Estrogen equivalent concentration of 13 branched para-nonylphenols in three technical mixtures by isomer-specific determination using their synthetic standards in SIM mode with GC-MS and two new diasteromeric isomers

Article abstract of DOI:10.1016/j.chemosphere.2007.09.049

Thirteen isomers of branched para-nonylphenols (para-NP) in three technical mixtures were isomer-specifically determined using their synthesized standards by SIM of structurally specific ions, m/z 135, 149 or 163 with GC-MS. Of the 13 isomers, four isomers, 4-(2,4-dimethylheptan-4-yl)phenol, 4-(4-methyloctan-4-yl)phenol, 4-(3-ethyl-2-methylhexan-2-yl)phenol (3E22NP) and 4-(2,3-dimethylheptan-2-yl)phenol synthesized for their determinations were first used as standard substances. The 13 isomers in the technical mixtures individually occurred at mass percent portion of more than 2%. The total mass percent portions in the mixtures from Tokyo Chemical Industry (TCI), Aldrich, and Fluka covered with 89 ± 2%, 75 ± 4% and 77 ± 2%, respectively. The abundance of 4-(3,6-dimethylheptan-3-yl)phenol in the three mixtures was the largest with 11.1 ± 2% to 9.9 ± 0.3%, while that of 4-(2-methyloctan-2-yl)phenol was the smallest with 2.9 ± 0.3% to 3.0 ± 0.2%. Additionally, structures of four new isomers of more than 1% portion present in a technical mixture were elucidated as two pairs of diastereomeric isomers: two types of 4-(3,4-dimethylheptan-4-yl)phenol (344NP) and those of 4-(3,4-dimethylheptan-3-yl)phenol (343NP). By estrogenic assay of 13 isomers with yeast estrogen screen system, the activity of 3E22NP was the highest, while that of 4-(3-methyloctan-3-yl)phenol was the least. Their relative activities to that of 3E22NP were individually calculated. Estrogenic equivalent concentrations of the three technical mixtures were predictively evaluated. The ratio of the EEC to the conventional concentration, total mass percent portions of the 13 isomers in technical mixtures were 0.208 for TCI, 0.206 for Aldrich and 0.205 for Fluka. The predicted estrogenic activity of measured concentration of para-NP in technical mixtures was approximately 5-fold greater than the measured estrogen agonist activity.

Full text of DOI:10.1016/j.chemosphere.2007.09.049

Available online at www.sciencedirect.com
Chemosphere 70 (2008) 1961–1972
www.elsevier.com/locate/chemosphere
Estrogen equivalent concentration of 13 branched para-nonylphenols
in three technical mixtures by isomer-specific determination
using their synthetic standards in SIM mode with GC–MS
and two new diasteromeric isomers
a,
a
b
b
c
Takao Katase *, Keiji Okuda , Yun-Seok Kim , Heesoo Eun , Hideshige Takada ,
d
d
e
e
Taketo Uchiyama , Hiroaki Saito , Mitsuko Makino , Yasuo Fujimoto
a
College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan
National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaragi 305-8604, Japan
b
c
Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
d
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajousui, Setagayaku, Tokyo 156-0045, Japan
e
Received 6 February 2007; received in revised form 27 August 2007; accepted 27 September 2007
Available online 13 November 2007
Abstract
Thirteen isomers of branched para-nonylphenols (para-NP) in three technical mixtures were isomer-specifically determined using their
synthesized standards by SIM of structurally specific ions, m/z 135, 149 or 163 with GC–MS. Of the 13 isomers, four isomers, 4-(2,4-
dimethylheptan-4-yl)phenol, 4-(4-methyloctan-4-yl)phenol, 4-(3-ethyl-2-methylhexan-2-yl)phenol (3E22NP) and 4-(2,3-dimethyl-
heptan-2-yl)phenol synthesized for their determinations were first used as standard substances. The 13 isomers in the technical mixtures
individually occurred at mass percent portion of more than 2%. The total mass percent portions in the mixtures from Tokyo Chemical
Industry (TCI), Aldrich, and Fluka covered with 89 2%, 75 4% and 77 2%, respectively. The abundance of 4-(3,6-dimethylheptan-
3-yl)phenol in the three mixtures was the largest with 11.1 2% to 9.9 0.3%, while that of 4-(2-methyloctan-2-yl)phenol was the small-
est with 2.9 0.3% to 3.0 0.2%. Additionally, structures of four new isomers of more than 1% portion present in a technical mixture
were elucidated as two pairs of diastereomeric isomers: two types of 4-(3,4-dimethylheptan-4-yl)phenol (344NP) and those of 4-(3,4-dim-
ethylheptan-3-yl)phenol (343NP). By estrogenic assay of 13 isomers with yeast estrogen screen system, the activity of 3E22NP was the
highest, while that of 4-(3-methyloctan-3-yl)phenol was the least. Their relative activities to that of 3E22NP were individually calculated.
Estrogenic equivalent concentrations of the three technical mixtures were predictively evaluated. The ratio of the EEC to the conven-
tional concentration, total mass percent portions of the 13 isomers in technical mixtures were 0.208 for TCI, 0.206 for Aldrich and
0.205 for Fluka. The predicted estrogenic activity of measured concentration of para-NP in technical mixtures was approximately 5-fold
greater than the measured estrogen agonist activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Single nonylphenol isomers; Technical nonylphenol; Estrogen equivalent concentration; GC–MS
1. Introduction
Nonylphenol (NP) is widely used as plastic additive and
antioxidant; a derivative of NP, nonylphenol ethoxylate
*
(NPE) is commonly used as a nonionic surfactant in deter-
gents, paints, emulsifying agents, pesticides herbicides as
Corresponding author. Tel./fax: +81 466 84 3720.
E-mail address: katase@brs.nihon-u.ac.jp (T. Katase).
0045-6535/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2007.09.049

1962
T. Katase et al. / Chemosphere 70 (2008) 1961–1972
well as a dispersing agent for industrial applications such as
production of paper, fiber, metal and agriculture chemicals
(White et al., 1994; Nimrod and Benson, 1996; Khim et al.,
1999). NPEs are produced by the addition of ethylene oxide
to NP and may have 1–20 ethoxy units per molecule.
Although the estrogenic activity in vitro of NP was reported
to be 10ꢀ6 times less than 17b-estradiol (E2) at the mini-
mum (Jobling and Sumpter, 1993) to 2 · 10ꢀ3 times less
Although Zhang et al. used an internal standard for deter-
mination, the single NP was not used as standard sub-
stance. We preliminary studied on variation in estrogenic
activity of the isomers isolated from fractions of a commer-
cial NP by high-performance liquid chromatography
(HPLC) (Kim et al., 2004) and an estrogen-equivalent con-
centration of NP isomers in Ariake Sea water by the deter-
mination of individual GC-peaks based on the Horii’s
method (Kim et al., 2005a). The predicted estrogenic activ-
ity for measured concentrations of NP in Ariake Sea was
calculated to be 2.7–3.0-folds greater than the measured
estrogen agonist activity (Kim et al., 2005a). Therefore, a
reliable analytical method for each isomer using synthe-
sized standards of individual NP isomers is helpful for a
risk assessment. Fourteen single isomers of branched
para-NPs were confirmed to be present in technical NP
mixtures previously by Thiele et al. (2004), Ruß et al.
(2005) and Shioji et al. (2006). The 10 isomers were quan-
tified in two technical mixtures using the standards synthe-
sized by Ruß et al. (2005) although their detail
quantification was not described there. In our previous
works, 11 isomers were fractionated from the technical
mixtures by HPLC and a gas-chromatograph equipped
with a preparative fraction collector (GC-PFC) and their
structures were elucidated by NMR and MS (Kim et al.,
2004, 2005b). Furthermore, Uchiyama et al. (in press)
and Saito et al. (2007) in our research group synthesized
13 isomers to be used as standard substance for bioassay
and environmental analyses.
In the present study, the 13 isomers of branched para-
NPs in three technical mixtures were isomer-specifically
determined using their synthesized standard by selected
ion monitor (SIM) for GC–MS of m/z 135, and m/z 149
and m/z 163 of all the mass-fragments regarding their iso-
mers, which were contained more than 1% in the technical
mixtures and systematically determined by only the three
selected fragment ions. Moreover, estrogenic activities of
individual single isomers by recombinant yeast estrogen
screen assay (YES) were determined, then finally estrogen
equivalent concentration (EEC) of three technical commer-
cial NP was calculated for evaluation of risk assessments of
NP isomers. Additionally, structures of two new diastereo-
meric isomers, 4-(3,4-dimethylheptan-4-yl)phenol (344NP)
and 4-(3,4-dimethylheptan-3-yl)phenol (343NP) of more
than 1% portion present in a technical mixture were eluci-
dated by MS and NMR. Details for the identification of
diastereomers from commercial NP mixture and for their
synthesis by Friedel-Crafts reaction will be published else-
where in the near future.
at the maximum (Flouriot et al., 1995). Muller (2004)
¨
recently pointed out that xenoestrogens may interfere with
the endocrine systems of living organisms. Dodds and Law-
son (1937) first reported that propyl and propenyl phenols
are weakly estrogenic substances. Routledge and Sumpter
(1997) reported that the estrogenic activity of alkylphenols
for human estrogen receptor (hER) on the structure–activ-
ity relationships study was found to be dependent on the
number of carbon atoms in the alkyl chain, and branching
(tertiary > secondary = normal) of the alkyl group affects
estrogenicity. Nonylphenol isomers differing in estrogenic
activity were recently discussed by Preuss et al. (2006).
NP is manufactured by the alkylation of phenol with
nonene isomers. Therefore, it is expected that commercial
NP would be a mixture of para-substituted monoalkylphe-
nols with various isomeric and branched nonyl groups. The
mixture of nonene isomers was industrially synthesized by
trimerization of propylene (CH3CH@CH2) (Lee and Peart,
1995). Wheeler et al. (1997) first elucidated on the NP struc-
ture and idea of the alkyl group at the a-position in the side
chain. There are theoretically 211 kinds of isomers of NP
(Guenther et al., 2006), caused from the structure of the
nonyl side chain in NP. Ieda et al. (2005) reported that com-
prehensive two-dimensional gas chromatography combined
with mass spectrometry enabled tentative identification of
102 components of NP from a technical mixture although
they did not clear if there are 102 or less different isomers.
Nevertheless, commercially available technical NPs were
used as standard samples for biological activity test and
analyses of environmental samples.
Total NPs were determined mass-spectrometrically in
environmental samples by using NP-specific ions (m/z
107, 121, 135, 149, 177 and 220) (Isobe et al., 2001; Isobe
and Takada, 2004; Nakada et al., 2004), Nakada et al.,
2004), and an isomer-specific quantification method for a
few individual NP isomers using selected ions, m/z 121
for peak 1, m/z 135 for peaks 2, 3, 5, 7 and 11, m/z 149
for peaks 4, 6, 9 and 13, m/z 163 for peaks 8 and 10, m/z
191 for peak 12 by gas chromatography–mass spectrome-
try (GC–MS), was developed (Horii et al., 2004). Both of
the above-mentioned methods by Isobe and Horii were,
however, assumed that each single peak contains only
one isomer, and that all isomers have the same response
to mass spectrometric detector and also flame ionization
detector (FID). Both the methods were, therefore, not for
isomer-specific determination regarding NP isomers but
for individual peaks on chromatogram by GC–MS. Enan-
tioselective separation and determination of two NP iso-
mers were recently reported by Zhang et al. (2007).
2. Experimental
2.1. Materials
2.1.1. Standards of synthetic para-NP isomers
For preparing calibration curves, following 13 para-NP
isomers synthesized in our laboratory were used: 4-(2,4-

T. Katase et al. / Chemosphere 70 (2008) 1961–1972
1963
dimethylheptan-4-yl)phenol (244NP), 4-(2,4-dimethylhep-
tan-2-yl)phenol (242NP), 4-(2,6-dimethylheptan-2-yl)phe-
nol (262NP), 4-(3,6-dimethylheptan-3-yl)phenol (363NP),
4-(4-ethyl-2-methylhexan-2-yl)phenol (4E22NP), 4-(3,5-
dimethylheptan-3-yl)phenol [353NP (NP-E, locally
named)], 4-(2,5-dimethylheptan-2-yl)phenol (252NP), 4-
(3,5-dimethylheptan-3-yl)phenol [353NP (NP-G, locally
named, diastereomer of NP-E)], 4-(4-methyloctan-4-
yl)phenol (44NP), 4-(3-ethyl-2-methylhexan-2-yl)phenol
(3E22NP), 4-(2,3-dimethylheptan-2-yl)phenol (232NP), 4-
(3-methyloctan-3-yl)phenol (33NP) and 4-(2-methyloctan-
2-yl)phenol (22NP). The 13 NP isomers were synthesized
by two different synthetic methods, A and B (Uchiyama
et al., in press; Saito et al., 2007). The method A using 4-
benzyloxyacetophenone as a starting material was used
for NP isomers having two methyl groups at a-carbon.
The benzyl group, a protective group of phenolic hydroxyl
was interestingly deprotected on conversion of the tertiary
hydroxyl group to the methyl by the action of (CH3)3Al/
TiCl4. 4E22NP and 252NP could be synthesized by this
method A. The method B, which is convenient for the syn-
thesis of NP isomer if requisite nonylalcohol is commer-
cially available or readily accessible synthetically, was
achieved by Friedel–Crafts alkylation of phenol with the
corresponding nonylalchohols (Vinken et al. 2002; Ruß
et al., 2005). Ten branched NP isomers, 244NP, 242NP,
262NP, 363NP, 353NP (diastereomer synthesized as mix-
ture), 44NP, 3E22NP, 232NP, 33NP and 22NP were syn-
thesized by this method B. The 3E22NP, 232NP and
22NP were synthesized by both methods A and B. Their
purities were more than 98% by GC (Table 1). The ratio
of diastereomers synthesized as mixture used in the present
study was 353NP (NP-E): 353NP (NP-G) = 0.918:1 by GC
determination.
Two new diastereomeric isomers, 344NP and 343NP,
which could be chromatographically separated each other
as four isomers, were used for structural elucidation by
NMR and MS. The four isomers, 244NP, 44NP, 232NP
and 3E22NP, which have not been synthesized by Lalah
et al. (2001) who synthesized 262NP and Vinken et al.
(2002) who synthesized 262NP, 363NP and 353NP, and
Thiele et al. (2004), Ruß et al. (2005) and Shioji et al.
(2006) as shown in Table 1, were first used as the standard
substance in the present work.
NP isomers were recently named by nomenclature of
IUPAC. However, considering that there are 211 theoreti-
cally possible constitutional isomers of para-nonylphenols
(Robinson et al., 1976), these abbreviations are unsuitable
for describing the complete system in order to get designa-
tions for all these isomers by Guenther et al. (2006) devel-
oped a practicable numbering system. It is important that
the branching is indicated in the name as the estrogenic
effect of para-nonylphenols is dependent on the structure
of the side chain, that is, the branched and bulky side
chains have more effect. Appendix A in the present study,
therefore, was written down both names by IUPAC and
Guenther et al. (2006).
2.1.2. Analytical sample of technical para-NPs
Three kinds of technical para-NP (CAS. No. 84852-15-
3), Tokyo Chemical Industry Co. Ltd. (TCI) (Product
No. N0300, Lot. No. FGE01), Fluka (Product No.
74430, Lot. No. 1092230) and Aldrich (Product No.
29085-8, Lot. No. LU00504CU) used in the present study
Table 1
Thirteen synthetic para-nonylphenol isomers present in technical mixtures in this study
Synthetic isomers (Saito et al., 2007 and Uchiyama et al., in press)
Previous synthetic isomers
IUPAC name
Abbreviation Local
ID
GC retention time
(min)
Purities by
GC
Shioji et al.
(2006)
Ruß et al.
(2005)
Thiele et al.
(2004)
4-(2,4-Dimethylheptan-4-yl)phenol
4-(2,4-Dimethylheptan-2-yl)phenol
4-(2,6-Dimethylheptan-2-yl)phenol
4-(3,6-Dimethylheptan-3-yl)phenol
4-(4-Ethyl-2-methylhexan-2-
yl)phenol
244 NP
242 NP
262 NP
363 NP
4E22 NP
NP-A
NP-B
NP-C0
NP-C
NP-D
8.369
8.459
8.533
8.543
8.560
>98
>98
>98
>98
>98
n.s.
+
+
n.s.
+
n.s.
+
+
+
+
+
n.s.
n.s.
+
+
4-(3,5-Dimethylheptan-3-yl)phenol
(NP-E)
353 NPa
NP-E
8.610
>98
n.s.
+
+
4-(2,5-Dimethylheptan-2-yl)phenol
4-(3,5-Dimethylheptan-3-yl)phenol
(NP-G)
252 NP
353 NPa
NP-F
NP-G
8.622
8.673
>98
>98
+
n.s.
+
+
n.s.
+
4-(4-Methyloctan-4-yl)phenol
4-(3-Ethyl-2-methylhexan-2-
yl)phenol
44 NP
3E22 NP
NP-H
NP-I
8.776
8.781
>98
>98
+
n.s.
n.s.
n.s.
+
n.s.
4-(2,3-Dimethylheptan-2-yl)phenol
4-(3-Methyloctan-3-yl)phenol
4-(2-Methyloctan-2-yl)phenol
4-Nonylphenolb
232 NP
33 NP
22 NP
4- NP
NP-M
NP-N
NP-O
–
8.979
9.045
9.068
>98
>98
>98
>99.5
+
+
+
–
n.s.
+
+
+
+
+
–
10.454
–
n.s.: Not synthesized.
a
Diastereomeric pair synthesized as mixture.
Purchased from Dr. Ehrenstorfer GmbH, Germany.
b

1964
T. Katase et al. / Chemosphere 70 (2008) 1961–1972
were purchased from each company. Anthracene-d10 pur-
chased from Supelco (Product No. 44-2456) was used as
injection internal standard (IIS). 4-Nonylphenol, the so-
called normal nonylphenol was purchased from Dr. Ehren-
storfer GmbH, Germany.
2.3. Estrogenic activity
Twelve single NP isomers were tested for estrogenic
activity by the YES (Kim et al., 2004) based on the method
by Routledge and Sumpter (1997). The yeast was kindly
supplied by Dr. Sumpter, Brunel University, UK. In this
system, the hER is expressed in a form capable of binding
to estrogen-responsive sequence (ERE). The yeast cells also
contain expression plasmids carrying the reporter gene,
lacZ, which is regulated by the ERE. Activation of the
receptor by binding of ligand causes expression of the
reporter gene lacZ which produces the enzyme b-galactosi-
dase. The activity of the estrogen-inducible b-galactosidase
was measured by the coloration of chlorophenyl-red-b-
galactopyranoside (CPRG). Each of single NPs were
diluted with dimethyl sulfoxide and added to the yeast cul-
ture media in wells of microtiter plates. These plates were
incubated for two days at 28 ꢁC. Then CPRG were added
to wells and incubated for 90 min. Color development
caused by absorption was measured at 540 and 620 nm
and the difference in the measurements was assumed to rep-
resent the activity of b-galactosidase which correlated well
with the estrogenicity of 17b-estradiol (E2), which was used
as a standard.
The amounts of color development by absorbance were
plotted against the molar concentrations of sample to give
a dose–response curve. From this curve, the minimal effec-
tive concentration was calculated as that gives the half of
the maximum effect. Under our conditions, the minimal
effective concentrations varied 20–50% depending on sam-
ples. We therefore carried out 4–6 independent experiments
and calculated the mean value. For comparison of the activ-
ities between the single NP samples, the minimal effective
concentration of each sample was compared with that of
E2, which was included in all the assay plates as the standard.
Estrogen-equivalent concentration (EEC) is defined as
follows:
2.1.3. Other reagents
2,2,4-Trimethylpentane (isooctane) (Kanto Chemical
Co. Inc.) in special grade was used after distillation as sol-
vent for chemicals.
2.2. Analytical procedures
2.2.1. Selected ions by structural types for determination of
NP isomers
Three kinds of fragment ions, m/z 135 for seven isomers,
242NP, 262NP, 4E22NP, 252NP, 3E22NP, 232NP and
22NP, m/z 149 for four isomers, 363NP, 353NP (NP-E),
353NP (NP-G) and 33NP, and m/z 163 for two isomers,
244NP and 44NP were selected for isomer-specific determi-
nation of structurally different types of 13 NP isomers by
GC–MS.
2.2.2. Calibration curves of single NP isomers by GC–MS
Isooctane solutions of 13 synthetic isomers for making
calibration curves were individually prepared at 0.05, 0.2,
0.6, 1.2 and 2.4 mg lꢀ1. Anthracene-d10 isooctane solution
of 1 mg lꢀ1 as IIS was added into each screw vial for mak-
ing calibration–curve-solutions.
The GC–MS analysis was carried out on an Agilent
5973Network quadrupole mass spectrometer fitted with
an Agilent 6890N gas chromatograph (Agilent Technol-
ogy). The fused silica capillary column with 30 m,
0.25 mm i.d., and 0.25 lm film thickness (HP-5 MS,
J&W Scientific) was used with helium as the carrier gas
at 100 kPa. For GC–MS, ionization potential was at
70 eV. The temperatures of the ion source and the trans-
fer-line were 240 ꢁC and 310 ꢁC, respectively. Target com-
pounds were measured based on SIM mode on the
following quantitation ions; m/z = 107, 121, 135, 149,
163, 177, 191 and 220 for NP, and m/z = 188 for anthra-
cene-d10. The data handling were carried out using an Agi-
EEC ¼ A ꢁ B
where A the mass percent portion in technical NP mixture,
B the estrogenic activity of single NP isomers relative to E2.
2.4. NMR and EI-MS analyses
lent
Chemstation
software
G1701DA
(Agilent
Technology). The injection port was maintained at
300 ꢁC, and the sample of 1 ll was injected with splitless
mode followed by purge 2 min after the injection. The col-
umn oven temperature was held at 70 ꢁC for the initial
2 min, then programmed at 30 ꢁC minꢀ1 to 180 ꢁC,
2 ꢁC minꢀ1 to 200 ꢁC, 30 ꢁC minꢀ1 to 310 ꢁC, and held
for 10 min.
1H and 13C NMR spectra were recorded on a JEOL
(Tokyo, Japan) JNM-LA600 FT-NMR in CDCl3 contain-
ing tetramethylsilane as an internal standard. EI-MS analy-
ses were carried out with a JEOL JMS-GCMATE.
3. Results and discussion
3.1. Chromatograms and mass spectra of single NP isomers
and technical NP
2.2.3. Sample preparation of technical NPs
As analytical samples, adequate amount of isooctane
solution of three technical NPs were prepared and finally
anthracene-d10 isooctane solution as IIS was added for
determining by GC–MS.
Typical gas-chromatograms of 13 single NP isomers and
the technical NP are shown in Fig. 1. Approximately
5.8 mg lꢀ1 of a mixture of the single isomers and 10 mg lꢀ1

T. Katase et al. / Chemosphere 70 (2008) 1961–1972
1965
Fig. 1. Chromatograms of synthetic NP isomers and technical NP mixture from Aldrich For operational conditions, see text 2-2. Peaks A–N indicate
A = 244NP, B = 242NP, C0 = 262NP, C = 363NP, D = 4E22NP, E = 353NP, F = 252NP, G = 353NP (diastereomer of E), H = 44NP, I = 3E22NP,
M = 232NP, N = 33NP and O = 22NP, and for peaks 7 [344NP (J)], 8 [343NP (K)], 9 [344NP (L)] and 12 [343NP (P)], see text 3-3 and Appendix B.
of a technical mixture from Aldrich were used for injection
to GC–MS. The chromatograms were drawn by total ion
monitor summarized with m/z 107, 121, 135, 149, 163,
177, 191 and 220. As shown in Table 1, the retention times
of each peak of the isomers on chromatogram were indi-
cated differently. The peaks due to 13 synthetic isomers
were, however, overlapped partly as shown in Fig. 1 (closed
square), and chromatographically behaved similarly to
those of technical NP mixture (open square). The peak
No. 3 overlapped with three isomers, and the peaks No.
4, 6 and 11 individually overlapped with two isomers. As
shown in Fig. 2, the peaks, No. 6 and 11 are new. Relation-
ship between mass spectra and structure of NP were
already shown in earlier study (Ieda et al., 2005). However,
their spectra were not corresponding to anthropogenic sin-
gle isomers. There are not significant meanings of mass
spectra for mixture of single isomers. For example, the big-
gest problem is that the spectrum of 4-(1,4-dimethy-1-eth-
ylpentyl)phenol, the so-called NP 3 shown in Fig. 6 in a
reference from Ieda et al., 2005 but IUPAC-named 4-
(3,6-dimethylheptan-3-yl)phenol (363NP) should not have
such a strong fragment ion m/z 135 as shown in mass spec-
trum of 363NP(c) in Appendix A. In the present study,
more comprehensive mass spectra of the 13 single NP iso-
mers and corresponding chromatographic peaks No. 1–12
of technical NP mixture (Fig. 1) are shown in Appendix A.
The eight above-mentioned strong fragment ions in mass
spectrum of each peak on the chromatogram were selected
for total ion monitoring. The 13 NP isomers were classified
into three branched structural types: One type having
intensive fragment of m/z 135 was for seven isomers,
242NP, 262NP, 4E22NP, 252NP, 3E22NP, 232NP and
22NP, another one having that of m/z 149 was for four iso-
mers, 363NP, 353NP (NP-E), 353NP (NP-G) and 33NP,
and the other one having that of m/z 163 was for two iso-
mers, 244NP and 44NP. As shown in the following section,
new 344NP and 343NP had the intensive fragment of
m/z 149 and that of m/z 163, respectively. Single isomers

1966
T. Katase et al. / Chemosphere 70 (2008) 1961–1972
and relationship of quantitations between individual iso-
mers and selected ions is summarized in Fig. 1 (bottom).
Four peaks 3, 4, 6 and 11 were found to overlap with more
than two isomers. The peak 3 that overlapped with 262NP,
363NP and 4E22NP was determined by using the selected
ions at m/z 149 for 363NP, and at m/z 135 for 262NP
and 4E22NP. In order to quantify 262NP and 4E22NP,
the inseparable peak 3 was split into peak 3-1 and peak
3-3 by vertical dotted line as shown in Fig. 3 (top). When
a 100 m-column (Petrocol DH, Supelco) was used, both
peaks of 3-1 and 3-3 could be experimentally separated
completely but also time-consuming. It took 90 min that
only one sample could be determined. The peak 4 that
overlapped with 353NP (NP-E) and 252NP could be deter-
mined by the ions at m/z 149 for 353NP (NP-E) and m/z
135 for 252NP, respectively. The peak 6 which consists of
44NP and 3E22NP could be determined at m/z 163 for
44NP and at m/z 135 for 3E22NP, while the peak 11 which
consists of 33NP and 22NP could be determined at m/z 149
for 33NP and at m/z 135 for 22NP. The four other single
peaks were determined at m/z 163 for 244NP, at m/z 135
for 242NP (corresponding to peak 2), at m/z 149 for
353NP (NP-G, corresponding to peak 5) and at m/z 135
for 232NP (corresponding to peak 10), respectively.
Fig. 2. Multiplicity of chromatographic peaks 6 and 11 of technical NP
mixture. For numbers on peaks, see Fig. 1.
The isomer, 3E22NP was the most estrogenic-active iso-
mer in our previous study (Uchiyama et al., in press).
Therefore, the important peak corresponding to 3E22NP
was peak 6 which consists of 44NP and 3E22NP. The most
important NP-isomer, 3E22NP could be determined at
m/z 135, while 44NP could be determined at m/z 163.
corresponding to peaks No. 7, 8, 9 and 12 on the chro-
matogram in Fig. 1 were not yet synthesized but already
elucidated as 344NP (NP-J), 343NP (NP-K), 344NP (NP-
L, diastereomer of NP-J) and 343NP (NP-P, diastereomer
of NP-K), respectively.
3.2. Selected ion chromatograms for the determination
3.3. Amounts of NP isomers in three technical NP mixtures
by using calibration curves of synthetic NP-isomer standards
by GC–MS
Three kinds of fragment ions, m/z 135, 149 and 163 were
selected for the determination of structurally different types
of 13 NP isomers by GC–MS (Fig. 3). Three ion chromato-
grams by m/z 135, 149 and 163 are shown in Fig. 3. These
possible fragment structures are shown in Fig. 1 (middle),
Typical calibration formulas of single NP-isomer stan-
dards by GC–MS are shown in Table 2. Quadratic
regression curves were applied for calibration curves
Fig. 3. Ion chromatograms by structurally selected ions for quantitation of NP isomers. For alphabets and numbers on peaks, see Fig. 1. E and G, 8 and
12, and 7 and 9 are individually diastereomeric pairs. For 8, 12, 7 and 9, see Fig. 1.

T. Katase et al. / Chemosphere 70 (2008) 1961–1972
1967
Table 2
Calibration formula, coefficient of determination, and limits of quantitation and detection of para-nonylphenol isomers
NP isomers Quadratic regression equation
Linear regression equation
Calibration formulab
Coefficient of determination of
calibration curves (R2)
Quantitation
Detection
limit (mg lꢀ1
Coefficient of determination of
calibration curves (R2
)
limit (mg lꢀ1
)
)
244 NP
242 NP
262 NP
363 NP
4E22 NP
353 NP
(NP-E)a
252 NP
353 NP
(NP-G)a
44 NP
3E22 NP
232 NP
33 NP
22 NP
4-NP
0.01045x2 + 0.02517X + 0.00005 0.99998
0.02313x2 + 0.24463X ꢀ 0.01225 0.99943
0.02998x2 + 0.16158X ꢀ 0.00518 0.99988
0.01429x2 + 0.06397X ꢀ 0.00190 0.99986
0.02293x2 + 0.09679X + 0.00108 0.99993
0.01890x2 + 0.06884X ꢀ 0.00179 0.99960
0.064
0.012
0.012
0.032
0.012
0.016
0.019
0.004
0.004
0.010
0.004
0.005
0.98263
0.99696
0.99312
0.99122
0.99063
0.98904
0.01173x2 + 0.23607X ꢀ 0.01476 0.99657
0.008
0.016
0.002
0.005
0.99575
0.99288
0.01650x2 + 0.09286X ꢀ 0.00364 0.99963
0.00641x2 + 0.04274X ꢀ 0.00226 0.99938
0.00421x2 + 0.20962X ꢀ 0.01383 0.99690
0.04651x2 + 0.25754X ꢀ 0.00668 0.99996
0.01881x2 + 0.07085X ꢀ 0.00328 0.99955
0.01186x2 + 0.03309X + 0.00027 0.99993
0.04740x2 ꢀ 0.02231X + 0.00722 0.99627
0.064
0.012
0.012
0.008
0.012
0.011
0.019
0.004
0.004
0.002
0.004
0.003
0.99438
0.99675
0.99345
0.98887
0.98599
0.90518
a
Diastereomeric pair synthesized as mixture.
Y = aX2 + bX + C, Y = (peak area of NP-isomer)/[(peak area of IIS (mg lꢀ1))], X = concentration of individual solution of NP isomers (mg lꢀ1).
b
because coefficients of determination (R2) of quadratic
regression curves were better than those of linear regres-
sion curves. The R2 of quadratic regression curves were
between 0.99627 and 0.99998, while those between of lin-
ear curves between 0.90518 and 0.99696 (Table 2). The
calibration curves for determination of the concentration
between 0.05 and 2.4 mg lꢀ1 of the standards were
applied to their determination of technical NP mixtures.
Coefficients of variation of determination in triplicate
were between 2% at 2.4 mg lꢀ1 for 363NP and 28% at
0.05 mg lꢀ1 for 232NP. For environmental samples of
water and sediments, however, concentration of standard
solution between 0.0024 and 0.05 mg lꢀ1 for all isomers
was found to be able to be determined in our experiment.
Quantitation limit with noise ratio of more than 10 was
between 0.008 mg lꢀ1 for 252NP and 33NP, and
0.064 mg lꢀ1 for 244NP and 44NP, and detection limit
with noise of more than 3 was between 0.0024 mg lꢀ1
for 252NP and 33NP, and 0.019 mg lꢀ1 for 244NP and
44NP (Table 2). All the coefficients of variation were less
than 10% in triple determinations of the NP isomers in
three technical NPs.
As shown in Table 3, the four isomers, 244NP, 44NP,
3E22NP and 232NP were first determined in technical
Table 3
Portions of para-nonylphenol isomers in three technical mixtures
IUPAC name
Abbreviation
Mass % in technical mixture
TCI
Aldrich
Fluka
Flukab
Acrosb
4-(2,4-Dimethylheptan-4-yl)phenol
4-(2,4-Dimethylheptan-2-yl)phenol
4-(2,6-Dimethylheptan-2-yl)phenol
4-(3,6-Dimethylheptan-3-yl)phenol
4-(4-Ethyl-2-methylhexan-2-yl)phenol
4-(3,5-Dimethylheptan-3-yl)phenol (NP-E)
4-(2,5-Dimethylheptan-2-yl)phenol
4-(3,5-Dimethylheptan-3-yl)phenol (NP-G)
4-(4-Methyloctan-4-yl)phenol
244 NP
242 NP
262 NP
363 NP
4E22 NP
353 NPa
252 NP
353 NPa
44 NP
6.1 0.1
3.8 0.1
7.0 0.2
4.4 0.1
9.9 0.3
5.9 0.3
7.4 0.8
7.2 0.7
6.0 0.7
3.6 0.3
4.4 0.4
6.4 0.3
5.2 0.5
3.4 0.3
3.8 0.2
7.9 0.2
4.4 0.3
10.1 0.3
7.3 0.2
7.4 0.5
7.1 0.5
6.1 0.4
3.3 0.1
4.3 0.3
7.4 0.1
5.0 0.3
2.9 0.3
n.d.
14
5
13
6
n.d.
12
2
9
3
9.4 0.2
3.9 0.3
11.1 0.3
8.2 0.4
8.8 0.4
7.7 0.3
7.3 0.3
3.2 0.1
5.4 0.1
9.2 0.4
5.9 0.2
3.0 0.2
10
7
10
3
10
n.d.
n.d.
n.d.
4
10
n.d.
n.d.
n.d.
2
4-(3-Ethyl-2-methylhexan-2-yl)phenol
4-(2,3-Dimethylheptan-2-yl)phenol
4-(3-Methyloctan-3-yl)phenol
3E22 NP
232 NP
33 NP
4-(2-Methyloctan-2-yl)phenol
22 NP
2
1
Total
89
2
75
4
77
2
71
52
n.d., not determined.
a
Diastereomeric pair.
Quantified by Ruß et al. (2005).
b

1968
T. Katase et al. / Chemosphere 70 (2008) 1961–1972
mixtures. The portions of the para-NP isomers deter-
mined in TCI sample were between 11.1 0.3% for
363NP and 3.0 0.2% for 22NP. The tendency of those
in the two other Fluka and Ardrich was almost similar to
that of TCI. Total 13 mass percent portion in the NP
mixture from TCI, Aldrich and Fulka covered with
89 2%, 75 4% and 77 2%, respectively. The residual
percent portions will cover with several not-yet-deter-
mined isomers. In our study, peaks 7, 8, 9 and 12 shown
in Fig. 1 will correspond to those isomers. They were two
pairs of diastereomeric isomers and their structures of
these corresponding isomers by MS and NMR were elu-
cidated as two types of 344NP (NP-J) for peak 7 and
344NP (NP-L) for peak 9, and those of 343NP (NP-K)
for peak 8 and 343NP (NP-P) for peak 12. Their isomers,
however, were not synthesized yet but chromatographi-
cally separated. More than 1 mass percent of 344NP
and 343NP in the technical NP mixture were estimated
by semiquantification comparing with responses of neigh-
bor peaks of single NP isomers. The spectral data of MS
and NMR and purities of these fractions are listed in
Appendix B. Information on detail fractionation will be
described elsewhere by Makino et al.
3.4. Estrogenic activity
There was some deference in estrogenic relative activity
to E2 between fractions by HPLC and GC-PFC, and the
estrogenic activity of n-NP was considerably lower when
compared with that of a technical mixture from a commer-
cial source (Kim et al., 2004, 2005b). The difference in
estrogenic activity for hER between n-NP and technical
NP was found before (Routledge and Sumpter, 1997).
The relative estrogenicity of twelve isomers of para-NP
by YES is given (Table 4). The 3E22NP exhibited the great-
est estrogenic activity, which was 25 · 10ꢀ4 as potent as E2.
The relative potency of 33NP was the least. In our previous
work (Kim et al., 2005b), the greatest estrogenic activity of
NP-7 exhibited 19 · 10ꢀ4 as potent as E2. The fraction NP-
7, which corresponded to GC-peak 6 in the present work,
was found to contain both 44NP and 3E22NP as shown
in Figs. 1 and 2. As shown in Table 4, the estrogenic activ-
ity of 44NP in the present work was fairly lower when com-
pared with that of 3E22NP. Therefore, the present method,
in which 44NP and 3E22NP could be separately deter-
mined, is important especially from a viewpoint of estro-
genic study.
More than 1% portions of para-NPs in technical mix-
tures have been studied in our work. However, a less than
1% mass portion of the para-NP, 4-(2,5,5-trimethylhexan-
2-yl)phenol (2552NP) was quantified in both mixtures from
Fluka and Acros (Ruß et al., 2005). In our study, the
2552NP having retention time of 8.010 min under the con-
ditions as described in text, 2-2 was also found at less than
1% portion in a technical mixture. It depends on their estr-
ogenicities in the further studies, whether or not the small
portions of the isomers present in technical mixtures will
be determined.
3.5. Estrogenic equivalent concentration
The pattern of relative portions of para-NP isomers var-
ied among three technical mixtures (Table 3). The EEC of
para-NP isomers in the technical mixture was calculated
because individual isomers exhibited different activities
(Table 4). The EEC of 13 para-NP in technical mixtures
was predicted, base on the use of portion of the para-NP
and their respective potencies, to be 0.205–0.208 times less
Table 4
Estrogen-equivalent concentration (EEC) of branched para-nonylphenol isomers in technical NP
NP isomers
Relative activity of NP-isomers:
EEC %
TCI
to 17b-estradiol(E2), ·10ꢀ4
to 3E22 NP (NP-I)
Aldrich
Fluka
244 NP
242 NP
262 NP
363 NP
4E22 NP
353 NP (NP-E)a
252 NP
353 NP (NP-G)a
44 NP
3E22 NP
232 NP
33 NP
3.62
3.89
6.35
2.42
7.71
3.58
3.23
3.58
2.75
25.3
4.39
1.37
6.6
0.143
0.154
0.251
0.096
0.305
0.142
0.128
0.142
0.109
1.000
0.173
0.0543
0.261
–
0.87
1.45
0.98
1.06
2.51
1.24
0.98
1.03
0.35
5.36
1.59
0.32
0.79
18.5
0.208
0.54
1.08
1.11
0.95
1.81
1.05
0.92
0.85
0.39
4.43
1.11
0.28
0.88
15.4
0.206
0.54
1.22
1.10
0.96
2.22
1.04
0.91
0.87
0.35
4.26
1.29
0.27
0.75
15.8
0.205
22 NP
Total
EEC %
Mass percent portionb
a
–
–
–
Diastereomeric pair synthesized as mixture,and for their activity, it is assumed for diastereomer of 353NP to be same. For information on assay, see
Section 2.3 (estrogenic activity).
b
For mass percent portion, see Table 3.

T. Katase et al. / Chemosphere 70 (2008) 1961–1972
1969
than the activity actually measured in technical mixtures.
Some more effective analytical data for fish and water, mar-
ine sediment should be collected by the present method in
the near future.
and Government, the Special Coordination Funds for Pro-
moting Science and Technology, a grant to promote multi-
disciplinary research projects, and a grant for Scientific Re-
search (TU 17510049), from the Ministry of Education,
Culture, Sports, Science, and Technology.
Acknowledgements
Appendix A. Mass spectra of synthetic NPs and
corresponding GC-peaks
The authors are extremely grateful to Dr. Sumpter,
Brunel University, UK for supplying recombinant yeast
cells. This study was supported in part by a grant for Effec-
tive Promotion of Joint Research with Industry, Academia,
Mass spectra of single isomers, left and corresponding
peaks on chromatogram, right.

1970
T. Katase et al. / Chemosphere 70 (2008) 1961–1972
Appendix B. Spectral data of EI-MS and NMR, and purities
for four fractions of a technical mixture from TCI
EI-MS m/z (relative intensity): 220 (M+), 163 (base). 1H
NMR (500 MHz, CDCl3, d): 0.73 (3H, t, J = 7.0 Hz,
CH3), 0.67–0.81 [3H, m, CH3CH2CH2 and CH3CH(H)],
0.78 [3H, t, J = 6.5 Hz, CH3], 0.88 [3H, d, J = 6.7 Hz,
CH3], 1.02–1.08 [1H, m, CH3CH(H)], 1.13 (3H, s, CH3),
1.49–1.62 [3H, m, CH3CH2CH2 and CH3CH2CH(CH3)],
4.54 [1H, brs, OH], 6.75 [2H, d, J = 8.6 Hz, CH = ·2],
7.11 [2H, d, J = 8.6 Hz, CH = ·2]. 13C NMR (125 MHz,
CDCl3, d): 13.1 (CH3), 13.4 (CH3), 14.9 (CH3), 17.6
(CH2), 18.3 (CH3), 24.6 (CH2), 43.3 (CH2), 43.8 (C),
B.1. 4-(3,4-Dimethylheptan-4-yl)phenol [344NP (NP-J)],
corresponding to peak 7
HO

T. Katase et al. / Chemosphere 70 (2008) 1961–1972
1971
45.5 (CH), 114.5 (CH · 2), 127.9 (CH · 2), 140.7 (C), 152.8
[1H, m, CH3CH2CH(H)], 1.12 [3H, s, CH3], 1.41–1.45
[1H, m, CH3CH(H)CH2], 1.49–1.52 [1H, m, CH3CH2-
CH(H)], 1.57–1.70 [3H, m, CH3CH2 and CH3CH2
CH2CH], 4.58 [1H, brs, OH], 6.75 [2H, d, J = 8.6 Hz,
CH = ·2], 7.10 [2H, d, J = 8.6 Hz, CH = ·2]. 13C NMR
(125 MHz, CDCl3, d): 8.9 (CH3), 14.4 (CH3), 14.8 (CH3),
18.1 (CH3), 21.7 (CH2), 32.2 (CH2), 33.8 (CH2), 43.4
(CH), 43.8 (C), 114.5 (CH · 2), 128.2 (CH · 2), 140.1 (C),
152.8 (C). Composition based on GC analysis (OV-
1701, 0.25 mm · 50 m): P (75%) and seven NPs are less
than 10%.
(C).
Composition based on GC analysis (OV-1701,
0.25 mm · 50 m): J (71%), and four NPs (each one is less
than 8%).
B.2. 4-(3,4-Dimethylheptan-3-yl)phenol [343NP (NP-K)],
corresponding to peak 8
HO
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