Determination of Dodecanol and Short-Chained Ethoxylated Dodecanols by LC–MS/MS (with Electrospray Ionization) After Their Derivatization (with Phenyl Isocyanate)

Article abstract of DOI:10.1007/s11743-017-2015-z

Abstract: This report describes the application of LC–MS/MS for the separation of dodecanol (C₁₂OH) and homogenous fatty alcohols ethoxylated (AE) containing a dodecyl moiety and 1–9 ethoxy groups. These ethoxylates and free alcohol were deriva

Full text of DOI:10.1007/s11743-017-2015-z

J Surfact Deterg (2017) 20:1421–1432
DOI 10.1007/s11743-017-2015-z
ORIGINAL ARTICLE
Determination of Dodecanol and Short-Chained Ethoxylated
Dodecanols by LC–MS/MS (with Electrospray Ionization) After
Their Derivatization (with Phenyl Isocyanate)
Joanna Zembrzuska1
Received: 15 November 2016 / Accepted: 23 August 2017 / Published online: 9 September 2017
Ó The Author(s) 2017. This article is an open access publication
Introduction
Abstract This report describes the application of LC–MS/
MS for the separation of dodecanol (C12OH) and
homogenous fatty alcohols ethoxylated (AE) containing a
dodecyl moiety and 1–9 ethoxy groups. These ethoxylates
and free alcohol were derivatized for LC–MS/MS analysis
with phenyl isocyanate (PIC). The derivatives of analytes
with PIC were separated using a C18 column. Gradient
elution with a mixture of ethyl acetate and acetonitrile
(5 mM) was employed. The described determination
method is characterized by low detection limits (range
from 0.005 lg L-1 for: C12OH, C12EO2–7 to 1 lg L-1 for
C12EO1) and quantification limits (range from 0.01 lg L-1
for: C12EO5–7 to 2 lg L-1 for C12EO1). The developed and
validated method was used in combination with liquid–
liquid extraction (using ethyl acetate) in order to identify
and quantitatively determine the C12OH and C12EO1–9
present in environmental samples collected from Warta
river water in Poznan.
Non-ionic ethoxylated surfactants are common pollutants
of the aquatic environment due to their multiple uses as
components in the manufacture of laundry detergents,
industrial cleaning products and polyurethanes, solubilizers
in enhanced oil recovery, emulsifiers in pharmaceutical
preparations, additives in cosmetic creams and lotions as
well as dispersing and wetting agents [1].
Aliphatic alcohol ethoxylates (AE) are the most
important group of non-ionic surfactants and are com-
monly found in surface water and wastewater [2]. This is
associated with the scale of their production, which reached
1 billion tons in 2012 [3]. These compounds are charac-
terized by rapid degradation and low toxicity of some of
their biodegradation products, including the formation of
free alcohol, ethoxylated polyols and short chain ethoxy-
lated alcohols.
It has been established that AE are degraded according
to two dominant mechanisms. The first, which is predom-
inant, is central fission, which results in the formation of a
hydrophobic part (free fatty alcohol) and hydrophilic
polyethylene glycols (PEGs) [4, 5]. The second mechanism
is the stepwise shortening of the ethoxyl chain by removal
of single ethoxyl groups [5], mechanism occurring in the
environment is associated with X-oxidation of the alkyl
and ethoxyl part [5, 6]. As a result, ethoxylated alcohols
with different ethoxyl chain length, free alcohols and
polyethylene glycols are present in wastewater as well as
surface water [5, 7].
Keywords Alcohol ethoxylates Á LC–MS/MS Á
Derivatization Á LLE Á River water Á Phenyl isocyanate Á
Dodecanol
Electronic supplementary material The online version of this
article (doi:10.1007/s11743-017-2015-z) contains supplementary
material, which is available to authorized users.
Analytical methods for the determination of AE include
gas chromatography [8], normal-phase high-performance
liquid chromatography and reversed-phase HPLC with
isocratic and gradient elution [9–11]. Separation of
ethoxylated surfactants has been performed using capillary
& Joanna Zembrzuska
Joanna.Zembrzuska@put.poznan.pl
1
Institute of Chemistry and Technical Electrochemistry,
Poznan University of Technology, Berdychowo 4,
60-965 Poznan, Poland
123

1422
J Surfact Deterg (2017) 20:1421–1432
gel electrophoresis (CGE), capillary zone electrophoresis
(CZE) [12, 13], micellar electrokinetic chromatography
(MEKC) [1], non-aqueous capillary electrophoresis
(NACE) [14] and capillary electrochromatography (CEC)
[1].
for determination of free alcohols and low ethoxymers. The
group of Ramis-Ramos [34] has published interesting
applications for the following analytical characterization
reagents: maleic anhydride, phthalic anhydride, and
diphenic anhydride. Sparham et al. [35] and Cassani et al.
[36] determined the total amount of AE in the form of
1-naphthoyl chloride derivatives using the LC–MS/MS
technique. A quantitative determination of dodecanol and
C12EO1–6 using the LC–MS/MS technique in MRM mode
was described by Zgola-Grzeskowiak et al. [18]. In this
case, 1-naphthoyl chloride was also used as the derivati-
zation agent.
Different detectors can be used for determination of
ethoxylated surfactants. The most popular are the ultravi-
olet detector (UV) [15–17], fluorescence detector [10, 18],
flame ionization detector (FID) [19], evaporative light-
scattering detector (ELSD) [20, 21], corona-charged aero-
sol detector (CAD) [1], MS detector and MS/MS detector
[6, 11, 18, 22–26].
The lack of a chromophore in the AE molecule in many
cases requires the use of a suitable derivatization processes
to improve the sensitivity when UV and fluorescence are
used. In this case, the most commonly used derivatization
agents include 3,5-dinitrobenzoyl chloride [15], naphthyl
isocyanate [10, 15], diphenic anhydride [27], naphthoyl
chloride [10], phenyl isocyanate [16, 17, 28].
Phenyl isocyanate (PIC) was proposed previously as a
derivatizating agent. PIC was used to derivatize AE in raw
and treated sewage and river water [16] and was also often
used to derivatize poly(ethylene glycol)s [1, 3]. Detection
of AE derivatives with phenyl isocyanate was conducted
using an UV detector at 240 nm [16, 33]. There are cur-
rently no reports regarding detection with the use of MS/
MS techniques in the MRM mode. This technique allows
for sensitive and selective determination of free alcohol
and single ethoxylated alcohols, due to the use of MRM
mode.
The use of a MS or MS/MS detector for quantitative or
qualitative determination of AE, which contain two or
more ethoxy groups in their structure, does not require the
derivatization process [23]. Unfortunately, due to their
structure, the free alcohol and AE with a single ethoxy
group (1 or 2) are unstable in the ESI ion source and
therefore it is not possible to observe them in the mass
spectrum. Therefore, studies regarding the determination of
AE after biodegradation processes focused only on the
percentage content of these compounds [7]. Most publi-
cations are focused on the evaluation of AE exposure in
environmental compartments. In almost all of these meth-
ods, free alcohols (AO) and low ethoxymers are not
detected.
The use of derivatization during LC–MS/MS determi-
nation allows to increase the mass of the pseudomolecular
ion, improves its stability in the ion source and enhances
the ionization efficiency. As a result, it is possible to
determine weakly ionized analytes.
In studies in which PIC was used during the derivati-
zation of AE for their chromatographic determination with
UV detection in wastewater, it was observed that signals of
derivatives of other contaminants, which were also present
in the sample, are visible on the chromatogram. The signals
often prevented or hampered the separation of AE [16].
This problem is eliminated by employing the LC–MS/MS
method working in the MRM mode.
As a result, the above-mentioned compounds should be
subjected to a derivatization process prior to their intro-
duction into the LC–MS or LC–MS/MS system, in order to
allow for the determination of their presence and subse-
quent analysis of their quantitative changes during the
biodegradation process. Such an approach allows one to
determine the content of specific AE including a 12 carbon
atom hydrophobic chain connected with a hydrophile
which included from 0 to 9 ethoxyl groups.
The aim of this study was to explore the possibility of
using PIC as a derivatization agent for quantitative deter-
mination of free alcohol and AE with 1–9 ethoxy groups
using the LC–MS/MS technique.
The selection of the C12EOx series is not random, since
this series is formed both from renewable and petrochem-
ical sources. Therefore, it is an adequate fingerprint which
describes the level of water contamination with non-ionic
surfactants [37]. Another reason to direct the focus on the
C12EO1–9 series and dodecanol is the availability of
homologous reference standards of this series. Unfortu-
nately, the reference standards for homologues with a
higher amount of ethoxyl groups in the compound are not
available to date.
Over the years, chemical derivatization techniques were
developed to enable the ionization of various analytes in
LC–MS [27]. Numerous methods employing LC–MS have
been developed for AE analysis without derivatization
[6, 24, 25, 29, 30] and after derivatization [7, 29, 31, 32].
There are several derivatization agents described in the
literature which may be employed for the determination of
ethoxylates with ESI-LC–MS. Dunphy et al. [33] used
2-fluoro-N-methylpyridinium salts as derivatizating agents
123

J Surfact Deterg (2017) 20:1421–1432
1423
Optimization of the Derivatization Procedure
Experimental
The following parameters were studied during the opti-
mization of the derivatization procedure: time of the
derivatization process (from 10 to 60 min, with a 10-min
interval), temperature (from 30 to 90 °C with a 10 °C
interval) and the amount of the derivatization agent relative
to AO and AE (1:1 with a 25, 50 and 100% excess). Each
of the samples with optimized parameters was prepared in
three replicates.
Reagents and Chemicals
Individual alcohol ethoxylates C12EOX (x = 1; 2; 3; 4; 5;
6; 7; 8; 9) and C12OH used as standards were obtained from
Fluka (Germany), and C12EO3 was from Sigma-Aldrich
(ST. Louis, MO, USA). All standards were of high purity
grade ([98%). MS-grade acetonitrile and MS-grade
methanol were from Sigma-Aldrich (ST. Louis, MO,
USA). Ammonium acetate used as mobile phase additive
was purchased from Sigma-Aldrich (ST. Louis, MO,
USA). Water was prepared by reverse osmosis in a
Demiwa system via double distillation in a quartz appara-
tus. Only freshly distilled water was used. Phenyl iso-
cyanate ([99%) used as a derivatization agent was
purchased from Fluka (Germany).
Liquid–Liquid Extraction (LLE)
The procedure of liquid–liquid extraction of AE was pre-
viously described by Zembrzuska et al. [23]. 15 g of
sodium chloride and 0.1 g of sodium hydrogen carbonate
were added to 50 mL of the water sample. The sample was
extracted with three portions (10, 10 and 5 mL respec-
tively) of ethyl acetate. An aliquot (5 mL) of the combined
extracts was evaporated under a gentle stream of nitrogen,
reconstituted in acetonitrile and derivatized. This proce-
dure was employed in order to separate and determine the
C12OH and C12EO1–9 in water samples collected from the
Warta river. The water samples were collected from the
Warta river (in Poznan at the measurement and control
point on St. Roch Bridge) in accordance with the Polish
standard PN EN ISO 566-6:2003. The collecting point was
situated upstream of the effluent of the Kozieglowy sewage
treatment plant. Samples were collected from the main
stream of the river from a depth of 1.0 m. Immediately
after transportation to the laboratory in the clean amber
glass bottles, the samples were filtered to remove sus-
pended solids using quantitative paper filters, and then
subjected to liquid–liquid extraction using ethyl acetate as
the extracting agent. The bottles and paper filters were
seasoned by an addition of a portion of water in order to
prevent adsorptive loss. The extraction was performed
Ethyl acetate, sodium chloride and hydrogen carbonate
(purchased from POCh Gliwice Poland) used for liquid–
liquid extraction (LLE) were of analytical grade.
Standard stock solutions (of each compound) were
prepared in acetonitrile and kept at 4 °C.
Derivatization
PIC (25.5 lL) was added to a sample containing 10 lg of
C12OH and C12EO1–9 in 200 lL of acetonitrile. The solu-
tion was heated at 70 °C for 30 min. Then, 10 lL of
methanol was added and the sample was heated again at
70 °C for 30 min. Finally, the solution was evaporated by
increasing the temperature to 80 °C. The dry residue was
re-suspended in the mobile phase to a final volume of 1 mL
and filtered through a syringe PTFE filter before subjecting
it to LC–MS/MS. Examples of the derivatization reactions
of C12OH and C12EO1 with phenyl isocyanate are shown in
Fig. 1a, b, respectively.
Fig. 1 Derivatization reactions of C12OH (a) and C12EO1 (b) with phenyl isocyanate
123

1424
J Surfact Deterg (2017) 20:1421–1432
immediately after collection of samples (the sampling point
was near the laboratory).
LOQ taking into account the concentration factors from
river water extraction procedure and the sample dilution
factors.
Samples were collected in March 2015. Fifty milliliters
of each water sample was extracted. The extraction of
water and water spiked standard mixture at three different
concentrations (2, 4 and 8 lg L-1) was carried out. The
samples used for the procedure of extraction and analysis
of river water samples were prepared in 3 replicates.
In order to check linearity, mixtures of standards were
prepared with the concentration of each component in the
range of 0.005–1000 lg L-1
.
Results and Discussion
Liquid Chromatography-Tandem Mass
Spectrometry
Optimization of MS/MS Conditions
LC analysis was performed using the UltiMate 3000 RSLC
chromatographic system from Dionex (Sunnyvale, CA,
USA). Five lL samples were injected into an analytical
column 100 mm 9 2.1 mm I.D packed with 1.9 lm
Hypersil Gold C18 RP from Thermo Scientific (USA). The
column was heated to 35 °C. The mobile phase was
composed of 5 mM ammonium acetate in water (A) and
acetonitrile (B), at a flow rate of 0.2 mL/min. The gradient
elution was: 0 min 40% B, 10 min 60% B, 20 min 100%
B, 25 min 100% B. The LC system was connected to the
API 4000 QTRAP triple quadrupole mass spectrometer
produced by AB Sciex (Foster City, CA, USA). The Turbo
Ion Spray source operated in positive ion mode. Dodecanol
and all alcohol ethoxylates were detected using multiple
reaction monitoring mode (MRM). ESI ion source param-
eters were: curtain gas 10 psi, nebulizer gas 40 psi, auxil-
iary gas 45 psi, temperature 350 °C, ion spray voltage
5500 V and collision gas set to medium. Table 1 shows the
MS/MS parameters used for quantitative dodecanol and AE
determination. In the optimized conditions, the [M?H]?
The first stage of the study aimed to determine the type of
ions which could be for detection of C12OH and AEO1–9
derivatives with PIC. By using ammonium acetate as an
addition to the phase, it was possible to obtain [M?NH4]?
ions as well as [M?H]? ions. In case of C12OH and AEO1
only the presence of [M?H]? ions was observed in the
mass spectrum. Both adducts were visible on the mass
spectrum for the remaining AE. Therefore, MRM chro-
matograms with protonated or ammoniated adducts as
precursor ions were obtained initially (Fig. 1S—see Sup-
porting Information). The peaks of adducts which served as
pseudomolecular ions in quantitative and qualitative ana-
lyzes of actual samples were based on the chromatographic
peak areas for specific derivatives of AEO2–9. In case of
C12EO4, C12EO6, C12EO7 these were [M?NH4]? ions,
whereas [M?H]? were used for C12EO2, C12EO3, C12EO5,
C12EO8 and C12EO9.
Fragmentation of AE Derivatives with PIC
adducts of C12OH, C12EO1–3 and C12EO8–9
,
and
Fragmentation of protonated or ammoniated derivatives of
C12EO1–9 homologues and C12OH was investigated. Col-
lision conditions were optimized automatically by the
spectrometer program and are given in Table 1. The
results, together with chromatograms with MRM detection,
are shown in Figs. 2 and 3. In case of C12OH and C12EO1
only the detection of protonated adducts was possible and a
single transfer between the pseudomolecular ion and the
fragmentation ion was determined.
[M?NH4]? adducts of C12EO4–7 were used as precursor
ions.
Method Validation
The limits of detection (LOD) for each derivative of AE
homologues and AO, defined as the concentrations which
yielded S/N ratios greater than or equal to 3, and the limits
of quantification (LOQ), defined as the concentrations
which yielded S/N ratios greater than or equal to 10, were
determined for AO and AE homologues in the mobile
phase (the solvent used for the introduction of dodecanol
and ethoxylates into the LC–MS/MS system) [38]. In order
to determine LOD and LOQ, the peak height above the
baseline was measured (signal). The peak-to-peak (mini-
mum to maximum) value of baseline noise away from the
peak tails for 60 s before the peak was measured [39]. Each
analysis was carried out in 3 replicates.
The fragmentation spectra of ammoniated ions (Fig. 3)
differ from the spectra of protonated ions due to the pres-
ence of one additional fragment, i.e. the formation of a
protonated ion. The signals with the highest intensity,
which are visible on the fragmentation spectra, originate
from the protonated ions of AE derivatives with PIC
[M?H]?, e.g. C12EO5 - m/z = 526 (Figs. 2, 3); whereas
the signals with lower intensity occur due to the loss of
phenyl isocyanate or subsequent ethoxy groups from the
AE molecule. The signal at m/z = 164, which is present on
every spectrum, occurs due to the loss of a fragment of the
phenyl isocyanate –OCH2CH2– unit, e.g. in case of C12EO5
Method limits of detection (MDL) and method limit of
quantification (MQL) were calculated based on LOD and
123

J Surfact Deterg (2017) 20:1421–1432
1425
Table 1 MS/MS parameters for the acquisition of dodecanol and alcohol ethoxylates
Compound Precursor ion [M?H]? m/z Declustering potential [V] MRM 1a
Collision energy [V] MRM 2b
Collision energy [V]
C
C
C
C
C
C
C
C
C
C
12OHc
12EOc1
12EOc2
12EOc3
12EO4
12EOc5
12EO6
12EO7
12EOc8
12EOc9
306
350
394
438
482
526
570
614
658
702
76
90
306 ? 138 17
–
–
–
–
350 ? 164 19
394 ? 164 23
438 ? 319 17
482 ? 271 13
526 ? 407 19
570 ? 451 19
614 ? 495 21
658 ? 540 21
702 ? 584 27
106
96
394 ? 275 15
438 ? 164 25
482 ? 120 41
526 ? 164 35
570 ? 164 27
614 ? 460 23
658 ? 164 29
702 ? 164 27
41
71
86
136
121
126
Compound Precursor ion [M?NH4]? m/z Declustering potential [V] MRM 1a
Collision energy [V] MRM 2b
Collision
energy [V]
C12EO2
411
455
499
543
587
631
675
719
48
56
411 ? 395 11
411 ? 275 20
C12EO3
C
12EOc4
C12EO5
C
12EOc6
C
12EOc7
C12EO8
C12EO9
455 ? 320 19
499 ? 364 23
543 ? 527 20
587 ? 452 26
631 ? 496 28
675 ? 541 28
719 ? 584 29
455 ? 439 14
499 ? 164 23
543 ? 408 24
587 ? 571 21
631 ? 164 22
675 ? 164 35
719 ? 164 37
50
110
70
96
99
90
a
b
c
Detection and quantification transitions
Confirmation transitions
In the majority of experiments, complexes of AOH and AE were used as precursor ions
this corresponds to a transfer from m/z = 526 to m/z = 164
(loss of a unit with m/z = 362). The repeatedly appearing
signal at m/z = 77 originates from the cleavage of the
benzene ring in the PIC unit, whereas the signal at m/
z = 45 originates from HOCH2CH2Á ethoxyl groups.
in temperature did not significantly impact the increase or
decrease in the peak area. It was also observed that the
temperature of 70 °C is optimal for each of the studied
C12EO1–2 homologues as well as C12OH, since the
derivatization process at this temperature value resulted in
the highest peak areas. This is also the only value which
allowed for maximum peak areas in the case of C12EO6.
Therefore, the derivatization reaction was carried out at the
temperature of 70 °C during subsequent studies.
The next optimization step was focused on the influence
of time on the derivatization process. The derivatization
reaction was conducted for periods of 10, 20, 30, 40, 50
and 60 min. Based on the obtained peak area values for all
the studied compounds (which are shown on Fig. 5), it was
established that time = 30 min is optimal for the deriva-
tization process. After this time, the maximum values of
peak areas were achieved for each component present in
the studied mixture. It was also observed that in the case of
C12OH, C12EO2, C12EO3, C12EO4, C12EO5, C12EO6 and
C12EO7 the reaction time did not influence the efficiency of
the derivatization process. In the case of the remaining
homologues, a reaction time lower than 30 min resulted in
Optimization of the Derivatization Procedure
In order to ensure that the derivatization procedure is
repeatable and efficient, it has been optimized in terms of
reaction temperature, reaction time and the amount of the
derivatization agent. All the studies were conducted for a
mixture of C12OH and C12EO1–9
.
During the first step, the influence of temperature on the
formation of derivatives was analyzed. The reaction was
carried out at temperature values ranging from 30 to 90 °C
with a 10 °C interval. It was observed that the derivatiza-
tion process may be conducted at 30 °C for C12OH,
C12EO2, C12EO3, C12EO4, C12EO5, C12EO7, C12EO8 and
C12EO9, since the highest peak areas were obtained for
these compounds at these temperature values (Fig. 4). For
each compound, with the exception of C12EO6, the increase
123

1426
J Surfact Deterg (2017) 20:1421–1432
Fig. 2 Mass chromatograms of MRM pairs and fragmentation spectra originating from dodecanol and AE with PIC for protonated ions [M?H]?
at the concentration of 1 g mL-1
decreased peak areas. Based on the results obtained during
this step, a derivatization time of 30 min was used during
subsequent studies.
areas of each compound. Based on the analysis of Fig. 6, it
was also observed that in case of C12OH, C12EO2, C12EO3,
C12EO5, C12EO6 and C12EO7 maximum peak areas may be
achieved during derivatization without any excess of PIC
and that these values do not change when excess of the
derivatization agent is used. In case of the remaining
compounds an excessive amount of PIC is necessary in
order to achieve maximum peak areas. Based on the
obtained results, a 50% excess of PIC relative to each
mixture component was used during subsequent studies
(the reaction was carried out at a 1:1.5 ratio).
The last optimization step was focused on the influence
of the amount of derivatization agent relative to the studied
mixture. The experiments were carried out using equimolar
ratios of each component of the mixture relative to PIC
(1:1) and different molar ratios: 1:1.25 (25% excess of
PIC), 1:1.5 (50% excess of PIC) and 1:2 (100% excess of
PIC). On the basis of peak areas obtained for all studied
compounds (Fig. 6), it was established that a 50% excess of
the derivatization agent allowed to achieve maximum peak
123

J Surfact Deterg (2017) 20:1421–1432
1427
Fig. 3 Mass chromatograms of MRM pairs and fragmentation spectra originating from dodecanol and AE with PIC for ammoniated ions
[M?NH4]? at the concentration of 1 g mL-1
Method Validation
limit of quantification) values for river water samples
ranged from 0.0008 lg L-1 for C12EO5 and C12EO6 to
0.1 lg L-1 for C12EO1, whereas MDL (method limit of
detection) values ranged from 0.0007 lg L-1 for C12EO8
to 0.06 lg L-1 for C12EO1. After considering the injection
volume, which amounted to 10 lL, the MDL values range
from 0.007 pg for C12EO8 to 0.6 pg for C12OH. The MDL
values obtained for river water are at least one order of
magnitude lower compared to those obtained by Crescenzi
et al. [30]. It should be noted, that the MDL depends on the
matrix as well as separation and concentration techniques
which were used during sample preparation. Crescenzi
et al. [30] separated the ethoxylates using SPE on a GCB
column, while the LOD and LOQ were determined against
an internal standard (C10EO6) based on a single chro-
matographic peak for the complete C12EOx series obtained
Linearity, LOD and LOQ were determined for [M?H]?
ions and [M?NH4]? ions (see Table 2). Linearity was
tested by analyzing free dodecanol samples and ethoxy-
lated alcohol at different concentrations, ranging from
0.005 to 1000 lg L-1. Good linearity was achieved for all
compounds, with correlation coefficients [0.97 (see
Table 2). In the studied concentration range only C12EO8
and C12EO9 exhibited a single linearity range for proto-
nated adducts. The remaining protonated or ammoniated
adducts of C12OH and AE derivatives exhibited two lin-
earity ranges in the studied concentration range. The LOD
ranged from 0.005 lg L-1 for C12OH and C12EO2–7 to
1 lg L-1 for C12EO1. The LOQ ranged from 0.01 lg L-1
for C12EO5–7 to 2 lg L-1 for C12EO1. The MQL (method
123

1428
J Surfact Deterg (2017) 20:1421–1432
Fig. 4 Influence of the
temperature of derivatization
reaction on the peak areas for
derivatives of C12OH and
C12EO1–9 with PIC. Asterisk the
bars in the graph are standard
error bars
Fig. 5 Influence of the time of
derivatization reaction on the
peak areas for derivatives of
C
12OH and C12EO1–9 with PIC.
Asterisk The bars in the graph
are standard error bars
123

J Surfact Deterg (2017) 20:1421–1432
1429
Fig. 6 Influence of the amount
of derivatization agent of
derivatization reaction on the
peak areas for derivatives of
C12OH and C12EO1–9 with PIC.
Asterisk The bars in the graph
are standard error bars
Table 2 Validation data for determination of C12OH and C12EO1–9
Compound
Calibration curve
range (lg L-1
Correlation
coefficient (r2)
Calibration curve
range (lg L-1
Correlation
coefficient (r2)
LOD
(lg L-1
MDL
(lg L-1
LOQ
(lg L-1
MQL
(lg L-1
)
)
)
)
)
)
C12OH
[M?H]?
0.05–200
0.9984
0.9997
0.9988
0.9983
0.9992
0.9988
0.9992
0.9992
0.9971
0.9982
200–1000
0.8707
0.9943
0.9882
0.9918
0.9900
0.9691
0.9706
0.9928
–
0.005
1
0.0004
0.06
0.05
2
0.004
0.1
C12EO1
[M?H]?
2–200
0.05–200
0.02–200
0.05–200
0.01–200
0.01–200
0.01–200
0.05–1000
0.01–1000
200–1000
200–1000
200–1000
200–1000
200–1000
200–1000
200–1000
–
C12EO2
[M?H]?
0.005
0.005
0.005
0.005
0.005
0.005
0.01
0.0005
0.0004
0.0004
0.0004
0.0004
0.0006
0.0007
0.0004
0.05
0.02
0.05
0.01
0.01
0.01
0.05
0.01
0.005
0.002
0.004
0.0008
0.0008
0.001
0.004
0.007
C12EO3
[M?H]?
C12EO4
[M?NH4]?
C12EO5
[M?H]?
C12EO6
[M?NH4]?
C12EO7
[M?NH4]?
C12EO8
[M?H]?
C12EO9
[M?H]?
–
–
0.005
123

1430
J Surfact Deterg (2017) 20:1421–1432
in TIC (total ion current) mode. The identification of the
mixture was conducted by analysis of mass spectrum for
this peak. Therefore, the determined LOD and LOQ values
as well as concentrations in the actual samples are for the
sum of the total C12EOx series, not for specific ethoxylated
alcohols in the C12EOx series, as in this study. The pre-
sented study employed classic LLE technique with ethyl
acetate, and the LOD, LOQ values as well as concentra-
tions of C12OH and C12EO1–9 in river water were deter-
mined using the analyzed compounds as standards (the
areas of chromatographic peaks). Each of the determined
ethoxylated alcohols was a separated, single peak, due to
the use of single homologues and the MRM mode during
their detection with the MS/MS technique and prior
derivatization with the use of PIC. The only limitation of
the presented methodology is the lack of single homo-
logues with more than 9 ethoxyl groups in the structure of
the C12EOx compound series on the market, since such
compounds are also present in surface water as confirmed
by literature references [2].
and 8 lg L-1) river water samples (River Warta, Poznan,
Poland). Ethyl acetate was used for LLE. Separation and
determination was performed in accordance with sections
Derivatization and Liquid chromatography-tandem mass
spectrometry. The results are shown in Table 3. The con-
centration values of specific analytes present in the non-
spiked Warta river water samples were determined using
the multiple standard addition method [37].
All of the studied analytes were identified in the Warta
river water samples. The highest concentrations were
determined for dodecanol (10 lg L-1) whereas the lowest
were observed for C12EO6 and C12EO7 (0.06 lg L-1). The
obtained concentrations of C12EO2–C12EO9 homologues
were at a similar level compared to the results described by
Zembrzuska et al. regarding the monitoring of Warta river
during a period from October 2011 to September 2012 [37].
The authors determined non-derivatized AE using LC–MS/
MS. It was observed that the obtained recovery rates for
higher additions of the model mixture were higher compared
to recovery rates obtained for lower additions of the model
mixture. It was also observed that in case of additions of 4
and 8 lg L-1, the recovery rates increased along with the
increase of the ethoxy chain length in the surfactant mole-
cule. The same dependencies, namely higher concentrations
of AE containing single ethoxyl groups in the compound and
free alcohol, were observed by other research groups [7, 40],
which indicates that central fission is the dominating
biodegradation mechanism and the source of free alcohol in
surface water and treated wastewater. Since the alcohol
ethoxylates C12–13 with low EO groups are characterized by
high toxicity [7, 41], their monitoring is crucial for proper
environmental protection. The observation of their concen-
tration in the environment is associated with the use of sen-
sitive, selective analytical methods which allow one to
precisely determine their content. The LC–MS/MS in MRM
mode may be a solution for determination of AE derivatives
with PIC. Employing this technique allows one to eliminate
the additional peaks associated with the presence of deriva-
tives of other contaminants in the river water samples, which
were observed during UV detection [16].
The LOD values are not equal for all the studied
ethoxylated alcohol homologues. Such differences result
from different responses of the detector, which are influ-
enced by ionization. Such dependencies were observed
during direct determination (without derivatization) of
these compounds using the LC–MS/MS technique [23].
Perhaps the length of the ethoxyl chain influences the
ionization process. With the increase of the ethoxyl chain
length up to a certain value more stable complexes are
formed. These observations were also described by Cres-
cenzi et al. [30]. The authors observed that the response of
the detector increased exponentially with the increase of
the amount of ethoxyl groups in the compounds from 1 to
6. This dependency was not observed for ethoxylated
alcohols with a number of ethoxyl groups higher than 6.
The determined LOD and LOQ values for AE deriva-
tives with phenyl isocyanate were lower by two orders of
magnitude compared to values obtained for the direct
determination method of these compounds using LC–MS/
MS in the form of protonated adducts [23]. Additionally,
the LOD values obtained in the framework of this study
were lower by one order of magnitude compared to LOD
values obtained for derivatives of and C12EO1–6 with
1-naphthoyl chloride using the same technique [18].
Due to the low level of analyte and the complex matrix,
the use of derivatization allowed to us increase the sensi-
tivity and range of determined AE (with 1–2 ethoxyl
groups and free alcohol) in comparison to detection of non-
derivatized compounds [23]. Furthermore, the use of
derivatization caused an increase in volatility during ion-
ization in the ESI source and changed the behavior of
compounds during their chromatographic separation,
which allowed to use the C18 column for separation of AE
based on the ethoxyl chain length in a reversed phase
system, which is not possible without derivatization, MS/
River Water Sample
In order to check whether the described method may be
used for determination of C12OH and C12EO1–9 in actual
environmental samples, the compounds were determined in
a non-spiked and spiked (at three concentration levels: 2, 4
123

J Surfact Deterg (2017) 20:1421–1432
1431
Table 3 Concentrations of C12OH and homogeneous AE in Warta River in March 2015, and recoveries of spikes of C12OH and C12EO1–9 Warta
River
Homologue River water
found
Spike 2 lg L-1
Spike 4 lg L-1
Found (lg
Spike 8lg L-1
Found (lg
(lg L-1
)
SD Found (lg
L-1
SD
Recovery
(%)
Recovery
(%)
Recovery
(%)
)
L-1
)
SD
L-1
)
SD
C12OH
10.00 0.28
2.19 0.04
1.20 0.03
0.56 0.01
0.55 0.01
0.36 0.02
0.06 0.01
0.06 0.01
0.24 0.01
0.13 0.01
10.32 0.28
16.0
28.0
49.0
51.5
63.5
74.5
63.5
61.0
71.0
69.0
12.04 0.33
3.98 0.07
4.37 0.11
3.84 0.07
3.84 0.07
3.87 0.18
3.07 0.07
3.15 0.11
3.37 0.02
3.61 0.08
51.0
44.8
79.3
82.0
82.3
87.8
72.3
77.3
78.3
87.0
16.24 0.35
8.66 0.15
8.53 0.23
7.98 0.11
7.72 0.11
7.51 0.34
6.84 0.13
6.79 0.22
6.87 0.06
6.54 0.15
78.0
80.9
91.6
92.8
89.6
89.4
84.8
84.1
82.9
80.1
C12EO1
C12EO2
C12EO3
C12EO4
C12EO5
C12EO6
C12EO7
C12EO8
C12EO9
2.75 0.07
2.18 0.06
1.59 0.03
1.82 0.03
1.85 0.08
1.33 0.03
1.28 0.04
1.66 0.01
1.51 0.03
ionic surfactants in elements of the aquatic environment. Tenside
Surf Det. 2015;52:380–5.
MS detection in MRM mode and the use of expensive
chromatographic columns [23].
´
´
3. Plata MR, Contento AM, Rıos A. Analytical characterization of
PEG polymers by MEKC. Electrophoresis. 2010;31:679–87.
4. Patterson SJ, Scott CC, Tucker KBE. Non-ionic detergent
degradation. I. Thinlayer chromatography and foaming properties
of alcohol polyethoxylates. J Am Oil Chem Soc. 1967;44:407–12.
5. Zembrzuska J, Budnik I, Lukaszewski Z. Parallel pathways of
ethoxylated alcohol biodegradation under aerobic conditions. Sci
Total Environ. 2016;557–558:612–9.
Concluding Remarks
The described method for determination of dodecanol and
C12EO1–9 in the form of derivatives with phenyl isocyanate
using the LC–MS/MS technique is characterized by lower
LOD and LOQ values compared to other determination
methods of these compounds, in the form of derivatives
with other derivatization agents [18] as well as non-
derivatized form using LC–MS/MS [23]. In combination
with liquid–liquids extraction using ethyl acetate, this
method is an appropriate and sensitive analytical tool for
the monitoring of environmental water samples.
¨
6. Schroder HF. Tracing of surfactants in the biological wastewater
treatment process and the identification of their metabolites by
flow injection-mass spectrometry and liquid chromatography-
mass spectrometry and –tandem mass spectrometry. J Chro-
matogr A. 2001;926:127–50.
7. Eadsforth CV, Sherren AJ, Selby MA, Toy R, Eckhoff WS,
McAvoy DC, Matthijs E. Monitoring of environmental finger-
prints of alcohol ethoxylates in Europe and Canada. Ecotoxicol
Environ Saf. 2006;64:14–29.
8. Asmussen C, Stan HJ. Determination of non-ionic surfactants of
the alcohol polyethoxylate type by means of high temperature gas
chromatography and atomic emission detection. J High Res
Chromatog. 1998;21:597–604.
Acknowledgements This work was performed with the financial
support of 03/31/DSPB/0338 from the Polish Ministry of Science and
Higher Education.
ˇ
9. Jandera P, Holcapek M, Theodoridis G. Investigation of chro-
matographic behavior of ethoxylated alcohol surfactants in nor-
mal-phase and reversed-phase systems using high-performance
liquid chromatography-mass spectrometry. J Chromatogr A.
1998;813:299–311.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
10. Zanette M, Marcomini A, Marchiori E, Samperi R. High-per-
formance liquid chromatographic-fluorescence determination of
aliphatic alcohol polyethoxylates and poly(ethylene glycol)s in
aqueous samples. J Chromatogr A. 1996;757:159–74.
11. Gomez V, Ferreres L, Pocurull E, Borrull F. Determination of
non-ionic and anionic surfactants in environmental water matri-
ces. Talanta. 2011;84:859–66.
References
12. Heinig K, Vogt C, Werner G. Separation of nonionic surfactants
by capillary electrophoresis and high-performance liquid chro-
matography. Anal Chem. 1998;70:1885–92.
13. Koike R, Kitagawa F, Otuska K. Separation of fatty alcohol
ethoxylates by capillary zone electrophoresis and micellar elec-
trokinetic chromatography. J Sep Sci. 2009;32:399–407.
´
´
1. Plata MR, Contento AM, Rıos A. Analytical characterization of
alcohol–ethoxylate substances by instrumental separation tech-
niques. TrAC. 2011;30:1018–34.
2. Kopiec D, Zembrzuska J, Budnik I, Wyrwas B, Dymaczewski Z,
Komorowska-Kaufman M, Lukaszewski Z. Identification of non-
123

1432
J Surfact Deterg (2017) 20:1421–1432
´
´
ˇ
´
14. Arias M, Bauza R, Rodrıguez J, Castaneda G, Rıos A. Rapid
characterization of fatty alcohol ethoxylates by non-aqueous
capillary electrophoresis. Electrophoresis. 2008;29:3060–8.
ethoxylates as alcohol ethoxy sulfate derivatives by liquid chro-
matography-mass spectrometry.
2004;7(2):195–202.
J
Surfactants Deterg.
15. Lemr K. Homologue separation of linear alcohol ethoxylates by
high-performance liquid chromatography.
1996;732:299–305.
16. Nitschke L, Huber L. Analysis of ethoxylated alcohol surfactants
in water by HPLC. Fresenius J Anal Chem. 1993;345:585–8.
17. Schmitt TM, Allen MC, Brain DK, Guin KF, Lemmel DE,
Osburn QW. HPLC determination of ethoxylated alcohol sur-
factants in wastewater. J Am Oil Chem Soc. 1990;67:103–9.
30. Crescenzi C, Di Corcia A, Samperi R. Determination of nonionic
polyethoxylate surfactants in environmental waters by liquid
chromatography/electrospray mass spectrometry. Anal Chem.
1995;67:1797–804.
31. Iwasaki Y, Nakano Y, Mochizuki K, Nomoto M, Takahashi Y,
Ito R, Saito K, Nakazawa H. A new strategy for ionization
enhancement by derivatization for mass spectrometry. J Chro-
matogr B. 2011;879:1159–65.
J Chromatogr A.
´
´
18. Zgola-Grzeskowiak A, Grzeskowiak T. Solid-phase extraction
combined with dispersive liquid-liquid microextraction, fast
derivatization and high performance liquid chromatography-tan-
dem mass spectrometry analysis for trace determination of short-
chained dodecyl alcohol ethoxylates and dodecyl alcohol in
environmental water samples. J Chromatogr A. 2012;1251:40–7.
19. Trathnigg B. Determination of MWD and chemical composition
of polymers by chromatographic techniques. Prog Polym Sci.
1995;20:615–50.
32. Zu C, Praay HN, Bell BM, Redwine OD. Derivatization of fatty
alcohol ethoxylate non-ionic surfactants using 2-sulfobenzoic
anhydride for characterization by liquid chromatography/mass
spectrometry. Rapid Commun Mass Sp. 2010;24:120–8.
33. Dunphy CJ, Pessler DG, Morrall SW. Derivatization LC/MS for
simultaneous determination of fatty alcohol and alcohol ethoxy-
late surfactants in water and wastewater samples. Envir Sci Tech.
2001;35:1223–30.
´
´
34. Escrig-Domenech A, Simo-Alfonso EF, Herrero-Martinez JM,
Ramis-Ramos G. Derivatization of hydroxyl functional groups
for liquid chromatography and capillary electroseparation.
J Chromatogr A. 2013;1296:140–56.
20. Cretier G, Podevin C, Rocca J-L. Analysis of aliphatic alcohol
ethoxylates in terms of alkyl and ethylene oxide chain lengths by
reversed-phase liquid chromatography with evaporative scatter-
ing detection. J Chromatogr. 2000;874:305–10.
35. Sparham CJ, Bromilow ID, Dean JR. SPE/LC/ESI/MS with
phthalic anhydride derivatization for the determination of alcohol
ethoxylate surfactants in sewage influent and effluent samples.
J Chromatogr A. 2005;10662:39–47.
36. Cassani G, Tibaldi F, Donato G, Andriollo N. Determination of
alcohol ethoxylates derivatized with Naphthoyl chloride, in waste
water treatment plant influent, effluent and sludge samples by
liquid chromatography mass spectrometry. J Surfactants Deterg.
2011;14:139–50.
´
´
21. Escrig-Domenech A, Simo-Alfonso EF, Ramis-Ramos G.
Determination of four major surfactant classes in cleaning
products by reversed-phase liquid chromatography using serially
connected UV and evaporative light-scattering detection. Anal
Chim Acta. 2016;932:106–13.
22. Nowicka D, Budnik I, Zembrzuska J. Biodegradation of alcohol
ethoxylates by bacterial consortium from industrial wastewater.
Tenside Surfact Det. 2013;50:31–5.
23. Zembrzuska J, Budnik I, Lukaszewski Z. Separation and deter-
mination of homogenous fatty alcohol ethoxylates by liquid
chromatography with multistage mass spectrometry. J Sep Sci.
2014;37:1694–702.
37. Zembrzuska J, Budnik I, Lukaszewski Z. Monitoring of selected
non-ionic surfactants in river water by liquid chromatography-
tandem mass spectrometry. J Environ Manage. 2016;169:247–52.
38. Morrall SW, Dunphy JC, Cano ML, Evans A, McAvoy DC, Price
PB, Eckhoff WS. Removal and environmental exposure of
alcohol ethoxylates in US sewage treatment. Ecotoxicol Environ
Saf. 2006;64:3–13.
39. Wells G, Prest H, Russ IV CW (2011) Signal, noise, and detec-
tion limits in mass spectrometry. Technical Note. Agilent Tech-
nologies Wilmington USA
40. Belanger SE, Dorn PB, Toy R, Boeije G, Marshall SJ, Wind T,
Van Compernolle R, Zeller D. Aquatic risk assessment of alcohol
ethoxylates in North America and Europe. Ecotoxicol Environ
Saf. 2006;64:85–99.
41. Wong DCL, Dorn PB, Chai EY. Acute Toxicity and structure-
activity relationships of nine alcohol ethoxylate surfactants to
fathead minnow and Daphnia magna. Environ Toxicol Chem.
1997;16:1970–6.
´
´
24. Lara-Martın PA, Gonzalez-Mazo E, Browwnawell BJ. Environ-
mental analysis of acohol ethoxylates and nonylphenol ethoxylate
metabolites by ultra-performance liquid chromatography-tandem
mass spectrometry. Anal Bioanal Chem. 2012;402:2359–68.
´
25. Krogh KA, Bu¨gel Mogensen B, Halling-Sørensen B, Cortes A,
Vejrup KV. Analysis of alcohol ethoxylates and alkylamine
ethoxylates in agricultural soils using pressurized liquid extrac-
tion and liquid chromatography-mass spectrometry. Anal Bioanal
Chem. 2003;376:1089–97.
26. Ma Q, Ma W, Chen X, Wang Z, Bai H, Zhang L, Li W, Wang Ch,
Li X. Comprehensive analysis of fatty alcohol ethoxylates by
ultra-high pressure hydrophilic interaction chromatography cou-
pled with ion mobility spectrometry mass spectrometry using a
custom-designed sub-2 lm column. J Sep Sci. 2015;38:2182–91.
`
`
27. Mico-Tormos A, Bianchi F, Simo-Alfonso EF, Ramis-Ramos G.
Determination of fatty alcohol ethoxylates with diphenic anhy-
dride derivatization and liquid chromatography with spec-
Joanna Zembrzuska graduated from the Poznan University of
Technology in 1999 and she finished her Ph.D. in 2003 there. Since
then, she has been working as an Assistant Professor at the Poznan
University of Technology. Her research interests include determina-
tion of non-ionic surfactants and pharmaceutical residues in environ-
mental by LC–MS/MS and selenium speciation.
trophotometric detection.
anhydrides. J Chromatogr A. 2009;1216:3023–30.
A comparative study with other
´
28. Ionescu M, Petrovic ZS, Wan X. Ethoxylated soybean polyols for
polyurethanes. J Polym Environ. 2010;18:1–7.
29. Cassani G, Pratesi C, Faccetti L, Pravettoni S, Nucci G, Andrioll
N, Valtorta L, Matheson L. Characterization of alcohol
123