Hair analysis as a useful procedure for detection of vapour exposure to chemical warfare agents: simulation of sulphur mustard with methyl salicylate
Abstract
Chemical warfare agents (CWA) are highly toxic compounds which have been produced to kill or hurt people during conflicts or terrorist attacks. Despite the fact that their use is strictly prohibited according to international convention, populations' exposure still recently occurred. Development of markers of exposure to CWA is necessary to distinguish exposed victims from unexposed ones. We present the first study of hair usage as passive sampler to assess contamination by chemicals in vapour form. This work presents more particularly the hair adsorption capacity for methyl salicylate used as a surrogate of the vesicant sulphur mustard. Chemical vapours toxicity through the respiratory route has historically been defined through Haber's law's concentration-time (Ct) product, and vapour exposure of hair to methyl salicylate was conducted with various times or doses of exposure in the range of incapacitating and lethal Ct products corresponding to sulphur mustard. Following exposure, extraction of methyl salicylate from hair was conducted by simple soaking in dichloromethane. Methyl salicylate could be detected on hair for vapour concentration corresponding to about one fifth of the sulphur mustard concentration that would kill 50% of exposed individuals (LCt50). The amount of methyl salicylate recovered from hair increased with time or dose of exposure. It showed a good correlation with the concentration-time product, suggesting that hair could be used like a passive sampler to assess vapour exposure to chemical compounds. It introduces great perspectives concerning the use of hair as a marker of exposure to CWA. Copyright © 2014 John Wiley & Sons, Ltd.
Introduction
Chemical warfare agents (CWA) are defined by the North-Atlantic Treaty Organization (NATO) as chemical compounds that can be used in military operations to incapacitate, injure, or kill people owing to their toxic properties.1 CWA have been known since Antiquity. The first report of their use dates from the siege of Kirrha in 600 BC, when Athenians contaminated water with Hellebores' roots.2 However, they were extensively employed for the first time during World War I with the use of chlorine, phosgene, or sulphur mustard among others.2 More recently, their use have been reported during international conflicts (Iraq-Iran war, 1980s), terrorist attacks (sarin attack in the Tokyo subway, 1995) or civil wars (Syria, 2013).3
NATO classifies CWA in several classes according to their effects on organism and refers to them by abbreviations. For example, blistering agents cause severe burns and their most known representatives are distilled mustard (HD) commonly called sulphur mustard (SM), and lewisite (L).2
- ICt50 refers to the Ct product leading to an incapacitating effect in half of the persons exposed.
- LCt50 corresponds to the Ct product for which half of the exposed population dies.
After exposure to CWA, priority must be given to providing care to the contaminated persons, as CWA's effects on organism are severe, and quickly observable (within minutes for vapour exposure to nerve agents2) except for insidious chemicals like SM and the low volatility organophosphate VX whose effects may arise a few hours after exposure.7, 9 It is thus necessary to develop markers of exposure that can be quickly analyzed in order to help rescue teams identify contaminated victims and prioritize decontamination.
Colourimetric tubes, flame photometry, photoionization, or ion mobility spectrometry are examples of procedures already used in the field.10 These techniques require liquid or air samples or swabs from exposed surfaces (skin or material) in order to detect potential contaminants. However, they do not allow identification and/or quantitation of contamination on each potentially exposed victim.
Mostly used in medicine or the forensic field to detect and/or quantitate consumption, by monitoring the endogenous concentration of a substance,11, 12 it has also been proven that hair is able to trap molecules after external exposure.13, 14 Besides, with an average surface area of 0.40 m2.g-1,15 corresponding to a total surface ranging from 2 to 80 m2 depending on the hair length, in comparison to the skin surface (1.5–2 m2 for an average 70-kg man), which is in most cases not fully exposed to air, hair's ability to retain molecules from external atmosphere is pertinent.
The objective of this work is to study the hair sorption capacity of chemicals in vapour form, in order to validate hair as an efficient trap for chemicals and CWA, especially the blistering agent SM.
SM is an alkylating agent and a rather volatile compound (vapour pressure = 0.11 mmHg at 25°C). With a high contaminating power and a long persistence on surfaces, impregnation is very quick, and skin absorption is almost immediate.7, 9 Principal physical and chemical characteristics of SM are resumed in Table 1 (inspired by Bartelt-Hunt et al.16).
| Compound | CAS number | Formula | Molecular mass (g.mol-1) | Vapour pressure (mmHg, at 25°C) | log Ko/w | Previous works using simulant |
|---|---|---|---|---|---|---|
| Sulphur mustard (SM) | 505-60-2 | C4H8Cl2S | 159.07 | 0.11 | 2.41-2.55 | |
| 2-chloroethyl ethyl sulfide | 693-07-2 | C4H9ClS | 124.63 | 3.4 | 2.2 | Willis et al.18 Gephart et al.20 |
| 2-chloroethyl methyl sulfide | 542-81-4 | C3H7ClS | 110.6 | 8.98 | 1.62 | Fox et al.24 Wagner and MacIver25 |
| Chloroethyl phenyl sulfide | 5535-49-9 | C8H9ClS | 172.67 | 0.0186 | 3.58 | Gephart et al.20 Wagner and MacIver25 |
| Diethyl malonate | 105-53-3 | C7H12O4 | 160.17 | 0.27 | 0.96 | Willis et al.18 |
| Diethyl sulfide | 352-93-2 | C4H10S | 90.19 | 54.2* | 1.8-1.9 | Vorontsov et al.23 |
| Methyl salicylate (MeS) | 119-36-8 | C8H8O3 | 152.15 | 0.04 | 2.55 | Willis et al.18 Feldman19 Gladish21 Leppert et al.22 |
Median lethal concentration-time product (LCt50) at respiratory level for SM was determined from on-field observations and animal experimentations to range from 900 to 1500 mg.min.m-3.8, 9, 17 Concerning its incapacitating thresholds, it has been reported that first ocular, cutaneous and respiratory symptoms occur at 12–70, 50 and 100–500 mg.min.m-3, respectively.7
Storage and use of SM are restricted to military infrastructures. Therefore, potential surrogates for SM have been proposed and studied.16, 18-24 List of common simulants for SM and their main physical and chemical properties are represented in Table 1.
Choice for a suitable simulant of SM to conduct a vapour exposure experiment was guided by two criteria: similarity with the physical properties of sulphur mustard, and minimum toxicity of the candidates. Hair incorporation capacity of chemicals was shown to mainly depend on the lipophilicity (which can be represented by octanol/water partition coefficient Ko/w) of the compound.11, 29 Consequently, for chemical vapour exposure of hair, we considered that the most important parameters to take into account were chemical vapour pressure (Vp) and Ko/w. As can be seen in Table 1, methyl salicylate, which is a low toxicity chemical, shows Vp and Ko/w quite similar to SM.
Despite the obvious differences in chemical structures (Figure 1), methyl salicylate (MeS) has proven to reliably mimic the physical behaviour of SM.19, 30, 31 Besides, MeS is one of the least toxic surrogates for SM.
Materials and methods
Chemicals and materials
Natural blonde hair from Secher-Fesnoux (Chaville, France) was used to conduct the exposure experiments.
MeS from Acros Organics was provided by Sigma-Aldrich (Saint-Quentin Fallavier, France) and internal standard (IS) methyl salicylate-d4 (MeS-d4) by Cluzeau Info Labo (Sainte-Foy-La-Grande, France). Polytetrafluoroethylene (PTFE) rubber was purchased at VWR (Fontenay-sous-Bois, France). SupraSolv® dichloromethane for chromatography and acetone GR for analysis were obtained from Merck (Darmstadt, Germany). N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was provided by Macherey-Nagel (Hoerdt, France).
A 1182 µg.mL-1 stock solution of MeS was prepared by weighing exactly 50 μL MeS and diluting them in 50 mL dichloromethane. This solution was diluted 1000 times to give a 1182 ng.mL-1 methyl salicylate working solution.
Standard solution of internal standard (IS solution) was prepared weighing exactly 1 μL MeS-d4 and diluting them in 20 mL acetone, giving a 58.6 µg.mL-1 solution.
Experimental design
Two sets of experiments were conducted: the first one intended to evaluate the influence of exposure dose (10 to 60 µg) with an exposure duration fixed to 30 min (Table 2); the second aimed at evaluating the influence of exposure time (5 to 60 min) with an exposure dose fixed to 60 µg (Table 3). Time and dose conditions were determined in order to be in a range around the median lethal concentration-time product (LCt50) of SM.
| Experiment | Methyl salicylate concentration in vapour phase (µg.L-1) | Ct (mg.min.m-3) | Mean % recovered |
|---|---|---|---|
| A | 49.3 | 1460–1480 | 12 ± 2 |
| B | 20.1 | 590–610 | 13 ± 5 |
| C | 10.0 | 290–310 | 10 ± 5 |
| D | 5.1 | 140–160 | ND |
| E (blank) | 0 | 0 | ND |
- ND: not detected
| Experiment | Exposure time (min) | Ct (mg.min.m-3) | Mean % recovered |
|---|---|---|---|
| F | 60 | 2960–3450 | 13 ± 2 |
| G | 45 | 2220–2540 | 12 ± 5 |
| H | 30 | 1470–1640 | 9 ± 5 |
| I | 15 | 740–860 | 6 ± 3 |
| J | 5 | 240–290 | 3 ± 2 |
Each experiment was repeated three times (n = 3) at different days. To avoid any evaporation loss, MeS stock solutions were freshly prepared for each experiment.
Exposure experiments were performed with a 1 L sealed two-neck round-bottom flask (exact volumes were determined by weighing water-full tanks). A photoionization detector (ppbRAE 3000 from RAE Systems, Vernaison, France) was used to ensure that no leak could result in a significant loss of MeS.
Around 90 mg hair were exactly weighed and tied with PTFE rubber to form a lock and hung to the central cap with the help of the PTFE rubber. Via the lateral cap, determined volume of MeS stock solution was loaded to the bottom of the tank, and then immediately vaporised by heating for 1 min at 80°C. The tank was then placed in a 25°C water bath in order to maintain a constant temperature during the exposure experiment, and left for the determined time of exposure.
Sample preparation
Right after exposure, hair was extracted through gentle agitation in 5 ml dichloromethane for 10 min. A 1 mL aliquot of the extract was then spiked with 10 μL of IS solution. 100 μL were transferred out, enriched with 50 μL BSTFA and heated 30 min at 55°C in order to complete the derivatization.32
In order to ensure that no unwanted contamination took place, blank experiments were conducted with each set of experiment by extracting and derivatizing non-exposed hair. MeS has never been detected in these blank samples.
Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis
Analyses were conducted on GC System 7890A equipped with a 7000 MS triple quadrupole from Agilent Technologies, Inc. (Santa Clara, California, USA). One μL was injected in splitless mode with the multimode inlet temperature program held at 60°C for 1 min at the beginning of the run, and finally increased to 300°C with a rate of 720°C.min-1. Elution of target compounds was performed on a HP-5 MS column [30 m × 0.250 mm (0.25 µm film thickness); Agilent Technologies, Inc.] with helium 99.9999% (Air Liquide, Paris, France) as carrier gas at a flow rate of 1 mL.min-1. The oven programming was set as followed: initial temperature of 50°C was held for 1 min then increased from 50 to 80°C at 120°C.min-1, held at 80°C for 2 min, increased from 80 to 300°C at 20°C.min-1, and finally held at final temperature for 4.5 min. Total run lasted 18.75 min. Ionization was operated in electron impact mode (70 eV). Other mass spectrometer parameters were transfer line temperature (300°C), source temperature (230°C) and quadrupole temperature (150°C). Detection was operated in multiple reaction monitoring (MRM) mode. Two transitions per compound and corresponding collision energies (CE) were optimised as following: derivatized MeS m/z 209.0 → 179.0, CE = 10 eV (quantitation), and m/z 209.0 → 161.0, CE = 20 eV (qualification); derivatized MeS-d4 m/z 213.0 → 183.1, CE = 10 eV (quantitation), and m/z 213.0 → 89.0, CE = 20 eV (qualification).
Data analysis and processing were realized with MassHunter QQQ Quantitative Analysis software (version B.05.00) from Agilent Technologies, Inc. Graphs and fitting were computed with SigmaPlot version 11.0 (Systat Software, San José, CA, USA).
Calibration curves were constructed with 5 points covering a concentration range of 6 to 1182 ng.mL-1 by subsequent dilution of 1182 ng.mL-1 working solution (with respective concentrations 1182, 283.7, 70.9, 23.6, and 5.9 ng.mL-1). Each calibration solution was spiked to 586 ng.mL-1 of MeS-d4, and silylated following previously described protocol. Quantitation was made by plotting the peak-area ratio of MeS and MeS-d4 versus MeS concentration. Linearity was assumed as correlation coefficients were superior to 0.99 and back-calculation of calibration solutions showed residues less than 15%. Intercepts were confirmed to be not significantly different from 0 according to the Student test with a risk of 5%. In the same way, slopes were confirmed to be significantly different from 0. Inter-day method precision was assessed by replicate injections of standard solutions (n = 8) over two weeks and was expressed as the relative standard deviation (RSD). RSD on the calculated concentrations of the replicates was less than 15%.
Investigation of matrix effect was performed by spiking blank matrix (dichloromethane extract of non-exposed hair; n = 6) with MeS. RSD for these points was less than 15%, too.
Blank control was performed all over the process to avoid cross contamination or carryover.
According to the Commission decision 2002/657/EC,33 peak identification was confirmed by making sure that the relative retention times and the relative abundance of the two investigated transitions correspond to those of the calibration standards. Relative retention times of the hair extracts were consistent with those of the standard analyte with a tolerance of 0.5%. Fragments relative abundances for the calibration standards were all over 50%, and relative abundances for the hair extracts were within the 20% tolerance.
Results and discussion
For the experiments set at fixed duration (Set 1), MeS was recovered from hair extracts, except for the lowest exposure dose of 6 µg (experiments D). Expectedly, MeS content in hair increased with exposure dose (Figure 2) and time (Figure 3).
Maximum MeS percentage recovered from hair in comparison to the initial dose is 15% for a 30 min exposure to 20 μL of stock solution. As shown in Tables 2 and 3, mean MeS percentages recovered from hair represented 3 to 13% of the MeS dose. Rest of MeS is assumed to mostly stay in gaseous phase.
Surface adsorption on hair has already been investigated with other organic molecules, i.e. cannabinoids13 or cocaine.14 Hair was shown to trap external vapour contamination, as target molecules were retrieved after usual extraction. Concerning the cocaine experiments, a kinetic study was conducted for times varying from 0 to 60 min, and showed that detected amount of cocaine increased linearly with the exposure time.14 It confirms our observation that time is crucial when assessing the intensity of exposure.
Isothermal sorption of gases in equilibrium or non-equilibrium state has been widely investigated in the literature.34-37
(1)Modelling data with Eqn (1) resulted in a power model with a correlation coefficient R2 = 0.9992.
(2)Best fitting is obtained with this model, as represented on Figure 4.
(3)
Plotting t/qt versus t resulted in a straight line (Figure 5) with a correlation coefficient of R2 = 0.9837. From Eqn (3), we could calculate the maximum content of MeS in hair at equilibrium qm to be 113 ng.mg-1, and the adsorption rate k to be 0.03406 min-1.
By using all experiments data, variation of MeS content recovered from hair was represented in function of the Ct product (Figure 6).
According to the Ct products, experiments could be ordered in function of the intensity of exposure from the lower exposure to the most severe one as following: J < C < B < I < A < H < G < F. This evolution is concomitant with the recovered MeS content evolution in hair. Maximum amount of MeS in hair was measured with 60 min exposure to a 56 µg initial dose: it represented 13 ± 2% of initial dose.
Our results could be fitted with a single rectangular 2-parameter hyperbola equation, showing a promising correlation coefficient R2 = 0.8807 (Figure 6). Model used for the fitting resembles the ones used for radioligands saturation binding curves in neurology,43 except that bound quantity is expressed in function of radioligand concentration. Nevertheless, radioligand concentration could be considered as an expression of exposure intensity, just as Ct product is. In the same way, the outline of the curve shows a saturation effect, proving that hair could be characterized by its adsorption properties such as its number of sorption sites. These results could also underline the importance of time for assessing the exposure.
As we only proceeded of simple soaking of hair in solvent during the extraction procedure, we assumed that surface adsorption alone was taken into account, as dichloromethane soaking is currently used for clean-up procedures of hair in forensic analyses in order to eliminate non-endogenously incorporated compounds.44 Our results showed that part of the initial dose was recoverable from hair extraction, that part increasing with exposure time (when hair was exposed to constant doses, Figure 4).
With a rather small lock of hair, MeS exposure was detected. Our results underlined the interest of hair as a trap surface, showing its great specific surface could be an advantage in low dose exposure. Besides, as extraction and detection conditions can surely be improved, improving the detection level is conceivable. Surface area of non-damaged hair has been shown to be 0.40 m2.g-1.15 With their 35 m2.g-1 specific surface area, Tenax® tubes have already been investigated as a suitable trap for chemical warfare agents,45 but they can be expensive and need a controlled throughput in order to give quantitative results. In this way, they are more appropriate to detect presence in the atmosphere, not personal contamination of people. Despite its less extended surface area, hair still can represent a valuable tool for contamination detection of each person.
Assuming that our results can be reliably transposed to a sulphur mustard exposure, our experimental exposure conditions were at high concentration-time products, as this preliminary study was designed in order to evaluate the feasibility of using hair as exposure marker. Our results showed that high intensity exposure above 1500 mg.min.m-3, corresponding to the observation of severe effects (death of more than half of the exposed population), was related to a saturation phase (max. 90 ng.mg-1 of hair, less than 0.1‰ of total mass).
Below 12 mg.min.m-3, no effect would normally be observed due to SM exposure. In this range of Ct products, no detection in hair was possible with our current detection conditions.
In between these two areas, a linear part could be observed on the MeS content = f (Ct) curve. This correlation curve would allow us to extrapolate the exposure intensity (Ct value) from the MeS measurement in hair, in the Ct range (12–1500 mg.min.m-3) where SM exposure induces the first incapacitating effects.
Intensity of exposure could also be expressed through AEGLs (Acute Exposure Guideline Levels). These threshold values are developed by the National Advisory Committee for the Development of Acute Exposure Guideline Levels for Hazardous Substances. AEGLs aim to describe concentration limits and inherent risks for general population when exposed once to chemicals during periods ranging from 10 min to 8 h.46 Three levels correspond to various intensity and severity of toxic effects induced by exposure17: AEGL-1 is the vapour concentration above which general population could experience non disabling and reversible effects; AEGL-2 is the vapour concentration above which general population could experience irreversible or long-lasting effects; AEGL-3 is the vapour concentration above which general population could experience life-threatening health effects or death. For sulphur mustard and for 30 min exposure, AEGLs range from 0.13 (AEGL-1) to 2.7 mg.m-3 (AEGL-3).17, 46
In the case of this study, for the 30-min-exposure experiments (Set 1), all MeS concentrations in the vapour phase (Table 2) were above these values, showing that acute exposure could be assessed through the analysis of hair. That approach of intensity exposure, seeming more stringent than Ct products, encourages us to carry on studies on lower concentrations in order to reach the AEGL-1 range.
Conclusion and perspectives
To the best of our knowledge, this study is the first report of CWA simulant adsorption on hair. CWA adsorption onto other surfaces has already been investigated with textile and skin by using MeS as surrogate as well.19, 21
Our results showed that external contamination of hair with vaporised MeS was detected with simple hair extraction (dichloromethane soaking), and with a rather little quantity of hair. This result is encouraging for the use of hair as a passive sampler for chemical compounds, especially chemical warfare agents, and identification of exposed individuals.
Although MeS is a suitable surrogate for the physical behaviour of sulphur mustard, chemical interactions would have to be studied, with the use of 2-chloroethyl ethyl sulphide as another simulant. Supplementary work would also imply to lower our current detection limits by improving extraction for example and thus reach assessment of contamination below the various exposure limits determined for CWA (Ct products and AEGLs). Moreover, no desorption phenomenon was taken into account in our study. After a certain amount of time after exposure, target molecules could be released from hair, leading to a possible secondary contamination. Therefore it would be pertinent to realize desorption studies after exposure of hair to target molecules.
Acknowledgements
This research was supported by the Direction Générale de l'Armement (DGA) and the Centre National de la Recherche Scientifique (CNRS) in France through a PhD thesis grant. The authors acknowledge the French research federation ECCOREV.
