*2.3. IT-SPME Procedure*

The setup used in this work corresponded to that developed for in-valve IT-SPME [19,20]. The stainless-steel injection loop of a six-port injection valve was replaced with an extractive capillary. Several gas chromatography capillary columns (0.32 mm i.d.) were tested as extractive capillaries. The columns used were TRB-5, TRB-20, TRB-35, TRB-50 (Teknokroma, Barcelona, Spain) and Zebron ZB-WAXplus (Phenomenex, Torrence, CA, USA). For coating details, see Table 2. Segments from 30 to 90 cm of these columns were directly tested for IT-SPME. Capillary connections to the valve were facilitated by the use of 2.5-cm sleeves of 1/16 in polyether ether ketone (PEEK) tubing; 1/16 in PEEK nuts and ferrules were used to complete the connections. In load valve position, 1800 μL of sample was manually passed through the capillary column by means of a 1000-μL precision syringe. A clean-up step was also carried out by processing 120 μL of ultrapure water after the sample loading. Finally, when the valve was manually rotated to the injection position, the analyte was desorbed in dynamic mode from the coating of the extractive capillary and transferred to the analytical column by the mobile phase. The valve was maintained in this position until the end of the chromatogram.


**Table 2.** Characteristics of capillary columns employed during the in-tube solid-phase microextraction (IT-SPME).

#### *2.4. Shooting and Collection of GSRs from Hands*

Test shots were carried out by police officers in an indoor range at Police Headquarters of Valencian Community (Valencia, Spain) under typical shooting practice conditions. Personal information was not recorded. The shots were fired with 9-mm Heckler & Koch pistols, model USP Compact (Oberndorf/Neckar, Germany), which is the most commonly used firearms among police forces in Spain. Each volunteer police officer fired a total number of 25 shots (regulatory number of shots). Only one of these police officers fired 12 shots because his pistol jammed. In order to avoid contamination, each police officer fired with his own firearm and did not touch other surfaces with their hands during the analysis. Gunshot residue samples were collected from the shooters' hands immediately after discharging the firearm. Sampled zones of the hands are shown in Figure 1. For each police officer, both hands, right and left (palm and back), were sampled after shooting. Two techniques for GSR collection from hands were carried out: swabbing and tape lifting. Swabbing was performed by scrubbing the hand with one of the tips of a cotton swab, which was stored in a 5-mL glass vial with a fitted cap to prevent contamination from other compounds in the air. Note that cotton swabs were not moistened in any solvent before sampling. The tape lift kit consisted of a metal stub equipped with a carbon adhesive tape inserted in a plastic vial with a tightly fitted cap. For the sampling, the metal stub was passed over the surface of the hand and then was returned to the vial. Once all the collected samples were placed back into their vials and capped, they were transported to the lab and were stored at room temperature awaiting analysis. A total of 11 shooters were sampled by swabbing and the other five shooters were sampled by tape lifting, which consisted of a total of 21 swab samples and six tape samples (see Analysis of Samples in the Results and Discussion section for identifying the samples). Additional swab samples from each volunteer police officer before test shots were also analyzed as blanks (hands were not previously washed).

**Figure 1.** Schematic diagram of the steps for DPA analysis: ( **A**) web and palm of the hand sampling (zone sampling in blue), (**B**) vortex-assisted extraction, and ( **C**) IT-SPME-capillary liquid chromatography (CapLC) system.

#### *2.5. Sample Treatment for DPA Analysis*

Several solvents (water, acetone, ethanol), samplers (cotton swabs, carbon-based tapes, PDMS-TEOS based samplers), extraction techniques (non-assisted, ultrasound-assisted, and vortex-assisted extraction) and time extraction (up to 20 min) were tested in order to find the proper sampling procedure. Three μL of 10 μg/mL in 2 mL of water with different (A) extraction modalities and (B) sample collectors were assayed by IT-SPME-CapLC-DAD. Each sample was analyzed in triplicate and all assays were carried out at ambient temperature.

In order to obtain the solid DPA from standard solutions, a volume (3 μL) of DPA solution (10 μg/mL) in acetonitrile (ACN) was deposited on a glass slide. Then, the solvent was evaporated to dryness at room temperature and solid DPA was collected carefully by scrubbing the glass slide with a cotton swab. After sample collection, the tip of the cotton swab was placed into a storage vial containing 2 mL of water, so that the cotton was completely wetted. Diphenylamine was extracted from the swab under vortex condition for 20 s at ambient temperature. Next, the swab was used in the analysis of inorganic residues, and 1800 μL of the solution was loaded into the IT-SPME capillary of the LC system. The same procedure was used for the other samplers assayed. The complete procedure for the DPA analysis is shown in Figure 1.

#### *2.6. IGSRs Analysis by SEM/EDX and Optical Microscopy*

In order to confirm the presence of GSRs in cotton swabs, the gunpowder grains were visually and microscopically identified before chromatographic analysis. For the IGSR particle analysis from the tape lift kits and from cotton swabs, SEM images, EDX spectrums, and X-ray maps were carried out. For cotton swab samplers, besides metallization with Au/Pd coating, silver lac was used for painting the sample. Magnification varied between 50 and 500× according to the particle size. Once the particle was located, an elemental analysis was carried out to determine the major components of the particle. The size, shape, and morphology of the particles were also recorded.

### **3. Results and Discussion**

#### *3.1. Optimization of the IT-SPME and Chromatographic Conditions*

Experiments were performed in order to optimize the DPA extraction by IT-SPME, as well as the subsequent chromatographic analysis. Initially, two mobile-phase compositions in isocratic elution were tested, 60:40 and 70:30 ACN: water (*v*/*v*). As can be seen in Figure 2A, both compositions were adequate to desorb DPA from the IT-SPME extractive capillary. However, a decrease in retention time and narrower peaks were achieved with the increase of ACN and flow rate of the mobile phase. A gradient elution program (See Section 2.2 for optimum conditions) with 100% of ACN during 4 min was employed as cleaning solvent.

**Figure 2.** Effect of (**A**) acetonitrile percentage and flow rate of mobile phase (800 μL at 7 ng/mL of DPA, TRB-35, 30 cm); (**B**) nature of the IT-SPME phase (800 μL at 5 ng/mL of DPA, capillary length 30 cm, optimum mobile phase); (**C**) capillary length (800 μL at 5 ng/mL of DPA, TRB-35, optimum mobile phase); and (**D**) sample volume processed (5 ng/mL of DPA, TRB-35, capillary length 90 cm, optimum mobile phase) in IT-SPME versus peak area of DPA. For more details, see the main text.

In-tube solid-phase microextraction was performed using a capillary column as the loop of the injection valve. The analytes were extracted during sample loading and were transferred to the analytical column with the mobile phase by changing the valve position. This configuration was advantageous in order to achieve suitable limit of detection (LOD) for detecting DPA deposited on shooters' hands. Herein, several assays were carried out to optimize the extraction step. The nature of the extractive phase, the length of the capillary column, and the volume of the sample processed were evaluated. Five phases for IT-SPME were assayed: 5, 20, 35, 50% diphenyl–95, 80, 65, 50% polydimethylsiloxanes, respectively, and 100% polyethylene glycol (PEG) (See Table 2). Figure 2B compares the analytical response (mean peak area) for DPA (5 ng/mL) with the different capillaries (30-cm length) when the volume of standard processed was 800 μL. As can be seen, the TRB-35 phase provided higher analytical responses for DPA. This suggests that the higher percentage of diphenyl groups in the extractive phase led to an increase in analytical response. It can be deduced that extraction involves π–π interactions with DPA, whose structure possesses two aromatic rings. However, TRB-50 provided a decrease on the peak area, and this effect was attributed to the increment on the polarity of the extractive phase, and so the affinity towards the DPA decreased (log Kow = 3.5). The same effect may occur by PEG capillary due to its higher polarity. Thus, the TRB-35 capillary column was selected as the best extractive phase for further experiments.

The effect of the capillary length on the analytical response (peak area) was also studied by processing 800 μL of working solution of DPA (5 ng/mL) with TRB-35 capillaries of 30, 60, and 90 cm. Figure 2C shows the increment of the analytical response with the length of the capillary, thus, the amount of analyte extracted also increased. The peak area for DPA improved 40% and 47% with the capillary columns of 60 and 90 cm, respectively, compared with the capillary of 30 cm. Capillaries longer than 90 cm did not improve the analytical response. The TRB-35 of 90 cm was chosen as the optimal capillary column length.

Sample volumes processed up to 4 mL at 5 ng/mL of DPA solution were studied. The results obtained are depicted in Figure 2D. As can be seen, a remarkable increase of analytical response (peak area) with the increase of the sample volume was observed up to 2 mL. The signal increased very slightly from 2 to 4 mL, and 2 mL was chosen as the optimum sample volume for further experiments. However, it was found that the swabs used in the present study absorbed about 125 μL of contact solution. According to this observation, further experiments were carried out by processing 1800 μL remaining in the vial.

The extraction efficiencies of the proposed methodology were estimated by comparing the amount of analyte extracted, which is the amount of the analyte transferred to the analytical column, with the total amount of analyte passed through the extraction capillary. The amount of analyte extracted was established from the peak areas in the resulting chromatograms and from the calibration equations constructed through the direct injection of 72 μL of analyte standard solutions of different concentrations. This volume is the inner volume of the TRB-35 capillary of 90 cm used for IT-SPME. The absolute extraction efficiency obtained was 7% which is in accordance with those reported for this technique [19,20]. Although low extraction efficiencies (absolute recoveries) were achieved by IT-SMPE, the analytical responses were improved significantly owing to the large volumes of sample that can be processed through the capillary column. In addition, a clean-up step was tested after sample loading by introducing 120 μL of nanopure water before changing the valve to the inject position. Significant loss of analyte was not observed; thus, clean-up was applied in order to remove fibers or compounds from cotton which could remain inside the capillary column. It was also tested to filter the solutions of DPA extracted from cotton swab through 0.45-μm nylon membranes. Nevertheless, the analyte was retained on the nylon filter. Hence, samples were not filtered before injection.

#### *3.2. Optimization of DPA Extraction from Hands*

### 3.2.1. DPA Extraction from Collector

The first step considered to optimize DPA extraction was to find an appropriate extraction procedure for DPA from the collector. For this aim, non-assisted, ultrasound-assisted, and vortex-assisted extraction of the analyte from a cotton swab sampler were tested. Three μL of 10 μg/mL DPA working solution (prepared in ACN to favor evaporation) was spread on a glass slide. After it was air evaporated to dryness, a dry cotton swab was used to collect DPA from the slide. The tip of the cotton swab was introduced into a vial containing 2 mL of water under the three abovementioned extraction modes for 5 min (See Section 2.6 for more details). Vortex-assisted extraction offered the best results in terms of both analytical response (peak area) and relative standard deviations (RSDs), as can be seen in Figure 3A. For evaluating the extraction efficiencies, the peak area ratios between non-assisted and assisted extractions were calculated, ratios of 2 and 5 were obtained for ultrasound and vortex, respectively. Moreover, the results provided a satisfactory RSD of 9% for extraction by vortex but not by ultrasounds with 32% of RSD. From these results, we concluded that the best extraction of DPA from the sampler was vortex-assisted extraction.

**Figure 3.** Comparison of peak areas obtained for standard solution (3 μL of 10 μg/mL in 2 mL of water, 15 ng/mL) with different ( **A**) extraction modalities with dry swab samplers, (**B**) sample collectors, and ( **C**) solvents to wet cotton swabs (at 10 ng/mL): dry (a), water (b), acetone (c), and ethanol (d), together with normalized spectra (inset) of DPA (black dashed line) and unknown compounds (black solid line), and ( **D**) extraction time with dry swab samplers. For other experiment details, see main text.
