Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 574–580

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

A case study demonstrating the migration of diethyl phthalate from

an ancillary component to the drug product
Gagandeep Singh ∗,1 , Ramarao Gollapalli 1 , Alejandro Blinder, Felix Gallo, Milan Patel
Research and Development, Akorn Pharmaceuticals, 50 Lakeview Parkway, Suite 112, Vernon Hills, IL, 60061, USA

a r t i c l e i n f o a b s t r a c t

Article history: Phthalates are chemical compounds employed as plasticizers in the plastic industry and have been
Received 17 August 2018 reported to migrate into drug products. The extent of their migration into the drug product depends
Received in revised form upon various factors including the chemical nature of the migrant and the permeability of its packaging
11 November 2018
container. Migration of semi-volatile phthalates such as Diethyl phthalate (DEP) into drug products is
Accepted 12 November 2018
often related to the primary and secondary packaging but due to its chemical nature, it could also migrate
Available online 13 November 2018
from an ancillary component. Therefore, it is not only important to screen the primary and secondary
components, but also the ancillary materials that are used during the handling of drug products. In our
Keywords:
Pharmaceutical packaging
study, we discovered an ancillary material (scotch tape) to be the source of DEP found in an ophthalmic
Phthalates drug product using orthogonal mass spectroscopy techniques (GC–MS and LC–MS). It is evident from
Diethyl phthalate our data that DEP migrated from the scotch tape into the drug product crossing the physical barriers
Ancillary material provided by the primary (LDPE container closure system) and secondary packaging (carton and label).
Migrants The tape was used as an ancillary material to wrap the packaged drug product units together for storage
Extractables and leachables in the stability chamber. The primary and the secondary packaging of the drug product did not exhibit
Low-density polyethylene any traces of DEP. The aim of this report is to demonstrate how a chemical compound can migrate into
Mass spectroscopy
the drug product from an ancillary source (which is not a part of its packaging) and adulterate a drug
product. The impact of ancillary materials on drug products should be evaluated appropriately prior to
their implementation.
© 2018 Published by Elsevier B.V.

1. Introduction Leachables and migrants often arise from polymeric materials

The purity of a drug is of paramount importance for the safety,
quality and efficacy of the drug. The impurities detected in the
drug product should be adequately identified and qualified for
toxicological risks [1,2]. The most common impurities encoun-
tered in drug product are degradants of active ingredients or
excipients, leachables and migrants. Among them, leachables and
migrants represent foreign chemical entities. Leachables are chem-
ical compounds that leach into the packaged drug product from
its packaging system during its storage or use [3]. Leachables are
typically derived either from production contact surfaces (manu-
facturing equipment) or packaging (primary and secondary) of the
drug product due to the direct contact. On the other hand, in a phar-
maceutical field, migrants are described as chemical compounds
that accumulate in drug product after crossing a physical barrier,
such as that provided by the primary and secondary packaging [4].
∗ Corresponding author.
E-mail address: gagandeep.singh@akorn.com (G. Singh).
1
These authors contributed equally.
related to packaging or manufacturing components of the drug
product [5]. Phthalates belong to a class of organic compounds
which are known to migrate. Phthalates are dialkyl or alkyl aryl
esters of 1,2- benzenedicarboxylic acid used as plasticizers in poly-
mer manufacturing [6]. However, due to the fact that phthalates
are not chemically bonded to the polymer, it causes their migra-
tion from the polymers into the air especially under rigorous
temperature and humidity conditions, from where they tend to dis-
solve after contact with liquids [6–9]. The composition of primary
packaging of the drug product also significantly affects its suscep-
tibility to migrants such as phthalates. For example, metal and
glass containers have superior barriers compared to plastics and
are generally more resistant to migrants [10]. Furthermore, softer
amorphous polymers such as Low-density polyethylene (LDPE) are
more susceptible to migrants as compared to rigid polymers. How-
ever, LDPE containers are generally employed in the packaging
of ophthalmic products owing to their desirable features, includ-
ing ease of dispensing the drug product. Therefore, even though
adequate precautions are taken, migrants such as phthalates are
often encountered in ophthalmic drug products. However, in the
pharmaceutical setting, if phthalates are detected at safety concern

https://doi.org/10.1016/j.jpba.2018.11.031
0731-7085/© 2018 Published by Elsevier B.V.
 G. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 574–580
levels during drug development, attempts should be made to find
out their origin and to avoid their formation. Furthermore, there are
guidelines from Product Quality Research Institute (PQRI) [3], USP
<1664> and ICH M7 that provide guidance to establish thresholds
and limits for controlling leachables/migrants in the drug products.
Migration of diethylhexyl phthalate (DEHP) from PVC bags into
intravenous cyclosporine solutions has been previously reported
[11]. Other reports exist which describe the presence of phtha-
lates in medical devices [12–15]. Diethyl phthalate (DEP) is among
the most commonly used low-molecular-weight phthalates whose
annual production between 2004 and 2005 in US reached 100 mil-
lion pounds [7]. DEP is commonly used in food/pharmaceutical
packaging, medical devices, adhesives etc [16]. and has been
reported to migrate into their surroundings [1].The toxic effects of
DEP are well documented especially the range of toxicities in gas-
trointestinal and cardiovascular systems along with reproductive
toxicities [17–19]. Accumulation of low molecular weight phtha-
lates such as DEP in drug products is often attributed to primary and
secondary packaging but due to its semi-volatile nature, it could
also migrate from an ancillary component. Therefore, it is not only
important to screen primary and secondary components, but also
the auxiliary materials that are used during the handling of drug
product units. Furthermore, the migration of phthalates (such as
DEP) from an ancillary material to drug products is often discussed
but rarely proven.
In this case study, we discovered an ancillary material (scotch
tape) to be the source of diethyl phthalate found in an ophthalmic
drug product using mass spectroscopy. It is evident from our data
that DEP migrated from the scotch tape into the drug product cross-
ing the physical barriers provided by the primary and secondary
packaging. The tape was used to wrap the drug packaging units
together for storage in the stability chamber. The primary and the
secondary packaging of the drug product did not exhibit any traces
of DEP. The identification was achieved using orthogonal mass
spectroscopy techniques (GC–MS and LC–MS). The aim of this paper
is to demonstrate how a chemical compound can migrate from an
ancillary source (which is not a part of its packaging) and can adul-
terate the drug product. The use of ancillary materials and their
impact on drug products should be evaluated appropriately prior
to their implementation. Clear and defined procedures should be
established for the process, packaging and handling of drug prod-
ucts in order to mitigate adulteration of the drug product due to
foreign compounds.
2. Materials and methods
2.1. Chemicals and materials
ACS grade ammonium acetate, formic acid, sodium phosphate
monobasic and sodium bicarbonate were obtained from Fisher
Scientific, Fairlawn, NJ, USA. HPLC grade methanol (MeOH) and
acetonitrile (ACN) and were also obtained from Fisher Scien-
tific. Ethanol (purity, ≥99%), Diethyl phthalate (purity, ≥99%) and
Dichloromethane (≥99.9% grade) were purchased from Sigma-
Aldrich, St Louis, MO, USA. The ultra-pure water used was
purified by a MilliQ water system (Millipore, France). Scotch tape
(item#UNV83412, Lot#61,022) was obtained from Universal Tape
Co., Windham, NH, USA.
2.2. Sample and standard preparation for LC analysis
Ophthalmic product (in house), 0.05%, 3 M CRT (Controlled room
temperature, 25 ◦ C/40% Relative Humidity), 3 M INT (Intermedi-
ate conditions, 30 ◦ C/65% Relative Humidity), 3 M ACC (Accelerated
conditions, 40 ◦ C/25% Relative Humidity) and 3 M retained sample
575
were used as drug product samples. 1 mL of sample was trans-
ferred into a 10 mL volumetric flask, which was then diluted to
volume with diluent (ACN: Water 50:50) and vortexed for 30 s. The
entire sample solution was transferred into an appropriate cen-
trifuge tube and centrifuged at a speed of 4000 rpm for 10 min. The
centrifuge tube was removed carefully so that the oil layer at the
bottom remained separated. 1 mL of the supernatant liquid was
drawn using a disposable pipette and transferred into a HPLC vial
and analyzed using LC Method-1.
Standard Preparation: Weighed and transferred approximately
15 mg of Diethyl phthalate into a 100 mL volumetric flask. Dis-
solved and diluted to volume with acetonitrile and mixed well. The
concentration of DEP in the stock solution was ∼150 ␮g/mL. The
working standard solution was prepared by pipetting 1 mL of the
stock standard into a 100 mL volumetric flask. Diluted to volume
with diluent (ACN: Water 50:50) and mixed well. The concentra-
tion of DEP in the working standard solution was approximately
1.5 ␮g/mL. The working standard solution was transferred into a
HPLC vial and analyzed using LC Method-1.
2.3. Standard preparation for LC–MS analysis
The working standard solution was prepared by pipetting 1 mL
of the stock standard into a 100 mL volumetric flask. Diluted to
volume with ethanol and mixed well. The concentration of DEP in
the working standard solution was approximately 1.5 ␮g/mL.
2.4. Sample preparation for GC–MS analysis
5 mL of the drug product solution was transferred into a sep-
aratory funnel and liquid-liquid extraction was carried out using
dichloromethane. It is to be noted that DEP is fairly soluble
(∼1 mg/mL) in aqueous solutions and dichloromethane is com-
monly used as an extractant for DEP in liquid-liquid extraction
involving aqueous solutions [20]. The organic (dichloromethane)
fraction was collected and concentrated to ∼1 mL by blowing nitro-
gen gas over the solution, transferred to a GC vial and analyzed
using GC–MS method-1.
2.5. LC method-1
The chromatographic separation was achieved on an Agilent
High Pressure Liquid Chromatography system 1200 equipped with
DAD detector (Agilent Technologies, Santa Clara, CA, USA), using
gradient elution (flow rate: 0.5 mL/min) on a Zorbax Rapid Res-
olution HD Extend C18 column 100 × 2.1 mm, 1.8 ␮m (Agilent,
Part # 758700-902 K). Ammonium acetate (10 mM, pH 8.5) was
used as solvent A and acetonitrile as solvent B. An elution gra-
dient was applied, 0-0.5 min: %B = 35, 0.5–5.5 min: %B = 35–45,
5.5–6.5 min: %B = 45–48, 6.5–8.0 m in. %B = 48–70, 8.0–8.1 min:
%B = 70-35, 8.1–14.0 min: %B = 35. The UV data was collected at
240 nm. The injection volume of the method was 4 ␮L and the
analytical column was maintained at 40 ◦ C.
2.6. LC–MS method-1
The LC–MS accurate mass analysis was performed on a liquid
chromatographic system model 1290 (Agilent Technologies, Palo
Alto, CA, USA) coupled to a 6460 triple quadrupole mass spec-
trometer equipped with electrospray ionization interface (ESI).
The chromatographic separation was achieved using a gradient
elution (flow rate: 0.4 mL/min) on a Zorbax Extend C18 column
100 × 2.1 mm, 1.8 ␮m (Agilent, Part # 758700-902). Water was
used as solvent A and acetonitrile as solvent B. An elution gradient
was applied, 0–10.0 m in. %B = 5–95, 10.0–12.0 min. %B = 95, 12.0-
12.1: % B = 95-5, 12.1–15.0: %B = 5. The UV data was collected at
576 G. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 574–580
237 nm. The injection volume of the method was 20 ␮L and the ana-
lytical column was maintained at 40 ◦ C. MS analysis was performed
in positive mode using an ESI interface. The gas temperature was
set to 300 ◦ C and sheath gas temperature to 250 ◦ C at a flow rate of
11 L/min. The nebulizer pressure was set to 45 psi and the capillary
voltage to 4000 V.
2.7. GC–MS method-1
GC–MS analysis was performed on an Agilent Gas Chromatog-
raphy system 7890 A (Agilent Technologies, Santa Clara, CA, USA),
equipped with a Mass Spectrometry triple-axis 5975C detector.
Agilent Mass Hunter software was used for data collection and
instrument control. The chromatographic separation was achieved
using an Agilent HP-5MS column, 30 m × 0.25 mm, 0.25 ␮m (Agi-
lent, Serial # USN723024 H). Helium was employed as a carrier gas
at a flow rate of 2.0 mL/min. A splitless injector was used and the
injector temperature was set to 300 ◦ C. The MS transfer line was
maintained at 250 ◦ C and the ionization mode was EI (Electron
Impact) at 70 eV. The initial oven temperature was 40 ◦ C, main-
tained for 5 min, followed by a temperature ramp to 280 ◦ C @
10 ◦ C/min, maintained for 3 min, and another temperature ramp
to 320 ◦ C @15 ◦ C/min, maintained for 5 min.
2.8. Extractable study on primary packaging components (LDPE
bottle, cap and tip)
The extractable study was performed using three types of
extraction solvents: i) Buffer pH 3 (0.1 M Sodium Phosphate); ii)
Buffer pH 9 (0.1 M Sodium bicarbonate); and iii) Ethanol and Water
(40:60 v/v). Approximately 5 g of test articles were weighed into
separate glass containers containing 200 mL of each extraction sol-
vent. After 30 min of sonication, the units were placed in an oven
maintained at a temperature of 50 ◦ C for four days. Control samples
were prepared similarly by placing the same volume of extraction
solvents in glass bottles without test articles. After four days, the
Fig. 1. Representative GC–MS Total Ion Chromatograms (TICs) of a control drug product sample (top), 3 M accelerated sample (middle) and the intermediate sample (bottom).
samples were taken out and injected as is for LC–MS analysis using
LC–MS method 1. For GC–MS analysis, each extraction solvent was
subjected to solvent extraction using dichloromethane. The organic
layers for each test article were combined and the resulting solution
was concentrated to ∼1 mL by blowing nitrogen gas over the top
of the solution, transferred to a GC vial and analyzed using GC–MS
method-1.
2.9. Extractable study on secondary packaging components and
ancillary material (Carton, label and tape)
Equal amounts (∼2 cm × 6 cm) of each test article (carton, label
and tape) were isolated into separate 20 mL scintillation vials. 5 mL
of ethanol was transferred into the scintillation vials, sonicated
for 1 h and the vials stood for 1 h with intermittent shaking. The
resulting solutions were transferred to individual HPLC vials and a
GC vials to analyze using LC–MS method 1 and GC–MS method 1
respectively.
3. Results and discussion
In this study, the unknown impurity was detected in an oph-
thalmic product (packaged in a LDPE container) at a level of
∼20 ppm (␮g/mL) during the stability testing using HPLC analysis
(see LC Method-1 in the experimental section). The representative
LC-UV chromatogram of the drug product is shown in support-
ive Fig. S1. The active pharmaceutical ingredient (API) eluted at a
retention time of ∼7 min and the unknown impurity at a retention
time of ∼4 min. The drug product sample was also analyzed using
GC–MS (see GC–MS Method-1 in the experimental section) and the
NIST library search proposed that the unknown impurity could be
a known plasticizer; Diethyl phthalate (DEP) which was confirmed
upon comparison with a reference standard using LC Method-1.
Refer to supportive Fig. S2 and Fig. S3 for relevant chromatograms.
It is crucial to determine the source of an impurity during drug
development in order to control the impurity’s concentration in
 G. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 574–580
Fig. 2. Overlay of the representative TICs (LC–MS) of the control, bottle, tip and cap extracts in positive mode.
the drug product. Furthermore, if an impurity such as DEP repre-
sents toxicological risks, its control becomes even more important
to ensure drug safety [21,22]. Therefore, a systematic study was
designed to determine DEP’s source so that it could be adequately
controlled. Initially, drug product samples stored under different
stability conditions were analyzed using GC–MS Method-1 and the
representative Total Ion Chromatograms (TICs) are presented in
Fig. 1. It is to be noted that the drug product sample stored under
intermediate conditions (30 ◦ C/65% Relative Humidity) exhibited
higher amounts of DEP as compared to the one stored under accel-
erated conditions (40 ◦ C/25% Relative Humidity). This observation
indicated that the higher humidity conditions were favoring DEP’s
accumulation in the drug product. The control drug product sample
of the same lot stored in a glass container in the stability chamber
did not exhibit a DEP peak which suggested that DEP might have
originated from a packaging component. Moreover, these results
suggest that the impurity was not drug product related.
As diethyl phthalate is commonly used as a plasticizer in the
plastic industry [21,23,24], the components of the container clo-
sure system were evaluated. Hence, the extractable study was
performed on the primary packaging components (LDPE bottle,
cap and tip) using an acidic buffer, a basic buffer and an organic
577
solvent mixture. The details of this study are outlined in the exper-
imental section. However, the extractable study performed on the
primary packaging suggested that none of the components con-
tained diethyl phthalate. The typical LC–MS TICs of the bottle, tip
and cap are shown in Fig. 2 and the corresponding GC–MS chro-
matograms are shown in supporting information Fig. S4.
It is known that compounds can migrate from the secondary
packaging of the drug product. Therefore, an extractable study was
also performed to screen secondary packaging components for the
DEP source using ethanol as an extraction solvent (see experi-
mental section for details). The secondary packaging components
included the carton and the label. The representative TICs (LC–MS)
are shown in the supporting information Fig. S5. None of these
components exhibited a DEP peak in their chromatograms. The
investigation was further continued and the articles which came
in contact with the drug product container during stability storage
were evaluated for any potential source of DEP. Review of the pack-
aging indicated that some ancillary materials (such as the scotch
tape and additional labels) were used to aid storage of samples
before they were placed into the stability chamber. The scotch tape
was used to wrap the drug product containers before placing them
into a stability chamber. Consequently, as a part of the investiga-
578 G. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 574–580

Fig. 3. (a) Overlay of the representative TICs (LC–MS) of Diethyl phthalate standard (top), and Tape extract (bottom) in positive mode. (b) Overlay of the MS spectra of Diethyl
phthalate peak in standard (top) and Tape extract (bottom).

tion, an extractable study was performed on the ancillary materials
using ethanol as an extraction solvent (Refer experimental sec-
tion for details). The resulting extracts were tested using LC–MS
Method-1 and GC–MS Method-1. Remarkably, the TICs (GC–MS
and LC–MS) of the scotch tape exhibited large amount of DEP. The
extracted mass spectra of the reference standard and the corre-
sponding peak in the sample confirmed the presence of DEP in the
adhesive tape. The relevant LC–MS chromatograms and mass spec-
tra are presented in Fig. 3a and b respectively. The corresponding
GC MS chromatograms and mass spectra are shown in supporting
Fig. S6.
As a proof of concept, drug product samples with and without
the scotch tape, were tested using LC Method-1. The representative
LC-UV chromatograms of both drug products along with the refer-
 G. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 574–580
Fig. 4. Representative UV chromatograms of the reference standard solution including DEP (top), drug product with tape (middle) and drug product without tape (bottom).
ence standard are shown in Fig. 4. As evident from the data, samples
wrapped with the scotch tape exhibited a DEP peak, whereas the
samples without the scotch tape did not. Based on our results, it
was concluded that DEP migrated from the scotch tape into the
drug product crossing the physical barrier provided by the primary
and the secondary packaging. It is to be noted that DEP has been pre-
viously reported to be a potential migrant. This study suggests that
any extraneous material should be screened prior to its implemen-
tation in the drug product handling or storage to ensure drug safety.
Well-defined procedures should be in place to define the materials
which can be used in the handling of drug products especially when
stored in a stability chamber.
4. Conclusion
The unknown peak observed in the drug product was identi-
fied as DEP. Based on our results, the source of the impurity was
attributed to the tape used to wrap the samples together. The
tape was found to contain a significantly high amount of Diethyl
phthalate as confirmed by orthogonal mass spectroscopy tech-
niques (GC–MS and LC–MS). Diethyl Phthalate was neither found
in the primary packaging (bottle, cap and tip) nor in the secondary
packaging (carton and label). This study shows that a chemical com-
pound can migrate from an ancillary component and accumulate
in the drug product. It is also recommended that any extraneous
material should be screened prior to its implementation in the drug
product handling or storage.
Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of the
manuscript.
Conflict of interest
The authors declare no competing financial interest.
579
Acknowledgements
We are grateful for the financial support from Akorn Pharma-
ceuticals Inc. We are also thankful to our team members for their
valuable suggestions throughout our work.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.jpba.2018.11.
031.
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