Microfibers represent one of the most significant groups of traces revealed at a crime scene. Due to their high transfer potential, they are considered valuable in forensic examinations, particularly in cases lacking other forms of physical evidence. Microfibers provide information regarding the type of fiber and the class of dye. Such information may confirm or exclude the suspect’s connection with a place or a victim. However, fiber analysis faces numerous challenges. Fibers revealed at the crime scene are typically extremely small, with only a few millimeters in length and a diameter below 25 µm [1]. The dye content inside the fiber is proportionally small and estimated at 2-200 ng [2], sometimes additionally unevenly distributed within the fiber. Effective examination of microfibers therefore requires the application of multiple, complementary and sensitive techniques to ensure reliable discrimination between fiber samples and to support robust forensic conclusions.

Polyester fibers are currently among the most widely used synthetic materials, in 2023 accounting for approximately 57% of total fiber production worldwide. Despite their widespread use, polyester fibers retain significant evidentiary value. This is largely due to the complexity of the dye mixtures used in their coloration, which can vary considerably between manufacturers, production batches, and end-use products. Disperse dyes are the principal class of colorants used for dyeing polyester materials [2]. They have been classified by the Colour IndexTM based on their method of application in the dyeing process [3], which accounts for their considerable structural diversity. Among them, azo dyes constitute the most prevalent subgroup (up to 70% [3]).

Azo dyes are characterized by at least one azo group (–N=N–) in structure. They constitute 60% of synthetic dyes produced worldwide, which makes them widely employed in textile, food, and pharmaceutical industries [4]. Their popularity as colorants is influenced by wide availability, cost-effectiveness, and the possibility of obtaining vibrant colors during the dyeing process, which enhances the product's visual appeal. However, certain azo dyes can be hazardous as the reductive cleavage of azo bonds leads to the formation of toxic and potentially carcinogenic aromatic amines. Therefore, their usage is limited in European countries.

Currently, the majority of established methodologies for analyzing disperse dyes derived from fibers employ high-performance liquid chromatography (HPLC) in conjunction with mass spectrometry. This approach has been extensively elaborated upon in the comprehensive review conducted by Śmigiel-Kamińska et al [2]. The reports indicate that HPLC methods are successful in the analysis of dyes extracted from fibers from 5 mm [5,6] to 10 mm [7] long. However, in the case of HPLC, the separation was relatively time-consuming, lasting from 20 to 67 minutes. To speed up the separation process and to make the sample throughput more intense, the application of ultra high-performance liquid chromatography (UHPLC) [8] or ultra high-performance supercritical fluid chromatography (UHPSFC) [9] was found suitable, offering separation time as short as 10 minutes. Several research groups suggests that the use of selective and sensitive detectors such as high resolution mass spectrometer (HRMS) [7,10], linear ion trap mass spectrometer with single reaction monitoring (LIT-MS, SRM) [5], or tandem mass spectrometry with multiple reaction monitoring (MS/MS, MRM) [6,9] may offer significant improvement of qualification and quantification parameters of methods.

Only a few reports of capillary electromigration methods (CEMs) [11] with spectrophotometric detection have been used for the analysis of disperse dyes extracted from threads [12] and single fibers [13], both with 10 mm length. The literature on the subject also indicates a single method where the capillary electrochromatography is hyphenated with MS(SIM) for the analysis of disperse dyes present in a commercially available dyeing agent [14]. The low number of published CE-MS methods may be explained by the poor solubility of disperse dyes in typically used water-based background electrolytes (BGE), making the separation challenging. Moreover, disperse dyes are non-ionic, and their electrokinetic separation requires the introduction of modifiers to BGE’s composition, which are usually poorly volatile and, therefore, incompatible with the mass spectrometers required for sensitive analysis.

To the authors' knowledge, to this date, there has been only one report of the successful application of the GC-MS technique to determine the Disperse Orange 37 dye [15]. Considering the high boiling points of disperse dyes and the potential risks associated with the evaporation or thermal decomposition of analytes under GC operating conditions, it has been suggested in the literature that gas chromatography-mass spectrometry (GC-MS) should be employed solely for identification purposes [16]. However, many publications discuss the use of GC-MS to determine the decomposition products of disperse azo dyes, particularly the potentially carcinogenic or toxic aromatic amines. They outline the context of environmental analyses [[17], [18], [19], [20], [21], [22], [23]] and quality control of textile products, which has also been raised in an international standard [24].

In the present study, the authors focus on identification of disperse dyes sourced from polyester fibers. This investigation is constrained to azo dyes, which are predominant due to their widespread application and popularity in the industry. The methodology includes multiple stages. The first stage is the extraction of dyes from polyester fibers, then the reduction of azo dyes according to the modified by authors' method presented in ISO 14362-1:2017(E), followed by the dispersive liquid-liquid microextraction (DLLME) of aromatic amines and their identification using GC-MS/MS in the multiple reaction monitoring (MRM) mode.

The utilization of MRM mode facilitates the acquisition of two critical identification parameters: retention time and MRM transitions. A significant advantage of the MRM measurement mode is its exceptional sensitivity, which enables the detection and identification of amines present in complex fiber extracts, even at significantly low concentrations.

In the current study, the analysis of real samples was precisely designed to simulate forensic comparative fiber examinations. In these instances, the forensic analyst possesses evidence collected from the crime scene, typically comprising a single fiber measuring only a few millimeters in length, alongside reference materials which are generally available in greater quantities. Throughout the analytical process, the characteristics of both the evidence and the reference fibers are systematically compared to ascertain (or negate) a shared origin for the fibers. The proposed methodologies are illustrated in Fig. 1.

Two possible cases were taken into account upon developing the method. The first involved the analyst having a relatively large amount of reference samples, specifically 5 cm of thread and 2 cm of single fiber, along with the evidence sample, which was a few centimeters of single fiber. Then, a selective method is used for analysis, dedicated to comparing two specific samples (evidence and reference, see Fig.1). The reference thread is analyzed in the SCAN mass spectrometry mode to identify the aromatic amines signal based on retention times and the acquired mass spectra. The next step is a selection of target ions of high intensity and the highest possible mass recorded on the MS spectrum for the optimization of collision energy for the MRM method. Finally, the optimized MRM method is used to analyze 2 cm-long single fibers from reference and evidence samples, and the identified signals are used to compare and differentiate the samples.

In the second scenario, when the analyst is tasked with comparing the properties of both evidence and reference samples, specifically in the form of single fibers, a universal methodology is employed. This methodology is developed through the analysis of commercially available fiber samples. Similar to the selective approach, the initial phase involves the identification of amines derived from dyes extracted from the fibers utilizing the SCAN measurement mode. Subsequently, the collision energy for MRM transitions is optimized. After a sufficient analysis of relevant real samples, the amines that are most frequently present in the fibers are established. This leads to the formation of the universal method, which is predicated on their characteristic retention times and MRM transitions, enabling a comparison and analysis of the evidence and reference samples.