- Email: [email protected]
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Accepted Manuscript Determination of metabolites of the UV filter 2-ethylhexyl salicylate in human urine by online-SPE-LC-MS/MS
Daniel Bury, Thomas Brüning, Holger M. Koch PII: DOI: Reference:
S1570-0232(18)31883-X https://doi.org/10.1016/j.jchromb.2019.02.014 CHROMB 21529
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
19 December 2018 6 February 2019 12 February 2019
Please cite this article as: D. Bury, T. Brüning and H.M. Koch, Determination of metabolites of the UV filter 2-ethylhexyl salicylate in human urine by online-SPE-LC-MS/ MS, Journal of Chromatography B, https://doi.org/10.1016/j.jchromb.2019.02.014
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ACCEPTED MANUSCRIPT Determination of Metabolites of the UV Filter 2-Ethylhexyl Salicylate in Human Urine by Online-SPE-LC-MS/MS Daniel Burya* , Thomas Brüninga, Holger M. Kocha
Institute for Prevention and Occupational Medicine of the German Social Accident Insurance,
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a
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Institute of the Ruhr-Universität Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789 Bochum,
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Germany
+49 (0)30 13001 4414
Fax:
+49 (0)30 13001 864414
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Tel.:
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[email protected]
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E-mail:
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* Corresponding author:
Further e-mail addresses:
[email protected]
Koch
[email protected]
Bury
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ORCID iD:
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Brüning
0000-0003-1283-3133
Brüning
0000-0001-9560-5464
Koch
0000-0002-8328-2837
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ACCEPTED MANUSCRIPT Abbreviations 1
Abstract The UV filter 2-ethylhexyl salicylate (EHS) is widely used in sunscreens and other personal care
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products (PCP). EHS has been detected in a variety of environmental matrices. However, data on
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the internal EHS exposure in humans is not available, due to the lack of exposure biomarkers and
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analytical methods for their determination. Here, we report a method for the determination of three oxidative EHS metabolites in human urine: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate 2-ethyl-5-oxohexyl
2- hydroxybenzoate
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(5OH-EHS),
(5oxo-EHS),
and
5-(((2-
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hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx-EPS). Urine samples are incubated with βglucuronidase and analyzed by liquid chromatography-electrospray ionization-triple quadrupole-
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tandem mass spectrometry, coupled with online sample clean- up and analyte enrichment using
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turbulent flow chromatography (online-SPE-LC-MS/MS). Quantification is performed by stable isotope dilution analysis, using deuterium- labeled standards of each of the three metabolites. The
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described method is precise (coefficient of variation <5% within-series and interday), accurate
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(mean relative recoveries between 96% and 105%), and sensitive, with limits of quantification (LOQ) of 0.01 µg/L (5cx-EPS), 0.05 µg/L (5OH-EHS), and 0.15 µg/L (5oxo-EHS). After dermal
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application of an EHS containing sunscreen to a human volunteer, we were able to quantify all three metabolites in urine samples collected post application, showing clear elimination kinetics. In spot urine samples from the general population (n = 35) we were able to quantify EHS biomarkers in 91% of all samples, with highest concentrations in individuals (n = 11) who stated use of PCPs containing UV filters within 5 days prior to sampling. We will apply the method for
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EHS: 2-ethylhexyl salicylate; 5OH-EHS: 2-ethyl-5-hydro xyhexyl 2-hydro xybenzoate; 5o xo-EHS: 2-ethyl-5oxohexy l 2-hydroxybenzoate; 5cx-EPS: 5-(((2-hydro xybenzoyl)o xy )methyl)heptanoic acid; PCP: personal care product; SPF: sun protection factor
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ACCEPTED MANUSCRIPT investigating human EHS metabolism and in future human biomonitoring studies for EHS exposure and risk assessment.
Keywords
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Ethylhexyl salicylate; UV filter; sunscreen; human biomonitoring; urinary metabolite; exposure
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assessment
1. Introduction
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2-Ethylhexyl salicylate (ethylhexyl salicylate, octyl salicylate, octisalate, EHS; CAS registry no.
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118-60-5; EC no. 204-263-4) is a UV-B filter, used in sunscreens worldwide [1]. Additionally, 2ethylhexyl salicylate (EHS) is used in other personal care products (PCP), such as perfumes, after
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shave, lipsticks, face creams, and make- up foundation [2–4]. The maximum permitted
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concentration in cosmetic products is 5%, both in the U.S.A. and the European Union [5,6]. EHS has been reported to cause allergic contact dermatitis in rare cases [7–10]. In vitro studies
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showed some anti-androgenic activity and no or only weak androgenic, estrogenic, and anti-
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estrogenic activity [11–14]. In 2018, EHS was included in the Community rolling action plan (CoRAP) of the European Union due to its potential endocrine activity [15].
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As a result of its use in personal care products, EHS has been detected in a variety of environmental matrices (including surface freshwater and sea water [16–20], beach sand [21], sediments [22–24], wastewater treatment plant effluents [16], and soil [25]), as well as in marine wildlife organisms [26,27], and also in indoor dust [28]. Thus, EHS exposure of the general population is very likely, both in view of its widespread use in PCPs and its presence in the environment. Accordingly, the determination of internal EHS exposure of humans is of high interest. Human biomonitoring (HBM) is a well-established tool 3
ACCEPTED MANUSCRIPT for exposure assessment, covering all exposure sources and uptake routes and applicable both to large scale population studies and individual exposure assessment [29–35]. However, exposure biomarkers for EHS and analytical methods for their determination have not been published yet. Accordingly, the aim of this study was the development of an analytical method for the
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determination of specific metabolites of EHS as biomarkers of exposure. The following
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metabolites were identified in a human oral dosing study (publication in preparation) and were
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chosen as target analytes: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5oxohexyl 2-hydroxybenzoate (5oxo-EHS), and 5-(((2- hydroxybenzoyl)oxy)methyl)heptanoic
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acid (5cx-EPS) (Figure 1). The presented method shall be applied in future HBM studies, both in
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the occupational and environmental field, and shall enable a robust exposure and risk assessment.
2.1. Chemicals and reagents
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2. Experimental
oxohexyl
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2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS; chemical purity 97%), 2-ethyl-52-hydroxybenzoate
(5oxo- EHS;
chemical
purity
>98%),
and
5-(((2-
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hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx- EPS; chemical purity >98%), as well as their
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respective deuterium- labeled analogs 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate-3,4,5,6-d4 (5OH-EHS-d4 ; chemical purity: 98%;
isotopic purity >97%),
2-ethyl-5-oxohexyl 2-
hydroxybenzoate-3,4,5,6-d4 (5oxo-EHS-d4 ; chemical purity: >98%; isotopic purity >98%), and 5(((2-hydroxybenzoyl-3,4,5,6-d4 )-oxy)methyl)heptanoic acid (5cx-EPS-d4 ; chemical purity: >98%; isotopic purity >98%) were synthesized by Dr. Vladimir Belov, Max Planck Institute for Biophysical Chemistry, Germany. The identities of all synthesized metabolite standards (labeled and non- labeled) were confirmed by ESI-MS, GC-MS (only for 5OH-EHS-d4 ) and 1 H and 4
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ACCEPTED MANUSCRIPT NMR, and their chemical purity was assessed by HPLC-UV. Acetonitrile, methanol and water CHROMASOLVT M LC-MS grade were purchased from Honeywell Riedel-de Haën (Seelze, Germany). Acetic acid puriss./Ph. Eur., formic acid p.a., and ammonium acetate BioXtra ≥98% were obtained from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was produced using
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an in-house Advantage A10 water purification unit (Merck Millipore, Darmstadt, Germany). β-
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glucuronidase (aryl sulfatase-free) from E. coli K12 (at least 140 u/mL at 37° C according to
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manufacturer) was purchased from Roche Diagnostics (Mannheim, Germany). Silanized HPLC
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vials (Macherey-Nagel, Düren, Germany) with silicone/PTFE screw caps (VWR, Leuven, Belgium) were used for LC-MS analysis.
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2.2. Standard solutions
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Stock solutions (approximately 1 g/L each, weighed exactly) of all three metabolite standards, both labeled and non-labeled, were prepared by weighing into volumetric flasks and dissolving
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and filling up with acetonitrile. For use as internal standards, the stock solutions of deuterium-
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labeled metabolites were diluted in acetonitrile (5OH-EHS-d4 and 5oxo-EHS-d4 : 5.00 mg/L each; 5cx-EPS-d4 : 25.0 mg/L and further diluted to 500 µg/L) and these dilutions were used to prepare
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an internal standard mix (5OH-EHS-d4 : 20 µg/L, 5oxo-EHS-d4 : 10 µg/L; 5cx-EPS-d4 : 1 µg/L).
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From the stock solutions of non- labeled metabolites, three mixed stock solutions were prepared (after preparation of appropriate intermediate dilutions) by serial dilution (a) 500 µg/L 5OH-EHS, 2.50 mg/L 5oxo-EHS, 250 µg/L 5cx-EPS; b) 25 µg/L 5OH-EHS, 125 µg/L 5oxo-EHS, 12.5 µg/L 5cx-EPS; c) 2.00 µg/L 5OH-EHS, 10.0 µg/L 5oxo-EHS, 1.00 µg/L 5cx-EPS). These mixed stock solutions were then used for the preparation of seven calibration standards in LC-MS grade water (0.02-10 µg/L 5OH-EHS, 0.1-50 µg/L 5oxo-EHS, 0.01-5 µg/L 5cx- EPS). All stock solutions, including intermediate dilutions and mixed solutions, were stored in glass flasks, capped with 5
ACCEPTED MANUSCRIPT screw caps with silicone/teflon seals. Aliquots of 300 µL of each calibration solution and pure water as blank solution were transferred into HPLC vials, immediately after preparation. All solutions were stored at -20 °C until further use. Stock solutions were stable for more than 1.5 years and calibration solutions for more than 12 weeks.
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2.3. Sample collection and preparation
Urine samples were collected in 250 mL PE containers and stored at -20 °C until analysis.
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Metabolites in urine were stable for more than 1.5 years. Urinary creatinine was determined by
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contract analysis (L.u.P. GmbH Labor und Praxisservice, Bochum, Germany). For analysis, 300 µL of homogenized (by repeated inversion) urine were transferred into an HPLC vial and 100 µL
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ammonium acetate buffer (1M, pH 6.0-6.4), 30 µL internal standard mix, and 6 µL β-
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glucuronidase, premixed with ammonium acetate buffer 1:1 (v/v), were added. Samples were homogenized by inverting several times and then incubated in a water bath at 37 °C for 3 h.
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Afterwards, 30 µL of formic acid were added, the samples were again homogenized and frozen at
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-20 °C overnight to precipitate cryophobic proteins. After thawing, samples were centrifuged (1900 g, 10 min) and the supernatant transferred into a new HPLC vial. Calibration solutions and
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quality control samples underwent the same treatment as urine samples.
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2.4. Chromatographic conditions A 1260 Infinity HPLC (Agilent Technologies, Waldbronn, Germany) was used for chromatographic separation and online sample cleanup. The instrument consisted of a G1311B quaternary low pressure gradient pump (used as loading pump for online-SPE), a G1312B binary high pressure gradient pump with G4225A degasser (used for chromatographic separation) a G1367E autosampler with G1330B thermostat, and a G1316A thermostated column compartment with 2-way 6-port switching valve. Instrument setup and chromatographic conditions were 6
ACCEPTED MANUSCRIPT identical to an HBM method for the UV filter octocrylene, previously described by our group [36]. A TurboFlow Phenyl (50 x 0.5 mm; Thermo Scientific, Franklin, MA, U.S.A.) turbulent flow chromatography (TFC) column was used for online sample cleanup and a Kinetex C18 column (150 x 3 mm, particle size 2.6 µm; with SecurityGuard ULTRA guard column;
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Phenomenex, Aschaffenburg, Germany) with superficially porous particles was used for HPLC.
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An inline filter (porosity 0.5 µm; 3.0 mm diameter; Phenomenex, Aschaffenburg, Germany),
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placed between autosampler and column compartment, was used to reduce contaminations of the online-SPE column. The injection volume was 100 µL. A needle wash with methanol/water
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80:20 (v/v) was performed for 10 s after sample aspiration. Autosampler and thermostated
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column compartment were kept at 6 °C and 25±1 °C, respectively. Eluents were water (eluent A) and acetonitrile (eluent B), both containing 0.05% acetic acid. The gradient of the loading pump
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is shown in table 1. The binary pump delivered eluent at a constant flow rate of 300 µL/min with
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the following gradient: Initial 30% B were kept for 2 min. Then, B was increased to 50% within 2 min. Afterwards, B was increased more slowly to 62% within 12 min and then to 95% within 2
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min. B was kept at 95% for 5 min, then decreased to initial conditions (30% B) within 0.5 min
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and kept for 8 min. The gradient delay volumes according to the manufacturer’s manuals were 600 to 800 µL for the binary pump and 870 to 1170 µL on the loading pump flow path (600 to
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900 µL for the quaternary pump and 270 µL for the autosampler). The 6-port valve was switched at 3 min to start the analyte transfer from the online-SPE column onto the HPLC column and again at 6 min to end analyte transfer and start the analytical separation and re-equilibration of the SPE column.
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ACCEPTED MANUSCRIPT 2.5. Mass spectrometric conditions Detection of EHS metabolites was performed with electrospray-tandem mass spectrometry (ESIMS/MS) in time-programmed multiple reaction monitoring (scheduled MRM) mode with polarity switching using a 4500 triple quadrupole mass spectrometer (Sciex, Darmstadt,
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Germany). The instrument gases (nitrogen) were set to: nebulizer gas, 40 psi; heater gas, 50 psi;
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curtain gas, 20 psi; collision gas, 6 arbitrary units. The source heater was kept at 450 °C.
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Electrospray voltages, entrance potential, and collision cell exit potential for +ESI/- ESI were 5.5 kV/-4.5 kV, 10 V/-10 V, and 12 V/-12 V, respectively. The target scan times were 0.200 s (+ESI)
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and 0.125 s (-ESI) with an MRM detection window of 60 s. For further MRM conditions see
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table 2. Declustering potentials and collision energies were manually optimized. Analyst 1.6
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(Sciex, Darmstadt, Germany) was used for instrument control and quantitative data analysis. 2.6. Calibration, validation and quality control
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Calibration was performed by weighted (concentration-1 ) linear regression, using the Analyst
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software. Method validation and quality assurance was performed according to the principles of the working group “Analyses in Biological Materials” of the Permanent Senate Commission for
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the Investigation of Health Hazards of Chemical Compounds in the Work Area of the German
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Research Foundation (Deutsche Forschungsgemeinschaft) [37]. Two quality control materials (Q low and Q high ) with different concentrations (table 3) of the three metabolites were prepared by mixing different urine samples (from an oral dosing study (publication in preparation) and, in case of Q low, from a pilot population (section 2.8)), containing native concentrations of the three metabolites. Native 5oxo- EHS concentrations were too low to achieve concentrations covering the calibration range without exceeding the calibration range for the other two metabolites. Thus, 5oxo-EHS concentrations were spiked (Qlow: 0.5 µg/L, Q high : 30 µg/L). These pooled urine 8
ACCEPTED MANUSCRIPT samples were frozen, thawed, and filtrated three times. The obtained materials were aliquoted (300 µL each) in HPLC vials and stored at -20 °C. To determine the method imprecision, this material was analyzed eight times within one analytical batch (within-series imprecision) and on eight different days (interday imprecision). After validation, the material was further analyzed in
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each batch for quality control purposes (quality control chart). For the determination of the
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method accuracy, eight different urine samples (0.21 to 2.59 g/L creatinine) were analyzed,
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spiked at three different concentration levels (prior to sample preparation; table 4) and without
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spiking. Relative recoveries were calculated after subtraction of native metabolite concentrations. 2.7. Pilot dermal application
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A commercial sunscreen containing 5% EHS (as listed on the product label) was applied to the
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whole body in a regular use scenario by one male volunteer (33 years of age, 90 kg body weight). A total amount of 10.9 g sunscreen was applied, corresponding to 543 mg EHS (6.03 mg/kg body
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weight). One week prior to the study and during the study, no known additional EHS exposure
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occurred (PCPs, containing EHS were completely avoided). To avoid non-dermal EHS uptake as good as possible, mouth contact with the sunscreen itself or with exposed hands was avoided.
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4.75 h after application, a shower was taken to simulate swimming activity and remove any
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sunscreen from the skin surface. Between the application and taking the shower, no food was touched or eaten.
2.8. Pilot population Spot urine samples from 35 volunteers (24 females and 11 males; age 23 to 59 (median 42)) without any known occupational EHS exposure were collected in April 2017. Urinary creatinine ranged from 0.09 to 2.31 g/L. Use of sunscreens, lipsticks with sun protection factor (SPF), and
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ACCEPTED MANUSCRIPT other PCP with SPF (such as day creams) was inquired via questionnaire. Of the 35 volunteers, 11 stated to have used products with sun protection factor within the last 5 days.
3. Results and discussion
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3.1. Mass spectrometry
The EHS metabolites were detected with ESI-MS/MS. 5cx-EPS was ionized both in positive and
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negative ion mode, but sensitivity in negative ion mode was much better. 5OH- EHS and 5oxo-
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EHS were poorly ionized in negative ion mode and much better in positive ion mode. Accordingly, 5cx-EPS was detected in ESI negative ion mode and 5OH-EHS and 5oxo-EHS
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were detected in ESI positive ion mode. Product ion spectra of all analytes (labeled and non-
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labeled), including schematic explanations of produced fragment ions are shown in figure 2. The observed mass signals can be interpreted as follows: The pseudo- molecular ion ([M-H]-) of 5cx-
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EPS (m/z 279) can eliminate the sidechain alkene 5- methyleneheptanoic acid to yield the
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fragment at m/z 137. This fragment can eliminate carbon dioxide to yield m/z 93. Further elimination of carbon monoxide from the aromatic ring yields the fragment at m/z 65. For the
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deuterium- labeled internal standard 5cx-EPS-d4 , corresponding fragments were found at +4 u.
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For 5OH-EHS, the pseudo- molecular ion ([M+H]+ ) can either eliminate the sidechain alkene 5methyleneheptan-2-ol or the sidechain diol 2-ethylhexane-1,5-diol to yield the aromatic fragments at m/z 139 and 121, respectively. The fragment at m/z 121 can also be formed by elimination of water from m/z 139. Corresponding fragments at +4 u were observed for 5OHEHS-d4 . 5OH-EHS can further eliminate salicylic acid (2-hydroxybenzoic acid) to yield a sidechain alkene fragment at m/z 129. This fragment can eliminate water to yield m/z 111 (an initial elimination of water from [M+H]+ would also be conceivable, but a corresponding signal
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ACCEPTED MANUSCRIPT at m/z 249 was not observed). Further elimination of propene from m/z 111 yields m/z 69. All of these sidechain fragments were also observed for 5OH-EHS-d4 . In case of 5oxo-EHS and 5oxoEHS-d4 , only two major fragments were observed. A sidechain alkene fragment (analogous to m/z 129 for 5OH-EHS and 5OH-EHS-d4 ) was observed at m/z 127. In analogy to 5OH-EHS and
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5OH-EHS-d4 , elimination of water from this sidechain alkene fragment (preceded by keto-enol
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tautomerism) yields m/z 109. Quantifier and qualifier MRM transitions were chosen according to
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their signal-to-noise (S/N) ratios in matrix and the absence of any interfering matrix peaks (see table 2). For example, in case of 5cx-EPS and 5cx-EPS-d4 , the most suitable ion traces were not
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the best in terms of absolute signal intensities, but were chosen based on their low noise levels
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and high resulting S/N ratios (figure 3).
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3.2. Method performance
Enzymatic β- glucuronidase/aryl sulfatase preparations are known to cleave ester moieties of
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certain metabolites, such as phthalate and terephthalate metabolites [38–40]. Accordingly, a pure
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β-glucuronidase, free of any aryl sulfatase/esterase activity, was chosen to avoid the cleavage of the ester moiety of EHS metabolites. We tested a ß-glucuronidase enzyme preparation with aryl
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sulfatase activity (type HP-2 from Helix pomatia; Sigma-Aldrich, Steinheim, Germany) and
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found indications of ester cleavage and degradation of the standards (data not shown). As a consequence of choosing a pure ß- glucuronidase, only free and glucuronidated metabolites are captured by this analytical approach; sulfates, which might also be formed, are not captured. To test for complete deconjugation of glucuronide conjugates we investigated incubation times of 1, 2, 3, 4, 5, 7, and 9 h in three urine samples with native metabolite levels (2.34-5.31 µg/L 5OHEHS, 0.82-2.56 µg/L 5oxo-EHS, 1.14-5.66 µg/L 5cx-EPS) and found no additional release of free metabolites with incubation times 2 h and longer (data not shown). For ruggedness purposes
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ACCEPTED MANUSCRIPT we therefore set the method’s incubation time to 3 h. We chose to calibrate in water instead of urine due to the high proportion of urine samples with metabolite concentrations above the limits of quantification (LOQ; see section 3.3) and the associated difficulty in finding metabolite- free and not unduly diluted urine. The slopes of calibration curves in urine (eight different urines
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tested – see section 2.6) and water were identical. Calibrations for all three metabolites were
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within the linear dynamic range. The limits of quantification (LOQ) were defined as a signal-to-
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noise (S/N) ratio of 10 in matrix. The LOQs obtained were 0.01 µg/L (5cx-EPS), 0.05 µg/L (5OH-EHS), and 0.15 µg/L (5oxo- EHS). Data on method precision is shown in table 3. Method
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imprecision (i.e. coefficient of variation) was < 5% both within-series and interday. The
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metabolite concentrations for Q low and Q high were chosen to cover the calibration range and reflect metabolite concentrations in both the dermal application (see below) and oral metabolism
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study (publication in preparation), and also in the general population (see below). Relative
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recoveries (table 4) were determined as described in section 2.6. Mean relative recoveries were between 96% and 106% for all three metabolites and all spiking levels with single values ranging
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from 85% to 115%. For the lowest spiking level, one urine samples was excluded from the
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evaluation for 5cx-EPS and 5OH-EHS, because native metabolite levels were by far exceeding the spiked concentrations. Including this urine sample, mean relative recoveries were between 94%
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and 106% with single values ranging from 80% to 132%. The presented method is based on an HBM method for the determination of biomarkers of the UV filter octocrylene, previously published by our group [36]. Combination of both methods for simultaneous analysis of OC and EHS biomarkers can be performed without interferences.
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ACCEPTED MANUSCRIPT 3.3. Applicability for human biomonitoring of EHS exposure To investigate the relevance of the analyzed EHS biomarkers for biomonitoring after EHS exposure, EHS metabolite levels were determined in urine samples collected after application of a sunscreen in a regular use scenario. Exemplary chromatograms from a urine sample collected
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40 h after the sunscreen application are shown in figure 3. Concentrations of all three EHS
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metabolites increased after application, reaching maximum levels around 9 h after application
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and decreasing slowly thereafter (figure 4; creatinine-adjusted concentrations). The excretion profile for all three metabolites was very similar, indicating similar elimination kinetics. The
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results show that all three EHS metabolites are suitable as biomarkers of EHS exposure,
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following (occupational or recreational) sunscreen use.
In addition to the dermal EHS exposure scenario, the applicability of the method for human
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biomonitoring of the general population (including background concentrations) was tested. For
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that purpose, 35 urine samples from the general population without known occupational EHS exposure were analyzed (table 5). 5cx-EPS was found above LOQ in 91%, 5OH-EHS in 49%,
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and 5oxo-EHS in 14% of all spot urine samples. Highest metabolite concentrations and highest
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detection rates were found in urine samples from individuals (n = 11) who had reported use of PCPs with SPF within 5 days prior to sampling. However, also in the subgroup (n = 24) who had
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reported no use of PCPs with SPF within five days prior to urine collection, 5cx-EPS was above LOQ in 88% of the samples (5OH-EHS: 38%, 5oxo-EHS: 4%). These results show that all three metabolites are suitable biomarkers of EHS exposure, with 5cx- EPS being the most sensitive biomarker, followed by 5OH-EHS and 5oxo-EHS.
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ACCEPTED MANUSCRIPT 4. Conclusions The method presented here enables the precise, sensitive, accurate, and rugged determination of three previously not described EHS metabolites in human urine samples. Results from a dermal pilot study (regular use scenario of an EHS containing sunscreen product) prove the applicability
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of all three metabolites as biomarkers of EHS exposure. Furthermore, we were able to quantify
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EHS metabolites in the majority of urine samples from a small pilot population, suggesting the
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method´s applicability for exposure biomonitoring of the general population, covering also background exposures (i.e. no recent use of sunscreens). This me thod, in conjunction with renal
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conversion factors for all three metabolites (to be determined in a human metabolism study, in
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preparation), will enable a robust exposure and risk assessment for EHS.
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Declarations of interest
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None.
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Funding
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The presented study and development of the analytical method applied herein are part of a largescale 10-year project on the advancement of human biomonitoring in Germany. This project is a
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cooperation agreed in 2010 between the German Federal Ministry for the Environme nt, Nature Conservation and Nuclear Safety (BMU) and the German Chemical Industry Association (VCI Verband der Chemischen Industrie e.V.) and is managed by the German Environment Agency (UBA). Experts from governmental scientific authorities, industry and science provide advice in the selection of substances to be investigated and during method development. The analytical method development and the human metabolism study are financed by the Chemie
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ACCEPTED MANUSCRIPT Wirtschaftsförderungsgesellschaft mbH while the first application of the novel methodology in a larger population study will be financed by the German Environment Agency.
Compliance with ethical standards
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Ethical approval
The study (oral and dermal dosage of 2-ethylhexyl salicylate; pilot population) was carried out in
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accordance with the Code of Ethics of the World Medical Association (1964 Declaration of
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Helsinki and later amendments) and has been approved by the ethical review board of the medical faculty of the Ruhr-University Bochum (IRB Reg. No.: 3867−10 and 4288−12). The
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obtained from each individual participant.
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study design was presented to the volunteers in written form and written informed consent was
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ACCEPTED MANUSCRIPT References [1] N.A. Shaath, Ultraviolet filters, Photochem. Photobiol. Sci. 9 (4) (2010) 464–469. https://doi.org/10.1039/b9pp00174c. [2] E. Manová, N. von Goetz, U. Hauri, C. Bogdal, K. Hungerbühler, Organic UV filters in
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[3] W. Uter, M. Gonçalo, K. Yazar, E.-M. Kratz, G. Mildau, C. Lidén, Coupled exposure to ingredients of cosmetic products: III. Ultraviolet filters, Contact Derm. 71 (3) (2014) 162–
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169. https://doi.org/10.1111/cod.12245.
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[5] U.S. Food & Drug Administration, 21 Code of Federal Regulations § 352.10.: 21 C.F.R. §
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352.10.
[6] European Parliament and the Council, Regulation (EC) No 1223/2009 of the European
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1223/2009, 2009.
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Parliament and the Council of 30 November 2009 on cosmetic products: Reg. (EC)
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Determination of Urinary Metabolites of the Emerging UV Filter Octocrylene by Online-
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ACCEPTED MANUSCRIPT B Analyt. Technol. Biomed. Life Sci. 1100-1101 (2018) 91–92.
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ACCEPTED MANUSCRIPT Figure captions Figure 1 – Metabolism scheme of EHS leading to the metabolites investigated. Phase II metabolites (conjugates) are not shown.
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Figure 2 – Product ion spectra of the metabolite standards. Product ion spectra of (from top
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to bottom) 5cx-EPS, 5OH-EHS, and 5oxo-EHS (left column), and their respective
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deuterium- labeled analogs (right column), including schematic explanations of produced fragment ions. 5cx-EPS was measured in ESI negative ion mode and 5OH-EHS and 5oxo-
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EHS in ESI positive ion mode.
Figure 3 – Exemplary chromatograms for all three EHS metabolites. Chromatograms of a
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calibration standard (left column) and a urine sample after dermal EHS application with
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native metabolite concentrations (right column) for 5OH-EHS (top), 5oxo-EHS (middle), and 5cx-EPS (bottom). Quantifier (continuous) and qualifier (dashed) transitions of
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metabolites (black) and internal standards (gray) are shown. Metabolite concentrations are
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given in the upper left corner.
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Figure 4 – EHS metabolite concentrations after sunscreen application. Creatinine-adjusted concentrations of 5OH-EHS (continuous black), 5oxo-EHS (dashed black), and 5cx-EPS (gray) in one male volunteer after whole body dermal application of 10.9 g sunscreen (corresponding to 543 mg EHS or 6.03 mg/kg body weight). Concentrations are plotted on a logarithmic scale.
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ACCEPTED MANUSCRIPT Table 1. Gradient for the loading pump.
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Eluent B [% ] 0 0 0 0 0 95 95 0 0 0 0
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Eluent A [% ] 100 100 100 100 100 5 5 100 100 100 100
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Flow rate [µL/min] 1500 1500 200 200 500 500 500 500 500 1500 1500
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Time [min] 0 3 3.1 6 7 10 21 22 28.5 29.5 31.5
ACCEPTED MANUSCRIPT Table 2. M RM conditions. For each analyte (including deuterium-labeled internal standards) the retention time (t R), ESI polarity, and precursor and product ions, as well as declustering potential (DP) and collision energy (CE) for the quantifier and qualifier (in parenthesis) transition are listed.
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DP [V] -60 -60 30 30 15 15
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ESI polarity negative negative positive positive positive positive
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tR [min] 14.7 14.6 15.6 15.5 17.7 17.6
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Analyte 5cx-EPS 5cx-EPS-d 4 5OH-EHS 5OH-EHS-d 4 5oxo-EHS 5oxo-EHS-d 4
MRM transitions m/z m/z product precursor 279 93 (137) 283 97 (141) 267 129 (111) 271 129 (125) 265 127 (109) 269 127 (109)
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CE [eV] -47 (-16) -47 (-16) 8 (14) 8 (35) 12 (23) 12 (22)
ACCEPTED MANUSCRIPT Table 3. Method precision. Mean concentrations and coefficients of variat ion are given for all three metabolites in both quality control materials (Qlow and Qhigh ).
5OH-EHS
Qlow Qhigh
0.101 6.30
2.4% 1.4%
5oxo-EHS
Qlow Qhigh
0.69 34.2
2.3% 2.0%
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Qlow Qhigh
0.098 6.17
3.4% 1.9%
0.64 32.4
3.6% 3.7%
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5cx-EPS
interday imprecision (n = 8) mean coefficient of concentration variation [µg/L] 0.155 3.7% 2.69 2.7%
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within-series imprecision (n = 8) mean coefficient of concentration variation [µg/L] 0.157 1.2% 2.68 1.3%
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ACCEPTED MANUSCRIPT Table 4. Method accuracy. Range of native metabolite concentrations and mean relat ive recoveries (ranges in parenthesis) at three spiking levels for eight urine samples (0.21 to 2.59 g/L creatinine). Spiked concentrations [µg/L] 0.1 0.5 2
5OH-EHS
0.1 0.5 2
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96 (85-106)a 103 (91-112) 101 (93-109)
1 97 (90-102)
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5oxo-EHS
102 (96-106)a 106 (102-115) 105 (100-111)
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5cx-EPS
Relative recovery [% ]
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Native concentrations [µg/L]
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ACCEPTED MANUSCRIPT Table 5. Pilot hu man bio monitoring population. Urine samples fro m the general population without known occupational EHS exposure were analy zed. Data for the whole population is shown, as well as data divided by individuals who d id not report any use of PCP with SPF (sunscreens, lip sticks, face creams, etc.) within 5 days prior to sampling, and those who did. Use of PCP with SPF (previous No use of PCP with SPF 5 days) (n = 11) (previous 5 days) (n = 24) Median Median Median concentration n > LOQ concentration n > LOQ concentration n > LOQ (range) [µg/L] (percentage) (range) [µg/L] (percentage) (range) [µg/L] (percentage) 5cx0.039 0.159 11 (100%) 0.028 21 (88%) 32 (91%) EPS ( LOQ.
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Total population (n = 35)
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ACCEPTED MANUSCRIPT Highlights Determination of three urinary metabolites of the UV filter 2-ethylhexyl salicylate
All three metabolites are specific side chain oxidation products
LC-MS/MS coupled with online sample clean-up using turbulent flow chromatography
After sunscreen use detection of all metabolites with clear excretion kinetics
Metabolites found >LOQ in 91% of urine samples from pilot population (n=35)
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Figure 1
Figure 2
Figure 3
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Daniel Bury, Thomas Brüning, Holger M. Koch PII: DOI: Reference:
S1570-0232(18)31883-X https://doi.org/10.1016/j.jchromb.2019.02.014 CHROMB 21529
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
19 December 2018 6 February 2019 12 February 2019
Please cite this article as: D. Bury, T. Brüning and H.M. Koch, Determination of metabolites of the UV filter 2-ethylhexyl salicylate in human urine by online-SPE-LC-MS/ MS, Journal of Chromatography B, https://doi.org/10.1016/j.jchromb.2019.02.014
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ACCEPTED MANUSCRIPT Determination of Metabolites of the UV Filter 2-Ethylhexyl Salicylate in Human Urine by Online-SPE-LC-MS/MS Daniel Burya* , Thomas Brüninga, Holger M. Kocha
Institute for Prevention and Occupational Medicine of the German Social Accident Insurance,
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a
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Institute of the Ruhr-Universität Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789 Bochum,
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Germany
+49 (0)30 13001 4414
Fax:
+49 (0)30 13001 864414
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Tel.:
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[email protected]
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E-mail:
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* Corresponding author:
Further e-mail addresses:
[email protected]
Koch
[email protected]
Bury
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ORCID iD:
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Brüning
0000-0003-1283-3133
Brüning
0000-0001-9560-5464
Koch
0000-0002-8328-2837
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ACCEPTED MANUSCRIPT Abbreviations 1
Abstract The UV filter 2-ethylhexyl salicylate (EHS) is widely used in sunscreens and other personal care
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products (PCP). EHS has been detected in a variety of environmental matrices. However, data on
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the internal EHS exposure in humans is not available, due to the lack of exposure biomarkers and
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analytical methods for their determination. Here, we report a method for the determination of three oxidative EHS metabolites in human urine: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate 2-ethyl-5-oxohexyl
2- hydroxybenzoate
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(5OH-EHS),
(5oxo-EHS),
and
5-(((2-
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hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx-EPS). Urine samples are incubated with βglucuronidase and analyzed by liquid chromatography-electrospray ionization-triple quadrupole-
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tandem mass spectrometry, coupled with online sample clean- up and analyte enrichment using
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turbulent flow chromatography (online-SPE-LC-MS/MS). Quantification is performed by stable isotope dilution analysis, using deuterium- labeled standards of each of the three metabolites. The
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described method is precise (coefficient of variation <5% within-series and interday), accurate
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(mean relative recoveries between 96% and 105%), and sensitive, with limits of quantification (LOQ) of 0.01 µg/L (5cx-EPS), 0.05 µg/L (5OH-EHS), and 0.15 µg/L (5oxo-EHS). After dermal
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application of an EHS containing sunscreen to a human volunteer, we were able to quantify all three metabolites in urine samples collected post application, showing clear elimination kinetics. In spot urine samples from the general population (n = 35) we were able to quantify EHS biomarkers in 91% of all samples, with highest concentrations in individuals (n = 11) who stated use of PCPs containing UV filters within 5 days prior to sampling. We will apply the method for
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EHS: 2-ethylhexyl salicylate; 5OH-EHS: 2-ethyl-5-hydro xyhexyl 2-hydro xybenzoate; 5o xo-EHS: 2-ethyl-5oxohexy l 2-hydroxybenzoate; 5cx-EPS: 5-(((2-hydro xybenzoyl)o xy )methyl)heptanoic acid; PCP: personal care product; SPF: sun protection factor
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ACCEPTED MANUSCRIPT investigating human EHS metabolism and in future human biomonitoring studies for EHS exposure and risk assessment.
Keywords
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Ethylhexyl salicylate; UV filter; sunscreen; human biomonitoring; urinary metabolite; exposure
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assessment
1. Introduction
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2-Ethylhexyl salicylate (ethylhexyl salicylate, octyl salicylate, octisalate, EHS; CAS registry no.
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118-60-5; EC no. 204-263-4) is a UV-B filter, used in sunscreens worldwide [1]. Additionally, 2ethylhexyl salicylate (EHS) is used in other personal care products (PCP), such as perfumes, after
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shave, lipsticks, face creams, and make- up foundation [2–4]. The maximum permitted
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concentration in cosmetic products is 5%, both in the U.S.A. and the European Union [5,6]. EHS has been reported to cause allergic contact dermatitis in rare cases [7–10]. In vitro studies
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showed some anti-androgenic activity and no or only weak androgenic, estrogenic, and anti-
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estrogenic activity [11–14]. In 2018, EHS was included in the Community rolling action plan (CoRAP) of the European Union due to its potential endocrine activity [15].
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As a result of its use in personal care products, EHS has been detected in a variety of environmental matrices (including surface freshwater and sea water [16–20], beach sand [21], sediments [22–24], wastewater treatment plant effluents [16], and soil [25]), as well as in marine wildlife organisms [26,27], and also in indoor dust [28]. Thus, EHS exposure of the general population is very likely, both in view of its widespread use in PCPs and its presence in the environment. Accordingly, the determination of internal EHS exposure of humans is of high interest. Human biomonitoring (HBM) is a well-established tool 3
ACCEPTED MANUSCRIPT for exposure assessment, covering all exposure sources and uptake routes and applicable both to large scale population studies and individual exposure assessment [29–35]. However, exposure biomarkers for EHS and analytical methods for their determination have not been published yet. Accordingly, the aim of this study was the development of an analytical method for the
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determination of specific metabolites of EHS as biomarkers of exposure. The following
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metabolites were identified in a human oral dosing study (publication in preparation) and were
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chosen as target analytes: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5oxohexyl 2-hydroxybenzoate (5oxo-EHS), and 5-(((2- hydroxybenzoyl)oxy)methyl)heptanoic
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acid (5cx-EPS) (Figure 1). The presented method shall be applied in future HBM studies, both in
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the occupational and environmental field, and shall enable a robust exposure and risk assessment.
2.1. Chemicals and reagents
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2. Experimental
oxohexyl
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2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS; chemical purity 97%), 2-ethyl-52-hydroxybenzoate
(5oxo- EHS;
chemical
purity
>98%),
and
5-(((2-
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hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx- EPS; chemical purity >98%), as well as their
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respective deuterium- labeled analogs 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate-3,4,5,6-d4 (5OH-EHS-d4 ; chemical purity: 98%;
isotopic purity >97%),
2-ethyl-5-oxohexyl 2-
hydroxybenzoate-3,4,5,6-d4 (5oxo-EHS-d4 ; chemical purity: >98%; isotopic purity >98%), and 5(((2-hydroxybenzoyl-3,4,5,6-d4 )-oxy)methyl)heptanoic acid (5cx-EPS-d4 ; chemical purity: >98%; isotopic purity >98%) were synthesized by Dr. Vladimir Belov, Max Planck Institute for Biophysical Chemistry, Germany. The identities of all synthesized metabolite standards (labeled and non- labeled) were confirmed by ESI-MS, GC-MS (only for 5OH-EHS-d4 ) and 1 H and 4
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ACCEPTED MANUSCRIPT NMR, and their chemical purity was assessed by HPLC-UV. Acetonitrile, methanol and water CHROMASOLVT M LC-MS grade were purchased from Honeywell Riedel-de Haën (Seelze, Germany). Acetic acid puriss./Ph. Eur., formic acid p.a., and ammonium acetate BioXtra ≥98% were obtained from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was produced using
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an in-house Advantage A10 water purification unit (Merck Millipore, Darmstadt, Germany). β-
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glucuronidase (aryl sulfatase-free) from E. coli K12 (at least 140 u/mL at 37° C according to
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manufacturer) was purchased from Roche Diagnostics (Mannheim, Germany). Silanized HPLC
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vials (Macherey-Nagel, Düren, Germany) with silicone/PTFE screw caps (VWR, Leuven, Belgium) were used for LC-MS analysis.
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2.2. Standard solutions
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Stock solutions (approximately 1 g/L each, weighed exactly) of all three metabolite standards, both labeled and non-labeled, were prepared by weighing into volumetric flasks and dissolving
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and filling up with acetonitrile. For use as internal standards, the stock solutions of deuterium-
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labeled metabolites were diluted in acetonitrile (5OH-EHS-d4 and 5oxo-EHS-d4 : 5.00 mg/L each; 5cx-EPS-d4 : 25.0 mg/L and further diluted to 500 µg/L) and these dilutions were used to prepare
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an internal standard mix (5OH-EHS-d4 : 20 µg/L, 5oxo-EHS-d4 : 10 µg/L; 5cx-EPS-d4 : 1 µg/L).
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From the stock solutions of non- labeled metabolites, three mixed stock solutions were prepared (after preparation of appropriate intermediate dilutions) by serial dilution (a) 500 µg/L 5OH-EHS, 2.50 mg/L 5oxo-EHS, 250 µg/L 5cx-EPS; b) 25 µg/L 5OH-EHS, 125 µg/L 5oxo-EHS, 12.5 µg/L 5cx-EPS; c) 2.00 µg/L 5OH-EHS, 10.0 µg/L 5oxo-EHS, 1.00 µg/L 5cx-EPS). These mixed stock solutions were then used for the preparation of seven calibration standards in LC-MS grade water (0.02-10 µg/L 5OH-EHS, 0.1-50 µg/L 5oxo-EHS, 0.01-5 µg/L 5cx- EPS). All stock solutions, including intermediate dilutions and mixed solutions, were stored in glass flasks, capped with 5
ACCEPTED MANUSCRIPT screw caps with silicone/teflon seals. Aliquots of 300 µL of each calibration solution and pure water as blank solution were transferred into HPLC vials, immediately after preparation. All solutions were stored at -20 °C until further use. Stock solutions were stable for more than 1.5 years and calibration solutions for more than 12 weeks.
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2.3. Sample collection and preparation
Urine samples were collected in 250 mL PE containers and stored at -20 °C until analysis.
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Metabolites in urine were stable for more than 1.5 years. Urinary creatinine was determined by
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contract analysis (L.u.P. GmbH Labor und Praxisservice, Bochum, Germany). For analysis, 300 µL of homogenized (by repeated inversion) urine were transferred into an HPLC vial and 100 µL
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ammonium acetate buffer (1M, pH 6.0-6.4), 30 µL internal standard mix, and 6 µL β-
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glucuronidase, premixed with ammonium acetate buffer 1:1 (v/v), were added. Samples were homogenized by inverting several times and then incubated in a water bath at 37 °C for 3 h.
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Afterwards, 30 µL of formic acid were added, the samples were again homogenized and frozen at
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-20 °C overnight to precipitate cryophobic proteins. After thawing, samples were centrifuged (1900 g, 10 min) and the supernatant transferred into a new HPLC vial. Calibration solutions and
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quality control samples underwent the same treatment as urine samples.
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2.4. Chromatographic conditions A 1260 Infinity HPLC (Agilent Technologies, Waldbronn, Germany) was used for chromatographic separation and online sample cleanup. The instrument consisted of a G1311B quaternary low pressure gradient pump (used as loading pump for online-SPE), a G1312B binary high pressure gradient pump with G4225A degasser (used for chromatographic separation) a G1367E autosampler with G1330B thermostat, and a G1316A thermostated column compartment with 2-way 6-port switching valve. Instrument setup and chromatographic conditions were 6
ACCEPTED MANUSCRIPT identical to an HBM method for the UV filter octocrylene, previously described by our group [36]. A TurboFlow Phenyl (50 x 0.5 mm; Thermo Scientific, Franklin, MA, U.S.A.) turbulent flow chromatography (TFC) column was used for online sample cleanup and a Kinetex C18 column (150 x 3 mm, particle size 2.6 µm; with SecurityGuard ULTRA guard column;
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Phenomenex, Aschaffenburg, Germany) with superficially porous particles was used for HPLC.
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An inline filter (porosity 0.5 µm; 3.0 mm diameter; Phenomenex, Aschaffenburg, Germany),
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placed between autosampler and column compartment, was used to reduce contaminations of the online-SPE column. The injection volume was 100 µL. A needle wash with methanol/water
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80:20 (v/v) was performed for 10 s after sample aspiration. Autosampler and thermostated
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column compartment were kept at 6 °C and 25±1 °C, respectively. Eluents were water (eluent A) and acetonitrile (eluent B), both containing 0.05% acetic acid. The gradient of the loading pump
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is shown in table 1. The binary pump delivered eluent at a constant flow rate of 300 µL/min with
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the following gradient: Initial 30% B were kept for 2 min. Then, B was increased to 50% within 2 min. Afterwards, B was increased more slowly to 62% within 12 min and then to 95% within 2
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min. B was kept at 95% for 5 min, then decreased to initial conditions (30% B) within 0.5 min
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and kept for 8 min. The gradient delay volumes according to the manufacturer’s manuals were 600 to 800 µL for the binary pump and 870 to 1170 µL on the loading pump flow path (600 to
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900 µL for the quaternary pump and 270 µL for the autosampler). The 6-port valve was switched at 3 min to start the analyte transfer from the online-SPE column onto the HPLC column and again at 6 min to end analyte transfer and start the analytical separation and re-equilibration of the SPE column.
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ACCEPTED MANUSCRIPT 2.5. Mass spectrometric conditions Detection of EHS metabolites was performed with electrospray-tandem mass spectrometry (ESIMS/MS) in time-programmed multiple reaction monitoring (scheduled MRM) mode with polarity switching using a 4500 triple quadrupole mass spectrometer (Sciex, Darmstadt,
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Germany). The instrument gases (nitrogen) were set to: nebulizer gas, 40 psi; heater gas, 50 psi;
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curtain gas, 20 psi; collision gas, 6 arbitrary units. The source heater was kept at 450 °C.
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Electrospray voltages, entrance potential, and collision cell exit potential for +ESI/- ESI were 5.5 kV/-4.5 kV, 10 V/-10 V, and 12 V/-12 V, respectively. The target scan times were 0.200 s (+ESI)
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and 0.125 s (-ESI) with an MRM detection window of 60 s. For further MRM conditions see
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table 2. Declustering potentials and collision energies were manually optimized. Analyst 1.6
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(Sciex, Darmstadt, Germany) was used for instrument control and quantitative data analysis. 2.6. Calibration, validation and quality control
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Calibration was performed by weighted (concentration-1 ) linear regression, using the Analyst
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software. Method validation and quality assurance was performed according to the principles of the working group “Analyses in Biological Materials” of the Permanent Senate Commission for
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the Investigation of Health Hazards of Chemical Compounds in the Work Area of the German
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Research Foundation (Deutsche Forschungsgemeinschaft) [37]. Two quality control materials (Q low and Q high ) with different concentrations (table 3) of the three metabolites were prepared by mixing different urine samples (from an oral dosing study (publication in preparation) and, in case of Q low, from a pilot population (section 2.8)), containing native concentrations of the three metabolites. Native 5oxo- EHS concentrations were too low to achieve concentrations covering the calibration range without exceeding the calibration range for the other two metabolites. Thus, 5oxo-EHS concentrations were spiked (Qlow: 0.5 µg/L, Q high : 30 µg/L). These pooled urine 8
ACCEPTED MANUSCRIPT samples were frozen, thawed, and filtrated three times. The obtained materials were aliquoted (300 µL each) in HPLC vials and stored at -20 °C. To determine the method imprecision, this material was analyzed eight times within one analytical batch (within-series imprecision) and on eight different days (interday imprecision). After validation, the material was further analyzed in
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each batch for quality control purposes (quality control chart). For the determination of the
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method accuracy, eight different urine samples (0.21 to 2.59 g/L creatinine) were analyzed,
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spiked at three different concentration levels (prior to sample preparation; table 4) and without
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spiking. Relative recoveries were calculated after subtraction of native metabolite concentrations. 2.7. Pilot dermal application
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A commercial sunscreen containing 5% EHS (as listed on the product label) was applied to the
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whole body in a regular use scenario by one male volunteer (33 years of age, 90 kg body weight). A total amount of 10.9 g sunscreen was applied, corresponding to 543 mg EHS (6.03 mg/kg body
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weight). One week prior to the study and during the study, no known additional EHS exposure
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occurred (PCPs, containing EHS were completely avoided). To avoid non-dermal EHS uptake as good as possible, mouth contact with the sunscreen itself or with exposed hands was avoided.
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4.75 h after application, a shower was taken to simulate swimming activity and remove any
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sunscreen from the skin surface. Between the application and taking the shower, no food was touched or eaten.
2.8. Pilot population Spot urine samples from 35 volunteers (24 females and 11 males; age 23 to 59 (median 42)) without any known occupational EHS exposure were collected in April 2017. Urinary creatinine ranged from 0.09 to 2.31 g/L. Use of sunscreens, lipsticks with sun protection factor (SPF), and
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ACCEPTED MANUSCRIPT other PCP with SPF (such as day creams) was inquired via questionnaire. Of the 35 volunteers, 11 stated to have used products with sun protection factor within the last 5 days.
3. Results and discussion
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3.1. Mass spectrometry
The EHS metabolites were detected with ESI-MS/MS. 5cx-EPS was ionized both in positive and
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negative ion mode, but sensitivity in negative ion mode was much better. 5OH- EHS and 5oxo-
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EHS were poorly ionized in negative ion mode and much better in positive ion mode. Accordingly, 5cx-EPS was detected in ESI negative ion mode and 5OH-EHS and 5oxo-EHS
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were detected in ESI positive ion mode. Product ion spectra of all analytes (labeled and non-
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labeled), including schematic explanations of produced fragment ions are shown in figure 2. The observed mass signals can be interpreted as follows: The pseudo- molecular ion ([M-H]-) of 5cx-
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EPS (m/z 279) can eliminate the sidechain alkene 5- methyleneheptanoic acid to yield the
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fragment at m/z 137. This fragment can eliminate carbon dioxide to yield m/z 93. Further elimination of carbon monoxide from the aromatic ring yields the fragment at m/z 65. For the
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deuterium- labeled internal standard 5cx-EPS-d4 , corresponding fragments were found at +4 u.
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For 5OH-EHS, the pseudo- molecular ion ([M+H]+ ) can either eliminate the sidechain alkene 5methyleneheptan-2-ol or the sidechain diol 2-ethylhexane-1,5-diol to yield the aromatic fragments at m/z 139 and 121, respectively. The fragment at m/z 121 can also be formed by elimination of water from m/z 139. Corresponding fragments at +4 u were observed for 5OHEHS-d4 . 5OH-EHS can further eliminate salicylic acid (2-hydroxybenzoic acid) to yield a sidechain alkene fragment at m/z 129. This fragment can eliminate water to yield m/z 111 (an initial elimination of water from [M+H]+ would also be conceivable, but a corresponding signal
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ACCEPTED MANUSCRIPT at m/z 249 was not observed). Further elimination of propene from m/z 111 yields m/z 69. All of these sidechain fragments were also observed for 5OH-EHS-d4 . In case of 5oxo-EHS and 5oxoEHS-d4 , only two major fragments were observed. A sidechain alkene fragment (analogous to m/z 129 for 5OH-EHS and 5OH-EHS-d4 ) was observed at m/z 127. In analogy to 5OH-EHS and
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5OH-EHS-d4 , elimination of water from this sidechain alkene fragment (preceded by keto-enol
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tautomerism) yields m/z 109. Quantifier and qualifier MRM transitions were chosen according to
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their signal-to-noise (S/N) ratios in matrix and the absence of any interfering matrix peaks (see table 2). For example, in case of 5cx-EPS and 5cx-EPS-d4 , the most suitable ion traces were not
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the best in terms of absolute signal intensities, but were chosen based on their low noise levels
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and high resulting S/N ratios (figure 3).
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3.2. Method performance
Enzymatic β- glucuronidase/aryl sulfatase preparations are known to cleave ester moieties of
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certain metabolites, such as phthalate and terephthalate metabolites [38–40]. Accordingly, a pure
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β-glucuronidase, free of any aryl sulfatase/esterase activity, was chosen to avoid the cleavage of the ester moiety of EHS metabolites. We tested a ß-glucuronidase enzyme preparation with aryl
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sulfatase activity (type HP-2 from Helix pomatia; Sigma-Aldrich, Steinheim, Germany) and
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found indications of ester cleavage and degradation of the standards (data not shown). As a consequence of choosing a pure ß- glucuronidase, only free and glucuronidated metabolites are captured by this analytical approach; sulfates, which might also be formed, are not captured. To test for complete deconjugation of glucuronide conjugates we investigated incubation times of 1, 2, 3, 4, 5, 7, and 9 h in three urine samples with native metabolite levels (2.34-5.31 µg/L 5OHEHS, 0.82-2.56 µg/L 5oxo-EHS, 1.14-5.66 µg/L 5cx-EPS) and found no additional release of free metabolites with incubation times 2 h and longer (data not shown). For ruggedness purposes
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ACCEPTED MANUSCRIPT we therefore set the method’s incubation time to 3 h. We chose to calibrate in water instead of urine due to the high proportion of urine samples with metabolite concentrations above the limits of quantification (LOQ; see section 3.3) and the associated difficulty in finding metabolite- free and not unduly diluted urine. The slopes of calibration curves in urine (eight different urines
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tested – see section 2.6) and water were identical. Calibrations for all three metabolites were
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within the linear dynamic range. The limits of quantification (LOQ) were defined as a signal-to-
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noise (S/N) ratio of 10 in matrix. The LOQs obtained were 0.01 µg/L (5cx-EPS), 0.05 µg/L (5OH-EHS), and 0.15 µg/L (5oxo- EHS). Data on method precision is shown in table 3. Method
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imprecision (i.e. coefficient of variation) was < 5% both within-series and interday. The
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metabolite concentrations for Q low and Q high were chosen to cover the calibration range and reflect metabolite concentrations in both the dermal application (see below) and oral metabolism
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study (publication in preparation), and also in the general population (see below). Relative
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recoveries (table 4) were determined as described in section 2.6. Mean relative recoveries were between 96% and 106% for all three metabolites and all spiking levels with single values ranging
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from 85% to 115%. For the lowest spiking level, one urine samples was excluded from the
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evaluation for 5cx-EPS and 5OH-EHS, because native metabolite levels were by far exceeding the spiked concentrations. Including this urine sample, mean relative recoveries were between 94%
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and 106% with single values ranging from 80% to 132%. The presented method is based on an HBM method for the determination of biomarkers of the UV filter octocrylene, previously published by our group [36]. Combination of both methods for simultaneous analysis of OC and EHS biomarkers can be performed without interferences.
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ACCEPTED MANUSCRIPT 3.3. Applicability for human biomonitoring of EHS exposure To investigate the relevance of the analyzed EHS biomarkers for biomonitoring after EHS exposure, EHS metabolite levels were determined in urine samples collected after application of a sunscreen in a regular use scenario. Exemplary chromatograms from a urine sample collected
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40 h after the sunscreen application are shown in figure 3. Concentrations of all three EHS
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metabolites increased after application, reaching maximum levels around 9 h after application
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and decreasing slowly thereafter (figure 4; creatinine-adjusted concentrations). The excretion profile for all three metabolites was very similar, indicating similar elimination kinetics. The
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results show that all three EHS metabolites are suitable as biomarkers of EHS exposure,
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following (occupational or recreational) sunscreen use.
In addition to the dermal EHS exposure scenario, the applicability of the method for human
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biomonitoring of the general population (including background concentrations) was tested. For
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that purpose, 35 urine samples from the general population without known occupational EHS exposure were analyzed (table 5). 5cx-EPS was found above LOQ in 91%, 5OH-EHS in 49%,
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and 5oxo-EHS in 14% of all spot urine samples. Highest metabolite concentrations and highest
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detection rates were found in urine samples from individuals (n = 11) who had reported use of PCPs with SPF within 5 days prior to sampling. However, also in the subgroup (n = 24) who had
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reported no use of PCPs with SPF within five days prior to urine collection, 5cx-EPS was above LOQ in 88% of the samples (5OH-EHS: 38%, 5oxo-EHS: 4%). These results show that all three metabolites are suitable biomarkers of EHS exposure, with 5cx- EPS being the most sensitive biomarker, followed by 5OH-EHS and 5oxo-EHS.
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ACCEPTED MANUSCRIPT 4. Conclusions The method presented here enables the precise, sensitive, accurate, and rugged determination of three previously not described EHS metabolites in human urine samples. Results from a dermal pilot study (regular use scenario of an EHS containing sunscreen product) prove the applicability
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of all three metabolites as biomarkers of EHS exposure. Furthermore, we were able to quantify
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EHS metabolites in the majority of urine samples from a small pilot population, suggesting the
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method´s applicability for exposure biomonitoring of the general population, covering also background exposures (i.e. no recent use of sunscreens). This me thod, in conjunction with renal
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conversion factors for all three metabolites (to be determined in a human metabolism study, in
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preparation), will enable a robust exposure and risk assessment for EHS.
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Declarations of interest
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None.
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Funding
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The presented study and development of the analytical method applied herein are part of a largescale 10-year project on the advancement of human biomonitoring in Germany. This project is a
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cooperation agreed in 2010 between the German Federal Ministry for the Environme nt, Nature Conservation and Nuclear Safety (BMU) and the German Chemical Industry Association (VCI Verband der Chemischen Industrie e.V.) and is managed by the German Environment Agency (UBA). Experts from governmental scientific authorities, industry and science provide advice in the selection of substances to be investigated and during method development. The analytical method development and the human metabolism study are financed by the Chemie
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ACCEPTED MANUSCRIPT Wirtschaftsförderungsgesellschaft mbH while the first application of the novel methodology in a larger population study will be financed by the German Environment Agency.
Compliance with ethical standards
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Ethical approval
The study (oral and dermal dosage of 2-ethylhexyl salicylate; pilot population) was carried out in
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accordance with the Code of Ethics of the World Medical Association (1964 Declaration of
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Helsinki and later amendments) and has been approved by the ethical review board of the medical faculty of the Ruhr-University Bochum (IRB Reg. No.: 3867−10 and 4288−12). The
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CE
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obtained from each individual participant.
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study design was presented to the volunteers in written form and written informed consent was
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[31] G. Schoeters, E. Govarts, L. Bruckers, E. Den Hond, V. Nelen, S. de Henauw, I. Sioen, T.S.
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human biomonitoring in Flanders - Time trends observed in the Flemish Environment and
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[32] B.K. Schindler, M. Esteban, H.M. Koch, A. Castano, S. Koslitz, A. Cañas, L. Casteleyn, M.
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Kolossa-Gehring, G. Schwedler, G. Schoeters, E.D. Hond, O. Sepai, K. Exley, L. Bloemen, M. Horvat, L.E. Knudsen, A. Joas, R. Joas, P. Biot, D. Aerts, A. Lopez, O. Huetos, A.
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Katsonouri, K. Maurer-Chronakis, L. Kasparova, K. Vrbík, P. Rudnai, M. Naray, C.
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Guignard, M.E. Fischer, D. Ligocka, B. Janasik, M.F. Reis, S. Namorado, C. Pop, I. Dumitrascu, K. Halzlova, E. Fabianova, D. Mazej, J.S. Tratnik, M. Berglund, B. Jönsson, A.
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Lehmann, P. Crettaz, H. Frederiksen, F. Nielsen, H. McGrath, I. Nesbitt, K. de Cremer, G. Vanermen, G. Koppen, M. Wilhelm, K. Becker, J. Angerer, The European COPHES/DEMOCOPHES project: Towards transnational comparability and reliability of human biomonitoring results, Int. J. Hyg. Environ. Health 217 (6) (2014) 653–661. https://doi.org/10.1016/j.ijheh.2013.12.002. [33] J. Angerer, U. Ewers, M. Wilhelm, Human biomonitoring: State of the art, Int. J. Hyg. Environ. Health 210 (3-4) (2007) 201–228. https://doi.org/10.1016/j.ijheh.2007.01.024. 20
ACCEPTED MANUSCRIPT [34] L.L. Needham, A.M. Calafat, D.B. Barr, Uses and issues of biomonitoring, Int. J. Hyg. Environ. Health 210 (3-4) (2007) 229–238. https://doi.org/10.1016/j.ijheh.2006.11.002. [35] M. Kolossa-Gehring, U. Fiddicke, G. Leng, J. Angerer, B. Wolz, New human biomonitoring methods for chemicals of concern-the German approach to enhance relevance, Int. J. Hyg.
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Determination of Urinary Metabolites of the Emerging UV Filter Octocrylene by Online-
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SPE-LC-MS/MS, Anal. Chem. 90 (1) (2018) 944–951.
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Quantitative Detection of Eight Phthalate Metabolites in Human Urine Using HPLC−APCI-
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di(2-ethylhexyl) terephthalate (DEHTP) in human urine by HPLC-MS/MS with on-line clean-up, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1011 (2016) 196–203. https://doi.org/10.1016/j.jchromb.2015.12.042. [40]H.M. Koch, F. Lessmann, S.H. Swan, R. Hauser, M. Kolossa-Gehring, H. Frederiksen, A.-M. Andersson, C. Thomsen, A.K. Sakhi, C.-G. Bornehag, J.F. Mueller, R.A. Rudel, J.M. Braun, V. Harth, T. Brüning, Analyzing terephthalate metabolites in human urine as biomarkers of exposure: Importance of selection of metabolites and deconjugation enzyme, J. Chromatogr. 21
ACCEPTED MANUSCRIPT B Analyt. Technol. Biomed. Life Sci. 1100-1101 (2018) 91–92.
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ACCEPTED MANUSCRIPT Figure captions Figure 1 – Metabolism scheme of EHS leading to the metabolites investigated. Phase II metabolites (conjugates) are not shown.
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Figure 2 – Product ion spectra of the metabolite standards. Product ion spectra of (from top
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to bottom) 5cx-EPS, 5OH-EHS, and 5oxo-EHS (left column), and their respective
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deuterium- labeled analogs (right column), including schematic explanations of produced fragment ions. 5cx-EPS was measured in ESI negative ion mode and 5OH-EHS and 5oxo-
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EHS in ESI positive ion mode.
Figure 3 – Exemplary chromatograms for all three EHS metabolites. Chromatograms of a
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calibration standard (left column) and a urine sample after dermal EHS application with
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native metabolite concentrations (right column) for 5OH-EHS (top), 5oxo-EHS (middle), and 5cx-EPS (bottom). Quantifier (continuous) and qualifier (dashed) transitions of
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metabolites (black) and internal standards (gray) are shown. Metabolite concentrations are
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Figure 4 – EHS metabolite concentrations after sunscreen application. Creatinine-adjusted concentrations of 5OH-EHS (continuous black), 5oxo-EHS (dashed black), and 5cx-EPS (gray) in one male volunteer after whole body dermal application of 10.9 g sunscreen (corresponding to 543 mg EHS or 6.03 mg/kg body weight). Concentrations are plotted on a logarithmic scale.
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ACCEPTED MANUSCRIPT Table 1. Gradient for the loading pump.
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Eluent B [% ] 0 0 0 0 0 95 95 0 0 0 0
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Eluent A [% ] 100 100 100 100 100 5 5 100 100 100 100
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Flow rate [µL/min] 1500 1500 200 200 500 500 500 500 500 1500 1500
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Time [min] 0 3 3.1 6 7 10 21 22 28.5 29.5 31.5
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DP [V] -60 -60 30 30 15 15
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ESI polarity negative negative positive positive positive positive
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tR [min] 14.7 14.6 15.6 15.5 17.7 17.6
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Analyte 5cx-EPS 5cx-EPS-d 4 5OH-EHS 5OH-EHS-d 4 5oxo-EHS 5oxo-EHS-d 4
MRM transitions m/z m/z product precursor 279 93 (137) 283 97 (141) 267 129 (111) 271 129 (125) 265 127 (109) 269 127 (109)
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CE [eV] -47 (-16) -47 (-16) 8 (14) 8 (35) 12 (23) 12 (22)
ACCEPTED MANUSCRIPT Table 3. Method precision. Mean concentrations and coefficients of variat ion are given for all three metabolites in both quality control materials (Qlow and Qhigh ).
5OH-EHS
Qlow Qhigh
0.101 6.30
2.4% 1.4%
5oxo-EHS
Qlow Qhigh
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0.64 32.4
3.6% 3.7%
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interday imprecision (n = 8) mean coefficient of concentration variation [µg/L] 0.155 3.7% 2.69 2.7%
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within-series imprecision (n = 8) mean coefficient of concentration variation [µg/L] 0.157 1.2% 2.68 1.3%
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ACCEPTED MANUSCRIPT Table 4. Method accuracy. Range of native metabolite concentrations and mean relat ive recoveries (ranges in parenthesis) at three spiking levels for eight urine samples (0.21 to 2.59 g/L creatinine). Spiked concentrations [µg/L] 0.1 0.5 2
5OH-EHS
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5cx-EPS
Relative recovery [% ]
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Native concentrations [µg/L]
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ACCEPTED MANUSCRIPT Table 5. Pilot hu man bio monitoring population. Urine samples fro m the general population without known occupational EHS exposure were analy zed. Data for the whole population is shown, as well as data divided by individuals who d id not report any use of PCP with SPF (sunscreens, lip sticks, face creams, etc.) within 5 days prior to sampling, and those who did. Use of PCP with SPF (previous No use of PCP with SPF 5 days) (n = 11) (previous 5 days) (n = 24) Median Median Median concentration n > LOQ concentration n > LOQ concentration n > LOQ (range) [µg/L] (percentage) (range) [µg/L] (percentage) (range) [µg/L] (percentage) 5cx0.039 0.159 11 (100%) 0.028 21 (88%) 32 (91%) EPS (
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Total population (n = 35)
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ACCEPTED MANUSCRIPT Highlights Determination of three urinary metabolites of the UV filter 2-ethylhexyl salicylate
All three metabolites are specific side chain oxidation products
LC-MS/MS coupled with online sample clean-up using turbulent flow chromatography
After sunscreen use detection of all metabolites with clear excretion kinetics
Metabolites found >LOQ in 91% of urine samples from pilot population (n=35)
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Figure 1
Figure 2
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Figure 4