Application of dispersive liquid-liquid microextraction and CE with UV detection for the chiral separation and determination of the multiple illicit drugs on forensic samples

A new method was developed for pre-concentration, chiral separation and determination of multiple illicit drugs on forensic samples using dispersive liquid-liquid microextraction (DLLME) and capillary electrophoresis (CE) with Ultra Violet (UV) 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 heroin, DL-methamphetamine, DL-3, 4-methylenedioxymethamphetamine and dl-ketamine from the prepared sample solution, was separated by centrifuging. 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 aqueous solution (internal standard) and introduced by electrokinetic injection into CE. Parameters affecting extraction efficiency were investigated and optimized. Under optimum conditions, linearity of the method was 0.15-6500 μg/L for all target analytes. The LODs (S/N=3) were 0.05-0.20 μg/L. Excellent repeatability (RSD ≤ 4.4%, n=5) was achieved. The feasibility of this method was demonstrated by analyzing spiked forensic samples. To our knowledge, it is the first time to combine DLLME with CE for chiral separation and determining illicit drugs on forensic samples.     

Segmental hair analysis for flunitrazepam and 7-aminoflunitrazepam in users: a comparison to existing literature

The availability of more quantitative data on flunitrazepam (FLU) and 7-aminoflunitrazepam (7AF) would aid in obtaining a better understanding of the interpretation of FLU concentrations in human hair. The purpose of this study was to provide concentrations of FLU and 7AF in hair segments of 22 FLU users. Quantitative data regarding hair concentrations of FLU and 7AF from various types of cases were also reviewed to give a comprehensive overview of the comparability of different studies. Three to six 1 cm segments of scalp hair from 22 FLU users were analyzed by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. FLU and its metabolite were confirmed in the hair segments from all cases. Concentrations of FLU and 7AF in the segments ranged from 0.01–0.16 ng/mg (median of 0.03) and 0.01–0.34 ng/mg (median of 0.09), respectively. Most cases had FLU and 7AF distributions along the hair segments that were suggestive of repeated drug use. A summary of the published concentrations gives valuable data and can assist forensic investigators in their estimations of drug use history and patterns.

Key points

• A method using LC–MS/MS to quantify flunitrazepam and its metabolite was described.
• Segmental analysis of flunitrazepam and its metabolite in human hair was reported.
• A comprehensive overview of quantitative data was given.

     

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.

     

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.

     

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.     

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 solvent microextraction to the analysis of amphetamines and phencyclidine in urine

A fast and simple method to detect some commonly abused illicit drugs, amphetamine, methamphetamine, 3,4-methylendioxy-amphetamine (MDA), 3,4-methylendioxy-methamphetamine (MDMA), 3,4-methylendioxy-N-ethylamphetamine (MDEA) and phencyclidine (PCP) in urine using solvent microextraction (SME) combined with gas chromatography (GC) analysis has been developed. The extraction is conducted by suspending a 2 μl drop of chloroform in a 2 ml urine sample. Following 8 min of extraction, the organic solvent is withdrawn into the syringe and injected into a GC with a pulsed discharge helium ionization detector (PDHID).The effects of different extraction solvents and times, pH and sample preparation were studied. The optimized method was capable of detecting drugs in urine at concentrations below Substance Abuse and Mental Health Services Administration (SAMHSA) established cut-off values for preliminary testing. Good linearity and reproducibility of extraction were obtained. The limits of detection were 0.5 μg/ml for amphetamine, 0.1 μg/ml for methamphetamine and MDA, 0.05 μg/ml for MDMA, 0.025 μg/ml for MDEA and 0.015 μg/ml for PCP. Relative standard deviation (R.S.D.) values ranged between 5 and 20% for the studied drugs.     

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.

     

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.     

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.

     

Sensory information in children’s statements of sexual abuse

The credibility of children’s statements of sexual abuse is a controversial issue in forensic psychiatry and psychology. Neurobiological and clinical laboratory studies show that real memories contain more information regarding sensory details than false memories. The goal of the present field study was to evaluate whether sensory information was present in children’s statements of sexual abuse, and whether this information was more often present in credible statements compared with non-credible statements. Sensory details were extracted from a sample of 96 statements of sexual abuse from children; 62 statements were considered credible and 34 statements were considered non-credible. This study showed that sensory information was present in 79% of children’s reports of child sexual abuse. Sensory information was significantly more often present in statements considered credible compared with non-credible statements (85.5%, P < 0.001), but there were large variations in the sense involved. Logistic regression analysis showed that the presence of at least one sensory detail may be a good predictor of credibility (odds ratio, OR = 23.484, P < 0.05). It seems appropriate to include sensory details when assessing the credibility of children’s statements of child sexual abuse, but it has not yet been demonstrated that use of such details significantly improves the validity of credibility assessments.KEYWORDS: Forensic sciences, forensic psychiatry, forensic psychology, credibility, sensory information, children’s statements, sexual abuse     

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.

     

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.

     

Analysis of Ecstasy Tablets Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

A method for the identification of 3,4‐methylenedioxymethamphetamine (MDMA) and meta‐chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D). Sample extraction, separation, and detection of “Ecstasy” tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE‐C⁴D and compared against routine gas chromatography‐mass spectrometry (GC‐MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C⁴D instead of traditional CE‐UV methods for in‐field analysis are also discussed.     

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.

     

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.

     

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.     

DNA analysis of the last Brazilian unknown soldier’s remains buried in Pistoia (Italy)

During World War II, many nations took part in the war. Among the supporters of the Alliance there was also Brazil. In August 1944, under the leadership of President Getúlio Vargas, Brazil declared war on Nazi Germany and took part in the Italian campaign by sending many troops to support the Allies in the Central Italy. Once the conflict was over, the deceased Brazilian soldiers were buried in Pistoia, a few kilometers from Florence. But only in 1960 the Brazilian government authorized the transfer of the dead soldiers to their homeland. Five years later, during the building of the Brazilian Military Votive Monument, still in the Pistoia cemetery, a last body was found but could not be identified: so he was buried as an “unknown soldier”. In December 2012, the Brazilian Embassy in Italy asked for performing forensic genetics analysis for identification purposes on the remains of this last unknown Brazilian soldier. After almost 70 years a complete short tandem repeats (STR) profile was obtained, useful for any relatives searching.Key points:

• Identification of the last Brazilian Unknown Soldier buried in Italy.
• DNA analysis on 70 years old skeletal remains.
• Brazilian soldier’s history during World War II.

     

Simultaneous quantification of opiates, cocaine and cannabinoids in hair

The present paper describes a sensitive method developed in our laboratory for the simultaneous analysis of opiates (morphine, codeine and monoacetylmorphine), cocainics (cocaine and benzoylecgonine) and cannabinoids (Δ⁹-tetrahydrocannabinol and 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid) in hair samples. After decontaminating the sample with dichloromethane, two consecutive hydrolyses were performed in order to achieve the best conditions for extracting the three kinds of drugs from the protein matrix. First the opiate and cocainic compounds were extracted by means of a soft acidic hydrolysis with 0.1 N HCl at 50 °C overnight and organic solvent extraction at pH 9.2. The cannabinoids need a stronger basic hydrolysis with 11.8 N KOH for 10 min at laboratory temperature. After adding maleic acid, the cannabinoids were extracted with an organic solvent. The derivatization was carried out with heptafluorobutyric anhydride and hexafluoropropanol. Calibration curves were linear between 0.5–100 ng/mg of hair. Recovery and reproducibility were assured. The quantification limits ranged between 0.04–0.26 ng/mg of hair. Seventy hair samples from known drug abusers were cut into 1-cm segments and analyzed by this method. The ranges of measured concentrations (ng/mg) were 0.31–89 for cocaine, 0.1–5.76 for benzoylecgonine, 0.34–45.79 for morphine, 0.45–39.59 for codeine, 0.09–48.18 for monoacetylmorphine, 0.06–7.63 for THC and 0.06–3.87 for THC---COOH. The results of sectional analyses agreed with the self reported drug histories. The usefulness of this method is in assessing earlier drug consumption, and also at the same time obtaining a chronological profile of the consumption of these three types of drugs.     

Determination of γ-hydroxybutyrate (GHB) and its precursors in blood and urine samples: A salting-out approach

γ-Hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose–response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 μL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na₂SO₄, incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC–MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC–FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.