A generic LC-MS/MS exposome method for the determination of xenoestrogens in biological matrices Karin Preindl1, Dominik Braun1, Georg Aichinger1, Sabina Sieri2, Mingliang Fang3, Doris Marko1, Benedikt Warth1* 1University of Vienna, Faculty of Chemistry, Department of Food Chemistry and Toxicology, Währingerstraße 38, 1090 Vienna, Austria 2Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3Nanyang Technological University, School of Civil and Environmental Engineering, 50 Nanyang Avenue, Singapore 639798, Singapore ABSTRACT: We are constantly exposed to a variety of environmental contaminants and hormones including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively re- ferred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are urgently needed but still not available. Herein, we describe an LC-MS/MS method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 µg/L, 0.03 to 14 µg/L, and 0.03 to 4.6 µg/L in urine, serum and breast milk, respec- tively. The applicability of the novel method was demonstrated in proof-of-principle experiments by analyzing urine from Austrian, and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods’ feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multi-class exposure assessment in the context of the expo- some paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class meas- urements and its potential impact in endocrine disruption and hormone related diseases including cancers.

The human endocrine system is an important signaling system caused by differing time windows of exposure or the applied, rather which regulates organ communication and behavior. In recent targeted, single class human biomonitoring (HBM) approaches years, attention has been drawn to the so-called endocrine disrupt- which did not consider mixture effects. Exposure to xenoestrogens ing chemicals (EDCs), compounds not produced by the body itself may also be a risk factor for hormone-related cancers including that can disturb the hormonal balance in various ways.1 One class those of breast and the reproductive tract, especially during the of EDCs are xenoestrogens, small molecules imitating or interfer- sensitive, early developmental stages.11-13 ing with endogenous estrogens, the most important female sex 2 The proper assessment of combinatory effects of multiple xenoes- hormones. Xenoestrogens are a highly diverse group of chemicals trogens are a current key challenge. Numerous studies investigating and can be produced either naturally, e.g. by plant (phytoestrogens) the potency of individual compounds or compound classes have and fungi (mycoestrogens), or synthetically as pesticides, plasticiz- 3 been published, while data on estrogenic mixtures is scarce. There ers, personal care product additives and other industrial chemicals. is a growing body of evidence that xenoestrogen combinations, In the past, research efforts typically focused on single representa- even at very low concentrations, can have potentiating14-17 and/or tives such as bisphenol A (BPA) and substance classes including antagonizing effects.18 Because of the variety of different structural parabens or phthalates. As a result, these chemicals may be banned classes, routes of exposure are divers; they may be inhaled through and replaced by less investigated analogues for which similar or 4 the ambient air, ingested via contaminated food or absorbed by the even more potent toxicological effects cannot be ruled out. skin. Due to these multiple exposure scenarios HBM is the best A suspected adverse health effect of xenoestrogens is reduced option for proper exposure assessment and should lead towards a fertility. It has been shown that high urinary concentrations of more comprehensive, exposome-scale assessment of environmental BPA, parabens and phthalate metabolites reduce positive outcomes risk factors in the etiology of chronic disease.19-20 of in vitro fertilization.5 A high uptake of phytoestrogens has been 6-8 To correlate exposure with potential health implications, ad- associated with negative effects on male fertility in some studies, vanced analytical assays for a simultaneous measurement of the while not in others.9-10 These controversial outcomes might be 1 different classes of xenoestrogens are essential, yet not readily achieve chromatographic separation. The column compartment and available. To date, the majority of multi-analyte methods focus on the autosampler were maintained at 40 °C and at 10 °C, respective- 21-23 single classes and the few existing multi-class methods typical- ly. LC-MS grade water with 0.3 mM ammonium fluoride (NH4F) ly contain no more than two different classes of xenoestrogens.24-28 as additive (eluent A) and acetonitrile (eluent B) were used as mo- The development of a method covering multiple classes of xenoes- bile phases. Aqueous ammonium fluoride solutions have to be trogens is a challenging task due to the diverse chemical properties handled with care as HF may outgas. Although, the NH4F concen- and low biological concentrations of such exposures. Single ana- tration in the eluent was very low, contact with acids (e.g. from lyte/class methods are not practical in large-scale epidemiological previously used eluent additives in the waste) was prevented. The studies involving the measurement of thousands of samples as system was operated at a flow rate of 0.4 mL/min with following proposed for future exposome-wide association studies (EWAS). gradient: 0-1 min: 5% B; rise to 18% B until min 1.8 and to 35% B Currently, a targeted multi-class method constitutes the most feasi- until min 4.2; rise to 48% B until min 13 and to 90% B until ble approach for the implementation of this challenging task. Ad- min 14; flush with 98% B from min 15.8 to min 17.6; re-equilibrate vantages of the targeted approach compared to non-targeted LC- with 5% B from min 17.7 to min 20. Multiple reaction monitoring HRMS screening, are the increased sensitivity and the improved (MRM) experiments were performed in positive and negative elec- ability to perform quantitative measurements. This work addresses trospray ionization (ESI) mode using fast polarity switching. The the need for a broad, quantitative LC-MS/MS method covering a detailed settings are described in the Supporting Information. multitude of estrogenic compounds in three relevant biological Quantification and data evaluation. To account for matrix ef- fluids. Following in-house validation, the method was applied to fects and potential retention time deviations in the different matri- biological samples for assessing early-life xenoestrogen exposures ces, matrix-matched reference standards were prepared by re- in first proof-of-principle experiments. solving evaporated ‘blank’ samples of urine, serum and breast milk EXPERIMENTAL SECTION with 200 µL (urine and serum) or 250 µL (breast milk) with solvent standards of six different concentrations. For quantification, the Chemicals and reagents. In this study, 75 analytes, 61 xenoes- fragment ion with the highest signal to noise ratio was chosen. A trogens and 14 endogenous estrogens, were included. Detailed second fragment ion was obtained for confirmation and ion ratio information regarding analytes and suppliers are provided in Table determination (Table S2). Methyl-, ethyl-, propyl-, butylparaben, S1 and Figures S2-S6. genistein, mono-n-butyl phthalate (MBP), perfluoroctanoic acid Sample collection. For validation experiments of human serum, (PFOA) and perfluoroctanesulfonic acid (PFOS) were evaluated by pooled male AB plasma derived serum was used (USA origin; internal standards in all matrices. Zearalenone (ZEN) and estradiol Sigma Aldrich, Vienna, Austria). For urine, a pooled sample was (E2) were evaluated by internal standard calibration in urine and obtained from a female volunteer who avoided foodstuff and bev- serum. 4-tert-octylphenol (4tOP) was evaluated by internal stand- erages stored in plastic containers, foods rich in phytoestrogens, ard calibration in serum and mono-2-ethylhexyl phthalate (MEHP) and cosmetics containing parabens for two days prior to sample was evaluated by internal standard calibration in urine. Data evalu- collection. For breast milk validation experiments, anonymized ation was carried out with TraceFinder™ software (version 4.1, pooled breast milk was provided by the Semmelweis Women’s Thermo Scientific). Linear calibration curves were created using 29 Clinic in Vienna, Austria, as previously reported. Urine and breast 1/x weighing. In case of ‘blank’ matrix contamination (not all milk were stored at -20 °C, serum at -80 °C until analysis. pooled samples for validation purpose were true blanks as the high To evaluate the applicability of the developed method, four urine sensitivity of the developed method allowed the identification of samples obtained from an Austrian mother-infant pair, as well as some trace level contamination), all concentrations were corrected. two samples from a female Austrian volunteer were analyzed. Validation. In-house method validation was performed accord- Moreover, five randomly selected breast milk samples from a pre- ing to the Commission Decision (EC) No. 657/200230 and evaluat- viously reported HBM study, focusing on mycotoxin exposure in ed in terms of selectivity, linearity, matrix effects (signal suppres- 29 Nigerian mothers, as well as four breast milk samples from two sion or enhancement, SSE), extraction recovery (RE), intermediate Austrian mothers were analyzed. All samples were stored at -20 °C precision (interday precision, RSDR), repeatability (intraday preci- until analysis and the studies approved by the respective ethic sion, RSDr) and limits of detection (LOD) and quantification committees in Austria (University of Vienna, No 00157) and Nige- (LOQ). Experiments were carried out three times with independent ria (Babcock University, No BUHREC294/16). sample preparation, calibration and measurements. Due to the Sample preparation. A volume of 200 µL urine or serum was absence of a suitable certified reference material, validation exper- spiked with 10 µL of internal standard solution and extracted with iments were performed with spiked ‘blank’ matrices. Blank matrix 790 µL ACN/MeOH (1/1) by sonication (10 min, 4°C). After pre- samples were spiked in triplicate at two levels (“low” and “high”) cipitating proteins in a freeze-out step (2 h, -20 °C), the samples by addition of 20 µL (for urine and serum) or 25 µL (for breast were evaporated and re-solved in 200 µL ACN/H2O (1/9). For milk) of a 10x concentrated multi-analyte stock. Spiking levels breast milk extraction, the protocol of Braun, et al. (2018)29 was aimed for 3x and 30x the LOQ values obtained in pre-experiments. slightly modified. Further details are reported in the Supporting In addition, three matrix blanks and three system blanks (LC-MS Information section. grade water instead of matrix) were extracted for each matrix. LC-MS/MS analysis. Measurements were performed on a Di- Measurement sequences consisted of external quality control sam- onex Ultimate 3000 UHPLC system coupled to a TSQ Vantage ples at the beginning and end of a sequence to evaluate the general triple quadrupole mass spectrometer, operated with a heated elec- performance of the system. Before and after each matrix batch, trospray ionization source (Thermo Scientific, Vienna, Austria). An solvent and matrix matched calibration sets were measured. Inter- Acquity HSS T3 column (1.8 µm, 2.1x100 mm) equipped with a mediate precision (RSDR) was determined by evaluation of the VanGuard pre-column (1.8 µm; Waters, Vienna, Austria) served to three validation batches (n=9 for each spiking level). Repeatability (RSDr) was determined by evaluation of the repeated measurement 2 of one validation batch (n=6 for each spiking level). Since the mass to charge ratio [m/z] and similar fragmentation patterns (e.g. 30 Commission Decision (EC) No. 657/2002 provides no RSDR zearalenol (ZEL) and zearalanol (ZAL) isomers, 16-/17-epiestriol limits for spikes with a concentration below 100 µg/L, the limit was and estriol, 2-/4-methoxy-estrogens, isobutyl- and butylparaben). set to 25%. Equally, the RSDr limit for spiked concentrations below For all analytes but isobutylparaben (iBP) and butylparaben (BP) 10 µg/L was set to 25%. To investigate selectivity, matrix blanks baseline separation was achieved. For iBP and BP, a resolution were manually inspected for potentially interfering peaks. Linearity factor RS of 0.7 was obtained. Although no baseline separation was was obtained by determination of the matrix matched calibration possible, two clearly separable peaks and linear calibration curves regression coefficient (R2), as well as by a manual inspection of the were generated (see Figure 3, Table S2). The lowest acceptable calibration curves. Signal suppression/enhancement (SSE) was retention time, according to EC No. 657/2002, is two times the determined as the ratio of the slope of the matrix matched calibra- retention time of the columns void volume. In this method analytes tion curve and the slope of the solvent calibration curve in percent. eluting after 1.75 min were accepted. Due to its hydrophilic charac- LOD and LOQ values were determined for each matrix by a signal ter, the paraben metabolite p-hydroxybenzoic acid (pHBA)36 elutes to noise ratio of three and ten, respectively. early in reversed phase chromatography and, despite highly aque- RESULTS AND DISCUSSION ous starting conditions (5% B), only in breast milk stable retention times higher than 1.75 min were achieved for pHBA. All other To the best of our knowledge, this is the first LC-MS/MS method analytes did not elute before min four. covering a vast number of known and suspected xenoestrogens including the most relevant endogenous estrogens. The 75 evaluat- Sample preparation and optimization. The majority of the few ed analytes included: 13 phytoestrogens, 12 mycoestrogens, seven existing multi-analyte methods, measuring more than one class of estrogenic compounds in biological matrices, rely on derivatization parabens, five UV- filter, seven bisphenols, five plasticizers, five 27, 37-38 industrial side products, three pesticides, two perflourinated alkyl- methods to increase sensitivity. We, however, aimed for a ated substances, one antimicrobial and one pharmaceutical as well simple and quick sample preparation protocol without time inten- as 14 endogenous estrogens, including precursors, phase I and II sive derivatization or sample dilution which would decrease sensi- metabolites. tivity, but nevertheless acceptable matrix effects and low LOD values. For exposome-scale studies involving the measurement of LC-MS/MS method development. To identify analytes suitable samples in a high throughput manner, a time and cost effective for a multi-class xenoestrogen method, the scientific literature, sample preparation is a prerequisite. These requirements were met EDC priority lists of the United Nations (UN), the European Food with our optimized protocol for serum and urine (Table 1 and Fig- Safety Authority (EFSA), the United States Environmental Protec- ure 1). For the particular challenging matrix breast milk, we tion Agency (EPA), and the European Commission (EC) were 29 adapted the protocol of Braun, et al. (2018) , which was originally screened. Analytes were selected based on their proven or strongly optimized for the extraction of mycotoxins including the estrogenic suspected estrogenicity, feasibility for LC-MS/MS analysis, and the ZEN and its metabolites α-/β-ZEL. We reduced the sample volume availability of commercial reference standards. In addition, some and exchanged the “filtering” to a “dry down and resolve” step in precursors (e.g. matairesinol, xanthohumol and isoxanthohumol) of order to overcome sample contamination with plastic components estrogenic compounds and in-house synthetized biotransformation from the filter material. This modifications led, at least for ZEN products (phase II metabolites of ZEN) were included. N- and α-/β-ZEL, to comparable extraction efficiencies (70-117% vs. butylbenzenesulfonamide, 2-naphthol and 2-tert-butylphenol were 29 82-106% in Braun, et al. (2018) ) but a higher variance. The re- selected based on the first application of cognitive compu- duced volume resulted in a relatively larger surface area which is in ting/artificial intelligence (AI) in exposome research from a list of contact with the reaction tube and the added salts, which potentially xenobiotics, ranked by the similarity to known estrogen receptor bind to or react with the analytes. The evaporation in turn, resulted agonists.31 These three compounds were selected as of their high in a difficult to dissolve pellet, containing one small fat drop, which scoring and (to date) no experimentally reported estrogenicity. could not be resolved again. We assume that this is a reason why Compound specific mass spectrometric parameters were opti- less than 50% of analytes yielded extraction recoveries above 80% mized by direct flow injection of a single solvent standard (5 mg/L) in breast milk (see Figure 1). utilizing a T-piece. All analytes have been tested thereafter in the Method validation and limitations of the method. Overall, 55 respective matrices in order to identify the best suited qualifier and and 53 out of 75 analytes were successfully validated in urine and quantifier ion (Table S2). Different eluent additives were tested in serum, and 31 out of 75 in breast milk. Selectivity was carefully order to increase sensitivity which was a major objective: ammoni- evaluated throughout method validation but also later during the um acetate, acetic acid and ammonium fluoride. Overall, ammoni- application of the method to naturally contaminated samples. um acetate resulted in the lowest signal intensities while acetic acid resulted in decent ionization for most analytes. However, neither of No interferences in a retention time window of 0.1 were detected. both enabled a satisfying ionization of endogenous estrogens. Pre- However, due to the high sensitivity of the developed assay we vious studies reported signal enhancement of steroids by addition detected a number of xenoestrogens in the “blank” material. 32-34 of NH4F to the aqueous mobile phase. It has been proposed that In “blank” serum 2-naphthol (approximately 9.6 µg/L), PFOA the basicity of the fluoride ion draws protons from the steroid and (enterolactone (

Table 1 Extraction efficiencies (RE), intermediate precision (RSDR), repeatability (RSDr) and limits of detection (LOD) and quantification (LOQ) in urine, serum and breast milk of analytes that have been successfully validated in at least one biological matrix. Analytes, not successfully evaluated in any matrix, are reported in Table S3 Urine Serum Breast milk a b e f h RE ± RSDR RE ± RSDR RSDr RE ± RSDR RE ± RSDR RSDr RE ± RSDR RE ± RSDR RSDr LOD LOQ LLc HLd LL/HL LL HL LL/HL LL HL LL/HL (U/S/M)g (U/S/M) [%] [%] [%] [%] [%] [%] [%] [%] [%] [µg/L] [µg/L] Phytoestrogens and metabolites 8-Prenylnaringenin 98 ± 21 103 ± 7 19/5 94 ± 15 76 ± 11 9/8 29 ± 77 74 ± 18 37/20 0.05/0.1/0.2 0.15/0.2/0.6 Coumestrol 96 ± 10 99 ± 6 14/2 86 ± 6 87 ± 7 3/5 83 ± 9 91 ± 3 10/10 0.15/0.4/0.3 0.5/1.3/1.0 Daidzein 112 ± 15 103 ± 6 15/4 90 ± 13 90 ± 6 9/4 86 ± 16 93 ± 4 12/9 0.15/0.1/0.05 0.5/0.4/0.15 Enterodiol 99 ± 12 101 ± 5 5/3 89 ± 7 91 ± 10 8/3 54 ± 28 56 ± 34 76/32 0.15/0.05/0.06 0.50/0.15/0.20 Enterolactone 102 ± 9 104 ± 3 6/2 85 ± 13 88 ± 13 8/5 88 ± 12 91 ± 6 10/5 0.15/0.2/0.1 0.50/0.7/0.4 Equol 103 ± 9 102 ± 6 5/4 94 ± 10 89 ± 12 5/7 70 ± 15 78 ± 9 14/10 0.2/0.2/0.1 0.5/0.7/0.4 Formononetin 105 ± 9 102 ± 7 7/3 90 ± 4 92 ± 5 3/2 87 ± 4 89 ± 3 7/8 0.05/0.015/0.02 0.15/0.050/0.06 Genistein 100 ± 21 94 ± 10 9/10 79 ± 17 88 ± 12 12/14 85 ± 13 84 ± 12 13/7 0.2/0.15/0.09 0.5/0.60/0.30 Glycitein 103 ± 13 105 ± 6 13/1 90 ± 12 94 ± 4 8/4 91 ± 13 92 ± 6 9/8 0.2/0.15/0.01 0.5/0.6/0.03 Isoxanthohumol 112 ± 20 102 ± 7 13/2 98 ± 9 94 ± 10 8/3 94 ± 12 83 ± 8 11/8 0.005/0.015/0.01 0.015/0.050/0.03 Matairesinol 102 ± 10 103 ± 9 13/6 86 ± 13 88 ± 10 22/5 90 ± 15 93 ± 5 15/6 1.0/0.6/0.750.009 3.0/2.0.030/2.5 0 Resveratrol 99 ± 22 107 ± 4 19/6 126 ± 21 81 ± 36 33/20 n.d.i 11 ± 80 n.d./63 2.0/4.8/7.5 3.0/16.0/25.0 Mycoestrogens and metabolites

Alternariol 101 ± 12 100 ± 5 13/7 88 ± 10 85 ± 8 6/4 77 ± 19 93 ± 3 20/8 0.5/0.6/0.35 1.5/2.0/1.20 Alternariol monomethyl ether 107 ± 10 105 ± 4 9/2 97 ± 12 96 ± 10 8/7 72 ± 21 78 ± 20 17/7 0.05/0.05/0.25 0.15/0.15/0.8 α-zearalanol (ZAL) 106 ± 8 100 ± 6 5/2 92 ± 8 94 ± 10 11/3 71 ± 13 80 ± 12 6/12 0.25/0.2/0.35 0.70/0.7/1.2 β-ZAL 108 ± 9 102 ± 7 6/2 95 ± 12 92 ± 7 9/3 73 ± 7 79 ± 4 10/9 0.25/0.2/0.2 0.70/0.6/0.6 α-zearalenol (ZEL) 105 ± 11 104 ± 7 9/3 95 ± 7 90 ± 10 4/3 70 ± 29 80 ± 20 6/10 0.3/0.3/1.0 0.8/1.0/3.0 β-ZEL 98 ± 16 104 ± 11 14/4 91 ± 17 93 ± 7 14/7 76 ± 17 79 ± 10 8/12 1.0/0.55/0.55 2.5/1.80/1.80 α-ZEL-14-glucuronide 136 ± 32 103 ± 6 8/6 92 ± 13 88 ± 7 7/10 77 ± 14 73 ± 15 37/22 12/2.7/0.2 40/9.0/0.6 (GlcA) β-ZEL-14-GlcA n.d.i 98 ± 12 n.d./6 90 ± 16 90 ± 5 11/5 79 ± 29 77 ± 15 35/18 45/2.4/0.3 140/8.0/1.0 Zearalanone 107 ± 9 102 ± 7 8/3 88 ± 9 87 ± 11 8/4 64 ± 31 82 ± 12 25/11 0.15/0.25/0.6 0.5/0.8/2.0 Zearalenone (ZEN) 98 ± 11 98 ± 11 11/7 90 ± 9 90 ± 7 14/7 109 ± 30 117 ± 28 34/26 0.15/0.25/0.5 0.5/0.8/1.5 ZEN-14-GlcA n.d. 101 ± 12 n.d./7 95 ± 19 90 ± 10 6/11 86 ± 22 87 ± 4 16/8 40/2.4/0.25 120/8.0/0.8 ZEN-14-sulfate 101 ± 7 100 ± 5 5/2 93 ± 4 92 ± 5 3/4 36 ± 67 38 ± 74 37/44 0.4/0.1/0.15 1.2/0.3/0.45 Personal care product ingredients, pharmaceuticals and metabolites Benzophenone 1 110 ± 11 101 ± 5 14/5 90 ± 7 92 ± 3 5/6 84 ± 15 89 ± 6 27/7 0.05/0.1/0.05 0.10/0.2/0.15 Benzophenone 2 100 ± 11 100 ± 6 6/3 91 ± 14 96 ± 16 9/6 87 ± 20 92 ± 4 10/6 0.1/0.1/0.06 0.3/0.3/0.2 Benzylparaben 94 ± 12 99 ± 5 21/4 89 ± 11 93 ± 14 16/6 55 ± 36 87 ± 13 29/13 0.05/0.03/0.08 0.10/0.09/0.25 Butylparaben 95 ± 5 94 ± 2 7/2 91 ± 6 91 ± 3 5/4 82 ± 13 86 ± 6 21/13 0.05/0.03/0.06 0.15/0.10/0.20 Ethylparaben 97 ± 13 97 ± 7 11/7 94 ± 9 93 ± 3 10/12 82 ± 12 82 ± 8 9/11 0.05/0.05/0.02 0.15/0.15/0.08 Isobutylparaben 97 ± 7 96 ± 2 5/3 90 ± 7 93 ± 10 8/3 83 ± 7 85 ± 6 9/8 0.05/0.03/0.05 0.15/0.10/0.20 Methylparaben 95 ± 9 99 ± 3 8/3 93 ± 5 92 ± 6 8/2 78 ± 12 77 ± 11 7/7 0.3/0.1/0.1 1.0/0.4/0.3 Propylparaben 96 ± 8 97 ± 4 6/3 88 ± 7 94 ± 5 10/5 82 ± 11 84 ± 4 4/4 0.06/0.06/0.06 0.3/0.2/0.2 Ethinylestradiol 101 ± 13 100 ± 4 8/3 90 ± 25 87 ± 19 6/7 79 ± 17 82 ± 9 20/6 0.5/0.6/1.7 1.5/2.0/5.9 Plastizicer/ plastic components and metabolites Bisphenol A (BPA) 102 ± 12 100 ± 6 12/2 95 ± 13 91 ± 18 9/4 86 ± 16 90 ± 4 11/9 0.2/0.6/0.3 0.7/2.0/0.9 BPAF 108 ± 12 107 ± 5 11/4 96 ± 19 87 ± 26 10/8 76 ± 9 79 ± 13 8/6 0.5/0.3/0.45 0.5/1.1/1.5 BPB 99 ± 7 102 ± 3 3/4 90 ± 12 94 ± 22 14/5 84 ± 9 90 ± 6 14/9 0.2/0.3/0.3 0.5/0.9/0.9 BPC 104 ± 13 101 ± 6 7/3 103 ± 16 90 ± 24 9/7 79 ± 19 85 ± 11 9/13 0.5/1.5/1.2 1.5/5.0/4.0 BPF 104 ± 9 100 ± 5 12/3 92 ± 15 90 ± 17 17/11 91 ± 20 90 ± 8 18/6 0.5/1.1/0.4 1.5/3.5/1.3 BPS 97 ± 20 102 ± 5 10/6 87 ± 14 92 ± 5 13/5 87 ± 10 91 ± 4 15/6 0.05/0.03/0.01 0.15/0.10/0.04 Mono-n-butylphthalate 93 ± 8 94 ± 5 9/5 158 ± 13 165 ± 16 9/9 85 ± 13 97 ± 8 12/10 0.8/0.3/0.4 2.2/1.0/1.3 Mono-2-ethylhexyl 96 ± 9 97 ± 5 7/6 180 ± 33 96 ± 10 17/5 56 ± 58 64 ± 33 44/13 1.5/n.d./12 4.0/n.d./40 phthalate N-butylbenzenesulfon- 102 ± 7 97 ± 3 6/3 98 ± 10 92 ± 5 11/4 82 ± 16 90 ± 4 7/7 0.8/1.1/1.0 2.2/3.7/3.5 amide Perfluorinated alkylated substances Perfluoroctanoic acid 101 ± 9 96 ± 6 5/2 91 ± 9 93 ± 4 8/2 12 ± 7 11 ± 6 7/2 0.1/0.09/0.06 0.3/0.30/0.20 Perfluoroctanesulfonic acid 84 ± 9 92 ± 7 6/5 84 ± 22 97 ± 15 18/11 80 ± 15 84 ± 16 20/12 2.5/2.4/1.4 2.5/8.0/4.6 Industrial side products and pesticides 2-Naphthol 85 ± 9 87 ± 4 6/2 94 ± 11 92 ± 16 6/8 74 ± 17 81 ± 8 28/12 0.25/0.1/0.7 0.80/1.4/2.4 Methiocarb 102 ± 9 96 ± 8 5/5 62 ± 11 51 ± 16 15/2 73 ± 18 82 ± 10 10/9 0.8/3.6/2.7 2.5/12.0/9.0 Prochloraz 103 ± 10 99 ± 6 17/3 92 ± 17 79 ± 12 18/12 71 ± 48 98 ± 73 146/36 0.01/0.01/0.1 0.03/0.03/0.3 Endogenous estrogens Estrone (E1) 101 ± 7 102 ± 5 6/3 91 ± 10 86 ± 14 9/5 82 ± 8 85 ± 9 9/11 0.1/0.15/0.2 0.3/0.50/0.6 Estradiol (E2) 105 ± 11 104 ± 9 11/6 90 ± 23 86 ± 14 18/11 92 ± 24 84 ± 12 27/14 0.5/1.0/1.4 1.5/3.2/4.6 E2-17-GlcA n.d. 111 ± 11 n.d./47 88 ± 12 87 ± 4 14/3 81 ± 19 90 ± 6 19/11 60/4.2/1.2 200/14.0/3.5 E2-3-sulfate 106 ± 16 103 ± 7 20/3 89 ± 2 90 ± 5 3/3 49 ± 24 50 ± 44 13/6 1.5/0.2/0.1 5.0/0.5/0.3 Estriol (E3) 90 ± 14 103 ± 7 13/6 94 ± 14 92 ± 9 11/7 82 ± 19 77 ± 10 19/8 1.0/0.3/0.9 3.0/1.0/3.0 16-Epiestriol 99 ± 11 101 ± 8 11/4 95 ± 8 88 ± 8 8/6 48 ± 50 48 ± 49 15/30 0.8/0.9/1.4 2.4/3.0/4.8 16-α-HydroxyE1 107 ± 8 99 ± 5 12/3 82 ± 13 83 ± 8 10/6 86 ± 14 85 ± 6 23/10 0.15/0.2/0.1 0.50/0.6/0.3 17-Epiestriol 102 ± 12 98 ± 7 10/4 95 ± 6 88 ± 9 11/7 54 ± 30 57 ± 54 11/46 1.0/0.9/1.1 3.5/2.8/3.8 2-MethoxyE1 99 ± 6 99 ± 5 6/2 91 ± 7 86 ± 11 6/1 74 ± 12 83 ± 9 9/8 0.2/0.3/0.6 0.7/0.9/1.9 2-MethoxyE2 103 ± 5 99 ± 7 6/5 86 ± 8 87 ± 12 9/5 63 ± 14 76 ± 14 12/13 0.3/0.4/0.6 1.0/1.2/2.0 4-MethoxyE1 99 ± 6 100 ± 4 5/5 90 ± 11 87 ± 15 5/8 76 ± 11 84 ± 9 3/10 0.05/0.05/0.10 0.15/0.15/0.30 4-MethoxyE2 101 ± 8 98 ± 4 6/1 91 ± 6 90 ± 12 4/5 75 ± 13 80 ± 12 14/10 0.15/0.1/0.2 0.50/0.3/0.6 a Extraction efficiency. b Intermediate precision. c Low spiking level. d High spiking level. e Repeatability. f Limit of detection. g Values in the following order: urine/serum/breast milk. h Limit of quantification. i Not determined due to either insufficient ionization or extraction recovery, too high background noise or invalid calibration caused by disturbing blank matrix contamination

4

Figure 1 Extraction efficiency (RE) and matrix effect (SSE) distribution of all 75 tested analytes in urine, serum and breast milk. Our LC-MS/MS system appeared to be heavily contaminated 0.05 µg/L (4-methoxy estrone) in urine and serum and 0.1 with dibutylphthalate and nonylphenol, which occurred in µg/L (16-α-hydroxy estrone and 4-methoxy estrone) in milk to every sample including the solvent blanks. One previous study 1.5 µg/L (E2-3-sulfate) in urine, 4.2 µg/L (E2-17-glucuronide) also reported background contamination with 4-nonylphenol in serum and 1.4 µg/L (E2) in milk. Levels of estradiol, the due to laboratory air contamination.39 We also observed a most prevalent endogenous estrogen, reach a maximum con- minimal system contamination of bisphenol AF. 4- centration around 0.4 µg/L in serum of ovulating premenopau- hydroxyestradiol exhibited carry over after highly concentrat- sal women,40 which is below our LOD value (0.5 µg/L, 1 µg/L ed standards across several injections. All successfully vali- and 1.1 µ/L in urine, serum and breast milk). Estrone (maxi- 2 41 dated compounds displayed a R value higher than 0.98. REs of mum values ca. 0.2 µg/L in serum) might be detectable. successfully validated analytes ranged from 94% (PFOS) to While rather sensitive for a non-tailor made method without 110% (benzophenone 1) in urine, 76% (8-prenylnaringenin) to chemical derivatization, the method may not be feasible for 103% (bisphenol C) in serum and 71% (α-zearalanone) to 97% the determination of endogenous estrogens in women not (MBP) in breast milk. pregnant or without any medical condition. However, in preg- In urine, the SSEs of the successfully validated analytes nant women, estradiol reach concentrations above 30 µg/L and estriol spikes about 15 µg/L, both clearly above the respective ranged from 66% (bisphenol S) to 263% (MBP). In serum and 41-42 breast milk the SSE of validated analytes ranged from 57% LOQ values. We also expect higher concentrations of (BPA) and 31% (2-methoxyestrone) to 145% and 144% (Es- endogenous estrogen metabolites in human gestation. The tradiol-17-glucuronide), respectively. SSE values are reported primary purpose of the newly developed method will be the in Table S4. Interestingly, 51% of all analytes in milk (com- investigation of the critical exposure windows during preg- pared to 7% in urine and 12 % in serum) experienced strong nancy and breast feeding, as these early- life exposures are likely to be of special relevance in the etiology of chronic signal suppression with an SSE <50% (Figure 1). Whereas, 13 17% and 16% of all analytes in urine and milk (7% in serum) disease later in life. The majority of all other included and experienced signal enhancement with and SSE above 110%. successfully validated compounds yielded LOD values below 1 µg/L which were generally deemed fit-for-purpose for the All analytes with R , RSD and RSD values which did not E R r first of its kind multi-analyte method. meet the criteria of the Commission Decision (EC) No. 657/200230 were regarded as not successfully validated (see Application to biological samples. For proof-of-principle Table 1, Table S3 and Table S5). In some cases (e.g. experiments in urine, six samples from three individuals were α/β-zearalenol-14-glucuronide in urine), the lowest spiking analyzed. Four samples resembled a mother-infant pair. We level was below the LOQ value and could not be properly did not detect any contamination source due to sample prepa- ration (clear system blank). The following compounds have evaluated in all three matrices. Some RE values slightly ex- ceeded the validation criteria in one spiking level (e.g. daidze- been detected in at least one urine sample: 2-naphthol, alter- in in urine and β-ZAL in breast milk). However, for these nariol-monomethylether, benzophenone 1 and 2, BPA, BPS analyte/matrix combinations at least a semi quantitative inter- methyl-, ethyl-, propyl-, butyl-, and isobutylparaben, daidzein, pretation is feasible and constitutes a valuable tool for com- enterodiol, enterolactone, equol, formononetin, genistein, bined exposure assessment. glyctein, isoxanthohumol, pHBA, MBP and MEHP. Concen- trations are reported in Table S6. Interestingly, adult females Although the ionization efficiency of endogenous estrogens showed a lower contamination of parabens, with an averaged is very weak and derivatization is typically required,27, 38 we sum of 2.6 µg/L than the two infant samples with an averaged yielded acceptable LODs through the addition of ammonium sum of 36.8 µg/L in urine. Parabens are metabolized in the fluoride to the aqueous mobile phase. LOD values of the suc- 36 liver via esterase hydrolysis and glucuronidation. cessfully validated endogenous estrogens ranged from 5

Figure 2 MRM-chromatograms demonstrating combined xenoestrogen exposure in infant urine (a, b) and breast milk (c) compared to matrix matched calibration standards. Numbers represent intensity (a, c) Methylparaben (MP) m/z 151.0 → 91.9, urine: 23.4 µg/L, breast milk: 28.3 µg/L; isotopic labeled MP (IS MP) m/z 157.0 → 97.9; ethylparaben (EP) m/z 165.0 → 91.9, urine: 5.5 µg/L, breast milk: 4.9 µg/L; IS EP m/z 171.0 → 97.9; propylparaben (PP) m/z 179.0 → 91.9, urine: 11.7 µg/L, breast milk: 2.8 µg/L; IS PP m/z 185.0 → 97.9, isobutylparaben (iBP) and bu- tylparaben (BP) m/z 193.0 → 92.0, urine: 1.1 µg/L and 2.8 µg/L, breast milk: n.d. and 1.7 µg/L; IS BP m/z 199.0 → 98.0 (b) Mono-n-butylphthalate (MBP) m/z 221.0 → 77.0, 24.9 µg/L; IS MPB m/z 225.0 → 79.0, benzophenone 2 m/z 244.9 → 134.9, 0.6 µg/L; 2-naphthol m/z 143.0 → 115.0, 1.5 µg/L; mono-2-ethylhexyl phthalate (MEHP) m/z 277.1 → 133.9, >200 µg/L; IS MEHP m/z 281.1 → 136.9, bisphenol A (BPA) m/z 227.0 → 132.9, 1.6 µg/L; IS BPA m/z 239.0 → 224.0 6

Infants have a lower plasma protein concentration and a re- est since xenoestrogens often appear at concentrations in duced metabolic performance resulting in a limited capacity of which a single compound itself has no significant toxicologi- drug detoxification via protein binding or glucuronidation cal effect, however, exposure to a mixture of several low-dose (reviewed by Lu and Rosenbaum (2014)43). Therefore, it is of xenoestrogens may lead to adverse outcomes as demonstrated particular importance to monitor and control environmental in recent in vitro and in vivo studies.14-17 The analysis of a exposures of un- and newborn babies. In this study, we did not limited number of real-world samples indicated that different include a de-conjugation step since we intentionally assessed classes of xenoestrogens co-occur and can be found in one sulfates and glucuronides (e.g. estradiol-3-sulfate or zeara- sample of urine or breast milk. These findings clearly demon- lenone-14-glucuronide) directly. In future studies, de- strate the need for large-scale epidemiological studies investi- conjugation of samples will be considered and likely result in gating potential correlations between exposure to xenoestro- an even higher number of positive samples. gens and health effects including the development of uterine Interestingly, we did not detect any phytoestrogens in infant or breast cancer. Further possible applications include the urine although mothers’ urine contained reasonable concentra- investigation of placental transfer and metabolism of estrogen- tions (averaged sum 62.4 µg/L). This leads to the assumption ic compounds as well as correlation studies between total that the phytoestrogen uptake of infants via breast milk is xenoestrogen exposure and early-onset puberty or infertility. moderate to low (detection of enterolactone, genistein and glycitein below LOQ in breast milk). Phytoestrogen concen- ASSOCIATED CONTENT trations are strongly dependent on the diet and can vary signif- Supporting Information icantly even in a particular individual.44 Our findings of per- Detailed description of the sample preparation protocol. Addi- sonal care product additives and bisphenols are in line with tional tables and figures, including chemicals and reagents with previous reports in urine, although some of our samples con- structures, LC-MS/MS parameters, the validation summary and a tained slightly higher amounts of methylparaben, benzophe- table of all quantitative measurements in the biological samples none-2 and bisphenol S.24, 26 One infant urine sample exhibited high concentrations of MEHP (>200 µg/L) and MBP AUTHOR INFORMATION (24.9 µg/L). MEHP and MBP are metabolites of di-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate, respectively45-46 Corresponding Author and it has been reported in a previous study that children typi- * Benedikt Warth: tel, +431427770806; e-mail, cally have a higher DEHP intake than adults.47 [email protected]. Figure 2 shows chromatograms of naturally contaminated urine and breast milk samples. To demonstrate the biological ACKNOWLEDGMENT feasibility of breast milk analysis, five Nigerian samples and We greatly acknowledge all participants of the studies in Nigeria four breast milk samples from two Austrian mothers were and Austria as well as Prof. Chibundu N. Ezekiel and the Sem- analyzed. The following compounds have been detected in at melweis Women Clinic in Vienna, Austria for providing breast least one breast milk sample: 2-naphthol, benzophenone 2, milk samples. We extend our thanks to Hannes Puntscher for BPS, methyl-, ethyl-, propyl-, and butylparaben, enterolac- technical support and helpful discussions. We also want to thank the staff of the Mass Spectrometry Centre (MSC) of the Universi- tone, genistein, glycitein, MBP, MEHP, PFOA and pHBA. ty of Vienna, where LC-MS/MS measurements have been per- BPA was detected in trace amounts in all breast milk samples, formed. This work was financed by the University of Vienna. but also in the system blank which leads to the assumption, that the samples were possibly contaminated with BPA during REFERENCES sample preparation. Consistently with our findings, parabens, 1. Commission, E., Towards a more comprehensive EU framework on phthalate metabolites, some phytoestrogens and PFOA have endocrine disruptors. European Commission: 2018; Vol. 2018. 24, 48-51 been identified in breast milk before. Interestingly, a 2. Singleton, D. W.; Khan, S. A., Xenoestrogen exposure and mechanisms previous study found more BPF, which was not detected at all of endocrine disruption. Frontiers in bioscience : a journal and virtual library 2003, 52 8, s110-8. in our samples, than BPS in breast milk. Nigerian samples 3. Paterni, I.; Granchi, C.; Minutolo, F., Risks and benefits related to did not contain parabens but methylparaben. alimentary exposure to xenoestrogens. Critical Reviews in Food Science and Nutrition 2017, 57 (16), 3384-3404. Every single biological sample contained 2-naphthol, which is 4. Rochester, J. R.; Bolden, A. L., Bisphenol S and F: A systematic review a metabolite of the carcinogenic naphthalene.53-54 The mean and comparison of the hormonal activity of bisphenol a substitutes. Environmental concentration in urine was 1.9 µg/L which is slightly lower Health Perspectives 2015, 123 (7), 643-650. 55 5. Mínguez-Alarcón, L.; Messerlian, C.; Bellavia, A., et al., Urinary than in a previous study of Korean individuals. In breast concentrations of bisphenol A, parabens and phthalate metabolite mixtures in milk, a mean of 7.7 µg/L was detected in our samples. To- relation to reproductive success among women undergoing in vitro fertilization. Environment International 2019, 126, 355-362. gether with 1-naphthol, 2-naphthol is usually used to estimate 6. Orzylowska, E. M.; Jacobson, J. D.; Bareh, G. M., et al., Food intake 53 polycyclic aromatic hydrocarbon exposure. However, it has diet and sperm characteristics in a blue zone: a Loma Linda Study. European to be considered that 2-naphthol showed only a low- Journal of Obstetrics & Gynecology and Reproductive Biology 2016, 203, 112-115. 7. Chavarro, J. E.; Toth, T. L.; Hauser, R., et al., Soy food and isoflavone abundance qualifier fragment due to its low molecular mass intake in relation to semen quality parameters among men from an infertility clinic. (2% of quantifier intensity). Therefore, not in all measured Human Reproduction 2008, 23 (11), 2584-2590. biological samples qualifier ions were detected. 8. Xia, Y.; Chen, M.; Zhu, P., et al., Urinary phytoestrogen levels related to idiopathic male infertility in Chinese men. Environ Int 2013, 59, 161-7. CONCLUSION AND OUTLOOK 9. Beaton, L. K.; McVeigh, B. L.; Dillingham, B. L., et al., Soy protein isolates of varying isoflavone content do not adversely affect semen quality in In this study a novel method for the identification of endog- healthy young men. Fertility and Sterility 2010, 94 (5), 1717-1722. enous and exogenous estrogens in the three human matrices 10. Gaskins, A. J.; Chavarro, J. E., Diet and fertility: a review. American journal of obstetrics and gynecology 2018, 218 (4), 379-389. urine, serum and breast milk was developed. For the first time, 11. Fernandez, S. V.; Russo, J., Estrogen and Xenoestrogens in Breast it is now possible to analyze these classes of estrogenic com- Cancer. Toxicologic pathology 2010, 38 (1), 110-122. pounds simultaneously, and thereby estimate the total estro- 12. Fucic, A.; Gamulin, M.; Ferencic, Z., et al., Environmental exposure to xenoestrogens and oestrogen related cancers: reproductive system, breast, lung, genic burden at an individual level. This is of particular inter- kidney, pancreas, and brain. Environmental Health 2012, 11 (Suppl 1), S8-S8. 7

13. Palmlund, I., Exposure to a xenoestrogen before birth: the Regioselective Attachment, Regiospecific Decompositions, Charge-Induced diethylstilbestrol experience. Journal of psychosomatic obstetrics and gynaecology Pathways, and Ion–Dipole Complex Intermediates. Journal of The American Society 1996, 17 (2), 71-84. for Mass Spectrometry 2012, 23 (9), 1558-1568. 14. Vejdovszky, K.; Schmidt, V.; Warth, B., et al., Combinatory estrogenic 35. Wang, W.; Cole, R. B., Enhanced collision-induced decomposition effects between the isoflavone genistein and the mycotoxins zearalenone and efficiency and unraveling of fragmentation pathways for anionic adducts of alternariol in vitro. Molecular nutrition & food research 2017, 61 (3). brevetoxins in negative ion electrospray mass spectrometry. Anal Chem 2009, 81 15. You, L.; Casanova, M.; Bartolucci, E. J., et al., Combined effects of (21), 8826-38. dietary phytoestrogen and synthetic endocrine-active compound on reproductive 36. Abbas, S.; Greige-Gerges, H.; Karam, N., et al., Metabolism of development in Sprague-Dawley rats: genistein and methoxychlor. Toxicological Parabens (4-Hydroxybenzoic Acid Esters) by Hepatic Esterases and UDP- sciences : an official journal of the Society of Toxicology 2002, 66 (1), 91-104. Glucuronosyltransferases in Man. Drug Metabolism and Pharmacokinetics 2010, 25 16. Rajapakse, N.; Silva, E.; Kortenkamp, A., Combining xenoestrogens at (6), 568-577. levels below individual no-observed-effect concentrations dramatically enhances 37. Wei, N.; Zheng, Z.; Wang, Y., et al., Rapid and sensitive determination steroid hormone action. Environ Health Perspect 2002, 110 (9), 917-21. of multiple endocrine-disrupting chemicals by ultrasound-assisted in situ 17. Vejdovszky, K.; Hahn, K.; Braun, D., et al., Synergistic estrogenic derivatization dispersive liquid–liquid microextraction coupled with ultra-high- effects of Fusarium and Alternaria mycotoxins in vitro. Archives of Toxicology 2017, performance liquid chromatography/tandem mass spectrometry. Rapid 91 (3), 1447-1460. Communications in Mass Spectrometry 2017, 31 (11), 937-950. 18. Aichinger, G.; Beisl, J.; Marko, D., The Hop Polyphenols Xanthohumol 38. Lee, C.; Kim, C. H.; Kim, S., et al., Simultaneous determination of and 8-Prenyl-Naringenin Antagonize the Estrogenic Effects of Fusarium Mycotoxins bisphenol A and estrogens in hair samples by liquid chromatography-electrospray in Human Endometrial Cancer Cells. Frontiers in nutrition 2018, 5, 85. tandem mass spectrometry. Journal of chromatography. B, Analytical technologies 19. Warth, B.; Spangler, S.; Fang, M., et al., Exposome-Scale Investigations in the biomedical and life sciences 2017, 1058, 8-13. Guided by Global Metabolomics, Pathway Analysis, and Cognitive Computing. Anal 39. Asimakopoulos, A. G.; Thomaidis, N. S., Bisphenol A, 4-t-octylphenol, Chem 2017, 89 (21), 11505-11513. and 4-nonylphenol determination in serum by Hybrid Solid Phase Extraction– 20. Niedzwiecki, M. M.; Walker, D. I.; Vermeulen, R., et al., The Precipitation Technology technique tailored to liquid chromatography–tandem mass Exposome: Molecules to Populations. Annual Review of Pharmacology and spectrometry. Journal of Chromatography B 2015, 986-987, 85-93. Toxicology 2019, 59 (1), 107-127. 40. Stricker, R.; Eberhart, R.; Chevailler, M. C., et al., Establishment of 21. Prasain, J. K.; Arabshahi, A.; Moore, D. R., et al., Simultaneous detailed reference values for luteinizing hormone, follicle stimulating hormone, determination of 11 phytoestrogens in human serum using a 2min liquid estradiol, and progesterone during different phases of the menstrual cycle on the chromatography/tandem mass spectrometry method. Journal of Chromatography B Abbott ARCHITECT analyzer. Clinical chemistry and laboratory medicine 2006, 44 2010, 878 (13), 994-1002. (7), 883-7. 22. Yu, J.; Wu, Q.; Qiao, S., et al., Simultaneous determination of 41. Estriol reference values. phytoestrogens and key metabolites in breast cancer patients’ urine by liquid https://www.questdiagnostics.com/testcenter/TestDetail.action?ntc=34883 (accessed chromatography–tandem mass spectrometry. Journal of Pharmaceutical and 19.03.2019). Biomedical Analysis 2009, 50 (5), 939-946. 42. Estradiol reference values. 23. Rocha, B. A.; da Costa, B. R.; de Albuquerque, N. C., et al., A fast https://www.healthcare.uiowa.edu/path_handbook/rhandbook/test748.html (accessed method for bisphenol A and six analogues (S, F, Z, P, AF, AP) determination in 19.03.2019). urine samples based on dispersive liquid-liquid microextraction and liquid 43. Lu, H.; Rosenbaum, S., Developmental pharmacokinetics in pediatric chromatography-tandem mass spectrometry. Talanta 2016, 154, 511-9. populations. The journal of pediatric pharmacology and therapeutics : JPPT : the 24. Azzouz, A.; Rascón, A. J.; Ballesteros, E., Simultaneous determination official journal of PPAG 2014, 19 (4), 262-276. of parabens, alkylphenols, phenylphenols, bisphenol A and triclosan in human urine, 44. Chávez-Suárez, K. M.; Ortega-Vélez, M. I.; Valenzuela-Quintanar, A. blood and breast milk by continuous solid-phase extraction and gas I., et al., Phytoestrogen Concentrations in Human Urine as Biomarkers for Dietary chromatography–mass spectrometry. Journal of Pharmaceutical and Biomedical Phytoestrogen Intake in Mexican Women. Nutrients 2017, 9 (10), 1078. Analysis 2016, 119, 16-26. 45. Koch, H. M.; Bolt, H. M.; Angerer, J., Di(2-ethylhexyl)phthalate 25. Heffernan, A. L.; Thompson, K.; Eaglesham, G., et al., Rapid, (DEHP) metabolites in human urine and serum after a single oral dose of deuterium- automated online SPE-LC-QTRAP-MS/MS method for the simultaneous analysis of labelled DEHP. Arch Toxicol 2004, 78 (3), 123-30. 14 phthalate metabolites and 5 bisphenol analogues in human urine. Talanta 2016, 46. Koch, H. M.; Christensen, K. L.; Harth, V., et al., Di-n-butyl phthalate 151, 224-33. (DnBP) and diisobutyl phthalate (DiBP) metabolism in a human volunteer after 26. Vela-Soria, F.; Ballesteros, O.; Zafra-Gómez, A., et al., A multiclass single oral doses. Arch Toxicol 2012, 86 (12), 1829-39. method for the analysis of endocrine disrupting chemicals in human urine samples. 47. Koch, H. M.; Preuss, R.; Angerer, J., Di(2-ethylhexyl)phthalate Sample treatment by dispersive liquid–liquid microextraction. Talanta 2014, 129, (DEHP): human metabolism and internal exposure-- an update and latest results. 209-218. International journal of andrology 2006, 29 (1), 155-65; discussion 181-5. 27. Kolatorova Sosvorova, L.; Chlupacova, T.; Vitku, J., et al., 48. Franke, A. A.; Halm, B. M.; Custer, L. J., et al., Isoflavones in breastfed Determination of selected bisphenols, parabens and estrogens in human plasma using infants after mothers consume soy. The American journal of clinical nutrition 2006, LC-MS/MS. Talanta 2017, 174, 21-28. 84 (2), 406-13. 28. Rocha, B. A.; de Oliveira, A. R. M.; Barbosa, F., A fast and simple air- 49. Franke, A. A.; Custer, L. J.; Tanaka, Y., Isoflavones in human breast assisted liquid-liquid microextraction procedure for the simultaneous determination milk and other biological fluids. The American journal of clinical nutrition 1998, 68 of bisphenols, parabens, benzophenones, triclosan, and triclocarban in human urine (6 Suppl), 1466s-1473s. by liquid chromatography-tandem mass spectrometry. Talanta 2018, 183, 94-101. 50. Völkel, W.; Genzel-Boroviczény, O.; Demmelmair, H., et al., 29. Braun, D.; Ezekiel, C. N.; Abia, W. A., et al., Monitoring Early Life Perfluorooctane sulphonate (PFOS) and perfluorooctanoic acid (PFOA) in human Mycotoxin Exposures via LC-MS/MS Breast Milk Analysis. Analytical Chemistry breast milk: Results of a pilot study. International Journal of Hygiene and 2018, 90 (24), 14569-14577. Environmental Health 2008, 211 (3), 440-446. 30. Commission, E., 2002/657/EC: Commission Decision of 12 August 51. Hartle, J. C.; Cohen, R. S.; Sakamoto, P., et al., Chemical Contaminants 2002 implementing Council Directive 96/23/EC concerning the performance of in Raw and Pasteurized Human Milk. Journal of Human Lactation 2018, 34 (2), analytical methods and the interpretation of results. European Commission: Vol. OJ 340-349. L 221, pp 8–36. 52. Dualde, P.; Pardo, O.; F. Fernández, S., et al., Determination of four 31. Warth, B.; Spangler, S.; Fang, M., et al., Exposome-Scale Investigations parabens and bisphenols A, F and S in human breast milk using QuEChERS and Guided by Global Metabolomics, Pathway Analysis, and Cognitive Computing. liquid chromatography coupled to mass spectrometry. Journal of Chromatography B Analytical Chemistry 2017, 89 (21), 11505-11513. 2019. 32. Fiers, T.; Casetta, B.; Bernaert, B., et al., Development of a highly 53. Preuss, R.; Angerer, J.; Drexler, H., Naphthalene—an environmental sensitive method for the quantification of estrone and estradiol in serum by liquid and occupational toxicant. International Archives of Occupational and chromatography tandem mass spectrometry without derivatization. Journal of Environmental Health 2003, 76 (8), 556-576. Chromatography B 2012, 893-894, 57-62. 54. Services, N. T. P. U. S. D. o. H. a. H., Toxicology and carcinogenesis 33. Owen, L. J.; Wu, F. C.; Keevil, B. G., A rapid direct assay for the studies of naphthalene (cas no. 91-20-3) in F344/N rats (inhalation studies). National routine measurement of oestradiol and oestrone by liquid chromatography tandem Toxicology Program technical report series 2000, (500), 1-173. mass spectrometry. Annals of Clinical Biochemistry 2014, 51 (3), 360-367. 55. Sul, D.; Ahn, R.; Im, H., et al., Korea National Survey for 34. Rannulu, N. S.; Cole, R. B., Novel Fragmentation Pathways of Anionic Environmental Pollutants in the human body 2008: 1-hydroxypyrene, 2-naphthol, Adducts of Steroids Formed by Electrospray Anion Attachment Involving and cotinine in urine of the Korean population. Environ Res 2012, 118, 25-30.

8

Supporting Information

Tables and Figures Tables

Table S1 Information on suppliers and additional data for used chemicals and reagents...... 11 Table S2 LCMS parameter. For some analytes retention time varied between matrices and batches. Ion ratio is given for first stated qualifier ion...... 13

Table S3 Extraction efficiencies (RE), intermediate precision (RSDR) and reparability (RSDr) of analytes which have not been successfully validated in urine, serum and breast milk. 4-tert-ctylphenol was evaluated with internal standard calibration in serum...... 15 Table S4 Signal suppression or enhancment (SSE), spiking concentrations, regression coeffiecients and linear range ..... 16 Table S5 Overview of validation outcome inclusive listing of parameters out of validation limits...... 23 Table S6 Concentrations in biological samples [µg/L]...... 24

Figures

Figure S1 Ion chromatogramm of PFOS in a matrix matched calibration standard (urine). blue: quantifier ion (m/z 498.8 → 79.9). red: qualifier ion (m/z 498.8 → 98.8). green: isotopic labeled PFOS (m/z 506.8 → 79.9)...... 10 Figure S2 Chemical structures of phytoestrogens with exact masses...... 18 Figure S3 Chemical structure of industrial side products, personal care product ingredients with one paraben metabolite (p-hydroxybenzoic acid), pesticides and one pharmaceuticals with exact masses...... 19 Figure S4 Chemical structures of endogenous estrogens and perfluorinated alkxlated substances with exact masses. .... 20 Figure S5 Chemical structures of plastic components, plasticizer and two plastizicer metabolites (Mono-n-butyl phthalate and Mono-2-ethylhexyl phthalate) with exact masses...... 21 Figure S6 Chemical structures of mycoestrogens with exact masses...... 22

9

Chemicals and reagents. Please find CAS numbers and supplier information in Table S1. Chemical structures of all analyzed compounds can be found in Figure S2-6. Particular attention should be drawn to the chemical structure of bisphenol C, since differing structures with this name are found in the literature. Nonylphenol was analyzed as a technical mixture as performed in previous studies. 1-3 Individual analytical standards were dissolved in appropriate solvent (ace- tonitrile (ACN), methanol (MeOH) or in rare cases dimethylsulfoxide (DMSO)) to a concentration of 1 or 2 mg/mL. A stock solution containing all analytes was prepared in ACN at 20x the concentration of the highest calibra- tion standard. Individual calibration solutions were prepared freshly from this multi-analyte stock before each experiment. Solid isotopic labeled standards were dissolved in MeOH or ACN. An internal standard (IS) mix was prepared in ACN (20-fold spiking concentration). All solutions were stored at -20 °C. Analytical grade perfluoroctanesulfonic acid (PFOS; Sigma Aldrich, Vien- na, Austria) consists of at least three different isomers, which could be sepa- rated with the prescribed method. Isomers of PFOS were integrated collec- tively, since no detailed breakdown of the structures and concentrations could be obtained from the supplier company. This practice has been conducted before, but it has to be considered that the ionization properties of the isomers Figure S3 Ion chromatogramm of PFOS in a matrix 4-6 matched calibration standard (urine). blue: quantifier ion may be differing. We suppose the last isomer peak resembles the linear (m/z 498.8 → 79.9). red: qualifier ion (m/z 498.8 → 98.8). structure, as it matches the retention time of the internal standard (see Figure green: isotopic labeled PFOS (m/z 506.8 → 79.9). S1). PFOS has been shown to change sex hormone levels in zebrafish.7 PFOA seems to be estrogenic in zebrafish.8 In rodents, PFOA seems to be estrogenic at low doses9 but not at high dosages in vivo.10 Both perfluorinated alkylated substances have been shown to enhance the estrogenic effect of estradiol in vitro.11 There is also some evi- dence that PFOS and PFOA could affect estrogen homeostasis during human pregnancy.12

LC-MS/MS method development. Due to no sufficient ionization during tuning, transitions of triclosan were obtained from litera- ture.13-14

Method validation. The relative recovery (RE), which corresponds to the extraction efficiency was calculated as follows: 푎푚표푢푛푡 푅 [%] = 푑푒푡푒푟푚𝑖푛푒푑 푏푦 푚푎푡푟𝑖푥 푚푎푡푐ℎ푒푑 푐푎푙𝑖푏푟푎푡𝑖표푛 × 100 퐸 푠푝푖푘푒푑 푎푚표푢푛푡

Sample preparation. For urine and serum, aliquots of 200 µL were prepared and 10 µL of internal standard solution added. For ex- traction, 790 µL ACN/MeOH (1/1) were added, followed by a vortexing and sonication (10 min, 4 °C) step. After a freeze out at -20 °C for two hours, the samples were centrifuged (10 min; 18,000 x g; 4 °C). The supernatant was dried down in a CentriVap Vacuum concentrator (Kansas City, MO, Labconco) at 4 °C and resolved in 200 µL ACN/H2O (1/9). Thereafter, the samples were centrifuged (10 min; 18,000 x g; 4 °C) and the supernatant transferred into an amber glass vial containing a glass insert. All steps were performed on ice. For breast milk, a modified approach of Braun, et al. (2018)15 was used. Therefore, 250 µL of breast milk were fortified with 12.5 µL of internal standard solution. A volume of 25 µL dilution solvent (ACN/H2O 1/9) was added. For extraction, 250 µL of acidified ACN (1% formic acid) were added and vortexed thoroughly (3 min). After addition of 100 mg of MgSO4 and 25 mg of NaCl, the samples were vortexed (3 min) and centrifuged (10 min; 18,000 x g; 4 °C). The supernatant was transferred to a new tube and kept at -20 °C for at least two hours. Then, the samples were centrifuged (10 min; 18,000 x g; 4 °C). The supernatant was dried down in a Vacuum concentrator at 4 °C and thereafter resolved in 250 µL ACN/H2O (1/9). After 10 min of centrifugation (18,000 x g; 4 °C), the supernatant was transferred into an amber glass vial containing a glass insert. All steps were performed on ice. Isotopic labeled standards (IS) were spiked at following concentrations: IS bisphenol A, IS genistein, IS Zearalenone, IS parabens: 5 µg/L; IS estradiol: IS mono-2-ethylhexyl phthalate, IS mono-n-butyl phthalate, IS perfluoroctanoic acid, IS PFOS: 10 µg/L; IS 4- tert-octylphenol: 50 µg/L, IS 4-hydroxybenzoic acid: 300 µg/L.

LC-MS/MS measurements. To obtain enough data points one acquisition run was divided into seven segments: segment 1 (6 min), segment 2 (1.2 min), segment 3 (1.5 min), segment 4 (1.3 min), segment 5 (2.0 min), segment 6 (3.4 min), segment 7 (4.6 min). All segments were operated in positive and negative mode with fast polarity switching except segment 3, which was operated in negative mode only. Electrospray settings were set as follows: spray voltage -/+3100 V, vaporizer temperature 340 °C, sheath gas pressure 50 au, ion sweep gas pressure 2.0 au (in pos mode)/ 2.5 au (in neg mode), auxiliary gas pressure 30 au and capillary temperature 315 °C. The H-ESI probe was set to position B. The LC flow was redirected to the waste from min 0.1 to min 0.5 via divert valve.

10

Table S2 Information on suppliers and additional data for used chemicals and reagents.

Compound CAS Catalogue number Supplier Lot number 13 C12-BPA (bisphenol A) 263261-85-0 720186-5.00mg Sigma Aldrich MBBB1923V 13 C18-ZEN (zearalenone) Romer Labs 13 C2-MBP (mono-n-butyl CLM-4590-MT-1.2 Cambridge Isotope Laboratories SDIC-008 phthalate) 13 C2-MEHP (mono-2- CLM-4584-MT-1.2 Cambridge Isotope Laboratories SDHC-027 ethylhexyl phthalate) 13 C3-E2 (estradiol) 1261254-48-1 719552-1mg Sigma Aldrich MBBC4575 13 C6-4tOP (4-tert- 1173020-24-0 33565 Sigma Aldrich BCBW5639 octylphenol) 13 C6-BP (butylparaben) 32125 Sigma Aldrich BCBT7363 13 C6-EP (ethylparaben) 32125 Sigma Aldrich BCBT7363 13 C6-MP (methylparaben) 32125 Sigma Aldrich BCBT7363 13 C6-pHBA (p- 267399-29-5 587869-10.0mg Sigma Aldrich MBBB9644V hydroxybenzoic acid) 13 C6-PP (propylparaben) 32125 Sigma Aldrich BCBT7363 13 C8-PFOA ES-5571 Cambridge Isotope Laboratories PR-28266 (perfluoroctanoic acid) 13 C8-PFOS ES-5571 Cambridge Isotope Laboratories PR-28266 (perfluoroctanesulfonic acid) 16-Epiestriol 547-81-9 E586500 TorontoResearchChemicals 29-AZC-158-2 16-α-Hydroxyestrone 566-76-7 H941900 TorontoResearchChemicals 3 CGF-676 17-Epiestriol 1228-72-4 E586510 TorontoResearchChemicals 20-THT-39-2 2 tert Butylphenol 88-18-6 B99405-50mL Sigma Aldrich SHBD7123V 2-Hydroxyestradiol 362-05-0 H941890 TorontoResearchChemicals 5-SBT 161-4 2-Methoxyestradiol 362-07-2 89242-1mg Sigma Aldrich BCBT7859 2-Methoxyestrone 362-08-3 73532-25mg Sigma Aldrich BCBS2816V 2-Naphthol 135-19-3 185507-5g Sigma Aldrich BCBV0179 3-Benzylidencampher 15087-24-8 91556-25mg Sigma Aldrich BCBT9983 4-Hydroxyestrone 3131-23-5 H941950 TorontoResearchChemicals 5KH 128-1 4-Methoxyestradiol 26788-23-8 M262630 TorontoResearchChemicals 4-YKZ-7-1 4-Methoxyestrone 58562-33-7 M226135 TorontoResearchChemicals 1SRE-175-1 4-Methylbenzyliden 36861-47-9 61547-50mg Sigma Aldrich BCBS7570V campher 4-octylphenol 1806-26-4 384445-1g Sigma Aldrich MKBW6803V 4-tert-octylphenol 140-66-9 442858 Sigma Aldrich LRAB8559 8-Prenylnaringenin 53846-50-7 75119 Sigma Aldrich BCBR2514V α-zearalanol Sigma Aldrich α -zearalenol Sigma Aldrich α -zearalenol-14- synthesized at the Technical University of Vienna and kindly provided by Dr. Mikula 16-17 glucuronide Alternariol 641-38-3 A575760 Sigma Aldrich Alternariol monomethyl 26894-49-5 A575770 TorontoResearchChemicals 10-CGS-13-1 ether Benzophenone 1 131-56-6 126217-100G Sigma Aldrich BCBN3480V Benzophenone 2 131-55-5 T16403-25g Sigma Aldrich MKBN6515V Benzyl butyl phthalate 85-68-7 442503 Sigma Aldrich LC26422V Benzylparaben 94-18-8 07389-100mg Sigma Aldrich BCBR26688V β-zearalanol 42422-68-4 Sigma Aldrich β -zearalenol 71030-11-0 Sigma Aldrich β -zearalenol-14- synthesized at the Technical University of Vienna and kindly provided by Dr. Mikula 16-17 glucuronide Bisphenol A 80-05-07 239658 Sigma Aldrich MKBS0991V Bisphenol AF 1478-61-1 90477-100mg Sigma Aldrich BCBV2156 Bisphenol B 77-40-7 50877-100mg Sigma Aldrich BCBS0967

11

Bisphenol C 79-97-0 68118-100mg Sigma Aldrich BCBV2464 (Dimethyl-bisphenol A) Bisphenol F 620-92-8 51453-100mg Sigma Aldrich BCBT7018 Bisphenol S 80-09-1 43034-100mg Sigma Aldrich BCBV2462 Butylparaben 94-26-8 PHR1022-1g Sigma Aldrich LRAB3701 Coumestrol 479-13-0 BML-S180-0005 Enzo Life sciences 11071708 d4-GEN 187960-08-3 D-6282 CDN Isotopes k-457 Daidzein 486-66-8 D7802 Sigma Aldrich 013M4027V Dibutylphthalate 84-74-2 18281-50mg Sigma Aldrich BCBV9940 E2-17-GlcA 15087-02-2 E1127 Sigma Aldrich 4856 Enterodiol 80226-00-2 45198-1MG-F Sigma Aldrich BCBT3858 Enterolactone 78473-71-9 45199-1mg-F Sigma Aldrich BCBT6193 Equol 94105-90-5 13184 Cayman Chemical Company 0433202-6 Estradiol (E2) 50-28-2 E8875 Sigma Aldrich SLBP6339V Estradiol-3-sulfate 4999-79-5 E9505-25mg Sigma Aldrich 067M4032V Estriol (E3) 50-27-1 E1253 Sigma Aldrich 115H0256 Estrone (E1) 53-16-7 46573-250mg Sigma Aldrich SZBE205XV Ethinylestradiol 57-63-6 E685100 TorontoResearchChemicals 1-TIM-118-1 Ethylparaben 120-47-8 PHR1011-1g Sigma Aldrich LRAB3628 Fenarimol 60168-88-9 45484-250mg Sigma Aldrich BCBW4607 Formononetin 485-72-3 94334-50mg Sigma Aldrich BCBT8620 Genistein (GEN) 446-72-0 1097 Extrasynthese Batch 24 ID 0421/0 Glycitein 40957-83-3 G635400 TorontoResearchChemicals 4-GRS-54-1 Isobutylparaben 4247-02-3 715077-25g Sigma Aldrich 23283 Isoxanthohumol 70872-29-6 1367S Extrasynthese Batch 01 ID: 1020/0 Matairesinol 580-72-3 40043-5MG-F Sigma Aldrich BCBW3093 Methiocarb 2032-65-7 36152-100mg Sigma Aldrich SZBF106XV Methylparaben 99-76-3 47889 Sigma Aldrich LRAB6911 Mono-2-ethylhexyl 4376-20-9 796832-500mg Sigma Aldrich MKCD4270 phthalate Mono-n-butyl phthalate 131-70-4 30751-100mg Sigma Aldrich BCBT4836 N butylbenzolsulfonamid 3622-84-2 B90653-250mL Sigma Aldrich MKCD8065 Nonylphenol 84852-15-3 46018-1g Sigma Aldrich BCBT5112 Octyl methoxycinnamate 5466-77-3 55529-100mg Sigma Aldrich BCBQ5953V Perfluoroctanoic acid 335-67-1 171468-5g Sigma Aldrich MKCC6736 (PFOA) Perfluorooctanesulfonic 1763-23-1 33607 Sigma Aldrich BCBW0899 acid p-Hydrobenzoic acid 99-96-7 H5376 Sigma Aldrich 090K8911 Prochloraz 67747-09-5 45631-250mg Sigma Aldrich BCBW4694 Propylparaben 94-13-3 PHR1010-1G Sigma Aldrich LRAB2246 Resveratrol 501-36-0 R5010 Sigma Aldrich SLBC6832V Tetrabrombisphenol A 79-94-7 11223-100mg Sigma Aldrich BCBW5493 Triclosan 3380-34-5 93453-100mg Sigma Aldrich BCBS1844 Xanthohumol 569-83-5 13496S Extrasynthese Batch03 ID 0524/0 Zearalanone 5975-78-0 Sigma Aldrich Zearalenone 17924-92-4 Sigma Aldrich Zearalenone-14-glucuronide synthesized at the Technical University of Vienna and kindly provided by Dr. Mikula 16-17 Zearalenone-14-sulfate kind gift from Prof. Berthiller from the University of Natural Resources and Life Sciences Vienna 18 Ammonium fluoride 12125-01-8 52481-50g Honeywell Fluka H0790 LCMS grade water 83645.320 VWR chemicals various LCMS grade acetonitrile 75-05-8 34967 Honeywell Riedel-del Haen various LCMS grade methanol 67-56-1 34966 Honeywell Riedel-del Haen various Magnesium sulfate, anhy- 7487-88-9 413485000 Acros organics A0379632 drous Natrium chloride 7647-14-5 9268.1 Roth 406250348 Human serum H4522 Sigma Aldrich SLBW8068

12

Table S3 LCMS parameter. For some analytes retention time varied between matrices and batches. Ion ratio is given for first stated qualifier ion. Compound RT U/S/M Precursor Ion Adduct Quantifier/ Qualifier Ions CE S-Lense IR U/S/M [min] [m/z] [m/z] [V] [%] Endogeneous estrogens Estrone (E1) 10.97 269.1 [M-H]- 145.0/142.9 39/58 100 34/34/34 Estradiol (E2) 9.41 271.1 [M-H]- 183.0/144.9 39/40 82 82/79/80 13 - C3-E2 9.41 274.1 [M-H] 186.0 39 82 E2-17-glucuronide (GlcA) 4.33-4.57/4.27/4.52 447.0 [M-H]- 84.7/271.0 28/30 110 6223/41/40 E2-3-sulfate 5.31/5.01/5.28 351.0 [M-H]- 182.9/271.2 36/35 110 1798/5/5 Estriol (E3) 5.60 287.0 [M-H]- 170.9/144.9 37/45 95 78/69/71 16-Epiestriol 6.59 287.0 [M-H]- 144.9/170.9 45/37 95 92/95/91 16-α-HydroxyE1 6.65 285.0 [M-H]- 145.0/142.9 41/56 95 39/36/36 17-Epiestriol 6.87 287.0 [M-H]- 144.9/170.9 45/37 95 37/37/38 2-MethoxyE1 12.08 299.0 [M-H]- 284.0/283.0 24/38 79 3/2/2 2-MethoxyE2 10.49 301.1 [M-H]- 286.0/285.0/171.0 25/40/47 79 2/2/2 2-HydroxyE2 7.61/7.85/7.60 287.0 [M-H]- 146.9/255.0/128.9/161.0a 43/40/55/44 95 75/81/80 4-MethoxyE1 11.54 299.0 [M-H]- 284.0/283.0 24/36 79 72/70/71 4-MethoxyE2 9.86 301.1 [M-H]- 286.0/285.0/213.0 25/40/52 79 67/65/65 4-HydroxyE1 8.80 285.0 [M-H]- 160.9/158.9 40/55 120 17/19/17 Industrial side products and pesticides 2-tert-Butylphenol 13.66 149.0 [M-H]- 133.0/92.9 22/26 70 37/39/38 2-Naphthol 7.86 143.0 [M-H]- 115.0/113.9/124.9 26/39/31 75 2/2/2 4-Octylphenol (OP) 15.92 205.1 [M-H]- 105.9/118.9 21/45 83 2/2/6 4-tert-OP (4tOP) 15.55 205.1 [M-H]- 133.0/116.9/93.0 27/60/49 68 12/12/10 13 - C6-4tOP 15.55 211.1 [M-H] 139.0 27 68 Nonylphenol 15.90 219.1 [M-H]- 133.0/147.0/116.9 34/28/60 75 14/11/13 Fenarimol 12.44 330.9 [M+H]+ 267.9/188.9 23/50 100 31/30/29 Methiocarb 11.27 226.0 [M+H]+ 121.0/169.0 18/5 58 33/33/32 Prochloraz 15.06 375.9 [M-H]- 307.9/70.0 13/24 62 11/10/9 Mycoestrogens and metabolites Alternariol 7.35/7.29/7.35 257.0 [M-H]- 213.1/215.1 21/27 90 83/89/91 Alternariol monomethyl ether 11.8 271.1 [M-H]- 256.1/255.1/227.1 26/33/38 85 32/33/32 α-zearalanol (ZAL) 9.38 321.2 [M-H]- 277.2/303.2 23/24 90 25/24/25 α-zearalenol (ZEL) 9.72 319.2 [M-H]- 275.2/160.1 23/29 110 54/54/55 α-ZEL-14-GlcA 4.36-4.58/4.32/4.54 495.2 [M-H]- 113.1/319.3/175.0b 21/27/20 110 255/45/45 β-ZAL 8.06 321.2 [M-H]- 277.2/303.2 23/24 90 23/24/23 β-ZEL 8.27 319.2 [M-H]- 275.2/160.1 23/29 110 57/57/57 β-ZEL-14-GlcA 4.06-4.24/4.01/4.22 495.2 [M-H]- 113.1/319.3/175.0b 21/20 110 118/43/44 Zearalanone (ZAN) 11.96 319.2 [M-H]- 275.2/205.2/160.1 23/23/29 110 60/59/60 Zearalenone (ZEN) 12.15 317.0 [M-H]- 175.0/131.0 25/30 110 84/83/86 13 - C18-ZEN 12.15 335.0 [M-H] 185.0 25 110 ZEN-14-GlcA 4.65-4.89/4.59/4.85 493.2 [M-H]- 317.2/113.1/175.1c 27/21/18 115 49/27/32 ZEN-14-sulfate 5.53-5.83/5.45-5.55/5.78 397.1 [M-H]- 317.2/175.1 23/36 110 7/7/7 Personal care product ingredients, pharmaceuticals and metabolites 3-Benzylidencampher 15.66 241.0 [M+H]+ 91.0/181.0d 34/18 63 95/118/98 4-Methylbenzyliden campher 15.96 255.1 [M+H]+ 171.0/105.0 20/31 70 77/81/93 Benzophenone 1 9.45/9.41/9.46 213.0 [M-H]- 134.9/90.9 20/28 69 72/73/74 Benzophenone 2 6/5.92/6.02 244.9 [M-H]- 134.9/108.9 17/22 67 66/61/63 Benzylparaben 10.93 227.0 [M-H]- 91.9/135.9e 25/16 64 91/113/93 Butylparaben (BP) 10.56 193.0 [M-H]- 92.0/135.9 26/18 80 46/47/46 13 - C6-BP 10.56 199.0 [M-H] 98.0 26 80 Ethylparaben (EP) 6.61 165.0 [M-H]- 91.9/136.9 26/15 53 124/107/110 13

13 - C6-EP 6.61 171.0 [M-H] 97.9 23 53 Isobutylparaben 10.38 193.0 [M-H]- 92.0/135.9 26/18 80 54/51/52 Methylparaben (MP) 5.46 151.0 [M-H]- 91.9/135.9 21/15 47 64/65/64 13 - C6-MP 5.46 157.0 [M-H] 97.9 21 47 Octyl-methoxycinnamate (OMC) 16.52 291.1 [M+H]+ 160.9/178.9/132.9 18/5/31 48 34/33/17 p-Hydrobenzoic acid (pHBA) 1-2.8/0.77-1.56/2.20-2.18 136.9 [M-H]- 92.9/65.0 17/30 43 3/3/3 13 - C6-pHBA 1-2.8/0.77-1.56/2.20-2.18 142.9 [M-H] 98.9 17 43 Propylparaben (PP) 8.30 179.0 [M-H]- 91.9/135.9 24/16 60 54/52/51 13 - C6-PP 8.30 185.0 [M-H] 97.9 24 60 Triclosan 15.42 287.0/289.0 [M-H]- 35.0/35.0 10/10 60 118/91/49 Ethinylestradiol 10.72 295.1 [M-H]- 144.9/142.9 43/57 96 60/62/62 Perfluorinated alkylated substances Perfluoroctanoic acid (PFOA) 6.94-7.43/6.29-7.15 /7.3-7.43 412.9 [M-H]- 368.9/168.8 11/18 55 24/24/24 13 - C8-PFOA 6.94-7.43/6.29-7.15 /7.3-7.43 420.9 [M-H] 375.9 11 55 Perfluoroctanesulfonic acid 10.15-11.44/10-11.18/10.42-11.30 498.8 79.9/98.8/129.8 46/41/42 80 84/76/74 [M-H]- (PFOS) several isomers 13C -PFOS 10.15-11.44/10-11.18/10.42-11.30 506.8 79.9/98.8 46/41 80 8 [M-H]- several isomers Phytoestrogens and metabolites 8-Prenylnaringenin 12.13 339.0 [M-H]- 218.9/118.9 21/30 90 49/51/49 Coumestrol 7.00 266.8 [M-H]- 265.8/210.8 28/29 80 27/27/27 Daidzein 5.77/5.73/5.78 252.9 [M-H]- 222.9/207.9f 34/32 92 119/88/84 Enterodiol 5.81 301.1 [M-H]- 253.0/271.0 24/25 85 23/24/24 Enterolactone 7.31 297.0 [M-H]- 106.9/253.0 27/21 80 277/280/269 Equol 7.03 241.0 [M-H]- 120.9/118.9 17/22 70 96/97/98 Formononetin 8.40 267.0 [M-H]- 251.9/222.9 22/32 90 24/24/24 Genistein (GEN) 6.92/6.83-6.90/6.93 268.9 [M-H]- 132.9/131.9/158.9 30/42/29 91 49/50/51 d4-GEN 6.92/6.83-6.90/6.93 272.9 [M-H]- 136.9 30 91

Glycitein 5.95/5.84-8.94/5.96 285.0 [M+H]+ 241.9/269.9g 32/26 118 246/44/45 Isoxanthohumol 8.93 355.0 [M+H]+ 178.9/299.0 26/16 82 35/34/35 Matairesinol 7.10 357.0 [M-H]- 82.9/121.9 26/36 78 57/60/56 Resveratrol 5.58 227.0 [M-H]- 142.9/184.9h 26/21 80 123/103/117 Xanthohumol 15.12 355.0 [M+H]+ 178.9/299.0 26/16 82 50/50/62 Plastizicer/ plastic components and metabolites Benzyl butyl phthalate 15.62 313.1 [M+H]+ 91/204.9/148.9/65.0 30/5/5/58 50 26/21/25 Bisphenol A (BPA) 8.73 227.0 [M-H]- 132.9/212.0i 28/19 80 296/33/32 13 - C12-BPA 8.73 239.0 [M-H] 224.0 19 80 BPAF 12.47 334.9 [M-H]- 264.9/176.9 22/46 77 4/5/4 BPB 10.43 241.0 [M-H]- 211.0/212.0/226.0 30/12/19 66 56/60/56 BPC 12.23 255.1 [M-H]- 239.8/147.0 20/29 85 87/81/90 BPF 6.82 199.0 [M-H]- 93.1/105.1 27/26 83 60/61/59 BPS 5.38/5.3/5.38 248.9 [M-H]- 107.9/91.9 28/39 82 63/58/63 Dibutylphthalate 15.71 279.1 [M+H]+ 148.9/205.0/120.9/65.0 16/5/33/46 49 11/10/10 Mono-n-butylphthalate (MBP) 4.3-4.82/4.04-4.24/4.72 221.0 [M-H]- 77.0/71.0/176.9j 18/18/11 52 36/85/37 13 - C2-MBP 4.3-4.82/4.04-4.24/4.72 225.0 [M-H] 79.0 18 52 Mono-2-ethylhexyl phthalate 7.64-9.0/6.48-9.01/8.68-8.72 277.1 133.9/127.0 16/17 66 41/43/34 [M-H]- (MEHP) 13 - C2-MEHP 7.64-9.0/6.48-9.01/8.68-8.72 281.1 [M-H] 136.9 16 66 n-Butylbenzenesulfon-amid 9.48 212.0 [M-H]- 140.9/146.0 24/19 75 66/64/64 Tetrabrombisphenol A 15.48 542.6 [M-H]- 445.8/417.8k 32/39 90 83/88/n.d.l a serum: 161.0/146.9. b serum and milk: 319.3/113.1. c serum: 317.2/175.1 milk: 317.2/113.1. d serum: 181.0/91.0 e serum: 135.9/91.9. f serum and milk: 207.9/222.9. g serum and milk: 269.9/241.9. h serum: 184.9/142.9. i serum and milk: 212.0/132.9.j serum: 77.0/176.9. k milk: 417.8/445.8. l not determined

14

Table S4 Extraction efficiencies (RE), intermediate precision (RSDR) and reparability (RSDr) of analytes which have not been successfully validated in urine, serum and breast milk. 4-tert-ctylphenol was evaluated with internal standard calibration in serum.

Urine Serum Breast milk a b e f h RE ± RSDR RE ± RSDR RSDr RE ± RE ± RSDR RSDr RE ± RSDR RE ± RSDR RSDr LOD LOQ c d g LL HL LL/HL RSDR LL HL LL/HL LL HL LL/HL (U/S/M) (U/S/M) [%] [%] [%] [%] [%] [%] [%] [%] [%] [µg/L] [µg/L] Phytoestrogens and metabolites Xanthohumol 112 ± 33 141 ± 18 43/15 85 ± 20 58 ± 16 34/21 34 ± 26 42 ± 41 137/57 0.05/0.5/2.1 0.2/1.7/7 Industrial side products and pesticides 2-tert-Butylphenol n.d.i n.d. n.d. 16 ± 49 12 ± 35 26/9 56 ± 35 57 ± 17 28/22 n.d./15/17 n.d./50/55 4-octylphenol (OP) 31 ± 129 50 ± 95 24/15 106 ± 24 97 ± 35 10/16 128 ± 272 68 ± 114 n.d./345 40/3/210 130/10/700 4-tert-OP n.d. 10 ± 139 n.d./29 83 ± 16 71 ± 17 21/4 71 ± 112 72 ± 16 127/19 10/6/140 30/20/460 Fenarimol 100 ± 27 104 ± 9 23/9 68 ± 38 86 ± 11 44/10 32 ± 163 78 ± 31 105/25 0.1/0.3/0.3 0.1/1.1/1.0 Nonylphenol 10 ± 313 32 ± 133 n.d./20 112 ± 21 82 ± 27 16/23 n.d. n.d. n.d. 15/1.9/16.5 50/6.3/55 Personal care product ingredients, pharmaceuticals and metabolites 3-Benzylidencampher n.d. 12 ± 116 n.d./13 38 ± 29 31 ± 29 23/14 n.d. 40 ± 88 n.d. 180/65/570 600/215/1900 4-Methylbenzyliden 9 ± 153 21 ± 97 50/31 61 ± 37 48 ± 26 34/10 16 ± 300 60 ± 117 n.d./200 130/30/480 450/100/1600 campher Octyl methoxycinnamate 34 ± 119 111 ± 32 90/43 91 ± 24 97 ± 34 27/20 444 ± 140 58 ± 125 n.d. 150/70/n.d. 500/240/n.d. p-hydrobenzoic acid 128 ± 15 102 ± 7 6/9 100 ± 15 95 ± 14 3/8 90 ± 22 96 ± 5 22/8 20/18/15 50/60/50 Triclosan 67 ± 73 92 ± 33 72/38 49 ± 144 92 ± 31 124/40 n.d. n.d. n.d. 3/3/n.d. 5/10/n.d. Plastizicer/ Plastic components and metabolites Benzylbutylphthalate 87 ± 20 69 ± 17 21/16 44 ± 35 48 ± 22 26/16 37 ± 330 35 ± 58 n.d./60 3/18/130 9/60/430 Dibutylphthalate 20 ± 91 44 ± 67 17/17 37 ± 44 33 ± 16 43/9 175 ± 239 56 ± 59 77/49 20/60/105 60/210/350 Tetrabrombisphenol A 99 ± 40 112 ± 20 40/17 64 ± 38 57 ± 17 31/23 n.d. n.d. n.d. 2/3/n.d. 6/10/n.d. Endogenous estrogens 2-Hydroxy estradiol 129 ± 40 118 ± 21 7/4 n.d. 92 ± 37 n.d./28 83 ± 17 90 ± 20 85/39 3.0/160/3.0 10/550/10 4-Hydroxy estrone 111 ± 20 103 ± 12 5/2 n.d. 60 ± 20 n.d./53 82 ± 12 87 ± 7 33/16 1.0/90/0.8 3.0/300/2.6 a Extraction efficiency. b Intermediate precision. c Low spiking level. d High spiking level. e Repeatability. f Limit of detection. g Values in the following order: urine/serum/breast milk. h Limit of quan- tification. i Not determined due to either insufficient ionization or extraction recovery, too high background noise or no possible calibration because of too high blank matrix contamination

15

Table S5 Signal suppression or enhancment (SSE), spiking concentrations, regression coeffiecients and linear range

Compound SSEa U/S/Mb Low/ high spike Regression coefficient R2 Rangec [%] [µg/L] U/S/M [µg/L] Endogeneous estrogens Estrone (E1) 98/75/33 3/30 0.999/0.998/0.997 0.3-100 Estradiol (E2) 91/81/34 6/60 0.999/0.997/0.993 0.6-200 E2-17-glucuronide (GlcA) 186/145/144 15/150 0.961/0.997/0.997 1.5-500 E2-3-sulfate 95/88/89 6/60 0.996/0.998/0.999 0.6-200 Estriol (E3) 94/89/105 6/60 0.998/0.997/0.998 0.6-200 16-Epiestriol 99/93/90 15/150 0.999/0.999/0.998 1.5-500 16-α-HydroxyE1 98/84/96 3/30 0.998/0.998/0.999 0.3-100 17-Epiestriol 101/84/81 15/150 0.999/0.999/0.997 1.5-500 2-MethoxyE1 118/78/31 9/90 0.999/0.997/0.997 0.9-300 2-MethoxyE2 117/81/31 9/90 0.998/0.995/0.994 0.9-300 2-HydroxyE2 404/6/195 30/300 0.997/0.912/0.989 U: 3-300 S/M: 6-2000 4-MethoxyE1 105/81/34 1.5/15 0.999/0.998/0.997 0.15-50 4-MethoxyE2 108/86/33 1.5/15 0.999/0.996/0.995 0.15-50 4-HydroxyE1 207/1/128 15/150 0.995/0.632/0.998 1.5-500 Industrial side products and pesticides 2-tert-Butylphenol 110/70/25 60/600 0.999/0.997/0.995 6-2000 2-Naphthol 113/69/83 15/150 0.998/0.997/0.998 1.5-500 4-octylphenol (OP) 32/51/1 30/300 0.98/0.986/0.799 U/S: 3-300 M: 3-1000 4-tert-OP 72/33/2 30/300 0.982/0.990/0.948 U: 3-300 S/M: 3-1000 Nonylphenol 44/49/2 15/150 0.963/0.960/0.682 U/S: 1.5-150 M: 1.5-500 Fenarimol 85/57/10 0.15/1.5 0.989/0.986/0.92 U: 0.015-1.5 S/M: 0.015-5 Methiocarb 89/51/34 9/90 0.993/0.991/0.991 S: 0.9-90 U/M: 0.9-300 Prochloraz 94/58/12 0.3/3 0.991/0.984/0.980 U/M: 0.03-3 S: 0.03-10 Mycoestrogens and metabolites Alternariol 102/86/87 1.5/15 0.996/0.997/0.997 0.15-50 Alternariol monomethyl ether 91/76/16 1.5/15 0.998/0.994/0.991 U/S: 0.15-50 M: 0.15-15 α-zearalanol (ZAL) 89/91/43 3/30 0.999/0.999/0.998 0.3-100 α-zearalenol (ZEL) 91/94/29 3/30 0.998/0.998/0.992 0.3-100 α-ZEL-14-GlcA 103/102/110 3/30 0.987/0.997/0.997 0.3-100 β-ZAL 87/81/69 3/30 0.999/0.998/0.999 0.3-100 β-ZEL 90/81/48 3/30 0.997/0.997/0.997 0.3-100 β-ZEL-14-GlcA 109/111/123 3/30 0.972/0.995/0.995 0.3-100 Zearalanone 86/97/24 3/30 0.997/0.998/0.993 0.3-100 Zearalenone (ZEN) 86/96/22 3/30 0.999/0.998/0.992 0.3-100 ZEN-14-GlcA 115/110/113 3/30 0.992/0.997/0.997 0.3-100 ZEN-14-sulfate 106/100/102 3/30 0.999/0.999/0.999 0.3-100 Personal care product ingredients, pharmaceuticals and metabolites 3-Benzylidencampher 87/53/2 150/1500 0.988/0.977/0.868 U: 15-1500 S/M: 15-5000 4-Methylbenzyliden campher 63/55/1 60/600 0.99/0.961/0.682 U: 6-600 S/M: 6-2000 Benzophenone 1 87/96/49 0.3/3 0.998/0.998/0.997 0.03-10 Benzophenone 2 104/77/138 1.5/15 0.998/0.997/0.998 0.15-50 Benzylparaben 85/92/32 0.15/1.5 0.996/0.996/0.991 0.015-5 Butylparaben 90/103/44 1.5/15 0.999/0.999/0.997 0.15-50 Ethylparaben 100/96/103 1.5/15 0.998/0.998/0.999 0.15-50 Isobutylparaben 83/86/47 1.5/15 0.998/0.999/0.997 0.15-50 Methylparaben 110/100/110 3/30 0.999/0.999/0.999 0.3-100 Octyl methoxycinnamate 18/97/0 150/1500 0.937/0.918/0.415 15-1500 p-Hydrobenzoic acid 122/129/204 90/900 0.963/0.996/0.996 S: 9-900 U/M: 9-3000 Propylparaben 84/91/75 1.5/15 0.998/0.999/0.999 0.15-50 Triclosan 55/49/1 3/30 0.937/0.981/0.440 U: 1-30 S/M: 0.3-100 Ethinylestradiol 102/72/31 15/150 0.998/0.994/0.995 1.5-500 Perfluorinated alkylated substances Perfluoroctanoic acid 101/102/74 1.5/15 0.999/0.998/0.999 0.15-50 Perfluoroctanesulfonic acid 115/65/84 9/90 0.997/0.991/0.997 0.9-300 Phytoestrogens and metabolites 8-Prenylnaringenin 94/82/14 0.3/3 0.991/0.997/0.972 U: 0.03-3 S/M: 0.03-10 Coumestrol 104/88/94 3/30 0.999/0.999/0.999 0.3-100 Daidzein 101/81/127 1.5/15 0.997/0.996/0.997 0.15-50 Enterodiol 93/77/100 1.5/15 0.998/0.999/0.999 0.15-50 Enterolactone 87/73/100 3/30 0.999/0.998/0.999 0.3-100 Equol 98/65/99 3/30 0.999/0.998/0.999 0.3-100 Formononetin 79/92/65 1.5/15 0.998/0.999/0.999 0.15-50 Genistein (GEN) 104/80/118 1.5/15 0.997/0.996/0.997 0.15-50 16

Glycitein 111/82/143 1.5/15 0.995/0.996/0.997 U/S: 0.15-50 M: 0.15-15 Isoxanthohumol 80/114/52 0.15/1.5 0.991/0.998/0.988 U/S: 0.015-5 M: 0.015-1.5 Matairesinol 109/78/126 6/60 0.998/0.994/0.999 0.6-200 Resveratrol 131/24/240 15/150 0.994/0.939/0.998 U: 1.5-150 S: 1.5-50 M: 1.5-500 Xanthohumol 32/66/5 1.5/15 0.970/0.887/0.945 U/S: 0.15-15 M: 0.15-50 Plastizicer/ Plastic components and metabolites Benzylbutylphthalate 75/40/2 15/150 0.988/0.924/0.774 U: 1.5-150 S/M: 1.5-500 Bisphenol A (BPA) 95/57/80 3/30 0.997/0.995/0.999 S: 0.3-30 U/M: 0.3-100 BPAF 76/61/20 1.5/15 0.995/0.994/0.991 S: 0.15-15 U/M: 0.15-50 BPB 100/59/57 3/30 0.999/0.994/0.995 S: 0.3-30 U/M: 0.3-100 BPC 103/61/40 6/60 0.998/0.993/0.995 S: 0.6-60 U/M: 0.6-200 BPF 104/68/108 6/60 0.997/0.997/0.998 0.6-200 BPS 66/83/103 0.3/3 0.997/0.997/0.997 0.03-10 Dibutylphthalate 77/37/3 30/300 0.985/0.954/0.842 U: 3-300 S/M: 3-1000 Mono-n-butylphthalate 263/91/76 6/60 0.998/0.997/0.997 S: 0.6-60 U/M: 0.6-200 Mono-2-ethylhexyl phthalate 90/101/6 6/60 0.998/0.755/0.958 M: 0.6-60 U/M: 0.6-200 n-Butylbenzenesulfonamid 98/94/74 6/60 0.998/0.998/0.996 0.6-200 Tetrabrombisphenol A 50/162/0 6/60 0.901/0.961/0.607 U/S: 6-60 M:0.6-200 a Signal suppression or enhancement. b urine/serum/breast milk c linear calibration range

17

Figure S4 Chemical structures of phytoestrogens with exact masses.

18

Figure S5 Chemical structure of industrial side products, personal care product ingredients with one paraben metabolite (p-hydroxybenzoic acid), pesticides and one pharmaceuticals with exact masses.

19

Figure S6 Chemical structures of endogenous estrogens and perfluorinated alkxlated substances with exact masses.

20

Figure S7 Chemical structures of plastic components, plasticizer and two plastizicer metabolites (Mono-n-butyl phthalate and Mono-2- ethylhexyl phthalate) with exact masses.

21

Figure S8 Chemical structures of mycoestrogens with exact masses.

22

Table S6 Overview of validation outcome inclusive listing of parameters out of validation limits.

a b Valsuccess not valid Valsuccess not valid Endogeneous estrogens c Estrone (E1) U/S/M 17-Epiestriol U/S M: RE, RSDR HL: RSDr d e Estradiol (E2) U/S M: LL : RSDr 2-MethoxyE1 U/S/M f g E2-17-glucuronide (GlcA) S/M U: LL: n.d. HL : RE , RSDr 2-MethoxyE2 U/S M: RE h E2-3-sulfate U/S M: RE HL: RSDR 2-HydroxyE2 - U: RE, RSDR S: LL: n.d.; HL: RSDR/r M: HL: RSDR/r Estriol (E3) U/S M: HL: RE 4-MethoxyE1 U/S/M

16-Epiestriol U/S M: RE, RSDR HL: RSDr 4-MethoxyE2 U/S/M 16-α-HydroxyE1 U/S/M 4-HydroxyE1 - U: LL: RE S: LL: n.d.; HL: RE, RSDr M: LL: RSDr Industrial side products and pesticides

2-tert-Butylphenol - U: n.d. S: RE, RSDR LL: RSDr M: RE, RSDR LL: RSDr Nonylphenol - U: RE, RSDR, RSDr S: LL: RE, RSDR HL: RSDr

M: RE, RSDR/r 2-Naphthol U/S M: LL: RSDr Fenarimol - U: LL: RSDR S: LL: RSDR/r M: RSDR/r LL: RE

4-Octylphenol (OP) - U: RE, RSDR LL: RSDr S: HL: RSDR/r M: RE, RSDR/r Methiocarb U/M S: RE 4-tert-OP - U: RE, RSDR/r S: RSDr HL: RE M: RE, RSDr LL: RSDR Prochloraz U/S M: RSDR/r Mycoestrogens and metabolites

Alternariol U/S/M β-ZEL U/S M: HL: RE

Alternariol monomethyl ether U/S M: HL: RE β-ZEL-14-GlcA S U: LL: n.d. M: HL: RE LL: RSDR/r α-zearalanol (ZAL) U/S/M Zearalanone U/S M: LL: RE, RSDR α-zearalenol (ZEL) U/S M: LL: RSDR Zearalenone (ZEN) U/S M: RSDR/r HL: RE

α-ZEL-14-GlcA S U: LL: RE, RSDR M: RSDr, HL: RE ZEN-14-GlcA S/M U: LL: n.d. β-ZAL U/S M: HL: RE ZEN-14-sulfate U/S M: RE, RSDR/r Personal care product ingredients, pharmaceuticals and metabolites

3-Benzylidencampher - U: RE, RSDR LL: RSDr S: RE, RSDR/r M: LL: n.d. HL: RE, RSDR/r Isobutylparaben U/S/M

4-Methylbenzyliden campher - U: RE, RSDR/r S: RSDR LL: RE, RSDr M: RE, RSDR/r Methylparaben U/S M: HL: RE Benzophenone 1 U/S M: LL: RSDr Octyl methoxycinnamate - U: RE, RSDR/r S: RSDR/r M: RE, RSDR/r

Benzophenone 2 U/S/M p-Hydrobenzoic acid - U: LL: RE S:too less retention M: LL: RSDr Benzylparaben U/S M: LL: RSDR/r Propylparaben U/S/M

Butylparaben U/S/M Triclosan - U: LL: RE, RSDR/r S: RSDR/r, LL: RE M: RE, RSDR/r Ethylparaben U/S/M Ethinylestradiol U/S M: LL: RE Perfluorinated alkylated substances

Perfluoroctanoic acid U/S M: RE Perfluoroctanesulfonic acid U/S/M Phytoestrogens and metabolites

8-Prenylnaringenin U/S M: LL: RE, RSDR/r Genistein (GEN) U/SM Coumestrol U/S/M Glycitein U/S/M

Daidzein S/M U: LL: RE Isoxanthohumol U/S/M Enterodiol U/S M: RE, RSDR/r Matairesinol U/M S: LL: RSDr

Enterolactone U/S/M Resveratrol U S: RSDr, LL: RE HL: RSDR, not in linear range M: RE RSDR/r Equol U/S M: HL: RE Xanthohumol - U: RE, LL: RSDR/r S: HL: RE LL: RSDr M: RE, RSDR/r Formononetin U/S/M Plastizicer/ Plastic components and metabolites

Benzylbutylphthalate - U: RSDr HL: RE S: RE, RSDR/r M: RE, RSDR/r BPS U/S/M Bisphenol A (BPA) U/S/M Dibutylphthalate - U: RE, RSDR HL: RSDr S: RE LL: RSDR/r M: RE, RSDR/r

BPAF U S: HL: RSDR M: HL: RE Mono-n-butylphthalate U/M S: RE BPB U/S/M Mono-2-ethylhexyl phthalate U S: LL: RE RSDR M: RE, RSDR LL: RSDr BPC U/S/M n-Butyl-benzenesulfonamide U/S/M

BPF U/S/M Tetrabrombisphenol A - U: HL: RE LL: RSDR/r S: RE, RSDr LL: RSDR M: n.d. a successfully validated in matrix. b parameters which are not within limits of the Commission decision Commission Decision (EC) No. 657/2002 19 and could not be successfully validated. c U: urine, S: serum, M: breast milk. d Low level spike. e Repeatabil- ity. f High level spike. g extraction recovery. h intermediate precision

23

Table S7 Concentrations in biological samples [µg/L].

Urine Breast Milk Urine Sys. Milk Sys. Sample name 1 2 1a 2a 5 6 blank blanka nig 1 nig 2 nig 3 nig 4 nig 5 aut 1 aut 2 aut 3 aut 4 blank blank Compound

Endogenous estrogens

Estrone (E1) n.d.b n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Estradiol (E2) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* E2-17-glucuronide (GlcA) n.d.*c n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. E2-3-sulfate n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Estriol (E3) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 16-Epiestriol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 16-α-HydroxyE1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 17-Epiestriol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 2-MethoxyE1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2-MethoxyE2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 2-HydroxyE2 n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 4-MethoxyE1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 4-MethoxyE2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 4-HydroxyE1 n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Industrial side products and pesticides

2-tert-Butylphenol -d ------n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 2-Naphthole 1.1e 1.0e 1.5 1.6 2.1 1.6 2.7 n.d. 7.9* 6.5* 10.5* 8.9* 11.1* 5.5* 4.1* 7.8* 9.0* 6.1* n.d.* 4-octylphenol (OP) n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 4-tert-OP n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Nonylphenol ------Fenarimol n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Methiocarb n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Prochloraz n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Mycoestrogens and metabolites

Alternariol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Alternariol monomethyl ether n.d. n.d. n.d. n.d. n.d.

3-Benzylidencampher n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* 4-Methylbenzyliden campher n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Benzophenone 1 n.d. n.d.

24

Isobutylparaben n.d. n.d. 1.08 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Methylparaben 1.63 2.51 23.39 22.16 n.d. n.d. n.d. n.d. n.d.* n.d.*

Perfluoroctanoic acid n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

8-Prenylnaringenin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Coumestrol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Daidzein 3.11* 0.48* n.d.* n.d.* 1.47* 30.95* n.d.* n.d.* n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Enterodiol 0.75

Benzylbutylphthalate n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* Bisphenol A (BPA) n.d. n.d. 1.6 n.d. n.d. n.d. n.d. n.d. 200 n.d. n.d. n.d. n.d. n.d. n.d.* n.d.* n.d.* n.d.* n.d.* n.d.* n.d.*

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References

1. Ademollo, N.; Ferrara, F.; Delise, M., et al., Nonylphenol and octylphenol in human breast milk. Environ Int 2008, 34 (7), 984-7. 2. Asimakopoulos, A. G.; Thomaidis, N. S., Bisphenol A, 4-t-octylphenol, and 4-nonylphenol determination in serum by Hybrid Solid Phase Extraction–Precipitation Technology technique tailored to liquid chromatography–tandem mass spectrometry. Journal of Chromatography B 2015, 986-987, 85-93. 3. Chen, M.; Zhu, P.; Xu, B., et al., Determination of nine environmental phenols in urine by ultra-high-performance liquid chromatography- tandem mass spectrometry. Journal of analytical toxicology 2012, 36 (9), 608-615. 4. Papadopoulou, E.; Sabaredzovic, A.; Namork, E., et al., Exposure of Norwegian toddlers to perfluoroalkyl substances (PFAS): The association with breastfeeding and maternal PFAS concentrations. Environment International 2016, 94, 687-694. 5. Riddell, N.; Arsenault, G.; Benskin, J. P., et al., Branched Perfluorooctane Sulfonate Isomer Quantification and Characterization in Blood Serum Samples by HPLC/ESI-MS(/MS). Environmental Science & Technology 2009, 43 (20), 7902-7908. 6. Keller, J. M.; Calafat, A. M.; Kato, K., et al., Determination of perfluorinated alkyl acid concentrations in human serum and milk standard reference materials. Analytical and Bioanalytical Chemistry 2010, 397 (2), 439-451. 7. Chen, J.; Wang, X.; Ge, X., et al., Chronic perfluorooctanesulphonic acid (PFOS) exposure produces estrogenic effects in zebrafish. Environmental pollution (Barking, Essex : 1987) 2016, 218, 702-708. 8. Benninghoff, A. D.; Bisson, W. H.; Koch, D. C., et al., Estrogen-like activity of perfluoroalkyl acids in vivo and interaction with human and rainbow trout estrogen receptors in vitro. Toxicological sciences : an official journal of the Society of Toxicology 2011, 120 (1), 42-58. 9. Yao, P. L.; Ehresman, D. J.; Rae, J. M., et al., Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid. Toxicology 2014, 326, 62-73. 10. Dixon, D.; Reed, C. E.; Moore, A. B., et al., Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay. Reproductive toxicology (Elmsford, N.Y.) 2012, 33 (4), 506-12. 11. Sonthithai, P.; Suriyo, T.; Thiantanawat, A., et al., Perfluorinated chemicals, PFOS and PFOA, enhance the estrogenic effects of 17β- estradiol in T47D human breast cancer cells. Journal of Applied Toxicology 2016, 36 (6), 790-801. 12. Wang, H.; Du, H.; Yang, J., et al., PFOS, PFOA, estrogen homeostasis, and birth size in Chinese infants. Chemosphere 2019, 221, 349- 355. 13. Haiba, E.; Nei, L.; Kutti, S., et al., Degradation of diclofenac and triclosan residues in sewage sludge compost. Agronomy Research 2017, 15 (2), 395-405. 14. Anumol, T.; Merel, S.; Clarke, B. O., et al., Ultra high performance liquid chromatography tandem mass spectrometry for rapid analysis of trace organic contaminants in water. Chemistry Central journal 2013, 7, 104-104. 15. Braun, D.; Ezekiel, C. N.; Abia, W. A., et al., Monitoring Early Life Mycotoxin Exposures via LC-MS/MS Breast Milk Analysis. Analytical Chemistry 2018, 90 (24), 14569-14577. 16. Mikula, H.; Hametner, C.; Berthiller, F., et al., Fast and reproducible chemical synthesis of zearalenone-14-β,D-glucuronide. World Mycotoxin Journal 2012, 5 (3), 289-296. 17. Mikula, H.; Weber, J.; Lexmuller, S., et al., Simultaneous preparation of alpha/beta-zearalenol glucosides and glucuronides. Carbohydrate research 2013, 373, 59-63. 18. Vendl, O.; Crews, C.; MacDonald, S., et al., Occurrence of free and conjugated Fusarium mycotoxins in cereal-based food. Food Additives & Contaminants: Part A 2010, 27 (8), 1148-1152. 19. Commission, E., 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. European Commission: Vol. OJ L 221, 8–36.

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