DSpace at VNU: Determination of 4-nonylphenol in water samples using 4-(2,6-dimethylhept-3-yl)phenol as new internal standard

Journal of Chromatography A, 1217 (2010) 2950–2955 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Determination of 4-nonylphenol in water samples using 4-(2,6-dimethylhept-3-yl)phenol as new internal standard Axel R Fischer a,∗ , Nham Thi Phuong Lan b , Cornelia Wiedemann a , Petra Heide a , Peter Werner a , Arndt W Schmidt c , Gabriele Theumer c , Hans-Joachim Knölker c,∗∗ a b c Institute of Waste Management and Contaminated Site Treatment, Technische Universität Dresden, Pratzschwitzer Str 15, D-01796 Pirna, Germany Hanoi University of Science, 334 Nguyen Trai, Hanoi, Viet Nam Department of Chemistry, Technische Universität Dresden, Bergstr 66, D-01069 Dresden, Germany a r t i c l e i n f o Article history: Received 24 November 2009 Received in revised form 18 February 2010 Accepted 24 February 2010 Available online March 2010 Keywords: Nonylphenol isomers Internal standard Gas chromatography–mass spectrometry a b s t r a c t A new method for determining the endocrine disrupting substance 4-nonylphenol (technical grade = mixture of isomers, 4-NP) from water samples has been developed by using 4-(2,6-dimethylhept3-yl)phenol (4-sec-NP) as model compound This branched monoalkylphenol is shown to serve as internal standard (IS) for the determination of technical 4-nonylphenol To the best of our knowledge, 4-(2,6dimethylhept-3-yl)phenol (racemic mixture) is a newly synthesized 4-nonylphenol isomer and has not been described elsewhere Recoveries have been determined by analyzing spiked water samples from distilled water, river water and wastewater Following acetylation, the compounds were enriched via solid phase extraction (SPE) Analyses of the compounds were performed by capillary column gas chromatography/mass spectrometry (GC/MS), operating in selected ion-monitoring (SIM) mode The recovery of technical 4-NP using either the newly prepared 4-sec-NP or 4-n-nonylphenol (4-n-NP) as IS have been compared 4-sec-NP showed slightly better results However, in the first series of experiments using wastewater, the yields for the derivatization of the two standard compounds were remarkably different The yield for derivatization of 4-n-NP was approximately 20%, probably due to the difficult matrix of the wastewater In contrast, the yield for the derivatization of 4-sec-NP was considerably higher (approximately 63%) This problem can be solved by increasing the concentration of the reagent used for derivatization For better control of the clean-up process, we recommend application of 4-sec-NP as internal standard, at least in water samples with complex matrices (e.g., high content of hydroxylated compounds) © 2010 Elsevier B.V All rights reserved Introduction Giger et al were the first to recognize the hazards which may arise from NPs for the environment and human health [1] Meanwhile, a lot of research has been done in this field [2] Nevertheless, many aspects have still not been fully investigated, e.g the exact composition of the technical 4-NP mixture and the potential different endocrine effects of these isomers Moreover, there is still no cost-efficient, reliable analytical method for the determination of nonylphenols in wastewater or river water The present study is directed towards solving this problem Isomeric 4-NP with branched alkyl groups are used in industry to prepare alkylphenol polyethoxylates, e.g for non-ionic surfac- ∗ Corresponding author Tel.: +49 3501 5300 28; fax: +49 3501 5300 22 ∗∗ Corresponding author Fax: +49 351 463 37030 E-mail addresses: axel rene.fischer@tu-dresden.de (A.R Fischer), hans-joachim.knoelker@tu-dresden.de (H.-J Knölker) 0021-9673/$ – see front matter © 2010 Elsevier B.V All rights reserved doi:10.1016/j.chroma.2010.02.063 tants [3] In the environment these ethoxylates are biodegraded mainly to the corresponding NPs [4], which often can be found in river waters and wastewater treatment plants in Germany [5,6] as well as in many other European countries like Sweden [7] Taking also the different enantiomers into account, a total number of 550 4-NP isomers exist [8] Considering only regio- and diastereoisomers, this number is reduced to 211 4-NPs However, it is assumed that technical 4-NP is a mixture of about 100 isomers [9] The 4-NP isomers are endocrine disruptors and often recalcitrant in the environment [10,11] Due to their hazardous properties 4-NP isomers are actually part of the “list of priority substances” of the European Union [12] The Directive 2003/53/EC implemented on 17th January 2005 allows application of 4-NP in additives only in concentrations of 100 Spectral absorption coefficient at 254 nm Table Recoveries and standard deviations (StD) of technical 4-NP with the internal standards 4-n-NP and 4-sec-NP Distilled water Elbe river Wastewater mL Ac2 O Wastewater mL Ac2 O Recovery [%] with 4-n-NP StD [%] n=4 125.8 95.2 184.6 5.6 4.4 32.8 (n = 8) 92.2 2.7 Fig Chromatogram of the NP isomers used in the present study and 262 were chosen for quantification of 4-sec-NP Although, this represents the molecular mass of all O-acetyl NP derivatives, only the 4-n-NP and 4-sec-NP isomers show a detectable peak for the molecular ion in the mass spectrum with a relative intensity of 20% and 4.7%, respectively Therefore, an overlap with peaks resulting from the other isomers present in 4-NP was not to be expected The presence of a significant peak for the molecular ion (m/z = 262) in the mass spectra of 4-NP isomers appears to depend on the branching of the nonyl side chain Considering the branching at the alpha-carbon atom of the nonyl chain, the 4-NPs are divided into three groups: primary (–CH2 –), secondary (–CHR1 –) and tertiary (–CR1 R2 –) derivatives [27] Presumably, most isomers of technical 4-NP have a tertiary alkyl side chain The retention Recovery [%] with 4-sec-NP 93.8 96.3 70.1 113.7 StD [%] n=4 1.5 2.1 8.0 (n = 8) 5.2 times of 4-sec-NP and 4-n-NP are different from those of the technical mixture which indicates their different structure Various research groups tried to identify the structure of the isomers of technical 4-NP Fries and Puttmann identified four different isomer groups using GC/MS [6] Thiele et al synthesized 10 isomers of 4NP [26] Finally, Ieda et al [25] identified in the technical mixture 12 main groups containing altogether 102 different NP isomers All of these authors found exclusively tertiary NP compounds Our assumption is based on the fact that the nonene isomers used for industrial production of the NP ethoxylates derive from trimerization of propylene [32] A higher degree of branching caused by a fast catalytical rearrangement during the reaction would stabilize the nonyl residue In order to confirm this assumption, it would be required to analyze all isomers of the technical mixture of 4NP However, identification of the structures based solely on mass spectra can be misleading Thiele et al [26] showed that the classification made by Wheeler et al [27] based on analysis of the mass spectra has not been correct in all cases Very recently, efforts have been made to predict the main fragments and the retention times of all 211 NP isomers for GC/MS determination [36] However, the present results not confirm these theoretical calculations, at least not for the new 4-sec-NP with the 2,6-dimethylhept-3-yl residue in the 4-position The mass spectrum of 4-sec-NP shows main fragments at m/z = 107 and 177, whereas the proposed main fragment m/z = 149 is only of little intensity (Fig 4) Moreover, the retention time of 4-sec-NP is not close to the mean value observed for all 4-NP isomers In contrast, the retention time of 4-sec-NP is similar to the least polar 4-NP isomers and to some 2-NPs The new 4-sec-NP isomer was used as IS in comparison to 4-nNP for analytical determination of technical 4-NP in water samples with difficult matrices Water from the Elbe river near Pirna, Germany, did not contain technical 4-NP above the determination limit (500 ng/L) On the other hand, one wastewater sample of the Fig Mass spectrum of 4-(2,6-dimethylhept-3-yl)phenol (4-sec-NP) 2954 A.R Fischer et al / J Chromatogr A 1217 (2010) 2950–2955 Table Derivatization yields of 4-n-NP and 4-sec-NP in different waters Distilled water Elbe river Wastewater mL Ac2 O Wastewater mL Ac2 O Yield [%] of deriv 4-n-NP StD [%] n=4 Yield [%] of deriv 4-sec-NP StD [%] n=4 83.2 70.7 19.7 9.1 5.3 4.6 (n = 8) 86.5 84.9 63.1 9.3 3.4 4.5 (n = 8) 88.7 2.4 97.8 0.6 influent of a communal wastewater treatment plant in Saxony, Germany, contained technical 4-NP in considerable amounts (ca ␮g/L) Table shows some properties of the used waters The results of the determination of 4-NP for a series of surface water samples have been validated by addition of 4-n-NP and the new 4-sec-NP isomer as internal standards applying the spiking technique (Table 2) In distilled water 4-NP was overestimated with the IS 4-n-NP The first results of wastewater experiments using 4-n-NP as IS were inaccurate The value obtained for concentration of 4-NP by this method was almost double of the real concentration The reason for the wrong calibration was probably an unsuitable derivatization of 4-n-NP, presumably due to the complex matrix The wastewater obviously contained compounds which influenced the derivatization Boyd proposed mL Ac2 O per liter aqueous solution [29] In our experiments, we used mL of the derivatization reagent Ac2 O per liter (approximately 0.01 mol/L) It was observed that 4-n-NP was derivatized only to a small extent leading to a considerable overestimation of the amount of technical 4-NP Technical 4-NP and 4-sec-NP have been derivatized with a higher yield (60–70%) For further experiments with wastewater the amount of the derivatization reagent (Ac2 O) was increased from to mL which had a positive effect on the yield of derivatization However, due to these experiences the yields for different derivatizations have been controlled by comparison of the peak areas for both, the derivatized and the non-derivatized internal standards The complex calculation of the non-derivatized 4-NP fraction was not performed since it can be roughly estimated based on the recovery For example, a recovery of 100% of 4-NP by using 4-n-NP as IS leads to the conclusion that both 4-NP and 4-n-NP have been derivatized to a similar extent Table shows the yields of the derivatization for both IS (4-n-NP and 4-sec-NP) using different water sources However, even in distilled water the derivatization was incomplete To some extent, the differences in the recoveries could be ascribed to this observation The incomplete derivatization is only noticeable if the yields differ significantly for different 4-NP isomers In complex matrices, this may be the case if the reagent used for derivatization, e.g acetic anhydride, is at least partially consumed by hydroxylated compounds as well as by the large amounts of water Depending on the structure of the alkyl chain, the endocrine impact of each isomer is different [18,19] Our results confirm the influence of the structure of the nonyl residue on the chemical behaviour The current guide of the European Union for the analysis of nonylphenols recommends derivatization to get improved results in case of poor chromatographic separation [37] This guide refers mainly to the work of Haller and Hansen who investigated three different suitable reagents for the derivatization of nonylphenol [38] However, acetylation and methylation have not been tested by these authors for derivatization of nonylphenol Considering the broad range of reagents known for derivatization of phenols, it is likely that many research groups are still using diverse methods for the derivatization of 4-NP Therefore, more research directed towards the improvement of NP analytics using 4-sec-NP as inter- nal standard by application of different methods (with or without derivatization) is still required The general problem of the quantification of 4-NP is the fact that the amount of each isomer within the entire mixture is unknown The response factors for the single isomers can differ and thus lead to inaccuracies of the calculation Moreover, different mixtures of technical 4-NP are being commercialized [6] Therefore, the accurate quantification of technical 4-NP in the environment is difficult Moreover, it can be assumed that the different isomers of technical 4-NP are affected to a different extent by biodegradation It has been reported that bacteria can differentiate between some isomers of technical 4-NP and also the estrogenic activity of the isomers is dependent on their structure [8,21] An approximate quantification of 4-NP can be made assuming that the response factors for all 4-NP isomers are nearly equal and thus, the correlation of the peak area with the concentration of each 4-NP isomer should be the same for all isomers Only the sharpest peaks of each 4-NP group are detected Therefore, quantification of isomers not providing sharp peaks in the chromatogram is not feasible In order to estimate the difference between the response factors of 4-n-NP and 4-sec-NP, a solution of acetonitrile/MeOH (1:1, 100 mL) containing equal amounts (2 mg) of 4-n-NP and 4-sec-NP was analyzed by GC–MS (SCAN mode) The relation between the resulting peak areas was 0.715:1.00 (mean value of 10 determinations) In consequence, there is a considerable difference between the two response factors of 4-n-NP and 4-sec-NP, which presumably applies to the response factors of other 4-NP isomers as well Therefore, the method widely used for the calculation of 4-NP concentrations may lead to wrong results when applied to environmental samples Conclusions A new method for quantitative determination of the endocrine disruptor 4-NP in water was elaborated using 4-sec-NP as new IS Quantifications based on the common IS 4-n-NP did not provide satisfactory results Using wastewater samples, the yield for derivatization (acetylation) of 4-n-NP has been very low, presumably due to matrix effects As a solution of this problem, we recommend 4-sec-NP as IS for quantitative determination of 4-NP in water samples Moreover, alternative methods of derivatization (e.g silylation or methylation) should be investigated for quantitative determination of 4-NP using 4-sec-NP as IS Acknowledgement We would like to thank the German Ministry of Education and Research for financial support of this work (Project Mega Fate II, ref no 10602067) References [1] W Giger, P.H Brunner, C Schaffner, Science 225 (1984) 623 [2] N Lubick, Environ Sci Technol 42 (2008) 6313 A.R Fischer et al / J Chromatogr A 1217 (2010) 2950–2955 [3] T Nakagawa, N Sugaya, K Sakurai, J Nakagawa, K Usukura, N Onda, Anal Sci Suppl 17 (2001) i1597 [4] M Sekela, R Brewer, G Moyle, T Tuominen, Water Sci Technol 39 (1999) 217 [5] C Hohne, W Puttmann, Environ Sci Pollut Res Int 15 (2008) 405 [6] E Fries, W Puttmann, J Environ 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Guidance Document No 19—Guidance on surface water chemical monitoring under the water framework directive, Annex II: Substance Guidance Sheets, nonylphenol, 44–45, ISBN 978-92-79-11297-3, ISSN 1725-1087, No Catalogue—KH-AN-09-019-EN-N, ©European Communities [38] R Haller, N Hansen, Nonylphenols—Experimental Work Including Ruggedness Test, Eurofins, Denmark, 2006, http://www.ecn.nl/docs/society/horizontal/ Report ruggedness NP.pdf  ... as IS for quantitative determination of 4-NP in water samples Moreover, alternative methods of derivatization (e.g silylation or methylation) should be investigated for quantitative determination. .. to environmental samples Conclusions A new method for quantitative determination of the endocrine disruptor 4-NP in water was elaborated using 4-sec-NP as new IS Quantifications based on the common... temperature was first held at 80 ◦ C for min, then increased to 200 ◦ C at a rate of 10 K/min and kept at this temperature for Subsequently, the temperature was increased to 300 ◦ C at a rate of