Article

An alternative method for the determination of estrogens in surface water and wastewater treatment plant effluent using pre-column trimethylsilyl derivatization and gas chromatography/mass spectrometry. Environ Monit Assess

State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing, 100085, China.
Environmental Monitoring and Assessment (Impact Factor: 1.68). 11/2008; 158(1-4):35-49. DOI: 10.1007/s10661-008-0563-4
Source: PubMed

ABSTRACT

A procedure using pre-column trimethylsilyl derivatization and gas chromatography/ mass spectrometry (GC/MS) was developed and applied in determining trace estrogens in complex matrix. Main conditions were optimized, including pH value, salinity of water sample, elution reagents, clean procedure, derivative solvent and temperature. The optimized method was used to determine steroid estrogens in surface water and effluents of wastewater treatment plant (WWTP). Low detection limits of 0.01, 0.03, 0.03, 0.07, 0.09 and 0.13 ng/l for DES, E1, E2, EE2, E3 and E(V), respectively were obtained under optimism condition. No apparent interferences appeared in chromatography in comparison with ultrapure water blank. Mean recovery ranged from 72.6% to 111.0% with relative standard deviation of 1.1-4.6% for spiked surface water, and from 66.6% to 121.1% with relative standard deviation of 1.5-4.7% for spiked effluent of WWTP. The results suggested that the optimized method provides a robust solution for the determination of trace steroid estrogens in complex matrix.

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Available from: Yiqi Zhou, Nov 19, 2015
Environ Monit Assess (2009) 158:35–49
DOI 10.1007/s10661-008-0563-4
An alternative method for the determination of estrogens
in surface water and wastewater treatment plant efuent
using pre-column trimethylsilyl derivatization and gas
chromatography/mass spectrometry
Yiqi Zhou ·Jun Zhou ·Yiping Xu ·
Jinmiao Zha ·Mei Ma ·Zijian Wang
Received: 21 April 2008 / Accepted: 11 September 2008 / Published online: 21 October 2008
© Springer Science + Business Media B.V. 2008
Abstract A procedure using pre-column trimeth-
ylsilyl derivatization and gas chromatography/
mass spectrometry (GC/MS) was developed and
applied in determining trace estrogens in complex
matrix. Main conditions were optimized, includ-
ing pH value, salinity of water sample, elution
reagents, clean procedure, derivative solvent and
temperature. The optimized method was used to
determine steroid estrogens in surface water and
efuents of wastewater treatment plant (WWTP).
Low detection limits of 0.01, 0.03, 0.03, 0.07, 0.09
and 0.13 ng/l for DES, E1, E2, EE2, E3 and
E
V
, respectively were obtained under optimism
condition. No apparent interferences appeared
in chromatography in comparison with ultrapure
water blank. Mean recovery ranged from 72.6%
to 111.0% with relative standard deviation of
Y. Zhou · Y. Xu · J. Zha · M. Ma · Z. Wang (
B
)
State Key Laboratory of Environmental Aquatic
Chemistry, Research Center for Eco-Environmental
Sciences, Chinese Academy of Sciences,
P.O. Box 2871, Beijing 100085, China
e-mail: wangzj@rcees.ac.cn
J. Zhou
Beijing Drainage Group Corporation,
Beijing 100038, China
Present Address:
Z. Wang
Shuangqing Rd 18, Haidian District,
Beijing 100085, PR China
1.1–4.6% for spiked surface water, and from
66.6% to 121.1% with relative standard deviation
of 1.5–4.7% for spiked efuent of WWTP. The
results suggested that the optimized method pro-
vides a robust solution for the determination of
trace steroid estrogens in complex matrix.
Keywords Determination ·Estrogen ·
Derivativation ·Gas chromatography mass
spectrometry ·Water
Introduction
It has been long concerned that the environmental
impacts of the discharge of sewage efuents to
water body. Attention in recent years has been
focused on substances in sewage, which contain
endocrine disrupting properties to wildlife and
human (Feigelson and Henderson 1996;Graham
et al. 2000; Xiao et al. 2001).
Steroid estrogens of natural and anthropogenic
origin have been identified as the majority con-
tributors to endocrine-disrupting activity in both
sewage efuent and surface water (Desbrow et al.
1998; Lai et al. 2000; Solé et al. 2001). Estrogens
are extremely potent compounds and estrogenic
effects have been observed in laboratory stud-
ies down to 1 ng/l (Desbrow et al. 1998;Zha
et al. 2008). So it is very important to determine
the concentration of estrogens in environmental
Page 1
36 Environ Monit Assess (2009) 158:35–49
water. Considering low volatility of steroid estro-
gens, LC/MS (Ferguson et al. 2001; López and
Barceló 2001;Huetal.2005; Cui et al. 2006)
and LC/MS/MS (Zhang and Henion 1999; Baronti
et al. 2000; Johnson et al. 2000;Xuetal.2004,
2005, 2006;Maurícioetal.2006; Matejicek et al.
2007) methods have recently been developed for
the determination of these estrogenic compounds.
However, at present, the application of LC/MS
and LC/MS/MS in environmental analyses of es-
trogenic steroids appears to be limited for their
complex operation and capital costs. Therefore,
as a sensitive analysis technique for determining
trace estrogens in water, GC/MS is used widely
in environmental analyses (Desbrow et al. 1998;
Larsson et al. 1999; Rodgers et al. 2000; Zhou et al.
2007).
It is necessary for estrogens to be derivative
before analyzed by GC/MS. A range of derivatives
used for the determination of the steroid estro-
gens has been included in a review by López
and Barceló (2001). Silylanized derivatives are
predominantly used to facilitate determination
by GC (Alda and Barceló 2001). Since GC/MS
has been an available tool, N,O-bis(trimethylsily)
trifluoroacetamide (BATFA) and N-methyl-
N(trimethylsily)trifluoroacetamide (MSTFA) are
widely used in the derivatization of estrogenic
steroids by reason of its facile and low cost, which
leads to the formation of trimethylsily derivative
(Xiao et al. 2001; Jeannot et al. 2002;Zuoand
Zhang 2005; Zhou et al. 2007). However, a great
deal of research has recently reported that forma-
tion of multiple trimethylsilyl derivatives in the
derivatization of 17α-ethinylestradiol with BSTFA
or MSTFA followed by gas chromatography-
mass spectrometry determination (Shareef et al.
2004; Zuo and Zhang 2005; Zhou et al. 2007).
Fortunately, it has been found that it is an
effective solution by adding trimethylsilylimidaz
(TMSI) in BSTFA or MSTFA reagent (Zhou
et al. 2007) or using pyridine as derivatization
solvent (Zuo and Zhang 2005). Even so, the deter-
mination of trace estrogens in complex matrix
such as efuents of WWTP is still a challenge for
environmental monitoring.
A pre-column trimethylsilyl derivatization and
gas chromatography/mass spectrometry method
for the determination of estrogens had been built
in our previous research (Zhou et al. 2007). In
order to realize much lower detection limit and
higher recovery for environmental water sample,
in this study, the pretreatment conditions were
optimized to assure better recovery and less ma-
trix disruptor, and the conditions of derivatization
were modified to enhance sensitivity. Based on the
development of the previously proposed method,
a more sensitive and precise method was built.
Materials and methods
Chemicals and materials
All solvents were of HPLC grade, purchased
from J.T. Baker (Philipsburg, NJ, USA). Deriva-
tization reagents, BSTFA+1%TMCS and TMSI,
were both purchased from Aldrich (Milwaukee,
WI, USA). Estrogen standards, including diethyl-
stilbestrol (DES), estrone (E1), 17-β-estradiol
(E2), ethinylestradiol (EE2), estriol (E3) and
estradiol-17-valerate (E
V
), were all obtained from
Aldrich (Milwaukee, WI, USA), whose struc-
tures and physicochemical properties were shown
in Table 1. Surrogate standard 17-β-estradiol-
16,16,17-d
3
(E2-d
3
) was produced by C/D/N Iso-
topes (Montreal, Canada). Pyrene-d
10
obtained
from Supelco Inc. (Bellefonte, PA, USA) was
used as the internal standard (IS). Anhydrous
sodium sulfate was purchased from Beijing chemi-
cal plant (Beijing, China) and roasted at 450
Cfor
12 h before used. Hydrochloric acid and sodium
hydroxide were purchased from Beijing chemical
plant (Beijing, China). Stock standard solutions
were prepared by dissolving solid standard in
methanol. Calibration standards were evaporated
to dryness under a gentle flow of nitrogen. Then
80 μl derivative reagent BSTFA + 1%TMCS +
0.5%TMSI was added and diluted to 200 μlusing
hexane, and reacted for 30 min at air bath of 40
C.
Silica gel (60/200 mesh, ultra pure) and neu-
tral aluminum oxide (50/200 mesh, ultra pure),
which were obtained from Acros Organics (New
Jersey, USA), heated at 180
C and 250
Cfor
12 hr, respectively, and then both cooled in a
desiccator and de-activated with 3% of deion-
ized (DI) water. The Oasis HLB cartridge (6 cc,
500 mg) was purchased from Waters Corp.
Page 2
Environ Monit Assess (2009) 158:35–49 37
Table 1 Structure and physico-chemical properties of analytes
Compound Structure Abbreviation
CAS
number
Molecular
weight
a
Water
solubility
a
(mg/l)
LogKow
a
Vapor pressure
a
(mm Hg)
Henry’s law
constant
a
(atm-m
3
/mole)
pKa
b
Diethylstilb
estrol
HO
OH
DES
000056-
53-1
268.36 12 5.07 1.41E-008 5.8E-012 na
Estrone
CH
3
O
HO
E
1
000053-
16-7
270.37 30 3.13 1.42E-007 3.8E-010 10.4
17ß-estadiol
CH
3
OH
HO
E
2
000050-
28-2
272.39 3.6 (27 ºC)
4.01 1.26E-008 3.64E-011 10.23
Ethynyl
estradiol
CH
3
HO
OH
EE
2
000057-
63-6
296.41 11.3 (27 ºC) 3.67 1.41E-008 7.94E-012 10.21
Estriol
CH
3
HO
OH
OH
E
3
000050-
27-1
288.39 441 2.45 1.97E-010 1.33E-012 na
17
ß-estadiol
acetate
HO
H
H
H
O
CH
3
O
CH
3
Ev na
d
365.50 na 6.41
c
na na na
a
Refer to Kuster et al. (2004, 2005)
b
Refer to Yu et al. (2004)
c
Refer to Lai et al. (2000)
d
Not available
(Milford, MASS, USA). Visi-prep
TM
-DLSPE
Vacuum Manifold with disposable flow con-
trol valve liners and a solid-phase extraction
(SPE) system were purchased from Supelco Inc.
(Bellefonte, PA, USA).
Extraction and clarifying
Water samples were pre-filtered using 0.8–2.0 μm
APFF fiberglass filters (Millipore, Bedford, MA,
USA) to eliminate particulate matter, and then
they were spiked surrogate standard (E2-d
3
).Ad-
just pH value using 6 mol/l hydrochloric acid
or 6 mol/l sodium hydroxide before extraction.
The Oasis HLB cartridge was conditioned with
5.0 mL tert-butyl methyl ether, 5.0 ml methanol
and 5.0 ml DI water respectively. Water sample
(4.0 l) was passed through cartridge at a flow rate
of 10.0 ml/min. Then cartridge was dried under a
gentle stream of nitrogen. The cartridge was then
washed by 5.0 ml of methanol/water (25/75, v/v),
5 ml DI water, 5 ml methanol/ammonia/DI water
(10/2/88, v/v), then dried under a gentle stream
of nitrogen. Subsequently, 10.0 ml of organic sol-
vent was used to elute SPE cartridge. Elution was
gathered by KD concentrator and dehydrated by
anhydrous sodium sulfate, then reduced to 0.5 ml
under a gentle stream of nitrogen with 40
C water
bath.
The concentrated solution was cleaned up us-
ing column chromatography. At first, concen-
trated solution was subjected to a glass column
(10 mm i.d.) containing 10 g of 1:1 alumina/silica
gel, and 2.0 ml of methanol/acetone (50/50) was
used to wash KD concentrator. Transfer the so-
lution to the glass column. Then 10.0 ml organic
solvent was used to elute the column. The elution
was evaporated to dryness under a gentle stream
of nitrogen.
Derivatization and analysis
The dry residues extracted from sample were re-
dissolved in 100 μl organic solvent, then 80 μl
derivativereagentBSTFA+1%TMCS+0.5%TMSI
was added and diluted to 200 μl. This mixture
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38 Environ Monit Assess (2009) 158:35–49
solution reacted in air bath at 40
C for 30 min.
Derivatized samples were analyzed using an
Agilent 6890 gas chromatograph and 5973 quad-
rapole mass spectrometer equipped with a non-
polar HP-5 MS 30 m × 0.25 mm capillary column
with 0.25 μm film (Agilent, USA). The injector
was set at 300
C, and the oven temperature
was programmed at 80
C for 1 min, ramped at
20
C/min to 200
C, then ramped at 10
C/min to
300
C and maintained the temperature for 7 min.
The carrier gas was helium with a constant flow
rate at 1 ml/min. The mass spectrometer was
operated in the electron impact ionization mode
at 70 eV with an interface temperature of 280
C
and a source temperature of 230
C. Positive
fragment ions were analyzed over 50–550 m/z
mass range in SCAN mode for qualitative
analysis. All control of GC and MS parameters
and analysis of data were performed by MSD
Productivity Chemstation Software Rev. D.00.00.
Qualification and quantitation
Qualitative analysis of target compounds was car-
ried out at the basis of retention time and mass
spectrum together in a full SCAN mode.
Quantitative analysis was performed in a se-
lected ion monitoring (SIM) mode using internal
standard calibration. Retention time, characteris-
tic ions and quantitative ion of DES, E1, E2, EE2,
E3, E
v
were shown in Table 2.
Initially, a series of injections of target com-
pounds in the concentration range from 20 ng/ml
to 10 μg/ml with constant internal standard
100 ng/ml were performed to determine the lin-
ear concentration range. Calibration curves were
performed by linear regression analysis.
Method validation
The recoveries and overall method reproducibility
were determined from triplicate analyses (N = 3)
of spiked samples. Surface water and efuent of a
wastewater treatment plant (WWTP) were spiked
with 10 ng/l of the composite standard solution
of six kinds of estrogen 24 h before analysis
(to prevent microorganism growth, 0.5% volume
methanol was added). Then the samples were
analyzed by the method described above. Surface
water was taken from a fish pool about 12,000 m
2
,
which lies in Haidian district in Beijing and efu-
ent (TOC 6.46 mg/l, TSS 5.40 mg/l, pH 7.28) was
collected from Lugouqiao WWTP donating about
219700 m
2
and designed to process 0.2 millions m
3
wastewater per day, which lies in Fengtai district
in Beijing.
The limit of detection (LOD) and limit of quan-
tification (LOQ) for this method were achieved by
concentrating 4 L water, and they were calculated
as the minimum amount of a compound present
in the sample that produced a signal to noise ratio
of three to nine, based on an injection of a 1 μl
aliquot of the final 0.2 ml extract solution.
Results and discussion
Optimization of the pre-concentration procedure
The sorbent of SPE cartridge and elution reagent
were very important factors for SPE method ef-
Table 2 Parameters of
quantification used for
selected estrogens
IS Internal standard
(pyrene-d
10
being as
internal standard), SS
surrogate standard
(17-β-estradiol-16,16,17-
d
3
being as surrogate
standard)
Compounds Abbreviation Retention Characteristic Quantification
time (min) ions (m/z)ion(m/z)
Internal standard IS 12.04 106, 212 212
cis-diethylstilbestrol cis-DES 12.65 412, 397, 383 412
trans-diethylstilbestrol trans-DES 13.47 412, 397, 383 412
Estrone E1 15.57 342, 218, 257 342
17-β-estradiol E2 15.99 285, 416, 129 416
Surrogate Standard SS 15.99 132, 419 419
17-a-ethynylestradiol EE2 16.81 285, 425, 440 425
Estriol E3 17.42 129, 345, 504 504
Estradiol-17-valerate E
v
18.97 428, 231, 244 428
Page 4
Environ Monit Assess (2009) 158:35–49 39
ficiency. In previous studies, C
18
SPE cartridge
was applied (Ternes et al. 1999; Rodgers et al.
2000; López and Barceló 2001) due to its small
interference of complex matrix sample. However,
during the concentration procedure, the cartridge
might be dry, which would affect the recovery
efciency. As a result, it is difcult to obtain good
reproducibility using C
18
cartridge. In comparison,
Oasis HLB cartridge was a very good choice be-
cause of its hydrophobic and hydrophilic groups,
which were more beneficial for enrichment of
steroid estrogen (Benijts et al. 2004; Hernando
et al. 2004;Becketal.2005;Pecketal.2007).
Acidity of water sample would change the dis-
tribution of target substances between water and
cartridge. It was necessary to optimize pH condi-
tion of the sample to achieve higher recovery of
target compounds. The effect of pH was examined
in the range of 1.3–13.4, and 50 ng/l target com-
pounds were fortified according to estrogen com-
posite standards. It was shown that the recovery of
DES increased from pH 1.3 to 5.0 and decreased
after pH 9 (Fig. 1a). Increasing pH value led to an
increase recovery of E2. Lower recovery of E
v
was
observed for pH 5–8. However, the recoveries of
E1, EE2 and E3 were pH-independent evidently.
HLB cartridge contains hydrophobic and hy-
drophilic function groups, which interacted with
steroid estrogen, including absorption and parti-
tion. The adjustment of water pH value would
change the speciation of estrogens and surface
property of sorbent, which would affect absorp-
tion or partition of analytes on cartridge. Table 1
suggested that the pK
a
of steroid estrogens was
about 10.0. When pH value varied between 3
and 4, steroid estrogens might present at neutral
form in water. These forms were beneficial for
hydrophobic partitioning on cartridge. Mean-
while, when water pH was higher than 4 or lower
than 3, it would lead to analyte and cartridge
carrying negative or positive charges respectively,
which would affect the absorption of analytes on
sorbent. Therefore, the pH condition was opti-
mized from pH 3 to pH 4. Previous studies also
showed that better retention could be acquired by
acidified water sample (Ternes et al. 1999; Isobe
et al. 2003; Quintana et al. 2004; Bila et al. 2007).
Salinity may also affect the extraction efciency
of organic pollutant by either liquid–liquid extrac-
tion (LLE) or solid phase extraction. Effect of
salinity was evaluated by adding sodium chloride
in this study. Sodium chloride was added 100 and
200 g/l respectively to evaluate their effects with
control. Figure 1b showed that the recoveries of
estrogens did not change with the concentration
of sodium chloride except for E3. It was found that
residual sodium chloride induced the dehydration
of cartridge more difcult, though the increase
concentration of sodium chloride enhanced the
recovery of E3. Incomplete removal of water led
to the fail of derivative reaction between steroid
estrogen compounds and BSTFA. In order to re-
Concentration of NaCL (g/l)
Recovery(%)
pH
ab
0 50 100 150 2001 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 1 Effect of a pH and b salinity on the recovery of analytes (methanol was used as elution)
Page 5
40 Environ Monit Assess (2009) 158:35–49
Table 3 Comparison of recovery by using different elution for silicon gel filling column (n = 2)
Solvent Recovery (%)
DES E1 E2 EE2 E3 EV
(mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD)
Ethyl acetate 132 ±5 143 ± 3.8 101 ± 4.4 104 ± 3.2 0 106 ± 4.9
Acetone/hexane 103 ± 2.4 122 ± 3.2 86.1 ± 2.1 85.7 ± 2.5 38.2 ± 6.1 83.9 ± 2.2
(65/35, v/v)
Dichloromethane000000
duce operation process and guarantee the success
of derivative reaction, no variation of the salinity
was performed.
HLB cartridge would capture a lot of com-
pounds during the concentration procedure due to
coextraction. The eluting reagent was optimized
to ensure a better recovery of target compound
and less matrix disrupting. Quintana et al. (2004)
reported 70–107% of recovery could be obtained
using ethyl acetate as elution, and 80–109%
of recovery was realized by mixture elution of
tert-butyl methyl ether and methanol (1/9, v/v)
(Jeannot et al. 2002). Therefore, comparing the
two-elution strategies, mixture solvent of tert-
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
0
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
0
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
0
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
0
b
c
d
Retention time (min)
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
0
2000000
4000000
6000000
8000000
Time-->
Abundance
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
0
2000000
4000000
6000000
8000000
Time-->
Abundance
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
0
2000000
4000000
6000000
8000000
Time-->
Abundance
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
0
2000000
4000000
6000000
8000000
Time-->
Abundance
a
b
c
d
Fig. 2 GC-MS chromatograms of a crude extract, b clarified with silica gel, c clarified with silica gel and neutral aluminum
oxide, d clarified with neutral aluminum oxide in SCAN Mode
Page 6
Environ Monit Assess (2009) 158:35–49 41
Table 4 Comparison of recovery using different elution for neutral aluminium oxide filling column (n = 2)
Solvent Recovery (%)
(acetone/methanol) DES E1 E2 EE2 E3 EV
(mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD)
3/1 27.7 ± 3.4 89.2 ±5.7 95.0 ± 7.3 79.2 ±4.6 0 89.4 ± 5.3
1/1 76.5 ± 2.2 118 ± 3.5 122 ± 4.6 83.2 ± 3.1 52.6 ± 1.2 93.5 ±4.1
butyl methyl ether/ methanol (1/9, v/v)was
selected as elution reagent in this study. The
coelution was tested with two different solvents
above and the results suggested that there was
less coelution in tert-butyl methyl ether/ methanol
(1/9, v/v).
Optimization of the clarification procedure
The effect of sorbent (10 g) of cleaning column
and elution reagents on recovery and clarifying
efciency were investigated in this study. And dif-
ferent elution solvents were selected to test silicon
gel and neutral aluminum oxide. Ethyl acetate,
acetone/hexane (65/35, v/v) and dichloromethane
(10 ml solvent) were tested for their recoveries
in the silicon gel test. Table 3 showed that in
the experiment of recovery efciency that used
ethyl acetate as elution, higher recoveries of 132 ±
5.0%, 143 ±3.8%, 101 ± 4.4%, 104 ±3.2%, 106 ±
4.9% for DES, E1, E2, EE2, E
v
respectively were
obtained except for E3. Acetone/hexane (65/35,
v/v) could enhance the recovery of E3 to 38.2 ±
DES E1 E2 EE2 E3 Ev
0
2000
4000
10000
15000
20000
25000
Peak area
Compounds
40
50
60
70
80
Fig. 3 Effect of the reaction temperature on peak area of
analytes (hexane used as solvent and reaction half an hour)
6.1, but decreased the recoveries of E2, EE2,
E
v
to 86.1 ± 2.1%, 85.7 ± 2.5%, 83.9 ± 2.2%.
Dichloromethane could not provide good recov-
eries for these estrogens at all. However, as shown
in Fig. 2b, the silicon gel could not remove matrix
disrupting effectively.
When neutral aluminum oxide was used as
sorbent, the acetone/methanol ratios of 3/1 (v/v)
and 1/1 (v/v) were tested respectively. Table 4
suggested that acetone/methanol of 3/1 could not
elute E3 at all and made a lower recovery for DES
(27.7 ±3.4). When 1/1 acetone/methanol was used
as elution, the recovery of E3 could attain 52.6 ±
1.2% and the recovery of DES, E1, E2, EE2 and
E
v
were 76.5 ± 2.2, 118 ± 3.5, 122 ± 4, 83.2 ± 3.1
and 93.5 ± 4.1 respectively. As shown in Fig. 2d,
neutral aluminum oxide could eliminate the ma-
trix disrupting completely. The lower recoveries
might be due to the strong absorption of polar
estrogens on aluminum oxide.
DES E1 E2 EE2 E3 Ev
0
5000
10000
15000
40000
60000
80000
100000
120000
140000
Peak area
Compounds
Hexane
Ethyl acetate
Methyl dichloride
Acetone
Acetone/hexane (1/1:v/v)
Fig. 4 Effect of reaction solvent on peak area of analytes
(reaction half an hour at 40
Cairbath)
Page 7
42 Environ Monit Assess (2009) 158:35–49
Fig. 5 a GC-MS
chromatograms of TMS
derivatives of analytes in
SCAN Mode (injection
1 μl 1000 ng/ml mixture
standard) b GC-MS
chromatograms of
trimethylsilyl (TMS)
derivatives of analytes,
surrogate and internal
standard in SIM Mode
(injection 1 μl50ng/ml
mixture standard)
Abundance
Diethylstibestrol
Estrone
17 -Estradiol
17 -Ethinylestradiol
Estriol
17 -Estradiol-17valerate
Rentention time (min)
Abundance
Diethylstibestrol
Estrone
-Estradiol
-Ethinylestradiol
Estriol
-Estradiol-17valerate
Rentention time (min)
Abundance
Diethylstibestrol
Estrone
17 -Estradiol
17 -Ethinylestradiol
Estriol
17 -Estradiol-17valerate
R
Abundance
Diethylstibestrol
Estrone
-Estradiol
-Ethinylestradiol
Estriol
-Estradiol-17valerate
Rentention time (min)
a
β
β
β
α
β
α
Combining the characteristics of silicon gel and
neutral aluminum oxide, a cartridge with silicon
gel and neutral aluminum oxide 1:1 as sorbent
were designed. Efuent of WWTP was used to
check the recovery and clarification efciency.
Higher recovery and better matrix elimination
were obtained simultaneously when acetone/
methanol (1:1) was used to elute cartridge. Less
disrupting peaks were found in Fig. 2c comparing
with Fig. 2b, which was clarified only with silica
gel. The recovery of DES, E1, E2, EE2, E3 and
E
v
are 97.5 ±4.2, 108 ± 3.6, 98.2 ± 6.5, 94.2 ±2.0,
88.1 ± 1.6 and 92.6 ± 2.8.
Optimization of the derivative condition
It was a novel solution by adding trimethylsilylim-
idaz (TMSI) in BSTFA or MSTFA reagent to deal
with the formation of multiple trimethylsilyl deriv-
atives in the derivatization of 17α-ethinylestradiol
Page 8
Environ Monit Assess (2009) 158:35–49 43
Fig. 5 (continued)
OH
D
D
D
OH
17 -estradiol-16,16,17-d3
m/z=419
Pyrene-d10
Cis-diethylstibestrol
Trans-diethylstibestrol
Estrone
17 -Estradiol
17 -Ethinylestradiol
17 -Estradiol-17valerate
Estriol
Retention time (min)
Abundance
OH
D
D
D
OH
17 -estradiol-16,16,17-d3
m/z=419
Pyrene-d10
Cis-diethylstibestrol
Trans-diethylstibestrol
Estrone
17 -Estradiol
17 -Ethinylestradiol
17 -Estradiol-17valerate
Estriol
Retention time (min)
Abundance
β
β
α
β
b
with BSTFA followed by gas chromatography-
mass spectrometry determination (Zhou et al.
2007). To obtain higher sensitivity and broader
linearity, the effects of temperature and solvent on
derivative reaction were examined. In the investi-
gation of the effects of temperature on chromato-
graphic peak area, hexane was chose as reaction
solvent. Figure 3 showed that the peak area of
analytes deceased when derivative reaction tem-
perature increased in the range of 40
Cto
80
C except for E
2
, which was temperature-
independent. In the experiments of effects of tem-
perature, the greatest variation of peak area was
found for E
1
. The ratios of variation of peak area
were about 1.36, 2.70, 1.17, 1.55, 1.31, 2.63 for
DES, E
1
,E
2
,EE
2
,E
3
and E
v
respectively. The
peak area of analytes did not change with time
after half an hour reaction at 40
C. The reaction
temperature was set to 40
C and the sensitivity
was enhanced about two times. The optimal tem-
perature in this study was obviously lower than
those that used in previous work with different
derivative reagent from 60
C to 100
C (Jeannot
et al. 2002; Shareef et al. 2004). Further lower
temperature was not suitable for the reaction. A
standard with 1,000 ng/ml EE
2
was injected to test
whether undesirable TMS derivative would pro-
duced, and it was suggested that no undesirable
by-products of EE
2
were traced in SCAN Mode.
At 40
C, different solvent, including hexane,
ethyl acetate, dichloromethane, acetone and ace-
tone/hexane (1:1, v/v) was tested on media effects.
As shown in Fig. 4, the largest peak area of analyte
was achieved when hexane was used as solvent.
Ethyl acetate led to the smallest peak area for
DES, EE2 and E3. Undesirable TMS derivative
Page 9
44 Environ Monit Assess (2009) 158:35–49
Fig. 6 Mass spectra of
TMS derivative of a
di-trimethylsilyl (TMS)
diethylstilbestrol (DES),
b mono-TMS estrone
(E1), c di-TMS 17
β-estradiol (E2),
d di-TMS
17α-ethinylestradiol
(EE2), e tri-TMS estriol
(E3), f mono-TMS 17
β-estradiol acetate (E
v
)
m/z
Relative abundance (%)
(a)
[M-29]
+
m/z=383
[M-29]
+
m/z=383
[M]
+
m/z=412
m/z
Relative abundance (%)
(a)
[M-29]
+
m/z=383
[M-29]
+
m/z=383
[M]
+
m/z=412
m/z
Relative abundance (%)
[M-15]
+
m/z=327
[M]
+
m/z=342
(b)
m/z
Relative abundance (%)
[M-15]
+
m/z=327
[M]
+
m/z=342
(b)
m/z
Relative abundance (%)
[M-131]
+
m/z=285
[M-15]
+
m/z=401
[M]
+
m/z=416
(c)
m/z
Relative abundance (%)
[M-131]
+
m/z=285
[M-15]
+
m/z=401
[M]
+
m/z=416
(c)
Page 10
Environ Monit Assess (2009) 158:35–49 45
Fig. 6 (continued)
m/z
Relative abundance (%)
[M-131]
+
m/z=285
[M-1
5
]
+
m/z=425
[M]
+
m/z=440
(d)
m/z
Relative abundance (%)
[M-131]
+
m/z=285
[M-1
5
]
+
m/z=425
[M]
+
m/z=440
(d)
/
Relative abundance (%)
[M]
+
m/z=504
(e)
[M-15]
+
m/z=489
Relative abundance (%)
[M]
+
m/z=504
(e)
[M-15]
+
m/z=489
m/z
Relative abundance (%)
[M-15]
+
m/z=413
[M]
+
m/z=428
(f)
m/z
Relative abundance (%)
[M-15]
+
m/z=413
[M]
+
m/z=428
(f
m/z
Page 11
46 Environ Monit Assess (2009) 158:35–49
Table 5 Average
recoveries and relative
standard (RSD) for 6
estrogens spiked 10 ng/l in
surface water and efuent
of WWTP (n = 3)
Estrogens Surface water Efuent of WWTP
Mean recoveries (%) RSD (%) Mean recoveries (%) RSD (%)
DES 80.7 4.5 66.6 3.3
E1 111.0 2.4 90.4 4.7
E2 81.3 4.4 85.3 1.5
EE2 107.5 3.7 121.1 2.6
E3 72.6 1.1 87.5 4.3
E
v
90.9 4.6 74.8 2.1
of EE2 was not present in above reactions. There-
fore, hexane was selected as reaction solvent.
Method performance
According to the optimized condition, water sam-
ples were pre-filtered using 0.8–2.0 μm APFF
fiberglass filters to remove particulate matter,
and then were spiked surrogate standard (E2-d
3
).
The pH value was adjusted to 3–4 using 6 mol/l
hydrochloric acid or 6 mol/l sodium hydroxide
before extraction. The Oasis HLB cartridge was
conditioned with 5.0 mL tert-butyl methyl ether,
5.0 ml methanol and 5.0 ml DI water respec-
tively. Water sample (4.0 l) was passed through
cartridge at a flow rate of 10.0 ml/min. Then car-
tridge was dried under a gentle stream of nitro-
gen. The cartridge was then washed by 5.0 ml of
methanol/water (25/75, v/v), 5 ml DI water, 5 ml
methanol/ammonia/DI water (10/2/88, v/v), then
dried under a gentle stream of nitrogen. Subse-
quently, 10.0 ml of methanol/ tert-butyl methyl
ether (1/9, v/v) was used to elute SPE cartridge.
Elution was gathered by KD concentrator and
dehydrated by anhydrous sodium sulfate, then re-
duced to 0.5 ml under a gentle stream of nitrogen
with 40
C water bath. The concentrated solution
was cleaned up using column chromatography.
At first, concentrated solution was subjected to a
glass column (10 mm i.d.) containing 10 g of 1:1
alumina/silica gel, and 2.0 ml of methanol/acetone
(50/50) was used to wash KD concentrator. Trans-
fer the solution to the glass column. Then 10.0 ml
methanol/ acetone (1/1, v/v) was used to elute
the column. The elution was evaporated to dry-
ness under a gentle stream of nitrogen. The dry
residues were redissolved in 100 μl hexane, then
80 μl derivative reagent BSTFA + 1%TMCS +
0.5%TMSI was added and diluted to 200 μl. This
mixture solution was reacted in air bath at 40
C
for 30 min and then analyzed by GC/MS.
Figure 5 showed the chromatograms of analytes
in SCAN and SIM mode. Figure 6 explained the
corresponding mass spectra on detail. Good chro-
matography separation was realized and char-
acteristic ion was obtained. The trimethylsilyl
(TMS) derivatives of DES, E1, E2, EE2, E3 and
E
v
were (a) di-TMS DES, (b) mono-TMS E1, (c)
di-TMS E2, (d) di-TMS EE2, (e) tri-TMS E3, (f)
mono-TMS E
v
. No undesirable TMS derivative
of EE2 was found. Instead, two isomers of DES
(cis-diethylstilbestrol and trans-diethylstilbestrol)
Table 6 Calibration curve, limit of detection (LOD) and limit of quantification (LOQ) for six estrogens fortified at 20, 40,
60, 80, 100, 500, 1,000 ng/ml
Compound Calibration curve R
2
LOD
a
(ng/l) LOQ
b
(ng /l)
DES y = 4173.5x 0.9979 0.01 0.03
E1 y = 1521.6x 0.9987 0.03 0.09
E2 y = 1454.0x 0.9951 0.03 0.09
EE2 y = 948.5x 0.9968 0.07 0.21
E3 y = 558.3x 0.9981 0.09 0.27
E
v
y = 343.4x 0.9981 0.13 0.39
a
Values estimated on a minimal S/N of 3
b
Values estimated on a minimal S/N of 9
Page 12
Environ Monit Assess (2009) 158:35–49 47
Table 7 Range, mean
and standard deviation
(SD) in surface water and
efuent of WWTP (ng/l)
nd No detection
Estrogens Surface water Efuent of WWTP
Range Mean SD Range Mean SD
DES nd nd–0.30 0.15 0.21
E1 1.60–2.00 1.80 0.30 1.90–3.00 2.45 0.78
E2 0.30–0.50 0.40 0.10 nd–2.00 1.00 1.41
EE2 nd–1.60 0.80 1.10 1.90–2.50 2.20 0.42
E3 nd 0.60–1.90 1.25 0.92
E
v
nd nd
were presented in Fig. 5b, while only the peak of
trans-DES appeared in Fig. 5a due to very small
ratio of cis-DES in total DES. Calculating its con-
centration on the basis of the correlation of total
peak area and total concentration, good linearity
was observed with correlation coefcients above
0.99 for concentrations from 0 to 1000 ng/ml and
a better linearity and instrument detection limit
(IDL) were obtained.
The procedure was applied in spiked surface
water and efuents of WWTP. Mean recovery
ranged from 72.6 to 111.0% with relative standard
deviation 1.1–4.6% for spiked surface water, and
66.6% to 121.1% with relative standard deviation
1.5–4.7% for spiked efuent of WWTP. Detailed
results were shown in Table 5. The limit of detec-
tion (LOD) was similar with the value calculated
from instrument detection limit (IDL). LOD of
analytes could even attain 0.01 ng/l (Table 6).
The method was applied to determine estro-
gens concentrations in surface water and waste-
water samples. The mean concentrations of E1,
E2, and EE2 in surface water ranged from 1.6
to 2.0, 0.3 to 0.5, no detection (nd) to 1.6 ng/l,
respectively. No DES, E3 and E
v
were detected in
surface water. The mean concentrations of DES,
E1, E2, EE2 and E3 in efuents of WWTP ranged
from nd to 3.0, 1.9 to 3.0, nd to 2.0, 1.9 to 2.5
and 0.6 to 1.9 ng/l respectively. Detail referred to
Table 7. Previous study widely reported the pres-
ence of steroid estrogens in surface water and
efuent of WWTPs. In river water sample, con-
centration of E1, E2 and EE2 were changed from
nd to 74 ng/l in United Kingdom (Fawell et al.
2001), Italy (Baronti et al. 2000) and China (Chen
et al. 2007). In efuent of WWTPs, common de-
tected value were nd to 82, nd to 64, nd to 42
and 0.4 to 39.1 ng/lfor E1, E2, EE2 and E3 in
Netherlands (Vethaak et al. 2005), Italy (Baronti
et al. 2000), U.K. (Desbrow et al. 1998; Johnson
and Sumpter 2001), Germany and Canada (Ternes
et al. 1999), Sweden (Larsson et al. 1999), Japan
(Nakada et al. 2007) and China (Wang et al. 2005;
Chen et al. 2007; Sun et al. 2008). Our results
in this study were within the range of previous
report.
Conclusions
An optimized condition for determining trace es-
trogen in surface water and wastewater treatment
plant efuent had been obtained in the current
work. The pH value of water sample was adjusted
to 3–4, and salinity was not altered. HLB cartridge
was conditioned with 10 mL methanol/ tert-butyl
methyl ether (1/9, v/v). Extract was clarified using
a glass column (10 mm i.d.) containing 10 g of 1:1
alumina/silica gel with 10 mL acetone/methanol
elution, then dried under gentle nitrogen. The
dried residuals were derivatized by TMSI and
BSTFA in hexane solvent at 40
C in air bath, then
analyzed by GC/MS.
Correlation coefcient of 0.9951 to 0.9987 was
determined. Detection limits were 0.01, 0.03, 0.03,
0.07, 0.09, 0.13 ng L
1
for DES, E1, E2, EE2, E3
and E
v
, based on a 4.0-l samples. Mean recovery
ranged from 72.6 to 111.0% with relative standard
deviation of 1.1–4.6% for surface water spiked,
and 66.6% to 121.1% with relative standard de-
viation of 1.5–4.7% for efuent of WWTP spiked.
Therefore, this method provided a robust solution
for the determination of trace steroid estrogen in
surface water and efuent of WWTP.
Acknowledgements This work was supported by Chinese
Academy of Sciences (KZCX1-YW-06) and National Basic
Research Program of China (2007CB407301).
Page 13
48 Environ Monit Assess (2009) 158:35–49
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    • "For assessment of over-all method recovery, seven 1-L deionized water samples were spiked to 100 ng À1 17b-estradiol and processed using the same SPE/LC–MS protocol as all samples. Overall recovery was 95 ± 16%, consistent with published results from other authors using the same SPE cartridges (Stavrakakis et al., 2009; Zhou et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: The fate and transport of endocrine disrupting chemicals (EDCs) in ambient river waters is a major concern associated with effluents from municipal wastewater treatment plants (WWTPs). This paper presents a methodology for quantifying the spatial distribution of EDCs in a river mixing zone. The core of the technical analysis is based on a two-dimensional steady-state analytical model characterized by ambient turbulence in the receiving water. This model was first calibrated with mass transport data from field measurements for a conservative substance (electrical conductivity) and then used to predict aqueous-phase EDC concentrations throughout a WWTP mixing zone. To demonstrate the usefulness of this methodology for water quality management purposes, the modeling framework presented in this paper was used to determine a lumped in-stream attenuation rate constant (k(d)=3 d(-1)) for 17β-estradiol under natural conditions. This rate constant likely accounts for the combined contributions of physical sorption, photolysis, microbial and chemical degradation, and the measured value is highly consistent with previously published results from bench-scale removal experiments.
    No preview · Article · Feb 2012 · Chemosphere
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    • "Limit of detection (LOD) and limit of quantification (LOQ) were achieved by 1,250 times concentration and 5,000 times concentration for influent and effluent from the WWTPs. They were calculated as the minimum amount of a compound present in the sample that produced a signal to noise ratio of six, based on a 1-μl aliquot injection of the final 0.4 ml extraction solution (Zhou et al. 2009). LOD and LOQ in water were 0.05–0.48 "
    [Show abstract] [Hide abstract] ABSTRACT: Concentration levels of six natural and anthropogenic origin steroid estrogens, namely, diethylstilbestrol (DES), estrone (E1), estradiol (E2), estriol (E3), ethinylestradiol (EE2), and estradiol-17-valerate (Ev), from different effluents in Beijing were assessed. Sampling sites include two wastewater treatment plants (WWTPs), a chemical plant, a hospital, a pharmaceutical factory, a hennery, and a fish pool. In general, concentrations of estrogens in the effluents varied from no detection (nd) to 11.1 ng/l, 0.7 to 1.2 × 10(3) ng/l, nd to 67.4 ng/l, nd to 4.1 × 10(3) ng/l, nd to 1.2 × 10(3) ng/l, and nd to 11.2 ng/l for DES, E1, E2, EE2, E3, and Ev, respectively. The concentration levels of steroid estrogens from different effluents decreased in the order of pharmaceutical factory and WWTP inlets > hospital > hennery > chemical factory > fish pool. This study indicated that natural estrogens E1, E2, and E3 and synthetic estrogen EE2 are the dominant steroid estrogens found in the different Beijing effluents. For source identification, an indicator (hE = E3/(E1 + E2 + E3)) was used to trace human estrogen excretion. Accordingly, hE in effluents from the hospital and WWTP inlets exceeded 0.4, while much smaller values were obtained for the other effluents. Human excretions were the major contributor of natural estrogens in municipal wastewater. Estimation results demonstrated that direct discharge was the major contributor of steroid estrogen pollution in receiving waters.
    Full-text · Article · May 2011 · Environmental Monitoring and Assessment
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    [Show abstract] [Hide abstract] ABSTRACT: We study pulse dynamics in one-dimensional heterogeneous media. In particular we focus on the case where the pulse is close to the singularity of codim 2 type consisting of drift and saddle-node instabilities in a parameter space. We assume that the heterogeneity is of jump type, namely one of the coefficients of the system undergoes an abrupt change at one point in the space. Depending on the height of this jump, the responses of pulse behavior are penetration, splitting, and rebound. Taking advantage of the fact that pulse is close to the singularity, the PDE dynamics can be reduced to a finite-dimensional system, which displays the three behaviors. Moreover it takes a universal form independent of model systems, and is valid for much more general heterogeneities such as bump, periodic, and random cases.
    Full-text · Article · Feb 2007 · Hokkaido Mathematical Journal
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