**1. Introduction**

Chemical and physical evidence such as gunshot residues (GSRs) from firearms discharge may provide valuable forensic information [1,2]. Gunshot residues are organic and inorganic components in nature, which can be deposited on a shooter's body, mainly onto the index fingers and thumbs of the hands, after discharging a firearm [3]. A suspect can be successfully identified if GSRs are reliably analyzed. Thus, the detection of these compounds plays an important role in the field of forensic science. Inorganic gunshot residues (IGSRs) are usually spherical particles mainly composed of Ba, Pb, and Sb [4]. Other elements such as Ca, Al, Cu, Fe, Zn, Ni, Si, and K can also be found [5], although they

are more prevalent in the environment than Pb, Ba, and Sb [6]. The size of these particles is usually from 0.5 μm to 10 μm, although sizes up to 100 μm have also been reported [7]. The presence of these metallic particles has been traditionally confirmed by scanning electron microscopy coupled to the energy dispersion X-ray (SEM-EDX) technique due to its non-destructive capability to perform both morphological and elemental analyses [8,9]. However, the analysis of IGSRs has its limitations. False positive results can be produced from inorganic particles derived from environmental and occupational sources [10–13], which is a problem when considering IGSRs as evidence in judicial proceedings in the forensic field. The analysis of organic gunshot residues (OGSRs) in the same sample could provide complementary information that could strengthen the probative value of a forensic sample. Organic components originate mostly from the propellant, and their composition depends on the commercial brand and ammunition type.

An important component of gun propellants is diphenylamine (DPA), which is used as a stabilizer in order to prevent the decomposition of explosive products like nitrocellulose and nitroglycerine, both of them present in many smokeless powders used as propellants [14]. Thus, this stabilizer may remain on a shooter's hands, and it may be used as an indicator of gunshot residues [15]. Diphenylamine detection could provide valuable evidence of firearm discharge for the identification of suspects in firearm-related crimes.

The low amount of DPA remaining on a shooter's hands requires highly-sensitive analytical techniques for its detection. In order to improve the sensitivity, many methods include off-line sample treatment, which involves time-consuming and tedious steps. Table 1 presents several methods used for extraction and determination of DPA that remains on the hands. The main drawback of the reported methods is the low detection limit required, taking into account the sampling and extraction process, time of analysis, and greenness of the procedure.


**Table 1.** Comparison of reported methods for determining diphenylamine (DPA) on a shooter's hands. The method proposed in this work was also included for comparison (On-line in-tube solid phase microextraction coupled to capillary liquid chromatography with diode array detection (IT-SPME-CapLC-DAD).


**Table 1.** *Cont.*

On-line sample pre-treatment has become an interesting alternative as green analytical chemistry indicates. In this context, our research group has successfully applied in-tube solid-phase microextraction (IT-SPME) in the analysis of a variety of analytes and matrices [19,20]. In-tube solid-phase microextraction typically uses a capillary column internally coated with extractive phase, which can be different in nature in function of the physical-chemical properties of the analytes [19,20], in order to extract, concentrate, and clean-up the sample. When IT-SPME is coupled to a miniaturized liquid chromatograph, important improvements in terms of sensitivity, selectivity, automation, and waste minimization can be achieved. Although mass spectrometry (MS) coupled to gas chromatography (GC) or liquid chromatography (LC) offers suitable sensitivity, the chromatographic techniques can present issues. Thermal degradation of DPA can occur by GC and the wide range of polarities of compounds present in GSRs can limit the LC. Some methods have also successfully identified DPA using several MS techniques without any chromatographic system such as tandem mass spectrometry (MS–MS) [16], desorption electrospray ionization-mass

spectrometry (DESI-MS) [17], nanoelectrospray ionization mass spectrometry (nESI-MS) [21], and ion mobility spectrometry (IMS) [22]. However, IT-SPME coupled to capillary liquid chromatography (CapLC) contributes to increase the sensitivity and sample clean-up in an on-line way. Additionally, the miniaturization of the LC technique (i.e., low column dimensions, low flow rates, low amount of wastes) contributes also to achieve improved sensitivity, which can permit the use of diode array UV-detectors (DADs), which cost less than an MS detector.

In the present work, a shooters' hands sampling was carried out using dry cotton swabs followed by short vortex-assisted extraction of DPA from cotton with water. Additionally, on-line IT-SPME-CapLC-DAD was employed, for the first time to our knowledge, for the DPA determination. Other samplers were also studied, but their extraction capacities were lower than that achieved by a dry cotton swab. Several parameters such as capillary length and coating, as well as extraction conditions, were optimized for the on-line system. On the basis of the results obtained, a new approach is proposed for the detection of DPA from shooters' hands, which integrates simple, rapid, and green extraction followed by on-line clean-up and preconcentration of samples. The method permits to carry out the analysis of IGSRs by SEM-EDX after the DPA extraction, in order to confirm the presence of inorganic gunshot residues on shooters' hands as well. Optical microscopy can be used for identifying particles with a spherical shape and size up to 20 μm in a cotton swab due to the presence of gunpowder particles, and it was proved that SEM-EDX can be applied after extracting DPA from the swab. The other aim of this work was to examine the morphology and elemental composition and distribution of GSR particles collected with the lift tape kits, the typical police collector, which provided lesser sensitivity in the DPA analysis (around fourteen times less). The possibility of environmental and occupational sources could be eliminated when DPA was found together with IGSRs. Both analyses can be used as evidence in judicial proceedings in the forensic field [23].

### **2. Materials and Methods**
