High-sensitivity determination of estrogens in fish plasma using chemical derivatization upstream UHPLC–MSMS

Article abstract of DOI:10.1016/j.steroids.2017.04.003

This article describes the development and validation of a sensitive LC–MSMS method for determination of estrogen in fish plasma. Dansyl chloride derivatization of the phenol functional group in estrogen was used to enhance the response to atmospheric pressure ionization leading to improve the sensitivity. Individual ¹³C internal standards were selected after comparison with deuterated standards. Liquid-liquid extraction (ethyl acetate or methyl tert-butyl ether) and protein precipitation (acetonitrile, methanol or acetone) were compared for the extraction and clean-up of estrogens from fish plasma. Ethyl acetate was selected as the best alternative with recovery ranging from 61 to 96% and matrix effect ranging from 88 to 106%. Limits of quantification ranged from 0.5 to 1?pg/mL showing a gain in sensitivity of 10,000 times over electrospray ionization of underivatized estrogens. Accuracy and precision were validated over three consecutive days and the method was applied to measure estrogen in sea lamprey (Petromyzon marinus) and lake trout (Salvelinus namaycush) plasma. Estrone and estriol were detected in fish below 1?ng/mL in plasma, justifying the need of a highly sensitive LC–MSMS quantification method.

Full text of DOI:10.1016/j.steroids.2017.04.003

Accepted Manuscript
High-sensitivity determination of estrogens in fish plasma using chemical de-
rivatization upstream UHPLC-MSMS
Ugo Bussy, Yu-Wen Chung-Davidson, Tyler J. Buchinger, Ke Li, Weiming Li
PII:
S0039-128X(17)30056-9
http://dx.doi.org/10.1016/j.steroids.2017.04.003
STE 8093
DOI:
Reference:
Steroids
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10 January 2017
7 April 2017
12 April 2017
Please cite this article as: Bussy, U., Chung-Davidson, Y-W., Buchinger, T.J., Li, K., Li, W., High-sensitivity
determination of estrogens in fish plasma using chemical derivatization upstream UHPLC-MSMS, Steroids (2017),
doi: http://dx.doi.org/10.1016/j.steroids.2017.04.003
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High-sensitivity determination of estrogens in fish plasma using chemical derivatization
upstream UHPLC-MSMS
Ugo Bussy, Yu-Wen Chung-Davidson, Tyler J. Buchinger, Ke Li and Weiming Li
*Address correspondence and reprint request to:
Dr. Weiming Li, Department of Fisheries and Wildlife, Michigan State University, Room 13
Natural Resources Building, 480 Wilson Road, East Lansing, MI 48824, USA
Email: liweim@msu.edu
Tel: +1-517-432-6705
Fax: +1-517-432-1699
Abstract: This article describes the development and validation of a sensitive LC-MSMS
method for determination of estrogen in fish plasma. Dansyl chloride derivatization of the phenol
functional group in estrogen was used to enhance the response to atmospheric ionization leading
13
to improve the sensitivity. Individual C internal standards were selected after comparison with
deuterated standards. Liquid-liquid extraction (ethyl acetate or methyl tert-butyl ether) and
protein precipitation (acetonitrile, methanol or acetone) were compared for the extraction and
clean-up of estrogens from fish plasma. Ethyl acetate was selected as the best alternative with
recovery ranging from 61 to 96 % and matrix effect ranging from 88 to 106 %. Limits of
quantification ranged from 0.5 to 1 pg/mL showing a gain in sensitivity of 10,000 times over
electrospray ionization of underivatized estrogens. Accuracy and precision were validated over
three consecutive days and the method was applied to measure estrogen in sea lamprey
(Petromyzon marinus) and lake trout (Salvelinus namaycush) plasma. Estrone and estriol were
detected in fish below 1 ng/mL in plasma, justifying the need of a highly sensitive LC-MSMS
quantification method.
Keywords: steroids, E1, E2, E3, LC-MSMS
1

1
Introduction
Estrogens are a group of sex steroids that regulate reproduction and many other functions in
vertebrates [1]. Although traditionally considered to be female hormones, estrogens are also
important for successful reproduction in males [2]. Estrone (E1), estradiol (E2) and estriol (E3)
are the main endogenous estrogens. Other molecules, such as estetrol (E4) or estrogen
metabolites, have also been reported [3, 4]. In general, systemic concentrations of estrogen are
very low [5]. For example, E2 concentrations in human (plasma) ranged from 1 to 1,000 pg/mL
[6]. Thus, sensitivity is especially important for methods dedicated to determine endogenous
estrogens.
Tools to quantify estrogens are particularly useful for studies in fish
endocrinology. As in most vertebrates, reproduction in fish is regulated, in part, by estrogens [7].
In addition, these tools will be critical for studies that address anthropogenic additions of
estrogens and estrogen-like compounds to the aquatic environment, and to the impact of
estrogenic compounds on aquatic organism endocrine systems [8-11].
Liquid chromatography tandem mass spectrometry is well-suited for the analysis of aquatic
organisms [12]. Unlike immunoassays, the LC-MSMS analysis of steroids in biological matrices
allows simultaneous analysis of several targets with better analytical performances [13].
Determination of steroids using LC-MSMS has been widely reported [14-16]. Sensitive detection
of steroids in biological matrices is mostly applied to endocrinological studies and to controlled
substance abuse (anti-doping) [17]. Despite the high performance of modern instruments,
concentrations of certain endogenous steroids such as estrogens remain under the limit of
quantification or show lower ionization efficiency under atmospheric pressure ionization
3

techniques (API), especially electrospray. To solve this problem, chemical derivatization is often
reported in the literature as estrogens usually show lower efficiency to atmospheric pressure
ionization compared to androstane in addition to lower concentration in vivo [18]. To enhance
API sensitivity, chemical derivatization has been implemented using various reagents with
functional groups such as 2-picolyamine [19], dansyl chloride [20], isocotinoyl acid [21],
quaternary aminooxy [22, 23] or 2-hydrazino pyridine [24] targeting carboxylic acid, phenol,
carbonyl or hydroxyl group in the steroid backbone, respectively. Dansyl chloride estrogen
derivatization has been reported for E1and E2 in rat serum [25]. Szarka et al. reported the use of
13C internal standard over deuterated estrogens [25]. Reagents involving similar chemical
reactions such as aminooxy quaternary ammonium (targeting carbonyl group) have recently been
developed [26] but are only suitable for E1 determination.
This study aimed to develop a sensitive analytical method to detect estrogen in fish plasma.
Dansyl chloride derivatives were investigated for their efficiency, cost and ease of
implementation for routine analysis. Deuterated and 13C labeled internal standard were
compared. Sample preparation was optimized using the liquid/liquid extraction and protein
precipitation techniques. Finally, this method was validated based on the FDA guidelines and
applied to plasma samples from sea lamprey (Petromyzon marinus), a fish with low systemic
concentrations of estrogens [27], and lake trout (Salvelinus namaycush), a fish with high
concentrations of estrogens [28].
4

2
Material and method
2.1 Chemicals and reagents
Estriol-d4 was purchased from Cambridge Isotope Laboratories (Cambridge Isotope
Laboratories, Inc. Tewksbury, MA, USA). Other chemicals were purchased from Sigma-Aldrich
(Sigma-Aldrich Co., Saint-Louis, MO, USA) except that the de-ionized water was prepared by a
Milli-Q system (EMD Millipore, Billerica, MA, USA).
Figure 1
2.2 Standard solution preparation
Non-labeled standards for each estrogen molecule and deuterated standards (estrone-d4, E1-d4;
estradiol-d4, E2-d4; estriol-d4, E3-d4) were solubilized in ethanol to obtain stock solution at the
13
concentration of 1mg/mL. C- labeled internal standards (estrone-13C3, E1-13C3; estradiol-13C3,
E2-13C3; estriol-13C3, E3-13C3) were purchased in solution at the concentration of 100 g/mL.
Stock solutions were stored at -20 °C away from light. Calibration curve samples were prepared
daily from stock solution by serial dilution and resulted in nine points (1, 2,5, 5, 10, 25, 50, 100,
250 and 500 pg/mL) with internal standards at 50 pg/mL. Quality control (QC) samples for
method validation for E1 and E3 were prepared at three concentrations (5, 50 and 500 pg/mL
with 50 pg/mL internal standard) for low, medium and high QC, respectively (LQC, MQC and
HQC). Endogenous concentrations in plasma used for method validation were approximately 10,
100 and 5 pg/mL for E1, E2 and E3, respectively. Due to the concentration of endogenous E2 in
real samples, E2 QC samples were 50, 500 and 5,000 pg/mL for LQC, MQC and HQC.
5

Table 1
2.3 Collection of fish plasma
Experimental animals were used with approval from the Michigan State University Animal Use
and Care Committee (AUF No.’s 08/12-148-00 and 03/14-054-00). Lake trout and sea lamprey
were provided by the U.S. Fish and Wildlife Service Sullivan Creek National Fish Hatchery and
Marquette Biological Station, and U.S. Geological Survey, Great Lakes Science Center,
Hammond Bay Biological Station. Male and female lake trout were 71.87 ± 0.83 and 78.92 ±
2.17 cm in total length (mean ± SEM), and 3.84 ± 0.16 and 5.94± 0.57 kg in body mass (mean ±
SEM). Male and female sea lamprey were 48.20 ± 0.92 and 51.14 ± 0.80 cm in total length, and
210.39 ± 13.95 and 265.61 ± 13.45 g in mass (mean ± SEM). Plasma was collected using
heparinized vacutainers, centrifuged at 9,000 x g for 5 min at 4 °C, and frozen at -80 °C.
2.4 Sample preparation
Liquid-liquid extraction and protein precipitation were evaluated for the extraction of estrogen
from sea lamprey plasma. Plasma (200 L) samples were spiked with 50 pg of internal standards
and vortexed. For protein precipitation, 1 mL of solvent (either acetonitrile, methanol or acetone)
was added and the sample vortexed. After incubation in a water-ice bath for one hour, samples
were centrifuged (10 minutes, 13,000 x g at 4 °C) and the supernatants transferred to a new tube.
For liquid/liquid extraction, 1.2 mL of solvent [methyl tert-butyl ether (MTBE) or ethyl acetate]
were added to the plasma and vigorously shaken for 20 min. Layer partition was achieved by
centrifugation (10 minutes, 4,000 x g at 4 °C) after the samples were placed at -20°C for one
6

hour. The organic layer was transferred to a new tube and the frozen aqueous layer discarded.
The supernatants were freeze-dried and stored upon derivatization.
2.4.1 Chemical derivatization
Extracts were reconstituted in 100 L of sodium hydrogen carbonate solution (100 mM, pH 10.5
adjusted with 0.1 M sodium hydroxide). Dansyl chloride was solubilized in acetone at the
concentration of 1 mg/mL and prepared daily. 100 L of the derivatization agent were added to
each sample. Samples were vortex and vigorously shaken for 10 minutes at 60 °C. Samples were
allowed to cool down at room temperature and subsequently 800 L of methanol:water (1:1)
were added to each sample. Samples were then vortexed, transferred to auto sampler vials and
stored at -20 °C until HUPLC-MSMS analysis.
Figure 2
2.5 UHPLC-MS/MS
Dansyl chloride derivatives were determined using a Waters UPLC H class Xevo TQ-S triple
quadrupole mass spectrometer (Waters, Milford, MA, USA). Several columns were investigated
for the chromatographic separation of E1, E2 and E3 derivatives (Waters BEH C18 columns
with different dimensions; 1.0 x 50 mm, 2.1 x 100 mm or 2.1 x 50 mm). The best separation was
achieved with Waters BEH C18 column (2.1 × 100 mm, 1.7 um particle size) and a binary
gradient between acetonitrile (solvent B) and 0.1 % formic acid (solvent A). Gradients were set
at 30 °C at a flow rate of 0.22 ml/min. The gradient was as follows (time in minute; % of A):
(initial; 50), (5; 50), (13; 10), (15; 1), (16; 1), (16.01; 50), and (20; 50), and triggered by a 10 L
7

injection. LC outlet from the first five minutes of the run were disposed. Electrospray in the
positive ion mode was set with the following parameters: capillary voltage, 3 kV; desolvation
gas flow, 800 L/h; cone gas flow, 150 L/h; source temperature, 150 ºC and desolvation
temperature, 600 ºC. QuantOptimize (Waters) software was used for the optimization of the cone
voltages and collision energies. MassLynx 4.1 was used to carry out the LC-MSMS experiment
and the data were processed using the TargetLynx software (Waters).
Figure 3
2.6 Method validation
Sample preparation was achieved by liquid/liquid extraction of sea lamprey plasma using ethyl
acetate. Limit of quantification (LOQ) and linearity were determined in standard solutions. Limit
of quantification was defined as the minimum concentration to reach a signal-to-noise ratio
greater or equal to ten. 1/χ least square regression of the ratio of standard area vs. internal
standard area was used to build the calibration curve and to determine the correlation coefficient.
All experiments for method validation and development were performed with 200 L sample
from pooled plasma from five sea lampreys to minimize individual contribution to the variation
13
between replicates. Matrix effect and recovery parameters were first evaluated on C3 internal
standards at the concentration of 50 pg/mL (MQC). Method validation experiments were
performed for the liquid/liquid extraction (with ethyl acetate) of three targets and three internal
standards at 50 pg/mL. Matrices were obtained by pooling together sea lamprey plasma in
phosphate buffer (0.01 M) to reach the concentration of 1g matrix material /mL. Samples were
made of 200 µL plasma. Recovery was calculated by the ratio of the measured peak area in the
8

matrix spiked pre-extraction over the peak area in the matrix spiked post-extraction. Matrix
effect was calculated by the ratio of the measured peak area in the matrix spiked post extraction
(minus the peak area in the blank matrix sample) over the peak area in the standard solution.
For the determination of accuracy and precision, 15 replicates per concentration were acquired
within the same day (intraday) or over three consecutive days (interday). Plasma aliquots (200
L) were spiked with 50 pg/mL of internal standards and targets (three different concentrations,
namely LQC, MQC and HQC). Blank samples consisted of plasma matrix spiked only with
internal standards. The samples were extracted with ethyl acetate and derivatized as described
above. LQC, MQC and HQC determined concentration were subtracted with the endogenous
concentration measured in blank sample. Accuracy was determined by the ratio of the
determined concentration over the spiked concentration. Precision was obtained by the
coefficient of variation within fifteen replicates. Intra- and inter-day retention time
reproducibility was determined at MQC concentration with fifteen replicates. The assay of
stability was conducted in plasma extracts spiked at MQC concentration and monitored for three
consecutive days at 4 °C away from light.
3
Results and discussion
3.1
Chemical derivatization and liquid chromatography tandem mass spectrometry
A highly sensitive method was developed for selective detection of estrogen dansyl derivatives.
Indeed, estrogens in sea lamprey are in minute amount [27, 29]. Conventional LC-MSMS
methods are not sensitive enough to detect their existence in sea lamprey samples. We first
9

developed a method based on previous experiments showing limit of quantification at 7.5, 10 and
20 ng/mL for E1, E2 and E3, respectively (Table 1). Using this method, no traces of estrogens
were observed in sea lamprey plasma. We reported a sensitive method for the determination of
neurosteroids in sea lamprey using the chemical derivatization of the carbonyl functional group
[22]. During these experiments estrone derivative was observed (data not shown). However, E2
and E3 do not contain similar functional group (carbonyl) to react with aminooxy derivatization
agent to form the oxime derivatives (Figure 1). For high-sensitivity analysis of estrogens in
biological matrices, chemical derivatization such as dansyl chloride has been reported to enhance
responses toward electrospray ionization [30]. We selected the dansyl chloride derivatization
agent for its ease of implementation, commercial availability and low cost that does not
compromise analytical performances. No other derivatives were evaluated. Tandem mass
spectrometry parameters were first optimized to reach the best sensitivity. The cleavage of the C-
S bond of the dansyl moiety led to the formation of an intense ion at m/z 171 [M+H]+. This
fragment has been observed in other studies [31] and is attributed to the N,N-
dimethylnaphtalene. Deuterated estrogen internal standards were used in initial method
development, as reported in other studies [31, 32]. Deuterated standards are usually more
available and affordable than 13C labeled standard. However, validation experiments showed
unexpectedly low precision and high inaccuracy with the deuterated standards. Similar
13
experiments implemented with C labeled internal standards achieved better performances (see
method validation section). Larger variations with deuterated standards were associated with
13
H/D exchange and the loss of E-d4 signal. Therefore, C labeled standard were preferred as
internal standards.
10

Although high sensitivity was achieved after MSMS parameter optimization, interferences were
observed during LC-MSMS experiments for shorter gradients (10 min) between E2 and E1-13C3.
A longer gradient of 20 minutes eliminated interferences by achieving a baseline separation. This
gradient was also free of carryover. The separation of dansylated estrogen is reported to be
challenging with reverse phase although alternatives such as phenyl hexyl column have shown
better performances [31]. Nonetheless, most of the reported methods used reverse phase columns
[33, 34]. The limit of quantification and linearity parameters (Table 1) were determined in
standard solution to avoid interferences with endogenous estrogens in sea lamprey plasma.
3.2 Sample extraction and clean-up
Sample preparation was optimized on sea lamprey plasma. The need to analyze and prepare large
number of samples at lower cost without compromising method performances favors protein
precipitation (PPT) or liquid/liquid extraction (LLE) techniques over solid phase extraction
(SPE). Extraction of estrogen from biological matrices have been reported with ethyl acetate
[30, 32], chlorobutane [33], MTBE [26, 35] or PPT combined with SPE [36]. Figure 2 shows the
recovery and matrix effect parameters for five different solvents (2 LLE and 3 PPT). Sample
preparation was optimized using 13C labeled internal standard to minimize interference of
endogenous estrogens. Three solvents employed in the PPT method (methanol, acetonitrile or
acetone) showed higher matrix effect and recovery parameters than LLE with ethyl acetate or
MTBE. In fact, strong ion suppression (80 %) and poor recovery (30 %) was observed when
using PPT. This was attributed to the matrix interferences during the chemical derivatization
process. Indeed , when using chemical derivatization, LLE is usually preferred for estrogen
extraction [37]. Recovery values were higher when ethyl acetate was used instead of MTBE.
11

Matrix effects were higher when using MTBE; however, no significant differences with ethyl
acetate were observed for E1 and E2. An important ion enhancement is observed for E3 when
using MTBE. Therefore, liquid/liquid extraction using ethyl acetate was selected for the sample
preparation of fish plasma. The validation of matrix effects and recovery parameters are shown
in Table 2. Recoveries ranged from 61 to 96 %. No significant difference was observed between
the estrogens and their respective internal standards. Matrix effects ranged from 88 to 106 %
(Table 2). Similar to the recovery, no significant difference was observed between the matrix
effects determined for the targeted estrogens and their respective internal standards. Therefore,
13
liquid/liquid extraction using ethyl acetate combined with C3 internal standards was validated
for the extraction of estrogens from fish plasma.
Table 2
3.3 Accuracy, precision and stability
The developed method was evaluated over three consecutive days (interday) and within a day
(intraday). Accuracy and precision parameters are summarized in Table 3. The validation
experiments were first set with a LQC at 0.5 pg/mL but the presence of endogenous steroids at
higher concentration (10, 100 and 5 pg/mL, for E1, E2 and E3, respectively) interfered with the
determination of the exogenous steroids spiked in a low concentration. Thus, LQC was set at 5
pg/mL, MQC at 50 pg/mL and HQC at 500 pg/mL for E1 and E3 while LQC was set at 50
pg/mL, MQC at 500 pg/mL and HQC at 5,000 pg/mL for E2. Acceptable range was 80 to 120 %
for LQC accuracy and ≤ 20 % for LQC precision while the range for MQC and HQC was set
from 85 to 115 % for accuracy and ≤ 20 % for precision. Intraday accuracy did not exceed the
12

acceptable range of ± 15 % except for E2 at MQC (117.2 ± 5.1) % and the variation was within
the acceptable range of 15 % except for E2 at MQC (116.1 ± 5.7) %. Intra- and inter-day
precision were all below the acceptable limit of 15 %.
Table 3
Stability was studied at MQC concentration for three consecutive days. No significant
differences were observed between three targeted estrogens and their respective internal
standard, supporting the choice of 13C labeled estrogen as internal standard. Moreover, no
significant differences were observed between the signal areas determined over three consecutive
days. This result confirms the stability of dansyl estrogen derivative (at 4 °C in the dark) and the
applicability of the method for routine analysis.
Figure 4
3.4 Analysis of estrogens in fish plasma
This method was applied to determine estrogens in fish plasma (sea lamprey and lake trout).
Concentrations are presented per mL of plasma (Figure 5). E1 was not detected in male lake
trout, but other estrogens were detected in both sexes in both species. E1 was detected in female
lake trout (111 ± 25 pg/mL; mean ± standard deviation) but not male lake trout. For sea lamprey,
E1 concentrations were significantly higher in females (160 ± 21 pg/mL) than males (64 ± 19
pg/mL). E2 concentration was higher in female lake trout (6930 ± 248) pg/mL than in male lake
trout (34 ± 12 pg/mL; Student-t test, p <0.001). However, no sex difference in E2 concentrations
was observed in the sea lamprey (3279 ± 591 pg/mL and 3072 ± 410 pg/mL for male and female
13

sea lamprey, respectively; p>0.05). E3 concentrations were lower than any other compound in all
groups. E3 concentration was higher in females (22 ± 4 pg/mL) compared to males (10 ± 2
pg/mL; p<0.001) in lake trout, and in sea lamprey (female, 88 ± 7 pg/mL; male, 47 ± 15 pg/mL;
p<0.001). Endogenous estradiol concentrations exceeded the range for which the method was
initially validated for. Therefore, the samples were determined after dilution (10 times). For
practicality, the method was re-validated with a range of concentrations that includes
endogenous concentrations (50-5,000 pg/mL). The other estrogens had lower endogenous
concentration, confirming the need for a sensitive and robust method for their determination in
fish plasma matrices.
Figure 5
4
Conclusion
A sensitive and reproducible method has been developed for determination of three endogenous
estrogens in fish plasma. Ethyl acetate has been shown as the most performant solvent for
extraction of estrogens from biological matrix (plasma). Recovery, matrix effect, accuracy,
precision and stability were validated and the method was applied to the determination of
biological samples. Estradiol was determined in relatively high concentration (over 1,000
pg/mL). However, estrone and estriol were determined at lower concentrations (5-500 pg/mL),
demonstrating the need for highly sensitive methods. Indeed, the method of chemical
derivatization using dansyl chloride was a thousand times more sensitive than conventional
electrospray methods. The simple sample preparation, the quick, easy and affordable chemical
derivatization and validated performances of this method render it a promising tool in
14

endocrinology and other studies required sensitive measurement of estrogens. This method could
also be extended to exogenous estrogen compounds to study their effects on fish.
Acknowledgments
The authors thank Professor Daniel Jones and Dr. Scott Smith of Michigan State University MS
Facility for helpful advice. This study was funded by the Great Lakes Fishery Commission.
YWCD was supported by seed funds from the office of Vice President for Research and
Graduate Studies and AgBio Station at Michigan State University. Luke Baker, Tyler Bruning,
Skye Fissette and Carrie Kozel assisted with sample collection.
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and Comparative Endocrinology, 1991. 81(1): p. 93-96.
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Correlates in Three Strains of Lake Trout. Transactions of the American Fisheries
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Sower, S.A. and C.B. Schreck, Steroid and thyroid hormones during sexual maturation of
coho salmon (Oncorhynchus kisutch) in seawater of fresh water. Gen Comp Endocrinol,
1982. 47(1): p. 42-53.
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Electrospray Ionization: Application in Trace Analysis of Ethinylestradiol in Rhesus
Monkey Plasma. Analytical Chemistry, 2002. 74(16): p. 4136-4144.
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single HPLC column for simultaneous quantitation by LC–MS/MS. Analytical and
Bioanalytical Chemistry, 2013. 405(10): p. 3399-3406.
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35.
36.
Farlow, D.W., X. Xu, and T.D. Veenstra, Quantitative measurement of endogenous
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Bichon, E., et al., Improvement of estradiol esters monitoring in bovine hair by
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17

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37.
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Chemistry, 2004. 50(2): p. 373-384.
18

Figure caption
Figure 1. A: Overview of the chemical structures of the targeted estrogens (E1, E2 and E3)
13
and their respective deuterium (E1-d4, E2-d4 and E3-d4) or C isotope labelled internal
standard. B: chemical derivatization of the phenol group of estrogens with dansyl chloride.
Figure 2. Recovery and matrix effect parameter for the extraction of exogenous estrogens
(13C3 labeled) from sea lamprey plasma using liquid/liquid extraction (MTBE and ethyl
acetate) and protein precipitation (methanol, acetonitrile or acetone).
Figure 3. Chromatograms of dansyl chloride derivatives of endogenous estrogens and
internal standards (13C3 labeled) in sea lamprey plasma after extraction with ethyl acetate.
Concentration were 10.9, 571.2 and 9.1 pg/mL for E1, E2 and E3 respectively.
Figure 4. Stability assay, monitoring three estrogen derivatives and their respective
internal standards over three consecutive days at 4 °C.
Figure 5. Determination of estrogens in 200 µL plasma from six males and six females in
sea lamprey and lake trout. For convenient reason, concentrations are displayed with
logarithm scale.
Table caption
Table 1. Tandem mass spectrometry parameters for the detection of estrogen dansyl
chloride derivatives. Cone voltage, collision energy and limit of quantification are CV, CE
and LOQ, respectively.
19

Table 2. Recovery and matrix effect parameters for the extraction of sea lamprey plasma
(estrogen at 50 pg/mL) with ethyl acetate.
Table 3. Intra- and inter-day accuracy and precision parameters for sea lamprey plasma
extracted with ethyl acetate. Estrogens were spiked at three concentrations (LQC, MQC
and HQC, respectively).
20

O
O
H
H
H
O
H
H
E1-13C3
E1-d4
A
E1
E2
E3
D
D
H
H
H
H
D
13C
H
H
H
H
H
13C
13C
H
HO
HO
HO
HO
OH
OH
D
OH
H
H
E2-13C3
E2-d4
D
D
D
H
H
13C
H
H
H
H
H
H
H
13C
13C
HO
HO
D
D
OH
H
OH
OH
H
H
E3-13C3
D
OH
E3-d4
H
H
OH
H
OH
D
13C
H
H
H
H
13C
13C
HO
HO
HO
D
B
O
S
O
Cl
O
S
NaHCO3
60 Celsius, 1 hour
N
O
+
O
HO
N
dansyl chloride
estrogen
estrogen derivative
Figure 1 A: Overview of the chemical structures of the targeted estrogens (E1, E2 and E3) and their respective deuterium (E1-d4, E2-d4
and E3-d4) or 13C isotope labelled internal standard. B: chemical derivatization of the phenol group of estrogens with dansyl chloride.
21

120.0
100.0
80.0
60.0
40.0
20.0
0.0
MTBE
ethyl acetate
methanol
acetonitrile
acetone
E1
E2
E3
160
140
120
100
80
MTBE
ethyl acetate
methanol
acetonitrile
acetone
60
40
20
0
E1
E2
E3
Figure 2 Recovery and matrix effect parameter for the extraction of exogenous estrogens (13C3 labeled) from sea lamprey plasma using
liquid/liquid extraction (MTBE and ethyl acetate) and protein precipitation (methanol, acetonitrile or acetone).
22

Figure 3 Chromatograms of dansyl chloride derivatives of endogenous estrogens and internal standards (13C3 labeled) in sea lamprey
plasma after extraction with ethyl acetate. Concentration were 10.9, 571.2 and 9.1 pg/mL for E1, E2 and E3 respectively.
23

120.0
100.0
80.0
60.0
40.0
20.0
0.0
targets
Internal
standards
1
2
3
4
5
6
7
8
9
10
11
E2
E3
E1
Figure 4 Stability assay, monitoring three estrogen derivatives and their respective internal standards over three consecutive days at 4
°C.
24

10000
1000
100
10
E1
E2
E3
1
Female lake trout
Male lake trout
Female sea lamprey Male sea lamprey
Figure 5 Determination of estrogens in 200 µL plasma from six males and six females in sea lamprey and lake trout. For convenience
reason, concentrations are displayed with logarithm scale.
25

Table 1 Tandem mass spectrometry parameters for the detection of estrogen dansyl chloride derivatives. Cone voltage, collision energy
and limit of quantification are CV, CE and LOQ, respectively.
paren
t
LOQ No
derivatization
pg/mL
fragment
CV
CE
LOQ
linearity
r2
pg/mL
5-500
V
V
pg/mL
0.5
0.9968
0.9959
0.9982
E1
E2
E3
504.4
506.3
522.4
171.1
171.1
171.1
60
36
7,500
10,000
25,000
5-5,000
5-500
20
40
36
36
0.5
1
E1-13C3
E2-13C3
E3-13C3
507.3
509.3
525.4
171.1
171.1
171.1
80
30
40
36
36
36
26

Table 2 Recovery and matrix effect parameters for the extraction of sea lamprey plasma (estrogen at 50 pg/mL) with ethyl acetate.
Recovery (%) Matrix effect (%)
E1
(90.9±20.4)
(96.5±23.3)
(104.7±6.8)
(105.3±5.6)
E1-13C3
E2
E2-13C3
(68.9±11.7)
(61.5±9.3)
(75.5±8.9)
(73.1±5.7)
(106.1±7.1)
(101.3±9.4)
(88.2±7.0)
(90.5±7.1)
E3
E3-13C3
27

Table 3 Intra- and inter-day accuracy and precision parameters for sea lamprey plasma extracted with ethyl acetate. Estrogens were
spiked at three concentrations (LQC, MQC and HQC).
intraday
MQC
Interday
MQC
LQC
5
HQC
500
LQC
5
HQC
500
50
(52.96±5.18)
105.9
50
(54.60±5.22)
109.2
spiked (pg/mL)
measured (pg/mL)
accuracy (%)
(4.48±0.65)
89.6
(472.65±38.29)
94.5
(4.90±0.63)
98.0
(455.35±33.81)
91.1
E1
E2
E3
13.1
10.4
7.7
12.6
10.4
6.8
precision (%)
50
500
5000
50
500
5000
spiked (pg/mL)
measured (pg/mL)
accuracy (%)
(48.82±4.29) (586.04±25.35) (5667.0±305.1) (52.68±6.55) (580.70±28.48) (5223.1±554.6)
97.6
117.2
5.1
113.3
105.4
13.1
116.1
104.5
11.1
8.6
5
6.1
500
5.7
50
precision (%)
50
5
500
spiked (pg/mL)
measured (pg/mL)
accuracy (%)
(4.60±0.50)
92.0
(49.38±6.00)
98.8
(505.40±21.43)
101.1
(4.76±0.59)
95.2
(45.56±3.47)
91.1
(505.60±21.56)
101.1
9.9
12.0
4.3
11.9
6.9
4.3
precision (%)
28

 Determination of estrogen using dansyl chloride derivatization and UHPLC-MSMS
 Full method validation for the determination of fish plasma matrix
 Application to real samples showed sex differences in endogenous estrogen
concentrations
29