Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by high performance liquid chromatography-electrospray ionization tandem mass spectrometry with phenazine derivatization

Article abstract of DOI:10.1016/j.jsbmb.2010.02.016

A sensitive and selective assay method for labile estrogen o-quinones, estrone (E₁)-2,3-quinone (Q), E₁-3,4-Q, estradiol (E₂)-2,3-Q and E₂-3,4-Q, based on the use of phenazine (Phz) derivatization with o-phenylenediamine and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were purified by solid phase extraction and analyzed by HPLC-ESI-MS/MS. The protonated molecule was observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode). In multiple reaction monitoring, the transition from [M+H]⁺ to m/z 231 was chosen for quantification. Calibration curves for the o-quinones were obtained using standard catechol estrogens after sodium metaperiodate treatment and Phz derivatization. Using this method, these four estrogen o-quinones were analyzed with the limit of quantification of 5ng/ml in acetonitrile (MeCN)-blank matrix (1:4, v/v), respectively, on a basis of the weight of catechol estrogens. Assay accuracy and precision for four estrogen o-quinones were 89.6-113.0% and 3.1-12.6% (5, 125 and 2000ng/ml in MeCN-blank matrix). Applications of this method enabled to determine the catalytic activities on hydroxylation and subsequent oxidation of E₁ and E₂ of Mushroom tyrosinase and rat liver microsomal fraction. It was confirmed by this method that tyrosinase exhibited 2- and 4-hydroxylation and further oxidation activities for catechols in the ring-A of estrogens. Whereas rat liver microsomal fraction possessed only 2- and 4-hydroxylation activities, and further oxidation activity for catechol estrogens was low.

Full text of DOI:10.1016/j.jsbmb.2010.02.016

Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
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Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by
high performance liquid chromatography–electrospray ionization tandem mass
spectrometry with phenazine derivatization
Kouwa Yamashita∗, Akina Masuda, Yuka Hoshino, Sachiko Komatsu, Mitsuteru Numazawa
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-1 Komatsushima 4-Chome, Aoba-ku, Sendai, Miyagi 981-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A sensitive and selective assay method for labile estrogen o-quinones, estrone (E1)-2,3-quinone (Q),
E1-3,4-Q, estradiol (E2)-2,3-Q and E2-3,4-Q, based on the use of phenazine (Phz) derivatization with o-
phenylenediamine and high performance liquid chromatography–electrospray ionization tandem mass
spectrometry (HPLC–ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were
purified by solid phase extraction and analyzed by HPLC–ESI-MS/MS. The protonated molecule was
observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode). In multiple
reaction monitoring, the transition from [M+H]+ to m/z 231 was chosen for quantification. Calibration
curves for the o-quinones were obtained using standard catechol estrogens after sodium metaperiodate
treatment and Phz derivatization. Using this method, these four estrogen o-quinones were analyzed with
the limit of quantification of 5 ng/ml in acetonitrile (MeCN)–blank matrix (1:4, v/v), respectively, on a
basis of the weight of catechol estrogens. Assay accuracy and precision for four estrogen o-quinones
were 89.6–113.0% and 3.1–12.6% (5, 125 and 2000 ng/ml in MeCN–blank matrix). Applications of this
method enabled to determine the catalytic activities on hydroxylation and subsequent oxidation of E1
and E2 of Mushroom tyrosinase and rat liver microsomal fraction. It was confirmed by this method that
tyrosinase exhibited 2- and 4-hydroxylation and further oxidation activities for catechols in the ring-A of
estrogens. Whereas rat liver microsomal fraction possessed only 2- and 4-hydroxylation activities, and
further oxidation activity for catechol estrogens was low.
Received 7 October 2009
Received in revised form 12 February 2010
Accepted 18 February 2010
Keywords:
Liquid chromatography–electrospray
ionization tandem mass spectrometry
o-Quinone
Immobilized iodoxybenzoic acid
Phenazine derivative
Tyrosinase
Rat liver microsomes
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
formation of estrogens to o-quinone and further oxidized products
[10]. 2,3-Quinones and 3,4-quinones provided different species of
It is known that estrogens exert inherent carcinogenic activities
by generating electrophilic molecular species such as o-quinones
or semiquinones that can covalently bind to DNA in addition to its
hormonal action to stimulate proliferation in receptor-mediated
cancer initiation [1–4]. In estrogen metabolism, hydroxylation of
aromatic A-ring with CYPs enzymes and subsequent oxidation of
catechols to the corresponding o-quinones have been hypothesized
to be leading cause of generation of electrophilic molecules [5,6]
(Fig. 1). Catechol estrogens are typically methylated by catechol
O-methyl-transferase to afford monomethyl ethers, however non-
methylated 2,3- and 3,4-catechols are transformed with peroxidase
or tyrosinase to the reactive o-quinones that are proposed to attack
nucleophilic groups on DNA via Michael addition [7–9]. It was also
demonstrated that tyrosinase was responsible for the direct trans-
adducts [4,9,11], and these facts were another interest in relation
to the existence of the difference in carcinogenic activity between
these o-quinones.
Most of studies in this field were focused on the investigation
with the purpose of obtaining insight into the possible mode of
binding mechanism of such o-quinones to DNA or glutathione by
analyzing the formed adduct qualitatively [3,4,7,12–16]. Synthesis
and characterization of estrogen o-quinones in a preparative scale
was investigated by Abul-Hajji and Dwivedy et al using 1H nuclear
magnetic resonance (NMR) spectroscopy and high performance
liquid chromatography (HPLC) with ultraviolet (UV) detection
[3,17]. In those reports, it was demonstrated that 3,4-quinones
were more stable than 2,3-quinones by the measurement of their
half-lives in the solution. Gelbke and Knuppen demonstrated the
specific conversion of 2-hydroxyestrogens to the corresponding
2,3-quinones under various conditions and its stability, and inves-
tigated further transformation to phenazine (Phz) derivatives to
stabilize o-quinones [18]. And it was also confirmed that prepared
estrogen 2,3-quinone was relatively unstable and not obtained
∗
Corresponding author. Tel.: +81 22 727 0079; fax: +81 22 727 0137.
E-mail address: kyama@tohoku-pharm.ac.jp (K. Yamashita).
0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2010.02.016

142
K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
Fig. 1. Possible metabolic pathway of estrone and estradiol with various enzymic systems involving o-quinone formation E1 (estrone), E2 (estradiol), 2-OH-E1/E2 (2-
hydroxyestrone/estradiol), 4-OH-E1/E2 (4-hydroxyestrone/estradiol), 2-MeO-E1/E2 (2-methoxyestrone/estradiol), 4-OH-E1/E2 (4-methoxyestrone/estradiol), E1/E2-2, 3-Q
(estrone/estradiol-2,3-quinones) and E1/E2-3,4-Q (estrone/estradiol-3,4-quinones).
by crystalline form, however 2,3-quinones survive in chloroform
solution for several hours [18]. The Phz derivatization was also
used to the identification of 2-hydroxyestrone in human urine
after oxidation [19] and other natural product [20]. However, the
feasibility and selectivity of this derivatization reaction in the dis-
criminative determination between o-quinone and hydroquinone,
especially in a microscale, have been unclear. In this report, we
described an assay method for the selective quantification of
estrogen o-quinones in a microscale based on the use of high per-
formance liquid chromatography–electrospray ionization tandem
mass spectrometry (HPLC–ESI-MS/MS) involving in situ generation
of o-quinones from catechols and its trapping by derivatization
with o-phenylenediamine. A Phz derivative of deuterated estrogen
was synthesized and used as an internal standard for quantifica-
tion. The present method was applied to the determination of the
catalytic activities of Mushroom tyrosinase and rat liver micro-
somal fraction in the conversion of estrone and estradiol to their
corresponding o-quinones.
and ␤-nicotineamide adenine dinucleotide phosphate (reduced
form) (NADPH) were purchased from Sigma–Aldrich (St. Louis,
MO, USA). 2,3-Dihydroxyestra-1,3,5(10)-trien-17-one (2-OH-E1:
2-hydroxyestrone, 3), estra-1,3,5(10)-triene-2,3,17␤-triol (2-OH-
E2: 2-hydroxyestradiol, 4), 3,4-dihydroxy-estra-1,3,5(10)-trien-
17-one (4-OH-E1: 4-hydroxyestrone, 5), estra-1,3,5(10)-triene-
3,4,17␤-triol (4-OH-E2: 4-hydroxyestradiol, 6) were obtained
from Steraloids (Newport, RI, USA). o-Phenylenediamine, sodium
metaperiodate (NaIO4), N, N-dimethylformamide (DMF), dimethyl-
sulfoxide (DMSO), 36% hydrochloric acid (HCl), ethyl acetate
(EtOAc), hexane, 95% ethanol (EtOH) and NaIO4 were obtained
from Nacalai Tesque (Kyoto, Japan). These solvents and reagents
were of analytical grade. HPLC grade methanol (MeOH), ace-
tonitrile (MeCN), acetic acid (AcOH) and ultra-pure water were
purchased from Wako (Osaka, Japan). Immobilized iodoxybenzoic
acid (IBX-polystyrene beads) was obtained from Nova Chemi-
cals (Calgary, Canada). Bond Elut C18 (200 mg, 3 ml) and Bond
Elut SI (100 mg, 1 ml) cartridges were obtained from Varian (Palo
Alto, CA, USA). Pre-coated plate for thin layer chromatogra-
phy (TLC) (Kiesel Gel 60 F254, 20 cm × 20 cm, 0.5 mm thickness)
was obtained from Merck KGaA (Darmstadt, Germany). Freshly
prepared solutions of catechol estrogens were used for each
experiment. Rat liver microsomal fraction (Four weeks old, male,
Wistar strain, 20 mg protein/ml) used in this study was pre-
pared [21] and donated by Dr. Yorihisa Tanaka (Department of
Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical
University).
2. Experimental
2.1. Materials and reagents
3-Hydroxyestra-1,3,5 (10)-trien-17-one (E1: estrone, 1), estra-
1,3,5(10)-triene-3,17␤-diol (E2: estradiol, 2a), estra-1,3,5(10)-
triene-3,17␤-diol 17-acetate (E2-17-OAc: estradiol 17-acetate,
2b), Mushroom tyrosinase (EC 1.18.14.1, 5500 U/mg protein)

K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
143
2.2. Preparation of phenazine (Phz) derivatives (11, 12a, 13,
14a) (Fig. 2)
2.3. Synthesis of internal standard (IS)
[16,16,17␣-2H3] E2-17␤-OAc (15 mg) was prepared in our lab-
oratory from [16,16,17␣-2H3] E2 by two steps of acetylation with
acetic anhydride and pyridine, and partial hydrolysis of 3-acetate
with NaHCO3 in MeOH. [16,16,17␣-2H3] E2-17␤-OAc was subse-
quently treated with IBX-polystyrene beads, o-phenylenediamine
and 5% KOH in methanol to afford deuterium labeled compounds
15 (1.5 mg) and 16 (1.3 mg), after preparative TLC purification using
EtOAc–hexane (1:2, v/v) as a developing solvent. Deuterium distri-
butions were as follows: 15: d0; 0.5%, d1; 3.7%, d2; 22.5%, d3; 70.0%,
d4; 3.3%, 16: d0; 0.2%, d1; 2.8%, d2; 22.9%, d3; 70.9%, d4; 3.2%. Com-
pound 16 was used as an IS for the simultaneous determination of
four estrogen o-quinones.
Phz derivatives of four estrogen o-quinones, E1-2,3-Q-Phz (11),
E2-2,3-Q-Phz (12a), E1-3,4-Q-Phz (13) and E2-3,4-Q-Phz (14a),
were prepared from the corresponding catechol estrogens by
treatment with NaIO4 in DMF followed by the cyclization with o-
phenylenediamine as described by Gelbke [18,19]. Derivatives 11
and 13 were also prepared by the direct oxidation of 1 with immo-
bilized IBX-polystyrene beads [22–24] followed by the treatment
of crude o-quinones with DMF solution of o-phenylenediamine.
Derivatives 12a and 14a were prepared from 2b in the similar man-
ner as 11 and 13. In the preparation of these derivatives, 12b and
14b were initially prepared as intermediates from 2b. Treatment of
12b or 14b with 5% KOH/MeOH gave 12a or 14a. Each Phz deriva-
tive was purified by preparative TLC using EtOAc–hexane (1:2, v/v)
as a developing solvent. Structures of each Phz derivative were
confirmed by the measurement of 1H NMR spectra (JEOL JNM-400
spectrometer; 400 MHz; CDCl3 solution, Tokyo, Japan), UV spec-
tra (Shimadzu MPS-2450 UV–vis spectrophotometer, Kyoto, Japan)
and high resolution mass spectra (HR-MS) (JEOL JMS-700 double
focusing mass spectrometer) in an electron ionization mode.
2.4. Preparation of blank matrix
Rat liver microsome suspension (20 mg protein/ml) was treated
at 100 ◦C for 15 min. Denaturated microsome suspension was
diluted with 50 mM phosphate buffer (pH 7.4, 40 ml) and then re-
suspended. The diluted microsome suspension was used as blank
matrix.
2.5. High performance liquid chromatography (HPLC)
HPLC was run on a Waters 2695 separation module equipped
with a Waters 2487 UV detector (Waters, Milford, MA, USA). The
column was an X-Bridge C18 (150 mm × 4.6 mm I.D., 5 ␮m, Waters)
and used at an ambient temperature. The mobile phases consisted
of MeOH–H2O–AcOH (45:55:0.1, v/v/v) for o-quinones or cate-
chols and MeCN–H2O–AcOH (70:30:0.1, v/v/v) for Phz derivatives
were used at a flow rate of 1 ml/min with an isocratic elution.
Wavelength of an UV detector was set at 257 nm for the detec-
tion of Phz derivatives and 280 nm for o-quinones and catechols,
respectively.
2.6. HPLC–electrospray ionization tandem mass spectrometry
(ESI-MS/MS)
HPLC–ESI-MS/MS was run on a Finnigan TSQ Quantum triple-
stage quadrupole mass spectrometer (Thermo Fischer Scientific,
San Jose, CA, USA) equipped with an ESI ion source and a Sur-
veyor auto-sampler and a MS-pump (Thermo Fischer Scientific).
The columns was an X-Bridge C18 (100 mm × 2.1 mm I.D., 5 ␮m,
Waters) connected to a guard cartridge (C18, 10 mm × 2 mm I.D.,
5 ␮m) in a guard-cartridge holder with the mobile phase consisted
of MeCN–H2O–AcOH (70:30:0.1, v/v/v) at a flow rate of 200 ␮l/min.
Elution was performed with an isocratic mode and at an ambi-
ent temperature. The general ESI/MS conditions were as follows:
spray voltage, 4500 V (positive); sheath gas, nitrogen, 35 arbitrary
unit (gas pressure); auxiliary gas, nitrogen, 15 arbitrary unit (gas
pressure); ion transfer capillary temperature, 350 ◦C; collision gas
argon, 1.5 mTorr (gas pressure). Transitions of Phz derivatives for
each o-quinone in multiple reaction monitoring (MRM) were as fol-
lows: E1-2,3-Q-Phz (11) and E1-3,4-Q-Phz (13); m/z 357 → m/z 231
(collision energy, 60 V), E2-2,3-Q-Phz (12a) and E2-3,4-Q-Phz (14a);
m/z 359 → m/z 231 (collision energy, 60 V), IS (16): m/z 362 → m/z
231 (collision energy, 60 V).
2.7. Generation of o-quinones (7, 9, 8a and 10a) from 2- and
4-hydroxyestrogens (3, 5, 4 and 6) with NaIO4 in a microscale
To a mixed solution of catechol estrogens (50 ␮l) (3, 4, 5 and
6: 50 ␮g each/ml) in MeCN–blank matrix (1:4, v/v) was added
10% NaIO4 (20 ␮l) and AcOH (5 ␮l), and the resulting mixture was
Fig. 2. Chemical structures of estrogens and phenazine derivatives used in this
study.

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K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
allowed to stand at room temperature for 2 min. The reaction mix-
ture was immediately analyzed by HPLC (ꢀ 280 nm). A mixture
of catechol estrogens (50 ␮l) (3, 4, 5 and 6) at two different con-
centrations (200 ng and 2000 ng/ml: MeCN–blank matrix, 1:4, v/v)
were prepared and oxidized with 10% NaIO4, and then submitted
to LC–ESI-MS/MS analyses.
Within assay precision was expressed as relative standard devia-
tion of analytical results for each concentration of sample. Accuracy
was determined and expressed as a percentage of analytical as
recovery rates of the measured concentrations against the spiked-
concentration for each o-quinone (catechol estrogen-equivalent
weight).
2.12. Determination of o-quinone formation from estrone and
estradiol by tyrosinase and rat liver microsomes
2.8. Trapping of o-quinones with o-phenylenediamine
To a solution of estrogen o-quinone generated as described
above (Section 2.7) was added o-phenylenediamine (15 mg/200 ␮l
in DMF, w/v), and the resulting mixture was allowed to stand at
room temperature for 30 min. The reaction mixture was diluted 5%
HCl (2 ml) and then the transferred onto Bond Elut C18 (200 mg,
pre-conditioned with 3 ml EtOAc, 3 ml MeOH and 3 ml water). The
cartridge was subsequently washed with 5% HCl (3 ml × 3), H2O
(3 ml × 3) and 35% MeCN (3 ml × 3) and the Phz derivative was
eluted with EtOAc (2 ml). To this eluate was added hexane (4 ml)
and the resulting mixture was passed through Bond Elut SI (100 mg,
1 ml). Eluate was evaporated to dryness and the residue was dis-
solved in EtOAc (100 ␮l) and then subjected to LC–ESI-MS/MS.
Absolute recovery rates in the processes of o-quinone genera-
tion from catechol estrogen followed by the Phz derivatization were
assessed by comparing the peak area of the prepared Phz deriva-
tive to that of peak area obtained from the corresponding synthetic
standards of each Phz derivative using 16 as an internal standard.
The substrate solution (E1 and E2: 10 ␮g each in DMSO 10 ␮l)
was added to the tyrosinase solution (500 ␮g in 50 mM phosphate
buffer, pH 7.4) (1 ml), and the resulting mixture was incubated
at 37 ◦C. At 30 min after starting incubation, a portion of sam-
ple (50 ␮l) was taken out and the sample was treated with
o-phenylenediamine (15 mg/200 ␮l DMF, v/v) and AcOH (5 ␮l) at
room temperature for 30 min. After addition of IS (16, 200 ng/20 ␮l
MeCN), the reaction mixture was diluted with 5% HCl (2 ml) and
the Phz derivative was extracted, purified and analyzed as men-
tioned above. Pre-treatment samples of incubation mixture (50 ␮l)
with 15% NaIO4 (15 ␮l) was prepared to estimate the formation of
catechol estrogens in the medium.
Similarly, the substrate solution (E1 and E2: 10 ␮g each in DMSO
10 ␮l) was added to the mixture of rat liver microsomes (500 ␮g)
and NADPH (10 ␮g) in 50 mM phosphate buffer (pH 7.4) (1 ml), and
the resulting mixture was incubated at 37 ◦C. At 30 min after start-
ing incubation, a portion of sample (50 ␮l) was taken out and the
sample was treated and analyzed as mentioned above.
2.9. Calibration curves
3. Results and discussion
The mixed solution of four catechol estrogens (50 ␮l) (3, 4, 5
and 6: 0, 2.5, 5.0, 12.5, 50, 125, 625, 2500 ng each/ml: MeCN–blank
matrix, 1: 4, v/v) was subsequently treated with 10% NaIO4 (20 ␮l)
and AcOH (5 ␮l) at room temperature for 2 min, and then with o-
phenylenediamine (15 mg/200 ␮linDMF, v/v)at roomtemperature
for 30 min. After addition of IS (16, 200 ng/20 ␮l MeCN), the reaction
mixture was diluted 5% HCl, purified by solid phase extraction and
then analyzed as mentioned above. Calibration curve for each o-
quinone was obtained by assigning the catechol-equivalent weight
to x and the peak area ratio of the Phz derivatives to the corre-
sponding IS to y. Subsequently, a linear regression was performed
for constructing calibration curve for each component.
3.1. Preparation of Phz derivatives of estrogen o-quinones
Phz derivatives of four estrogen o-quinones (11, 12a, 13, 14a)
were prepared as standards. o-Quinones have been generally
obtained by oxidation of catechol estrogens using various oxidants
such as activated manganese dioxide, sodium iodate or NaIO4 as
described by Gelke et al. [18,19]. In this study, we employed direct
oxidation of E1 or E2 with IBX-polystyrene beads [22–24]. Treat-
ment of E1 with IBX-polystyrene beads in DMF solution resulted
in the formation of 7 and 9 as a mixture. IBX-polystyrene beads
were easily removed by filtration and o-quinone intermediates thus
obtained were then treated with o-phenylenediamine in DMF solu-
tion. Preparative TLC purification of crude product gave the Phz
derivatives 11 and 13 as pure materials using EtOAc–hexane (1:2,
v/v) as a developing solvent.
Direct oxidation of E2 with IBX-polystyrene beads resulted
in the formation of the mixture of 8a and 10a. In addition to
the formation of 8a and 10a, the production of 7 and 9 as by-
products was inevitable by further oxidation at 17␤-OH by this
reagent showing complicated HPLC and TLC patterns. Then 12a
and 14a were prepared using 2b as a starting material. Oxida-
tion of 2b with IBX-polystyrene beads followed by the treatment
with o-phenylenediamine resulted in the formation of 12b and 14b.
Alkaline hydrolysis of 12b and 14b followed by the purification with
TLC using EtOAc–hexane (1:2, v/v) as a developing solvent gave 12a
and 14a as pure materials. 1H NMR, HR-MS and UV spectral mea-
surement supported the structures of 11, 12a, 12b, 13, 14a and 14b,
respectively.
2.10. Assay specificity
The mixed solution of four catechol estrogens (3, 4, 5 and
6: 500 ng each/50 ␮l: MeCN–blank matrix, 1:1, v/v) was directly
treated with o-phenylenediamine (15 mg/200 ␮l DMF, w/v) and
AcOH (5 ␮l) at room temperature for 30 min. The mixture was
purified and analyzed in the similar manner as mentioned above.
The peak areas corresponding to the retention time (tR) of each
Phz derivative of o-quinone were recorded by LC–ESI-MS/MS and
compared to those of peak areas obtained for the samples by pre-
treatment of catechols (500 ng each) with NaIO4, and then data
were expressed as relative peak area.
2.11. Assay accuracy and precision
To determine the assay accuracy and precision, the mixed solu-
tion known amounts of catechol estrogens (3, 4, 5 and 6: 5, 125
and 2000 ng each/ml: MeCN–blank matrix, 1:1, v/v) was prepared
and a portion of solution (50 ␮l) was subsequently treated with
10% NaIO4 (20 ␮l) and AcOH (5 ␮l) at room temperature for 2 min,
and then with o-phenylenediamine (15 mg/200 ␮l in DMF, v/v) at
room temperature for 30 min. After addition of IS (200 ng/20 ␮l
MeCN), the reaction mixture was purified as mentioned above.
3.2. HPLC–ESI-MS/MS
In ESI-MS, the Phz derivatives of these estrogen o-quinones gave
protonated molecule ([M+H]+) as the base peaks accompanying
the appearance of adduct ion of [M+H + MeCN]+ in each deriva-
tive. The ESI-mass spectra and product ion spectra of the Phz

K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
145
Fig. 3. Representative ESI-mass spectra (upper) and product ion mass spectra (bottom) of the Phz derivatives of estrogen o-quinones. (A) E1-2,3-Q-Phz (11), (B) E2-2,3-Q-Phz
(12a), (C) E1-3,4-Q-Phz (13) and (D) E2-3,4-Q (14a). Spray voltage: 4500 V, polarity: positive.
derivatives E1-2,3-Q-Phz (11), E2-2,3-Q-Phz (12a), E1-3,4-Q-Phz
(13) and E2-3,4-Q-Phz (14a) were shown in Fig. 3A–D. The frag-
mentation pattern of the base peak ion of each derivative under
various levels of collision energy was examined and the ion at m/z
231 (formed by cleavage of both C9–C11 and C8–C14 bonds) was
observed as common product ion for each Phz derivative of o-
quinone with relatively high intensity. Then the transition from
[M+H]+ to m/z 231 was selected for quantification of each Phz
derivative in MRM. Using reversed phase ODS-column (X-Bridge
C18, 100 mm × 2.1 mm I.D., 5 ␮m), the Phz derivative of estrogen
o-quinones were eluted in the order 12a, 11, 14a and 13, respec-
tively. Retention times for each of the Phz derivatives were as
follows (mobile phase; MeCN–H2O–AcOH = 70:30:0.1, v/v/v, flow
rate; 0.2 ml/min): E2-2,3-Q-Phz (12a, tR = 3.4–3.5 min), E1-2,3-Q-
Phz (11, tR = 3.7–3.8 min), E2-3,4-Q-Phz (14a, tR = 5.4–5.5 min) and
E1-3,4-Q-Phz (13, tR = 6.0–6.1 min). All Phz derivatives of estrogen
o-quinones were successfully separated using this mobile phase
constitution.
3.3. Generation and derivatization of o-quinones in a microscale
o-Quinone
generation
and
derivatization
with
o-
phenylenediamine in a microscale was examined using catechol
estrogens, 3, 4, 5 and 6. In order to optimize these processes,
we investigated the reaction conditions with some variations
of constituents (AcOH: 1, 2, 5 and 10 ␮l, 10% NaIO4: 10, 20 and
30 ␮l, o-phenylenediamine: 5, 10, 15, 30 mg in DMF 200 ␮l).
The highest yields of the Phz derivatives from each catechol
estrogen were obtained using the following reagent mixture;
AcOH: 5 ␮l, 10% NaIO4 20 ␮l and o-phenylenediamine: 15 mg
Table 1
Absolute recovery rates in the processes of o-quinone formation and phenazine derivatization from catechol estrogens.
Concentrationa
Absolute recovery rateb (%)
E1-2,3-Q-Phz(11)
E2-2,3-Q-Phz(12a)
E1-3,4-Q-Phz(13)
E2-3,4-Q-Phz(14a)
200 ng/ml
2000 ng/ml
60.6 16.2
66.3 6.3
70.7 9.1
76.0 2.9
36.3 8.0
34.5 2.5
55.7 7.7
54.4 0.8
Reaction conditions were as follows:
o-Quinone formation; steroids (50 ␮l), AcOH (5 ␮l), 10% NaIO4 (20 ␮l), 2 min.
Phenazine derivatization: o-phenylenediamine 15 mg/200 ␮l DMF, 30 min.
a
Concentrations of catechol estrogens in MeCN–blank matrix (1:4, v/v).
Data were calculated on a basis of catechol estrogen-equivalent weight and expressed as the mean value of three experiments.
b

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K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
Table 2
Table 3
Relative peak area of the generated o-quinones from catechols with or without NaIO4
Assay accuracy and precision.
after phenazine derivatization by LC–ESI-MS/MS.
Analytes
Amounta (ng/ml)
Accuracy (%)
RSD (%)
Analytes
Spiked (ng)a
500
NaIO4 treatment
Relative peak areab,c
Added
Found (mean SD)b,c
+
1.0
0.0035
E1-2,3-Q (7)
5
125
2000
5.0 0.5
115.5 14.5
2260.0 108.5
100.0
92.4
113.0
10.0
12.6
4.8
E1-2,3-Q (7)
−
500
500
500
+
−
1.0
0.0052
E2-2,3-Q (8a)
E1-3,4-Q (9)
5
125
2000
5.5 0.5
126.0 15.1
2019.5 103.5
110.0
100.8
101.0
9.1
11.9
5.1
E2-2,3-Q (8a)
E1-3,4-Q (9)
+
−
1.0
0.0053
+
1.0
0.0079
E2-3,4-Q (10a)
5
125
2000
5.5 0.6
112.0 10.0
2084.0 126.0
110.0
89.6
104.2
10.9
8.9
6.0
−
a
Spiked amount was expressed on a basis of the weight of catechol estrogens.
Peak areas were determined as their Phz derivatives.
Data were presented as the mean values of three experiments.
b
c
5
125
2000
5.5 0.5
116.5 12.0
2152.5 65.5
110.0
93.2
107.6
9.1
10.3
3.1
E2-3,4-Q (10a)
a
Added and found values were expressed on a basis of the weight of catechol
estrogens.
b
in 200 ␮l DMF. The absolute recovery rates of each catechol
estrogen were summarized in Table 1. The absolute recovery
rates of 11, 12a, 13 and 14a from 3, 4, 5 and 6 (2000 ng/ml) were
66.3 6.3%, 76.0 2.9%, 34.5 2.5% and 54.4 0.8%, respectively.
Similar recovery rates were obtained for the samples with lower
concentration (200 ng/ml). The absolute recovery rates in the
processes with the optimized condition were varied from 35%
to 76%, however, the overall yields in these processes for each
catechol estrogen were reproducible in these concentrations.
Data were obtained from four experiments.
o-Quinone concentrations were determined as their Phz derivatives.
c
3.4. Calibration curves
Calibration curves were constructed for each derivative (11,
13, 12a and 14a) by means of catechol estrogens (3, 4, 5 and
6) after NaIO4 oxidation and Phz derivatization using 16 as an
internal standard. Each calibration curve, as determined by sim-
Fig. 4. Typical MRM chromatograms of o-quinones formed from E1 and E2 after incubation with tyrosinase (500 ␮g/ml in 50 mM phosphate buffer, pH 7.4, 37 ◦C for 30 min,
substrate concentration: 10 ␮g each/ml) as their Phz derivatives. (A) Direct derivatization, (B) pre-treatment with 10% NaIO4 at room temperature for 2 min. Spray voltage:
4500 V, collision energy: 60 eV. MRM transitions for each component were as follows: E1-2,3-Q-Phz (11) and E1-3,4-Q-Phz (13); m/z 357 → m/z 231, E2-2,3-Q-Phz (12a) and
E2-3,4-Q-Phz (14a); m/z 359 → m/z 231 and IS (16): m/z 362 → m/z 231.

K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
147
ple linear regression analysis, exhibited excellent linearity for
the weight range of 2.5–2500 ng/ml (each catechol estrogen-
equivalent weight/ml in MeCN–blank matrix, 1:4, v/v) with
regression coefficient of more than 0.999 and small y-intercept.
the limit of quantifications (LOQs) for each estrogen o-quinone
as Phz derivatives in the real sample (MeCN–blank matrix; 1:4,
v/v) were estimated to be 5 ng/ml with acceptable accuracy and
precision (>15%). The LOQ of this method enabled to analyze
spontaneously generated estrogen o-quinones in the medium
obtained by the incubation of E1 and E2 with Mushroom tyrosi-
nase or rat liver microsomal fraction at a protein concentration of
0.5 mg/ml.
The Phz derivatives of estrogen o-quinones were detected by
HPLC–UV method with high sensitivity (10 pg on-column, synthetic
standard, data not shown) because of the large ε value of inherent
Phz moiety. However, the LOQ in the real sample (MeCN–blank
matrix; 1:4, v/v) was limited to be approximately 25 ng/ml due to
the co-existing impurities originated from the excess derivatizing
agent even after the use of solid phase purification. The typical
HPLC–UV chromatogram of the Phz derivatives of generated o-
quinones from catechol estrogens in the blank matrix was shown
in Supplementary materials, which can be found in the electronic
version of this article.
3.5. Assay specificity
A degree of Phz derivatives formation from catechol estrogens
(hydroquinones) with o-phenylenediamine without oxidation pro-
cess by NaIO4 was assessed by LC–ESI-MS/MS. Results were shown
in Table 2. The relative peak areas corresponding to the retention
times for 11, 13, 12a and 14a in the samples obtained from catechol
estrogens (500 ng) were 0.0035–0.0079 when compared to those
of the peak area of the same samples pre-treated with NaIO4. This
result suggested that the Phz derivatization was specific for estro-
gen o-quinones and enabled to determine o-quinones selectively
even in the presence of hydroquinones.
3.6. Assay accuracy and precision
Results were given in Table 3. Assay accuracy and precision (rel-
ative standard deviation, RSD) ranged from 89.6 to 113.0% and 3.1
to 12.6%, respectively. These results indicated that the assay accu-
racy and reproducibility of this study were found to be satisfactory.
The pure Phz derivatives (11, 12a, 13, 14a) were detected with
injected amount of 500 fg (signal-to-noise ratio of >5), however,
3.7. Catalytic activities of tyrosinase and rat liver microsomes on
o-quinones formation from estrone and/or estradiol
Fig. 4 showed the typical MRM chromatograms of estro-
gen o-quinones formed in the medium after incubation of E1
or E2 with Mushroom tyrosinase after Phz derivatization. As
Fig. 5. Typical SRM chromatograms of o-quinones formed from E1 and E2 after incubation with rat liver microsomes (500 ␮g/ml in 50 mM phosphate buffer, pH 7.4, 37 ◦C for
30 min, substrate concentration: 10 ␮g each/ml) as their Phz derivatives. (A) Direct derivatization, (B) pre-treatment with 10% NaIO4 at room temperature for 2 min. Spray
voltage: 4500 V, collision energy: 60 eV. MRM transitions for each component were as follows: E1-2,3-Q-Phz (11) and E1-3,4-Q-Phz (13); m/z 357 → m/z 231, E2-2, 3-Q-Phz
(12a) and E2-3, 4-Q-Phz (14a); m/z 359 → m/z 231 and IS (16): m/z 362 → m/z 231.

148
K. Yamashita et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 141–148
shown in Fig. 4A, the direct derivatization of the incubation mix-
ture of E1 and E2 with tyrosinase resulted in the formation of
the Phz derivatives E1-2,3-Q-Phz (11) and E2-2,3-Q-Phz (12a)
as major products accompanying the production of E1-3,4-Q-
Phz (13) and E2-3,4-Q-Phz (14a) in a small quantity (3.8–6.2%,
w/w, of 11 and 12a). Pre-treatment of the incubation mixture
with NaIO4 prior to Phz derivatization resulted in the slight
increase of 11 and 12a formation (119–137% of direct derivati-
zation) but the tremendous increase of 13 and 14a formations
(890–913% of direct analysis) (Fig. 4B). These results indicated
that tyrosinase possessed the catalytic activities for both 2-
and 4-hydroxylation of the ring-A of estrogens and subsequent
oxidation of catechols to 2,3-quinones. The catalytic activities
of tyrosinase for 4-hydroxylation were 25–50% in comparison
with those of 2-hydroxylation activities. The catalytic activity
for the oxidation of 3,4-catechols to the corresponding 3,4-
quinones was low in contrast to the corresponding oxidation
activity for 2,3-catechols. As shown in these results, the high
regio-specificities of this enzyme were observed in the oxidation
processes between 2,3-catechols and 3,4-catechols to their corre-
sponding o-quinones.
On the other hand, the direct derivatization of the incubation
mixture of E1 and E2 with rat liver microsomes resulted in the
detection of 11, 12a, 13 and 14a with only trace levels (Fig. 5A),
however the significant increases in the formations of 2,3-quinones
and 3,4-quinones were observed after pre-treatment of the incu-
bation mixture with NaIO4 (Fig. 5B). These results indicated that rat
liver microsomal fraction possessed mainly the catalytic activities
of 2- and 4-hydroxylation of the ring-A of estrogens, which might
be attributable to CYP1A1, CYP1A2, CYP3A4 and CYP1B1 activities
[25], and further oxidation activities of catechols to the correspond-
ing o-quinones were estimated to be low in this enzyme system.
These results also suggested that rat hepatic microsomal enzymes
seem not to convert primarily produced catechol estrogens to the
corresponding o-quinones inherently.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jsbmb.2010.02.016.
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A sensitive and selective assay method of estrogen o-quinones
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This work was supported in part by a High Technology Research
Center Project from the Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan.