5991 4685EN

5991 4685EN
Determination of Endocrine-Disrupting
Chemicals in Drinking Water at
Sub ng/L Levels Using the Agilent
6495 Triple Quadrupole Mass
Spectrometer
Application Note
Authors
Abstract
László Tölgyesi
This Application Note demonstrates the precise, accurate, and robust
Agilent Technologies Sales and
determination of endocrine-disrupting chemicals (EDCs) in drinking water at low
Services GmbH & Co. KG, Chemical
concentrations (ng/L) using the new Agilent 6495 Triple Quadrupole LC/MS system
Analysis Group,
operated in dynamic multiple reaction monitoring (DMRM) and fast polarity
Hewlett-Packard-Str. 8, 76337
(positive/negative) switching mode. The increased sensitivity of the instrument
Waldbronn, Germany
was used to streamline the analysis using direct large volume injection of tap
Dan-Hui Dorothy Yang, Bernhard
Wuest, and Anabel Fandino
Agilent Technologies, Inc.
5301 Stevens Creek Blvd,
Santa Clara, CA 95051-7201,
USA
water samples instead of more tedious and time-consuming sample enrichment by
solid phase extraction (SPE) methods.
Introduction
The presence of EDCs in the aquatic
system has raised concerns about the
aquatic environment and its relation
to human health1. Excreted EDCs from
humans and animals enter raw sewage
and reach wastewater treatment plants
either through direct discharge into the
human effluent or agricultural runoffs.
These compounds, if not removed
during the various treatment processes
(for example, oxidation and activated
carbon), may lead to contamination
of the drinking water system. EDCs in
sufficient concentrations can interfere
with the endocrine system and cause
adverse health effects in an organism or
its progeny. Recently, both estrogens and
androgens have received considerable
attention since they promote feminization
and masculinization in fish2.
EDC levels in municipal water supplies
are regulated by several government
agencies down to part-per-trillion (ppt)
levels (EPA Method 539, EPA Method
1698)3,4. Such low concentrations (ng/L
or pg/L) pose significant analytical
challenges. Sample enrichment is often
necessary using solid phase extraction
(SPE) or liquid liquid extraction (LLE)
where detection is performed using low
to mid-range triple quadrupole mass
spectrometers. Furthermore, both SPE
and LLE require large sample quantities,
high consumption of solvents, and
laborious procedures5.
This Application Note describes
improvements to a previously published
application note, and demonstrates how
the increased sensitivity of the 6495
Triple Quadrupole LC/MS can be used
to simplify the analytical workflow5.
Several modifications of the triple
quadrupole mass spectrometer have
resulted in better analytical performance.
Improvements include new front-end
ion optics for increased precursor ion
transmission, a newly designed curved
Honeywell (Catalog number 230-4 and
015-4, respectively). Ultrapure water
was obtained from a Milli-Q Integral
system equipped with LC-Pak Polisher
and a 0.22-μm membrane point-of-use
cartridge (Millipak). Ammonium
fluoride was purchased from Fluka
(338869-25 g), from which a 5 M stock
solution was prepared by dissolving
the appropriate amount of ammonium
fluoride in Milli-Q water. The EPA 539
calibration stock standard was purchased
from RESTEK (p/n 31998) containing
the target hormone compounds in a
single mix standard at the following
concentrations: androstenedione
(99.9 µg/mL), equilin (200.0 µg/mL),
17-b-estradiol (250.0 µg/mL), estriol (E3)
(200.0 µg/mL), estrone (E1)
(200.0 µg/mL), 17-a-ethinylestradiol
(EE) (351.0 µg/mL), and testosterone
(100.0 µg/mL). The chemical structures of
these hormones are given in Figure 1.
and tapered collision cell providing
enhanced MS/MS spectral fidelity, a
new ion detector operating at dynode
accelerating voltages up to 20 kV, and a
new tune algorithm for enhanced speed
and sensitivity. In addition, the 6495 Triple
Quadrupole Mass Spectrometer uses
the proven Agilent JetStream Ionization
source in combination with a dual stage
ion funnel and hexabore capillary for more
efficient ion generation and sampling.
The enhanced sensitivity enables large
injection volumes of water samples so
that sample enrichment is no longer
required to meet the Limit of Detection
(LOD) requirements at sub ng/L.
Experimental
Reagents and chemicals
All reagents and solvents were of
HPLC-MS or analytical grade. Methanol
and acetonitrile were purchased from
O
OH
H
H
H
H
H
Estrone
H
H
H
HO
17-b-estradiol
Estriol
OH
O
OH
H
H
OH
H
HO
HO
H
H
H
O
HO
Androstenedione
Testosterone
OH
H
H
HO
Ethynyl estradiol
Figure 1. Structures of hormones.
2
H
H
O
H
OH
Equilin
H
Dilutions
Method
The EPA 539 calibration stock standard
was used to prepare the working standard
in methanol:water (1:1 v/v) solution.
A binary gradient method was used with
0.4 mM ammonium fluoride as Solvent A,
and methanol:acetonitrile (1:1, v/v) as
Solvent B. An Agilent Poroshell Phenyl
Hexyl 2.1 × 100 mm, 2.7-µm column
(p/n 695775-912) was used at a flow rate
of 0.4 mL/min; the gradient is detailed
in Table 2. The column temperature
was maintained at 40 °C and injection
volume was 900 µL (draw/eject speed:
1,000 µL/min; needle wash in vial:
100 % methanol). MS parameters
and compound specific acquisition
Compound concentrations in the working
standard were as follows:
•
Androstenedione: 10 ng/mL
•
Equilin: 20 ng/mL
•
17-b-Estradiol (E2): 25 ng/mL
•
Estriol (E3): 20 ng/mL
•
Estrone (E1): 20 ng/mL
•
17-a-Ethynylestradiol (EE):
35 ng/mL
•
settings are presented in Tables 3 and 4,
respectively. Cycle time was adjusted to
500 ms for the dynamic MRM acquisition
mode, and the minimum and maximum
dwell times were 34.1 and 249.1 ms.
MRM transitions for the target hormone
compounds and associated collision
energies were automatically determined
in positive and negative mode using
MassHunter Optimizer Software by
flow injection analysis. Ion source
parameters were optimized using the
final chromatographic method with
MassHunter Source Optimizer tool.
Table 1. Calibration series prepared in tap water (Santa Clara, CA).
Testosterone: 10 ng/mL.
Level
Testosterone
(ng/L)
Androstenedione
(ng/L)
Equilin
(ng/L)
E3
(ng/L)
E1
(ng/L)
EE
(ng/L)
E2
(ng/L)
Using the working standard, the
calibration series detailed in Table 1 was
prepared in tap water (source: Santa
Clara, CA, USA).
6
10
10
20
20
20
35.1
25
5
5
5
10
10
10
17.55
12.5
4
1
1
2
2
2
3.51
2.5
3
0.5
0.5
1
1
1
1.76
1.25
Instrumentation
2
0.2
0.2
0.4
0.4
0.4
0.7
0.5
1
0.1
0.1
0.2
0.2
0.2
0.35
0.25
Chromatographic separation was carried
out using an Agilent HPLC system
consisting of an Agilent 1260 Binary
Pump (G1312B), an Agilent 1260 Infinity
Standard Autosampler (G1329B equipped
with a 900-µL loop), sample cooler
(G1330B), and an Agilent 1290 Infinity
Thermostatted Column Compartment
(G1316C). The HPLC system was coupled
to an Agilent G6495 Triple Quadrupole
Mass Spectrometer.
Agilent MassHunter Data Acquisition
for Triple Quadruple Mass Spectrometer
(version B 07.00) was used for data
acquisition, while Agilent MassHunter
Qualitative (version B 06.00) and Agilent
MassHunter Quantitative software
(version B 07.00) were used for data
processing.
Table 3. MS parameters – Positive and negative
polarity.
Table 2. Gradient table.
Time (min)
%B
Parameter
0.0
1
Gas temperature
210 °C
4.2
1
Gas flow
15 L/min
5.5
35
Nebulizer
45 psi
12.0
95
Sheath gas temperature
375 °C
12.1
1
Sheath gas flow
12 L/min
Stop time
16
Capillary voltage
3,500/4,000 ± V
Post time
6
Nozzle voltage
0/0 ± V
Delta EMV
250/250 ± V
HP RF voltage
190/190 ± V
LP RF voltage
80/100 ± V
3
Value
Results and Discussion
Increased method performance
Table 4. DMRM parameters for the target compounds.
Compound name
Precursor MS1
ion
Res
Product MS2
ion
Res
RT
RT
window
CE
(V)
CAV
(V)
17-a-ethynylestradiol
295.2
Widest
145
Wide
12.4
1
47
2
−
17-a-ethynylestradiol
295.2
Widest
159
Wide
43
2
−
17-b-estradiol
271.2
Widest
183
Unit
17-b-estradiol
271.2
Widest
145
Unit
Androstenedione
287.2
Unit
108.9
Unit
Androstenedione
287.2
Unit
96.9
Unit
Equilin
267.1
Unit
265.1
Unit
Equilin
267.1
Unit
Estriol
287.2
Widest
Estriol
287.2
Instrument Detection Limit (IDL)
and Lower Limit of Quantitation
(LLOQ)
Estrone
Estrone
IDL refers to the minimum amount of
analyte required to produce a signal
that is statistically distinguishable
from background noise with a given
confidence level. This approach helps to
avoid ambiguity related to the variation
in the chemical noise and the different
ways in which signal-to-noise (S/N) are
determined. To test the sensitivity of the
instrument and the feasibility of detecting
hormones in drinking water at sub ppt
level, target compounds were diluted in
tap water using the working standard
according to Table 1.
The mathematical formula for the IDL
calculation is described below, where t
corresponds to 99 % (1–a) confidence
level at n–1 degrees of freedom
(n = number of replicate injections, eight
in this case), and RSD % is the relative
standard deviation (precision) of signal
response at the amount measured, from n
replicate injections6.
(RSD%
100 ) @ amount_measured
12.7
1
1
1
143.1
Unit
171.2
Widest 10.8
1
Widest
145
Widest
269.2
Unit
145
Unit
269.2
Unit
143
Unit
Testosterone
289.2
Unit
108.9
Unit
Testosterone
289.2
Unit
96.9
Unit
×10 2
12.7
12.4
1
1
Polarity
47
2
−
51
2
−
26
2
+
24
2
+
28
4
−
42
2
−
44
2
−
50
2
−
43
2
−
61
2
−
28
2
+
22
2
+
269.2 & 145.0, 269.2 & 143.0
Ratio = (92.5 %)
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
6.0
IDLLCMS = t @ SD = t @
12.2
12.5
Counts
The 6495 Triple Quadrupole LC/MS
design enhancements have demonstrated
a significant increase in ion transmission.
In addition, the detector design provides
signal gains especially for negative ions.
Figure 2 shows the response of 0.25 pg
estrone on the 6495 Triple Quadrupole
LC/MS. The increases in response
comparing with a 6490 instrument,
ranged from a factor of 2 to 5 for the
seven hormones discussed in this study.
6.5
7.0
Acquisition time (min)
7.5
8.0
Figure 2. Signal response of the Agilent 6495 Triple Quadrupole LC/MS System (0.25 pg estrone on
column).
4
Table 5 shows the RSD % and IDL as well
as the spike concentrations at which the
calculations were made.
Figure 3 shows an overlay of MRM
chromatograms of the target compounds
(quantifier and qualifier transitions) to
illustrate the separation efficiency of the
method.
LLOQ values were calculated (S/N > 10)
for the quantifier (peak-to-peak), area
RSD % < 20 and accuracy values within
80–120 %. The observed LLOQ values
demonstrated very good correlation with
the IDL values (data not shown) that
ranged from 0.1 to 1.75 ng/L.
Table 5. The calculated IDL and RSD % values.
Compound name
Concentration (ng/L)
RSD%
IDL (ng/L)
17-a-Ethynylestradiol
1.8
14.8
0.78
17-b-estradiol
0.5
13.5
0.20
Androstenedione
0.2
4.3
0.03
Equilin
0.2
3.7
0.02
Estriol
1.0
5.6
0.17
Estrone
0.2
7.2
0.04
Testosterone
0.1
10.3
0.03
×10 4
8.5
Testosterone
8.0
7.5
7.0
6.5
6.0
Counts
5.5
Estrone
5.0
Androstenedione
4.5
4.0
3.5
Equilin
3.0
17-a-ethynylestradiol
2.5
2.0
1.5
Estriol
1.0
17-b-estradiol
0.5
0
10.4
10.6
10.8
11.0
11.2
11.4
11.6
11.8
12.0
Acquisition time (min)
12.2
12.4
12.6
12.8
13.0
Figure 3. Chromatogram of calibration Standard 5 including all seven hormones (quantifier and qualifier transitions) in overlaid representation to illustrate the
separation efficiency of the method.
5
The chromatograms at or close to the
LLOQ levels for all seven hormones are
shown in Figure 4.
*12.424 minutes
1151
17-aEthynylestradiol
×10 2
8
– MRM (271.2 & 145.0) Op5ppt
*12.147 minutes
2136
5
Counts
Counts
2.5
17-bEstradiol
6
Counts
×10 3
4
3
– MRM (287.2 & 145.0) Op1ppt
*10.730 minutes
4536
×10 2
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
– MRM (269.2 & 145.0) Op1ppt
*12.634 minutes
1439
Estrone
10.6 10.8 11.0 11.2
Acquisition time (min)
Equilin
4
3
12.2 12.4 12.6 12.8
Acquisition time (min)
+ MRM (289.2 & 96.9) Op1ppt
*12.337 minutes
4776
Testosterone
Counts
Counts
10.4
12.4 12.6 12.8 13.0
Acquisition time (min)
×10 3
2.0
1.75
1.5
1.25
1.0
0.75
0.5
0.25
0
5
0
12.0 12.2 12.4 12.6
Acquisition time (min)
Counts
Estriol
×10 2
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
6
– MRM (267.1 & 265.1) Op1ppt
*12.522 minutes
2168
1
0
11.8
×10 2
2
0.5
1
12.2 12.4 12.6 12.8
Acquisition time (min)
1.5
1.0
2
12.0
2.0
+ MRM (287.2 & 96.9) Op2ppt
*12.692 minutes
8063
Androstene
dione
Counts
– MRM (295.2 & 159.0) Op5ppt
×10 2
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
12.0
12.4 12.6 12.8 13.0
Acquisition time (min)
12.2 12.4 12.6
Acquisition time (min)
12.8
Figure 4. Extracted quantifier MRM transitions for all seven hormones at close to LLOQ level
Linearity
Linearity was assessed with spiked tap
water samples covering a concentration
range of 2 orders of magnitude (Table 1).
Calibration curves for equilin and
17-b-estradiol are shown in Figure 5.
×10 5
2.5
2.25
The equations of the linear fit and the
corresponding correlation coefficients (R2)
for all target analytes are listed in Table 6.
In each case, a weight factor of 1/x was
applied.
Equilin - 6 Levels, 6 Levels used, 44 Points, 44 Points used, QCs
y = 12471.659549 *x – 683.699386
R 2 = 0.99650697
Type: Linear, Origin: Ignore, Weight: 1/x
Responses
Responses
2.0
1.75
1.5
1.25
1.0
0.75
0.5
0.25
0
0
2
4
6
8
10 12
14
Concentration (ng/L)
16
18
×10 4
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
-0.5
20
17-β-estradiol - 6 Levels, 6 Levels used, 44 Points, 44 Points used,...
y = 2106.472502 *x – 247.242686
R 2 = 0.99560663
Type: Linear, Origin: Ignore, Weight: 1/x
0
Figure 5. Calibration curves of equilin and 17-β-estradiol in spiked tap water samples.
6
2
4
6
8 10 12 14 16 18
Concentration (ng/L)
20 22 24 26
Table 6. Linear regression parameters.
Linear equation
R2
17-a-Ethynylestradiol
y = 804.12x – 255.39
0.996
17-b-estradiol
y = 2106.47x – 247.24
0.996
Androstenedione
y = 43246.58x + 2273.65
0.995
Equilin
y = 12471.66x – 683.70
0.997
Estriol
y = 2257.79x + 277.07
0.997
Estrone
y = 8749.02x – 656.93
0.996
Testosterone
y = 65307.71x – 2825.52
0.994
Conclusion
References
A fast and simple LC/MS/MS method
for the sensitive, precise and accurate
quantitation of the hormones regulated
by the EPA Method 539 has been
developed using an Agilent 6495 Triple
Quadrupole Mass Spectrometer. The
enhanced instrument’s sensitivity was
demonstrated based on signal response
precision with instrument detection limits
(IDLs) ranging from 0.02 to 0.78 ng/L.
Sub ng/L IDLs were achieved while using
a streamlined analytical workflow with
direct injection of tap water samples
instead of time-consuming offline-solid
phase extraction.
1. Flores-Valverde, A.M.; Horwood, J.;
and Hill, E.M. Disruption of the
steroid metabolome in fish caused
by exposure to the environmental
estrogen 17alpha-ethinylestradiol.
Environ. Sci. Technol. 2010, 44(9)
pp 3552-8.
2. Jenkins, R., et al., Identification of
androstenedione in a river containing
paper mill effluent. Environ. Toxicology
and Chem. 2001, 20(6) pp 1325-1331.
3. EPA, Method 1698: Steroids and
Hormones in Water, Soil, Sediment,
and Biosolids by HRGC/HRMS. 2007.
7
4. EPA, EPA Method 539 – Determination
of Hormones in Drinking Water by
Solid Phase Extraction (SPE) and
Liquid Chromatography Electrospray
Ionization Tandem Mass Spectrometry
(LC-ESI-MS/MS), O.o.W.M. 140),
Editor. 2010.
5. Hindle, R., Improved Analysis of
Trace Hormones in Drinking Water
by LC/MS/MS (EPA 539) using
the Agilent 6460 Triple Quadrupole
LC/MS, Agilent Technologies,
publication number 5991-2473EN,
2013.
6. Parra, N.P; Taylor, L. Why Instrument
Detection Limit (IDL) is a Better Metric
for Determining The Sensitivity of
Triple Quadrupole LC/MS Systems,
Agilent Technologies Technical
Overview, publication number
5991-4089EN, 2014.
www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc., 2014
Published in the USA, June 24, 2014
5991-4685EN
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