Increasing numbers of drug-facilitated sexual assaults (DFSAs) have been reported recently and have become a social concern. In DFSAs, biological specimens (urine, blood, and hair) are analyzed to prove the victim's drug exposure, which can lead to proof of the sexual crime. Hair is the only specimen that can provide firm evidence of drug ingestion in cases of long delays (more than a week) in reporting the crime. Furthermore, detailed sectional hair analysis of a single hair strand enables to estimate the victim's drug-use history (date and amount of intake). Several recent studies have demonstrated high-sensitivity methods using mass spectrometry to detect sub-pg/mg concentrations of hypnotics in hair, and additionally illustrated the incorporation pathways of drugs and detailed distribution patterns in the hair after intake. Based on these findings, hair testing for hypnotics has been put to practical applications since 2016. In this paper, we review the practical concepts and usefulness of hair testing for hypnotics while introducing the current situation of DFSAs.
In this study, the excretion profiles of etizolam (EZ) and its main metabolites including conjugates in urine have been investigated to obtain fundamental information for proving the drug intake in the cases of drug-related crimes. Urine specimens were collected from three volunteers during about 200 h after a single oral ingestion of EZ (1 mg). Three glucuronides (including diastereomers) of 8-α-hydroxyetizolam (EZ-M3) and 1-hydroxyetizolam (EZ-M6) along with free EZ-M3 and EZ-M6 were detected for the first time directly as metabolites of EZ using liquid chromatography-tandem mass spectrometry with a C18 semi-micro column. EZ, EZ-M3 and EZ-M6 were quantified using the authentic standards, and urinary concentrations of the glucuronides were calculated based on the increased amounts of EZ-M3 and EZ-M6 after enzymatic hydrolysis. The quantitative results demonstrated that the ratios of amounts of EZ-M3 and EZ-M6 including their glucuronides excreted in urine to the dose ranged about 18-22% and 9.5-14%, respectively, although that of the unchanged EZ was less than 0.3%. The glucuronide of EZ-M6 reached the highest concentration (more than 200 ng/mL) of metabolites in the early period (within 12 h) after intake. However, in a latter period (24 h or later) after intake, the concentration of EZ-M3 or its glucuronides was higher than that of glucuronide of EZ-M6 in most of the samples. The conjugation percentages of EZ-M3 and EZ-M6 were calculated to be approximately 50% and 98% or higher, respectively. This study suggested that the detectable duration of EZ-M3 (approximately one week after intake, Limit of detection: 0.05 ng/mL) is longer than those of EZ and EZ-M6. In addition, the enzymatic hydrolysis prior to analysis is expected to prolong the detectable duration of EZ-M3 slightly.
Blood acetaldehyde levels are often measured to elucidate individual differences and pharmacokinetics of alcohol metabolism due to the gene polymorphisms of aldehyde dehydrogenase (ALDH2). Blood acetaldehyde levels are of great interest in the field of addictive behaviors, particularly drunk driving behaviors. Conventional blood acetaldehyde concentration measurements are direct and derivatization analyses. Because direct analysis is problematic in that acetaldehyde disappears during storage due to its high volatility and reactivity, derivatization analysis is often performed for acetaldehyde determination in biological samples. The conventional derivatizing method using 2,4-dinitrophenylhydrazine for determination of aldehydes unfortunately produces interfering substances from ketones and carboxylic acids. Therefore, we applied the derivatization method using 9,10-phenanthrenequinone (PQ), reported recently for the interference-free determination of aldehydes. As a result of optimizing the derivatization conditions, a linear calibration relation-ship (R2=0.9994) in the range of 3-100 μM indicated strong results. The limit of detection and quantification was 1 μM and 3 μM, respectively. The intra- and inter-day precision were both below 13.0% and accuracy was in the range of −8.5% to 2.5%. The extraction recoveries of acetaldehyde in human plasma ranged from 80.4% to 106.8%. The derivative was stable at 4 ℃ for 2 days. When acetaldehyde was added in human plasma and derivatized with PQ, acetaldehyde could be quantified by liquid chromatography-mass spectrometry. However, after drinking alcohol, acetaldehyde was measured in healthy subjects with ALDH2*1/*2, but not with ALDH2*1/*1. Acetaldehyde was difficult to detect in subjects with ALDH2*1/*1, as they are rapid metabolizers of acetaldehyde resulting in low concentrations of acetaldehyde. Acetaldehyde was detected in those subjects with ALDH2*1/*2, however, the measured acetaldehyde levels varied between the analytical methods.
The concealed information test (CIT) is a psychophysiological memory-detection technique. The judgment to assess whether an examinee recognizes crime-relevant information is based on the difference in autonomic responses between the crime-relevant information and the crime-irrelevant information. The present study examined the within-questionnaire consistency of the differential physiological responses, which is a key point in the above judgment. The mock theft CIT data obtained from 167 participants were modeled into three possible models, and Bayesian estimation of parameters and model selection were performed. Results of model selection implied that participants in the encoding group were not clearly divided into high consistency responders and non-responders. Bayesian estimates of parameters revealed that the within-questionnaire consistency of the differential physiological responses had different characteristics among the physiological measures. Most participants showed high consistency for heart rate and respiratory speed, while some participants show low consistency for skin conductance response, respiratory amplitude, and respiratory rate.
DNA quantification is an important process in DNA typing. Today, quantitative real-time PCR (qPCR) targeting the autosomal D17Z1 region, which cannot distinguish between male and female DNA, is the main method for forensic human DNA quantification for the Japanese police. For more efficient testing Y-STR type, we examined the forensic evaluation (sensitivity, repeatability, specificity, inhibition, comparison with commercial qPCR reagent and simulated mixture study) of qPCR assays targeting Y-chromosomal Sex determining region Y (SRY) and DYZ5 regions. We evaluated the effectiveness of simple and practical male DNA quantification methods in male and female mixture samples. The sensitivity of SRY and DYZ5 assays were 0.015 ng/μL and 0.0015 ng/μL. Both assays could quantify male DNA in the presence of high concentrations of female DNA. The SRY assay had the same sensitivity as the commercial qPCR reagent, but had more resistant to inhibitors. The DYZ5 assay tended to have higher DNA concentration than the commercial qPCR reagent and the SRY assay, but could detect trace amounts of male DNA. The reason for this is that DYZ5 region is multi-copy locus and there are individual differences in the copy number. The DYZ5 assay was considered to be one of the effective methods for proving the presence of male DNA in the sample. Therefore, we consider that these assays can select the regions according to purpose and utilized to forensic applications.
Non-destructive discrimination of white paper sheets of 9 different notepads was tried by analyzing the color of the images. All of the paper sheets of these notepads were scanned to obtain reflection, transmission, and fluorescence images using the scanner with multiple light sources. The scanning condition was 300 ppi and 24 bit RGB color, namely 256 gradations. We examined the brightness values of the R, G, and B channels in the width of 640 pixels and the height of 1020 pixels, a card size, from each of the obtained three images.
Since the brightness values were uneven within one paper, the most frequent values of those were used for comparison of the notepads. The most frequent values were nine kinds for one paper because each of the obtained three images had three channels. The most frequent values had values with a range in one notepad. Therefore, those ranges were nine kinds for one notepad.
As a result, comparing nine ranges of the most frequent values of one notepad with other notepads, 9 different notepads could be discriminated from each other by some of nine ranges. Thus, this study indicates that the image analysis using the most frequent values of the brightness values is useful for non-destructive discrimination of white paper sheets in forensic science.
We previously typed Y-STRs of 1299 Japanese samples using the Yfiler Plus kit and found three cases with a low peak of DYS533. In this study, we sequenced the flanking regions of DYS533 in these three samples and found a single nucleotide substitution at the position 111 bp upstream from the DYS533 repeat region. Because our previous study revealed that the Y-chromosomes of these three samples belong to haplogroup D-M57*, the mutation of the single nucleotide is likely to have occurred on the common ancestral Y-chromosome of these samples. The single nucleotide substitution may have caused the low peak of DYS533 in the three samples.
Saliva presumptive tests, which use the blue starch agarose plate method, are simple and sensitive tests; however, they are not suitable for crime scene investigations or urgent cases. The aim of this study was to evaluate the forensic application of a 2-chloro-4-nitrophenol (CNP)-based saliva presumptive test using the α-amylase assay kit which is a commercial brewing analysis kit. The sensitivity and specificity of the saliva presumptive test were validated using diluted saliva stains and various body fluid stains. The forensic applicability of the test was evaluated using mock forensic samples to compare results from the CNP-based method and blue starch agarose plate method. The α-amylase activity was qualitatively detected from diluted saliva stains (4 μL) and different donor-derived saliva stains (n=21) after reaction at room temperature for 10 min. All mock forensic samples produced concordant results from the two saliva presumptive tests. Our results indicate that the CNP-based saliva presumptive test combined with the α-amylase assay kit is rapid, simple, and useful for on-site examination.
Cable tie is one type of evidence submitted to forensic science laboratories, due to its potential as a tool used in crime. In this report, ten commercially available cable tie samples were analyzed and compared to study their potential for forensic discrimination. Samples were first evaluated through examination of their physical properties, shape, size, and traces from the mold. As a result of these examinations, 9 groups of the possible comparison pairs were discriminated. The chemical compositions of the samples were subsequently characterized through the use of infrared spectroscopy and matrix assisted laser desorption ionization mass spectrometry. With this result, it was possible to discriminate the pair that could not originally be discriminated.