Determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction and capillary electrophoresis with PDA detection

A new method was developed for pre-concentration and determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction (DLLME) and capillary electrophoresis (CE) with photodiode array detection. The method was based on the formation of tiny droplets of an organic extractant in the prepared sample solution using water-immiscible organic solvent (chloroform) dissolved in water-miscible organic dispersive solvent (isopropyl alcohol). The organic phase, which extracted eight drugs of abuse from the prepared urine solution, was separated by centrifugation. The sedimented phase was transferred into a small volume CE auto-sampler vial with 10 µL of 1% HCl methanol solution and evaporated to dryness. The residue was reconstituted in lidocaine hydrochloride (internal standard) aqueous solution and introduced by electrokinetic injection into CE. Under the optimum conditions, acceptable linear relationship was observed in the range of 3.0–500 ng/mL with the correlation coefficient (r) of 0.9982–0.9994 for spiked urine samples. The limit of detection (LOD) (S/N = 3) was estimated to be 1.0 ng/mL. A recovery of 75.7%–90.6% was obtained for spiked samples. The mean relative error (MRE) was within ±7.0% and the relative standard deviation (RSD) was less than 6.9%. The proposed DLLME-CE procedure offers an alternative analytical approach for the sensitive detection of drugs of abuse in real urine samples.

Key points

• The dispersive liquid-liquid microextraction (DLLME) was involved for the determination of drugs in urine with capillary electrophoresis with photodiode array detection (CE-PDA).
• Good linearity, sensitivity, recovery and precision were achieved.
• The proposed method was eco-friendly with microliter scale solvent consumption.

     

Age estimation of individuals aged 5–23 years based on dental development of the Indonesian population

Dental development can be used to estimate age for forensic purposes. However, most of the currently available methods are less reliable for the Indonesian population due to population variability. This study presents a new method and evaluates other methods that utilize dental development to estimate the age of Indonesian people. Panoramic radiographs of 304 young Indonesian people aged 5–23 years old were analysed for deciduous tooth root resorption, permanent tooth calcification, and eruption. The extent of tooth root resorption was determined based on AlQahtani’s modified Moorrees et al. method. Tooth calcification was classified based on a modified Demirjian et al. method. Tooth eruption was evaluated based on AlQahtani’s modified Bengston system. The sequence of tooth root resorption, and permanent tooth calcification and eruption were grouped into 19 age categories (from 5–23 years old) in an atlas. The differences between males and females, between maxillary and mandibular teeth, and between right and left teeth were also analysed. There were minimal significant differences of tooth development between males and females, and between the right and left teeth (P > 0.05), while the maxillary and mandibular dental development was significantly different (P < 0.05). The newly developed atlas showed the development of the right side of maxillary and mandibular tooth of combined sex of Indonesian population. Another 34 panoramic radiographs of known-age and sex individuals from Indonesia were assessed using the newly developed Atlas of Dental Development in the Indonesian Population, Ubelaker’s Dental Development Chart, The London Atlas of Human Tooth Development and Eruption by AlQahtani, and the Age Estimation Guide-Modern Australia population by Blenkin-Taylor. Accuracy was assessed by comparing estimated age to actual chronological age using the Bland-Altmand test. Results show that the smallest range of error was found in the Atlas of Dental Development in the Indonesian Population (−0.969 to 1.210 years), followed by The London Atlas of Human Tooth Development and Eruption by AlQahtani (−2.013 to 1.990 years), the Age Estimation Guide-Modern Australia population by Blenkin-Taylor (−2.495 to 2.598 years), and the Dental Development Chart by Ubelaker (−2.960 to 3.289 years). These findings show that the Atlas of Dental Development constructed in this study performs better than the other three methods and presents greater accuracy of age estimation in the Indonesian population.

Key points

• Dental development such as deciduous tooth root resorption, permanent tooth calcification, and tooth eruption can be used to estimate age for forensic purposes.
• The development of the teeth are influenced by genetic, ethnicity, and sex, therefore an age estimation method must be constructed based on the same population.
• There were minimal significant differences in tooth development between male and female, and between right and left teeth, but there was significant difference between maxillary and mandibular teeth.
• The Atlas of Dental Development in the Indonesian Population constructed in this study allowed more accurate age estimation of the Indonesian sample than the other methods tested.

Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1886648.     

Application of Cameriere’s method for dental age estimation in children in South China

The aim of this study was to evaluate the applicability of Cameriere’s European formula for age estimation in children in South China and to adapt the formula to establish a more suitable formula for these children. Moreover, the performance of dental age estimation based on Cameriere’s method combining the developmental information of permanent teeth (PT) and third molar (TM) was also analysed. Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed. The samples of Group A were divided into training dataset 1 and test dataset 1, and the samples of Group B were also divided into training dataset 2 and test dataset 2. A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere’s European formula and the South China-specific formula was conducted with the test dataset 1. Additionally, a PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2. The Cameriere’s European formula underestimated chronological age with a mean difference (ME) of −0.47 ± 1.11 years in males and −0.69 ± 1.19 years in females. However, the South China-specific formula underestimated chronological age, with a mean difference (ME) of −0.02 ± 0.71 years in males and −0.14 ± 0.73 years in females. Compared with PT model and TM model, the PT and TM combined model obtained the smallest root mean square error (RMSE) of 1.29 years in males and 0.93 years in females. In conclusion, the South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.

Key points

• Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed.
• A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere’s European formula and the South China-specific formula was conducted with the test dataset 1.
• A PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2.
• The South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.

     

Sequencing of human identification markers in an Uyghur population using the MiSeq FGxTM Forensic Genomics System

Massively parallel sequencing (MPS) offers a useful alternative to capillary electrophoresis (CE) based analysis of human identification markers in forensic genetics. By sequencing short tandem repeats (STRs) instead of determining the fragment lengths by CE, the sequence variation within the repeat region and the flanking regions may be identified. In this study, we typed 264 Uyghur individuals using the MiSeq FGx™ Forensic Genomics System and Primer Mix A of the ForenSeq™ DNA Signature Prep Kit that amplifies 27 autosomal STRs, 25 Y-STRs, seven X-STRs, and 94 HID-SNPs. STRinNGS v.1.0 and GATK 3.6 were used to analyse the STR regions and HID-SNPs, respectively. Increased allelic diversity was observed for 33 STRs with the PCR-MPS assay. The largest increases were found in DYS389II and D12S391, where the numbers of sequenced alleles were 3–4 times larger than those of alleles determined by repeat length alone. A relatively large number of flanking region variants (28 SNPs and three InDels) were observed in the Uyghur population. Seventeen of the flanking region SNPs were rare, and 12 of these SNPs had no accession number in dbSNP. The combined mean match probability and typical paternity index based on 26 sequenced autosomal STRs were 3.85E−36 and 1.49E + 16, respectively. This was 10 000 times lower and 1 000 times higher, respectively, than the same parameters calculated from STR repeat lengths.

Key Points

• Sequencing data on STRs and SNPs used for human identification are presented for the Uyghur population.
• STRinNGS v.1.0 was used to analyse the flanking regions of STRs.
• The concordance between PCR-CE and PCR-MPS results was 99.86%.
• Detection of sequence variation in STRs and their flanking regions increased the allelic diversity.

     

Identification of a female murder victim found in Burgenland,Austria in 1993

In 1993, the skeletal remains of a female corpse were found in Burgenland, Austria. Initial identification of the approximately 25–35-year-old female appeared impossible, but the case was reopened 23 years later. By applying biogeochemical isotope methods to her body tissues, the geographical origin of the unknown corpse could be predicted. The results of the C, N, S, H, Sr, and Pb isotope analyses suggested that the female did not originate from Europe and most likely spent her youth in the northern Caribbean. Using these findings, the police were able to identify the woman within 2 weeks. The female came from the Dominican Republic and resided in Austria for only a short period before she was murdered. This case shows that isotope biogeochemistry investigations can provide the police with crucial information that enables unknown persons to be identified.

Keypoints

• C-N-S-H and Sr-Pb isotope analyses were applied to human remains associated with a cold case.
• It was possible to determine the region of origin of the unknown deceased individual as the northern Caribbean.
• After 23 years, the murder victim was successfully identified.

     

Swedish rape offenders — a latent class analysis

Sweden has witnessed an increase in the rates of sexual crimes including rape. Knowledge of who the offenders of these crimes are is therefore of importance for prevention. We aimed to study characteristics of individuals convicted of rape, aggravated rape, attempted rape or attempted aggravated rape (abbreviated rape+), against a woman ≥18 years of age, in Sweden. By using information from the Swedish Crime Register, offenders between 15 and 60 years old convicted of rape+ between 2000 and 2015 were included. Information on substance use disorders, previous criminality and psychiatric disorders were retrieved from Swedish population-based registers, and Latent Class Analysis (LCA) was used to identify classes of rape+ offenders. A total of 3 039 offenders were included in the analysis. A majority of them were immigrants (n = 1 800; 59.2%) of which a majority (n = 1 451; 47.7%) were born outside of Sweden. The LCA identified two classes: Class A — low offending class (LOC), and Class B — high offending class (HOC). While offenders in the LOC had low rates of previous criminality, psychiatric disorders and substance use disorders, those included in the HOC had high rates of previous criminality, psychiatric disorders and substance use disorders. While HOC may be composed by more “traditional” criminals probably known by the police, the LOC may represent individuals not previously known by the police. These two separated classes, as well as our finding in regard to a majority of the offenders being immigrants, warrants further studies that take into account the contextual characteristics among these offenders.

Key points

• Rape, aggravated rape, attempted rape or attempted aggravated rape (rape+) are increasing in Sweden.
• The majority of those convicted of rape+ are immigrants.
• LCA identifies two classes of rape+ offenders: LOC and HOC.

     

Reflections on variability in the blood–breath ratio of ethanol and its importance when evidential breath-alcohol instruments are used in law enforcement

Variability in the blood–breath ratio (BBR) of alcohol is important, because it relates a measurement of the blood-alcohol concentration (BAC) with the co-existing breath-alcohol concentration (BrAC). The BBR is also used to establish the statutory BrAC limit for driving from the existing statutory BAC limits in different countries. The in-vivo BBR depends on a host of analytical, sampling and physiological factors, including subject demographics, time after end of drinking (rising or falling BAC), the nature of the blood draw (whether venous or arterial) and the subject’s breathing pattern prior to exhalation into the breath analyzer. The results from a controlled drinking study involving healthy volunteers (85 men and 15 women) from three ethnic groups (Caucasians, Hispanics and African Americans) were used to evaluate various factors influencing the BBR. Ethanol in breath was determined with a quantitative infrared analyzer (Intoxilyzer 8000) and BAC was determined by headspace gas chromatography (HS-GC). The BAC and BrAC were highly correlated (r = 0.948) and the BBR in the post-absorptive state was 2 382 ± 119 (mean ± SD). The BBR did not depend on gender (female: 2 396 ± 101 and male: 2 380 ± 123, P > 0.05) nor on racial group (Caucasians 2 398 ± 124, African Americans 2 344 ± 119 and Hispanics 2 364 ± 104, P > 0.05). The BBR was lower in subjects with higher breath- and body-temperatures (P < 0.05) and it also decreased with longer exhalation times into the breath-analyzer (P < 0.001). In the post-absorptive state, none of the 100 subjects had a BBR of less than 2 100:1.     

Deposition of 7-aminoflunitrazepam and flunitrazepam in hair after a single dose of Rohypnol

In recent years, there has been a notable increase in the number of reports on drug-facilitated sexual assault. Benzodiazepines are the most common so-called "date-rape" drugs, with flunitrazepam (Rohypnol) being one of the most frequently mentioned. The aim of this study was to determine whether flunitrazepam and its major metabolite 7-aminoflunitrazepam could be detected in hair collected from ten healthy volunteers after receiving a single 2 mg dose of Rohypnol using solid phase extraction and NCI-GC-MS. Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault in order to reveal drug use. Ten healthy volunteers (eight women and two men, 21 to 49 years old) participated in the study. The following hair samples were collected from each volunteer: one before flunitrazepam administration, and 1, 3, 5, 14, 21, and 28 days after. In five volunteers, 7-aminoflunitrazepam was detected 24 h after flunitrazepam administration and remained in hair throughout the entire 28-day study period (0.6-8.0 pg/mg). In two cases, 7-aminoflunitrazepam appeared in hair 21 days after drug intake (0.5-2.7 pg/mg), and in two subjects 14 days later (0.5-5.4 pg/mg). In one volunteer, 7-aminoflunitrazepam was detected on day 14 and 21 but concentrations were below the quantitation limit. Flunitrazepam was detected in some samples but all concentrations were below the quantitation limit (0.5-2.3 pg/mg).     

Analysis of sensitivity and specificity: precise recognition of neutrophils during regeneration of contused skeletal muscle in rats

In this report, we applied the TissueFAXS 200 digital pathological analysis system to rapidly and accurately identify neutrophils during regeneration of contused skeletal muscle, and to provide information for follow-up studies on neutrophils to estimate wound age. Rat injury model was established, and skeletal muscle samples were obtained from the control group and contusion groups at 1, 1.5, 2, 3, 4, and 6 h, as well as at 1, 3, 5, and 15 d post-injury (n = 5 per group). The expression of nuclei and neutrophils was detected by hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining. A total of 20 injury site areas of 0.25 mm² (0.5 mm × 0.5 mm) were then randomly selected at all time points. A TissueFAXS 200 digital pathological analysis system was used to identify the positive and negative numbers. Knowledge of five professional medical workers were considered the gold standard to measure the false positive rate (FPR), false negative rate (FNR), sensitivity, specificity, and area under the curve (AUC) of receiver operating characteristic (ROC) curves. As a result, with a staining area of neutrophils from 8 µm² to 15 µm², the FPR was 4.28%–12.14%, the FNR was 12.42%–64.08%, the sensitivity was 35.92%–87.58%, the specificity was 87.86%–95.72%, the Youden index was 0.316–0.754, the accuracy was 82.80%–88.30%, and the AUC was 0.771–0.826. The AUC was largest when the cut-off value of the staining area was 12 µm². Our results show that this software-based method is more accurate than the human eye in evaluating neutrophil infiltration. Based on the sensitivity and specificity, neutrophils can be accurately identified during regeneration of contused skeletal muscle. The TissueFAXS 200 digital pathological analysis system can also be used to optimize conditions for different cell types under various injury conditions to determine the optimal cut-off value of the staining area and provide optimal conditions for further study. Furthermore, it will provide evidence for forensic pathology cases.     

Fingerprint analysis for the determination of hand origin (right/left) using the axis slant in whorl patterns

Fingerprints are frequently encountered during both civil and criminal investigations. Fingerprints possess numerous characteristics that assist with personal identification. Determining the hand of origin (right or left) for an individual fingerprint would help reduce investigation time and potentially eliminate certain suspects. In this study, we collected a total of 2 900 single digit fingerprints from 290 individuals, and the whorl axis slant was examined in the 743 whorl pattern fingerprints (385 from the right hand and 358 from the left hand). A slant towards the right side was present in 81.82% of samples from the right hand, while a slant towards the left side was observed in 80.73% of samples from the left hand. After applying a chi-square test to the dataset, the results were found to be statistically significant for the determination of hand origin. Our results suggest that the whorl axis slant in a fingerprint is indicative of hand origin (right or left).

Key points

• Single digit fingerprints with whorl pattern were analyzed.
• Whorl “axis slant” was used to determine the hand origin.
• Right axis slant would indicate the right hand of the print.
• Left axis slant would indicate the left hand of the print.

     

Determination of barbiturates in hair samples by using a validated UHPLC-HRMS method: application in investigation of drug-facilitated sexual assault

In recent years,benzodiazepines and benzodiazepine-like drugs are the most common substances associated with drug-facilitated sexual assaults(DFSA);however,barbiturates are also detected occasionally.Segmental hair analysis provides useful information on the historic pattern of drug use,enabling differentiation between single exposure in DFSA cases and chronic use.However,sensitive and specific methods for barbiturate analysis in hair samples are needed.Herein,we present an ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry(UHPLC-HRMS)method for qualitative and quantitative determination of seven barbiturates in hair samples.Firstly,a hair strand was decontaminated and then freeze-milled in liquid nitrogen.Next,50mg of powdered hair was extracted with methanol in an ultrasonic bath for 10 min in the presence of 10 ng phenobarbital-d5.The supernatant was dried under nitrogen gas,and the pellet was dissolved in 100 μL mobile phase.Afterwards,10 μL of the suspension was injected into the UHPLC-HRMS system.The present method involved two UHPLC conditions for determination of barbiturates(I)and identification of the structural isomers amobarbital and pentobarbital(II).This method showed satisfactory linearity in a range of 0.02–20.00 ng/mg for UHPLC conditions I and II,both with a high determination coefficient(0.9991–0.9999).The selectivity,intra-and interday precision,accuracy and matrix effect of the method were acceptable.Next,the validated method was applied to investigate an authentic DFSA case.Hair samples(black,approximate 25cm long)were collected 3 months after the assault,and the proximal segments(0–5 cm from the root;each segment was 1 cm long)were analysed.Amobarbital was detected at a concentration of相似文献   

Isolation and identification of an isomeric sildenafil analogue as an adulterant in an instant coffee premix

The proliferation of adulterated health foods and beverages in the market demands a comprehensive analytical strategy to identify the adulterants, particularly those of isomeric phosphodiesterase 5 (PDE5) inhibitors. An instant coffee premix (ICP) purchased from an online retailer was flagged for suspected adulteration through PDE5 inhibition assay. The ICP was then analysed using suspected-target and non-targeted screenings of a liquid chromatography-quadrupole time-of-flight mass spectrometry. Based on these findings, a PDE5 inhibitor initially assigned as compound X was isolated from the ICP by employing a liquid chromatography-diode array detection before its structural elucidation with liquid chromatography-ultraviolet (LC-UV) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The suspected-target screening matched the protonated molecule ([M + H]⁺) precursor ion of compound X at m/z 499.2310 with two suspected analytes that are structural isomers of one another. The fragmentation patterns of compound X were comparable to those analogues in the dithiocarbodenafil group through the non-targeted screening. These findings, complemented by the LC-UV and NMR spectroscopy data, together with the chromatographic separation of related structural isomers, conclude the identity of compound X. To our best knowledge, this is the first study to report the presence of 3,5-dimethylpiperazinyl-dithiodesmethylcarbodenafil in an ICP sample.

Key points

• The herbal-based male sexual performance products’ lucrative market has instigated their rampant adulteration, particularly with PDE5 inhibitors.
• The adulterated products may also contain analogues of the approved PDE5 inhibitors, which usually passed into the market undetected as they are not included in the routine targeted screening procedure.
• The present study detected, isolated, and identified an isomeric sildenafil analogue from an instant coffee premix sample using rapid qualitative assay and comprehensive analytical analysis.
• This paper highlighted the applicability of the established strategies for routine casework, particularly in a forensic drug testing laboratory.

     

Virtual anthropology: a preliminary test of macroscopic observation versus 3D surface scans and computed tomography (CT) scans

Virtual anthropology (VA) is based on applying anthropological methods currently used to analyse bones to 3D models of human remains. While great advances have been made in this endeavour in the past decade, several interrogations concerning how reliable these models are and what their proper use should be remain unanswered. In this research, a fundamental assumption of VA has been investigated: if the way we perceive and apply an anthropological method is truly similar when looking at bones macroscopically and through various 3D media. In order to answer, 10 skulls of known age and sex were scanned using a computed tomography (CT) scanner and a 3D surface scanner. Two observers separately applied a defined staging method to eight suture sites on these skulls, first looking at the bone macroscopically, then at the 3D surface scan, and finally on the CT scan. Two rounds of observation were carried out by each observer. Intra- and inter-observer error were evaluated, and two sample t-tests used to evaluate if the different types of medium used yielded significantly different observations. The results show a high degree of inter-observer error, and that data obtained from 3D surface scans differ from macroscopic observation (confidence level 95%, P ≤ 0.05). CT scans, in these settings, yielded results comparable to those obtained through macroscopic observations. These results offer many possibilities for future research, including indications on the kind of anthropological methods and anatomical landmarks that might be reliably transferable to the virtual environment. All current methods used in traditional anthropology should be tested, and if they prove unreliable, new techniques to analyse bones from virtual models should be developed.

Key points

• Large discrepancies between observation on dry bones and computer-generated 3D models (surface scans or CT scans) could lead to the re-evaluation of the suitability of traditional anthropological methods for application on 3D models.
• This preliminary study evaluates whether macroscopic, 3D surface scans, and CT scans viewings generate different observations.
• The results indicate that the data are not always coherent across all three media of observation.
• Explanations include the aspect given to the bone by the 3D software, differences between handling bones in real life versus on a computer, and level of expertise of the observers.

     

Identification of adrafinil and its main metabolite modafinil in human hair. Self-administration study and interpretation of an authentic case

For several years, the misuse of stimulant substances is increasingly observed both in the field of sport, to improve the functions of the body and therefore to be more performant, and also by non-athletes to make life more tolerable on a daily basis. Adrafinil, 2-((diphenylmethyl)sulfinyl)-N-hydroxyacetamide, is a drug designed for the treatment of narcolepsy by promoting an awakened state, and to treat alertness and neurological symptoms in the elderly. It is primarily metabolized in vivo to an active form, i.e. modafinil, 2-((diphenylmethyl)sulfinyl)acetamide. The World Anti-Doping Agency (WADA) banned these two drugs in sports in 2004. The authors report an authentic case involving adrafinil and modafinil. The laboratory was requested to test for adrafinil in a hair strand collected from a woman found in possession of vials of adrafinil and suspected of trafficking. A specific method was developed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unlike modafinil (varying from 6.8 to 13.9 ng/mg), adrafinil was not identified in the strand. The interpretation of the results was difficult because this is the first case describing human hair analysis. In order to be able to interpret the results, a self-administration study was conducted after an oral administration to a volunteer (200 mg) whose beard hair was collected 10 days after administration. The analysis of this specimen highlighted the presence of adrafinil at 0.8 ng/mg and modafinil at 0.5 ng/mg. These results demonstrate the dual identification of both compounds after a single consumption, even after administration of a low dose. According to these results, the analysis of the hair strand from the authentic case does not match with a consumption of adrafinil, in accordance with abuse of modafinil alone. Intelligence considered that this was a trafficking case of adrafinil, with no self-consumption.     

Social media video analysis methodology for sarin exposure

As social media becomes increasingly ubiquitous, many events are recorded and released on social media platforms, including chemical weapon attacks. We develop an objective tool in order to evaluate brief and unstructured social media videos for analysing sarin exposure from a civilian medical pathology perspective. We developed and validated this new questionnaire using a standardized procedure that includes content domain specification, item pool generation, content validity evaluation, a pilot study, and assessment of reliability and validity. In total, 51 sarin attacks and 48 matched videos were analysed. Cronbach’s α for all 20 items was 0.75, which indicates adequate internal reliability. The test–retest reliability was 0.96, which indicates good internal reliability. The inter-observer intraclass correlation coefficient was 0.97. After verifying sampling adequacy with the Kaiser–Meyer–Olkin measure and the factorability of the items with Barlett’s test of sphericity, a factor analysis was performed. According to the principal axis factoring, a six-factor solution explained 51.86% of the total variance. The receiver-operating characteristic curve analysis showed that the Video Score Questionnaire has a sensitivity of 0.817, a specificity of 0.478, and an efficiency of 65.3. Therefore, the Video Score Questionnaire is reliable and valid for evaluating sarin attacks from brief and unstructured social media videos.

Key points

• Chemical weapons are still used as a method of warfare.
• Social media videos are an important source of information.
• We developed a validated scale which can analyse sarin exposure in short and unstructured videos.

     

Segmental determination of ethyl glucuronide in hair: a pilot study

Ethyl glucuronide (EtG) is a minor metabolite of ethanol. Its detection in hair is more and more studied in both clinical and forensic context for the purpose of alcohol abuse monitoring. In this pilot study, hair specimens from 15 patients included in a treatment program after alcohol abuse cessation, were segmented and analyzed for EtG. The results were then compared to their self-reported past alcohol consumption and to their blood biomarkers values (GGT, MCV, ASAT, ALAT). EtG concentrations measured in hair varied from 8 to 261 pg/mg. The pattern of EtG concentration detected in the different hair segments matched with the drinking history of patients, displaying variations (increase and decrease) in alcohol consumption and also time of cessation. Results also demonstrated the existence of a significant correlation (r(p)=0.5357; p=0.0390) between EtG concentration in hair and the amount of alcohol intake. Variations in the EtG concentrations with respect to hair segments may provide an overview of the drinking history of patients. Moreover, EtG concentration in hair may help to estimate the daily alcohol intake.     

Forensic isoscapes based on intra-individual temporal variation of δ18O and 206Pb/207Pb in human teeth

Isotopic signatures used in the georeferencing of human remains are largely fixed by spatially distinct geologic and environmental processes. However, location-dependent temporal changes in these isotope ratios should also be considered when determining an individual’s provenance and/or trajectory. Distributions of the relevant isotopes can be impacted by predictable external factors such as climate change, delocalisation of food and water sources and changes in sources and uses of metals. Using Multi-Collector Inductively-Coupled Plasma Mass Spectrometer (MC-ICP-MS) analyses of ²⁰⁶Pb/²⁰⁷Pb in tooth enamel and dentin from a population of 21 ± 1-year-old individuals born circa 1984 and isotope ratio mass spectrometry (IRMS) of δ¹⁸O in their enamel, we examined the expected influence of some of these factors. The resulting adjustments to the geographic distribution of isotope ratios (isoscapes) found in tooth enamel and dentin may contain additional useful information for forensic identification, but the shifts in values can also impact the uncertainty and usefulness of identifications if they are not taken into account.

KEY POINTS

• Isoscapes of ²⁰⁶Pb/²⁰⁷Pb and δ¹⁸O used for geolocation are not static.
• Within a few years, the enamel and dentin of a person may exhibit measurable differences in ²⁰⁶Pb/²⁰⁷Pb even without changing locations.
• Changes in climatic patterns tied to rising temperatures are more significant than the direct effect of increasing temperature on δ¹⁸O fixed in tooth bioapatite.
• Third molar (M3) enamel mineralisation includes material incorporated from before formal amelogenesis takes place.

     

Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system

With the development and maturation of massively parallel sequencing (MPS) technology, the mitochondrial genome (mitogenome) sequencing is increasingly applied in the forensic field. In this study, we employed the strategy of short overlapping amplicons for the whole mitogenome, library preparation with tagmentation using the Nextera® XT DNA Library Preparation Kit, sequencing on the MiSeq FGx^TM Forensic Genomics System and analyzing data using the mitochondrial(mtDNA) MSR Plug-in and the mtDNA Variant Analyzer. A total of 27 libraries and 56 libraries were sequenced in a run using MiSeq Reagent Kit v2 and v3, respectively. Results showed more than 1800 × of averaged depth of coverage (DoC) at each position. Concordant haplotypes of 9947 A and 2800 M were obtained at 32 variants. Cross-reactivity was observed with 1 ng primate DNA and 10 ng non-primate DNA but could be easily distinguished. Full and accurate variants were obtained from at least 50 pg input DNA and from minor contributors between 19:1 and 1:19 mixed ratios with known reference profiles. More than 86% variants were detected from ≥200-bp degraded samples but its haplotype was assigned to more ancestral haplogroup. Further, a total of 3 962 variants were observed at 613 nucleotide positions from 103 Xibe mitogenomes with 25:1 ratio of transitions to transversions. Two new transversions (C13735A and A14755C) and two tri-alleles at nps 9824 and 16092 were identified. There were 103 unique mitogenome haplotypes from 103 Chinese Xibe that were assigned to 79 haplogroups. Haplogroup D was the preponderant top-level haplogroup in Xibe followed by F, B, M, A, N, G, C, Z, Y, HV and J. Random match probability (RMP) and haplotype diversity (HD) of the whole mitogenome was calculated as 0.0097 and 1.0000, respectively. Compared with HVS-I only, RMP decreased 33.56%, while the number of haplotypes and HD increased 15.73% and 0.49%, respectively. Principal component analysis (PCA) showed that Xibe was clustered to East and Southeast Asian. As a whole, this MPS strategy is suitable for the whole mitogenome sequencing especially for degraded samples and can facilitate generating mitogenome data to support the routine application in forensic sciences. {"type":"entrez-protein","attrs":{"text":"EMP00726","term_id":"464458599"}}EMP00726 is the first whole mitogenome dataset from Xibe contributed to the EMPOP.Supplemental data for this article are available online at.     

Sex estimation in a Turkish population using Purkait’s triangle: a virtual approach by 3-dimensional computed tomography (3D-CT)

Sex estimation is considered one of the first steps in the forensic identification process. Morphological and morphometrical differences between males and females have been used as means for morphoscopic and metric methods on both cranial and postcranial skeletal elements. When dry skeletal elements are not available, virtual data can be used as a substitute. The present research explores 3-dimensional (3D) scans from a Turkish population to test a sex estimation method developed by Purkait (2005).Overall, 296 individuals were used in this study (158 males and 138 females). Purkait’s triangle parameters were measured on computed tomography (CT) scans obtained from both right and left femora of each patient at the Bakirkoy Dr. Sadi Konuk Training Research Hospital (Istanbul, Turkey). Intra- and inter-observer errors were assessed for all variables through technical error of measurements analysis. Bilateral asymmetry and sex differences were evaluated using parametric and non-parametric statistical approaches. Univariate and multivariate discriminant function analyses were then conducted.Observer errors demonstrated an overall agreement within and between experts, as indicated by technical error of measurement (TEM) results. No bilateral asymmetries were reported, and all parameters demonstrated a statistically significant difference between males and females. Fourteen discriminant models were generated by applying single and combined parameters, producing a total correct sex classification ranging from 78.4% to 92.6%. In addition, over 67% of the total sample was accurately classified, with 95% or greater posterior probabilities.Our study demonstrates the feasibility of 3D sex estimation using Purkait’s triangle on a Turkish population, with accuracy rates comparable to those reported in other populations. This is the first attempt to apply this method on virtual data and although further validation and standardisation are recommended for its application on dry bone, this research constitutes a significant contribution to the development of population-specific standards when only virtual data are available.

Key points

• CT analysis using Purkait’s triangle is a suitable tool for assessment of sex in unidentified individuals.
• The best overall estimation rate was achieved with the F11 model, with around 92% of accuracy.
• The results suggested 78.4% to 92.6% correct sex identification rates.
• More research is needed to expand the sample set and verify the results.

     

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.