Transcript Benzotriazoles and benzothiazoles in human urine: method

Alexandros G. Asimakopoulos1,2, Nikolaos S.
Thomaidis1, Kurunthachalam Kannan2
1Laboratory
of Analytical Chemistry, Department of Chemistry,
University of Athens, Panepistimioupolis Zografou, Athens,
Greece.
2Wadsworth
Center, New York State, Department of Health,
and Department of Environmental Health Sciences, School
of Public Health, State University of New York at Albany,
Albany, NY, USA.
Target analytes measured in human urine
BTRs :
BTRs:
BTHs:
BTHs:
1. 1H-benzotriazole
(1H-BTR)
2. 1-hydroxy-benzotriazole
(1-OH-BTR)
3. Tolyltriazole
(TTR, a mixture of isomers of
4-methyl-1H-benzotriazole [4-Me-1H-BTR]
and 5-methyl-1H-benzotriazole [5-Me-1H-BTR])
4. Xylyltriazole
(XTR, or 5,6-dimethyl-1H-benzotriazole
[5,6-diMe-1H-BTR])
5. 5-chloro-1H-benzotriazole
(5-Cl-1H-BTR)
6. 4-/5-hydroxy-benzotriazole
(4-OH-BTR and 5-OH-BTR)
1. benzothiazole
(BTH)
2. 2-hydroxy-benzothiazole
(2-OH-BTH)
3. 2-methylthio-benzothiazole
(2-Me-S-BTH)
4. 2-amino-benzothiazole
(2-amino-BTH)
5. 2-thiocyanomethylthio-benzothiazole
(2-SCNMeS-BTH)
6. 2-morpholin-4-yl-benzothiazole
(2-M-BTH)
“BUT WHY MEASURE THESE
CHEMICALS IN URINE?”
 No human biomonitoring method had ever been reported and validated.
.
 As small polar compounds, they are expected to be excreted through urine.
 In some applications they are used in high concentrations
(e.g. BTR and TTR are used in dishwasher detergents on the order of tens or hundreds μg/mL
(ppm); BTH is reported used in foodstuffs at levels up to 0.5 (μg/mL) ppm including nonalcoholic and alcoholic beverages, soft and hard candy, baked goods, meat products,
gravies, soups, milk products, and cheese)
 BTRs and BTHs have been detected in wastewater and other receiving environmental media
(Tap water, surface water).
 The occurrence of 10 BTRs & BTHs was shown in indoor dust.
 10 ng/g of BTH was previously reported in human atherosclerotic aortas.
Human Exposure is inevitable
Protocol for extraction from human urine
2 mL of urine
Add benzotriazole-d4 and atrazined5 as internal standards.
Incubation at 37°C for 24 h
Add 4 mL of 1M ammonium
acetate that contained 291.4 units
of β-glucuronidase (Helix Pomatia)
(0.77 g of ammonium acetate
dissolved in 10 mL of Milli-Q water
with 20 μL of β-glucuronidase
solution).
Dilution with Milli-Q water to
20 mL, pH adjusted to 3.00 (±
0.10), and vortex mixed.
SPE with Oasis® HLB
6cc/200 mg.
1.
2.
3.
4.
Conditioning with 10 mL of MeOH.
Equilibration with 10 mL of acidified Milli-Q water.
Washing with 2×5 mL of acidified Milli-Q water/MeOH (95:5% v/v).
Elution with 10 mL of MeOH/ACN (1:1 v/v)
Evaporation to near-dryness with nitrogen, reconstituted
with 200 μL of MeOH/ACN (1:1 v/v), vortex-mixed.
LC-ESI(+)MS/MS
qQq: ABSCIEX API 2000
Method performance (1/2)
Target analytes
Average absolute
recoveries %*
Target analytes
LLOQ*
(ng/mL)
1H-BTR
1-OH-BTR
TTR
XTR
5-Cl-1H-BTR
4-/5-OH-BTR
BTH
2-OH-BTH
2-Me-S-BTH
2-Amino-BTH
2-SCNMeS-BTH
2-M-BTH
85-99 %
111-127 %
93-116 %
92-107 %
95-99 %
83–99 %
20-23 %
88-122 %
23-39 %
110-115 %
88-101 %
80–117 %
1H-BTR
0.5
1-OH-BTR
2.0
TTR
0.2
XTR
0.2
5-Cl-1H-BTR
0.5
4-/5-OH-BTR
Semi-quantified
BTH
5.0
2-OH-BTH
2.5
2-Me-S-BTH
0.2
2-Amino-BTH
0.2
2-SCNMeS-BTH
0.5
2-M-BTH
0.5
*calculated from 2 concentration levels minimum;
In each level N = 6 replicates, k=2 days
 Instrumental calibration was verified by injecting matrix
matched calibration standards and showed an
excellent linearity (r > 0.99) (at least 67% of the matrixmatched calibration points were within the established
linear range)
MRM Chromatograms (1/3)
MRM Chromatograms (2/3)
MRM Chromatograms (3/3)
 The analytical parameters of 2-M-BTH were determined apart from BTH, since they co-eluted and
2-M-BTH cross talked to the MRMs of BTH. However, due to the very low detection rate and
concentrations of 2-M-BTH present in urine, cross-talk of 2-M-BTH did not pose a problem for
BTH measurement, and consequently separation was not needed and co-analysis of 2-M-BTH
and BTH was possible in human urine.
Occurrence of BTRs
Country/Target analytes
BTR
TTR
XTR
.
ΣΒΤRs
Japan
(N=36)
DR %
0.0 %
(0/36)
5.5 %
(2/36)
33.3 %
(12/36)
38.8 %
(14/36)
GM (ng/mL)
-
1.7
0.7
0.9
DR %
2.0 %
(1/49)
10.2 %
(5/49)
20.4%
(10/49)
26.5 %
(13/49)
GM (ng/mL)
-
1.6
0.2
0.4
DR %
8.6 %
(4/46)
0.0 %
(0/46)
28.2%
(13/46)
36.9 %
(17/46)
GM (ng/mL)
2.0
-
2.1
2.1
DR %
8.0 %
(2/25)
0.0%
(0/25)
68.0%
(17/25)
72.0 %
(18/25)
GM (ng/mL)
5.4
-
1.1
1.5
DR %
29.4%
(15/51)
3.9%
(2/51)
68.6%
(35/51)
76.4 %
(39/51)
GM (ng/mL)
1.4
3.2
1.5
2.1
DR %
12.0 %
(12/100)
43.0 %
(43/100)
3.0 %
(3/100)
GM (ng/mL)
1.2
0.7
1.1
0.9
DR %
0.0 %
(0/25)
8.0 %
(2/25)
4.0 %
(1/25)
12.0 %
(3/25)
GM (ng/mL)
-
0.8
-
0.6
Korea
(N=49)
India
(N=46)
Vietnam
(N=25)
China
(N=51)
Greece
(N=100)
U.S.
(N=25)
52.0 %
(52/100)
Occurrence of BTHs
BTH
2-ΟΗ-BTH
2-Amino-BTH
2-M-BTH
ΣΒΤHs
DR %
55.5 %
(20/36)
16.6 %
(6/36)
19.4 %
(7/36)
2.7 %
(1/36)
69.4 %
(25/36)
GM (ng/mL)
22.7
2.1
0.7
-
9.5
DR %
48.9 %
(24/49)
8.1 %
(4/49)
6.1 %
(3/49)
0.0 %
(0/49)
53.0 %
(26/49)
GM (ng/mL)
6.4
2.8
1.2
-
5.9
DR %
41.3 %
(19/46)
4.3 %
(2/46)
6.5 %
(3/46)
13.0 %
(6/46)
42.8 %
(24/46)
GM (ng/mL)
5.4
7.5
1.3
0.6
4.5
DR %
100.0 %
(25/25)
16.0%
(4/25)
4.0 %
(1/25)
0.0 %
(0/25)
100.0 %
(25/25)
GM (ng/mL)
10.4
2.2
-
–
10.7
DR %
37.2 %
(19/51)
15.6 %
(8/51)
0.0 %
(0/51)
3.9 %
(2/51)
50.9 %
(26/51)
GM (ng/mL)
7.6
2.7
-
0.8
5.6
DR %
29.0 %
(29/100)
15.0%
(15/100)
8.0%
(8/100)
2.0%
(2/100)
40.0%
(40/100)
GM (ng/mL)
5.1
2.3
0.2
1.6
3.4
DR %
24.0 %
(6/25)
8.0%
(2/25)
12.0%
(3/25)
0.0%
(0/25)
44.0%
(11/25)
GM (ng/mL)
5.7
-
1.0
–
2.8
Country/Target analytes
Japan
(N=36)
Korea
(N=49)
India
(N = 46)
Vietnam
(N = 25)
China
(N = 51)
Greece
(N = 100)
U.S.
(N = 25)
Estimation of Human Exposures
 Estimated daily intake (EDI; μg/day) = Urinary concentrations (μg/L) × 1.7 L/day
Estimated daily intake (EDI; μg/day)
ΣBTRs
ΣBTHs
Japan
(N=36)
range
0.2-15.3
0.7-313
GM
1.5
16.2
Korea
(N=49)
range
0.2-6.0
0.4-20.4
GM
0.7
10.0
India
(N=46)
range
0.2-22.3
1.0-39.4
GM
3.6
7.7
Vietnam
(N=25)
range
0.2-34.2
6.1-192
GM
2.6
18.2
China
(N=51)
range
0.2-41.7
0.7-69.2
GM
3.6
9.5
Greece
(N=100)
range
0.3-14.1
0.2-20.4
GM
1.5
5.8
U.S.
(N=25)
range
0.3-5.3
0.3-14.8
GM
1.0
4.8
Biotransformation of BTRs (1/2)
.
The overall detection rate of BTRs that we found in urine was:
XTR (27.4%) > TTR (16.3%) > BTR (10.2%) > 4-/5-OH-BTR (5.7%) >1-OH-BTR (not detected).
1-OH-BTR was not detected in human urine, which suggests that it is
found at concentrations below the LOD of the method and/or degraded
(metabolized) quickly after its formation to ring scission products, such
as (2-nitrosophenyl)diazene.
The metabolic transformation of BTR to 4-/5-OH-BTR was found to be
relatively low (<5% product formation) in rat liver, which is in keeping with
the low detection frequency of 4-/5-OH-BTR (5.7%) that we found in human
urine.
Biotransformation of BTRs (2/2)
Taking into consideration the metabolism of
benzimidazole, a compound structurally
similar to BTR, hydroxylation of BTR is highly
expected at positions 5- (primary) and/or 4through cytochrome P-450 enzymes.
Biotransformation of BTHs
 We found no clear evidence
of metabolism of BTH to the
more
polar
2-OH-BTH
derivative.
 For 2-mercaptobenzothiazole,
in particular, it was proposed
that, instead of ring scission,
conjugation of the side chain
functional group occurs.
•The results of urine analysis support conjugation of the side chain functional
group, since 2-SCN-MeS-BTH and 2-Me-S-BTH were not detected in human
urine. However, this may be the case for other BTH derivatives also.
Summary
 Urine samples are a potential source of specimens for human
biomonitoring studies to assess exposures of BTRs & BTHs.
 XTR and BTH were the more abundant chemicals with the
highest concentrations found in urine.
Our chromatograms from 332 samples indicate that nontarget analysis must be applied in future studies.
 Human toxicological studies are needed primarily for BTH
since exposure levels are high.
“The dose makes the poison”
Acknowledgements
We thank Drs. Hyeon Seo Cho, Tu Binh Minh, and
Haruhiko Nakata for urine samples from Korea, Vietnam
and Japan,respectively.
This study was funded by a grant (1U38EH00046401)
from the Centers for Disease Control and Prevention
(CDC, Atlanta, GA) to Wadsworth Center, New York State
Department of Health.
References