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High-sensitivity determination of estrogens in fish plasma using chemical derivatization upstream UHPLC–MSMS
Accepted Manuscript 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, Weiming Li PII: DOI: Reference:
S0039-128X(17)30056-9 http://dx.doi.org/10.1016/j.steroids.2017.04.003 STE 8093
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
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: [email protected] 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 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. 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 13
C 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
13
C 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 2.1
Material and method 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 concentration of 1mg/mL.
13
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 between replicates. Matrix effect and recovery parameters were first evaluated on
13
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 3.1
Results and discussion 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 CS 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,Ndimethylnaphtalene. 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
13
C labeled standard. However, validation experiments showed
unexpectedly low precision and high inaccuracy with the deuterated standards. Similar experiments implemented with 13C labeled internal standards achieved better performances (see method validation section). Larger variations with deuterated standards were associated with H/D exchange and the loss of E-d4 signal. Therefore,
13
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
13
C 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, liquid/liquid extraction using ethyl acetate combined with
13
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
13
C 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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Figure caption 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
13
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
A
H O
E1 H
H OH
D
D D
H
D
H H
OH
H D OH D H OH
E3-d 4 D H
H
H
13 C
H
13 C
H OH
E3-13C3
HO
D
H OH
H
13 C
H
HO
HO
13 C
HO D
H OH
E3
H
13 C
13 C
HO
H H
E2-13C3
H
H
H
HO
13 C
HO
E2-d 4
H O
E1-13C3 13 C
HO
H H
H
H
H OH
E2
D D
H
D H
H HO
H O
E1-d 4
H H
OH H
13 C
B O
O Cl S +
NaHCO 3 HO
N
60 Celsius, 1 hour
O S O O
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
Recovery / %
100.0 MTBE
80.0
ethyl acetate
60.0
methanol
40.0
acetonitrile
20.0
acetone
0.0 E1
E2
E3
160 140 Matrix effect / %
120
MTBE
100
ethyl acetate
80
methanol
60
acetonitrile
40
acetone
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
targets
40.0
Internal standards
20.0
0.0
1
2
E1
3
4
5
6
E2
7
8
9
10
E3
11
Figure 4 Stability assay, monitoring three estrogen derivatives and their respective internal standards over three consecutive days at 4 °C.
24
Estrogen concentrations per mL of plasma / pg/mL log scale
10000
E1 E2 E3
1000
100
10
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
fragment
LOQ No derivatization pg/mL
CV
CE
LOQ
linearity
r2
V
V
pg/mL
0.9968
7,500
E1
504.4
171.1
60
36
0.5
pg/mL 5-500
E2
506.3
171.1
20
36
0.5
5-5,000
0.9959
10,000
E3
522.4
171.1
40
36
1
5-500
0.9982
25,000
13
E1- C3
507.3
171.1
80
36
E2-13C3
509.3
171.1
30
36
525.4
171.1
40
36
13
E3- C3
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)
(104.7±6.8)
E1-13C3
(96.5±23.3)
(105.3±5.6)
E2
(68.9±11.7)
(106.1±7.1)
E2-13C3
(61.5±9.3)
(101.3±9.4)
E3
(75.5±8.9)
(88.2±7.0)
13
(73.1±5.7)
(90.5±7.1)
E3- C3
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
HQC
LQC
MQC
HQC
5
50
500
5
50
500
(4.48±0.65)
(52.96±5.18)
(472.65±38.29)
(4.90±0.63)
(54.60±5.22)
(455.35±33.81)
accuracy (%)
89.6
105.9
94.5
98.0
109.2
91.1
precision (%)
13.1
10.4
7.7
12.6
10.4
6.8
spiked (pg/mL) E1
measured (pg/mL)
50
500
5000
50
500
5000
(48.82±4.29)
(586.04±25.35)
(5667.0±305.1)
(52.68±6.55)
(580.70±28.48)
(5223.1±554.6)
accuracy (%)
97.6
117.2
113.3
105.4
116.1
104.5
precision (%)
8.6
5.1
6.1
13.1
5.7
11.1
5
50
500
5
50
500
(4.60±0.50)
(49.38±6.00)
(505.40±21.43)
(4.76±0.59)
(45.56±3.47)
(505.60±21.56)
accuracy (%)
92.0
98.8
101.1
95.2
91.1
101.1
precision (%)
9.9
12.0
4.3
11.9
6.9
4.3
spiked (pg/mL) E2
measured (pg/mL)
spiked (pg/mL) E3
Interday
LQC
measured (pg/mL)
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
S0039-128X(17)30056-9 http://dx.doi.org/10.1016/j.steroids.2017.04.003 STE 8093
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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: [email protected] 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 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. 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 13
C 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
13
C 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 2.1
Material and method 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 concentration of 1mg/mL.
13
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 between replicates. Matrix effect and recovery parameters were first evaluated on
13
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 3.1
Results and discussion 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 CS 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,Ndimethylnaphtalene. 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
13
C labeled standard. However, validation experiments showed
unexpectedly low precision and high inaccuracy with the deuterated standards. Similar experiments implemented with 13C labeled internal standards achieved better performances (see method validation section). Larger variations with deuterated standards were associated with H/D exchange and the loss of E-d4 signal. Therefore,
13
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
13
C 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, liquid/liquid extraction using ethyl acetate combined with
13
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
13
C 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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18
Figure caption 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
13
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
A
H O
E1 H
H OH
D
D D
H
D
H H
OH
H D OH D H OH
E3-d 4 D H
H
H
13 C
H
13 C
H OH
E3-13C3
HO
D
H OH
H
13 C
H
HO
HO
13 C
HO D
H OH
E3
H
13 C
13 C
HO
H H
E2-13C3
H
H
H
HO
13 C
HO
E2-d 4
H O
E1-13C3 13 C
HO
H H
H
H
H OH
E2
D D
H
D H
H HO
H O
E1-d 4
H H
OH H
13 C
B O
O Cl S +
NaHCO 3 HO
N
60 Celsius, 1 hour
O S O O
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
Recovery / %
100.0 MTBE
80.0
ethyl acetate
60.0
methanol
40.0
acetonitrile
20.0
acetone
0.0 E1
E2
E3
160 140 Matrix effect / %
120
MTBE
100
ethyl acetate
80
methanol
60
acetonitrile
40
acetone
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
targets
40.0
Internal standards
20.0
0.0
1
2
E1
3
4
5
6
E2
7
8
9
10
E3
11
Figure 4 Stability assay, monitoring three estrogen derivatives and their respective internal standards over three consecutive days at 4 °C.
24
Estrogen concentrations per mL of plasma / pg/mL log scale
10000
E1 E2 E3
1000
100
10
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
fragment
LOQ No derivatization pg/mL
CV
CE
LOQ
linearity
r2
V
V
pg/mL
0.9968
7,500
E1
504.4
171.1
60
36
0.5
pg/mL 5-500
E2
506.3
171.1
20
36
0.5
5-5,000
0.9959
10,000
E3
522.4
171.1
40
36
1
5-500
0.9982
25,000
13
E1- C3
507.3
171.1
80
36
E2-13C3
509.3
171.1
30
36
525.4
171.1
40
36
13
E3- C3
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)
(104.7±6.8)
E1-13C3
(96.5±23.3)
(105.3±5.6)
E2
(68.9±11.7)
(106.1±7.1)
E2-13C3
(61.5±9.3)
(101.3±9.4)
E3
(75.5±8.9)
(88.2±7.0)
13
(73.1±5.7)
(90.5±7.1)
E3- C3
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
HQC
LQC
MQC
HQC
5
50
500
5
50
500
(4.48±0.65)
(52.96±5.18)
(472.65±38.29)
(4.90±0.63)
(54.60±5.22)
(455.35±33.81)
accuracy (%)
89.6
105.9
94.5
98.0
109.2
91.1
precision (%)
13.1
10.4
7.7
12.6
10.4
6.8
spiked (pg/mL) E1
measured (pg/mL)
50
500
5000
50
500
5000
(48.82±4.29)
(586.04±25.35)
(5667.0±305.1)
(52.68±6.55)
(580.70±28.48)
(5223.1±554.6)
accuracy (%)
97.6
117.2
113.3
105.4
116.1
104.5
precision (%)
8.6
5.1
6.1
13.1
5.7
11.1
5
50
500
5
50
500
(4.60±0.50)
(49.38±6.00)
(505.40±21.43)
(4.76±0.59)
(45.56±3.47)
(505.60±21.56)
accuracy (%)
92.0
98.8
101.1
95.2
91.1
101.1
precision (%)
9.9
12.0
4.3
11.9
6.9
4.3
spiked (pg/mL) E2
measured (pg/mL)
spiked (pg/mL) E3
Interday
LQC
measured (pg/mL)
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