Tosylation of endocrine-disruptive alkylphenolic compounds in a solid-phase reaction system consisting of C18-modified silica gel and aqueous buffer solution

Article abstract of DOI:10.1248/cpb.52.1470

The tosylation reaction of endocrine-disruptive alkylphenolic compounds in a solid-phase aqueous system was investigated with the aim of developing an environment-friendly and efficient derivatization method for HPLC analyses of environmental samples. The

Full text of DOI:10.1248/cpb.52.1470

1470
Notes
Chem. Pharm. Bull. 52(12) 1470—1472 (2004)
Vol. 52, No. 12
Tosylation of Endocrine-Disruptive Alkylphenolic Compounds in a
Solid-Phase Reaction System Consisting of C18-Modified Silica Gel and
Aqueous Buffer Solution
Bunji UNO* and Noriko OKUMURA
Gifu Pharmaceutical University; Mitahora-higashi, Gifu 502–8585, Japan.
Received June 10, 2004; accepted September 2, 2004
The tosylation reaction of endocrine-disruptive alkylphenolic compounds in a solid-phase aqueous system
was investigated with the aim of developing an environment-friendly and efficient derivatization method for
HPLC analyses of environmental samples. The phenols were rapidly and efficiently converted to the tosyl deriva-
tives on a commercially available ODS solid-phase cartridge by passing an aqueous buffer solution through it.
The solid-phase aqueous tosylation system has been incorporated into a preconcentration step performed by
solid-phase extraction from environmental water.
Key words tosylation reaction; solid-phase reaction; environmental sample; solid-phase extraction; high-performance liquid
chromatography
Experimental
Recently, much attention has been paid to synthetic
organic reactions in aqueous media instead of in organic sol-
vents such as silica gel-dispersal and polymer-dispersal aque-
ous systems from the point of view of environmental conser-
Chemicals Tosyl chloride as a derivatization reagent was purchased
from Nacalai Tesque and used as received without further purification. The
samples used were alkylphenols [4-tert-butylphenol, 4-sec-butylphenol,
4-(1,1-dimethylpropyl)phenol,
4-(1,1,3,3-tetramethylbutyl)phenol,
and
vation.1—3) The results suggest that the total synthetic process 4-nonylphenol] which are recognized as estrogenic compounds.10) They were
of the best available grade from Nacalai Tesque and Tokyo Chemical Indus-
try and were used as received. The ODS powder was purchased from
Nacalai Tesque and was used in the solid-phase disperse system as a solid-
phase catalyst.
in aqueous systems is simple and the interface between the
solid phase and the aqueous liquid provides specificity and
diversity in the synthetic reactions. On the other hand, many
attempts at chromatographic determination of analytes cou-
pled with the derivatization reaction have been made to over-
come the problems of low detection and insufficient separa-
tion.4) However, the derivatization techniques utilizing the
solid phase and solid phase-dispersed reactions have only
been discussed in terms of efficiency in the analytical proce-
dure, that is, efficient conversion of the analytes to the deriva-
tives, high recovery, and shortening of the analysis time.5—9)
In particular, trace analyses of biological samples have been
developed coupled with on-column derivatization and micro
solid-phase extraction techniques.6,7) However, environmental
analyses still essentially require an enrichment step such as
preconcentration and a derivatization step to achieve low de-
tection limits prior to chromatographic separation.10) The
solid-phase aqueous reaction is a desirable method for envi-
Preparation of the Standard of Tosyl Phenols The standard of the
tosyl derivatives of the alkylphenols was synthesized by addition of a phos-
phate buffer (pH 12) to a mixture of equimolar tosyl chloride and the phe-
nols in CH3CN. The reaction mixture was kept at 60 °C for 30 min and then
extracted with ether. The crystals of the tosyl phenols were obtained by
evaporation of the solvent from the extract after washing with 0.1 mol/l of
KOH solution. The structure and purification of the tosyl derivatives were
determined by mass spectrometry and elemental analyses.
Preparation of the Solid-Phase Extraction Cartridge Used for the To-
sylation Reaction Waters Sep-Pak® cartridges packed with ODS were
used for the solid-phase tosylation field of the phenols. Tosyl chloride was
adsorbed to ODS in the cartridges prior to solid-phase extraction (SPE) of
alkylphenols as follows. A 1-ml aliquot of a CH3CN solution containing
5 mg of tosyl chloride was run in the inlet of the cartridges, and then the car-
tridges were washed with 20 ml of water.
HPLC Apparatus and Conditions The chromatographic separation
was performed with a Hewlett-Packard (HP) 1050 series HPLC unit
equipped with an UV-Vis detector set at 250 nm. The tosyl phenols were
ronmental analyses from the point of view of environmental separated on a Cosmosil 5C8-MS packed column (4.6ꢀ150 mm) with a
mobile phase consisting of a water–CH3CN mixture (1 : 1) at a flow rate of
1.0 ml/min. A 10-ml aliquot of the sample solutions was injected into the
HPLC column. Data collection and manipulation were performed using HP
conservation as well as efficiency in the analytical procedure.
The derivatization reagents characterized by an active sul-
fonyl chloride to the phenolic hydroxyl group such as tosyl
chloride (4-methylbenzenesulfonyl chloride), dansyl chlo-
Chem Station software for HPLC analyses.
ride, and ferrocenesulfonyl chloride are entirely used for pre-
column derivatization to overcome the disadvantage of the Results and Discussion
background response. Precolumn dansylation is applied to
Tosylation Reaction in Solution Derivatization of phe-
the HPLC analysis of endocrine-disruptive alkylphenolic nols with sulfonyl chlorides such as tosyl chloride and dansyl
compounds in environmental water.10) The solid-phase reac- chloride is usually carried out in aqueous acetone saturated
tion of the sulfonyl chlorides is important in improving the with sodium carbonate with a long reaction time at a rela-
analytical methods. In this study, we have developed a direct tively high temperature.4) The optimal conditions are set with
tosylation method for alkylphenolic compounds preconcen- regard to pH in the reaction mixture, which significantly
trated in a C18-modified silica gel (ODS) cartridge used for affects the competitive rates of the labeling reaction and the
their enrichment step using a heterogeneous system consist- hydrolysis of sulfonyl chlorides. The reaction mixture was
ing of an aqueous medium and ODS instead of organic sol- prepared by the addition of 1.0 ml of an acetone solution of
vents, as a prototype of the solid-phase derivatization method tosyl chloride to a mixture of 1.0 ml of the aqueous sample
of the phenols with sulfonyl chlorides.
solutions and 1.0 ml of a buffer solution. The dependence of
∗ To whom correspondence should be addressed. e-mail: uno@gifu-pu.ac.jp
© 2004 Pharmaceutical Society of Japan

December 2004
1471
pH of the buffer solutions and reaction time on the reaction
yields is shown in Figs. 1 and 2, respectively. The buffer
solutions with pH less than 7 did not allow the reaction to
proceed smoothly. On the other hand, hydrolysis of tosyl
chloride prevented the derivatization reaction from proceed-
ing with high yield at pH higher than 12. The reaction at
60 °C quickly gave high yields of the labeling product com-
pared with that at room temperature. The reaction yields of
81.6% and 78.5% were obtained at 60 °C for 15 min and at
room temperature for 20 min, respectively, using the carbon-
ate buffer (pH 10) for 10ꢁ5 mol/l of 4-tert-butylphenol. The
other phenols were also converted to the tosyl derivatives
under the same conditions in yields of 72.0—82.2%. The
results indicate that the mild conditions in the usual tosyla-
tion system in solution do not give the excellent yields of the
tosylated products.
Fig. 1. Dependence of Yields of the Tosylation Reaction of 4-tert-
Butylphenol on pH of the Buffer Solutions in the Usual Solution (ꢀ) and
ODS Solid-Phase Systems (ꢁ)
The reaction yields were estimated for the reaction time of 20 min at room tempera-
ture in the solution system and for 5 min at room temperature in the solid-phase system.
Tosylation Reaction on ODS Solid Phase The solid
phase-dispersed aqueous reaction has been preliminarily
investigated in a heterogeneous system consisting of tosyl
chloride adsorbed on ODS and a aqueous buffer solution of
the phenols. The results suggested that ODS is suitable for
solid-phase catalysis for the tosylation reaction of phenols,
probably owing to good retention of both the phenols and the
tosylated product.
The tosylation reaction in the ODS solid-phase aqueous
system was examined in the following procedure in consider-
ation of coupling with SPE of phenols. An aqueous sample
solution (10 ml) of alkylphenols was flowed into the ODS
cartridge containing tosyl chloride, followed by a run of a
buffer solution as a pH control and a reaction initiator. The
reaction mixture on ODS was eluted with 10 ml of CH3CN.
Figures 1 and 2 show the dependence of the yield of the
solid-phase tosylation against 4-tert-butylphenol on pH of
the buffer solutions and on the reaction time, respectively.
The phenols were tosylated quickly and easily under aqueous
solid-phase conditions compared with the reaction in solu-
tion. The results indicate that the derivatization of the solid
phase is completed within a few minutes at room tempera-
ture using the buffer solution of pH 12. We realized that the
tosylation reaction proceeded more effectively and selec-
tively on ODS under mild conditions than in solution. The
optimal conditions for the ODS aqueous reaction system are
set at the reaction time of 5 min using the buffer solution of
pH 12 at room temperature. The other phenols were also de-
rivatized into the tosylated products under the same condi-
tions in high yields of 89.3—98.2%.
Fig. 2. Dependence of the Reaction Yields of the Tosylation Reaction of
4-tert-Butylphenol on the Reaction Time in an Aqueous Acetone Solution of
pH 10 (ꢂ at Room Temperature, ꢃ at 60 °C), and in the Solid-Phase System
at Room Temperature Using a Buffer Solution of pH 12 (ꢁ)
The calibration curves were linear over a range of
1.0ꢀ10ꢁ2 to 3.0ꢀ10ꢁ6 mol/l for the phenols in water sample
solutions with correlation coefficients between 0.9981 and
0.9997. The wide dynamic range indicates that the derivati-
zation is carried out with high yields regardless of the con-
centrations of the sample solutions. The detection limit
(6.0ꢀ10ꢁ7 mol/l) is the same as that of the standards of the
tosyl phenols.
SPE Followed by Direct Derivatization It is well
known that commercially available ODS cartridges for
SPE are suitable for the extraction and concentration of
endocrine-disruptive alkylphenolic compounds from environ-
mental water.10) An additional advantage of the solid-phase
derivatization is to simplify the analytical procedure, com-
bined with an enrichment step by SPE of the samples. The
Fig. 3. Typical Chromatogram Obtained for 4-tert-Butylphenol 10ꢁ8 mol/l
Spiked in Tap Water
The phenol was preconcentrated 100-fold on the ODS cartridge used for the tosyla-
tion reaction. Peak 1 corresponds to tosylated 4-tert-butylphenol.
alkylphenolic compounds in environmental water were
extracted using the cartridge and were simultaneously and
easily converted into the tosyl derivatives in the same car-
tridge with aqueous basic buffer solution. Figure 3 shows a
typical chromatogram obtained for 10ꢁ8 mol/l of 4-tert-
butylphenol in tap water using the present method coupled
with 100-fold enrichment. The phenols at concentrations less
than the detection limit give clear chromatograms without
interference from a large amount of 4-methylbenzenesulfonic
acid (an aqueous decomposition product of tosyl chloride)

1472
Vol. 52, No. 12
Table 1. Recovery of 4-tert-Butylphenol Spiked in Tap Water Measured with the Present Solid-Phase Tosylation Method
4-tert-Butylphenol
Sample volume
(ml)
Preconcentration ratio
Recovery
(%)
RSD
(%)
(mol/l)
5.0ꢀ10ꢁ5
5.0ꢀ10ꢁ6
3.6ꢀ10ꢁ6
5.0ꢀ10ꢁ7
2.5ꢀ10ꢁ8
100
100
1000
1000
10000
10
10
100
100
1000
97.5
96.5
87.2
83.0
72.3
2.1 (nꢂ10)
3.0 (nꢂ5)
4.5 (nꢂ5)
6.7 (nꢂ5)
18.5 (nꢂ3)
RSD, relative standard deviation.
and are easily quantified by the present derivatization for highly sensitive fluorometric and electrochemical detec-
method. Table 1 lists the recovery of 4-tert-butylphenol (10ꢁ5 tion, respectively.
to 10ꢁ8 mol/l) spiked in tap water. The enrichment in the
Acknowledgment This work was supported by a Grant-in-Aid for
Scientific Research (No. 15590046) from the Japan Society for the Promo-
tion of Science.
ratio of less than 100 gave high recovery with the relative
standard derivation (RSD) of 2.1—3.0%. However, an
increase in the ratio lowered the recovery, probably due to
References
insufficient retainment of the phenols resulting from the
prolonged run of a large volume of the sample water into the
SPE cartridge. The recovery of the other phenols spiked in
tap water (10ꢁ5 to 10ꢁ6 mol/l) was also examined with the
present solid-phase tosylation method involving an enrich-
ment step at a ratio of less than 100. High recovery rates
(90.2—96.6%) were obtained for all sample solutions with a
RSD of 3.1—5.2% (nꢂ5).
1) Bartoli G., Bartolacci M., Bosco M. Foglia G., Giuliani A., Marcan-
toni E., Sambri L., Torregiani E., J. Org. Chem., 68, 4594—4597
(2003).
2) Manabe K., Mori Y., Wakabayashi T., Nagayama S., Kobayashi S.,
J. Am. Chem. Soc., 122, 7202—7207 (2000).
3) Tanaka H., Kubota J., Itogawa S., Ido T., Kuroboshi M., Shimamura
K., Uchida T., Synlett, 2003, 951—954 (2003).
4) Danielson N. D., Targove M. A., Miller B. E., J. Chromatogr. Sci., 26,
362—371 (1988).
In conclusion, we have developed a new approach to
derivatize alkylphenolic compounds into detectable products
linked by the sulfonyl group on ODS. The aqueous solid-
phase derivatization is easily coupled with SPE of the sam-
ples, and it is expected that trace analyses of endocrine-dis-
ruptive alkylphenolic compounds in environmental samples
can easily be recognized in an environment-friendly way,
coupled with the precolumn derivatization using dansyl chlo-
ride and ferrocenesulfonyl chloride as derivatization reagents
5) Saito K., Horie M., Nose M., J. Chromatogr. A, 595, 163—168 (1992).
6) Paleologos E. K., Kontominas M. G., Anal. Chem., 76, 1289—1294
(2004).
7) Ferreira V., Cullere L., Lopez R., Cacho J., J. Chromatogr. A, 1028,
339—345 (2004).
8) Tsuchiya H., Tatsumi M., Takagi N., Koike T., Yamaguchi H., Hayashi
T., Anal. Biochem., 155, 28—33 (1986).
9) Ruiter C. D., Otten R. R., Brinkman U. A. Th., Frei R. W., J. Chro-
matogr., 436, 429—436 (1988).
10) Uno B., Kato Y., Miwa S., Anal. Sci., 18, 685—687 (2002).