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A biomarker approach to measuring human

dietary exposure to certain phthalate diesters
W. A. C. Anderson , L. Castle , M. J. Scotter , R. C. Massey & C. Springall
Published online: 10 Nov 2010.

To cite this article: W. A. C. Anderson , L. Castle , M. J. Scotter , R. C. Massey & C. Springall (2001) A biomarker
approach to measuring human dietary exposure to certain phthalate diesters, Food Additives & Contaminants,
18:12, 1068-1074, DOI: 10.1080/02652030110050113

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 Food Additives and Contaminants , 2001, Vol. 18, No. 12, 1068 ±1074

A biomarker approach to measuring human dietary

exposure to certain phthalate diesters

W. A. C. Andersony, L. Castley*, M. J. Scottery, Introduction

R. C. Masseyy and C. Springall‡
y Ministry of Agriculture Fisheries and Food, Central Science
Laboratory, Sand Hutton, York, UK; ‡ TNO-BIBRA International Diesters of phthalic acid, known colloquially as
Ltd., Carshalton, Surrey, UK.
phthalates, are used in a wide range of industrial
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products. They are produced in large quantities every

(Received December 2000; revised January 2001; accepted year with, for example, production of 1150 ktonnes
February 2001) predicted for the year 2000 in western Europe (Barrett
1996). As a consequence of this extensive production
Three groups of eight volunteers were administered and moderate persistence, they are well-known envir-
stable isotope-labelle d phthalate diesters in a single onmental contaminants. They have been found at low
dose and the amount of the correspondin g phthalate levels in foods, particularly fatty foods such as dairy
monoesters excreted in the urine was measured. products because they are fat-soluble (Sharman et al.
Amongst the phthalates administered were the symme- 1994, Page and Lacroix 1995). Human exposure to
trical dibutyl-, di-2-ethyl- and diisooctyl- phthalates phthalates has received much public attention and
along with the unsymmetrical benzylbutylphthalate . their e€ ects on human health have been widely de-
The control group received no dose, the low dose group bated (MRC 1995, Sharpe et al. 1995, Harris et al.
received 168±255 mg of each phthalate and the high dose 1997). Despite a lack of evidence for any link between
group received 336 to 510 mg of each phthalate. The adverse trends in human reproductive health and the
excreted phthalate monoesters were measured by LC± presence of these substances in the environment,
MS following hydrolysi s of conjugates. The bulk of concerns are still expressed that phthalates may have
phthalate monoester was excreted in the ®rst 24 hour a detrimental e€ ect on human sexual development
period following the dose. For dibutylphthalate , 64% (Colon et al. 2000).
and 73% on a mole basis of the low and high dose
respectively was excreted as monobutylphthalate . For Such concerns have, for example, encouraged the
dioctlyphthalat e (sum of the 2-ethylhexyl and the Commission of the European Union to prohibit the
isooctyl species) the yield was 14 and 12% of the low marketing of toys and childcare articles, intended to
and high dose excreted as monooctylphthalate . For be placed in the mouth by children of less than three
benzylbutylphthalate , 67% and 78% was eliminated years of age, containing more than 0.1% by weight
as monobenzylphthalat e and only 6% (measured for of six phthalates including di-isononylphthalat e
the high dose only) was eliminated as monobutylphtha - (DINP), di-(2-ethylhexyl)phthalate (DEHP), di-n-oct-
late. These conversion factors can be used in future ylphthalate (DNOP), diisodecylphthalat e (DIDP),
studies to assess exposure to phthalate esters via meas- benzylbutylphthalat e (BBP, also known as butyl-
uring urinary levels of the monoester metabolites. benzylphthalate) and dibutylphthalate (DBP) (CEU
2000). Japan is also considering an initiative to ban
DEHP-plasticized vinyl gloves for food handling
Keywords : phthalates, monoesters, urinary metabo- (Tsumura et al. 2001a, 2001a,b).
lites, exposure, LC±MS
Phthalates are in part eliminated in urine as their
monoesters (®gure 1) (Ikeda et al. 1980, Albro et al.
1982, Thomas 1982, Egestad et al. 1992,). A very
recent US study has reported levels of phthalate
* To whom correspondence should be addressed. e-mail: l.castle@ monoesters in the urine of 289 adults (Blount et al.
csl.gov.uk 2000). These workers reported high urinary levels of
Food Additive s and Contaminant s ISSN 0265±203X print/ISSN 1464±5122 online # British Crown Copyright 2001
http://www.tandf.co.uk/journals
DOI: 10.1080/0265203011005011 3
 Measuring human dietary exposure to phthalates 1069

Diester Phthalate monoester

O O

R R
O metabolism O
+ ROH
O OH
R
O O

metabolism
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Other breakdown products Conjugate

Urinary excretion
Figure 1. Metabolic excretion of phthalates.

monoethylphthalate and monobenzylphthalate sug- for DBP, BBP, and DOP (dioctylphthalate). Other
gesting higher than expected exposure to the parent phthalates were included in this volunteer study
diesters diethylphthalate (DEP) and BBP. Lower but e€ orts to measure their monoester metabolites
levels of monoethylhexyl-, mono-n-octyl-, monocy- have not succeeded yet and these will be reported
clohexyl-, and monoisononyl phthalates were also separately.
reported, indicating relatively lower exposure to
DEHP, DNOP, dicyclohexylphthalat e (DCHP), and
DINP respectively. However, despite these very inter-
esting ®ndings, no exact calculations could be made
by these workers as to what these excretion levels Experimental
corresponded to in terms of overall exposure to the
parent phthalates (Kohn et al. 2000). This was be-
cause quantitative data on any correlation between Synthesis of labelled phthalates
phthalate monoester elimination and phthalate expo-
13
sure were lacking for humans. Rather, conversion C-DBP, 13 C-DIOP and 13 C-DEHP were synthesized
factors could at best be inferred by reference to using a literature method (Castle et al. 1987). d4 -BBP
animal ADME (adsorption, distribution, metabolism was used in the deuterated form, to avoid confusion
and excretion) studies. with the metabolite of 13 C-DBP, and it was purchased
from Cambridge Isotope Laboratories Limited (MA,
The work reported here describes a quantitative USA). The 13 C labelling was at the carboxy position
biomarker method for correlating human urinary and the deuterium labelling was on the aromatic ring.
phthalate monoester elimination with exposure to
the corresponding phthalate diester. This was The labelled phthalate monoester metabolites, 13 C-
achieved by conducting an ethically approved chal- and d4 -monobutylphthalate (MBP), d4 -monoben-
lenge study using three groups of volunteers (8 people zylphthlate (MBeP) and 13 C-monoethylhexylphtha-
in each) dosed with isotopically labelled phthalates. late (MEHP), were synthesized from the labelled
By analysing subsequent 24-hour urine collections for phthalic acid which was dehydrated to form the
labelled phthalate monoesters and correlating this anhydride and then reacted with a limiting amount
to the dose, a biomarker correlation was determined of the corresponding alcohol.
 1070 W. A. C. Anderson et al.

Challenge study details since they were four mass units removed from any
unlabelled background monoesters. Speci®c method-
ology will be published elsewhere.
The full challenge protocol has been described (Ayesh
and Pallett 1997). The phthalate dose was spiked into
margarine and administered on toast as breakfast to
twenty-four volunteers (eight each of blank, `low’,
and `high’ spikes, table 1). The dose administered did Results and discussion
not exceed the daily exposure ®gures estimated from
dietary surveys (MAFF 1987, 1996). (p-Aminobenzoic
acid (PABA) tablets were used by the volunteers as a Synthesis results
urine collection integrity check. Urinary levels of
PABA were determined using a published method The chemical purity of the phthalate monoesters and
(Berg et al. 1985). 24-Hour urine samples were col- diesters was assessed using nuclear magnetic reso-
lected by the volunteers 1 day before the dose, 1 day nance spectroscopy (NMR) and GC±MS analysis
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after the dose, and subsequently 2 and 6 days after the (table 2). The diesterphthalate clean-up procedure
dose. Boric acid was used as a preservative for the worked well for 13 C-DBP but was less e€ ective for
13
urine (approximately 2 g per 2 litre collection vessel) C-DIOP and 13 C-DEHP. The major impurity was
and the samples were stored at 20°C between collec- unreacted alcohol which was not a concern for the
tion and analysis. The collection vessels were made of volunteer study.
plastic (polyethylene).

Day 1 results (24 hours prior to dosing)

Phthalate monoester extraction and analysis
Background levels of (unlabelled) MBP, MBeP, and
The extraction method was based on that used by MOP (monooctylphthalate) were detected in most of
industry to estimate occupational exposure to phtha- the urine samples. There was no measurable inter-
lates. Conventionally, this involves enzymic hydroly- ference in the respective 13 C- or d4 -channels after
sis of the glucuronide conjugates in urine, solvent correction for natural-abundance 13 C-contribution.
extraction and clean-up, derivatization and gas chro-
matography (GC, GC±MS) (Albro et al. 1973, Dirven
et al. 1993a). In this work, high performance liquid
chromatography±mass spectrometry (LC±MS) was
Rate of excretion
used in place of GC±MS since no derivatization is
required for LC. An LC±MS approach has been used It was anticipated that the labelled phthalate mono-
successfully before (Blount et al. 2000). For the esters would be eliminated over the ®rst 24 h (Dirven
determination of the 13 C-labelled monoesters, it was et al. 1993b) with a minimal amount determined on
necessary to also measure any unlabelled phthalate subsequent days. This proved to be correct with no
monoesters in the urine samples and correct the labelled phthalate excretion measurable in the second
labelled analyte result for the natural-abundance or sixth day collections. Background levels of (un-
13
C-contribution from this source. A similar correc- labelled) MBP, MBeP, and MOP were observed,
tion was not required for the d4 -labelled monoesters however, in most of the samples tested.

Table 1. Low and high doses for the volunteer study. Table 2. Phthalate monoester and diester purity.
Phthalate Low’ Dose `High’ Dose Phthalate Purity (%) Monophthalate Purity (%)
dosed (mg) (mg)
13 13
C-DBP 99 C-MBP 92
13
C-DBP 255 510 d4 -BBP 99 d4 -MBP 99
13
C-DEHP 190 380 d4 -MBeP 99
13 13
d4 -BBP 253 506 C-DEHP 34 C-MEHP 98
13 13
C-DIOP 168 336 C-DIOP 60 MIOP 95
 Measuring human dietary exposure to phthalates 1071

Day 0 results (24 h immediately following dosing) 64% and 73%, respectively that appeared rapidly, in
the ®rst 24-hour urine collection.
Two of the twenty-four urine collections were rejected
as possibly being incomplete, on the basis that the
PABA levels were outside the boundaries of 60% to d4 -BBP metabolism and elimination as d4 -MBeP
120% for acceptable recovery. Phthalate monoester and d4 -MBP
levels were not corrected for PABA recovery. One
other urine sample was lost, due to spillage by the The unsymmetrical BBP may be eliminated as either
volunteer. MBP (loss of benzyl alcohol) or as MBeP (loss of
13
C-MBP, d4 -MBP, d4 -MBeP and 13 C-MOP were butanol, ®gure 3). On average 140 mg and 323 mg of
observed in the urine from the low and high dose MBeP was eliminated, with RSD (n ˆ 7 volunteers)
values of 39% and 26% for the low and high doses
groups and not in the urine of the control group.
respectively (®gure 4). On a molar basis this repre-
The results were corrected for analytical batch
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sents excretion yields of 67% and 78% of BBP

recovery and natural-abundance 13 C contribution
excreted as MBeP.
from background phthalate monoester levels.
These background levels presumably arise in partici- The formation and excretion of MBP was much lower
pant’s urine from their normal diet, from cosmetics and could only be measured for the high dose group,
or other consumer products, or from occupational with an average of 20 mg excreted and a relatively high
exposure. Finally, the amount of labelled phthalate RSD of 59%. This conversion of BBP to MBP was
monoester excreted was calculated as the product of just 6% on a molar basis (®gure 5).
the concentration determined times the volume of the These results for BBP identi®ed MBeP as the main
24-hour urine sample from each volunteer. metabolite eliminated rapidly, within 24 h. As
expected, the butyl ester link of BBP was cleaved
preferentially to the benzyl ester link and that
there was little further metabolism before MBeP
13 13 was eliminated.
C-DBP metabolism and elimination as C-MBP

On average 129 mg and 298 mg of MBP was eliminated 13 13

with relative standard deviation (RSD) values (n ˆ 6 C-DOP metabolism and elimination of C-MOP
and 7 volunteers) of 29% and 28% for the low
(255 mg) and high (510 mg) doses respectively (®gure The monoesters 2-ethylyhexyl- and isooctyl- were
2). On a molar basis this represents excretion yields of found to co-elute under the LC conditions employed

500
450
400
350
MBP (ug)

300
250
200
150
100
50
0
0 1 2 3 4 5 6 7 8
Volunteer
13 13
Figure 2. The elimination of C-MBP from high (&) and low (^) doses of C-DBP.
 1072 W. A. C. Anderson et al.

Butylbenzylphthalate Monobenzylphthalate
O Conversion rate O
ca. 70 %
O O

d4 O CH3 d4 OH

O O

Conversion rate <6%

Monobutylphthalate Other breakdown products ca. 24 %

O

OH
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d4 O CH3

Figure 3. Excretion products (as conjugates) of the unsymetrical BBP.

450
400
350
300
MBeP (ug)

250
200
150
100
50
0
0 1 2 3 4 5 6 7 8
Volunteer
Figure 4. The elimination of d4 -MBeP from high (&) and low (~) doses of d4 -BBP.

45
40
35
30
MBP (ug)

25
20
15
10
5
0
0 1 2 3 4 5 6 7 8
Volunteer
Figure 5. The elimination of d4 -MBP from the high dose of d4 -BBP.
 Measuring human dietary exposure to phthalates 1073

and they could not be disentangled on the basis of would therefore re¯ect exposure for only the day
mass fragmentation using the single-stage LC±MS before sampling.
available for the work. Consequently, they were
measured together and the results reported here are A dose-elimination relationship was determined for
an average for the two octyl species. LC±MS±MS DBP, BBP, and DOP. Excretion yields (mole basis) of
may o€ er possibilities to distinguish between the the phthalate monoester metabolites for these com-
isomeric monoalkylphthalates and this technique will pounds were consistent with levels of approximately
be investigated in future work. Excretion data for 69% for DBP to MBP, 73% BBP to MBeP, 6% BBP
MOP are presented in ®gure 6. On average 29 mg and to MBP, and 13% for DOP to MOP. The fact that
61 mg of MOP was eliminated, with RSD (n ˆ 7 BBP is cleaved preferentially to the benzyl ester
volunteers) values of 35% and 46% for the low and means that (unlabelled) metabolites from BBP and
high doses respectively. On a molar basis this repre- DBP could be measured independently in any general
sents excretion yields of 14% and 12% respectively population survey without major confounding e€ ects.
that appeared rapidly, in the ®rst 24-hour urine
The between-individual variability in excretion yield
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collection.
is considered to be acceptable, with excretion yields
These yields agree with the literature data, obtained at (not PABA-corrected) showing RSD’s of 28% to
higher dose levels commensurate with occupational 59% (mean for all monoesters, 37%).
exposure. In total, between 10% and 50% of DEHP
is excreted in urine within 24 h and some 10% is Background levels of MBP, MBeP, and MOP were
excreted as monoethylhexylphthalat e (Dirven et al. readily detected in the urine samples of most volun-
1993). A higher ®gure of about 18% was reported in a teers for all days before and after labelled phthalate
similar study (Albro et al. 1982). administration. Although this was a confounding
factor here and required correction for the natural
13
C contribution to the LC±MS channels monitored,
it gave con®dence that this would be a feasible analy-
sis to apply in a general population survey. This has
Conclusions subsequently been con®rmed by the excellent work of
the research group of Brock (Blount et al. 2000, Kohn
et al. 2000).
The absence of detectable levels of labelled phthalate
monoesters in samples collected after the ®rst 24-h In summary, the results from this study indicate that
post dosing indicates that the phthalates were meta- a urinary biomarker approach to quantify human
bolized and cleared rapidly. Urinary metabolites of dietary exposure to phthalates is feasible for DBP,
phthalates measured in any future general survey BBP and DEHP/DIOP, and it provides the conver-

120

100

80
MOP (ug)

60

40

20

0
0 1 2 3 4 5 6 7 8
Volunteer
13 13
Figure 6. The elimination of C-MOP from high (&) and low (~) doses of C-DOP.
 1074 W. A. C. Anderson et al.

sion factors necessary to interpret analytical ®ndings zer di(2-ethylhexyl)phthalate in human urine samples.
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Jongeneelen, F. J., 1993b, Metabolites of the plasticiser di(2-
ethylhexyl)phthalate in urin samples of workers in polyvi-
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