Original

Chromatography, Vol.23 No.2 (2002)

Original

Highly Sensitive Time−Resolved Fluorometric Determination for
Alkylphenols by High Performance Liquid Chromatography
Using β−Diketonate Europium Chelate
Takeshi Matsuya1*, Kazuhiko Ohtake1, Nobuhiro Hoshino1, Minoru Ogasawara1,
Tatsuyuki Harita1, Shinsuke Arao1 and Kazuko Matsumoto2
1

Iatron Laboratories, Inc. 1460−6, Mitodai, Mito, Takomachi, Katori−gun, Chiba 289−2247, Japan2

Department of Chemistry, Advanced Research Center for Science & Engineering, Waseda University 3−4−1,
Ohkubo, Shinjuku−ku, Tokyo 169−8555, Japan
Received for Review April 28, 2002.

Accepted May 29, 2002.

Abstract
A time−resolved fluorescence detection system by HPLC for alkylphenols has been developed using a europium chelate fluorescent labeling
reagent, 4−(1,1,1,2,2−pentafluoro−3,5−dioxo−pentyl)−[1,11’−biphenyl]−4’−sulfonyl−chloride (PDB−SCl). The labeling reaction was on tophenolic hydroxyl group of alkylphenols, and the derivatized alkylphenols were separated on a HPLC column. After separation, EuCl3, TOPO
(tri−n−octylphosphine oxide), and Triton X−100 were introduced to the eluent, and the fluorescence of the europium chelate was measured
with the time−resolved phosphorescence detector. The detection limit for 4−nonylphenol in the time−resolved fluorometric HPLC method
was 4 to 65 times lower than that of the commonly used LC−MS method. The detection limits for 4−nonylphenol in the river water sample
was 0.99 ng/L. The average recovery from river water samples was 64.8%. The precision of the method ranged from 2.5 to 5.9%. Using thismethod, the concentration of 4−nonylphenol in a river water sample was 4.8 ng/L.
key words: alkylphenols; time−resolved fluorescence; HPLC; Eu chelate; river water

Even these methods are quite sensitive, still higher sensitivity has

Introduction

been required for one environmental monitoring system.

Nonylphenol polyethoxylates (NPEO) are important nonionicsurfactants which have been used commercially for many years as

Fluorescent europium chelates exhibit large Stokes shifts (~emulsifiers and solubilizers in pharmaceutical [1] and agrochemi-

290 nm) with no overlap between the excitation and emission spec-

cal formulations, [2] cosmetics [3] and various biotechnologicaltra. These chelates have a very narrow (10−nm bandwitdth) emis-

processes [4] due to their favorable physicochemical characteris-sion spectra at 615 nm. In addition, their long fluorescence life-

tics. The main source of nonylphenol in the environment has beentimes (600−1000 µs) allow use of microsecond time−resolved fluo-

recognized as a biodegradation product of NPEO [5], and non-rescence measurements, which further reduces the observed back-ylphenol has been reported to cause a number of estrogenic re-ground signals. [14−16] We have constructed the time−resolved

sponses in a variety of aquatic organisms. [7]fluorometric detection system for HPLC by using the fluorescent β

Gas chromatography/mass spectrometry (GC/MS) [8,9] and,−diketonate europium (Eu) chelate label, and the time−resolved

more recently, liquid chromatography/mass spectrometry (LC/MS)

fluorometric HPLC method was applied to the analysis of estro-[10−13] have been reported for the environmental nonylphenol.

gens. [15,16]*To whom correspondence should be addressed: Tel/fax, +81−479−76−3666, +81−479−76−3663
e-mail :matsuya@iatron.co.jp―７３―

Chromatography, Vol.23 No.2 (2002)

In this paper, the description is on the analysis of 4−nonylphe-

PDB−SCl was performed as follows. To an alkylphenol solution

nol in a river water sample by using a β−diketonate Eu chelate(22 µl) was added 20 mM carbonate buffer of pH 10 (8 µl) andlabel, 4 − (1, 1, 1, 2, 2−pentafluoro−3, 5−dioxo−pentyl) − [1, 11’−12.5 µg/ml PDB−SCl (5 µl). The solution was incubated for 30

biphenyl]−4’−sulfonyl−chloride (PDB−SCl)*, and time−resolved

min at 70℃ in the dark, and 10% HCl (5 µl) was added to the reac-

fluorometric HPLC system. The detection limit for alkylphenols

tion mixture.

was compared with that of the commonly used LC/MS method.
[10,11,13]
Materials and Methods
Time−resolved fluorometric HPLC
Chromatography was performed with a Shimadzu (Kyoto, Japan) LC−10 ATVP HPLC pump, a Rheodyne (Cotati, CA, USA)
Model 7725 I syringe−loading sample injector valve (10−µl loop).
The time−resolved phosphorescence detector(TRPD) equippedwith 16 µl flow cell (Tosoh, Tokyo, Japan) was used for measuring
the fluorescence of the Eu chelates in HPLC. The operation and thedata recording were performed with a PC computer, which wasconnected to TRPD via a RS−232 C cable. The TRPD and the
Figure 1. Derivatization of 4−nonylphenol with PDB−SClcomputer software for time−resolved fluorescence measurement
was Hamamatsu Photonics K.K. (Hamamatsu, Japan) made and designed for only analysis. Teflon tubings of 0.25 mm I.D. were gen-Analysis

erally used for all the flow circuit connections. The measurement

fluorometric HPLC

ofprederivatizedalkylphenols

bytime−resolved

conditions were as follows: excitation at 350 nm, emission at 615

The PDB−S derivatives (10 µl) were chromatographed on a

nm, the excitation gate time of 0.01 ms, the delay time of 0.1 ms,

reversed−phase column, YMC−Pak TMS (150×4.6 mm I.D., parti-

and the signal gate time of 1.8 ms, and the excitation interval of 2

cle size 5 µm, YMC, Kyoto, Japan), by isocratic elution with aceto-ms.

nitrile−water (70:30, v/v) containing 0.05% of trifluoroacetic acidas the mobile phase. The flow rate of the mobile phase was set atReagents and chemicals
A β−diketone europium chelate reagent, PDB−SCl was synthesized and purified as described previously.[16] Organic solventsand distilled water of HPLC grade (Kanto Chemicals Co., Tokyo,
Japan) were used without further purification. Alkylphenols,bisphenol−A, t−butylphenol, 4−nonylphenol, and 4−octylphenol
were purchased from Wako Pure Chemicals Industries (Osaka, Japan). The stock solutions of each alkylphenol (1000 µg/ml) wereprepared in acetonitrile. The stock solutions were stored in screw−
capped centrifuge tubes at −20℃ in the dark. A Sep−Pak C18 cartridge was purchased from Waters (Massachusetts, USA), and waspreconditioned with 20 ml of acetonitrile, 5 ml of distilled water, 5
ml of ethylacetate, and 5 ml of distilled water prior to use. A Sep−
Pak NH2 cartridge (Waters) was also with 10 ml of methanol, 5 mlof ethylacetate, 10 ml of methanol, and 5 ml of ethylacetate prior to
Figure 2. Schematic representation of the post−column Eu derivatization HPLC system
P1, Shimadzu HPLC Pump (LC−10 ATvp); P2, Shimadzu HPLC Pump (LC−10 ATvp); RP column, TMS (4.6mm I.D.×150 mm, YMC); TRPD, time−resolved phosphorescence detector (Hamamatsu Photonics K.K.).

use. All other chemicals were of analytical reagent grade.
Prederivatization of alkylphenols
The prederivatization of 4−nonylphenol with PDB−SCl was
shown in Figure 1. The derivatization of the alkylphenols with

*PDB−SCl (CDPP): same compound as used previously [16], but the nomenclature
is changed to indicate the reactive group, sulfonyl chloride group.
―７４―

Chromatography, Vol.23 No.2 (2002)

Takeshi Matsuya, Kazuhiko Ohtake, Nobuhiro Hoshino, Minoru Ogasawara,
Tatsuyuki Harita, Shinsuke Arao and Kazuko Matsumoto

0.6 ml/min and the column temperature was ambient (24±3℃）．

gun, Ibaraki, Japan) in August 2001. The samples were kept at 4℃

3+and analyzed within 48 h in order to keep the microbiological deg-

The postcolumn derivatization of Eu in HPLC was used asradation to a minimum. No preservative was added.

described previously. [16] The eluent from the LC column was
mixed with a solution containing 0.2 mM EuCl3, 0.2 mM TOPO,

Clean−up of the river samples

and 1% Triton X−100 in distilled water by the mixing tee (GL Sciences, Tokyo, Japan), delivered by a Shimadzu LC−10 ATVPThe clean−up procedure [16], which was described previ-

HPLC pump. The flow rate was at 0.7 ml/min. The generated

ously, was applied to 4−nonylphenol analysis as shown in Figure 3.

fluorescence was measured with the TRPD. The HPLC system is

The sample water was first filtered through a 48−mm Whatmann

shown in Figure 2.

(Maidstone, UK) GF/C filter with a pore size of 1.2 µm, and to 500
ml of the filtrate was concentrated by means of Figure 3. The resulted residue was dissolved into 0.2 ml of acetonitrile, and was

Sampling of the river water

processed for PDB−SCl labeling.

The river water samples were taken from Tone River (Inashiki-

Figure 3. Water sample preparation method using solid−phase extraction―７
５―

Chromatography, Vol.23 No.2 (2002)

and 1.60 ng/ml for 4−nonylphenol in the pure water sample. The

Results and Discussion

detection limits, which were obtained from the peak height of theTime−resolved fluorometric HPLC profile of the PDB−S−derivat-

PDB−S−derivatized alkylphenols, were defined as the concentra-

ized alkylphenols

tion of 3 SD of the baseline signal. The fluorescence intensity of

The HPLC profile of the PDB−S−derivatized bisphenol−A

the peak area of the alkylphenols (each 250 ng/ml) were 72,691 for(1.38 ng), the PDB−S−derivatized t−butylphenol (1.38 ng), thebisphenol A, 41,894 for t−butylphenol, 41,589 for 4−octylphenol,

PDB−S−derivatized 4−octylphenol (1.38 ng), and the PDB−S−

and 42,007 for 4−nonylphenol. The fluorescence intensity of thederivatized 4−nonylphenol (1.38 ng) are shown in Figure 4. The

PDB−S−derivatized bisphenol−A was approximately 1.7 times−
3

peak at 2.0 min is the hydrolyzed form of PDB−SCl (PDB−SO ).

compared to those of the PDB−S−derivatized t−butylphenol, 4−oc-

The PDB−S−derivatized bisphenol−A, t−butylphenol, 4−octylphe-tylphenol, and 4−nonylphenol. It was suggested that the two phe-nol, and 4−nonylphenol were eluted at 6.8 min, 9.7 min, 20.7 min,nolic hydroxyl groups of bisphenol A were derivatized with PDB−

and 24.9 min, respectively.

SCl, since bisphenol−A has two phenolic hydroxyl groups in the

The PDB−S derivatives of the alkylphenols in the final reac-

molecule. The relative standard deviations of the peak intensities oftion mixture were stable, and still gave constant time−resolved

the alkylphenols at 75 ng/ml for 5−times measurement were 1.8 tofluorescence intensities after standing for at least 24 h in the dark at

2.5%.

room temperature.
Recovery test and detection limit for 4−nonylphenol in river water
sampleThe standard addition and recovery measurement were carriedout by using 500 ml of the river water sample according to theclean−up procedure described in Materials and Methods. After
solid−phase extraction and labeling reaction, the solution was analyzed. The recovery was calculated from the difference of the intensities of added and non−added samples. The chromatograms of
the spiked or non−spiked sample are shown in Figure 5. The average recovery (n=3) of 4−nonylphenol (33.6 ng/L) in river water
samples was 64.8%, and the method precision was 5.9%. The re-Figure 4. Time−resolved fluorometric HPLC chromatogram of
PDB−S−derivatized alkylphenols
Peaks and amounts (ng per injection volume 10 µl); 1,
reagent blank (PDB−SO3−); 2, bisphenol−A (1.38 ng);3, t−butylphenol (1.38 ng); 4, 4−octylphenol (1.38 ng);
5, 4−nonylphenol (1.38 ng); asterisk, impurity from
POB-SCI.
Calibration curves and detection limits for alkylphenols in purewater sample
In time−resolved fluorometric HPLC, good linearity of thecalibration curves was obtained in the concentration range from 5
Figure 5. HPLC chromatograms of the concentrated river water
samples with or without spiking of 4−nonylphenol
River water samples (500 ml) spiked with or without
4−nonylphenol were extracted as described in the text.
1, 4−nonylphenol; asterisk, peaks from river water materials.

ng/ml to 500 ng/ml (bisphenol−A, y = 72.9 x, r2 = 1.000; t−bu22tylphenol, y = 30.0 x, r = 1.000; 4−octylphenol, y = 18.9 x, r =1.000; 4−nonylphenol, y = 18.6 x, r2 = 1.000). The detection limits
for alkylphenols were determined to be 0.49 ng/ml for bisphenol−
A, 1.20 ng/ml for t−butylphenol, 1.57 ng/ml for 4−octylphenol,―７６―Chromatography, Vol.23 No.2 (2002)

Takeshi Matsuya, Kazuhiko Ohtake, Nobuhiro Hoshino, Minoru Ogasawara,
Tatsuyuki Harita, Shinsuke Arao and Kazuko Matsumoto

covery of 4−nonylphenol from the river water sample was low

river water. On the other hand, the signals for other alkyl phenols

when the clean−up procedure for estrogens was applied to the clean

did not be detected in the river water.

−up for 4−nonylphenol. Therefore, the internal standard wasneeded for the correction of the recovered value of 4−nonylphenol.

Comparison of the detection limits for 4−nonylphenol with other

The lower recovery of 4−nonylphenol in the clean−up procedure

methods
In the measurement of the pure water sample, the detection

was due to the steps using Sep−Pak NH2 cartridge.
The peaks from the river water sample did not interfered tolimit for 4−nonylphenol in the time−resolved fluorometric HPLC

the peak for 4−nonylphenol, and the baseline signals at the reten-

was 30.9 times lower than that in a micro HPLC with UV detection

tion time for 4−nonylphenol were low as well as the signals in the(data not shown). Careri et al. [17] reported on the determination

measurement of the 4−nonylphenol standard (Figure 3). Most ofof alkylphenols including 4−nonylphenol by HPLC with UV detec-

the peaks from the river water sample were eluted within 20 min.

tion using a semi−microcolumn (250×2.1 mm I.D.). The detection

Therefore, the highly sensitive detection of 4−nonylphenol was

limit for nonylphenol in a groundwater was 120 ng/ml, which was

also possible in the river water sample. In the present method, 500

48.6 times higher than that of the present method. Thus, the detec-

ml of the river water sample was concentrated to 200 µl with thetion limit for alkylphenol in the present method was approximately

concentration factor of 2500, the detection limit for 4−nonylphenol

30 to 50 times lower than those of the commonly used microin the river water sample was 0.99 ng/L.

HPLC methods.
The detection limit for 4−nonylphenol in the present method
was also compared to those of MS methods in Table 1. The detec-

Measurement of 4−nonylphenol in the original river water
In the present method, the amounts of 4−nonylphenol in thetion limits of 4−nonylphenol in reference 11, 13, and 10 are 160 ng

river water sample were determined by means of standard addition.

/L, 1000 ng/L, and 10000 ng/L, respectively, where however the

The water sample was concentrated according to the procedure de-water samples were concentrated by solid−phase extraction prior toscribed in Materials and Methods, and the prepared samples were

the analysis. Therefore, the detection limits for 4−nonylphenol cor-

introduced into the HPLC system after addition of different con-responding to the concentration of the measured solution would becentrations of standard 4−nonylphenol. The linear correlation co-

160 ng/ml for reference 11, 60 ng/ml for reference 13, and 10 ng/

efficients were more than 0.998 in the analysis of the river water

ml for reference 10. In the present method, the detection limit for 4

sample. The line was extrapolated to the x−axis, and the concen-−nonylphenol in the measured solution was 2.47 ng/ml. The detec-

tration of the intersecting point with the axis was read. It was sug-tion limit of 4−nonylphenol in the present methods is approxi-

gested that 4.8 ng/L of 4−nonylphenol was dissolved in the original

mately 4 to 65 times lower than those of the methods. [10,11,13]

Table 1. Comparison of the detection limits for 4−nonylphenol with other methods
methoddetection limita(ng/L)

concentration
factordetection limitb(ng/ml)analytereference

LC−MS

160

1,000

160

river water

11

LC−MS

1,000

60

60

wastewater13semi−microcolumn
LC−MS

10,000on−line extraction(injection volume;100 µl)

10river water

10

0.99

2,500

2.47

river water

this workab

The concentration corresponding to the original water sample.
The concentration in the measured solution.
ylphenol in river water sample, and the detection limit was 0.99 ng/
L. The previously developed clean−up procedure for estrogens [16]Conclusion

was applied to that of 4−nonylphenol, and the materials from the

The time−resolved fluorometric HPLC method for alkylphe-river water did not interfered to the 4−nonylphenol analysis.

nols including bisphenol−A, t−butylphenol, 4−octylphenol and 4−

Therefore, the present method allows the determination of 4−

nonylphenol was more sensitive than the commonly used LC/MS

nonylphenol in river water.

methods. The system was applied to the measurement of 4−non-―７７―Chromatography, Vol.23 No.2 (2002)[8]

Acknowledgment

Proc. 24 th Meeting of Japan Society for Environmental
Chemistry. Tokyo, Japan, July 1984

The present work is financially supported by CREST (Core[9]

Research for Evolutional Science and Technology) of Japan Sci-

Ding, W.H. and Chen, C.T. J. Chromatogr. A 1999, 862,
113−120.

ence and Technology Corporation (JST). The Grant−in−Aid for[10] Motoyama, A., Suzuki, A., Shirota, O. and Namba, R. Rapid

COE Research, Ministy of Education, Culture, Sports, Science andCommun. Mass Spectrom. 1999, 13, 2204−2208.

Technology (MEXT) is also acknowledged.

[11] Takino, M., Daishima, S. and Yamaguchi, K. J. Chromatogr. A 2000, 904, 65−72.

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