A Generalized Approach to Forensic Dye Identification: Development and Utility of Reference Libraries.

While color is arguably the most important optical property of evidential fibers, the actual dyestuffs responsible for its expression in them are, in forensic trace evidence examinations, rarely analyzed and still less often identified. This is due, primarily, to the exceedingly small quantities of dye present in a single fiber as well as to the fact that dye identification is a challenging analytical problem, even when large quantities are available for analysis. Among the practical reasons for this are the wide range of dyestuffs available (and the even larger number of trade names), the low total concentration of dyes in the finished product, the limited amount of sample typically available for analysis in forensic cases, and the complexity of the dye mixtures that may exist within a single fiber. Literature on the topic of dye analysis is often limited to a specific method, subset of dyestuffs, or an approach that is not applicable given the constraints of a forensic analysis. Here, we present a generalized approach to dye identification that (1) combines several robust analytical methods, (2) is broadly applicable to a wide range of dye chemistries, application classes, and fiber types, and (3) can be scaled down to forensic casework-sized samples. The approach is based on the development of a reference collection of 300 commercially relevant textile dyes that have been characterized by a variety of microanalytical methods (HPTLC, Raman microspectroscopy, infrared microspectroscopy, UV-Vis spectroscopy, and visible microspectrophotometry). Although there is no single approach that is applicable to all dyes on every type of fiber, a combination of these analytical methods has been applied using a reproducible approach that permits the use of reference libraries to constrain the identity of and, in many cases, identify the dye (or dyes) present in a textile fiber sample.

Reversible Data Hiding in FTIR Microspectroscopy Images with Tamper Indication and Payload Error Correction.

Fourier transform infrared (FTIR) microspectroscopy images contain information from the whole infrared spectrum used for microspectroscopic analyses. In combination with the FTIR image, visible light images are used to depict the area from which the FTIR spectral image was sampled. These two images are traditionally acquired as separate files. This paper proposes a histogram shifting-based data hiding technique to embed visible light images in FTIR spectral images producing single entities. The primary objective is to improve data management efficiency. Secondary objectives are confidentiality, availability, and reliability. Since the integrity of biomedical data is vital, the proposed method applies reversible data hiding. After extraction of the embedded data, the FTIR image is reversed to its original state. Furthermore, the proposed method applies authentication tags generated with keyed Hash-Based Message Authentication Codes (HMAC) to detect tampered or corrupted areas of FTIR images. The experimental results show that the FTIR spectral images carrying the payload maintain good perceptual fidelity and the payload can be reliably recovered even after bit flipping or cropping attacks. It has been also shown that extraction successfully removes all modifications caused by the payload. Finally, authentication tags successfully indicated tampered FTIR image areas.

Validation of a new 96-well plate spectrophotometric method for the quantification of compound 48/80 associated with particles.

A new, simple, inexpensive, and rapid 96-well plate UV spectrophotometric method was developed and validated for the quantification of compound 48/80 (C48/80) associated with particles. C48/80 was quantified at 570 nm after reaction with acetaldehyde and sodium nitroprusside in an alkaline solution (pH 9.6). The method was validated according to the recommendations of the ICH Guidelines for specificity, linearity, range, accuracy, precision, and detection and quantification limits (DL and QL). All the validation parameters were assessed in three different solvents, i.e., deionized water, blank matrix of chitosan nanoparticles, and blank matrix of chitosan/alginate nanoparticles. The method was found to be linear in the concentration range of 5 to 160 µg/ml (R(2)>0.9994). Intraday and interday precision was adequate, with relative standard deviation lower than those given by the Horwitz equation. The mean recoveries of C48/80 from spiked samples ranged between 98.1% and 105.9% for calibration curves done with the blank matrices and between 89.3% and 103.3% for calibration curves done with water, respectively. The DL were lower than 1.01 µg/ml and the QL were lower than 3.30 µg/ml. The results showed that the developed method is sensitive, linear, precise, and accurate for its intended use, with the additional advantages of being cost-effective and time-effective, allowing the use of small-volume samples, and the simultaneous analysis of a large number of samples. The proposed method was already successfully applied to evaluate the loading efficacy of C48/80 chitosan-based nanoparticles and can be easily applied during the development of other C48/80-based formulations.

Detection of counterfeit Viagra® by Raman microspectroscopy imaging and multivariate analysis.

During the past years, pharmaceutical counterfeiting was mainly a problem of developing countries with weak enforcement and inspection programs. However, Europe and North America are more and more confronted with the counterfeiting problem. During this study, 26 counterfeits and imitations of Viagra® tablets and 8 genuine tablets of Viagra® were analysed by Raman microspectroscopy imaging. After unfolding the data, three maps are combined per sample and a first PCA is realised on these data. Then, the first principal components of each sample are assembled. The exploratory and classification analysis are performed on that matrix. PCA was applied as exploratory analysis tool on different spectral ranges to detect counterfeit medicines based on the full spectra (200-1800 cm⁻¹), the presence of lactose (830-880 cm⁻¹) and the spatial distribution of sildenafil (1200-1290 cm⁻¹) inside the tablet. After the exploratory analysis, three different classification algorithms were applied on the full spectra dataset: linear discriminant analysis, k-nearest neighbour and soft independent modelling of class analogy. PCA analysis of the 830-880 cm⁻¹ spectral region discriminated genuine samples while the multivariate analysis of the spectral region between 1200 cm⁻¹ and 1290 cm⁻¹ returns no satisfactory results. A good discrimination of genuine samples was obtained with multivariate analysis of the full spectra region (200-1800 cm⁻¹). Application of the k-NN and SIMCA algorithm returned 100% correct classification during both internal and external validation.

Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation.

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So(2)) in vivo. We implemented a system for in vivo noninvasive measurements of So(2). A laser light was focused onto areas of 15-30 microm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So(2) was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm(-1) range (oxidation state marker band nu(4)), as well as from the nu(19) and nu(10) bands (1,500- to 1,650-cm(-1) range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary path length and Hb concentration did not affect So(2) estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So(2) estimations and So(2) measured by CO-oximetry was highly significant for nu(4), nu(10), and nu(19) bands. The method allowed So(2) determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So(2) determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.