/mL with a limit of detection （LOD） of 0.05 μg/mL. The mass concentration of sulfide ion was less than 0.05 μg/mL in blank blood from different sources such as healthy subjects and dead cases. In 3 sulfide poisoning cases, sulfide ion was detected in the blood samples of 6 victims, and the mass concentration range was 1.02-3.13 μg/mL.@*CONCLUSIONS@#This study establishes a method for investigation of sulfide ion in blood which has been applied successfully to the cases of fatal sulfide poisonings.

Evaluation of Brodifacoum-induced Toxicity by Metabonomics Approach Based on HPLC-TOF-MS / 法医学杂志

OBJECTIVES@#To analyse the metabolic changes in urine of rats with brodifacoum intoxication, and to reveal the molecular mechanism of brodifacoum-induced toxicity on rats.@*METHODS@#By establishing a brodifacoum poisoning rats model, the urine metabolic profiling data of rats were acquired using high performance liquid chromatography-time of flight mass spectrometry （HPLC-TOF-MS）. The orthogonal partial least squares analysis-discrimination analysis （OPLS-DA） was applied for the multivariate statistics and the discovery of differential metabolites closely related to toxicity of brodifacoum.@*RESULTS@#OPLS-DA score plot showed that the urinary metabolic at different time points before and after drug administration had good similarity within time period and presented clustering phenomenon. Comparing the urine samples of rats before drug administration with which after drug administration, twenty-two metabolites related to brodifacoum-induced toxicity were selected.@*CONCLUSIONS@#The toxic effect of brodifacoum worked by disturbing the metabolic pathways in rats such as tricarboxylic cycle, glycolysis, sphingolipid metabolism and tryptophan metabolism, and the toxicity of brodifacoum is characterized of accumulation effect. The metabonomic method based on urine HPLC-TOF-MS can provide a novel insight into the study on molecular mechanism of brodifacoum-induced toxicity.

Analysis of GHB and Its Precursors in Urine and Their Forensic Application / 法医学杂志

OBJECTIVE@#To establish the method to analyze γ-hydroxybutyric acid (GHB) and its precursors 1,4-butanediol (1,4-BD) and gamma-butyrolactone (GBL) in urine through LC-MS/MS and provide evidence for related cases.@*METHODS@#GHB-d6 and MOR-d3 were used as the internal standard. The urine sample was separated by LC after protein precipitation with methanol. The electrospray ion source was for ionization. Each compound was detected through multiple-reaction monitoring (MRM) mode.@*RESULTS@#The limits of detection of GHB and its precursors 1,4-BD and GBL were 0.1, 0.1 and 2 μg/mL. The accuracy was 87.6%-98.1%. The intra-day and inter-day precisions were less than 15% and matrix effects were higher than 80%.@*CONCLUSION@#The method is high sensitive, simple, rapid, specific and with high reliability. This study has provided technical support and basic data for forensic cases involving GHB.

Chlorpyrifos Determined in Human Blood by UPLC-MS/MS and Its Application in Poisoning Cases / 法医学杂志

OBJECTIVE@#To determine the chlorpyrifos in human blood by liquid chromatography-tandem mass spectrometry and to validate its application in poisoning cases.@*METHODS@#The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 MGII column (250 mm x 2.0 mm, 5 μm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammonium acetate) and solvent B (methanol with 2 mmol/L ammonium acetate) at 5:95 V:V).@*RESULTS@#The linear ranged from 5 to 500 ng/mL (r = 0.998 7). The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 2 ng/mL and 4 ng/mL, respectively. For this method, the precision and accuracy of intra-day and inter-day were < 10% and 97.44%-101.10%, respectively. The results in stability test of long-term frozen were satisfied. The matrix effect, recovery and process efficiency were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively.@*CONCLUSION@#This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.

Method development and validation for determining 1,3-butadiene in human blood by gas chromatography-mass spectrometry and head-space gas chromatography / 法医学杂志

To develop a simple, validated method for identifying and quantifying 1,3-butadiene (BD) in human blood by gas chromatography-mass spectrometry (GC-MS) and head-space gas chromatography (HS-GC). BD was identified by GC-MS and HS-GC, and quantified by HS-GC. The method showed that BD had a good linearity from 50 to 500 microg/mL (r > 0.99). The limits of detection and quantification were 10 microg/mL and 50 microg/mL, respectively. Both the intra-day precision and inter-day precision were < 6.08%, and the accuracy was 96.98%-103.81%. The method was applied to an actual case, and the concentration of BD in the case was 242 microg/mL in human blood. This simple method is found to be useful for the routine forensic analysis of acute exposure to BD.

Measurement of acetonitrile in blood and urine by head-space gas chromatography / 法医学杂志

OBJECTIVE@#To establish the method for measurement of acetonitrile in blood and urine by head-space gas chromatography.@*METHODS@#DB-ALC1 (30 m x 320 microm x 1.8 microm) and DB-ALC2 (30 m x 320 microm x 1.2 microm) capillary column were used to measure the acetonitrile in blood and urine with the isopropanol as internal standard reference.@*RESULTS@#The limits of detection of acetonitrile in both blood and urine were 0.5 microg/mL, with a linear range of 5-1000 microg/mL (r = 0.999).The accuracy of this method was 93.2%-98.0%. The RSD for the intra-day and inter-day were less than 3.7%.@*CONCLUSION@#The method is capable for measurement analysis of acetonitrile in blood and urine.

Screening and confirmation of psychotropic drugs in blood and urine by HPLC-LTQ Orbitrap MS / 法医学杂志

OBJECTIVE@#To establish a screening and confirmation method for psychotropic drugs and their metabolites in human blood and urine by HPLC-LTQ Orbitrap MS.@*METHODS@#The samples were pretreated with Sirocco protein precipitation plate, and then analyzed by HPLC-LTQ Orbitrap MS. The method was validated in terms of the limit of detection (LOD). An accurate mass database was created for psychotropic drugs screening.@*RESULTS@#The LOD for most of 56 determined compounds was < or = 0.1 ng/mL. The accurate mass database included the accurate mass information of 61 psychotropic drugs.@*CONCLUSION@#The method is accurate, rapid, sensitive and the database is suitable for psychotropic drugs screening and confirmation.

Application of liquid chromatography-high resolution mass spectrometry in toxicological screening / 法医学杂志

Due to the diversity of toxicologically relevant substances, the uncertainty of target compounds and the specificity of samples, toxicological screening techniques have always been valued by the forensic toxicologists. Depending on its powerful separation ability, superhigh resolution and accurate mass measurement, combined with the two levels spectrum database matching and abundance ratio of isotope ion, the liquid chromatography-high resolution mass spectrometry (LC-HRMS) analyzers have increasingly advantage in screening and identification of chemical compound. This review focuses on the applications of LC-HRMS in screening and identification of drug-of-abuse, prescription drugs, pesticide and stimulant. The prospect of LC-HRMS in forensic toxicology analysis is also included.

Determination of Hg in biological samples by inductively coupled plasma mass spectrometry / 法医学杂志

OBJECTIVE@#To establish an inductively coupled plasma mass spectrometry (ICP-MS) method for determination of Hg in biological samples.@*METHODS@#The samples were digested with microwave digestion instrument. ICP-MS was applied to detect Hg in blood, urine and hair specimens by using 115In as an internal marker. The ability of gold to eliminate the memory effect of mercury was investigated with the gold amalgamate produced by gold and mercury.@*RESULTS@#The limits of detection were in the 0.01 microg/L, and the accuracy of the method ranged from 97.0% to 107.1%. The concentration of gold was 10 microg/L and the memory effect of mercury was resolved.@*CONCLUSION@#The method is accurate, rapid, sensitive and suitable for the cases of mercury poisoning and the clinical diagnosis and monitoring for patients with mercury poisoning.

Simultaneous determination of sixteen antibiotics in human urine with ultra performance liquid chromatography-tandem mass spectrometry / 法医学杂志

OBJECTIVE@#To develop a method for simultaneous determination of sixteen antibiotics in human urine by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).@*METHODS@#With Piperacillin as an internal standard, the target antibiotics in urine samples were enriched and purified by Oasis HLB solid phase extraction (SPE) cartridges, then separated in a ZORBAX SB-C18 column with a gradient elution of mobile phase of 0.1% formic acid water and acetonitrile, finally analyzed with multiple reaction monitoring (MRM) mode.@*RESULTS@#The limits of detection (LOD) for these sixteen antibiotics were in the range of 0.05-10.0 ng/mL and the limits of quantification (LOQ) in the range of 0.25-20.0 ng/mL. Within the related linear range, the related coefficient (r) of sixteen antibiotics were all more than 0.995. Accuracies for these antibiotics were ranged from 82.0%-119.3%, the within-day precision were less than 13.9%.@*CONCLUSION@#The developed method is sensitive, specific and appropriate for the analysis of antibiotics in forensic toxicology and therapeutic drugs monitoring.