Analysis of Nonylphenol Isomers in a Technical Mixture and in Water

Environ. Sci. Technol. 2005, 39, 7202-7207

Analysis of Nonylphenol Isomers in a Technical Mixture and in Water by Comprehensive Two-Dimensional Gas Chromatography-Mass Spectrometry TERUYO IEDA,† YUICHI HORII,‡ GERT PETRICK,§ NOBUYOSHI YAMASHITA,‡ NOBUO OCHIAI,† AND K U R U N T H A C H A L A M K A N N A N * ,| Gerstel K.K., 2-13-18, Nakane, Meguro-ku, Tokyo 152-0031, Japan, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan, Leibniz Institute of Marine Sciences at Kiel University (IFM-GEOMAR), Dusternbrooker Weg 20, D-24105 Kiel, Germany, and Wadsworth Center, New York State Department of Health, and Department of Environmental Health and Toxicology, School of Public Health, State University of New York at Albany, Empire State Plaza, Post Office Box 509, Albany, New York 12201-0509

Nonylphenol (NP) is a degradation product of nonylphenol polyethoxylate surfactants and has been reported to occur in water and sediments from urban areas. Technical NP is composed of several structural isomers, and conventional gas chromatography-mass spectrometry techniques have tentatively identified up to 22 components. Isolation and characterization of individual isomers in a technical NP mixture is important, because of the differences in estrogenic and bioaccumulation potential among the isomers. In this study, comprehensive two-dimensional gas chromatography (GC × GC) combined with mass spectrometry (MS) enabled tentative identification of 102 components of NP from a technical mixture. GC × GC-MS was also used to quantify two NP isomers in river water samples. This is the first study to use GC × GC-MS to characterize NP isomers in technical mixtures and for quantitative analysis of NP in river waters.

Introduction Nonylphenolpolyethoxylates (NPEOs) are among the most commonly used nonionic surfactants worldwide. NPEOs degrade in wastewater treatment systems to form a variety of products, including nonylphenol (NP), by sequential deethoxylation (1, 2). NP, as found in most environmental samples, including sediments, is a mixture of isomers due to branching of the C-9 group. Studies using capillary gas chromatographic separation have reported the occurrence of 8-12 isomers of NP (3, 4). Gas chromatography-mass * Corresponding author phone: (518)474-0015; fax: (518)473-2895; e-mail: [email protected]. † Gerstel K.K. ‡ National Institute of Advanced Industrial Science and Technology. § Leibniz Institute of Marine Sciences at Kiel University. | Wadsworth Center and SUNY at Albany. 7202

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spectrometric (GC-MS) and/or GC-Fourier transform infrared spectroscopic (FTIR) separation of technical NP mixtures enabled identification of several NP isomers (5, 6). These studies also reported the occurrence of additional NP isomers, which remain uncharacterized. NP has been reported to elicit a number of estrogenic responses, in a variety of aquatic organisms and in bioassay systems (7, 8). Structural characterization of NP isomers is of great interest, because only a few of the isomers or fractions have thus far been shown to elicit estrogenic potential (810). Furthermore, log Kow values for representative NP isomers range from 4.7 to 5.6, suggesting differences in bioconcentration potential among isomers (2). To date, toxicity of individual isomers has not been reported, due to the inability to separate individual isomers from the complex mixtures in which they occur. This situation may be analogous to that for polychlorinated biphenyls, which comprise 209 structural isomers, of which only a few are highly toxic. Gas chromatography-mass spectrometry (GC-MS) techniques have been extensively applied to the characterization of technical NP isomers. However, structural elucidation of the isomers by mass spectral interpretation is limited, due to the lack of separation of all of the isomers present in technical NP mixtures by GC-MS techniques. Separation and characterization of individual NP isomers would enable the identification of those isomers that are bioaccumulative and toxic. This knowledge might permit the purification and elimination of these congeners in the industrial production of technical NP mixtures. Comprehensive two-dimensional gas chromatography (GC × GC) has been shown to be useful in the analysis of complex samples and environmental pollutants (11, 12). GC × GC offers a significantly greater peak capacity than does conventional GC, and it thereby provides the potential for improved resolution of target compounds in a single analysis. Recent developments in two-dimensional GC have enabled the separation of compounds present in complex mixtures such as polychlorinated biphenyls, diesel fuel, and essential oils (13). Nevertheless, GC × GC methods developed for the analysis of complex samples have been largely qualitative (14); only a few reports have been published on quantitative analysis (15, 16). In this study, we demonstrate the separation of individual NP isomers in a technical mixture using GC × GC-MS. Further, we demonstrate the use of GC × GC in the quantification of two NP isomers in river water samples using GC × GC-MS. To our knowledge, this is the first study to characterize a technical NP mixture using GC × GC-MS, and the first to quantify selected NP isomers in waters using this technique.

Materials and Methods Reagents and Solvents. All solvents used were high-purity, pesticide grade (Wako Pure Chemical Industries, Tokyo, Japan). p-NP standard mixture (97.4% purity) was obtained from Tokyo Kasei Kogyo, Tokyo, Japan. p-NP is a technical NP product that can likely contain trace amounts of octylphenols, 2-NPs, and decylphenols. A deuterated NP mixture, 4-NP-d4 (95%; Kanto Chemical Company, Tokyo, Japan), was used as an internal standard. Two NP isomers, 4-(1,1,4-trimethyl-hexyl)-phenol and 4-(1,1-dimethyl-2-ethylpentyl)-phenol, were synthesized in our laboratory (17), referred hereafter as NP5 and NP7, and were also used in this study. NP5 and NP7 were used for the quantification. Instrumentation. An Agilent 6890A GC interfaced with a 5973A MS (Agilent Technologies, Palo Alto, CA) equipped with a Zoex KT2003 system (Zoex Inc., Lincon, NE) was used. 10.1021/es050568d CCC: $30.25

 2005 American Chemical Society Published on Web 08/13/2005

FIGURE 2. Schematic illustration of thermal desorption GC × GC-MS used in this study. MSD ) mass spectrometric detector; GC ) gas chromatograph; MFC ) mass flow controller; N2 ) nitrogen gas.

FIGURE 1. Sampling locations of water in rivers in Tokyo, Japan. The first-dimension column was a 30 m × 0.25 mm i.d. DB-5 (95% dimethyl, 5% diphenyl polysiloxane; Agilent Technologies), with film thickness of 1 µm. Optimization of separation for NP isomers was evaluated using five different seconddimension columns, DB-WAX (poly(ethylene glycol); Agilent; 2 m × 0.1 mm i.d. × 0.1 µm film thickness), DB-17 (50% methyl and 50% phenyl polysiloxane; Agilent; 2 m × 0.1 mm i.d. × 0.1 µm), DB-1701 (14% cyanopropylphenyl, 86% dimethyl polysiloxane; Agilent; 2 m × 0.1 mm i.d. × 0.1 µm), DB-225 (50% cyanopropylmethyl, 50% phenylmethyl polysiloxane; Agilent; 2 m × 0.1 mm i.d. × 0.1 µm), and Rt-βDEX (permethylated β cyclodextrin; 2.0 m × 0.18 mm × 0.18 µm film thickness; Restek Corp., Bellefonte, PA), while DB-5 was used as the first-dimensional column. The columns were directly coupled by using a glass press-fit connector with polyimide resin. A Zoex KT2003 loop type modulator was used. The modulation period was 4 s, and the hot gas duration time was 250 ms. The optimized oven temperature program (for DB-WAX) was as follows: initial temperature 40 °C, increased at 30 °C/min to 205 °C, and then increased at 3 °C/min to 250 °C (held for 10 min). Helium was used as the carrier gas at a flow rate of 2.3 mL/min (constant pressure mode). The MS was operated in the scan mode using electronimpact (EI) ionization (70 eV). The scan range was set from m/z 105 to m/z 170 (24.51 Hz). 4-Nonylphenol mixture (97.4% purity, 248 ppm) dissolved in hexane was injected manually using a cold injection system (CIS, Gerstel, Mu ¨ lheim an der Ruhr, Germany). Extraction and GC × GC-MS Analysis of Water. River water samples were collected from the Hanami, Ayase, and Takatsu Rivers in Tokyo, Japan (Figure 1). Water samples were collected in a stainless steel bucket, and transferred into amber glass bottles. Samples were stored at 5 °C until analysis, which was usually within a week. Water samples were not filtered prior to analysis. A stir bar sorptive extraction (SBSE)-thermal desorption (TDS) system was used for extraction and preparation of water samples (18). Details of this method have been reported elsewhere (18). Stir bars coated with poly(dimethylsiloxane) (0.5 mm film thickness, 20 mm length) were obtained from Gerstel (Mu ¨ lheim an der Ruhr, Germany). Sixty milliliters of water sample was poured into a 60-mL glass vial. The vials were stirred for 120 min at 1000 rpm, at room temperature. After extraction, stir bars were removed from sample vials with tweezers and were dried briefly with a lint-free tissue paper. The stir bars were then transferred into an empty glass thermal desorption tube and desorbed using a TDS (Gerstel) equipped with an auto sampler (TDSA, Gerstel). Stir bars were thermally desorbed

by programming the TDS from 20 °C (held for 1 min) to 250 °C (held for 3 min), at a rate of 60 °C/min. Desorbed compounds were cryo-focused into a programmable temperature vaporization (PTV) inlet at -100 °C for subsequent GC × GC-MS analysis. After desorption, the PTV inlet was programmed from -100 to 280 °C (held for 10 min), at a rate of 12 °C/s. Injection was performed in the splitless mode. For water samples, separations were performed on a DB-5 (30 m × 0.25 mm i.d. × 1 µm film thickness, Agilent) as the first dimension column and SP-WAX (1 m × 0.1 mm i.d. × 0.1 µm film thickness; Supelco; confirmed to have retention properties similar to DB-WAX, but withstands high temperatures) as the second-dimension column. The oven temperature was programmed from 40 °C (held for 2 min) to 250 °C at a rate of 10 °C/min (held for 15 min), and then to 280 °C at a rate of 10 °C/min (held for 24 min). Helium was used as a carrier gas at 2.3 mL/min (constant-pressure mode). The MS was operated in the scan mode (m/z 105 to m/z 170) using electron-impact ionization. Quantification. NP5 and NP7 were spiked into Milli-Q water at 5, 10, 25, 50, and 100 ng/L and were passed through the analytical method described for water. The limit of detection (LOD) of NP5 and NP7 was estimated on the basis of a signal-to-noise ratio of 3. The LOD of NP mixture was estimated by using a similar method for the lowest peak. 4-NP-d4 was used as an internal standard. Peak volume was used to quantify NP. Data were analyzed by GC Image software (Version 1.5, Zoex Corp.) for quantification (19).

Results and Discussion Separation of NP Isomers. Separation and identification of components in the complex NP mixture was achieved by interfacing GC × GC with MS. A schematic illustration of the GC × GC-MS system with TDS is shown in Figure 2. Because GC × GC greatly improves resolution and thus presents relatively pure compound to the MS, the mass spectrum obtained for individual peaks is relatively pure. The resulting component spectra enabled accurate interpretation of identity. Nonetheless, very closely related isomers can still coelute in the GC × GC analysis. Therefore, optimization of analytical conditions, including modulation time, desorption time, and the choice of the first- and second-dimension columns to improve the separation of components, is necessary. The choice of first- and second-dimension columns plays a role in the separation of individual isomers in complex mixtures. Using DB-5 (low polarity) as the firstdimension column, and either DB-1701 (mid polar), DB-17 (mid polar), Rt-βDEX (polar, shape-selective), DB-225 (polar), or DB-WAX (polar) as the second-dimension column, we examined the separation of NP isomers in technical mixtures. Two-dimensional GC × GC-MS total ion chromatograms for NP isomers separated using each of DB-1701, DB-17, VOL. 39, NO. 18, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. GC × GC-MS chromatograms of nonylphenol isomers separated using DB-5 as the first dimension column and DB-1701, DB-17, Rt-βDEX, and DB-225 as the second dimension column.

FIGURE 4. GC × GC-MS chromatograms of nonylphenol isomers separated using DB-5 as the first dimension column and DB-WAX as the second dimension column; structures and mass spectra of identified peaks are illustrated in Figure 6. NP1; NP2; NP3; NP4; NP5; NP6; NP7; NP8; NP9; NP10; NP11; and NP12 correspond to peaks denoted as 3,3′; 4; 4′; 5; 6; 7; 8; 9,9′; 10,10′; 10,10′; 10,10′; and 11,11, respectively. One-dimensional GC-MS chromatogram is shown at the top.

FIGURE 5. GC × GC-MS chromatograms of nonylphenol isomers separated using DB-5 as the first dimension column and DB-225 as the second dimension column; structures and mass spectra of identified peaks are illustrated in Figure 6. NP1; NP2; NP3; NP4; NP5; NP6; NP7; NP8; NP9; NP10; NP11; and NP12 correspond to peaks denoted as 1,1′; 2; 3; 4; 5; 6; 7; 8; 9,9′; 10,10′; 11,11′; and 12,12, respectively. One-dimensional GC-MS chromatogram is shown at the top.

Rt-βDEX, and DB-225 as the second-dimension column are shown in Figure 3. The first-dimension retention is shown on the x-axis, and the second-dimension retention is on the y-axis. Components that are fully resolved from the complex mixtures are seen as individual spots (blobs) or peaks in the two-dimensional plane. The number of peaks separated from

the NP mixture varied as a function of the polarity of the second-dimension column. A combination of low-polar DB-5 with polar DB-WAX column resolved 102 peaks of NP isomers (Figure 4). Identification of individual peaks was not possible, due to the lack of pure standards. However, 13 of the peaks were tentatively identified, based on the information available

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FIGURE 6. Structures of nonylphenol isomers determined by NMR and mass spectrum (ref 17). Isomers NP5 and NP7 were quantified by using newly synthesized chemicals. NP1; NP2; NP3; NP4; NP5; NP6; NP7; NP8; NP9; NP10; NP11; and NP12 correspond to peaks denoted as 1,1′; 2; 3; 4; 5; 6; 7; 8; 9,9′; 10,10′; 11,11′; and 12,12, respectively, on DB-225 column as shown in Figure 5. in the literature (17, 20). Although DB-WAX as the seconddimension column could resolve the NP mixture better than could DB-225, we instead used separation from DB-225 for identification purposes (Figure 5), because the elution pattern of NP for this column resembled the pattern reported in the literature earlier (17, 20). This facilitated the identification of 13 NP peaks in the mixture (Figure 6). While earlier studies

have separated around 20 components of NP in technical NP mixtures, using GC × GC-MS, we found 102 peaks in technical NP mixtures. Quantification of NP by GC × GC. Most of the GC × GC applications in the literature have been qualitative characterizations of complex mixtures; only a few reports have been published on quantitative analysis. In the published quanVOL. 39, NO. 18, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Recoveries of NP7 and 4-n-NP-d4 Spiked into Milli-Q Water and River Water Samples

compound

amount spiked (ng/L)

NP7 4-n-NP-d4

100 100

Milli-Q river water water recovery recovery RSD (%) (%) (n ) 3) RSD (%) (%) (n ) 3) (n ) 6) 76.4 74.6

7.27 8.04

70.4 62.4

2.23 8.04

TABLE 2. Instrumental Calibration, Limit of Detection (LOD), and Limit of Quantitation (LOQ) of Nonylphenol Isomers Using GC × GC-MS compound

correlation coefficient [r]a LOD (ng/L)b LOQ (ng/L)c

m/z

NP5 107, 135 NP7 107, 135 d total NP 107, 121, 135, 149, 163

0.9979 0.9949 0.991

0.19 0.31 0.7

0.63 1.03 2.31

a Linear ranges of the calibration curves were between 5 and 100 ng/L. b LOD was based on S/N ratio of 3. c LOQ was based on S/N ratio of 10. d Total NP was determined from the sum of the areas under m/z 107, 121, 135, 149, and 163.

TABLE 3. Concentrations (ng/L) of NP Isomers and Total NP in River Waters as Determined by GC × GC-MS and GC-MS study this study (GC × GC-MS) (refs 17, 23) GC-MS

Ayase River

Takatsu River

5.5

13.3

2.6

NP7 total NP NP5

7.9 163.6 3.9

11.1 167.4 54

0.8 42 170

NP7 total NP

9.3 180

76 994

compound NP5

Hanami River

440 5400

titative GC × GC applications (21, 22), peak heights, peak areas, or peak volumes have been compared between samples and standards. In our study, peak volume has been used to quantify NP concentrations in river water samples. NP concentrations in river water samples collected from the Hanami, Ayase, and Takatsu Rivers in Tokyo, Japan, were determined using GC × GC-MS. For this quantification, DB-5 and DB-WAX columns were used as the first- and seconddimension columns, respectively. A calibration curve was prepared for two pure individual NP isomers, NP5 and NP7, and the internal standard, 4-NP-d4. These isomers and the mixture were spiked into Milli-Q water at 5-100 ng/L and passed through the analytical procedure involving SBSETDS (18). Recoveries of NP7 and 4-NP-d4 were greater than 70% (Table 1). GC × GC-MS showed an excellent linearity between 5 and 100 ng/L for NP isomers (r2 > 0.995) (Table 2). Precision was assessed by replicate analyses of water samples fortified at 100 ng/L. The results showed good precision, with relative standard deviation between analyses less than 10% (RSD ) 7.2-9.5%). High selectivity and specificity of the GC × GC-MS resulted in a low background and improved sensitivity. The LOD for NP using this method (60 mL water) was between 0.19 and 0.7 ng/L (Table 2). Total NP, NP5, and NP7 concentrations in Hanami river water samples were 163.6, 5.5, and 7.9 ng/L, respectively. These values were comparable to values reported on the basis of the Japanese Industrial Standard method. However, the concentrations measured in the Ayase and Takatsu Rivers were significantly lower than the concentrations reported in previous studies (Table 3). This finding may be due to high 7206

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particulate content in water samples from these two rivers. The SBSE method has not been optimized for samples containing a large amount of particles. Furthermore, the SBSE method was not used in previous studies; GC-MS was used in the identification of NP isomers in previous studies. In summary, separation of 102 peaks of NP isomers in a technical NP mixture was made possible by use of GC × GC-MS. While 13 of the NP peaks have been structurally identified, further characterization will require pure standards or NMR analysis of peaks chromatographically separated using two-dimensional GC analysis. GC × GC-MS analysis can also be used in the analysis of NP isomers from water samples.

Acknowledgments We thank Dr. S. E. Reichenbach (Lincon, NE), Dr. T. Katase, Dr. T. Uchiyama (Nihon University, Japan), Dr. S. Nakamura (Yokogawa A. S. Inc., Japan), Dr. T. Oshima (Sapporo Breweries Ltd., Japan), and the staff of the AIST (Tsukuba, Japan) and Gerstel K.K. (Tokyo, Japan) for their critical suggestions. This study was funded in part by MEXT Japan (2004-2006).

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Received for review March 22, 2005. Revised manuscript received June 16, 2005. Accepted July 18, 2005. ES050568D

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