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

This branched monoalkylphenol is shown to serve as internal standard IS for the determination of technical 4-nonylphenol.. For analysis of the technical mixture 4-NP, the linear non-bran

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Journal of Chromatography A

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / c h r o m a

Determination of 4-nonylphenol in water samples using

4-(2,6-dimethylhept-3-yl)phenol as new internal standard

Axel R Fischera,∗, Nham Thi Phuong Lanb, Cornelia Wiedemanna, Petra Heidea, Peter Wernera,

Arndt W Schmidtc, Gabriele Theumerc, Hans-Joachim Knölkerc,∗∗

a Institute of Waste Management and Contaminated Site Treatment, Technische Universität Dresden, Pratzschwitzer Str 15, D-01796 Pirna, Germany

b Hanoi University of Science, 334 Nguyen Trai, Hanoi, Viet Nam

c 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 3 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-dimethylhept-3-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-(2,6-dimethylhept-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 chro-matography/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 differ-ent 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

1 Introduction

Giger et al were the first to recognize the hazards which may

arise from NPs for the environment and human health[1]

Mean-while, 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

dif-ferent 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).

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 diastereoiso-mers, 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 envi-ronment[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 <0.1% by mass[13] The analytical determination of both polyethoxylates and 4-NP

is often established in environmental laboratories[14] During the last years, successful efforts have been made to synthesize single isomers of 4-NP Aim of these efforts was the investigation of the properties of single NP isomers (e.g toxicity) and their

identifica-0021-9673/$ – see front matter © 2010 Elsevier B.V All rights reserved.

tion within the commercial mixture[15–20] It has been shown

that biodegradation and endocrine effects are different for the

sin-gle 4-nonylphenol isomers Therefore, experiments using only the

technical mixture (4-NP) are not sufficient[8,21]

For analysis of the technical mixture (4-NP), the linear

(non-branched) isomer 4-n-NP is often used as standard [22,23]

However, structure and shape of this molecule with a linear alkyl

chain differ markedly from the isomers in the technical mixture

The determination of 4-NP using 4-n-NP as internal standard leads

sometimes to an underestimation of the concentration of 4-NP due

to a stronger discrimination of the analyte as compared to the IS

[24] Moreover, the possible dependency of the response factors of

4-NP and NP quantification standards on the concentration must

be taken into account as well In conclusion, a 4-nonylphenol with

a branched alkyl chain would represent a better internal standard

for the determination of 4-NP However, it is required that the

iso-mer used as new IS is not contained in the technical 4-nonylphenol

mixture (4-NP) An insufficient chromatographic separation of this

branched 4-NP isomer from the isomers of the technical mixture

would also prevent its use as IS

The highly branched 4-NP isomers often contain a tertiary

car-bon atom in the alkyl chain which is linked to the aromatic ring

[25–27] Herein, we describe the synthesis of a 4-NP isomer with a

secondary alkyl side chain (4-sec-NP) and its suitability to serve

as IS for the determination of 4-NP The new internal standard,

introduced by the present study, shows a much better cost

perfor-mance than deuterated octylphenol which has been used as IS in

an alternative study[28] Due to better chromatographic separation

phenols and alkylphenols are often derivatized, for example with

N,O-bis(trimethylsilyl)acetamide[28]or acetic anhydride prior to

the analysis[29,30] In the present study, acetylation was chosen for

derivatization of the phenols as this represents a routine method in

our laboratory for analyzing alkyl phenols including 4-NP[31,32]

2 Materials and methods

2.1 Synthesis of the 4-sec-nonylphenol isomer

General: All reactions were carried out in dry solvents under an

argon atmosphere Diethyl ether (Et2O) and dichloromethane were

dried using a solvent purification system (MBraun-SPS, M Braun

Inertgas-Systeme GmbH, Garching, Germany) Chemicals were

used as received from commercial sources Flash chromatography:

Merck KG, Darmstadt, Germany, silica gel (0.040–0.063 mm)

Melt-ing points IA9100, Electrothermal, Essex, UK IR spectra: Nicolet

Avatar 360 FT-IR, Thermo Fisher Scientific, Waltham, USA;  in

cm−1 NMR spectra: Bruker, Billerica, USA, DRX 500;ı in ppm, J

in Hz Mass spectra: Finnigan MAT-95, Thermo Fisher Scientific,

Waltham, USA, ionization potential: 70 eV Elemental analyses:

EuroEA3000, EuroVector, Milan, Italy

1-(4-Hydroxyphenyl)-2-methylpropan-1-one was prepared

according to a literature procedure[33]

Aluminium trichloride (42.7 g, 320 mmol) and anhydrous

CH2Cl2 (200 mL) were added to phenyl isobutyrate (24.7 g,

150 mmol) at−5◦C After standing at room temperature for 3 d,

the reaction was quenched by addition of ice water and HCl (2 M,

200 mL) The aqueous solution was extracted four times with Et2O,

the combined organic layers were dried over sodium sulfate and

the solvent was removed Purification of the residue by flash

chromatography (SiO2; petroleum ether–Et2O, 5:1) afforded

1-(4-hydroxyphenyl)-2-methylpropan-1-one as colourless oil, yield:

14.3 g (58%) (Fig 1)

IR (ATR): = 3280, 2972, 2934, 2874, 1651, 1597, 1574, 1512,

1439, 1382, 1352, 1280, 1217, 1153, 1107, 980, 844, 762, 692,

599 cm−1.1H NMR (500 MHz, CDCl ):ı = 1.21 (d, J = 6.8 Hz, 6 H),

Fig 1 Synthesis of 1-(4-hydroxyphenyl)-2-methylpropan-1-one.

3.56 (septett, J = 6.8 Hz, 1 H), 6.96 (d, J = 8.8 Hz, 2 H), 7.84 (br s, 1 H), 7.92 (d, J = 8.8 Hz, 2 H).13C NMR and DEPT (125 MHz, CDCl3):

ı = 19.37 (2 CH3), 34.99 (CH), 115.63 (2 CH), 128.33 (C), 131.14 (2 CH), 161.14 (C), 205.18 (C O) MS (EI): m/z (%) = 164 (6) [M+], 121 (100), 93 (9), 65 (7) HRMS: m/z [M+] calcd for C10H12O2: 164.0837; found: 164.0832 Elemental analysis calcd for C10H12O2: C 73.15,

H 7.37; found: C 73.57, H 7.43

A solution of 1-bromo-3-methylbutane (18.8 g, 120 mmol) in

Et2O (100 mL) was added to a suspension of magnesium turnings (3.0 g, 120 mmol) in anhydrous Et2O (30 mL) The rate of addition was controlled to keep the solvent boiling After the addition was completed, the mixture was heated at reflux for 20 min A solution

of 1-(4-hydroxyphenyl)-2-methylpropan-1-one (8.2 g, 50 mmol) in

Et2O (25 mL) was added at room temperature to the freshly pre-pared Grignard reagent and the mixture was heated at reflux for 1 h After cooling to room temperature, HCl (1 M) (60 mL) was added slowly and the mixture was stirred at room temperature for 3 h Additional HCl (1 M) was added until the mixture was acidic The aqueous layer was separated and extracted three times with Et2O The combined organic layers were dried over sodium sulfate and the solvent was evaporated Purification of the residue by flash chromatography (SiO2; dichloromethane) gave a mixture of iso-meric nonenylphenols as light yellow oil, yield: 10.8 g (99%) The solution of isomeric nonenylphenols (1.0 g, 4.58 mmol) in MeOH (20 mL) was added to a suspension of 10% palladium on activated charcoal (100 mg) in MeOH (10 mL) and stirred under

a hydrogen atmosphere (1 atm) for 24 h The mixture was fil-tered through a short pad of Celite (Et2O) and the solvent was evaporated Purification of the residue by flash chromatography (SiO2; petroleum ether–Et2O, 20:1) gave a colourless oil, which crystallised to afford 4-(2,6-dimethylhept-3-yl)phenol, colourless crystals, yield: 930 mg (92%), m.p 44–45◦C (Fig 2)

IR (ATR): = 3327, 3022, 2953, 2928, 2869, 1612, 1598, 1511,

1466, 1383, 1366, 1223, 1172, 825 cm−1.1H NMR (500 MHz, CDCl3):

ı = 0.69 (d, J = 6.7 Hz, 3 H), 0.79 (d, J = 6.6 Hz, 3 H), 0.80 (d, J = 6.6 Hz,

3 H), 0.86–1.01 (m, 2 H), 0.90 (d, J = 6.7 Hz, 3 H), 1.43–1.53 (m, 2 H), 1.68–1.76 (m, 2 H), 2.15 (ddd, J = 10.7, 7.3, 4.1 Hz, 1 H), 4.59 (br s, 1 H), 6.74 (d, J = 8.5 Hz, 2 H), 6.95 (d, J = 8.5 Hz, 2 H).13C NMR and DEPT (125 MHz, CDCl3):ı = 20.44 (CH3), 21.01 (CH3), 22.27 (CH3), 22.90

Fig 2 Synthesis of 4-(2,6-dimethylhept-3-yl)phenol.

(CH3), 28.10 (CH), 30.66 (CH2), 33.50 (CH), 37.10 (CH2), 52.36 (CH),

114.71 (2 CH), 129.50 (2 CH), 136.96 (C), 153.35 (C) MS (EI): m/z

(%) = 220 (12) [M+], 177 (63), 149 (7), 121 (11), 107 (100) HRMS:

m/z [M+] calcd for C15H24O: 220.1827; found: 220.1827 Elemental

analysis calcd for C15H24O: C 81.76, H 10.98; found: C 81.67, H

11.31

2.2 Chemicals

The water used in the experiments was purified in a reagent

grade water system (Millipore) Standards of 4-NP (technical

grade mixture) and 4-n-NP (p.a grade) were received from

Sigma–Aldrich Acetic anhydride, sodium hydroxide, potassium

carbonate and sodium sulfate were also p.a grade (Merck,

Ger-many) Prior to use, sodium sulfate was activated at 100◦C for 2 h

All solvents were purchased at p.a grade (Merck, Germany) Stock

solutions of 4-NP, 4-n-NP and 4-sec-NP were prepared in MeOH

and stored at 4◦C

2.3 Derivatization and solid phase extraction (SPE)

For calibration experiments – from determination limit

(500 ng/L) to 33␮g/L – stock solutions of technical 4-NP in distilled

water were prepared first 4-n-NP and 4-sec-NP isomers were used

as IS After calibration, surface water samples of the Elbe river near

Pirna, Germany, as well as wastewater samples, the influent of a

wastewater treatment plant in Saxony, Germany, have been used

For derivatization a pH≥ 11.0 is necessary However, at this pH

small amounts of iron precipitated from river water and

wastew-ater Therefore, the filtration was carried out subsequent to the

increase of the pH value

1 L of each water sample was poured into a brown bottle

Afterwards, 1 mL of NaOH 32% and 25 mL of K2CO3 (1.0 M) were

added Stirring (500 rpm) was stopped after 10 min Then, the water

sample was filtered through a cellulose acetate filter (0.45␮m)

pur-chased from Sartorius Stedim Biotech GmbH, Goettingen, Germany

Afterwards, the water was spiked with a mixture of 4-sec-NP,

4-n-NP and technical 4-4-n-NP Under stirring, 1 mL of Ac2O was injected

The stirring was stopped for 10 min Subsequently, the water

sam-ples (1 L volume) were applied to the cartridges at a flow rate

of approximately 25 mL/min for distilled water and river water

and 15 mL/min for wastewater The cartridges contained 200 mg

LiChrolut RP-18 (Merck KG, Darmstadt, Germany) in the top and

100 mg LiChrolut EN in the bottom, separated by a frit The columns

were prepared for analyses by rinsing them sequentially with 5 mL

of distilled water, 5 mL of MeOH, and with 5 mL of distilled water

After the extraction, the cartridges were dried by a slight nitrogen

stream and eluted with 1.9 mL of acetonitrile The extract was dried

with sodium sulfate and moved into a small brown glass vial

2.4 Gas chromatography

An HP 6890 GC with MSD 5973 was used for analyses of NP

The GC was equipped with a DB-5MS column, 30 m× 0.25 mm

i.d (phenyl-arylene-polymer virtually equivalent to a

(5%-phenyl)-methylpolysiloxane; Agilent Technologies; made in USA) with the

film thickness of 0.25␮m The injector/autosampler (7683 Series,

Agilent Technologies) was operated in splitless mode by the front

EPC split/splitless inlet at a temperature of 250◦C The

pres-sure inside the inlet was adjusted at 64,673 Pa and total flow of

48.6 mL/min at setpoint The purge flow of split venting was set

at 45.0 mL/min after 2 min Injection volume was 2␮L The

wash-ing between two sequenced sample vials was carried out for six

times by solvents (n-hexane and MeOH, respectively) The carrier

gas used was helium with a flow rate of 1 mL/min (constant

pres-sure of 64,673 Pa) and the average velocity inside the column was

approximately 37 cm/s Mass spectra were obtained at 70 eV and monitored from m/z = 29 to 550 (SCAN mode) For quantification, the selective ion-monitoring mode (SIM) has been used The GC oven temperature was first held at 80◦C for 1 min, then increased

to 200◦C at a rate of 10 K/min and kept at this temperature for

2 min Subsequently, the temperature was increased to 300◦C at a rate of 30 K/min and then kept at this temperature for 5 min Concentrations of 4-NP in all water samples were calculated by comparing the sum of the peak areas of all isomer peaks relative

to the peak areas of the internal standards 4-n-NP and 4-sec-NP, respectively Blanks were performed on each day of extraction 2.5 Quantification procedure

The quantification of technical 4-NP is difficult because the com-position of the mixture is unknown and peak detection of the pure single isomers is impossible Therefore, to avoid a superimposition

of single isomer peaks, five groups of 4-NP isomers with different main fragments (121, 135, 149, 163 and 177) have been identified

by one peak each, which showed the best chromatographic separa-tion Detected masses for the internal standards were 107, 220 and

262 (4-n-NP) and 220 and 262 (4-sec-NP) Recoveries have been determined by addition of the TIC areas of the acetyl derivatives These TIC areas of the single NP isomers have been determined from the relative main fragments by using correction factors C according

to a well known published method[6]:

areaquantification ion The TIC full scan areas of the isomers were calculated by ana-lyzing both the area of the main fragments of the five 4-NP isomer groups and the mass distribution at the top of each peak in the gas chromatogram This approach is based on the assumption that the top of each peak shows the correct percentage distribution of the relative mass spectrum

The areas of the internal standards 4-n-NP and 4-sec-NP were used for calculating the 4-NP concentrations by using the IS method with the following equation:

Conctech NP= Cn× Concis×R1

f ×AAn

is

+ Cn+1× Concis ×R1

f ×AAn+1

is + where Conctech NP is the concentration of technical 4-NP in the samples, Cn is the correction factor for each isomer, Concisis the concentration of the IS, Rf is the response factor, An is the area

of one main fragment of an isomer of technical 4-NP (e.g area at m/z = 135) and Aisis the area of the main fragment of the IS, respec-tively The response factors for each concentration range have been determined anew before each measurement

3 Results and discussion

Fig 3 shows a chromatogram of all isomers: technical 4-NP, 4-n-NP and the new 4-sec-NP According to the new nomen-clature system [34] the new isomer 4-sec-NP used in this study has No 146 (4-[1-isopropyl-4-methylpentyl]phenol = 4-[2,6-dimethylhept-3-yl]phenol)

The technical 4-NP showed additional small peaks interfering with the retention time of the 4-sec-NP isomer These could be either isomers of 2-NP, which are also mentioned in the commercial description of the technical 4-NP mixture[35], or other 4-sec-NPs Despite the fact that these 2-isomers are only impurities in 4-NP

a disturbance of some main quantification masses could not be excluded Therefore, the peaks of the molecular ions at m/z = 220

Table 1

Selected properties of the used waters.

Conductivity [␮S/cm] Chem O 2 demand [COD, mg/L] Biol O 2 demand [BOD 5 , mg/L] pH a SAC 254 [m −1 ]

a Spectral absorption coefficient at 254 nm.

Table 2

Recoveries and standard deviations (StD) of technical 4-NP with the internal standards 4-n-NP and 4-sec-NP.

Recovery [%] with 4-n-NP StD [%]

n = 4

Recovery [%] with 4-sec-NP StD [%]

n = 4

Wastewater

1 mL Ac 2 O

Wastewater

5 mL Ac 2 O

Fig 3 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

branch-ing at the alpha-carbon atom of the nonyl chain, the 4-NPs are

divided into three groups: primary (–CH2–), secondary (–CHR1–)

and tertiary (–CR1R2–) derivatives[27] Presumably, most isomers

of technical 4-NP have a tertiary alkyl side chain The retention

times of 4-sec-NP and 4-n-NP are different from those of the tech-nical 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

4-NP[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 trimeriza-tion 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

4-NP However, identification of the structures based solely on mass spectra can be misleading Thiele et al.[26]showed that the classi-fication 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 frag-ments and the retention times of all 211 NP isomers for GC/MS determination[36] However, the present results do 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-n-NP for analytical determination of technical 4-4-n-NP in water samples with difficult matrices Water from the Elbe river near Pirna, Ger-many, did not contain technical 4-NP above the determination limit (500 ng/L) On the other hand, one wastewater sample of the

Table 3

Derivatization yields of 4-n-NP and 4-sec-NP in different waters.

Yield [%] of deriv 4-n-NP StD [%]

n = 4

Yield [%] of deriv 4-sec-NP StD [%]

n = 4

Wastewater

1 mL Ac 2 O

Wastewater

5 mL Ac 2 O

influent of a communal wastewater treatment plant in Saxony,

Germany, contained technical 4-NP in considerable amounts (ca

4␮g/L).Table 1shows 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

derivatiza-tion Boyd proposed 2 mL Ac2O per liter aqueous solution[29] In

our experiments, we used 1 mL of the derivatization reagent Ac2O

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

deriva-tization reagent (Ac2O) was increased from 1 to 5 mL which had a

positive effect on the yield of derivatization However, due to these

experiences the yields for different derivatizations have been

con-trolled by comparison of the peak areas for both, the derivatized

and the non-derivatized internal standards The complex

calcula-tion of the non-derivatized 4-NP fraccalcula-tion was not performed since

it can be roughly estimated based on the recovery For

exam-ple, 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 3 shows the yields of the

derivati-zation for both IS (4-n-NP and 4-sec-NP) using different water

sources

However, even in distilled water the derivatization was

incom-plete 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

iso-mers 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 accu-rate 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 quantifica-tion 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 fac-tors 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 determina-tions) In consequence, there is a considerable difference between the two response factors of 4-n-NP and 4-sec-NP, which presum-ably 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 envi-ronmental samples

4 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 deriva-tization (acetylation) of 4-n-NP has been very low, presumably due to matrix effects As a solution of this problem, we recom-mend 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 quanti-tative 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)

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