Supporting Information. Sweetened Swimming Pools and Hot Tubs
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1 Supporting Information Sweetened Swimming Pools and Hot Tubs Lindsay K Jmaiff Blackstock, Wei Wang, Sai Vemula, Benjamin T Jaeger and Xing-Fang Li * Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada, T6G 2G3 Corresponding author telephone: (78) ; fax: (78) ; and xingfang.li@ualberta.ca 1
2 Materials and Methods Reagents The acesulfame-k (ACE) standard was obtained from Supelco (Bellefonte, PA), and the deuterated isotopic internal standard acesulfame-k-d4 (ACE-d4) was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Working stocks were prepared in methanol and stored in a -2 C freezer. LCMS-grade formic acid (FA, 49 51%) was obtained from Sigma- Aldrich (St. Louis, MO). Water and methanol used in this study were Optima LCMS-grade, purchased from Fisher Scientific (Fair Lawn, NJ). Optimized HPLC-MS/MS Conditions The mobile phase gradient program linearly increased B from 5% to 95% in 2 min; maintained B at 95% until 6 min; returned to 5% B between 6. and 6.1 min; and remained at 5% for column equilibration until run completion at 15 min. Optimized conditions for negative electrospray ionization (ESI) were: curtain gas, 45 psi; collision gas, high; gas 1, 5 psi; gas 2, 8 psi; ion spray voltage, -45 V; temperature, 4ºC; and dwell time, 75 msec. The optimized declustering potential (DP), collision energy (CE), and cell collision exit potential (CXP) for all transitions are listed below > 81.9 = DP -6, CE -19, CXP > 77.9 = DP -6, CE -44, CXP > 85.9 = DP -53, CE -2, CXP > 77.9 = DP -54, CE -46, CXP -1 Method Development and Validation Limit of Detection Calculation The instrument limit of detection (LOD) was calculated as three times the standard deviation of a.5 ng/l standard divided by the slope. This agrees with the observed chromatographic signal-tonoise ratio of 4.3 obtained for a.5 ng/l ACE standard. The method LOD was calculated as three times the standard deviation of the method blank signal divided by the slope. LCMS-grade water was filtered through.45-µm PVDF filters and analyzed as method blanks. Typical chromatograms displaying signal response from the method blank, LOD standard, and the ACE- 2
3 d4 sample spike are shown in Figure S4. The linear range for ACE was.5 to 1 ng/l; a typical calibration curve is shown in Figure S5. Reproducibility Intraday and interday reproducibility was evaluated for a 5.-ng/L standard prepared in LCMS grade Optima water (Figure S6). Within the same day, the %RSD for the determined concentrations from triplicate analyses was less than 2%. Over four non-consecutive days, a oneway ANOVA test determined that p=.564, indicating no significant difference between the mean concentrations reported on different days. The retention time for ACE and ACE-d4 in LCMS-grade, tap, and recreational waters were 4.4 ±.1 minutes (N=558 over 4 days), respectively. Therefore this method is stable over a multi-day analysis on a shared instrument. Matrix Effects The matrix effects for recreational waters were evaluated in two ways. First, HT5 was used as a representative complex sample to determine whether recreational water matrix effects had an impact on signal intensity. The sample was diluted to 1/5, 1/1, 1/2, 1/4, and 1/8 and analyzed. The concentration of ACE was plotted against the dilution factor resulting in an R 2 value of.994 (Figure S7), indicating that the matrix did not affect the analyte intensity. Based on the concentration range of ACE in swimming pool and hot tub samples obtained from preliminary screening, we chose a dilution factor of 1/2 to be used for all samples to ensure the analyte was within the linear range, to eliminate carry over, and to reduce residue buildup in the LC column and ESI source. Second, a spike recovery study was conducted with three representative samples: SP2, SP4, and HT8 (Table S4). Each sample was diluted to 1/2 and split into six portions; three were spiked with 5 ng/l ACE. The non-spiked samples represented the matrix. Recoveries ranged from 86 to 91% indicating that the matrix did not affect the recovery of ACE at 1/2 dilution. 3
4 Calculations Calculation 1: Total ACE present The following calculation estimates the total mass of ACE in SPx. The values used were the known pool volume (V1) and average concentration of ACE determined over all 15 days of collection (C1). V1 = Swimming Pool Volume = 11 US Gal 42 L C1 = Average ACE in pool = 156 ng/l 156 ng L 1 µg 1 ng 1 mg 1 µg 1 g 1 mg = g 1.56E 7 L of ACE 1.56E 7 g L 42 L =. 655 g 65 mg ACE Calculation 2: Total Volume of Urine The following calculation estimates the volume of urine in SPx. The values used were the known pool volume (V1) and average concentration of ACE determined on the final day of collection (C1), along with the determined average concentration of ACE in Canadian urine (C2, Figure S3). V1 = Swimming Pool Volume = 11 US Gal 42 L C1 = Average ACE in pool = 156 ng/l C2 = Average ACE in Adult Urine = 2 36 ng/ml 2 36 ng/l C1V1 = C2V2 V2 = C1V1 C2 V2 = 156 ng 42 L L 2 36 ng L V2 3 L Urine Calculations 1 and 2 were repeated for SPz, which had twice the total volume of SPx (22 US Gal) and an average ACE concentration of 21 ng/l over the collection period (Table S3). 4
5 Dissolved Organic Carbon (mg/l) Acesulfame (ng/l) Acesulfame (ng/l) Figures and Tables 2 15 a RF1 RF2 RF3 RF4 RF5 H1 H2 H3 b RF6 RF7 RF8 RF9 RF1 RF11 RF12 P1 Figure S1. Average ACE concentration (N=3) detected in tap water samples collected from each sampling location in (a) City 1 and (b) City 2. The combined average of ACE concentration in each city s tap water was found to be statistically different using an unpaired Student s t-test (p<.1) T = Tap Water Control * p.5 SP = Swimming Pool * * p.1 HT = Hot Tub p.1 RF = Recreational Facility H = Hotel * * * * * * 13.6 * * * 6.3 * * Blank T SP1 SP2 HT1 HT2 T SP3 HT3 T SP4 SP5 T SP6 T SP7 T SP8 HT4 T SP9 HT5 T SP1 RF1 RF2 RF3 RF4 RF5 H1 H2 H3 Figure S2. Average dissolved organic carbon (DOC) concentration detected 1 in tap and recreational water samples (N=3) collected from City 1. The average ACE in each sample type was compared using an unpaired Student s t-test. (1) Total Organic Carbon in Water EPA Method (accessed Feb 15, 217) 5
6 ACE Intensity (cps) MRM Transition (162/81.9 Da) ACE-d4 Intensity (cps) MRM Transition (166/85.9 Da) Counts per second (cps) ACE: 162.1>81.9 ACE-d4: 166.2>86 ACE(2): 162.1>77.9 ACE-d4(2): 166.2> Retention time, min Figure S3. A sample chromatogram showing ACE and ACE-d4 internal standard transitions detected in a pooled human urine (N=2) sample at 1 times dilution a. LCMS Grade Method Blank:.5 ng/l ACE 4 b. 1 ng/l ACE-d4 Optima H 2 O Filtered LCMS S/N=4.3 S/N=17 Grade Optima H 2 O (PDVF.45µm) Retention Time (minutes) Figure S4. Typical chromatograms showing the signal intensity of (a) ACE in a blank sample, method blank sample, and a.5 ng/l ACE standard and (b) a 1 ng/l ACE-d4 internal standard. 6
7 Acesulfame (ng/l) ACE/ACE-d4 Peak Area Ratio y =.98x -.15 R² = Concentration ACE (ng/l) Figure S5. A typical ACE calibration curve prepared with 1-ng/L ACE-d4 spiked ACE standards ranging from.5 to 1 ng/l in 9:1 LCMS H 2 O:MeOH ± ± ± ±. 215 June July July July 15 One-way ANOVA p=.56 Intraday N=3 Figure S6. Interday and intraday variation of a 5-ng/L ACE standard prepared in LCMS grade water (1% MeOH with 1-ng/L ACE-d4 spike). 7
8 Acesulfame (ng/l) y = 11x R² = Dilution Factor Figure S7. Recreational water matrix dilution and corresponding ACE response, the sample HT5 was diluted to 1/5, 1/1, 1/2, 1/4, and 1/8 and the concentration of ACE was determined. Table S1. Average ACE fold increase in swimming pools and hot tubs compared to input tap water concentration; fold increase = (A-B)/B, where A = SP or HT, B = T. City 1 City 2 RF1 SP1 98 RF6 SP11 14 SP2 165 RF7 SP12 5 H1 343 SP13 36 H2 513 RF8 SP14 36 RF2 SP3 93 SP15 15 HT3 7 RF9 SP16 12 RF3 SP4 12 HT6 7 SP5 9 RF1 SP17 39 RF4 SP6 6 HT7 5 RF5 SP7 6 RF11 SP18 34 H1 SP8 29 SP19 1 HT4 9 RF12 SP2 75 H2 SP9 27 HT8 199 HT5 571 P1 SP21 1 H3 SP1 4 8
9 Table S2. Average concentration of ACE in SPx, SPz, and corresponding input tap water samples over 15 non-consecutive days. Sample Day # Acesulfame, ng/l Tap Water (N=3) SPx (N=6) SPz (N=6) Avg. ± St. Dev. %RSD Avg. ± St. Dev. %RSD Avg. ± St. Dev. %RSD 1 16 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 7 4 Table S3. Average concentration of ACE in SPx and SPz over three weeks of sample collection as well as the corresponding estimated total mass of ACE and total urine content. Pool ACE Average (ng/l, N=9) ACE Standard Deviation (ng/l, N=9) ACE % Relative Standard Deviation ACE Estimated Total Mass (mg) Estimated Total Urine (L) SPx SPz
10 Table S4. Percent spike recovery for ACE in three representative recreational sample matrices. Sample % Spike Recovery ϕ % RSD RF1 SP2 87% 4% RF2 SP4 86% 5% RF12 HT8 91% 8% %R=((F-I)/A)*1% Where: F=[Matrix+Spike]; I=[Matrix]; A=[Spike] ϕ Each sample was diluted to 1/2 and then split into 6 portions. Three portions were spiked with 5 ng/l ACE; all portions were spiked with 1 ng/l ACE-d4 internal standard for quantification. 1
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