Article pubs.acs.org/ac
Skin Permeation of Organic Gunshot Residue: Implications for Sampling and Analysis Jordan Wade Moran and Suzanne Bell* C. Eugene Bennett Department of Chemistry, West Virginia University, 1600 University Avenue, Oglebay Hall Room 208, Morgantown, West Virginia 26506, United States S Supporting Information *
ABSTRACT: Traditional gunshot residue (GSR) analysis is based on detection of particulates formed from metals found in the primer. Recent concerns regarding the interpretation of GSR evidence has led to interest in alternatives such as the organic constituents (organic gunshot residue, OGSR) found in propellants. Previous work has shown OGSR to be detectable on hands for several hours after a firing event, and given the lipophilic nature of these compounds, it was expected that losses due to secondary transfer (an issue with GSR particulates) would be negligible. However, other loss mechanisms have been identified, specifically skin permeation and evaporation. This paper describes experimental and modeling studies used to elucidate characteristics of skin permeation of 5 compounds present in OGSR. Pharmaceutical methods were adapted to characterize skin permeation using a skin surrogate and Franz diffusion cells. The amount of compounds deposited on skin after an authentic firing event (1 and 2 shots) was experimentally determined and applied for the permeation experiments. A fully validated selected ion monitoring GC/MS method was developed for quantitative analysis, and easily accessible online tools were employed for modeling. Results showed that OGSR residues should be detectable on skin for many hours after a firing event of as few as one or two shots, with detection capability being a function of the efficacy of sampling and sample preparation and the instrumental method employed. The permeation rates of the OGSR compounds were sufficiently different to suggest the potential to develop methods to approximate time-since-deposition.
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methods; (3) estimate experimentally the rate of loss from the surface of the skin due to evaporation and absorption; (4) compare these findings to skin permeation models used in pharmaceutical and toxicological applications; (5) estimate how long OGSR will be detectable using simple noninvasive sampling methods; and (6) evaluate the implications of the results in terms of analytical methodology and potential evidentiary value. This study focused on five commonly studied OGSR components (Figure 1). The lipophilic nature of these compounds suggests that, when deposited on the hands, they will adhere. Consequently, organic residues are expected to be less prone to secondary transfer and this was demonstrated in previous work by our laboratory.3 However, this study also indicated that persistence was limited to periods ranging from 3 to 24 h. Absent deliberate removal by hand washing, only two loss mechanisms are feasible: evaporative loss and dermal absorption. These mechanisms must be characterized and understood before this type of physical evidence can be
unshot residue (GSR) consists of particulates formed from primer ingredients that vaporize and condense after a weapon is discharged. These particulates have a distinctive morphology (relatively smooth and rounded) and size (on the order of 1−10 μm). The other distinguishing characteristic of these particulates is the presence of lead (Pb), barium (Ba), and antimony (Sb).1,2 The preferred method for analysis of GSR is collection from the hands using carbon tape stub with analysis using scanning electron microscopy (SEM) coupled with X-ray spectroscopy. Particulate evidence is by nature transitory and subject to secondary transfer,3−8 a factor that complicates interpretation of findings. This has led to reconsideration of organic compositional analysis as an alternative target compound set. These compounds arise from unburnt propellant and associated compounds, collectively referred to as organic gunshot residue (OGSR). The energetic compounds in small arms propellants are nitrocellulose and nitroglycerin, although the energetics are usually not primary target compounds for the analysis of OGSR. Rather, ancillary compounds such as stabilizers are the focus, as was the case in this work.1−3,9−19 The goals of this research were to (1) estimate the amount of OGSR deposited on the hands during an authentic firearm discharge; (2) estimate the efficiency of skin sample collection © 2014 American Chemical Society
Received: April 4, 2014 Accepted: May 16, 2014 Published: May 16, 2014 6071
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by the diffusivity in, and the thickness of various skin layers.24 The corneocytes are tightly packed, proteinaceous, and subject to changes in hydration which can impact permeation rates.20 Compounds traverse the SC by partitioning via trans- and intercellular pathways. Once compounds reach lower skin layers, protein binding processes can contribute to convective transport into the blood,21 creating a sink. A detailed skin crosssection figure is provided in the Supporting Information. A typical and constant hydration level of 15−20% was assumed for this project.25 Skin permeation models have been developed for toxicological and pharmacological applications. In the typical scenario, a drug entrained in a solvent (vehicle) is placed in contact with the skin or skin surrogate and partitioning begins, influenced by concentration and characteristics of the drug and vehicle. However, the deposition that occurs after a weapon is fired does not fit directly into such a model. When a weapon is fired, a cloud of residues are produced, a portion of which is deposited on the hands. The deposited materials include solids (particles of unburnt propellant and particles formed from the primer) and vapor-phase compounds such as propellant ingredients and combustion products. The unburned propellant particles are often large enough to be distinguishable by the naked eye. Each of these particles could be considered as a system consisting of a vehicle (the bulk propellant) and the target compounds although modeling this scenario with current tools is unrealistic. It is also unnecessary for practical reasons; these particles would be subject to secondary transfer in the same sense as GSR particulates and thus are not of interest here. Rather, our attention focused on direct vapor deposition on the skin, no vehicle, from a single finite dosing event. Fick’s First Law of Diffusion is the starting point for most permeation models:
Figure 1. Compounds evaluated and descriptors used in modeling. VP is vapor pressure.
exploited. The key factors for such utilization will be the efficacy of sample collection from the skin, the efficiency of the sample extraction and preparation, and the instrumental limits of detection and quantitation. Underlying all of these issues is the elapsed time since deposition which will determine the degree of evaporative and absorptive loss of each target compound. The primary barrier to percutaneous skin absorption is the outer stratum corneum (SC) which is ∼20−40 μm thick. The SC (Figure 2) consists of layers of flattened cells (corneocytes20,21) organized in a brick-and-mortar structure.21−23 These cells contain keratin structures and lipids encased within the lipid cell membrane.20,21 Permeation is a complex process that incorporates diffusion (driven by a concentration gradient) and convective transport,20 controlled
J = −D
∂C ∂x
(1)
where J represents the rate of transfer (flux) in a given area; C is the concentration of the deposited compound; D is the coefficient of diffusion; and x is the distance of penetration. In a finite dose model, the concentration gradient changes over time due to absorption, evaporation, and removal to a sink such as entering general circulation. Other complicating factors are reservoir effects and skin metabolism. There are numerous papers and review articles that describe skin permeation modeling and dermal/epidermal transport processes, and the interested reader is directed to these for more information.21,24,26−40 The permeation coefficient, Kp (cm/h) represents the rate of dermal uptake of the compound as a function of time. Because this rate depends in part on the characteristics of the vehicle (not present in the deposition of OGSR), a better descriptor is the steady state flux (Jss, μg/cm2/h), which is vehicle independent:
Jss = K p × C
(2)
Models describing skin permeation have been derived from experimental data, mathematical and pharmacokinetic compartment models, QSPR (quantitative structure permeation relationships),31,34,39,41,42 and Gaussian models.21,42 A central goal of this project was to measure the flux rate of each compound experimentally and compare it to modeling predictions. Two online calculators provided by the U.S. Department of Health, Centers for Disease Control (CDC),
Figure 2. Stratum corneum layer of skin. 6072
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were utilized. The first calculator tool estimates skin permeability (Kp) using three paradigms24,43 and two molecular descriptors: molecular weight and the octanol/water partition coefficient (Kow).44 The Frasch model employs a random walk methodology to model diffusion of a drug from an aqueous vehicle into the SC and accounts for the heterogeneity of the SC structure (Figure 2). The Potts and Guy model and the Robinson model are relatively simple in comparison and are based on regression equations derived from experimental data. One review concluded that, of the latter two models, the Robinson model best captured the effect of molecular weight on skin permeation.24 The objective here was not to critique the models per se but to determine which model best agreed with experimental data. The second resource used was the Finite Dose Skin Permeation Calculator maintained by the CDC/National Institute for Occupational Safety and Health (NIOSH).45 This utility incorporates several models32,40,46,47 including one that describes direct skin disposition of volatile compounds40,47 and provides for many types of calculations including flux rates and concentration profiles. Example screenshots for simulations run for this project are shown in the Supporting Information. The molecular descriptors required for this modeling are molecular weight, Kow, vapor pressure, melting and boiling points, number of aromatic ring systems, and number of multiple bonds. Human hand skin is difficult to obtain, let alone in quantities sufficient to perform the number of experiments required in the present study. Skin from other areas, such as that associated with breast reduction surgery, is available, but comparability is questioned given that skin characteristics vary by location. Because of this well-known limitation, a frequently used analogue for the stratum corneum is a polydimethylsiloxane (PDMS) membrane.42,48 In addition to convenience and minimal biosafety issues, PDMS offers a realistic and proven skin analogue.42,48−50
for the exposed skin surface area is dependent upon the volume of the receptor chamber of the FDC. The value of the surface area associated with a 5 mL receptor chamber as used in these experiments is 0.64 cm2. A previous publication describes the experimental setup and calculations in greater detail.52 A rigorously validated gas chromatography/mass spectrometry (GC/MS) protocol was utilized for detection and quantitation of the target compounds. GC/MS has been previously used to detect OGSR components recovered from physical evidence;1,15,19,58−61 however, until now, a method has not been developed and fully validated for the analysis of dermally absorbed OGSR. The validation protocols and figures of merit used here were based on guidelines from the Standard Practices for Method Validation in Forensic Toxicology by the Scientific Working Group for Forensic Toxicology (SWGTOX)62 and the Validation of Analytical Procedures: Text and Methodology Q2(R1) from The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH, www. ich.org).63 Complete details of the validation are presented in the Supporting Information. Determination of Modeling Parameters. Given that the goals in this project were 2-fold (establish the utility of PDMS as a skin model for OGSR absorption and to estimate relative penetration rates of key compounds), a single source for obtaining molecular descriptors was desired to facilitate comparisons. For the Kow values, the EPA’s Estimation Programs Interface (EPI) Suite was utilized. If peer-reviewed experimental data exists within the software database (as is the case for DPA), these values are produced. In other cases, the value is estimated via calculation. Vapor pressure values were obtained from experimental values or estimations collated at the ChemSpider (http://www.chemspider.com/) Web site. Peer reviewed experimental data was used whenever available. Values used in the models are given in Figure 1 and Table 1.
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Table 1. Modeling Parameters
EXPERIMENTAL SECTION Franz diffusion cells (FDCs) were used for the skin permeation studies.51−54 FDCs are composed of two chambers, an upper donor chamber and a lower receptor chamber. The tissue or membrane being used is placed between the two chambers. The target analyte(s) are placed on the surface of the tissue or membrane and then partition into and diffuse through tissue or membrane toward the receptor chamber which contains phosphate buffered saline (PBS). A diagram of a FDC along with detailed descriptions is provided in the Supporting Information. A surface loading method (delivery via solution in solvent that is allowed to evaporate) was used to initiate the diffusion experiments. The only transdermal parameter that can be calculated with a surface loading technique is the steady state flux (Jss, μg cm−2 h−1).44,55−57 This flux is calculated by quantitation of the cumulative amount of the permeant collected in the receptor fluid (μg) as a function of time since deposition.44 A line is fitted to the steady state portion of the curve. From the regression equation, the steady state flux (eq 2) is calculated: slope Jss = (3) surface area where the slope is the value from the linear regression equation of the cumulative amount permeated vs time graph. The value
compound
molecular weight
H-bond sites
MPa
BPb
double bonds
rings
DPA EC DMP 2NDPA 4NDPA
169.23 268.36 194.18 214.22 214.22
1 3 4 4 4
53 127 6 74 133
302 379 283 354 211
6 7 5 7 7
2 2 1 2 2
a
Melting point. bBoiling point.
Reagents, Materials, and Instrumentation. A 1000.0 ppm OGSR stock solution that consisted of DMP, DPA, EC, 2NDPA, and 4NDPA was prepared in LC/MS grade methanol (Sigma-Aldrich, St. Louis, MO) from analytical-grade neat materials (Sigma-Aldrich). A 20.0 ppm solution of the internal standard, nitrobenzene-d5 (Sigma-Aldrich), was prepared from a 1000.0 ppm stock solution using the same solvent. Chromasolv methanol (Sigma-Aldrich), anhydrous methanol (Sigma-Aldrich), LC Chromasolv methanol (>99.9%, Fluka, St. Louis, MO), and OmniSolv methanol (EMD, Billerica, MA) were used for the selectivity (specificity) study. A separate 1000.0 ppm stock solution of the same OGSR compounds was prepared in acetone (VWR, Philadelphia, PA) for spiking and recovery studies as described below. GC/MS grade hexane (Sigma-Aldrich) was used in extractions. For all firearm studies, 6073
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Table 2. OGSR Recovery Data DMP # shots fireda average concentration (ppm) %RSD calculated deposited dose (ppm) experimentally determined dose (ppm) μg deposited on PDMS a
1 0.74 0.3
DPA 2 0.74 0.3
1 0.93 2.5
0.78 0.90 0.09
EC 2 0.93 1.0
0.99 1.15 0.115
1 1.6 0.7
2NDPA 2 1.6 0.9
1.65 1.78 0.178
1 0.62 6.3
4NDPA 2 0.63 4.2
0.67 0.73 0.073
1 1.36 1.9
2 1.37 1.1 1.42 1.55 0.155
n = 15 shots for each series.
NY) were performed using FDCs (Permegear, Hellertown, PA). The thickness of these membranes correlates to the thickness of the stratum corneum and the epidermis. The FDCs had a receptor compartment of 5.0 mL, and the surface area of the exposed silicone membranes was 0.64 cm2. Sheets of PDMS were cut to appropriate circular sizes (1 in. O.D.) using a punch. The FDCs were allowed to equilibrate overnight prior to all experiments. The receptor compartments were filled with PBS, making sure air underneath the membranes was removed. The solutions were continuously stirred and maintained at 37 °C. To minimize evaporation from the donor and receptor compartments, the donor cells and receptor arms were covered with Parafilm. 100.0 μL of the prepared deposited dose solution in acetone was applied to the surface of each membrane, and the acetone was allowed to evaporate until all visible traces were gone (∼1 min). At intervals of 0, 2, 4, 8, 12, 16, and 24 h, 500.0 μL samples were removed from each receptor compartment. The same volume of fresh PBS buffer was added back to the receptor compartments to maintain constant volume. Samples were removed, and fresh buffer was added using gastight syringes. 500.0 μL of each sample obtained from the FDC experiment was transferred to 2 mL Costar microcentrifuge tubes (Corning Incorporated, Corning, NY). Liquid/liquid extraction (LLE) was performed by adding an equal volume of hexane (500.0 μL) to the microcentrifuge tubes. The mixture was vortexed and centrifuged for 10 min. The upper hexane layer was removed and placed in 2 mL glass vials (Phenomenex, Torrance, CA) and allowed to evaporate. The remaining OGSR was reconstituted with 500.0 μL of a 20.0 ppm internal standard: methanol solution for GC/MS analysis. A thorough LLE efficiency study, broken down by steps, was conducted, and details are provided in the Supporting Information. Repeatability was deemed acceptable for this application (
