Article pubs.acs.org/est
Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
First Detection of Photoinitiators and Metabolites in Human Sera from United States Donors Runzeng Liu* and Scott A. Mabury Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada
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ABSTRACT: Photoinitiators (PIs), including benzophenones (BZPs), thioxanthones (TXs), and amine co-initiators (ACIs), are commonly used in photopolymerization systems, and their contamination in foodstuffs and the environment is attracting attention. Although humans are likely exposed to PIs, no data on human burdens of these chemicals are available. In this study, 18 PIs were detected in 50 individual human serum samples with concentrations of ΣPIs (sum of the detected PIs) from 423 to 2870 pg/mL (geometric mean, GM: 836 pg/mL). ΣBZPs (231−1240 ng/g,; GM: 593 pg/mL) were the dominant components, while ΣTXs (21.0−1431 ng/g; GM: 145 pg/mL) and ΣACIs (11.3−976 ng/g; GM: 48.5 pg/mL) were much lower. Data analysis found significantly higher concentrations of most PIs in the male sera than in the female (p < 0.05). ΣPIs (2921−4139 ng/g; GM: 3621 pg/ mL) were also detected in five pooled serum samples, each from at least 1000 donors, indicating the prevalent human burdens of PIs in a large population. Human liver S9 biodegradations of representative PIs, 2-isopropylthioxanthone (2-ITX) and 2,4diethylthioxanthone (DETX), were conducted. Hydroxylation, sulfoxide, and sulfone metabolites of DETX and 2-ITX were identified by high resolution mass spectrometry in human liver S9 incubation systems. With synthesized standards, the sulfoxide and sulfone metabolites were successfully detected in the human serum samples, which contributed substantially to total human burdens. The ubiquitous presence of PIs in human sera indicates significant human exposure to PIs, although photopolymerization reaction has been generally considered a green technology.
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INTRODUCTION Photopolymerization reactions are a green technology characterized by low energy requirements, low operation temperatures, and solvent-free systems without release of organic compounds.1 Photoinitiators (PIs) are chemicals crucial to the photopolymerization process of synthesizing polymers. The function of PIs is to generate active substances, such as free radicals, and initiate polymerization of monomers and oligomers to generate polymers during the optical absorption process.2 The most frequently used PIs are grouped into the following three families: benzophenones (BZPs), thioxanthones (TXs), and amine co-initiators (ACIs).3,4 These PIs are mainly added in light sensitive materials including UV curable printing inks, adhesives, coatings, and resin, which are used in plenty of industrial applications including primers, overprint vanishes, pigmented paints, clear topcoats, and so on.5 The burgeoning applications of photopolymerization in various techniques, such as UV curable printing inks and UV curable resin three-dimensional (3D) printing,6 which are widely used in Europe, North America, and China,4,7,8 suggest the production and use of PIs will likely increase in the future. PIs first caught attention of the public when migration of 2isopropylthioxanthone (2-ITX) from printing inks on food packaging materials to foodstuffs was found. In 2005, 2-ITX © XXXX American Chemical Society
was identified in baby formula at concentrations as high as 300 μg/L, resulting in withdrawal of more than 30 million liters of milk from commercial markets.9 Subsequent studies showed many other PI congeners such as benzophenone (BP), 4methylbezophenone (4-MBP), 2-methyl-4′-(methylthio)-2morpholinopropiophenone (PI-907), 2-ethylhexyl-4(dimethylamino)benzoate (EHDAB), and 2,4-diethylthioxanthone (DETX) were also present in foodstuffs (juice, milk powder, muesli, cheese, and chocolate) at concentrations up to part-per-million (ppm) levels.10−12 A recent study demonstrated that PIs were not totally consumed during the 3D printing process, leading to high residual concentrations of BZPs (ppm levels) in UV cured 3D printing products.7 The widespread use of PIs has also triggered increasing awareness of their pollution in the environment. Elevated concentrations of these chemicals were reported in indoor dust and sewage sludge collected across China, with total concentrations of PIs up to more than 5 μg/g.7 Received: Revised: Accepted: Published: A
May 8, 2018 July 10, 2018 July 31, 2018 July 31, 2018 DOI: 10.1021/acs.est.8b02457 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Native standards of the PI analytes were obtained from TCI (Tokyo, Japan). Isotope-labeled benzophenone (BP-d10) and 2-isopropyl-d7-thioxanthone (2-ITX-d7) were purchased from Sigma-Aldrich (Oakville, Canada). The purities of all target analytes were >95%. Stock solutions at concentrations of 1000 μg/mL were individually prepared in acetonitrile (ACN), while intermediate solutions were prepared weekly from the stock solutions by methanol (MeOH) dilution. Centrifuge tubes (Polypropylene, 15 mL) were obtained from VWR (Mississauga, Canada). HPLC-grade ACN, MeOH, and methyl-tert-butyl ether (MTBE) were from Sigma-Aldrich (Oakville, Canada). Formic acid (FA) was supplied by Caledon (Georgetown, Canada). Ultrapure water (H2O, 18.2 MΩ × cm) was generated by Purelab flex (Veolia Water Technologies, Mississauga, Canada). Human liver S9 was from Sigma-Aldrich (Oakville, Canada); the NADPH regeneration system was from Promega (Madison, WI). Serum Samples. Fifty human serum samples were obtained from Golden West Biologicals, Inc. (Temecula, CA). The samples were collected in the U.S. in 2016 and 2017 from donors varying in age (19−65 years old) and gender. The 50 individual serum samples were equally from male and female donors. In order to detect the PIs in a large composite population, five pooled serum samples, each containing at least 1000 individual donors, were also included in this study. The serum samples used in production of the pooled sera were originally drawn for transfusion use. Once the products expired for transfusion use, they were used to generate pooled serum. The basic information on the samples, such as age and gender, is included in Table S2. Calf serum was purchased from SigmaAldrich (Oakville, ON, Canada) to use as a recovery matrix. All the serum samples were stored at −20 °C. Sample Preparation and Instrumental Analysis. A liquid−liquid extraction method was developed to extract the target PIs from the serum samples. In order to avoid contamination in the sample pretreatment, all tubes were washed twice with MTBE before use. Briefly, sample extraction involved mixing 5 mL of serum (spiked with 1 ng of BP-d10 and 1 ng of 2-ITX-d7) with 5 mL of MTBE and shaking at 500 rpm for 20 min, after which the mixture was centrifuged for 5 min and MTBE was transferred to another tube; the extraction was performed 3 times. The combined MTBE was dried under a stream of nitrogen and solvent exchanged into 0.5 mL of MeOH. The samples were then centrifuged at 3000 rpm for 5 min to remove the suspended particles. After centrifugation, 2 μL aliquots were injected into the instrument. The quantification of target PIs was performed on a Waters ultrahigh performance liquid chromatograph (UPLC) coupled to a Xevo triple-quadrupole mass spectrometer (MS/MS, Milford, USA). Electrospray ionization (ESI) was operated in the positive mode. The source and desolvation temperatures were set to 120 and 400 °C, respectively. Desolvation gas flow was 800 L/h; cone gas flow was 120 L/h. Details of multiple reaction monitoring parameters are shown in Table S3. A Waters ACQUITY BEH C18 analytical column (2.1 × 100 mm, 1.7 μm) was heated to 60 °C with a flow rate of 0.3 mL/ min. The flow gradient was initiated at a composition of 40:60 (MeOH/H2O, v/v, 0.1% formic acid in each phase), held for 3 min, linearly ramped to 100% MeOH in 7 min, and kept for another 5 min. Quality Assurance/Quality Control. Recoveries of the target analytes in the spiked serum samples (200 pg/mL of each analyte) were 52−98%, with relative standard deviations
Toxicological studies suggested adverse effects of some PIs on biota and humans. Using yeast hormone bioassays, TX compounds, including 2-ITX and DETX, were found to have antiandrogenic and antiestrogenic activities.13,14 2-ITX may also affect the mobility and rigidity of biological membranes by strong interaction with the cellular lipid bilayer.15 In addition, the arylhydrocarbon receptor can be activated by 2-ITX, perhaps due to the structural similarity to 2,3,7,8-tetrachlorodibenzo-p-dioxin.13 Estrogenic activities of ACIs (EHDAB and PI-907) and BZPs, including methyl-2-(benzoyl)benzoate (MBB), 1-hydroxycyclohexyl phenyl ketone (PI-184), and 2,2dimethoxy-2-phenylacetophenone (PI-651), were also reported using in vitro assays.16,17 BP was demonstrated to have carcinogenic activity in mice and rats.18 4,4′-Bis(dimethylamino) benzophenone (Michler’s ketone, MK) was also found to be a potential carcinogen.19 Though no mandatory regulations are available, European Council of the Paint, Printing Inks, and Artists’ Colors Industry and Japan Printing Ink Makers Association have voluntarily placed MK on a list of substances not recommended in printing inks to be used on paper or paperboard food packaging materials.20,21 Detection of anthropogenic chemicals in human sera can directly demonstrate the human exposure to these chemicals and is the first step for human health risk assessment. The large use of some anthropogenic additives, such as bisphenol A (BPA) and benzophenone-type UV filters, has led to their ubiquitous presence in humans.22,23 Furthermore, previous studies found the potential relationships between health issues and human burdens of chemical contaminants.24,25 Though humans are likely exposed to PIs via many pathways, including food intake and indoor dust ingestion, no data for their occurrence in humans are found. In the present study, 50 individual and five pooled serum samples were collected from United States donors to provide an initial impression of the occurrence, concentrations, and composition profiles of PIs in humans. Using high resolution mass spectrometry, in vitro metabolism pathways of some representative PIs were explored. Finally, some metabolites were quantified in human sera using the synthesized standards in our lab.
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METHODS Materials. The analyte abbreviations, structures, and other relevant data are shown in Figures 1 and S1, and Table S1.
Figure 1. Structures and abbreviations of the most detected PIs. B
DOI: 10.1021/acs.est.8b02457 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Table 1. Descriptive Statistics of the Measured PI Concentrations (pg/mL) in the Investigated Serum Samplesa average proportion (%) compounds
GMb
Benzophenones (BZPs) BP 246 PI-651 101 4-MBP 64.3 MBB 11.0 PBZ 3.54 PI-184 19.4 EAQ 14.7 benzil 11.8 ΣBZPs 593 Amine Co-initiators (ACIs) MK 0.93 MEK 4.43 EDMAB 5.49 EHDAB 1.03 PI-369 1.68 PI-907 19.9 DMAB 4.68 ΣACIs 48.5 Thioxanthones (TXs) 2-ITX 35.4 DETX 63.3 2-Cl-TX 17.0 ΣTXs 145 ΣPIs 836
median
range
quantification frequency (%)
in each analogue
in ΣPIs
253 137 60.6 3.50c 2.80c 18.0c 14.0c 11.0c 569
37.0−855
