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MS quantification of benzyl salicylate on skin and hair: A novel chemical simulant for human decontamination studies
Journal Pre-proofs GC-MS/MS quantification of benzyl salicylate on skin and hair: a novel chemical simulant for human decontamination studies Thomas James, Samuel Collins, Richard Amlôt, Tim Marczylo PII: DOI: Reference:
S1570-0232(19)30995-X https://doi.org/10.1016/j.jchromb.2019.121818 CHROMB 121818
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
31 July 2019 20 September 2019 24 September 2019
Please cite this article as: T. James, S. Collins, R. Amlôt, T. Marczylo, GC-MS/MS quantification of benzyl salicylate on skin and hair: a novel chemical simulant for human decontamination studies, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121818
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
GC-MS/MS quantification of benzyl salicylate on skin and hair: a novel chemical simulant for human decontamination studies
Authors: Thomas Jamesa, Samuel Collinsa, Richard Amlôtb and Tim Marczyloa a
Centre for Radiation, Chemicals and Environmental Hazards (CRCE), Public Health
England, Chilton, OX11 0RQ b
Emergency Response Department Science & Technology, Public Health England, Salisbury,
SP4 0JG.
Corresponding Author: Thomas James, CRCE, Public Health England, Chilton, OX11 0RQ [email protected]
1
Abstract Human studies investigating the efficacy of emergency decontamination protocols for chemical incidents require the use of non-hazardous chemical simulants. Methyl salicylate (MeS) has almost exclusively been used for this purpose. Whilst MeS is a simulant of the chemical warfare agent (CWA) sulphur mustard, it is not an ideal simulant for many other chemical threats with greater persistence and lower volatility. Benzyl salicylate (BeS) has been investigated here as a low toxicity simulant for lower volatility, persistent chemical threat agents and toxic industrial chemicals (TICs). To evaluate the suitability of BeS as a simulant for human decontamination studies a gas chromatography-triple quadrupole mass spectrometry method was designed, optimised and validated, for the analysis of human skin and hair. Quantification was achieved using isotope-dilution, EI and collision-induced dissociation and multiple reaction monitoring for both qualifier and quantifier ion transitions. The mass transitions were m/z 285 → 91 and m/z 210 → 181, respectively for the quantifier and qualifier ions of BeS, and m/z 289 → 91 and m/z 214 → 185 for the quantifier and qualifier ions for the BeS-d4 internal standard, respectively. The method exhibited excellent coefficients of determination (R2 = 0.9992 to 0.9999) with LOD and LOQ values at 0.023 ng/ml and 0.23ng/ml. Across three Quality Controls (QCs), 11.5 ng/ml, 115 ng/ml and 1150 ng/ml) average accuracy (intra-day 95.6% to 100.3%, inter-day 98.5% to 104.91%) and precision (intra-day RSD (%) 2% to 13.7%, inter-day RSD (%) 3.3% to 8.8%) were determined. The validated method was applied in a proof of principle volunteer study for the determination of BeS recovered from skin and hair. The average total BeS recovery after 70 minutes was 37.9% from skin and there was a significant increase between baseline and post-intervention levels for hair. These data demonstrate that BeS is an appropriate simulant for persistent chemicals and that the analytical method employed here is suitable for BeS analysis in human studies. Keywords: chemical warfare agent, simulant, benzyl salicylate, methyl salicylate, gas chromatography-mass spectrometry
2
1
Introduction
The decontamination of casualties following chemical exposure is a primary concern for first responders. Determining the efficacy of decontamination interventions for highly toxic chemical threats under realistic conditions necessitates using non-toxic chemical simulants that mimic the physicochemical properties of the chemical threat(s) of concern with human volunteers. Methyl salicylate (MeS, CAS 119-36-8) has low toxicity and similar physicochemical properties to sulphur mustard [1, 2]. It has been used in the majority of human decontamination studies [3-5], however, due to its relatively high vapor pressure only a small proportion of the applied dose of MeS remains on human skin at the end of a study [6, 7]. This complicates interpretation of decontamination efficacy, or the perceived effectiveness of multi-step or prolonged interventions that may be necessary for particularly hazardous or persistent chemical agents. Novel simulants that remain detectable on the skin or hair for longer intervention studies and thus mimic more-persistent agents of concern are needed. Benzyl salicylate (BeS, CAS 118-58-1), the benzyl ester of salicylic acid, is commonly used in perfumes and cosmetics for its fragrance and UV absorbing properties [8]. BeS has a vapour pressure of 0.01 Pa at 25°C, compared to 4.6 Pa at 25°C for MeS, meaning it is more persistent and will not evaporate so readily under typical human decontamination study conditions. The acute dermal LD50 of BeS in rabbits is 14.15 g/kg [9]. The relatively low vapour pressure and low toxicity means that BeS could be an ideal simulant for more prolonged decontamination studies. Furthermore, BeS could be a simulant for persistent toxic industrial chemicals such as malathion and dimethyl methylphosphonate or chemical warfare agents such as VX and Novichok which have comparable vapour pressures. We aimed to develop and validate a GC-MS/MS method for the quantification of BeS in human skin and hair samples and conducted a proof of principle study to determine the suitability of BeS as a persistent chemical simulant for human decontamination studies.
3
2 2.1
Materials and Methods Reagents and Materials
Benzyl salicylate (99.9%) was purchased from Sigma (Gillingham, UK). The internal standard benzyl 2-hydroxybenzoate-3,4,5,6-d4 (isotopic purity 98%) was purchased from Qmx Laboratories Ltd. (Thaxted, UK). Application of BeS to skin was conducted using a Gilson Microman M10 positive displacement pipette. For application of BeS to hair, a Revell Master Class Compressor attached to a Renegade Series Krome Gravity Feed airbrush (Badger Air-brush, USA) was used. All other associated reagents and materials are outlined in James et al. [10]. 2.2
Preparation of standards
A working solution (0.46 mg/ml) of BeS in DCM was serially diluted in DCM to generate 0.23, 2.3, 23, 230 and 2300 ng/ml calibration standards, and three QC standards at 1.15, 115 and 1150 ng/ml. Derivatisation master mix incorporating internal standard (2 µg/ml), pyridine and BSTFA/TMCS in a ratio of 10:95:95, respectively was prepared immediately prior to sample derivatisation. The final concentration of internal standard in all calibrations, QCs and samples was 50 ng/ml. All solutions were stored in sealed headspace vials, in the dark at room temperature and were cooled on ice before use. 2.3
Standard/sample derivatisation
Calibrations, QCs and samples (20 µl) were mixed with 20 μl of the derivatisation master mix in a 2 mL GC vial with fused 200ul glass insert prior to heating at 55 °C for 60 min; an increase in derivatisation time of 30 min over the previous MeS method using non-insert vials [10]. 60 min was experimentally determined to be the minimum derivatisation time that resulted in 100% derivatisation of BeS (data not shown).
4
2.4
Instrumentation
BeS analysis was undertaken using a previous MeS method [10] with select modifications. The oven temperature was held at 70 °C for 1 min then ramped to 250 °C at 45 °C/min. At 5 min, the GC oven continued ramping to 290°C at 15°C/min. At 7 min, the inlet pressure changed to 2 psi and the pneumatics control module (PCM) increased to 45 psi, reversing the flow of helium through the phaseless post-column and implementing a backflush that voided 12 column volumes in 2.6 min. Multiple reaction monitoring was carried out between 4.7 and 7 min to detect both BeS and the internal standard co-eluting at 6.2 min. Nitrogen collision gas was supplied at a pressure of 10psi and a flowrate of 1.5ml/min. For BeS, the quantifier and qualifier transitions were m/z 285 → 91 (collision energy, CE 10 eV) and m/z 210 → 181(CE 10 eV), respectively. For BeS-d4 the quantifier and qualifier transitions were m/z 289 → 91 (CE 10eV) and m/z 214 → 185 (CE 20eV), respectively. All transitions and collision energies were determined by systematic product ion scans within which Q2 voltages were incrementally adjusted until the optimal signal to noise ratio for that transition was achieved. 2.5
Validation
Linearity was assessed by the coefficient of determination (r2) of multiple five-point calibration curves (0.23 to 2300 ng/ml; n=8). Determination of matrix effects were conducted by analysing QCs in sample-spiked solvent (hair or D-Squame discs) and referencing them against “non-spiked” QCs. Accuracy and precision were validated through inter and intra-day replicate analyses of QCs, and sensitivity was based on the limits of detection (LOD, S/N 3) and quantification (LOQ, S/N 10). Reproducibility was assessed using ten replicate samples. Recovery was assessed using four methods; recovery from BeS-spiked D-Squame discs, recovery from BeS-spiked hair, recovery from BeS-dosed skin and a time course from BeSdosed skin (sample collections at 15, 30 and 60 min). All validation data are shown in supplementary materials 1.
5
2.6
Proof of principal study in human volunteers
2.6.1
Application of BeS to skin
To demonstrate the applicability of the analytical methods, a selection of no decontamination intervention (control) samples were analysed from two human studies. Ethical approval was independently granted by Public Health England’s Research Ethics and Governance Group. Two groups of participants (skin study n= 5 male, n = 5 female, hair study n = 6 male, n = 4 female) were used for the studies. Participants were aged between 18 and 60 and in good health. 2µl of BeS was applied to the left shoulder blade in a pre-marked area. At T+70 min the application site was tape stripped sequentially using six individual D-Squame discs. Every two discs were combined into 10ml DCM to create three samples for each participant (Discs 1+2, Discs 3+4 and Discs 5+6, n=30 total). Samples were diluted 100-fold with DCM prior to analysis.
2.6.2
Application of BeS to hair
A baseline sample of hair ~5-10 hairs from the posterior vertex of the head was collected from each participant to detect BeS on the hair before trial application. The cut hairs were combined in one pre-weighed vial containing 10 ml DCM. For hair application, 500 µl of BeS was sprayed out of a gravity fed airbrush onto the back of the participants’ heads from a distance of 10 cm using a pressure of 10 psi and at maximum spray gun velocity. After 70 min, a hair sample was taken. The back of the head was divided into a three row, five column grid configuration surrounding the application area. From each sector, a sample of ~5-10 hairs was collected and combined in a preweighed vial containing 10 ml DCM.
6
All hair samples were diluted 100-fold 24h after collection. The total mass of hair was determined by mass balance following the evaporation of DCM in a fume cupboard for one week.
3 3.1
Results and Discussion Adaptation and optimisation of the analytical method
Method development started with elongating the oven ramp of the original published method for MeS [10], and it was found that when the 45°C/min ramp was extended to 290°C, BeS and BeS-d4 eluted slightly later (full scan mode employed between 4.7 and 10 min) than that reported for MeS with a retention time of 5 min but with a broad peak width. The initial oven ramp was reduced to a rate of 15°C/min to a temperature of 290°C and the new retention time of BeS and BeS-d4 was 6.2 min, with a greatly decreased peak width. Finally, the previously employed backflush method was introduced at 7min once BeS and BeS-d4 had completely eluted. To identify suitable MRM transitions, the largest and most abundant peak in the mass spectrum of each analyte was chosen as the first ions to fragment. Quadrupole 1 was set to single ion monitoring (SIM) mode, and the ions were fragmented at 10eV in the collision cell. This was repeated in increments of 5eV up to 40eV. The transition that produced the largest product peak was chosen as the quantifier transition of each molecule (figure 1). This process was then repeated using the second most abundant ion in the full scan, and this produced the optimal transition for qualifier ions for both compounds. The common quantifier product ion of both BeS and BeS-d4 of m/z 91 indicates this fragment is not derived from the deuterium-containing salicylate ring, therefore it is believed that m/z 91 is due to the fragmentation of a methyl benzene ion [CH2-C6H5]+ from the parent molecule. Additionally, this fragment is not present in the MeS product ion spectra, highlighting that the fragment is likely to have originated from the benzyl side chain on the salicylic acid base structure.
7
3.2
Validation
All validation results are summarised in Table A, presented in Supplementary Material 1. 3.3
Proof of principal volunteer study
3.3.1 Skin Samples BeS was detected in all samples. The mean top layer (discs 1+2) recovery was calculated as 28.6% of the applied dose, which was consistent with the validation data (top layer recovery of 28.9% after 60min, see supplementary material 1). Mean recoveries from layers 3+4 and 5+6 were 6.9% and 2.4%, respectively, resulting in a total mean recovery of 37.9% (figure 2). These data confirm that BeS is more persistent on skin than MeS (14.7% recovery after only 30 min) [10] and could be a useful simulant for determining decontamination efficacy for multiple sequential interventions at longer timescales. Further work is required to determine the fate of the unaccounted dose (~60%). Volatilisation, skin penetration beyond the recoverable layers of the stratum corneum or spread outside of the collection area are possibilities. 3.3.2 Hair Samples Baseline (n=10) and post-control intervention (n=10) hair samples were collected. Table 1 summarises the recovery of BeS from hair. Compared to baseline levels (mean 0.16 µg/mg), the mass of BeS recovered post BeS-application increased for all participants (mean 47.6 µg/mg) (Figure 3). Particularly large increases were observed for participants 3 and 10 who had the shortest hair among the participants. This allowed for small amounts of hair containing large amounts of simulant to be recovered, while participants with longer hair would have had a smaller simulant to hair mass ratio. This may also explain the large discrepancies between post-baseline concentrations. Baseline levels of BeS varied by 2 orders of magnitude (0.008-0.594 µg/mg hair) (Table 1) possibly reflecting the presence of BeS in consumer products such as shampoo. Although the participants were instructed to avoid hair care products for up to 24h before the study, it is possible that residual BeS may have been present. Residual levels were however mostly
8
2 or more orders of magnitude lower than post application levels (except participants 7 and 8). Following application, a ~150-fold difference between the lowest and highest BeS concentrations recovered was detected (Table 1). Possible reasons for this variability include, hair type, length, density and study conditions on the day of sampling (temperature, humidity). Low post-intervention recovery may be attributed to occlusion by non-exposed hair especially in long-haired participants. While every care was taken to ensure hair was collected from the correct zone, it’s possible that the top layers of hair moved, reducing the amount of contaminated hairs within the application zone collected. Attempts were made to mitigate this issue by using multi-increment sampling to obtain a better estimate of the average BeS concentration. Sampling may be improved by increasing the number of increments or by using fluorescent markers to aid in contamination visualisation. Despite this limitation, a clear increase in BeS concentration was still seen between pre-and postapplication for all participants, indicating that BeS is an effective simulant for assessment of contamination in levels in hair, even after 70 min.
4
Conclusion
A method was successfully adapted, optimised and validated for the analysis of BeS. Skin recovery during validation and the proof of concept human study was higher for BeS than previously described for MeS. The method was shown to accurately quantify levels of BeS on hair before and after simulant application, with good recovery at 70 min. These data show that BeS can be a valuable chemical simulant for higher persistence agents, or when studying decontamination interventions for extended time periods in human volunteer studies.
9
Acknowledgements The authors would like to thank Louise Davidson, Natalie Williams, Emily Orchard and Felicity Southworth for their assistance in collecting the samples in the human study. This paper is based on independent research commissioned and funded by the National Institute for Health Research (NIHR) Policy Research Programme (PR‐ST‐1015‐10016). Richard Amlôt is part‐funded by the NIHR Health Protection Research Unit in Emergency Preparedness and Response at King's College London in partnership with Public Health England (PHE) and Tim Marczylo is part-funded by the NIHR Health Protection Research Unit in Health Impacts of Environmental Hazards. The views expressed in the publication are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care, ‘arms’ length bodies or other Government departments.
10
References
1.
2.
3.
4.
5. 6.
7. 8. 9. 10.
T. James, S. Wyke, T. Marczylo, S. Collins, T. Gaulton, K. Foxall, R. Amlôt, R. Duarte-Davidson, Chemical warfare agent simulants for human volunteer trials of emergency decontamination: A systematic review. J. Appl. Toxicol. 38 (2018) 113121. S.L. Bartelt-Hunt, D.R.U. Knappe, M.A. Barlaz, A Review of Chemical Warfare Agent Simulants for the Study of Environmental Behavior. Crit. Rev. Environ. Sci. Technol. 38 (2008) 112-136. P. Ribordy, D. RockseẤn, U. Dellgar, S.Å. Persson, K. Arnoldsson, H. EkÃlsen, S. HaÂggbom, O. Nerf, Å. Ljungqvist, D. Gryth, O. Claesson, Mobile decontamination units-room for improvement?, Prehosp. Disaster. Med. 27 (2012) 425-431. S. Torngren, S.A. Persson, A. Ljungquist, T. Berglund, M. Nordstrand, L. Hagglund, L. Rittfeldt, K. Sandgren, E. Soderman, Personal decontamination after exposure to stimulated liquid phase contaminants: functional assessment of a new unit, J. Toxicol. Clin. Toxicol. 36 (1998) 567-573. R. Amlôt, H. Carter, L. Riddle, J. Larner, R.P. Chilcott, Volunteer trials of a novel improvised dry decontamination protocol for use during mass casualty incidents as part of the UK’S Initial Operational Response (IOR), PLoS One. 12 (2017) e0179309. R.P. Chilcott, et al., Evaluation of US Federal Guidelines (Primary Response Incident Scene Management [PRISM]) for Mass Decontamination of Casualties During the Initial Operational Response to a Chemical Incident. Ann. Emerg. Med. 73 (2019) 671-684. Orchids Project. http://www.orchidsproject.eu, (2009) (accessed 17 June 2019) D. Belsito, et al., A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food Chem. Toxicol. 45 (2007) S318-S361. Cosmetic Ingredient Review Expert Panel, Safety Assessment of Benzyl Salicylate As Used in Cosmetics. https://www.cir-safety.org/supplementaldoc/safetyassessment-benzyl-salicylate-used-cosmetics-0 (2019) (accessed on 17 June 2019) T. James, S. Collins, R. Amlôt, T. Marczylo, Optimisation and validation of a GC– MS/MS method for the analysis of methyl salicylate in hair and skin samples for use in human-volunteer decontamination studies. J. Chromatogr. B. 1109 (2019) 84-89.
11
Figure Captions Figure 1. Diagram A shows the mass fragmentation of 285 m/z (BeS) and diagram B shows the fragmentation of 289 m/z (BeS-d4). Both fragmentations occurred through collision induced dissociation in Q2 at 10eV. 91 is the dominant ion produced for both BeS and BeSd4. Figure 2. n = 3 D-Squame disc pairs (1 + 2, 3 + 4 and 5 + 6) for 10 participants (total N = 30) showing percentage recovery of BeS through three skin penetration levels. The top two layers (discs 1-4) can be regarded as the “non-bioavailable” fraction, while the bottom layer (discs 5 and 6) is considered the bioavailable fraction. Box and whisker plot shows median and inter–quartile range, together with the maximum and minimum values. Figure 3. N=10 baseline, N=10 post-intervention samples (70min) for BeS recovery from hair. The data is displayed on a logarithmic scale. Box and whisker plot shows median and inter–quartile range, together with the maximum and minimum values.
12
Figures Figure 1
A
B
Figure 2
Percentage of applied dose
100
10
1
Disc 1+2 Discs 3+4 Discs 5+6
Total
13
Concentration (ug/mg hair)
Figure 3
1000 100 10 1 0.1 0.01 0.001
Baseline
Post application
14
Table 1: Recovery of BeS from control participants’ hair. Each post application sample is much higher in concentration than baseline samples, however the recovered concentration of BeS is highly variable amongst participants. Participant
1
2
3
4
5
6
7
8
9
10
Baseline
0.015
0.011
0.008
0.181
0.067
0.050
0.594
0.204
0.022
0.496
5.001
45.86
147.02
33.57
84.63
40.613
1.281
3.138
6.456
108.30
(µg/mg) Postapplication (µg/mg)
15
S1570-0232(19)30995-X https://doi.org/10.1016/j.jchromb.2019.121818 CHROMB 121818
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
31 July 2019 20 September 2019 24 September 2019
Please cite this article as: T. James, S. Collins, R. Amlôt, T. Marczylo, GC-MS/MS quantification of benzyl salicylate on skin and hair: a novel chemical simulant for human decontamination studies, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121818
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
GC-MS/MS quantification of benzyl salicylate on skin and hair: a novel chemical simulant for human decontamination studies
Authors: Thomas Jamesa, Samuel Collinsa, Richard Amlôtb and Tim Marczyloa a
Centre for Radiation, Chemicals and Environmental Hazards (CRCE), Public Health
England, Chilton, OX11 0RQ b
Emergency Response Department Science & Technology, Public Health England, Salisbury,
SP4 0JG.
Corresponding Author: Thomas James, CRCE, Public Health England, Chilton, OX11 0RQ [email protected]
1
Abstract Human studies investigating the efficacy of emergency decontamination protocols for chemical incidents require the use of non-hazardous chemical simulants. Methyl salicylate (MeS) has almost exclusively been used for this purpose. Whilst MeS is a simulant of the chemical warfare agent (CWA) sulphur mustard, it is not an ideal simulant for many other chemical threats with greater persistence and lower volatility. Benzyl salicylate (BeS) has been investigated here as a low toxicity simulant for lower volatility, persistent chemical threat agents and toxic industrial chemicals (TICs). To evaluate the suitability of BeS as a simulant for human decontamination studies a gas chromatography-triple quadrupole mass spectrometry method was designed, optimised and validated, for the analysis of human skin and hair. Quantification was achieved using isotope-dilution, EI and collision-induced dissociation and multiple reaction monitoring for both qualifier and quantifier ion transitions. The mass transitions were m/z 285 → 91 and m/z 210 → 181, respectively for the quantifier and qualifier ions of BeS, and m/z 289 → 91 and m/z 214 → 185 for the quantifier and qualifier ions for the BeS-d4 internal standard, respectively. The method exhibited excellent coefficients of determination (R2 = 0.9992 to 0.9999) with LOD and LOQ values at 0.023 ng/ml and 0.23ng/ml. Across three Quality Controls (QCs), 11.5 ng/ml, 115 ng/ml and 1150 ng/ml) average accuracy (intra-day 95.6% to 100.3%, inter-day 98.5% to 104.91%) and precision (intra-day RSD (%) 2% to 13.7%, inter-day RSD (%) 3.3% to 8.8%) were determined. The validated method was applied in a proof of principle volunteer study for the determination of BeS recovered from skin and hair. The average total BeS recovery after 70 minutes was 37.9% from skin and there was a significant increase between baseline and post-intervention levels for hair. These data demonstrate that BeS is an appropriate simulant for persistent chemicals and that the analytical method employed here is suitable for BeS analysis in human studies. Keywords: chemical warfare agent, simulant, benzyl salicylate, methyl salicylate, gas chromatography-mass spectrometry
2
1
Introduction
The decontamination of casualties following chemical exposure is a primary concern for first responders. Determining the efficacy of decontamination interventions for highly toxic chemical threats under realistic conditions necessitates using non-toxic chemical simulants that mimic the physicochemical properties of the chemical threat(s) of concern with human volunteers. Methyl salicylate (MeS, CAS 119-36-8) has low toxicity and similar physicochemical properties to sulphur mustard [1, 2]. It has been used in the majority of human decontamination studies [3-5], however, due to its relatively high vapor pressure only a small proportion of the applied dose of MeS remains on human skin at the end of a study [6, 7]. This complicates interpretation of decontamination efficacy, or the perceived effectiveness of multi-step or prolonged interventions that may be necessary for particularly hazardous or persistent chemical agents. Novel simulants that remain detectable on the skin or hair for longer intervention studies and thus mimic more-persistent agents of concern are needed. Benzyl salicylate (BeS, CAS 118-58-1), the benzyl ester of salicylic acid, is commonly used in perfumes and cosmetics for its fragrance and UV absorbing properties [8]. BeS has a vapour pressure of 0.01 Pa at 25°C, compared to 4.6 Pa at 25°C for MeS, meaning it is more persistent and will not evaporate so readily under typical human decontamination study conditions. The acute dermal LD50 of BeS in rabbits is 14.15 g/kg [9]. The relatively low vapour pressure and low toxicity means that BeS could be an ideal simulant for more prolonged decontamination studies. Furthermore, BeS could be a simulant for persistent toxic industrial chemicals such as malathion and dimethyl methylphosphonate or chemical warfare agents such as VX and Novichok which have comparable vapour pressures. We aimed to develop and validate a GC-MS/MS method for the quantification of BeS in human skin and hair samples and conducted a proof of principle study to determine the suitability of BeS as a persistent chemical simulant for human decontamination studies.
3
2 2.1
Materials and Methods Reagents and Materials
Benzyl salicylate (99.9%) was purchased from Sigma (Gillingham, UK). The internal standard benzyl 2-hydroxybenzoate-3,4,5,6-d4 (isotopic purity 98%) was purchased from Qmx Laboratories Ltd. (Thaxted, UK). Application of BeS to skin was conducted using a Gilson Microman M10 positive displacement pipette. For application of BeS to hair, a Revell Master Class Compressor attached to a Renegade Series Krome Gravity Feed airbrush (Badger Air-brush, USA) was used. All other associated reagents and materials are outlined in James et al. [10]. 2.2
Preparation of standards
A working solution (0.46 mg/ml) of BeS in DCM was serially diluted in DCM to generate 0.23, 2.3, 23, 230 and 2300 ng/ml calibration standards, and three QC standards at 1.15, 115 and 1150 ng/ml. Derivatisation master mix incorporating internal standard (2 µg/ml), pyridine and BSTFA/TMCS in a ratio of 10:95:95, respectively was prepared immediately prior to sample derivatisation. The final concentration of internal standard in all calibrations, QCs and samples was 50 ng/ml. All solutions were stored in sealed headspace vials, in the dark at room temperature and were cooled on ice before use. 2.3
Standard/sample derivatisation
Calibrations, QCs and samples (20 µl) were mixed with 20 μl of the derivatisation master mix in a 2 mL GC vial with fused 200ul glass insert prior to heating at 55 °C for 60 min; an increase in derivatisation time of 30 min over the previous MeS method using non-insert vials [10]. 60 min was experimentally determined to be the minimum derivatisation time that resulted in 100% derivatisation of BeS (data not shown).
4
2.4
Instrumentation
BeS analysis was undertaken using a previous MeS method [10] with select modifications. The oven temperature was held at 70 °C for 1 min then ramped to 250 °C at 45 °C/min. At 5 min, the GC oven continued ramping to 290°C at 15°C/min. At 7 min, the inlet pressure changed to 2 psi and the pneumatics control module (PCM) increased to 45 psi, reversing the flow of helium through the phaseless post-column and implementing a backflush that voided 12 column volumes in 2.6 min. Multiple reaction monitoring was carried out between 4.7 and 7 min to detect both BeS and the internal standard co-eluting at 6.2 min. Nitrogen collision gas was supplied at a pressure of 10psi and a flowrate of 1.5ml/min. For BeS, the quantifier and qualifier transitions were m/z 285 → 91 (collision energy, CE 10 eV) and m/z 210 → 181(CE 10 eV), respectively. For BeS-d4 the quantifier and qualifier transitions were m/z 289 → 91 (CE 10eV) and m/z 214 → 185 (CE 20eV), respectively. All transitions and collision energies were determined by systematic product ion scans within which Q2 voltages were incrementally adjusted until the optimal signal to noise ratio for that transition was achieved. 2.5
Validation
Linearity was assessed by the coefficient of determination (r2) of multiple five-point calibration curves (0.23 to 2300 ng/ml; n=8). Determination of matrix effects were conducted by analysing QCs in sample-spiked solvent (hair or D-Squame discs) and referencing them against “non-spiked” QCs. Accuracy and precision were validated through inter and intra-day replicate analyses of QCs, and sensitivity was based on the limits of detection (LOD, S/N 3) and quantification (LOQ, S/N 10). Reproducibility was assessed using ten replicate samples. Recovery was assessed using four methods; recovery from BeS-spiked D-Squame discs, recovery from BeS-spiked hair, recovery from BeS-dosed skin and a time course from BeSdosed skin (sample collections at 15, 30 and 60 min). All validation data are shown in supplementary materials 1.
5
2.6
Proof of principal study in human volunteers
2.6.1
Application of BeS to skin
To demonstrate the applicability of the analytical methods, a selection of no decontamination intervention (control) samples were analysed from two human studies. Ethical approval was independently granted by Public Health England’s Research Ethics and Governance Group. Two groups of participants (skin study n= 5 male, n = 5 female, hair study n = 6 male, n = 4 female) were used for the studies. Participants were aged between 18 and 60 and in good health. 2µl of BeS was applied to the left shoulder blade in a pre-marked area. At T+70 min the application site was tape stripped sequentially using six individual D-Squame discs. Every two discs were combined into 10ml DCM to create three samples for each participant (Discs 1+2, Discs 3+4 and Discs 5+6, n=30 total). Samples were diluted 100-fold with DCM prior to analysis.
2.6.2
Application of BeS to hair
A baseline sample of hair ~5-10 hairs from the posterior vertex of the head was collected from each participant to detect BeS on the hair before trial application. The cut hairs were combined in one pre-weighed vial containing 10 ml DCM. For hair application, 500 µl of BeS was sprayed out of a gravity fed airbrush onto the back of the participants’ heads from a distance of 10 cm using a pressure of 10 psi and at maximum spray gun velocity. After 70 min, a hair sample was taken. The back of the head was divided into a three row, five column grid configuration surrounding the application area. From each sector, a sample of ~5-10 hairs was collected and combined in a preweighed vial containing 10 ml DCM.
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All hair samples were diluted 100-fold 24h after collection. The total mass of hair was determined by mass balance following the evaporation of DCM in a fume cupboard for one week.
3 3.1
Results and Discussion Adaptation and optimisation of the analytical method
Method development started with elongating the oven ramp of the original published method for MeS [10], and it was found that when the 45°C/min ramp was extended to 290°C, BeS and BeS-d4 eluted slightly later (full scan mode employed between 4.7 and 10 min) than that reported for MeS with a retention time of 5 min but with a broad peak width. The initial oven ramp was reduced to a rate of 15°C/min to a temperature of 290°C and the new retention time of BeS and BeS-d4 was 6.2 min, with a greatly decreased peak width. Finally, the previously employed backflush method was introduced at 7min once BeS and BeS-d4 had completely eluted. To identify suitable MRM transitions, the largest and most abundant peak in the mass spectrum of each analyte was chosen as the first ions to fragment. Quadrupole 1 was set to single ion monitoring (SIM) mode, and the ions were fragmented at 10eV in the collision cell. This was repeated in increments of 5eV up to 40eV. The transition that produced the largest product peak was chosen as the quantifier transition of each molecule (figure 1). This process was then repeated using the second most abundant ion in the full scan, and this produced the optimal transition for qualifier ions for both compounds. The common quantifier product ion of both BeS and BeS-d4 of m/z 91 indicates this fragment is not derived from the deuterium-containing salicylate ring, therefore it is believed that m/z 91 is due to the fragmentation of a methyl benzene ion [CH2-C6H5]+ from the parent molecule. Additionally, this fragment is not present in the MeS product ion spectra, highlighting that the fragment is likely to have originated from the benzyl side chain on the salicylic acid base structure.
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3.2
Validation
All validation results are summarised in Table A, presented in Supplementary Material 1. 3.3
Proof of principal volunteer study
3.3.1 Skin Samples BeS was detected in all samples. The mean top layer (discs 1+2) recovery was calculated as 28.6% of the applied dose, which was consistent with the validation data (top layer recovery of 28.9% after 60min, see supplementary material 1). Mean recoveries from layers 3+4 and 5+6 were 6.9% and 2.4%, respectively, resulting in a total mean recovery of 37.9% (figure 2). These data confirm that BeS is more persistent on skin than MeS (14.7% recovery after only 30 min) [10] and could be a useful simulant for determining decontamination efficacy for multiple sequential interventions at longer timescales. Further work is required to determine the fate of the unaccounted dose (~60%). Volatilisation, skin penetration beyond the recoverable layers of the stratum corneum or spread outside of the collection area are possibilities. 3.3.2 Hair Samples Baseline (n=10) and post-control intervention (n=10) hair samples were collected. Table 1 summarises the recovery of BeS from hair. Compared to baseline levels (mean 0.16 µg/mg), the mass of BeS recovered post BeS-application increased for all participants (mean 47.6 µg/mg) (Figure 3). Particularly large increases were observed for participants 3 and 10 who had the shortest hair among the participants. This allowed for small amounts of hair containing large amounts of simulant to be recovered, while participants with longer hair would have had a smaller simulant to hair mass ratio. This may also explain the large discrepancies between post-baseline concentrations. Baseline levels of BeS varied by 2 orders of magnitude (0.008-0.594 µg/mg hair) (Table 1) possibly reflecting the presence of BeS in consumer products such as shampoo. Although the participants were instructed to avoid hair care products for up to 24h before the study, it is possible that residual BeS may have been present. Residual levels were however mostly
8
2 or more orders of magnitude lower than post application levels (except participants 7 and 8). Following application, a ~150-fold difference between the lowest and highest BeS concentrations recovered was detected (Table 1). Possible reasons for this variability include, hair type, length, density and study conditions on the day of sampling (temperature, humidity). Low post-intervention recovery may be attributed to occlusion by non-exposed hair especially in long-haired participants. While every care was taken to ensure hair was collected from the correct zone, it’s possible that the top layers of hair moved, reducing the amount of contaminated hairs within the application zone collected. Attempts were made to mitigate this issue by using multi-increment sampling to obtain a better estimate of the average BeS concentration. Sampling may be improved by increasing the number of increments or by using fluorescent markers to aid in contamination visualisation. Despite this limitation, a clear increase in BeS concentration was still seen between pre-and postapplication for all participants, indicating that BeS is an effective simulant for assessment of contamination in levels in hair, even after 70 min.
4
Conclusion
A method was successfully adapted, optimised and validated for the analysis of BeS. Skin recovery during validation and the proof of concept human study was higher for BeS than previously described for MeS. The method was shown to accurately quantify levels of BeS on hair before and after simulant application, with good recovery at 70 min. These data show that BeS can be a valuable chemical simulant for higher persistence agents, or when studying decontamination interventions for extended time periods in human volunteer studies.
9
Acknowledgements The authors would like to thank Louise Davidson, Natalie Williams, Emily Orchard and Felicity Southworth for their assistance in collecting the samples in the human study. This paper is based on independent research commissioned and funded by the National Institute for Health Research (NIHR) Policy Research Programme (PR‐ST‐1015‐10016). Richard Amlôt is part‐funded by the NIHR Health Protection Research Unit in Emergency Preparedness and Response at King's College London in partnership with Public Health England (PHE) and Tim Marczylo is part-funded by the NIHR Health Protection Research Unit in Health Impacts of Environmental Hazards. The views expressed in the publication are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care, ‘arms’ length bodies or other Government departments.
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T. James, S. Wyke, T. Marczylo, S. Collins, T. Gaulton, K. Foxall, R. Amlôt, R. Duarte-Davidson, Chemical warfare agent simulants for human volunteer trials of emergency decontamination: A systematic review. J. Appl. Toxicol. 38 (2018) 113121. S.L. Bartelt-Hunt, D.R.U. Knappe, M.A. Barlaz, A Review of Chemical Warfare Agent Simulants for the Study of Environmental Behavior. Crit. Rev. Environ. Sci. Technol. 38 (2008) 112-136. P. Ribordy, D. RockseẤn, U. Dellgar, S.Å. Persson, K. Arnoldsson, H. EkÃlsen, S. HaÂggbom, O. Nerf, Å. Ljungqvist, D. Gryth, O. Claesson, Mobile decontamination units-room for improvement?, Prehosp. Disaster. Med. 27 (2012) 425-431. S. Torngren, S.A. Persson, A. Ljungquist, T. Berglund, M. Nordstrand, L. Hagglund, L. Rittfeldt, K. Sandgren, E. Soderman, Personal decontamination after exposure to stimulated liquid phase contaminants: functional assessment of a new unit, J. Toxicol. Clin. Toxicol. 36 (1998) 567-573. R. Amlôt, H. Carter, L. Riddle, J. Larner, R.P. Chilcott, Volunteer trials of a novel improvised dry decontamination protocol for use during mass casualty incidents as part of the UK’S Initial Operational Response (IOR), PLoS One. 12 (2017) e0179309. R.P. Chilcott, et al., Evaluation of US Federal Guidelines (Primary Response Incident Scene Management [PRISM]) for Mass Decontamination of Casualties During the Initial Operational Response to a Chemical Incident. Ann. Emerg. Med. 73 (2019) 671-684. Orchids Project. http://www.orchidsproject.eu, (2009) (accessed 17 June 2019) D. Belsito, et al., A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food Chem. Toxicol. 45 (2007) S318-S361. Cosmetic Ingredient Review Expert Panel, Safety Assessment of Benzyl Salicylate As Used in Cosmetics. https://www.cir-safety.org/supplementaldoc/safetyassessment-benzyl-salicylate-used-cosmetics-0 (2019) (accessed on 17 June 2019) T. James, S. Collins, R. Amlôt, T. Marczylo, Optimisation and validation of a GC– MS/MS method for the analysis of methyl salicylate in hair and skin samples for use in human-volunteer decontamination studies. J. Chromatogr. B. 1109 (2019) 84-89.
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Figure Captions Figure 1. Diagram A shows the mass fragmentation of 285 m/z (BeS) and diagram B shows the fragmentation of 289 m/z (BeS-d4). Both fragmentations occurred through collision induced dissociation in Q2 at 10eV. 91 is the dominant ion produced for both BeS and BeSd4. Figure 2. n = 3 D-Squame disc pairs (1 + 2, 3 + 4 and 5 + 6) for 10 participants (total N = 30) showing percentage recovery of BeS through three skin penetration levels. The top two layers (discs 1-4) can be regarded as the “non-bioavailable” fraction, while the bottom layer (discs 5 and 6) is considered the bioavailable fraction. Box and whisker plot shows median and inter–quartile range, together with the maximum and minimum values. Figure 3. N=10 baseline, N=10 post-intervention samples (70min) for BeS recovery from hair. The data is displayed on a logarithmic scale. Box and whisker plot shows median and inter–quartile range, together with the maximum and minimum values.
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Figures Figure 1
A
B
Figure 2
Percentage of applied dose
100
10
1
Disc 1+2 Discs 3+4 Discs 5+6
Total
13
Concentration (ug/mg hair)
Figure 3
1000 100 10 1 0.1 0.01 0.001
Baseline
Post application
14
Table 1: Recovery of BeS from control participants’ hair. Each post application sample is much higher in concentration than baseline samples, however the recovered concentration of BeS is highly variable amongst participants. Participant
1
2
3
4
5
6
7
8
9
10
Baseline
0.015
0.011
0.008
0.181
0.067
0.050
0.594
0.204
0.022
0.496
5.001
45.86
147.02
33.57
84.63
40.613
1.281
3.138
6.456
108.30
(µg/mg) Postapplication (µg/mg)
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