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: (780) 492-5094; fax: (780) 492-7800; and e-mail:
xingfang.li@ualberta.ca

1
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 -20 C freezer. LCMS-grade formic acid (FA, 4951%) 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 6.0 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, 50 psi; gas 2, 80 psi; ion spray voltage, -4500 V; temperature, 400C;
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.
162.1 > 81.9 = DP -60, CE -19, CXP -10
162.1 > 77.9 = DP -60, CE -44, CXP -7
166 > 85.9 = DP -53, CE -20, CXP -10
166 > 77.9 = DP -54, CE -46, CXP -10

Method Development and Validation

Limit of Detection Calculation
The instrument limit of detection (LOD) was calculated as three times the standard deviation of a
0.5 ng/L standard divided by the slope. This agrees with the observed chromatographic signal-to-
noise ratio of 4.3 obtained for a 0.5 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 0.45-m PVDF filters and analyzed as method blanks. Typical
chromatograms displaying signal response from the method blank, LOD standard, and the ACE-

2
d4 sample spike are shown in Figure S4. The linear range for ACE was 0.5 to 1000 ng/L; a
typical calibration curve is shown in Figure S5.

Reproducibility
Intraday and interday reproducibility was evaluated for a 5.0-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 one-
way ANOVA test determined that p=0.564, 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 0.1 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, 1/10, 1/20, 1/40, and 1/80 and
analyzed. The concentration of ACE was plotted against the dilution factor resulting in an R2
value of 0.994 (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 1/20 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 1/20 and split
into six portions; three were spiked with 50 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 1/20 dilution.

3
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 = 110 000 US Gal 420 000 L
C1 = Average ACE in pool = 156 ng/L
1 1 1
156 = 1.56 7
1000 1000 1000

1.56 7 420 000 = 0. 0655

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 = 110 000 US Gal 420 000 L
C1 = Average ACE in pool = 156 ng/L
C2 = Average ACE in Adult Urine = 2 360 ng/mL 2 360 000 ng/L

11 = 22
11
2 =
2

156 420 000
2 =

2 360 000

Urine

Calculations 1 and 2 were repeated for SPz, which had twice the total volume of SPx (220 000
US Gal) and an average ACE concentration of 210 ng/L over the collection period (Table S3).

4
Figures and Tables
20
a.
12
Acesulfame (ng/L)

15 12

8 8
10 7 7
6 6

0
RF1 RF2 RF3 RF4 RF5 H1 H2 H3
20
b.
Acesulfame (ng/L)

15
15
13
15 12 13 12
13 12

10

0
RF6 RF7 RF8 RF9 RF10 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 citys tap water was found to be statistically different using an unpaired Students t-test
(p<0.001).

50
T = Tap Water Control ***
* p 0.05
SP = Swimming Pool 40.5
40 ** p 0.01
HT = Hot Tub
*** p 0.001
Dissolved Organic

RF = Recreational Facility
Carbon (mg/L)

30 H = Hotel
*** ***
***
*** ***
*** *** ** ***
20 ** 15.0 15.0
16.5 **
13.6 ** ***
11.4 12.0
*** 10.1 10.4 9.4 *** ***
*** *** *** ***
7.2 7.4 7.4 *** 7.2
10 5.5 4.8 5.1 5.3 5.6 6.7 6.3 5.9 4.8
0.9
0
Blank T SP1 SP2 HT1 HT2 T SP3 HT3 T SP4 SP5 T SP6 T SP7 T SP8 HT4 T SP9 HT5 T SP10

RF1 RF2 RF3 RF4 RF5 H1 H2 H3

Figure S2. Average dissolved organic carbon (DOC) concentration detected1 in tap and
recreational water samples (N=3) collected from City 1. The average ACE in each sample type
was compared using an unpaired Students t-test.
(1) Total Organic Carbon in Water EPA Method 415.1.
https://www.epa.gov/sites/production/files/2015-06/documents/415_1dqi.pdf (accessed
Feb 15, 2017)

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 18000
ACE: 162.1>81.9

15000 ACE-d4: 166.2>86

ACE(2): 162.1>77.9
Counts per second (cps)

ACE-d4(2): 166.2>77.9
12000

9000

6000

3000

0
3 3.5 4 4.5 5
Retention time, min

Figure S3. A sample chromatogram showing ACE and ACE-d4 internal standard transitions
detected in a pooled human urine (N=20) sample at 100000 times dilution.
MRM Transition (166/85.9 Da)

1000 Method Blank: 4000

a. LCMS Grade 0.5 ng/L ACE b. 10 ng/L ACE-d4
MRM Transition (162/81.9 Da)

ACE-d4 Intensity (cps)

Optima H2O Filtered LCMS S/N=4.3 S/N=107

800 Grade Optima H2O
ACE Intensity (cps)

(PDVF 0.45m) 3000

600
2000
400
1000
200

0 0
1 3 5 1 3 5 1 3 5 1 3 5
Retention Time (minutes)

Figure S4. Typical chromatograms showing the signal intensity of (a) ACE in a blank sample,
method blank sample, and a 0.5 ng/L ACE standard and (b) a 10 ng/L ACE-d4 internal standard.

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 100
90
80
ACE/ACE-d4 Peak Area Ratio

70
60
50
40
30
y = 0.098x - 0.015
20 R = 0.998

10
0
0 200 400 600 800 1000
Concentration ACE (ng/L)

Figure S5. A typical ACE calibration curve prepared with 10-ng/L ACE-d4 spiked ACE
standards ranging from 0.5 to 1000 ng/L in 9:1 LCMS H2O:MeOH.

6.0
5.0
Acesulfame (ng/L)

5.2 0.1 5.2 0.1 5.1 0.1 5.2 0.0

4.0
3.0
2.0
1.0
0.0
2015 June 30 2015 July 3 2015 July 8 2015 July 15
One-way ANOVA p=0.056
Intraday N=3

Figure S6. Interday and intraday variation of a 5-ng/L ACE standard prepared in LCMS grade
water (10% MeOH with 10-ng/L ACE-d4 spike).

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 250

200
Acesulfame (ng/L)

150

100

50 y = 1010x + 4.48
R = 0.994

0
0 0.05 0.1 0.15 0.2 0.25
1 1 1 1 1.
80 40 20 10 5
Dilution Factor

Figure S7. Recreational water matrix dilution and corresponding ACE response, the sample HT5
was diluted to 1/5, 1/10, 1/20, 1/40, and 1/80 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 RF10 SP17 39
RF4 SP6 6 HT7 5
RF5 SP7 6 RF11 SP18 34
H1 SP8 290 SP19 10
HT4 9 RF12 SP20 75
H2 SP9 27 HT8 199
HT5 571 P1 SP21 10
H3 SP10 4

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Table S2. Average concentration of ACE in SPx, SPz, and corresponding input tap water
samples over 15 non-consecutive days.
Acesulfame, ng/L
Sample
Day # Tap Water (N=3) SPx (N=6) SPz (N=6)
Avg. St. Dev. %RSD Avg. St. Dev. %RSD Avg. St. Dev. %RSD
1 16 0.5 3 133 5 4 173 13 8
2 16 0.8 5 135 3 2 180 13 7
3 17 0.5 3 125 7 5 240 10 4
4 16 0.4 3 134 4 3 242 13 5
5 17 1.9 11 127 8 6 251 9 3
8 18 3.6 20 114 21 18 221 16 7
9 15 0.1 1 165 4 2 202 8 4
10 16 0.4 2 174 8 4 258 24 9
11 15 0.9 6 150 27 18 242 9 4
12 14 0.7 5 154 12 8 220 7 3
15 16 0.6 4 173 9 5 186 19 10
16 17 1.0 6 181 8 4 191 3 2
17 20 1.4 7 183 6 4 186 13 7
18 16 0.7 4 181 6 3 178 5 3
19 12 0.4 3 202 8 4 182 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.

ACE ACE ACE % ACE Estimated

Pool Average Standard Deviation Relative Standard Estimated Total Total Urine
(ng/L, N=90) (ng/L, N=90) Deviation Mass (mg) (L)
SPx 156 28 18 65 30

SPz 210 32 15 176 75

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Table S4. Percent spike recovery for ACE in three representative recreational sample matrices.
Sample % Spike Recovery % RSD
RF1 SP2 87% 4%
RF2 SP4 86% 5%
RF12 HT8 91% 8%
%R=((F-I)/A)*100%
Where: F=[Matrix+Spike]; I=[Matrix]; A=[Spike]

Each sample was diluted to 1/20 and then split into 6 portions. Three portions were
spiked with 50 ng/L ACE; all portions were spiked with 10 ng/L ACE-d4 internal standard
for quantification.

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