Forensic Discrimination of Dyed Hair Color: I. UV‐Visible Microspectrophotometry*†

Abstract: Current protocols for examining hair do not attempt to differentiate hair color using instrumental analysis. In this study, hair samples treated with 55 different red hair dyes were analyzed using UV‐visible microspectrophotometry between 200 and 700 nm. Using air as a background reference gave the best results, although mounting media such as glycerin could also be used. The contribution of the hair substrate is predominantly observed in the range of 300–400 nm while the dye peak is evident in the range of 425–550 nm. It was found that the presence of hair dye reduces the overall intrasample variability of the hair color. In addition, visual inspection and spectral interpretation showed that dyed hair exhibits distinct and discernable shades. The color of all samples was stable during storage and while all hair dyes fade with washing, significant fading of the color was only evident after daily washing for 3 weeks.     

Identification of human hair stained with oxidation hair dyes by gas chromatographic-mass spectrometric analysis.

This paper describes the gas chromatographic-mass spectrometric (GCMS) analysis of oxidation hair dyes from human hair. Diamines from the dyes were directly extracted from the hair in basic solution and aminophenols were extracted after neutralization. Both extracts were derivatised with trifluoroacetic anhydride and analysed by GCMS. Five components of oxidation hair dyes namely, p-phenylenediamine, toluene-2,5-diamine, o-aminophenol, m-aminophenol and p-aminophenol were clearly identified, whilst no other compounds originating from the hair dyes were detected. The presence and relative amounts of these dye components from hair extracts may assist in the discrimination of human hair especially in cases involving forensic science.     

Surface-enhanced Raman spectroscopy enables confirmatory detection of dyes on hair submerged in hypolimnion water for up to twelve weeks

Difficulties in the localization of bodies of homicidal or drowning victims in natural water result in their submergence for weeks if not months. Water insects and microbes drastically change the body's appearance, which significantly changes the determination of a victim's identity. DNA analysis is commonly used for identifying the decedent; however, this PCR-based approach is time-consuming and destructive of the evidence. Considering that nearly half of the people in the world dye their hair with a variety of permanent and semi-permanent dyes, one can expect that confirmatory identification of dyes on the body's hair can be used to shed light on the victim's identity. A growing body of evidence suggests that surface-enhanced Raman spectroscopy (SERS) can be used to detect and identify hair dyes. In this study, we investigated the extent to which SERS could be used to detect black and blue, permanent and semi-permanent dyes on hair submerged in hypolimnion water for up to twelve weeks. We found that SERS enabled 100% accurate identification of analyzed dyes on hair submerged in hypolimnion water for up to 8 weeks, whereas, on average, 87% accurate identification of the hair dyes could be achieved on hair exposed for 10 weeks and 50% for hair exposed 12 weeks in hypolimnion water. We also found that the aqueous environment caused progressive fading of some dyes, whereas other dyes showed substantial spectral transformations after prolonged submergence. Finally, we found that changes in the intensity of vibrational bands of dyes could be used to predict the duration of submergence of colored hair in hypolimnion water.     

Elucidation of the effect of heat exposure on hair colored by permanent and semipermanent colorants using surface-enhanced Raman spectroscopy

Confirmatory identification of hair colorants can be used to establish a connection between a suspect and the crime science or demonstrate the absence of such connections. A growing body of evidence shows that surface-enhanced Raman spectroscopy (SERS) could be a confirmatory, minimally destructive, and fully noninvasive analysis of hair colorants. In SERS, a signal that provide the information about the chemical structure of both permanent and semipermanent dyes present on hair is enhanced by a million-fold using noble metal nanostructures. However, it is unclear whether the information of hair colorants can be revealed if hair was contaminated or exposed to harsh environments such as sunlight and heat. In this work, we determine the effect of a short- and long-term heat exposure on SERS-based analysis of hair colored with blue and red permanent and semipermanent dyes. We found that short and especially long-term heat exposure at 220°C could alter chemical structure, and consequently SERS spectra, of permanent and semipermanent colorants. This thermal degradation of permanent dyes complicates their direct identification using SERS. We also found that partial least squares discriminant analysis can be used to overcome this issue allowing for highly accurate identification of both permanent and semipermanent dyes on colored hair that was exposed to 220°C for 6–12 min. These results show that heat exposure of colored hair should be strongly considered upon their SERS-based examination to avoid both false positive or false negative identification of chemical dyes.     

High-performance liquid chromatography-ultraviolet-visible spectroscopy-electrospray ionization mass spectrometry method for acrylic and polyester forensic fiber dye analysis

A critical point of comparison between a fiber collected from a crime scene and a fiber from a known source is the color. Fiber dye analysis using thin-layer chromatography or ultraviolet (UV)-visible (Vis) microspectrophotometry provides useful, although limited, data for comparison. High-performance liquid chromatography-electrospray ionization mass spectrometry (LC/MS) overcomes these limitations by integrating chromatography, ultraviolet-visible spectroscopy, and mass spectrometry into a single instrument. In order to evaluate the applicability of the LC/MS to forensic fiber dye analysis, a multi-stage chromatographic method using acidified water and acidified acetonitrile was developed that separated and identified a mixture of 15 basic and 13 disperse dye standards. The LC/MS also detected and analyzed dyes extracted from individual 0.5 cm acrylic and polyester fibers, demonstrating its applicability to this type of analysis. With regard to the analysis of disperse dyes in polyester fibers, the replacement of pyridine with acetonitrile in the extraction system allowed direct injection of the extracts into the LC/MS. The advantage of the LC/MS over other instrumental methods of textile dye analysis is demonstrated by the analysis and differentiation of three black acrylic fibers: two fibers had similar UV-Vis spectra but were differentiated with chromatography and two had similar UV-Vis spectra and chromatograms but were differentiated using the mass spectrometer.     

Quantitative Determination of Cannabinoids in Cannabis and Cannabis Products Using Ultra‐High‐Performance Supercritical Fluid Chromatography and Diode Array/Mass Spectrometric Detection

Ultra‐high‐performance supercritical fluid chromatography (UHPSFC ) is an efficient analytical technique and has not been fully employed for the analysis of cannabis. Here, a novel method was developed for the analysis of 30 cannabis plant extracts and preparations using UHPSFC /PDA ‐MS . Nine of the most abundant cannabinoids, viz . CBD , ?⁸‐THC , THCV , ?⁹‐THC , CBN , CBG , THCA ‐A, CBDA , and CBGA , were quantitatively determined (RSD s < 6.9%). Unlike GC methods, no derivatization or decarboxylation was required prior to UHPSFC analysis. The UHPSFC chromatographic separation of cannabinoids displayed an inverse elution order compared to UHPLC . Combining with PDA ‐MS , this orthogonality is valuable for discrimination of cannabinoids in complex matrices. The developed method was validated, and the quantification results were compared with a standard UHPLC method. The RSD s of these two methods were within ±13.0%. Finally, chemometric analysis including principal component analysis (PCA ) and partial least squares‐discriminant analysis (PLS ‐DA ) were used to differentiate between cannabis samples.     

GC‐MS and GC‐MS/MS in PCI Mode Determination of Mescaline in Peyote Tea and in Biological Matrices

Peyote, a cactus containing the hallucinogen mescaline, is used to induce altered states of consciousness in religious ceremonies or for recreational purpose. This study reports a case of an underage boy suspected of mescaline abuse. For this purpose, we analyzed a dark green liquid sample found in the bedroom of the boy whose urine and hair samples were collected shortly after the drink was found. A method by gas chromatography‐mass spectrometry tandem mass spectrometry (GC‐MS/MS) in positive chemical ionization mode was developed and validated in terms of linearity, specificity, accuracy, and sensitivity for mescaline determination at the low concentrations present in hair. GC‐MS analysis of the liquid identified mescaline, while urine was negative; GC‐MS/MS segmental hair analysis identified mescaline in the proximal segment (root to 2 cm), while the distal segments were negative. Although peyote was uncommonly encountered, its use was confirmed by segmental hair analysis that can provide long‐term information about drugs use.     

Raman spectroscopy and microspectrophotometry of reactive dyes on cotton fibres: Analysis and detection limits

A collaborative study on Raman spectroscopy and microspectrophotometry (MSP) was carried out by members of the ENFSI (European Network of Forensic Science Institutes) European Fibres Group (EFG) on different dyed cotton fabrics. The detection limits of the two methods were tested on two cotton sets with a dye concentration ranging from 0.5 to 0.005% (w/w). This survey shows that it is possible to detect the presence of dye in fibres with concentrations below that detectable by the traditional methods of light microscopy and microspectrophotometry (MSP). The MSP detection limit for the dyes used in this study was found to be a concentration of 0.5% (w/w). At this concentration, the fibres appear colourless with light microscopy. Raman spectroscopy clearly shows a higher potential to detect concentrations of dyes as low as 0.05% for the yellow dye RY145 and 0.005% for the blue dye RB221. This detection limit was found to depend both on the chemical composition of the dye itself and on the analytical conditions, particularly the laser wavelength. Furthermore, analysis of binary mixtures of dyes showed that while the minor dye was detected at 1.5% (w/w) (30% of the total dye concentration) using microspectrophotometry, it was detected at a level as low as 0.05% (w/w) (10% of the total dye concentration) using Raman spectroscopy. This work also highlights the importance of a flexible Raman instrument equipped with several lasers at different wavelengths for the analysis of dyed fibres. The operator and the set up of the analytical conditions are also of prime importance in order to obtain high quality spectra. Changing the laser wavelength is important to detect different dyes in a mixture.     

The importance of thin layer chromatography and UV microspectrophotometry in the analysis of reactive dyes released from wool and cotton fibers

Samples of reactively-dyed wool and cotton were obtained from a range of dye manufacturers, dye distributors and the Forensic Science Service (FSS) Fibre Data Collection. The wool fibers were red in color and had previously been compared using comparison microscopy (CM), visible range microspectrophotometry (VS) and thin layer chromatography (TLC). The cotton fibers were blue and black in color and had not been previously compared. Red, blue and black fibers were chosen because they are often encountered in casework. The usage of reactive dyes to color fibers has increased over the last 10-15 years and these are often seen in casework. Before techniques were available that allowed reactively-dyed fibers to be compared using TLC only CM and microspectrophotometry were routinely carried out. Many laboratories, who had a microspectrophotometer, only had a visible range instrument. It was therefore important to see which techniques provide additional information, that gives greater individuality to fibers, to that obtained from CM. The color was released from the wool and cotton fibres using alkaline hydrolysis and a cellulase enzyme respectively. Many of the red wool samples were differentiated from each other using CM. More differentiation was found using VS and even more when ultraviolet range microspectrophotometry (UV) or TLC was used. Two samples could only be differentiated using TLC because CM, VS and UV failed to separate them. The black cotton samples were predominately differentiated using CM but VS allowed for further differentiation. With the samples used in this project UV and TLC failed to separate the samples further. The blue cotton samples benefited from the use of CM, VS and either UV or TLC to reduce the number of matching pairs. All techniques aided differentiation although with this set TLC and UV proved to be complementary techniques. Results demonstrate that TLC and UV both yield important information over and above that obtained from CM and VS. Although in some parts of the project TLC and UV are complementary if the concentration of the dye in the fiber is not sufficient for TLC or the scientist doesn't wish to 'destroy' the fiber UV would be of more use than TLC.     

Determination of phenotype associated SNPs in the MC1R gene

Prediction of physical appearance based on genetic analysis is a very attractive prospect for forensic investigations. Recent studies have proved that there is a significant association between some genetic variants of the melanocortin 1 receptor (MC1R) gene and red hair color. The present study focuses on the potential forensic applicability of variation within this pigment-related gene. Sequencing of the complete MC1R gene was performed on a group of red-haired individuals and controls with different pigmentation. A major role in determination of red hair color is played by two MC1R variants--C451T and C478T. The optimized minisequencing assay for genotyping of the above positions and three other important red hair-related MC1R polymorphisms, C252A, G425A, and G880C was successfully applied to analyze typical forensic specimens. Determination of a homozygous or heterozygous combination can be a good predictor of both red hair color and fair skin of a subject.     

Source Determination of Red Gel Pen Inks using Raman Spectroscopy and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy combined with Pearson's Product Moment Correlation Coefficients and Principal Component Analysis

The potential combination of two nondestructive techniques, that is, Raman spectroscopy (RS) and attenuated total reflectance–fourier transform infrared (ATR‐FTIR) spectroscopy with Pearson's product moment correlation (PPMC) coefficient (r) and principal component analysis (PCA) to determine the actual source of red gel pen ink used to write a simulated threatening note, was examined. Eighteen (18) red gel pens purchased from Japan and Malaysia from November to December 2014 where one of the pens was used to write a simulated threatening note were analyzed using RS and ATR‐FTIR spectroscopy, respectively. The spectra of all the red gel pen inks including the ink deposited on the simulated threatening note gathered from the RS and ATR‐FTIR analyses were subjected to PPMC coefficient (r) calculation and principal component analysis (PCA). The coefficients r = 0.9985 and r = 0.9912 for pairwise combination of RS and ATR‐FTIR spectra respectively and similarities in terms of PC1 and PC2 scores of one of the inks to the ink deposited on the simulated threatening note substantiated the feasibility of combining RS and ATR‐FTIR spectroscopy with PPMC coefficient (r) and PCA for successful source determination of red gel pen inks. The development of pigment spectral library had allowed the ink deposited on the threatening note to be identified as XSL Poppy Red (CI Pigment Red 112).     

The Discrimination of Colored Acrylic,Cotton, and Wool Textile Fibers Using Micro‐Raman Spectroscopy. Part 1: In situ Detection and Characterization of Dyes

Raman spectroscopy has been applied to characterize fiber dyes and determine the discriminating ability of the method. Black, blue, and red acrylic, cotton, and wool samples were analyzed. Four excitation sources were used to obtain complementary responses in the case of fluorescent samples. Fibers that did not provide informative spectra using a given laser were usually detected using another wavelength. For any colored acrylic, the 633‐nm laser did not provide Raman information. The 514‐nm laser provided the highest discrimination for blue and black cotton, but half of the blue cottons produced noninformative spectra. The 830‐nm laser exhibited the highest discrimination for red cotton. Both visible lasers provided the highest discrimination for black and blue wool, and NIR lasers produced remarkable separation for red and black wool. This study shows that the discriminating ability of Raman spectroscopy depends on the fiber type, color, and the laser wavelength.     

Application of Raman Spectroscopy and Surface‐Enhanced Raman Scattering to the Analysis of Synthetic Dyes Found in Ballpoint Pen Inks*

Abstract: The applicability of Raman spectroscopy and surface‐enhanced Raman scattering (SERS) to the analysis of synthetic dyes commonly found in ballpoint inks was investigated in a comparative study. Spectra of 10 dyes were obtained using a dispersive system (633 nm, 785 nm lasers) and a Fourier transform system (1064 nm laser) under different analytical conditions (e.g., powdered pigments, solutions, thin layer chromatography [TLC] spots). While high fluorescence background and poor spectral quality often characterized the normal Raman spectra of the dyes studied, SERS was found to be generally helpful. Additionally, dye standards and a single ballpoint ink were developed on a TLC plate following a typical ink analysis procedure. SERS spectra were successfully collected directly from the TLC plate, thus demonstrating a possible forensic application for the technique.     

Structure elucidation of dyes that are formed in the colorimetric detection of the improvised explosive urea nitrate

Urea nitrate (uronium nitrate, UN) is a powerful, improvised explosive that can be easily made from urea and nitric acid. It is considered the most frequently used, illegal explosive in the Israeli arena, which is responsible for the loss of more than a hundred lives in terrorist incidents. Urea nitrate is a colorless, crystalline substance that looks very much like sugar. A sensitive color test for UN was developed recently. It is based on the formation of a red dye in the reaction between p-dimethylaminocinnamaldehyde and UN under neutral conditions. A similar reaction with p-dimethylaminobezaldehyde produces a yellow dye. The two dyes have been synthesized, and their structures determined by X-ray crystallography. Both dyes are protonated Schiff bases, prevailing in the crystal in a quinoid form. They are identical to the compounds, which are obtained in the colorimetric detection of urea with the same reagents, under strong acidic conditions, whose structures have been postulated in the literature, but never fully proved experimentally.     

Differentiation of unevaporated gasoline samples according to their brands, by SPME-GC-MS and multivariate statistical analysis

One of the aims of fire investigations is to identify associations among accelerants according to their source. In this study, 50 gasoline samples--representing five brands--were analyzed using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). Chemometric procedures, such as principal component analysis (PCA) and discriminant analysis (DA), were applied to a data matrix obtained by the target compound chromatogram method, to discriminate samples according to their brand. PCA was successful in finding a natural grouping of samples according to their brand, suggesting that aromatic compounds were more useful than aliphatics for the purpose of this study. DA, if applied to aromatic compounds, gave both a classification ability and a prediction ability of 100%. The outstanding results obtained by this work provide the basis of a data matrix that could be used in real cases of arson to link a sample of unevaporated gasoline to its brand or refinery.     

毛发中GHB的检测及评价

目的探索毛发中外源性GHB的检测及判断的可行性,为涉GHB的鉴定提供方法和依据。方法建立毛发中GHB的GC/MS分析方法,并通过动物实验,考察毛发中内源性GHB的质量分数范围、外源性GHB在毛发中的时间过程以及给药剂量、毛发颜色与毛发中GHB的质量分数关系。结果豚鼠和中国人黑色毛发中内源性GHB质量分数分别为(3.01±1.41)ng/mg(n=28)和(1.02±0.27)ng/mg(n=20);摄GHB后毛发中GHB质量分数明显增加且与给药剂量呈正相关性;GHB在毛干中呈窄带分布;深色毛发中GHB质量分数高于浅色毛发。结论毛发中GHB的检测适用于GHB滥用和中毒的法医毒物学鉴定;根据毛发中的GHB质量分数和毛发分段分析可判断GHB的来源。     

A Radiographic Study on the Utility of Cranial Vault Outlines for Positive Identifications

A standard method for positive identification is the use of antemortem and postmortem radiographic comparisons. The purpose of this research is to test the visual accuracy of antemortem and postmortem radiographic comparisons of cranial vault outlines and to evaluate their uniqueness using geometric morphometric methods. A sample of 106 individuals with varying levels of education and forensic case experience participated in a visual accuracy test. Of the 106 individuals, only 42% correctly assigned all of the radiographs, with accuracy rates ranging from 70 to 93% for each radiographic comparison. Vault shape was further examined using elliptic Fourier analysis, and paired t‐tests were computed on the first 10 principal components accounting for 100% of the variance, which found no significant differences. The visual accuracy test and elliptic Fourier analysis shows that vault outlines may not be unique enough for positive identifications when used as a sole indicator.     

X‐ray Diffraction and Rietveld Refinement in Deferrified Clays for Forensic Science

Soil vestiges might provide information about a crime scene. The Rietveld method with X‐ray diffraction data (RM‐XRD) is a nondestructive technique that makes it possible to characterize minerals present in the soils. Soil clays from the metropolitan region of Curitiba (Brazil) were submitted to DCB treatment and analyzed using XRD with CuK_α radiation in the step‐scan mode (0.02° 2θ/5 s). The GSAS+EXPGUI software was used for RM refinement. The RM‐XRD results, together with the principal component analysis (PCA) (52.6% total variance), showed the kaolinite predominance in most analyzed samples and the highest quartz contents in “site 1.” Higher anatase, and gibbsite and muscovite contents influenced discrimination, mainly in “site 3” and “site 1,” respectively. These results were enough to discriminate clays of four sites and two horizons using a reduced amount of sample showing that the technique can be applied to the investigation into soil vestiges.     

Rapid and non-destructive approach for characterization and differentiation of sealing wax using ATR-FTIR spectroscopy

Sealing wax is used for maintaining the integrity and authenticity of a document or physical evidence. Any tampering with the seal calls into question the overall integrity and authenticity of the tangible evidence or document. In these circumstances, determining the authenticity of the sealing material (physical and chemical) becomes imperative. In this study, ATR-FTIR spectroscopy supported by chemometrics has been used to differentiate sealing wax samples belonging to 12 different brands available across India. All the samples were first melted, cooled, and then analyzed using ATR-FTIR spectroscopy in the mid-infrared region (4000–600 cm⁻¹). The obtained spectra were first examined visually for the presence of different functional groups. Principal component analysis (PCA) and principal component analysis-linear discriminant analysis (PCA-LDA) were employed to analyze the sample clustering patterns and to categorize them into their respective groups, respectively. For classification, a PCA-LDA training model was applied, and it demonstrated 95.83% accuracy. The validation test resulted in an accuracy of 83.33%. PCA-LDA model offered 100% accurate prediction for samples on various substrates, including cloth, cardboard, and paper. A blind study was also performed using five unknown samples, which were accurately classified into their respective groups. PCA-LDA model will be helpful in providing investigative leads by linking a questioned sealing wax sample with its respective group.