New approach based on immunochemical techniques for monitoring of selective estrogen receptor modulators (SERMs) in human urine

Article abstract of DOI:10.1016/j.jpba.2018.04.027

Antiestrogenic compounds such as tamoxifen, toremifen and chlomifen are used illegally by athletes to minimize physical impacts such as gynecomastia resulting from the secondary effects of anabolic androgenic steroids, used to increase athletic efficiency unlawfully. The use of these compounds is banned by the World Anti-Doping Agency (WADA) and controls are made through analytical methodologies such as HPLC–MS/MS, which do not fulfil the sample throughput requirements. Moreover, compounds such as tamoxifen are also used to treat hormone receptor-positive breast cancer (ER +).Therapeutic drug monitoring (TDM) of tamoxifen may also be clinically useful for guiding treatment decisions. An accurate determination of these drugs requires a solid phase extraction of patient serum followed by HPLC–MS/MS. In the context of an unmet need of high-throughput screening (HTS) and quantitative methods for antiestrogenic substances we have approached the development of antibodies and an immunochemical assay for the determination of these antiestrogenic compounds. The strategy applied has taken into consideration that these drugs are metabolized and excreted in urine as the corresponding 4-hydroxylated compounds. A microplate-based ELISA procedure has been developed for the analysis of these metabolites in urine with a LOD of 0.15, 0.16 and 0.63 μg/L for 4OH-tamoxifen, 4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL established by WADA (20 μg/L).

Full text of DOI:10.1016/j.jpba.2018.04.027

Accepted Manuscript
Title: New approach based on immunochemical techniques for
MONITORING of Selective estrogen receptor modulators
(SERMs) in human urine
´
Authors: J.-Pablo Salvador, Ester Vila-Roca, Nuria Monfort,
Rosa Ventura, M.-Pilar Marco
PII:
DOI:
Reference:
S0731-7085(18)30204-8
https://doi.org/10.1016/j.jpba.2018.04.027
PBA 11926
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date:
Revised date:
Accepted date:
23-1-2018
16-4-2018
17-4-2018
´
Please cite this article as: J.-Pablo Salvador, Ester Vila-Roca, Nuria Monfort,
Rosa Ventura, M.-Pilar Marco, New approach based on immunochemical
techniques for MONITORING of Selective estrogen receptor modulators
(SERMs) in human urine, Journal of Pharmaceutical and Biomedical
Analysis https://doi.org/10.1016/j.jpba.2018.04.027
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NEW APPROACH BASED ON IMMUNOCHEMICAL TECHNIQUES FOR MONITORING OF
SELECTIVE ESTROGEN RECEPTOR MODULATORS (SERMS) IN HUMAN URINE
J.-Pablo Salvador2,1,*, Ester Vila-Roca1,2, Núria Monfort3, Rosa Ventura3,4, M.-Pilar Marco1,2
1Nanobiotechnology for Diagnostics group (Nb4D). IQAC- CSIC. Jordi Girona 18–26, 08034 Barcelona,
Spain.
2CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)
3Barcelona Antidoping Laboratory, IMIM (Hospital del Mar Medical Research Institute), Barcelona,
Spain
4Department of Expermerimental and Health Sciences, UPF, Pompeu Fabra University, Barcelona, Spain
Highlights

A class-selective antibody for the determination of the main metabolites of selective estrogen receptor
modulators (SERMS) is presented

The ELISA developed achieved the analysis of SERMS metabolites with a LOD of 0.15, 0.16 and 0.63 µg/L
for 4OH-tamoxifen, 4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL
established by WADA (20 µg/L).

The hapten design has been demonstrated as a powerful tool to produce antibodies with tailored
properties as has been demonstrated for the simultaneous determination of the main metabolites of
SERMs.
Abstract
Antiestrogenic compounds such as tamoxifen, toremifen and chlomifen are used illegally by athletes to
minimize physical impacts such as gynecomastia resulting from the secondary effects of anabolic
androgenic steroids, used to increase athletic efficiency unlawfully. The use of these compounds is
banned by the World Anti-Doping Agency (WADA) and controls are made through analytical
methodologies such as HPLC-MS/MS, which do not fulfil the sample throughput requirements.
Moreover, compounds such as tamoxifen are also used to treat hormone receptor-positive breast cancer
(ER+).Therapeutic drug monitoring (TDM) of tamoxifen may also be clinically useful for guiding
treatment decisions. An accurate determination of these drugs requires a solid phase extraction of patient
serum followed by HPLC-MS/MS. In the context of an unmet need of high-throughput screening (HTS)
and quantitative methods for antiestrogenic substances we have approached the development of
antibodies and an immunochemical assay for the determination of these antiestrogenic compounds. The
1

strategy applied has taken into consideration that these drugs are metabolized and excreted in urine as the
corresponding 4-hydroxylated compounds. A microplate-based ELISA procedure has been developed for
the analysis of these metabolites in urine with a LOD of 0.15, 0.16 and 0.63 µg/L for 4OH-tamoxifen,
4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL established by WADA (20
µg/L).
1. Introduction
Selective estrogen receptor modulators (SERMs) are a family of compounds that interacts specifically
with estrogen receptors in order to modulate its estrogenic-like action. This kind of compounds is
commonly used to treat breast cancer, being Tamoxifen the most frequently employed [1]. Structurally
similar compounds are toremifen and clomifen, showing a 2-chloroethyl and a chlorine group,
respectively, instead of the ethyl group on the allyl core of the tamoxifen.
The use of antiestrogenic compounds may compensate an extensive abuse of anabolic androgenic steroids
by athletes, which produces gynecomastia between other secondary effects [2]. For this reason, the use of
agents with anti-estrogenic activity has been banned in sports by the World Anti-Doping Agency
(WADA) [3], and anti-doping control laboratories must be able to detect the administration of the drug. In
the wake of this prohibition, several analytical methodologies have been developed mainly based on
chromatographic techniques coupled with mass spectrometry[4-8]. It is reported[4] that SERMs are
excreted mainly as 4-hydroxylated metabolites and their corresponding glucuronide or sulfate conjugates.
Although several advances have been accomplished towards highly sensitive techniques with minimum
sample pre-treatment, nowadays clean-up processes are still necessary before injection into the
chromatographic equipment. In parallel, immunochemical methods could support these analyses by
increasing the screening efficiency and reducing the number of samples that need to be analysed by
confirmatory chromatographic methods, thanks to their high sample throughput screening potential. It
has often been shown that immunoassays can provide the necessary reliability, low cost, simplicity,
selectivity and detectability to analyse small organic molecules in complex biological samples [9, 10].
Previously, our research group reported the production of antibodies with tailored properties for anabolic
steroid residues detection[11-13]. Regarding immunochemical detection of SERMs, to our knowledge no
antibodies have been produced. Other alternative strategies for the determination of tamoxifen include an
amperometric biosensor using conducting polymers such as polyaniline [14] and a surface plasmon
resonance (SPR) biosensor using an estrogen receptor[15]. Thus, here we present the development of
class- selective antibodies for hydroxylated SERMs and the development of an immunoassay for the
analysis of urine samples according to the WADA requirements for anti-doping testing.
2

2. Materials and Methods
2.1 Chemicals, Biochemicals and Instruments.
Polystyrene microtiter plates were purchased from Nunc (Maxisorp, Roskilde, Denmark). Washing steps
were carried out using a SLY96 PW microplate washer (SLT Labinstruments GmbH, Salzburg, Austria).
Absorbances were read using a SpectramaxPlus (Molecular Devices, Sunnyvale, CA) at a single
wavelength mode of 450 nm. The competitive curves were analyzed with a four-parameter logistic
equation using the software SoftmaxPro v2.7 (Molecular Devices) and GraphPad Prism v 4 (GraphPad
Software Inc., San Diego, CA). Unless otherwise indicated, data presented correspond to the average of at
least two well replicates. The immunoreagents employed has been performed with the support of the
ICTS “NANBIOSIS”, more specifically by the Custom Antibody Service (CAbS, CIBER-BBN, IQAC-
CSIC). Immunochemicals were obtained from Sigma Chemical Co. (St. Louis, MO). 4-hydroxyclomifene
was purchased by Synfine Research Inc. (Ontario, Canada) .All other chemicals were of analytical grade.
Biochemicals and immunochemicals were obtained from Sigma-Aldrich Co (St. Louis, MO).
2.2 Synthesis
The standards 4-hydroxytamoxifen, 4-hydroxytoremifen and the hapten (Z/E)1-[4-(2-dimethylamino-
ethoxy)-fenyl]-1-(4-hydroxy-phenyl)-6-phenyl-hept-6-enoic acid (hTAM) were synthesized following the
procedure described by Gauthier et al. [16] and Yu et al. [17] with slight modifications (see SI section
S2.3).
2.3 Preparation of immunoreagents for SERMs detection
The preparation and characterization of the immunogen hTAM-HCH (MA) and the corresponding
hTAM-BSA (MA) conjugate are described (see SI section S2.4), as well as the competitors hTAM-BSA
(AE) and hTAM-HRP (AE).The production of the antibodies As211-213 were performed according the
protocol of immunization for hTAM-HCH (see SI section S2.5).
2.4 Hydroxyl-SERMs ELISA
The appropriate dilutions of the antisera and the enzyme tracer (hTAM-HRP (AE)) and/or coated antigen
(hTAM-BSA (AE)) were established after two-dimensional checkerboard titration assays. All the buffers
used are described (see SI section S2.2).
ELISA As212/hTAM-HRP. The microtiter plates were coated with the antiserum As212 (1/12000 in a
coating buffer, 100µL/well) overnight at 4 ◦C. The next day the plates were washed four times with PBST
3

and the solutions of the different concentrations of the standards, cross-reactants, or samples were added
(50µL/well) followed by the solution of the enzyme tracer hTAM-HRP (0.125 µgmL-1 in PBST,
50µL/well). After 30 min at room temperature, the plates were washed again four times with PBST. The
substrate solution was added (100µL/well) and the enzymatic reaction stopped after 30 min at room
temperature with 4N H2SO4 (50µL/well). The absorbance was measured at 450 nm. The standard curve
was fitted to a four-parameter logistic equation according to the following formula: y = (A–B/[1–(x/C)D])
+ B, where A is the maximal absorbance, B is the minimum absorbance, C is the concentration producing
50% of the difference between the maximal absorbance and the minimum absorbance, and D is the slope
of the inflection point of the sigmoid curve. The cross-reactivity studies were performed in buffer and the
values were calculated according to the following equation: (IC50 4OH-TAM/IC50 other
compounds)×100.
2.5 Urine sample pretreatment
The urine samples (see SI section S2.6) were treated as previously described with slight modifications[4].
This method hydrolyzed simultaneously the sulfate and glucuronide conjugates in order to obtain the free
drug[11]. First, an enzymatic hydrolysis was performed by mixing the urine sample (1 mL) with PBS 0.4
M pH=7.0 (1 mL) and β-glucuronidase/sulfatase (5 µL) and incubating the mixture for 1h at 55ºC.
Afterwards, 5% carbonate buffer (500 µL) was added followed by tert-butyl methyl ether (3 mL) and the
mixture was shaken and centrifuged at 4000 rpm during 5 min. The organic layer was then separated and
evaporated with the aid of a N2 stream. Before the assay, the dried extract was reconstituted in 100 µL of
ethanol and 900 µL of PBST.
3. Results and discussion
The aim of this work has been to produce class-specific antibodies for the determination of SERM’s main
well known metabolites [7]. Hence, Z/E-4-hydroxy-tamoxifen, desmethyltamoxifen and trans-metabolite
E are the main metabolites of tamoxifen [18] and a similar pattern has been reported for toremifen[4] and
clomiphene[8], being the 4-hydroxylated (4-OH) metabolites and the corresponding sulfate and
glucuronide conjugate the most frequently found. For this reason, and with the aim to establish an
screening immunochemical method, we proposed a production of class-selective antibodies for these
metabolites.
The production of antibodies for the determination of small molecular weight molecules requires the
design of a hapten mimicking the target analyte. Figure 1 shows the chemical structure of the target
substances where it can be observed that they all share three common epitopes including the phenolic, the
aryl and the 4-(2-dimethylamino-ethoxy)-phenyl groups. Hence, the hapten had to be designed to
4

maximize the recognition of these chemical moieties, which was accomplished by introducing a linker at
the allylic position that differentiates them. Thus, hapten hTAM 7 ((Z/E)1-[4-(2-dimethylamino-ethoxy)-
phenyl]-1-(4-hydroxy-phenyl)-6-phenyl-hept-6-enoic acid, see figure 2) was proposed as the appropriate
chemical structure for production of class-selective antibodies against the 4-OH metabolites of this family
of SERMs.
On a first instance we addressed the synthesis of the 4-OH metabolites of tamoxifen and toremifen
following the procedure described by Gauthier et al.[16] and Yu et al. [17] with slight modifications (see
Figure 2). The reductive coupling of 4,4'-hydroxybenzophenone with the corresponding ethyl and 2-
chlolroethyl phenones employing the McMurry reaction allowed to obtain the 4-OH phenylbutenes 1 and
3, respectively. After the purification by flash chromatography, the ether group at position 4 was
introduced by reacting 1 and 3, with 2-dimethylaminoethyl chloride using NaH to generate the alkoxide
that would act as nuclephile, obtaining the 4-OH metabolites of tamoxifen and toremifen 2 and 4,
respectively, as a 1:1 mixture of E/Z isomers. The compounds were purified and characterized by
spectroscopic and spectrometric methods. According to the same procedure, the immunizing hapten
hTAM was synthesized but using methyl 5-benzylpentanoate on the McMurry reaction to obtain the
intermediate 5 (see figure 2) which was subsequently reacted with 2-dimethylaminoethy chloride, as
before, to introduce this functionality in one of the aromatic rings to obtain 6. Finally, the hapten hTAM
was obtained after the hydrolysis of the methyl ester as 1:1 mixture of E/Z isomers, according to
spectroscopic and spectrometric data.
The hapten hTAM-HCH was coupled to HCH and BSA by the mixed anhydride (MA) method, obtaining
a hapten density of 1 for the hTAM-BSA (MA) conjugate according to MALDI-TOF-MS analysis,
indicating that the corresponding HCH conjugate, with a greater number of lys residues available for
conjugation, would have sufficient hapten density to use it as immunogen, since it was prepared
simultaneously from the same batch of activated hapten. MALDI-TOF MS spectra of the HCH conjugate
could not be recorded because of the high molecular weight (> 2000 kDa). Additionally, the same hapten
was coupled to BSA and HRP using the ester active (AE) method of conjugation in order to minimize
undesired unspecific signals in the ELISA due to the presence of undesired secondary epitopes that could
have been formed during the preparation of the immunogen [19]. Hapten densities of 2 and 1 were
recorded for the hTAM-BSA (EA) and hTAM-HRP (EA) bioconjugates, respectively.
Three antisera raised against hTAM-HCH (MA, As211-213) was used to set-up competitive assays in
direct and indirect ELISA formats, using the above mentioned bioconjugate competitors. From these
studies the antibody/enzyme tracer combination As212/hTAM-HRP was selected based their suitable
5

analytical features (4OH-TAM, IC50 of 2.4 nM in buffer). Specificity studies showed that the
As212/hTAM-HRP assay recognized 4OH-TOR and 4OH-CLO with a cross reactivity of 165% and 7%,
respectively (in respect to 4OH-TAM, see table 1) Despite the low cross-recognition of 4OH-CLO, the
IC50 achieved (17.25 nM, 7.26 µg·L-1) was below the MRPLs established by the WADA for the whole
SERMs family[3] (20 µg·L-1). In the same way, the parent drugs tamoxifen (IC50 of 19.9 µg·L-1) and
toremifen (IC50 of 11.5 µg·L-1) could also be detected in compliance with the WADA requirements.
Other compounds tested, such as testosterone and estradiol, yielded negligible values.
Finally, we addressed the detection of SERMs metabolites in human urine. On a first instance, the
variability in the response of human urine samples collected from different healthy volunteers was
evaluated. As it can be observed in figure 3, the matrix effect is different among the different urine
samples. While for some samples a 10 times dilution with buffer minimized this effect for some others
this treatment was not enough. Higher dilution factors compromised the necessary detectability,
particularly for 4OH-CLO which was less recognized and therefore the LOD did not reach the MRPL.
For this reason, it was proposed to introduce a clean-up procedure to minimize the undesired matrix effect
variability. Moreover, since these metabolites are mainly excreted in urine as glucuronide and sulfate
conjugates [7] a hydrolysis step was also introduced.
For setting up the conditions, experiments were performed with a sample of pooled urine sample. The
urine was enzymatically hydrolyzed as previously described[13] and subsequently extracted with tert-
butyl methyl ether as reported before[4] for SERMs metabolites from urine. Following the extraction
protocol, the matrix effect was minimized, as can be observed in figure 4, where the ELISA performed
using pooled urine sample behave almost equal to the assay run in buffer. In the same way, when the
assay was performed using urine from a single volunteer. The analytical features of the assay run in urine
are summarized in table 2 where it can be observed the great detectability accomplished for all the
hydroxylated SERMs, reaching values of LOD of 0.04, 0.14 and 0.57 µg·L-1 for 4OH-TAM, 4OH-TOR
and 4OH-CLO, respectively. Finally, recovery studies were performed obtaining values of 24±8%,
40±6% and 50±15%, for 4OH-TAM, 4OH-TOR and 4OH-CLO, respectively. All the metabolites tested
are under the requirements for the MRPL established by the WADA, despite the recovery values.
To summarize, an analytical protocol has been established to screen SERMs in urine samples. It consist
on hydrolyzing of the urine samples, extracting them with tert-butyl methyl ether and evaporating them
under N2 stream. Before running the assay the samples are reconstituted with the assay buffer. The whole
process allowed the simultaneous determination of 24 samples per triplicate on a microplate in a single
run, together with the calibration curve. The total assay time of analysis is 2h and 20 min for several
6

microplates can be run at the same time. The assay protocol is simple in terms of the type of treatments
used and the immunochemical assay procedure and reduces the cost of the analysis. In respect to our
previous work [4], before the LC–MS/MS it was necessary to include a sold phase extraction (SPE)
procedure after the hydrolysis of the sample, and the analyses of the different urine samples have to be
done sequentially.
The urine samples collected from patients treated with toremifen (M2-8 and M10-21) and clomifen
(C066u3-5 and C066u6-10) were finally analyzed by ELISA. The excretion profiles of toremifen and
clomifen are shown in figure 5. Except for those samples collected at t=0, all samples could be quantified
(first sample collected at t=12 h). Urinary levels of 4-OH-toremifen remain high several days after
administration. Thus, the levels in patient M10-21 could be quantified after more than a week. Urinary
levels of 4-OH-clomifen could be quantified after more than 72 h for the case of volunteer C066u6-10.
Unfortunately, no additional samples were collected after this period; similarly, the urine collection was
stop earlier for voluenteers M2-8 or C066u3-5. Finally, just as a proof of concept a single volunteer was
treated with tamoxifen and the urine collected after 24 h, being possible to quantify the presence of the
drug in urine at 52.6 ± 7.6 µg·L-1.
4. Conclusions
A new methodology has been established for the detection of the main metabolites of SERMs in human
urine by an immunochemical method. The antibodies raised were produced according to a rational design
of the immunizing hapten that mimics the main metabolites of the most employed SERMs. The whole
process, consisting in an easy clean-up step of human urine and the subsequently immunochemical assay,
reached very good limits of detection; 0.04, 0.14 and 0.57 µg·L-1 for 4OH-TAM, 4OH-TOR and 4OH-
CLO, respectively. The LOD obtained are in compliance with the MRPL values recommended by the
WADA regarding the illegal use of this steroid by athletes. The fact of having class-selective antibodies
for hydroxylated SERM allows the detection of those prohibited compounds. Therefore, this developed
tool can be used as screening assay to select the positive samples for further confirmation by
chromatographic techniques. The assay demonstrates the applicability of this assay for the determination
of urine samples to establish the excretion profiles after tamoxifen, toremifen and clomifen uptake.
5. Acknowledgements
This work has been funded by SEA-on-a-chip project (FP7-OCEAN-2013-614168). The Nb4D group
(formerly Applied Molecular Receptors group, AMRg) is a consolidated research group (Grup de
Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i
Societat de la Informació de la Generalitat de Catalunya (expedient: 2017 SGR 1441). CIBER-BBN is an
7

initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation
2013-2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions are financed by the Instituto de
Salud Carlos III with assistance from the European Regional Development Fund. The ICTS
“NANOBIOSIS”, and particularly the Custom Antibody Service (CAbS, IQAC-CSIC, CIBER-BBN), is
acknowledged for the assistance and support related to the immunoreagents used in this work.
8

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spectroscopy, European Journal of Mass Spectrometry 14(3) (2008) 171-180.
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 [11] J.P. Salvador, F. Sanchez-Baeza, M.P. Marco, Preparation of Antibodies for the Designer Steroid
Tetrahydrogestrinone and Development of an Enzyme-Linked Immunosorbent Assay for Human
Urine Analysis, Analytical Chemistry 79(10) (2007) 3734-3740.
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 [12] J.P. Salvador, F. Sánchez-Baeza, M.P. Marco, Simultaneous immunochemical detection of
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
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Figure captions
Figure 1. Chemical structures of the 4-hydroxylated SERMs: 4-hydroxy-tamoxifen (4OH-TAM), 4-
hydroxy-toremifen (4OH-TOR) and 4-hydroxy-Clomifene (4OH-CLO) and the proposed immunizing
hapten (hTAM). The highlighted areas indicate the common epitopes of these substances, which have
been considered for designing the immunizing hapten. The aryl group is highlighted in blue, in red, the
phenolic group, and in green the 4-(2-dimethylamino-ethoxy)-phenyl group.
Figure 2. Synthetic pathways used for the preparation of the 4-hydroxylated metabolites 4OH-Tamoxifen
(2, R1= -CH2CH3) and 4OH-Toremifen (4, R1=-CH2-CH2-Cl), also the synthesis of the immunizing
hapten hTAM(7, R1=-(CH2)4 -COOMe).
Figure 3. Immunochemical response of different urine samples (Woman 1-5 and Man 1-5) from different
healthy volunteers at different dilution factors (from 1/1 to 1/10) in respect to the response of the assay
in PBST buffer (Amax). The urine samples samples were diluted in PBST and tested in the ELISA
as described in the experimental section
Figure 4. Calibration graphs showing the responds of the urine samples ( pooled urine sample and from
one volunteer) after sample treatment compared to the response in PBST buffer. As it can be observed
,after sample treatment both pooled urine sample and individual urine sample from a volunteer behave
identically as the assay buffer, demonstrating that the difference between different individuals have been
minimized. The points of the calibration curve corresponds to three well replicates run in the same ELISA
plate.
Figure 5. Excretion profiles of 4OH-TOR and 4OH-CLO of two individual and analysed by
As212/hTAM-HRP assay. The volunteers were treated of a single dose of xxxx, and the urine samples
were collected regularly during a period from 3 to 30 days. The results showed the average and SD of
three analysis made on different days. Each point were measure in three well replicates.
11

Salvador
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Figura
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Salvador et al. Figura 2
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Salvador et al. Figura 3
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Salvador et al. Figura 4
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Excretion profile 4OH-CLO
Excretion profile 4OH-TOR
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h
h
Salvador et al. Figura 5
16

Table 1. Recognition of structurally related chemicals, expressed by their IC50 and the percentage of
cross-reactivity with respect to (E/Z)4OH-Tamoxifen
IC50,
nM CR,
%
(µg·L-1)
(E/Z)4OH-Tamoxifen
Tamoxifen
1.19 (0.46)
100
2.2
1.2
4.2
165
0.6
7.0
53.65 (19.9)
(Z)3-Methoxy-4OH-Tamoxifen 102.9 (42.9)
Toremifen
28.5 (11.5)
0.72 (0.30)
213.6 (87.5)
17.25 (7.26)
(E/Z)4OH-Toremifen
Clomifen
(E/Z)4OH-Clomifen
Testosterone
Estradiol
>1000 (>288) <0.04
>1000 (>272) <0.04
17

Table 2. Analytical parameters for each hydroxylated-SERMs in the As212/hTAM-HRP assay (N=3)
As212/hTAM-HRP
Amin
4OH-TAM
0.050±0.006
1.23±0.08
4OH-TOR
0.054±0.016
1.44±0.16
-0.88±0.06
4.29±0.99
0.998±0.001
0.15±0.03
1.76±0.36
4OH-CLO
0.05±0.01
1.20±0.04
-0.69±0.04
31.14±4.99
0.99±0.01
0.57±0.13
12.38±1.56
Amax
Slope
-0.75±0.02
1.61±1.03
IC50, nM
R2
0.9991±0.0004
0.04±0.02
LOD, µg·L-1
IC50, µg·L-1
WR, µg·L-1
0.61±0.36
0.10±0.06/3.67±2.2 0.37±0.08/8.21±1.72 1.75±0.27/82.87±12.04
18