3.2.2. DPA Collecting

Several sampling tools were tested for DPA collection from shooters' hands. In a first attempt, 3 μL of 10 μg/mL DPA working solution was dropped on a glass slide. After it was air evaporated to dryness, solid DPA was collected by several sampling tools: adhesive tape lifts; PDMS-based devices at several PDMS: TEOS proportions (100:0, 50:50, and 70:30); and dry cotton swabs and wet cotton swabs with non-skin-toxic solvents such as water, acetone, and ethanol. According to the 24.

European Chemicals Agency (ECHA) database [24], methanol and acetonitrile were not used due to their harmfulness and toxicity in contact with the skin, respectively. After, the samplers were in contact with 2 mL of water under vortex conditions. Figure 3B compares the mean peak areas of DPA extracted from slides and their RSDs to determine the suitability of the several sampling devices tested. The dry cotton swab achieved the highest analytical response with suitable precision. The adhesive tape lift, which was used in the tape lift kits, showed an analytical response about 14 times lower than the dry cotton swabs. Similar loss of peak area was observed with the pure PDMS-based device. However, increases of analytical response were achieved when the TEOS proportion increased in the composition device. In the case of the PDMS: TEOS (30:70) device, the analytical response was improved by four times, compared with the response with the pure PDMS device. This effect can be attributed to the increment of the device hydrophilicity as a function of the TEOS amount, suggesting the improvement of analyte extraction from device to the aqueous solution. When the cotton swab was wet with water and ethanol, the analytical response decreased 80% and 97%, respectively, compared with the response obtained by a dry swab. It could be due to the wet swab spreading the analyte on the slide surface instead of collecting it; RSD > 30% were obtained indicating the difficulty in controlling the analyte collection. When acetone was used as the extractive solvent, DPA was not detected but a small chromatographic peak at a retention time slightly lower than that of the analyte was observed (Figure 3C). As can be confirmed by the spectra depicted in the Figure 3C inset, this peak could be differentiated from the analyte peak by retention time and spectrum, and it could correspond to some compound from the cotton swab. From these results, a dry cotton swab was chosen as the best sampling collector of DPA from shooters' hands for further work.

Peak areas of DPA were obtained for different extraction times under vortex-assisted extraction of the dry cotton swab sampler: 20 s, 2 and 5 min, as can be seen in Figure 3D and non-significant differences on peak areas were observed. Worse results were achieved with non-assisted and ultrasound-assisted extractions even under higher extraction times. Therefore, 20 s as extraction time was selected by using vortex to extract DPA from hands to suitable level in a short time frame.

#### 3.2.3. Effect of Extraction Solvent on the DPA Extraction from the Sampler

The capacity of three solvents to remove the DPA residues from cotton swabs was investigated: acetonitrile, ethanol, and water. Mixtures of 90:10 water, ACN and water, and ethanol and 100% water were tested. Fifty-one percent and 85% decreases in peak area were observed when ethanol and ACN, respectively, were present in the extraction solvent (See Figure 4). This suggests that the analyte was probably non-retained on the IT-SPME capillary column. Moreover, high peaks were observed at a retention time slightly lower than that corresponding to the analyte. These peaks were not detected when the analysis was carried out in solution, suggesting they were due to compounds extracted from cotton. Note that ethanol was the solvent which extracted more interfering compounds. However, water offered the best results in terms of extraction and reduced interferences, as well as it is a greener solvent. Hence, water was chosen as optimum extraction solvent.

**Figure 4.** Chromatograms of blanks (dashed lines) and standard solution of 5 ng/mL DPA (solid lines) obtained with different extraction solvents: water (a), 90:10 water: ethanol (b), and 90:10 water: acetonitrile (c). Experimental conditions were the optimized once (see main text for more explanation).

#### *3.3. Analytical Performance of DPA Determination*

Relevant analytical parameters such as calibration equations, linear working range, limit of detection (LOD), limit of quantification (LOQ), and precision are shown in Table 3, for both solution and swab-vortex extraction procedures. Satisfactory linearity for the working concentrations was achieved. The LODs and LOQs were calculated experimentally from solutions containing concentrations providing signal/noise of 3 and 10, respectively. Limit of detection and LOQ for the swab-vortex extraction were 0.15 ng/mL and 0.5 ng/mL, respectively. Converted into the equivalent amount of DPA injected onto the system, the LOD and LOQ were 0.3 ng and 1 ng, respectively. These results showed that the sensitivity reached with the proposed procedure is suitable for detecting DPA on shooters´ hands and the observed LODs improved the published ones shown in Table 1. The precision was suitable at the working concentration levels tested, with intra- and inter-day relative standard deviations of 9% and 15%, respectively (*n* = 4). The precision of the retention times was also estimated obtaining RSD values of 1.5% and 2.5% for intra- and inter-day, respectively (*n* = 3, concentration = 15 ng/mL). Satisfactory results for the study in solution were obtained as depicted in Table 3. To test the extraction efficiency of DPA from samples (including sample collection by cotton swab and extraction from swab to water), the peak area of solution obtained after extraction (2 μL of 10 μg/mL DPA spread on a glass slide followed by the protocol described in Section 2.6) was compared with the peak area obtained for the equivalent concentration in solution directly injected (5 ng/mL of DPA). The extraction efficiency estimated was 37 ± 5%. A recovery study of spiked samples at 10 ng/mL was performed and the value obtained was 108 ± 16%.

**Table 3.** Analytical data for DPA determination by IT-SPME-CapLC-DAD; a: ordinate, b: slope, sa and sb: standard deviation of the ordinate and slope, respectively, R2: determination coefficient. Limit of detection (LOD) and limit of quantitation (LOQ).


### *3.4. Analysis of Samples*

Several samples collected from hands of police officers after shooting tests (See Section 2.5) were analyzed by the optimized procedure. Additionally, the same procedure as described for shooting hands (See Section 2.6) was carried out for the hands of each police officer before shooting to obtain blank samples. The samples were analyzed without identification of volunteers. Figure 5 shows the chromatograms for the hands of a shooter (sample 2A) and a non-shooter and the UV-Vis spectra of a standard sample. Diphenylamine was identified in samples by their concordance between retention time (9.4 min) and UV–Vis spectra of DPA from the library. As can be seen in Figure 5, the chromatogram of a blank showed no peak interferences at the retention time of DPA.

**Figure 5.** Chromatograms obtained for sample 2A (black solid line) and blank of non-shooter's hand (black dashed line). The inserts correspond to the matching of the spectra of DPA found (blue line) in reference to the standard in the library (red line).

Quantification of the samples was carried out based on the regression equation previously obtained (See Table 3). Table 4 shows the samples screened and the quantification results. With a total of twenty-one swab samples and six tape kit samples, DPA was found and quantified in seventeen swab samples (81% of all swab samples analyzed). In the literature, few studies of DPA are focused on hands and LODs reported are higher to that provided by the proposed method (See Table 1). In this work, the amount of DPA found on hands exceeded LOQ, providing forensic evidence for the presence of DPA. The paired *t*-test was used to evaluate statistical differences between both hands of a shooter, left and right. The α value obtained at a 95% significant level was higher than 0.05 (*p*-value = 0.232). From these results, we can conclude that the results from both hands of a shooter were statistically equivalent.


**Table 4.** Samples screened and quantification of results of DPA on hands determined by the optimized extraction procedure followed by IT-SPME-CapLC-DAD. \* Tape lift kit samples quantified by a regression equation with a slope 14 times lower than that obtained for a regression equation by the cotton swab. \*\* On shooters' hands.

### *3.5. IGSR Particles' Identification*

As can be seen in Figure 6, the presence of GSR particles remaining on cotton swabs can be confirmed by naked eye and optical microscopy before chromatographic analysis. Clean fibers of the cotton swab can be seen after sampling a non-shooter's hand (See Figure 6A). However, gunpowder particles with a typical spherical shape and size up to 20 μm [8] were observed between cotton fibers (see red circles) after sampling a shooter's hand (Figure 6B). It is worth mentioning that this non-destructive microscopic analysis allows the subsequent DPA chromatographic analysis too.

Figure 7 shows the same cotton sample (sample 2A) shown in Figure 6 but characterized by SEM/EDX after DPA extraction. This was possible due to the presence of some gunpowder particles remaining on the cotton swabs after the DPA was extracted. Figure 7 shows a typical IGSR particle with a spherical shape and 38 μm size in accordance with References [6,7]. As can be observed in the elemental analysis, the predominant elements were Ba (46%) and Sb (44%), as reported in the literature for IGSRs [4]. Both inorganic and organic compounds were identified on shooters' hands by SEM/EDX and chromatography, respectively. Hence, the presence of GSRs on the hands of shooters was confirmed.

**Figure 6.** Visual and microscopic (10× magnification) inspection of cotton swab after sampling a non-shooter's hand (**A**) and after sampling a shooter's hand, sample 2A (**B**).

**Figure 7.** (**a**) Scanning electron microscopy SEM image and (**b**) Energy dispersion X-ray EDX spectra of inorganic gunshot residue found on a swab sample (sample 2A) after shooting.

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. Only particles which can be identified as GSR by their composition and morphology were selected for SEM/EDX analysis. Roughly 6–7 particles per sample were studied as can be seen in Table 5. As reported in Reference [8], this number of particles is approximately equivalent to the particles that can be recovered on a shooter's hand at a forensic scene. A portion between 3–40% of the total surface of the sample was explored to find this number of particles, depending on the sample. Figures 8 and 9 show the morphology and elemental data of particles found on adhesive tapes collected after shooting. Most of the particles observed were spherical. Less than 20% of particles found had an irregular shape, probably due to being distorted after shooting. As shown, particles had different surfaces such as smooth, bumpy or covered with craters with or without a metallic shine. More than 60% of the particles found had a smooth surface. Their morphology was an effect of conditions taking place during the firing. Particles can be perforated, capped, broken or stemmed. Results of the SEM/EDX analysis of GRS particles found on the tapes from shooters' hands are displayed in Table 5.

As observed in Table 5, most of the particles had the characteristic elemental composition of GSRs, which was mainly based on Pb, Sb, and Ba; 35 particles contained on average 61% Ba, 30% of Sb, and 9% Pb, and other two particles contained 95% Pb and 97% Sb, probably from bullets, shells or cartridges. Moreover, some particles also contained other elements such as Al, Cu, and Fe at trace levels. About 66% of samples contained traces of Cu, 20% Al, and 3% Fe, while 12% of them contained both Al and Cu. Nevertheless, these minority elements cannot be considered evidence of firing a gun. Even though these particles had similar elemental composition, their size varied over a range from 3 to 30 μm according to the bibliography [4,6,7].

**Table 5.** Summary of shape, surface, and elemental composition of GRS particles found on tape lift kits from shooters' hands.


**Figure 8.** SEM images (left) and EDX spectra (right) of non-spherical particles found on tapes used to collect GSRs after shooting a pistol: sample 22K.2 (**A**), sample 25N.3 (**B**), sample 21K.1 (**C**), sample 25N.2 (**D**).

**Figure 9.** (**A**) SEM image (**B**) overlay X-ray map of singles X-ray of Sb (**C**); Pb (**D**), and Ba (**E**) of particle found on a tape used to collect GSRs on hands after firing a gun.

Spatial distribution of the Sb, Pb, and Ba of GSR particles shown in Table 5 was observed by X-ray mapping using colors to represent the elemental distribution. In this case, Sb appears red, Pb is green, and Ba is blue. Figure 9 shows the X-ray mapping of sample 21K together with its corresponding SEM image. Figure 9B gives the merging of Figure 9C–E. As can be seen, the GSR particle presented the three elements Sb, Ba, and Pb together. Thus, these mapping results were in accordance with the previous elemental composition studied (see Table 5). The results obtained by SEM/EDX can be considered as indicative of IGSR particles on shooters' hands.
