Synthesis of DEHP metabolites as biomarkers for GC-MS evaluation of phthalates as endocrine disrupters

Article abstract of DOI:10.1016/j.bmc.2005.03.005

Phthalates are used primarily as plasticizers to make polyvinyl chloride (PVC) soft and flexible. In recent years the phthalate esters have attracted increasing attention as environmental and biomedical pollutants and, because of their toxicological characteristics. In particular, they are more and more recognized as endocrine disrupters. In this context, we describe herein an efficient synthetic pathway leading to a series of metabolites of di(2-ethylhexyl) phthalate (DEHP). Mono(2-ethylhexenyl) phthalate was used as starting material to obtain these products in good yield, large scale and GC-MS purity. The metabolites of DEHP were synthesized, for the first time, as biomarkers to verify their quantitative determination in human urine and serum by GC-MS analysis for studying the exposure to phthalates and establishing reference values.

Full text of DOI:10.1016/j.bmc.2005.03.005

Bioorganic & Medicinal Chemistry 13 (2005) 3461–3465
Synthesis of DEHP metabolites as biomarkers for
GC–MS evaluation of phthalates as endocrine disrupters
Francesca Nuti,a Sibylle Hildenbrand,b,* Mario Chelli,a
Roman Wodarzb and Anna Maria Papinia,*
aLaboratory of Peptide & Protein Chemistry & Biology, Department of Organic Chemistry, University of Florence and
CNR-ICCOM, Polo Scientifico, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Firenze, Italy
bDepartment of Occupational and Social Medicine, University of Tu¨bingen, Wilhelmstraße 27, D-72074 Tu¨bingen, Germany
Received 25 October 2004; revised 23 February 2005; accepted 1 March 2005
Available online 28 March 2005
Abstract—Phthalates are used primarily as plasticizers to make polyvinyl chloride (PVC) soft and flexible. In recent years the phtha-
late esters have attracted increasing attention as environmental and biomedical pollutants and, because of their toxicological char-
acteristics. In particular, they are more and more recognized as endocrine disrupters. In this context, we describe herein an efficient
synthetic pathway leading to a series of metabolites of di(2-ethylhexyl) phthalate (DEHP). Mono(2-ethylhexenyl) phthalate was
used as starting material to obtain these products in good yield, large scale and GC–MS purity. The metabolites of DEHP were
synthesized, for the first time, as biomarkers to verify their quantitative determination in human urine and serum by GC–MS anal-
ysis for studying the exposure to phthalates and establishing reference values.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
inhalation, ingestion, dermal and after medical proce-
dures. Its widespread use leads DEHP to be considered
as an ubiquitous ecological contaminant.1,2 Phthalates
are known as endocrine disrupters, an exogenous sub-
stance or mixture that alters function(s) of the endocrine
system, and consequently causes adverse health effects in
an intact organism or its progeny or (sub)populations.
Therefore, in recent years phthalate esters have attracted
increasing interest as environmental and biomedical pol-
lutants, and because of their toxicological characteristics.
It was previously demonstrated that phthalates have per-
oxisomal proliferative activity. They have teratogenic
and feminizing effects, at low levels, in rat and fish.3–5
They are rapidly metabolized to their respective monoes-
ters and further oxidative products, which are partially
glucuronidated and excreted through urine and feces.6–9
To our knowledge, the extent of exposure of humans to
phthalate esters has not yet been sufficiently investigated.
Few studies have focused on the exposure to DEHP
and to its metabolites8–12 of workers, of hemodialysis
patients,13–15 or of newborn infants,16 because of exchange
transfusions.17,18 Latini et al.19 confirmed a significant
and widespread presence of the phthalate DEHP and of
its metabolite MEHP, in the blood of newborn infants
in an Italian hospital. The DEHP is metabolized to
mono(2-ethylhexyl) 1,2-benzenedicarboxylate [mono(2-
ethylhexyl) phthalate, MEHP] and this compound can
Bis(2-ethylhexyl) 1,2-benzenedicarboxylate [di(2-ethylhexyl)
phthalate, DEHP], commonly called phthalate, is
used primarily as a plasticizer to make polyvinyl chloride
(PVC) soft and flexible. Blood bags, childrenÕs toys, vinyl
floor, wall covering and car underbody coating are prod-
ucts made with plasticized PVC. Non-polymeric uses of
phthalates as fixatives, detergents, lubricating oils and
solvents let find them in different products such as cosmet-
ics and wood finishes. Phthalates are also found in foods,
which are processed or packaged with plastic materials
and in dialysis and intravenous drip fluids as contami-
nants from the plastic tubing. The amount of the phtha-
late esters varies between 10% and 60% depending on
the product. Since phthalate esters are not chemically
bound to the polymer, they are released into the environ-
ment and persist for a long time both in water and in air
because of their stability. For that reason human popula-
tion is subjected to absorption of phthalates through
Keywords: DEHP metabolites; Phthalates; GC–MS analytes; Endo-
crine disrupters; Plasticizers.
*
Corresponding authors. Tel.: +39 055 4573561; fax: +39 055 4573584
(A.M.P.), fax: +49 7071 29 4362 (S.H.); e-mail addresses: sibylle.
hildenbrand@med.uni-tuebingen.de; annamaria.papini@unifi.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.005

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F. Nuti et al. / Bioorg. Med. Chem. 13 (2005) 3461–3465
be transformed, by x-1 oxidation reactions of the mono-
ester alkyl chain, also in mono(2-ethylhexenyl) 1,2-benz-
enedicarboxylate [mono(2-ethylhexenyl) phthalate, 2],
mono(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate
[mono(2-ethyl-5-hydroxyhexyl) phthalate, 5-OH-MEHP,
6],20 mono(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate
[mono(2-ethyl-5-oxohexyl) phthalate, 5-oxo-MEHP, 5],20
and mono(2-ethyl-5-carboxypentyl) 1,2-benzenedicarb-
oxylate [(mono(2-ethyl-5-carboxypentyl phthalate, 5-carb-
oxy-MEPP, 4].21 Biological monitoring of DEHP and
their metabolites, not only in urine but also in serum,
can be instrumental to investigate the environmental
exposure of members of the general population to phth-
alate esters.22 For that reason it is evident that the interest
in establishing reference tests by using synthetic biomark-
ers for quantitative determinations of such metabolites in
a pool of selected humans.
The 2-ethylhex-5-en-1-ol (1) was synthesized as de-
scribed in the literature.23 The crude monoester 2 was
used as starting material to achieve four oxidative
metabolites of MEHP (Scheme 1). The hydroboration–
oxidation process gave the mono(2-ethyl-6-hydroxy-
hexyl) 1,2-benzenedicarboxylate [mono(2-ethyl-6-hydroxy-
hexyl) phthalate, 6-OH-MEHP, 3] by an anti-
Markovnikov mechanism. Oxidation of the primary
alcohol 3 was undertaken by addition of an acidic aque-
ous solution containing chromic acid (JonesÕ reagent)24–26
to an acetone solution of the alcohol 3. Oxidation
occurred rapidly and lead to compound 4. The crude
acid was purified by several extractions in different
solvents. Moreover oxidation of the double bond in
compound 2, undertaken with catalytic amount of
PdCl2 in the presence of p-benzoquinone following the
Wacker reaction,27 lead to the derivative 5. The quinone
was necessary to reoxidize Pd(0) to Pd(II). The net
reaction employs alkene, water and p-benzoquinone.
Therefore, the catalyst could be recovered at the end
of the reaction. The quinone was purified by sublima-
tion before use. Compound 6 was obtained by an
efficient reduction of the oxo group of 5 with NaBH4
in an alcoholic solution. The excess of hydride was
destroyed with H2O. All the expected products were
obtained in good yield, with chemical purity >95%
Therefore, we decided to develop an efficient synthetic
pathway leading to a series of secondary metabolites
of DEHP to be used in GS–MS analysis for their biolog-
ical monitoring in human urine and serum.
2. Results and discussion
1
2.1. Synthesis of DEHP metabolites
and were characterized by H, 13C NMR and ESI-MS.
Before their use as analytes in GC–MS, all the metabo-
lites were transformed in the corresponding methyl
esters by treatment with diazomethane, prepared from
Phthalate 2 was obtained starting by a classical esterifi-
cation reaction of phthalic anhydride with alcohol 1.
Scheme 1. Reagents and conditions: (a) phthalic anhydride, pyridine (Py), under N2; (b) CH2N2, MeOH/ether (9:1); (c) 1 M B2H6 in THF, then
H2O2, OHÀ in THF; (d) CrO3, H2SO4 in acetone; (e) PdCl2, quinone in DMF/H2O (7:1); (f) NaBH4 in EtOH.

F. Nuti et al. / Bioorg. Med. Chem. 13 (2005) 3461–3465
3463
ethylhexenyl) 1,2-benzenedicarboxylate (2), methyl (2-
ethyl-5-hexenyl) 1,2-benzenedicarboxylate (2a), mono(2-
ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate (6-OH–
MEHP, 3), mono(2-ethyl-5-carboxypentyl phthalate (5-
carboxy-MEPP, 4), mono(2-ethyl-5-oxohexyl) 1,2-benz-
enedicarboxylate (5-oxo-MEHP, 5) methyl (2-ethyl-5-
oxohexyl) 1,2-benzenedicarboxylate (5a), mono(2-ethyl-
5-hydroxyhexyl) 1,2-benzenedicarboxylate (5-OH-MEHP,
6) in gram scale. Moreover, we demonstrated, for the
first time, that the oxidative derivatives of MEHP
(DEHP metabolites) can be used as analytes for their
determination in GC–MS in urine and serum samples.
As an example, these standard biomarkers can be pro-
posed for investigating if exposure to DEHP can have
toxicological risks for reproduction in humans.
Figure 1. The concentrations of 5-OH-MEHP (6), 5-oxo-MEHP (5)
and 5-carboxy-MEPP (4) in urine samples of 50 persons of the general
population in lg/L. The 90th percentiles of these analytes, calculated
assuming the underlying model to be a log-normal distribution, is
shown.
4. Experimental
4.1. General chemistry methods
N-methyl-N-nitroso-p-toluenesulfonamide,28 and in the
corresponding silyl ethers by N,O-bis(trimethylsilyl)tri-
fluoroacetamide (BSTFA).
1H and 13C NMR spectra were recorded in CDCl3 with
a Varian spectrometer at a frequency of 200 MHz and
50 MHz, respectively. Chemical shifts are reported in
parts per million relative to TMS and coupling con-
stants (J) in hertz (s, d, t, m and br for singlet, doublet,
triplet, multiplet and broad, respectively). FT-IR spectra
were obtained on a Perkin Elmer Spectrum BX II appa-
ratus. For mass analysis a ThermoFinnigan LCQ
Advantage ESI-MS spectrometer was used. Flash col-
umn chromatography (FCC) was accomplished on
2.2. Biological monitoring of DEHP metabolites in human
urine and serum by GC–MS
The secondary metabolites 4, 5 and 6 were demonstrated
to be useful as standards for GC–MS in order to quan-
tify the phthalate metabolites in urine of humans and to
establish reference values.
˚
SiO2 (ICN; 32–63 lm, 60 A). Thin layer chromatogra-
Preliminary data on the quantitative analysis of DEHP
and their metabolites were performed on a pool of 50
persons of the general population of south-west Ger-
many, using synthetic metabolites as standard biomark-
ers for GC–MS analysis. The study showed that in every
analyzed person 5-OH-MEHP (6), 5-carboxy-MEPP (4)
and 5-oxo-MEHP (5) were present in urine. The 90th
percentile of these metabolites, calculated assuming the
underlying model to be a log-normal distribution, is re-
ported in Figure 1. In particular, it was shown that be-
neath phthalic acid, these metabolites had relatively
high urinary concentrations, but in all serum samples
they were below the detection limit (0.1 lg/L). This
means that general population accumulates phthalates
from the environment. In fact, phthalate diesters are
omnipresent and contaminations of the extracts should
be avoided. MEHP was also detected in river water29
and in solutions that were stored in medical grade
PVC bags,30 while the oxidative metabolites of MEHP
were not present in the environment. In the context of
a study in environmental medicine, we demonstrated
that the oxidative metabolites of MEHP, measured in
urine, are suitable biomarkers for monitoring the expo-
sure of the general population to phthalate esters.
phies were carried out on Merck silica gel 60 F254 plastic
sheets. Dry solvents were distilled immediately before
use.
4.1.1. 1-Hydroxy-2-ethylhex-5-ene (1). This compound
was synthesized as described in the literature.23
4.1.2. Mono(2-ethylhexenyl) 1,2-benzenedicarboxylate (2).
2-Ethyl-5-hexen-1-ol (1) (2.2 g, 17.2 mmol), phthalate
anhydride (2.54 g, 17.2 mmol) and pyridine (1.6 mL)
were refluxed under nitrogen for 3 h. The mixture was
quenched with cold water and extracted with Et2O.
The organic phase was washed twice with a solution of
10% HCl to remove pyridine. Finally, the mixture was
extracted with 0.4 M K2CO3. The aqueous basic layer
was acidified to pH 1 with 1 M HCl and then extracted
with Et2O. After drying with Na2SO4 and evaporation
of the solvent under vacuum, compound 2 was obtained
as a colourless oil (4.48 g, 94% yield) and used without
further purification in the next step. Rf (Et2O/hexane
1:1, UV) = 0.15. ESI-MS: calcd for [M+H]+ 277.3.
1
Found 277.9. H NMR d 0.90 (3H, t, J = 8 Hz, CH3),
1.24–2.14 [7H, m, CH2@ CH(CH2)2CHCH2CH3], 4.25
(2H, d, J = 6 Hz, COOCH2), 4.96 (2H, dd, J = 10,
J = 1.8 Hz, CH2@), 5.02 (1H, br, OH), 5.76 (1H, m,
CH@), 7.61 and 7.81 (4H, 2 · m, aromatic CH). 13C
3. Conclusions
NMR
d
10.9 (CH3), 23.6 (CH3CH2), 29.9
(CH2CH@CH2), 30.9 (CH2CH2CH@ CH2), 38.1
(CH3CH2CH), 68.0 (COOCH2), 114.5 (CH@CH2),
128.6–133.4 (aromatic C), 138.5 (CH@CH2), 168.2
(COOCH2), 172.3 (COOH).
The described synthetic pathway represents the first effi-
cient synthesis leading to a series of DEHP metabolites.
By this methodology, it is possible to obtain mono(2-

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F. Nuti et al. / Bioorg. Med. Chem. 13 (2005) 3461–3465
4.1.3. Methyl (2-ethyl-5-hexenyl) 1,2-benzenedicarboxy-
late (2a). A fresh diethyl ether solution of CH2N2 was
(2H, m, CH2COOH), 4.22 (2H, m, OCH2), 6.25
(2 · COOH), 7.8–7.5 (4H, m, aromatic CH). 13C
NMR d 10.9 (CH3), 23.6 (CH2CH2COOH), 30.0
(CH2CH2CH2COOH), 34.2 (CHCH2CH3), 38.5
(CH2COOH), 67.7 (OCH2), 132.9–128.7 (aromatic C),
168.1 (COOCH2), 172.1 (CH2COOH), 179.8 (aromatic
C).
slowly added, at 0 ꢁC, to compound
2 (0.57 g,
2.06 mmol) in MeOH/ether (30 mL, 9:1) until the colour
of the solution remained yellow. The excess of CH2N2
was flushed out with a nitrogen stream and the solvent
removed under vacuum obtaining product 2a as a pale
yellow oil (0.59 g, 98% yield). Rf (AcOEt/petroleum
ether 2:1, UV) = 0.74. ESI-MS: calcd for [M+H]+
4.1.6. Mono(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxy-
late (5) and methyl (2-ethyl-5-oxohexyl) 1,2-benzenedi-
carboxylate (5a). Compound 2 (2.46 g, 8.9 mmol) or
compound 2a (2.58 g, 8.9 mmol) was added, drop by
291.3. Found 291.9. 1H NMR
J = 7.4 Hz, CH3), 1.4–2.15 [7H,
d
0.91 (3H, t,
m, CH2@
CH(CH2)2CHCH2CH3), 3.88 (3H, s, COOCH3), 4.24
(2H, d, J = 6 Hz, COOCH2), 4.96 (2H, dd, J = 8.2,
J = 1.8 Hz, CH2@), 5.76 (H, m, CH@), 7.61 and 7.81
(4H, 2 · m, aromatic CH). 13C NMR d 10.4 (CH3),
drop in 5 min, to
a solution of PdCl2 (16 mg,
0.089 mmol) and p-benzoquinone (1.06 g, 9.8 mmol) in
DMF/H2O (7:1, 24 mL). After stirring overnight at
room temperature, the reaction mixture was quenched
with 3 N HCl (60 mL) and extracted with ether. The or-
ganic phase was washed with 10% NaOH and brine. The
aqueous basic layer was acidified to pH 1 with 3 N HCl
and then re-extracted with Et2O. The crude product was
purified by FCC.
23.1
(CH2CH3),
29.7
38.1
(CH2CH@CH2),
(CH3CH2CH),
30.7
38.2
(CH2CH2CH@CH2),
(COOCH3), 68.0 (COOCH2), 114.4 (CH@CH2), 128.3–
133.4 (aromatic C), 138.0 (CH@CH2), 167.4 (COOMe),
168.1 (COOCH2).
4.1.4. Mono(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarb-
oxylate (3). B2H6 (1 M) in THF (5.5 mL) was added,
drop by drop at 0 ꢁC under nitrogen, to a solution of
compound 2 (1.52 g, 5.5 mmol) in anhydrous THF
(50 mL). After stirring at room temperature for 1 h, ex-
cess of hydride was destroyed with H2O (0.1 mL). After
5 min, 3 N NaOH (0.5 mL) and 30% H2O2 (0.5 mL)
were added and the reaction mixture was stirred for
1 h at 50 ꢁC. The mixture was extracted with Et2O,
washed with brine and dried over Na2SO4 and FeSO4.
The crude alcohol, isolated after evaporation of the sol-
vent under vacuum, was purified by FCC. Product 3 was
obtained as an oil (1.44 g, 89% yield). Rf (AcOEt/petro-
leum ether 1:1, UV, KMnO4) = 0.3. ESI-MS: calcd for
[M+H]+ 295.3. Found 295.4; calcd for [M+Na]+ 317.3.
Compound 5: oil (1.06 g, 41% yield). Rf (AcOEt/petro-
leum ether 2:1, UV) = 0.2. ESI-MS: calcd for [MÀH]+
291.3. Found 291.1. IR (KBr) cmÀ1: 3500–2500 (OH),
1
2964.5 (CH), 1714.2 (C@O). H NMR d 0.92 (3H, t,
CH3), 1.3 (2H, m, CH2CH3), 1.69 (3H, m,
CHCH2CH2CO), 2.19 (3H, s, COCH3), 2.52 (2H, m,
CH2CO), 4.2 (2H, m, OCH2), 7.64 and 7.8 (4H, m, aro-
matic CH). 13C NMR
d 11.1 (CH3CH2), 23.7
(CH3CH2), 24.7 (CH2CH2CO), 30.0 (aliphatic CH),
38.2 (CH3CO), 40.6 (CH2CO), 63.5 (OCH2), 128.7–
131.5 (aromatic C), 168.1 (COOCH2), 170.6 (COOH),
210.6 (CH3CO).
Compound 5a: oil (1.52 g, 56% yield). Rf (AcOEt/petro-
leum ether 2:1, UV) = 0.2. ESI-MS: calcd for [M+H]+
1
Found 317.3. H NMR d 0.90 (3H, t, CH3), 1.44 [9H,
1
m, CH3CH2CH(CH2)3CH2OH], 2.92 (1H, br, OH),
3.84 (2H, m, CH2OH), 4.23 (2H, m, COOCH2), 7.56
and 7.74 (4H, 2 · m, aromatic CH). 13C NMR d 11.1
(CH3), 22.0 (CH3CH2), 24.0 [CH2(CH2)2OH], 29.9
306.3. Found 306.9. H NMR d 0.92 (3H, t, CH3), 1.4
(2H, m, CH2CH3), 1.63 (3H, m, CHCH2CH2CO), 2.11
(3H, s, COCH3), 2.45 (2H, m, CH2CO), 3.88 (2H, m,
COOCH3), 4.2 (2H, m, OCH2), 7.54 and 7.7 (4H, m,
aromatic CH). 13C NMR d 10.9 (CH3CH2), 23.7
(CH3CH2), 24.7 (CH2CH2CO), 29.8 (aliphatic CH),
38.2 (COCH3), 40.7 (CH2CO), 52.5 (COOCH3), 63.5
(OCH2), 128.7–131.2 (aromatic C), 167.6 (COOCH2),
167.8 (COOCH3), 208.5 (CH3CO).
[CH2(CH2)3OH],
31.9
(CH2CH2OH),
38.9
(CHCH2CH3), 61.9 (CH2OH), 67.4 (COOCH2), 128.5–
132.9 (aromatic C), 168.6 (COOCH2), 170.1 (COOH).
4.1.5. Mono(5-carboxy-2-ethylpentyl) 1,2-benzenedicarb-
oxylate (4). JonesÕ reagent (1 M, 2 mL), prepared from
CrO3 (6.7 g) in 96% H2SO4 (6 mL) and H2O (50 mL),
was added, drop by drop at 15–20 ꢁC, to compound 3
(553 mg, 1.88 mmol) in anhydrous acetone (6 mL). The
precipitated reduced chromium salt was centrifuged
off. The solution was quenched by addition of H2O
and extracted with Et2O that was dried over Na2SO4.
Methanol or NaHSO3 was added to the brown solution
to eliminate residue Cr(VI) salts. The crude acid 4 was
extracted with 0.4 M K2CO3. The aqueous layer, acidi-
fied to pH 1 with 1 M HCl, was extracted with CHCl3
and dried over Na2SO4. Compound 4 (546 mg, 94%
yield) was obtained as a colourless oil by evaporation
of the solvent under vacuum. Rf (AcOEt/hexane 1:1,
UV) = 0.5. ESI-MS: calcd for [MÀH]+ 309.3. Found
309.0. 1H NMR d 0.90 (3H, t, CH3), 1.42 (4H, m,
(CH2)2CH2COOH), 1.65 (3H, m, CHCH2CH3), 2.38
4.1.7. Mono(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarb-
oxylate (6). A solution of NaBH4 (117 mg, 3.16 mmol)
in ethanol (2 mL) was slowly added to compound 5
(231 mg, 0.79 mmol). After stirring at room temperature
for 2 h, the mixture was quenched with H2O (1 mL). The
aqueous basic layer was acidified to pH 1 with concd
HCl and then extracted with n-hexane. The crude alco-
hol, isolated after concentration under vacuum, was
purified by FCC affording 6 as an oil (110 mg, 48%
yield). Rf (EtOAc/petroleum ether 2:1, UV,
KMnO4) = 0.3. ESI-MS: calcd for [M+H]+ 294.3.
1
Found 294.8. H NMR d 0.90 (3H, t, CH3), 1.25 (3H,
d, J = 6.2, CH3CHOH), 1.45 (6H, m, CH2CH2CHOH
and CH3CH2), 1.75 (1H, m, CHCH2CH3), 4.07 (2H,
m, OCH2), 4.36 (1H, m, CHOH), 6.25 (1H, br, OH),
7.41 and 7.74 (4H, m, aromatic CH). 13C NMR d 11.1

F. Nuti et al. / Bioorg. Med. Chem. 13 (2005) 3461–3465
3465
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66.7 (CHOH), 69.5 (OCH2), 128.8–131.8 (aromatic C),
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4.2. GC–MS analysis of DEHP metabolites in human
urine and serum
For enzymatic hydrolysis of the glucuronidated phtha-
late metabolites, the urine samples were incubated with
b-glucuronidase for 20 h. Thereafter, serum and urine
samples were acidified to pH 2–3, extracted twice with
ethyl acetate and derivatized with diazomethane and
BTSFA [N,O-bis(trimethylsilyl) trifluoroacetamide].
For separation of the analytes a 20 m capillary column
(DB1, J & W Scientific, 0.18 mm i.d. and 0.18 lm film
thickness) was used. The column oven temperature was
initially kept isothermal at 40 ꢁC for 1 min; followed by
an increase to 80 ꢁC at a rate of 20 ꢁC/min, to 200 ꢁC at
a rate of 10 ꢁC/min, to 280 ꢁC at a rate of 5 ꢁC/min and
kept at 280 ꢁC for 5 min. Quantitative analysis of DEHP
and its metabolites was performed by selected ion moni-
toring gas chromatography/mass spectrometry,14 operat-
ing in negative ion chemical ionization mode to monitor
the negative ion at m/z 148. The limit of detection was
approximately 0.1 lg/L for the DEHP metabolites.
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This study was supported by the German foundation
ÔLieselotte und Dr. Karl Otto Winkler-Stiftung fur
ArbeitsmedizinÕ. We express gratitude to Fondazione
Ente Cassa di Risparmio di Firenze (Italy), for finan-
cially supporting the Laboratory of Peptide & Protein
Chemistry & Biology of the University of Florence.
¨
Deutschen Gesellschaft fur Arbeitsmedizin und Umwelt-
¨
und Umweltmedizin e.V., Munchen, 2002, 535–536.
¨
medizin e.V., Deutsche Gesellschaft fur Arbeitsmedizin
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