Journal of Chromatography B, 949950 (2014) 99108

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Identication and quantication of 14 phthalates and 5 non-phthalate

plasticizers in PVC medical devices by GCMS
Pascal Gimeno , Sbastien Thomas, Claudine Bousquet, Annie-Francoise Maggio,
Corinne Civade, Charlotte Brenier, Pierre-Antoine Bonnet
Agence nationale de scurit du mdicament et des produits de sant (ANSM), Direction des Contrles (CTROL), 635 rue de la Garenne, 34740 Vendargues,
France

a r t i c l e

i n f o

Article history:
Received 3 October 2013
Accepted 26 December 2013
Available online 2 January 2014
Keywords:
Phthalates
Plasticizers
Medical devices
ISO 12787
Gas chromatographymass spectrometry

a b s t r a c t
A GC/MS method was developed for the identication and quantication of 14 phthalates: 8 phthalates
classied H360 (DBP, DEHP, BBP, DMEP, DnPP, DiPP, DPP and DiBP), 3 phthalates proposed to be forbidden
in medical devices (DnOP, DiNP and DiDP) and 3 other phthalates none regulated (DMP, DCHP and DEP)
which may interfere with hormone function. In order to identify and quantify other plasticizers that are
commonly used in PVC medical devices such as DEHP substitute, 5 non-phthalate plasticizers (ATBC,
DEHA, DEHT, TOTM, and DINCH) were included in this study. Analyses are carried out on a GC/MS system
with electron impact ionization mode (EI). The separation of plasticizers is obtained on a cross-linked 5%phenyl/95%-dimethylpolysiloxane capillary column 30 m 0.25 mm (i.d.) 0.25 m lm thickness using
a gradient temperature. Compounds quantication is performed by external calibration using an internal
standard. Validation elements on standard solutions were determined using the ISO 12787 standard
approach. Plasticizers are extracted from PVC medical devices using THF for dissolving the PVC part of
the sample followed by precipitation of the PVC by addition of ethanol. The supernatant is injected into a
GC/MS system after dilution in ethanol. Different validation elements, including extraction recoveries for
all compounds or for DEHP a cross-validation of the extraction process using the European pharmacopoeia
monograph 3.1.14 as reference method, are discussed. Results obtained on 61 medical devices in PVC and
12 raw materials used as plasticizers are given.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Phthalates are esters of ortho-phthalic acid. These compounds
have applications in many industrial sectors. They are used as plasticizers to improve exibility of plastics such as polyvinyl chloride
(PVC) in which they can represent up to 40% of the total mass [1].
As phthalates are not chemically bound to plastics, these compounds may be released from plastics used for medical devices
[26]. Suspicions of harmful effects of phthalates on human health
have recently been brought to the attention, even if phthalate

Abbreviations: DBP, dibutyl phthalate; DEHP, di(2-ethylhexyl) phthalate; BBP,

butylbenzyl phthalate; DMEP, di(2-methoxyethyl) phthalate; DnPP, di-n-pentyl
phthalate; DiPP, diisopentyl phthalate; DPP, n-pentyl isopentyl phthalate; DiBP,
diisobutyl phthalate; DiNP, di-isononyl phthalate; DiDP, di-isodecyl phthalate;
DnOP, di-n-octyl phthalate; DCHP, dicyclohexyl phthalate; DEP, diethyl phthalate;
DMP, dimethyl phthalate; ATBC, acetyl tributyl citrate; DEHA, bis(2-ethylhexyl)
adipate also named Dioctyladipate (DOA); DEHT, bis(2-ethylhexyl) terephthalate; TOTM, tris(2-ethylhexyl)trimellitate also abbreviated TEHTM; DINCH,
di-isononylcyclohexane-1,2-dicarboxylate.
Corresponding author. Tel.: +33 467913951; fax: +33 467913983.
E-mail address: pascal.gimeno@ansm.sante.fr (P. Gimeno).
1570-0232/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2013.12.037

esters toxicity is already known since 1950s [7]. Phthalates may

be involved in reprotoxicity, carcinogenesis, cardiotoxicity, hepatotoxicity and nephrotoxicity [810]. Experiments on animals have
shown that phthalates may also have inuence on immune and
allergic response, even if effects observed in rodents regarding
immune toxicity may not be relevant to humans based on much
lower human exposure and on the route of human exposure [11].
One of the main potential toxic effects of some phthalates experimentally observed concerns the human endocrine system [12,13].
A study published in 2013 reports signicant increased mono(2ethylhexyl)phthalate (MEHP) blood levels in pregnant women
upon recent exposure to perfusion materials [14]. According to the
Directive 2007/47/EC (7.5) requirement, manufacturers must indicate on the labelling of the medical device or on the medical device
itself the presence of any compound carcinogenic, mutagenic or
toxic to reproduction in accordance with Directive 67/548/EEC
(Annex I). Still nowadays, no target value exists beyond which
the phthalates or DEHP label must be mentioned on medical
devices.
In 2013, the only ofcial method for the determination of phthalates as ingredients in plastic materials used for medical devices
is mentioned in the European pharmacopoeia (monograph 3.1.14)

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P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

[1]. This ofcial method, focused exclusively on the determination

of DEHP as plasticizer, is based on the extraction of this phthalate
from PVC materials with diethyl ether using a reux condenser. The
extraction solvent (diethyl ether) is then completely evaporated
and the dry residue dissolved in toluene. The analysis is performed
using thin layer chromatography. As part of a recent study, published by our laboratory and focused on the determination of
phthalates in cosmetic products, analytical methods proposed in
the literature for the identication and separation of phthalates
were studied and reported [15]. Among analytical methods existing [1626], HPLC/UV is one of the methods used for the separation,
identication and quantication of phthalates [1618,2022]. Few
numbers of phthalates are simultaneously determined by these
methods. Indeed, most articles propose analytical methods for the
separation of 4 or 5 phthalates among which only 2 or 3 are classied H360 (BBP, DEHP and DBP). Only one HPLC/UV method [20]
proposes the separation of up to 7 phthalates (DMP, DEP, DPP,
DiBP, BBP, DBP and DEHP) among which 5 are classied H360.
Gas chromatography (GC) is also mentioned for the analysis of
phthalates [18,19,2326,39]. A 5% dimethylpolysiloxane column is
always used for the separation of up to 7 phthalates (DnOP, DPP,
DCHP, DEP, BBP, DBP, DEHP) among which only 4 are classied
[19]. When mass spectrometry is used for the analysis of phthalates, a capillary column of 30 m 0.25 mm 0.25 m is described,
the phthalate ionization is performed by electronic impact (EI) and
the detection obtained using SIM mode (m/z = 149). The sample
is injected either in splitless mode [18,25], in one-fth [19], onetwentieth [23] split mode or in injection thermal desorption (TD)
[24] using He as carrier gas. At least, a new technique based on
real time detection using thin lm micro-electromechanical system
(MEMS) semiconductor device for rapid detection of phthalates in
water and beverages has been recently published [27].
Complementary analytical methods dealing with the determination of DiDP and DiNP, phthalates proposed to be forbidden in
medical devices [28,29], were also examined. DiNP and DiDP are
commonly used as plasticizers in children toys and food packaging
can also be found in medical devices. Analyses are carried out by gas
chromatography with ame ionization detection [30,31] or mass
detection [3234] using characteristic ions such as m/z = 293 (for
DiNP) and m/z = 307 (for DiDP) or m/z = 149 (for both compounds).
As for other phthalates, split/splitless is a common injection
mode, even if other injection modes such as thermal desorption
[33,34] or large-volume injection are also reported [35]. The type
of chromatographic columns used (polydimethylsiloxanes, polydiphenylsiloxanes or cyanopropylpolydimthylsiloxanes) does not
appear to be essential for the determination of these phthalates.
DiNP and DiDP can also be quantied using liquid chromatography with UV detection after extraction with C8 [36] or phenyl [37]
SPE cartridge. The mobile phase used is often water/acetonitrile. As
DiNP and DiDP consist of several isomers, depending on the nature
of alcohols used for their synthesis, these phthalates are eluted as
broad mass peaks [30,33,34,38]. Their quantication is performed
on the sum of all mass peaks. Limits of quantication obtained for
these phthalates are always higher than that for other phthalates
eluted as a single peak [33,34]. Co-elutions and poor resolutions
[30,38] are commonly reported between DiNP and DiDP.
Among analytical methods found in the literature for the extraction of phthalates from plastic materials, some papers propose
protocols based on stirring of the plastic at room temperature in
an organic solvent such as dichloromethane [38] or acetone [36]
after having cut off the material into pieces. Another paper [39] proposes an extraction process in which plastic samples are crushed
into powder, after being frozen using liquid nitrogen or dry ice, and
then extracted by hexane using an ultrasonic bath and stirring the
content. Extraction process, based on the solubility of PVC in THF, is
also described for materials derived from food packaging, children

items (like toys), PVC medical devices or seals of metal lids used
for the closure of glass containers. PVC is rst dissolved in THF
and then precipitated by adding ethanol or methanol. Phthalates
contained in the supernatant solution are analysed using different chromatographic techniques [32,40,41]. The PVC solution in
THF can also be diluted in acetonitrile and injected onto a HPLC/MS
system after SPE purication [42]. Other methods describe extraction protocols similar to the technique described in the European
Pharmacopoeia. PVC-based materials are introduced into ether and
reuxed for 8 h using a reux condenser. A Soxhlet extraction is also
reported using either dichloromethane or cyclohexane for PVC children toys [31] and chloroform/methanol for food packaging [43]. An
automatic Soxhlet process allowing the simultaneous extraction of
6 samples is described for the determination of phthalates in sludge
and vegetables [44]. Procedures using special equipment to shorten
the extraction process are reported. Among these procedures are
supercritical uid extraction (SFE) [31], used for the determination of phthalates in children toys, accelerated solvent extraction
(ASE) with a dichloromethane/acetone mixture for inorganic materials [37] and pressurized solvent extraction (PSE) with methanol
for sediments [45]. The extraction assisted by microwave (MAE) is
reported as a possible extraction technique for the determination
of phthalates in soil or sediment using acetone/dichloromethane
[46], acetone [47] or other solvents [45]. A method allowing the
extraction of plasticizers, other than phthalates, contained in PVC
materials with methanol is also reported [48]. After the extraction
step, the analysis can be performed either by injecting the supernatant [32,38] or after a centrifugation step. The supernatant is then
evaporated to dryness and the residue dissolved in an organic solvent possibly after a previous ltration [41,42]. Purication using
C18 SPE cartridges [18,19,34] or more specic ones such as Oasis
[25,42] has also been considered.
2. Experimental
2.1. Materials and methods
2.1.1. Gas chromatography and mass spectrometry
All analyses are carried out on a Varian 3800 gas chromatograph interfaced with a Varian 1200 quadrupole mass spectrometer
(Palo Alto, USA). The column is an Agilent HP-5MS capillary
column (cross-linked poly 5% diphenyl/95% dimethylsiloxane);
30 m 0.25 mm (i.d.) 0.25 m lm thickness. The oven temperature is settled as follows: 100 C ramped to 200 C at 30 C min1 ,
then to 250 C at 3 C min1 held for 2.5 min, then to 270 C at
40 C min1 held for 2 min and then to 320 C at 80 C min1 held
for 5 min. One microliter of each extract is injected in split mode
one-twentieth using a Varian 8400 autosampler. The injection port
and transfer line temperatures are both set at 300 C. The ion source
temperature is set at 230 C, and the helium carrier gas ow rate
set at 1 mL min1 . The mass spectrometer is tuned on electron
impact ionization (Ei) at 70 eV. The solvent delay is set at 2.5 min
and the run time at 31.0 min. The acquisition is performed on fullscan (m/z = 40350) and on SIM mode. For the SIM mode, three
different ions are monitored for each plasticizer (ions are listed in
Table 1) according to the literature [49] and after preliminary injections using the full-scan mode. Retention times of each group are
determined after the injection of a standard solution.
2.1.2. Reference standards
The 14 phthalates and 5 non-phthalate plasticizers analysed are
listed in Table 2. Of these, 8 phthalates are classied H360 [50], 3
phthalates (DnOP, DiNP and DiDP) which may be used as plasticizers are proposed to be forbidden in France in medical devices
[28,29] and 3 other phthalates non-regulated may interfere with

P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

101

Table 1
Selected ions for SIM mode acquisition.
Analytes

RT (min)b

Ion 1 (m/z)

Ion 2 (m/z)

Ion 3 (m/z)

1st group
DMP

2.5
3.8

163

194

135

2nd group
DEP

4.0
4.6

149

177

105

3rd group
DiBP
DBP
DMEP

5.0
6.8
7.9
8.4

149
149
59

104
205
149

223
223
104

4th group
DiPP
DPP
DnPP

8.6
9.4
10.1
10.7

149
149
149

237
237
237

104
219
104

5th group
ATBC

11.5
12.3

185

129

259

6th group
BBP

13.5
14.4

149

91

206

7th group
DEHA

14.8
15.1

129

57

112

8th group
DCHP
DEHP

17.0
17.9
18.5

149
149

167
167

249
279

9th groupa
DnOP
DEHT
DINCH
DiNP
DiDP

19.0
22.7
22.9
22.9
24.0
25.4

279
261
155
293
307

149
149
127
149
149

167
167
281
127
167

10th group
TOTM

28.0
29.3

305

193

323

a
b

50
50
30

40

50
50
50
40

20

50

Partially co-eluted compounds.

Retention times should be determined after the injection of a standard solution.

Table 2
Phthalates and non-phthalate plasticizers considered.
CAS number
Phthalates
DBP (dibutyl phthalate)
DEHP (diethylhexyl phthalate)
BBP (butylbenzyl phthalate)
DMEP (di(2-methoxyethyl)
phthalate)
DnPP (di-n-pentyl phthalate)
DiPP (diisopentyl phthalate)
DPP (n-pentyl isopentyl
phthalate)a
DiBP (diisobutyl phthalate)
DnOP (di-n-octyl phthalate)
DiNP (di-isononyl phthalate)
DiDP(di-isodecyl phthalate)
DCHP (dicyclohexyl phthalate)
DEP (diethyl phthalate)
DMP (dimethyl phthalate)
Non-phthalates
ATBC (acetyl tributyl citrate)
DEHA (bis(2-ethylhexyl)
adipate)
DEHT (bis(2-ethylhexyl)
terephthalate)
TOTM (trioctyl trimellitate)
DINCH
(di-isononyl-cyclohexane1,2-dicarboxylate)
a

Dwell time (ms)

Mixed isomers (DiPP, DnPP and DPP).

84-74-2
117-81-7
85-68-7
117-82-8

Reference
[28,29,50]
[28,29,50]
[28,29,50]
[50]

131-18-0
605-50-5
84777-06-0

[50]
[50]
[50]

84-69-5
117-84-0
68515-48-0
26761-40-0
84-61-7
84-66-2
131-11-3

[50]
[28,29]
[28,29]
[28,29]
[51,52]
[51,52]
[51,52]

77-90-7
103-23-1

[53]
[53]

6422-86-2

[53]

3319-31-1
166412-78-8

[53]
[53]

hormone function [51,52]. In order to identify and quantify other

plasticizers that are commonly used in PVC medical devices such
as DEHP substitute [53], 5 non-phthalate plasticizers were added
to phthalates considered.
Standards with purity equal to or greater than that of DBP
(99.0%), DEHP (99.0%), BBP (98.0%), DMEP (99.4%), DCHP (99.7%),
DEP (99.5%), DMP (99.0%), DiBP (99.0%), DnOP (98.0%), DiDP (99.0%),
DEHT (96.0%), ATBC (98.0%), TOTM (99.0%), DEHA (99.0%) and 4,4 dibromobiphenyl (ISTD) were purchased from SigmaAldrich (St.
Quentin Fallavier, France); DPP (99.0% mixed of isomers DiPP,
DnPP and DPP), DiPP (99.5%), DnPP (99.0%), and DiNP (99.0% CAS
68515-48-0) were obtained from CIL Cluzeau Info Labo (SainteFoy-La-Grande, France). Actually, two DINPs are available on the
market: DINP1 with CAS number 68515-48-0 and DINP2 with CAS
number 28553-12-0, the major difference being that DINP2 consists
only of C9 isomers while DINP1 contains up to 10% C10 isomers and
exhibits a broad isomer distribution. DINCH (99.0%) comes from
BASF (Ludwigshafen, Germany). All organic solvents (purity more
than 99%) were obtained from different commercial sources (VWR,
Fluka, ChromaNorm, SigmaAldrich). Distilled water or water of
grade 1 in accordance with ISO 3696:1987 was used.
2.1.3. Standard solutions
Preparation and storage of standard solutions are performed
only with glass material to prevent the extraction of phthalates from plastics. The glassware is decontaminated before use
[35,16,54] by rinsing several times with organic solvents (THF,
ethanol). During assay, ethyl acetate [55] is regularly injected to
check and prevent carry over. A rst stock standard solution (Sm1) is prepared in ethanol by weighing 20.0 mg of each of the
following phthalates: DBP, DEHP, BBP, DMEP, DCHP, DEP, DMP,

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P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

DiBP, DnOP, DPP, DiPP and DnPP in a 20.0 mL volumetric ask

(C = 1000 g mL1 ). This stock solution can be used for at least 7
days stored in a refrigerator [15]. A second stock standard solution (S-m2) is prepared in ethanol by weighing 20.0 mg of each
of the following non-phthalate plasticizers: ATBC, DEHA, TOTM,
DEHT in a 20.0 mL volumetric ask (C = 1000 g mL1 ). This solution is prepared daily. Due to a lower sensitivity (the presence
of isomers multi-peaks), an independent stock standard solution (S-m3) is prepared in ethanol by weighing 20.0 mg of each
of the following compounds: DiDP, DiNP and DINCH in a 20.0 mL
volumetric ask (C = 1000 g mL1 ). This solution is also prepared
daily. From these stock standard solutions (S-m1, S-m2 and S-m3)
mixed standard solutions of all compounds are prepared ranging
from 2.5 g mL1 to 50 g mL1 for DiNP, DiDP and DINCH and
from 0.25 g mL1 to 5.0 g mL1 for other analytes by dilution
in ethanol. 4,4 -Dibromobiphenyl (retention time about 9.0 min)
is used as internal standard (ISTD) for the GCMS analyses at a
concentration of 10 g mL1 . The quantication of DiDP, DiNP and
DINCH is performed for each compound on the sum of the peak
areas corresponding to the different isomers.
2.1.4. Medical devices
A total of 61 medical devices, containing one or several parts
of PVC material, have been used to check the analytical method
described. Among medical devices considered most of them were
composed of extension lines, drip chambers and bags.
2.1.5. Plasticizers
Further to results obtained on medical devices, 12 raw materials
(Table 10) used as plasticizers from different manufacturer sources
were studied in order to check a possible correlation between the
presence of some phthalates in medical devices tested and the quality/nature of the plasticizer used. Each plasticizer was analysed
after complete solubilization in EtOH (1.0 g/10.0 mL).
2.1.6. Sample preparations
2.1.6.1. Standard protocol. From the PVC sample, 1.0 g is cut into
pieces of about 0.5 cm of length. The pieces are dissolved in 25.0 mL
of THF using a PTFE magnetic stir bar obtained from Dutsher
(tested phthalate free). A complete dissolution of the PVC sample
is obtained after stirring about 2030 min. The internal standard
is added to the preparation. The PVC is precipitated, as a white
powder, by dripping 75.0 mL of ethanol. After centrifugation, 4.0 mL
of supernatant is transferred into a 10.0 mL volumetric ask, and
ethanol is used to make up to volume. The nal concentration
of the internal standard in the injected solution corresponds to
10 g mL1 .
2.1.6.2. Extraction protocol used for the cross-validation of the DEHP
extraction using the European Pharmacopoeia monograph 3.1.14. The
extraction method used is adapted from the European Pharmacopoeia monograph 3.1.14 [1]. From the sample, 1.0 g is cut into
pieces of about 0.5 cm length and the internal standard solution
is added to the sample. The preparation is heated for 8 h with
100.0 mL of diethyl ether free of peroxide using a reux condenser.
The residual tubing is separated from the ether. The extraction
solvent (ether) is evaporated to dryness using a 50 C water bath.
The residue is dissolved in 50.0 mL of ethanol R. Depending on the
phthalate amount (DEHP) additional dilutions in ethanol may be
performed. The nal concentration of the internal standard in the
injected solution corresponds to 10 g mL1 .
2.2. ISO 12787 standard approach
Validation criteria of standard solutions were determined
using the ISO 12787 standard approach [56]. This standard denes

validation criteria to which analytical results obtained from the

analysis of cosmetic products should comply in order to give condence in performance, reliability and quality of the nal result. It
proposes an analytical approach for chromatographic analyses in
order to obtain an assay result and different validation elements
which can be determined for each sample (or sample groups) submitted to the analysis. In this manuscript for medical devices, only
the rst part of this ISO standard was used for the determination
of some validation criteria of standard solutions. Indeed, this ISO
standard is divided into three different parts. The rst part consists
of determining, using standard solutions, the main characteristics
of the analytical method used before performing tests on samples
(i.e. system conformity, limit of detection (LoD), limit of quantication (LoQ), linearity range, etc.). The second and third parts of
this standard, called screening step and assay, are specically
adapted to cosmetic matrices (using spiked preparations).

3. Results and discussion

3.1. Method development
An analytical method for the quantication of phthalates in cosmetic products was developed and published [15]. In order to adapt
this method to the quantication of phthalates in PVC medical
devices, the extraction step was completely changed and additional phthalates such as DiDP or DiNP proposed to be forbidden in
France in medical devices [28,29] were added. In order to identify
and quantify other plasticizers, which are commonly used in PVC
medical devices such as DEHP substitute, 5 non-phthalate plasticizers, most commonly found such as DINCH, ATBC, DEHT, DEHA and
TOTM, were added to the method [53]. After slight modications of
the GC oven programme used for cosmetics [15], see Table 3, results
pointed out a good separation of most added plasticizers (ATBC,
TOTM, DEHA) towards other analytes and for partially co-eluted
compounds the possibility to be specic on the detection of each
analyte, using specic m/z ions (Fig. 1). As DiNP, DiDP and DINCH
consist of different isomers, a low resolution between chromatographic peaks obtained has already been observed in the literatures
[30,38]. As already reported elsewhere the distribution of isomers
in DINCH remains constant and is very different from that of other
highly isomeric phthalates such as DINP, which can be obtained
with various degrees of branching and different CAS numbers from
several manufacturers [57].

3.2. Validation data obtained previously on standard solutions

for 11 phthalates [15]
As reported in Table 3, modications on the GC oven temperature appear only after the elution of DEHP (Tr = 18.5 min). For all
compounds eluted before DEHP, including DEHP, the chromatographic conditions used for the quantication of phthalates in
medical devices are identical to those developed for the determination of the same compounds in cosmetics (same chromatographic
column, same injection conditions, same solvent, same ISTD, etc.).
This similarity allows the use of validation data obtained previously
on standard solutions using ISO 12787 for all phthalates eluted
before modications (i.e. 11 phthalates: all phthalates considered
for cosmetics except DnOP). Results highlight a good resolution
between compounds (Rs > 1.5); quantication limits on standard
solutions LoQs 0.25 g mL1 (0.25 ng injected) which complies
with most assays purpose; acceptable linearity from 0.5 g mL1 to
5.0 g mL1 for all compounds considered; precision and accuracy
in agreement with in-house validation criteria [15].

P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

103

Table 3
GC/MS oven programme.
Oven programme
Ramp

Temperature (100 C)

Time

Total time (0 min)

Method used for cosmetics [15]

30 C min1
3 C min1
3 C min1
30 C min1

200 C
250 C
260 C
320 C

0 min
0 min
0 min
5 min

3.3 min
20.0 min
23.3 min
25.3 min

Method developed for medical devices

30 C min1
3 C min1
40 C min1
80 C min1

200 C
250 C
270 C
320 C

0 min
2.5 min
2.0 min
5.0 min

3.3 min
22.5 min
25.0 min
30.6 min

Table 4
Complementary data on specicity for compounds not mentioned in Ref. [15].
Compounds

RT (min)

Ion 1 (m/z)

Ion 2 (m/z)

Ion 3 (m/z)

Ion ratio 2/1 (%)

Ion ratio 3/1 (%)

DnOP
ATBC
DEHA
DEHT
TOTM
DINCH
DiNP
DiDP

22.7
12.3
15.1
22.9
29.3
22.9a
24.0b
25.4c

279
185
129
261
305
155
293
307

149
129
57
149
193
127
149
149

167
259
112
167
323
281
127
167

1850
40
50
130
30
30
1000
700

30
45
25
85
15
13
120
70

a
b
c

From 20.0 min to 25.0 min.

From 22.9 min to 25.5 min.
From 24.0 min to 27.0 min.

3.3. Complementary validation data obtained on standard

solutions for DnOP, DiNP, DiDP and non-phthalate plasticizers
(ATBC, DEHA, DEHT, TOTM, DINCH) using ISO 12787 (1st part)
In agreement with the ISO 12787 standard, assays using
standard solutions were performed. For DnOP, DiNP, DiDP and nonphthalate plasticizers, the detector calibration has been achieved
via the performance of a mass spectrometer auto-tune. The apparatus conformity was checked by injecting 6 times a standard
solution containing a mix of the 14 phthalates and the 5 nonphthalate plasticizers at a concentration of 50.0 g mL1 for DiNP,
DiDP and DiNCH and 5.0 g mL1 for all other compounds. The RSD
obtained for all compounds considered was 3.5% using the internal standard, in agreement with in-house validation criteria. Due
to a lack of references in the eld of medical devices, most inhouse criteria were established from different documents in the
eld of cosmetic and agro chemistry [5865].

3.3.1. Analytical conformity/specicity

The specicity of the detection is ensured by monitoring three
different ions for each compound using a mass spectrometer in SIM
mode (Table 4). The maximum permitted tolerances for relative ion
intensities should be in agreement with the 2002/657 Commission
Decision [63]. Nevertheless, a low specicity of the MS detection
(m/z = 149) was noticed (general issue for benzene derivatives).
System compliance is checked by injecting a standard solution corresponding to the higher calibration level. To ensure the absence
of interfering peaks or of a carryover, the dilution solvent (ethanol)
and each analyte separately are also injected at the highest concentration level. Results obtained do not point out any interference.
For co-eluted compounds (DEHT, DnOP, DINCH, DiDP and DiNP),
the specicity of the analysis is ensured by the quantier ion used
(specic to each analyte). For information, a chromatographic prole obtained in SIM mode at the middle range calibration level
(20.0 g mL1 for DiNP, DiDP and DINCH and 2.0 g mL1 for other
compounds) is given in Figs. 1 and 2.

3.3.2. Detection and quantication limits on the quantier ion

Different approaches exist for the determination of detection
limits. For this study, LoDs were estimated from calibration curves
obtained during the linearity assay [66,67]. Alternatively, these data
could be obtained experimentally using standard solutions; S/N
ratios 3 and percent CV cut-off of roughly 20% are commonly used
as specications. Using calibration curves, each LoD is calculated
from the y intercept (a) and the standard deviation of the y intercepts (SDa) according to the formula: LoD = a + 3 SDa. According to
this mathematical approximation, for DINCH, DiDP and DiNP, LODs
range from 0.07 g mL1 to 0.25 g mL1 (0.25 ng injected) and for
other analytes from 0.01 g mL1 to 0.06 g mL1 . In agreement
with our previous study [15], a common LoD on standard solution was estimated for DINCH, DiNP and DiDP at 0.3 g mL1 and
for all other compounds at 0.1 g mL1 . These limits correspond in
PVC samples, according to sample preparation, to detection limits roughly of 75 g g1 (0.008%) for DINCH, DiNP and DiDP and
25 g g1 for other analytes.
LoQs were evaluated using standard solutions. S/N ratios
obtained for low calibration levels are presented in Table 5. For
DINCH, DiDP and DiNP LoQs range from 0.1 g mL1 to 2.0 g mL1
(corresponding to an S/N ratio 10) and for all other compounds
from 0.01 g mL1 to 0.1 g mL1 . In agreement with our previous study [15], a common LoQ on standard solution was set for
DINCH, DiNP, and DiDP at 2.5 g mL1 and for all other analytes
at 0.25 g mL1 . These limits correspond to PVC samples to quantication limits roughly of 650 g g1 for DINCH, DiNP, DiDP and
65 g g1 for other analytes.
For assays, report limits were xed at 1000 g g1 in PVC samples for DINCH, DiNP, DiDP and 100 g g1 in PVC samples for other
analytes.

3.3.3. Calibration linearity standard accuracy

Calibration linearity is checked on standard solutions using
statistical software (AVA Aide la Validation Analytique version
3.1. distributed by Qualilab (45160 Olivet France) or Statgraphic

104

P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

Fig. 1. Example of chromatogram obtained on standard solutions (TIC).

Fig. 2. Co-eluted compounds separated using their specic m/z ions (SIM).

Table 5
Complementary data on S/N ratio obtained on the quantier ion for compounds not mentioned in Ref. [15].
S/N ratio
Compound

Quantier ion (m/z)

Solvent

0.025 g mL1

0.050 g mL1

0.10 g mL1

0.25 g mL1

0.50 g mL1

DnOP
ATBC
DEHA
DEHT
TOTM

279
185
129
261
305

0
0
0
0
0

0
21
24
28
18

8
42
32
56
35

20
98
78
115
74

58
284
157
423
268

114
584
372
810
529

Compound

Quantier ion (m/z)

Solvent

0.25 g mL1

0.50 g mL1

1.0 g mL1

2.5 g mL1

5.0 g mL1

DINCH
DiNP
DiDP

155
293
307

0
0
0

20
0
0

50
0
0

119
16
8

282
90
20

546
170
43

S/N ratio is obtained using the instrument software on peak-to-peak ratio mode.

P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

105

Table 6
Complementary data on calibration linearity for compounds not mentioned in Ref. [15].
Validation paramaters

DnOP

ATBC

DEHA

DEHT

TOTM

DINCH

DiNP

DiDP

Determination coefcient R2
Slope
Y-intercept
Variance homogeneity (COCHRAN)
Aberrant values (DIXON)
Y-intercept comparison with 0 (STUDENT)
Signicant slope existence (FISHER)
Validity of calibration curve (FISHER)

0.9962
0.0091
0.0013
NS
NO
S
HS
NS

0.9951
0.1299
0.0415
NS
NO
S
HS
NS

0.9965
0.2216
0.0351
NS
NO
S
HS
NS

0.9959
0.0237
0.0060
NS
NO
S
HS
NS

0.9928
0.1416
0.0357
NS
NO
S
HS
NS

0.9951
0.0554
0.1743
NS
NO
S
HS
NS

0.9818
0.0096
0.045
NS
NO
S
HS
NS

0.9850
0.0116
0.0646
NS
NO
S
HS
NS

NS, non signicant; S, signicant; HS, highly signicant.

Table 7
Complementary data on mean relative bias (%) for compounds not mentioned in Ref. [15].
Calibration levels
Compound

0.5 g mL1

1.0 g mL1

1.5 g mL1

2.0 g mL1

3.0 g mL1

4.0 g mL1

5.0 g mL1

DnOP
ATBC
DEHA
DEHT
TOTM

4.1%
24.6%
5.4%
18.7%
7.6%

5.8%
1.5%
1.3%
4.4%
1.7%

4.5%
0.6%
2.0%
2.9%
1.8%

3.4%
4.5%
1.4%
6.1%
1.4%

1.8%
3.7%
0.3%
0.8%
0.5%

0.2%
0.9%
1.0%
0.8%
2.7%

0.1%
2.4%
0.2%
1.7%
1.1%

Compound

5.0 g mL1

10.0 g mL1

15.0 g mL1

20.0 g mL1

30.0 g mL1

40.0 g mL1

50.0 g mL1

DINCH
DiNP
DiDP

20.9%
40.6%
39.7%

2.3%
4.4%
3.9%

3.2%
6.8%
6.2%

3.8%
6.9%
7.9%

3.8%
5.9%
3.6%

1.5%
0.9%
0.8%

1.0%
2.7%
3.1%

In-house validation criteria. First calibration level (close to LoQ) < 50%/other calibration levels < 10%.

Centurion version 16.1.05 (32 bits) distributed by StatPoint

Technologies, Inc. (USA)) (Table 6). Results obtained point out
a nonsignicant variance homogeneity (Cochrans test) for
non-phthalate plasticizers, DiDP, DiNP and DnOP. This variance
homogeneity (NS) allows a valid statistical process for linearity criteria. Determination coefcients (R2 ) obtained for each plasticizer
(>0.990 except for DiNP and DiDP > 0.980), Fishers tests, performed
to verify the good correlation between experimental values and the
regression model used (NS), as well as mean relative bias (absolute
bias/calibration level 100) in agreement with in-house specications (Table 7) point out an acceptable linear correlation for all
phthalates on the calibration range considered (0.55.0 g mL1
or 5.050.0 g mL1 ). As DiNP and DiDP are eluted as broad mass
peaks and as their limits of quantication are signicantly higher
than that of other compounds considered, determination coefcient (R2 ) > 0.9800 and mean relative bias (%) < 50% for the rst
calibration level (close to LoQ) were considered as acceptable. For
this study, the 0 value has not been forced through the origin and
the comparison of y-intercept with zero (student test) is signicant
(S). As for other phthalates considered previously [15], the linearity
may probably not be acceptable on a wider concentration range.

Table 8
Mean recoveries and RSD (n = 3) determined on spiked preparation.

3.4. Validation data obtained on real samples for all compounds

considered (14 phthalates + 5 non-phthalate plasticizers)

corresponds to the report limit value depending on compounds

considered. Tests were performed on 2 different PVC extension
lines using TOTM or ATBC as plasticizer. Recoveries (Table 8) were
calculated relative to the amount of plasticizer added before the
precipitation of the PVC. Mean recoveries determined on spiked
preparations range between 80% and 120% (RSD < 20.0%) for all
compounds considered except for DPP (78%) and DiDP (127%). DPP
and DiDP were not detected in PVC tubing during market survey (either as plasticizer or as contaminant). Recoveries between
80% and 120% were considered as acceptable regarding sample
preparation and trace level analysis. In accordance with internal
specications used [5864], these results indicate that there is no
signicant loss of analyte during the precipitation step of PVC.
These results might be extended to all PVC extension lines.

3.4.1. Extraction recoveries obtained for phthalates and

non-phthalate plasticizers
As PVC is completely soluble in THF, the extraction of all components, including phthalates, from the PVC part of the medical device
can be considered as total. In order to check the noninuence of the
PVC precipitation step (addition of EtOH) on the amount of analytes
initially extracted from the PVC, tests using spiked preparation have
been performed. Two preparations without the addition of analytes and three preparations with the addition of 100 ppm of each
compound (1000 ppm for DINCH, DiDP and DiNP) were performed
on a noncontaminated PVC extension line. 100 ppm or 1000 ppm

Compounds
DMP
DEP
DiBP
DBP
DMEP
DiPP
DPP
DnPP
BBP
DCHP
DEHP
DnOP
DiNP
DiDP
ATBC
DEHA
DEHT
TOTM
DINCH

Mean recovery
115%
115%
92%
90%
119%
98%
78%
92%
111%
98%
91%
91%
107%
127%
100%
116%
98%
120%
105%

RSD (n = 3)
4.2%
10.1%
2.3%
17.9%
13.8%
15.2%
9.7%
5.0%
16.9%
14.1%
2.1%
2.2%
8.4%
4.4%
3.5%
1.8%
6.9%
19.3%
3.7%

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P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

Table 9
Comparative results obtained for DEHP using Ph. Eur [1] and THF/EtOH.
Sample

DEHP result
Plasticizera

Expected amount

Extract. Ph. Eur. [1]

Extract. THF/EtOH

Sample 1A

DEHP

DEHP > 10 000 ppm

Sample 1B

DEHP

DEHP > 10 000 ppm

Sample 1C

DEHP

DEHP > 10 000 ppm

24.1%
RSD = 3.4%, n = 3
30.0%
RSD = 3.5%, n = 3
24.6%
RSD = 5.0%, n = 3

24.5%
RSD = 3.9%, n = 3
29.0%
RSD = 3.3%, n = 3
27.4%
RSD = 2.0%, n = 3

Sample 2A

DINCHb

100 < DEHP < 1000

Sample 2B

DINCHb

1000 < DEHP < 10 000

Sample 3A

TOTM

100 < DEHP < 1000

Sample 3B

TOTM

100 < DEHP < 1000

Sample 3C

TOTM

DEHP > 10 000 ppm

Sample 4A

ATBC

1000 < DEHP < 10 000

Sample 4B

ATBCb

1000 < DEHP < 10 000

Sample 5A

DiNPb

100 < DEHP < 1000

Sample 6A

DEHT

100 < DEHP < 1000

185 ppm
RSD = 11.0%, n = 3
1790 ppm
RSD = 10.6%, n = 3
220 ppm
RSD = 14.4%, n = 3
485 ppm
RSD = 11.5%, n = 2
(1970; 445; 525)
2.3%
RSD = 5.4%, n = 3
1445 ppm
RSD = 3.4%, n = 3
1720 ppm
RSD = 25.4%, n = 3
(2190; 1330; 1630)
350 ppm
RSD = 8.4%, n = 3
405 ppm
RSD = 6.8%, n = 2
(1060; 425; 385)

210 ppm
RSD = 12.1%, n = 3
2010 ppm
RSD = 1.7%, n = 3
245 ppm
RSD = 3.3%, n = 3
430 ppm
RSD = 19.3%, n = 2
(1760; 370; 490)
2.4%
RSD = 4.0%, n = 3
1390 ppm
RSD = 12.6%, n = 3
1800 ppm
RSD = 30.2%, n = 3
(2330; 1250; 1815)
380 ppm
RSD = 3.6%, n = 3
410 ppm
RSD = 5.9%, n = 2
(1270; 425; 390)

Difference %
+2%
3%
+10%
+14%
+12%
+11%
11%

+4%
4%
+5%

+9%
+1%

No sample using DEHA as plasticizer was found contaminated with a DEHP amount >100 ppm.
For samples using DINCH, ATBC and DINP the DEHP contamination may come from contaminations during the production of either plasticizer or PVC lines. Indeed, some
medical devices, containing several parts in PVC material, use different plasticizers (for stability reason blood bags often used DEHP as plasticizer).
b

3.4.2. Cross-validation of the DEHP extraction step using the

European Pharmacopoeia extraction method.
Twelve PVC medical devices extension lines using different
plasticizers and containing different amounts of DEHP were
assayed using the extraction method described in the European
Pharmacopoeia monograph 3.1.14 [1]. The residue after evaporation was dissolved in ethanol, instead of toluene, and the analysis
was performed by GC/MS, instead of TLC. Results obtained for
DEHP were compared to results obtained using THF/EtOH as
extraction process. In order to assess the absence of carryover
or cross-contamination for the 14 analytes considered, 3 control
samples assayed with <100 ppm of phthalates using the European Pharmacopoeia monograph were also analysed using the
THF/EtOH extraction process in addition to the 12 PVC medical
devices containing different amounts of DEHP. Results obtained for
these control samples conrmed the absence of the 14 phthalates
analysed at levels higher than the limit report (100 ppm/1000 ppm)
regardless of the method used. For the 12 PVC medical devices
containing different amounts of DEHP (Table 9), results point out a
good correlation between DEHP amount obtained using the European Pharmacopoeia and the THF/EtOH method regardless of the
plasticizer or the DEHP amount present in the sample. For the 3 rst
samples using DEHP as plasticizer (1A, 1B and 1C), results obtained
for the determination of DEHP with THF/EtOH are comparable to
results obtained using the European Pharmacopoeia monograph
extraction mode (difference 10%, RSD 5%, n = 3). For samples
containing DEHP as contaminant, at concentration levels ranging
from 100 to 10 000 g g1 , results point out as well a good correlation of DEHP amount using both methods (difference 15%) with
an acceptable repeatability of the assay for most samples studied
(RSD 15%, n = 3). Differences were observed for some samples (3B,
4B and 6A) between replicates due to a discrepancy in the DEHP
contamination between the different PVC extension lines analysed
for the same sample. This discrepancy in the DEHP contamination

of samples was conrmed with both analytical methods used

(THF/EtOH and the European Pharmacopoeia) and during the assay
of the 61 medical devices. Results obtained, using either THF/EtOH
or the European Pharmacopoeia for the extraction of DEHP from
PVC tubing, are comparable for the 12 different medical devices.
Results of this cross-validation appear satisfactory. The extraction
method THF/EtOH proposed in this manuscript appears suitable
for the determination of at least DEHP in PVC tubing.
3.4.3. Results obtained for the analyses of more than 60 PVC
medical devices
A total of 61 medical devices corresponding to 107 sample parts
(between 1 and 7 parts per sample) were used to assess the analytical method proposed. Results obtained point out a predominant
use of TOTM as plasticizer in more than half of the sample parts
analysed, followed by DINCH (16%), DEHA (11%). Other plasticizers were respectively used in less than 10% of the sample assayed
(Fig. 3). The chart given in Fig. 4 shows the samples distribution
according to DEHP or DEHT contamination. When several parts of
a sample were analysed, the sample result reported in the graph
Distribution of sample parts according to the plasticizer identified

4%
4%
7%

4%
TOTM
DiNCH

11%

DEHA
ATBC

54%
16%

DEHT
DiNP
More than 2 platicizers

Fig. 3. Distribution of PVC medical devices sample parts (%) according to the plasticizer identied.

P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

107

Distribution of samples (% ) according to DEHT / DEHP

contamination

31

70

63

55

60

30

50
40
30

< 100 ppm

100 -1000 ppm

> 1000 ppm

%

100 -1000 ppm

< 100 ppm

20
10

14

0
> 1000 ppm

DEHT
DEHP

Fig. 4. Distribution of PVC medical devices sample parts (%) according to the DEHT/DEHP content.

corresponds to the highest part content. For about 14% of samples

tested, the DEHP contaminant measured > 1000 ppm (0.1%). The
limit value of 0.1% corresponds to the limit of acceptable contamination according to the REACH regulation. Indeed, as DEHP is listed
as a substance of very high concern (SVHC) in REACH, its amount
in the nished product is limited to 0.1% (REACH Annex XVII, 51)
[68,69]. Depending on the plasticizer used, the DEHP or DEHT contamination may differ. Result obtained point out a predominant
presence of DEHT as contaminant (>0.01%) when TOTM is used as
plasticizer. Indeed, more than 90% of the samples contaminated
by DEHT (DEHT > 0.01%) used TOTM as plasticizer. This contamination level ranges from 1000 g g1 to 3000 g g1 when TOTM
is used as plasticizer and is near or less than 100 ppm when other
plasticizers are used. For DEHP, more than 65% of the samples contaminated (DEHP > 0.01%) also used TOTM as plasticizer. No other
compounds analysed were detected as contaminant at levels higher
than 1000 g g1 for DiNP, DiDP and DINCH or 100 g g1 for other
target compounds. According to the literatures [70,71], possible
contamination of TOTM by DEHP may result in DEHP levels higher
than 0.1% in the medical device. Indeed, the presence of DEHP as
impurity in TOTM is well known and originates from the presence
of ortho-phthalic acid as impurity in the trimellitic acid used for the
synthesis of TOTM. Similarly, the presence of para-phthalic acid in
the trimellitic acid used may explain the high level of DEHT contamination found in medical devices when TOTM is used as plasticizer.
The large level of contamination observed for DEHP/DEHT could
be explained by the use of raw materials (plasticizers) of low
purity.
3.4.4. Results obtained for the analyses of plasticizers
In order to investigate the origin of the DEHT/DEHP contaminations, different plasticizers coming from different sources were
analysed. Results obtained (Table 10) clearly highlight the presence
of DEHP and DEHT as impurities in some plasticizers. The amounts
of impurities depend on the nature and the purity of the plasticizer
used. According to results obtained, TOTM seems to be the plasticizer containing the highest amount of DEHP and DEHT impurities,
whereas for some other plasticizers (ATBC, DiNP or DiNCH), neither
DEHT nor DEHP was detected. These results are in agreement with
results obtained in PVC medical devices tested. The origin of DEHP
and DEHT contamination seems to be directly linked to the nature
of the plasticizer used [70,71].

Table 10
Plasticizers analysed.
Plasticizer

DEHP (ppm)

DEHT (ppm)

ATBC-1
DiNP-1
DiNP-2
DEHT-1
DiNCH-1
DiNCH-2
DEHA-1
DEHA-2
TOTM-1
TOTM-2
DiDP-1
DiDP-2

<5
<5
<5
85
<5
<5
160
270
920
720
50
40

<5
<5
<5
NA
<5
<5
15
70
2050 (0.2%)
3900 (0.4%)
<5
<5

NA, not applicable.

4. Conclusion
In 2013 different papers deal with the quantication of phthalates in plastic material using PVC as plastic but the only ofcial
method is the European pharmacopoeia (monograph 3.1.14). This
ofcial method, focused on the determination of DEHP, is based
on an extraction of this phthalate with diethyl ether using a reux
condenser.
In order to have a more modern method able to determine
more phthalates, a performant analytical method has been developed for the assay of 14 phthalates: 8 phthalates classied H360, 3
phthalates proposed to be forbidden in medical devices and 3 other
phthalates nonregulated which may interfere with hormone function. In order to identify and quantify other plasticizers commonly
used in PVC medical devices such as DEHP substitute, 5 additional
non-phthalate plasticizers were included in this study. This method
being far more exhaustive (14 phthalates + 5 non-phthalate plasticizers vs one phthalate), faster (1 h approximately vs 8 h), safer (use
of THF instead of diethyl ether), more specic and sensitive (use of
GCMS instead of TLC) than the ofcial method for DEHP points
out an acceptable separation of most compounds considered and
the possibility to be specic on the detection of each analyte, using
specic m/z ions, for compounds not enough separated. Validation
data obtained for all compounds considered highlight report limits (1000 g g1 for DINCH, DiNP, DiDP and 100 g g1 for all other
analytes) in PVC samples which comply with the limit of acceptable

108

P. Gimeno et al. / J. Chromatogr. B 949950 (2014) 99108

contamination level according to the Reach regulation (less than

1000 ppm); the absence of signicant loss of phthalates during the
precipitation step of PVC and for DEHP the suitability of the extraction process proposed (THF/EtOH) according to results obtained
during the cross-validation. The analytical method proposed was
tested on more than 60 PVC medical devices. Results point out a
predominant use of TOTM as plasticizer. When the plasticizer used
is TOTM, the content of DEHT obtained as contaminant in the PVC
medical devices ranges from 1000 ppm to 3000 ppm, whereas its
amount is generally <100 ppm when another plasticizer is used. For
DEHP, more than 65% of the samples contaminated (DEHP > 0.01%)
also used TOTM as plasticizer. Analysis performed on 12 plasticizers (raw materials) highlights that TOTM seems to be the plasticizer
containing the highest amount of DEHP and DEHT.
In order to update actual ofcial methods given for the assay of
DEHP in plastic material using PVC as plastic (monographs 3.1.1.1,
3.1.1.2 and 3.1.14) [1,72,73], this method has been presented and
proposed to the European Pharmacopoeia (working group 16).
Indeed, up to now only DEHP is listed in this Pharmacopoeia and
methods proposed are based on extraction of this phthalate with
diethyl ether using a reux condenser. The working group 16 is
currently working on the revision of those monographs in order to
update the analytical methods and to list additional plasticizers to
replace DEHP and/or to provide alternatives [74].
Acknowledgements
The authors gratefully acknowledge Nelly LASSU and Nathalie
LAYOUN who contributed to the present study.
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