Microchemical Journal 96 (2010) 391–396

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Microchemical Journal
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c

GC–MS method for the determination of paraben preservatives in the human breast
cancerous tissue
Govindaraj Shanmugam a, Babu Rajendran Ramaswamy a,⁎, Vijayalakshimi Radhakrishnan a, Hiroaki Tao b
a
Department of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirappalli-620024, India
b
National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba, Ibaraki-305-8569, Japan

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

Article history: The general presumption that the preservative laden personal care products may be one of the causative agents
Received 1 April 2010 for breast cancer, has remained a matter of controversy during this decade. Extensive studies have not been
Received in revised form 14 June 2010 carried out to either prove or disprove the role of preservatives in breast cancer incidences. In this study we have
Accepted 2 July 2010
developed a new method for the identification and quantification of the preservatives such as methyl paraben
Available online 31 July 2010
(MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP) in breast tissue using Gas
Chromatography and Mass Spectrometry (GC–MS). Tissue was extracted by using acetone:n-hexane mixture
Keywords:
PPCPs
(1:1 v/v) and derivatized with N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The extent of reaction
Paraben time and the amount of MSTFA to attain greater derivatization were optimized. The developed method yielded
MSTFA good recovery (mean ± SD) of 99.8± 5.1, 96± 4.4, 107 ± 17 and 113 ± 13% with relative standard deviations
GC–MS (RSDs) of 5.1, 4.6, 15.6 and 13%, and the limits of detection (LOD) of 2.02, 1.05, 1.71 and 3.75 ng g− 1 for MeP, EtP,
Cancerous tissue PrP and BuP, respectively. The method was successfully validated for the determination of parabens including
butyl paraben (log Kow = 3.57) in cancerous breast tissues; this could be a promising one for screening of breast
tissues and also the environment for paraben residues. As far as our knowledge goes this is the first GC–MS
method for the determination of parabens in human tissue.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction Most of the preservatives may be harmful to the consumers due to

The esters of p-hydroxybenzoic acid are commonly known as
parabens including methyl paraben, ethyl paraben, propyl paraben
and butyl paraben. They are widely used as antibacterial, antifungal
and preservative agents in food and beverages, and in pharmaceutical
and personal care products (PPCPs) [1,2]. Parabens are extensively
used in PPCP formulations because they have no perceptible odor or
taste, have neutral pH, and do not produce discoloration and cause
hardening [3]. Individually or in combination, they are used in over
13,200 formulations in nearly all types of cosmetics [4]. In a survey of
215 cosmetic products, 77% of the rinse-off cosmetics and 99% of
leave-on products contained parabens [5]. The products containing
parabens are applied on the skin, hair, scalp, lips, mucosae (oral,
ocular and vaginal) and nails, either occasionally or on a daily basis,
and their use may be continuing over a period of few decades. Thus,
the frequency and duration of paraben application by both oral and
topical ways may lead to continuous exposure of humans [4,6].
⁎ Corresponding author. Center for Marine Environmental Studies (CMES), Ehime
University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan. Tel./fax: + 81 89 927 8171.
E-mail addresses: indiradeebi@yahoo.com, ramaswamybr@gmail.com
(B.R. Ramaswamy).
their potency to induce allergic contact dermatitis. Based on in vitro
assays and in vivo animal models, it was shown that parabens possess
estrogenic activity [6]. Since estrogen is a major etiological factor in
the growth and development of human breast cancer in majority
cases, it has been proposed that the use of parabens in cosmetics,
particularly underarm deodorants and antiperspirants may contribute
to the rising incidence of breast cancer and they were detected in
human breast tumor tissues at low ng g− 1 levels [1].
Parabens have been widely detected in food and cosmetic
products [7], waters and wastewaters [2,8], and indoor dust [9]
using HPLC–CL, HPLC–MS/MS, LC–MS/MS, GC–MS, and Micellar
Electrokinetic Chromatography (MEKC); they were scarcely ana-
lyzed in human breast tissue [1], breast milk [10], serum[11,12] and
urine [13] by TLC, HPLC–MS/MS and LC–MS/MS techniques. Darbre
and Harvey [14] raised the issue of breast cancer caused by the
intentional use of under arm cosmetics, and warranted further
investigations in this line to obtain more information and develop a
database. However, significant effort has not been invested to
develop analytical techniques for the detection and quantification
of preservatives in breast cancer. Till date, only one set of data on the
occurrence of paraben preservatives in breast tissue is available [1].
Further, Ye et al. [13] pointed out that high frequency of detection of
free and conjugated methyl, ethyl, propyl and butyl parabens could
be valid biomarkers to assess the human exposure to these

0026-265X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2010.07.005
392 G. Shanmugam et al. / Microchemical Journal 96 (2010) 391–396
compounds. Moreover, all types of body care cosmetics applied to the
skin (not only underarm cosmetics) can be a source of local
estrogenic chemical input to the breast and therefore should be
considered in risk assessments [14]. Gas Chromatography–Mass
Spectrometry (GC–MS) is well suited for the identification of a large
number of potential steroids and metabolites due to its high
chromatographic resolution capacity and reproducible ionization
efficiency [15]. This study was aimed to develop a simple and suitable
method for the detection and quantification of paraben preservatives
in human tissue using GC–MS. Silylation of parabens with MSTFA
was adopted in the analysis to improve the performance of GC–MS
determination.
2. Experimental
2.1. Reagents and chemicals
The reference standards of MeP, EtP, PrP and BuP, and the
derivatizing reagent, N-Methyl-N-(trimethylsilyl) trifluoroacetamide
(MSTFA) were purchased from Sigma-Aldrich (USA). Acetone and
ethyl acetate of HPLC grade were procured from Qualigens Fine
Chemicals (Mumbai, India). Sodium sulfate (anhydrous) and glass
wool were obtained from HiMedia Laboratory Pvt. Ltd., (Mumbai,
India). Silica gel and florisil (60–120 mesh) were procured from
Merck Specialties Private Limited (Mumbai, India). The laboratory-
purified water was obtained from a water purification system (ELGA,
UK). To avoid potential contamination with parabens, the glassware
were sequentially washed with 10% soap solution (Labolene), tap
water and 50% hydrochloric acid (HCl), ultrapure water and acetone.
Then they were air dried and covered with aluminium foil and
sterilized in hot air oven (Heco, Chennai, India) at 200 °C for 12 h.
2.2. Standard solutions
The individual standard stock solutions were prepared by
accurately weighing 10 mg of methyl, ethyl, propyl and butyl
parabens separately and dissolving them in 100 mL of acetone:ethyl
acetate (1:1 v/v) (100 μg mL− 1). The working standard solutions for
calibration and for recovery spike were prepared at required
concentrations from the stock standard solutions, and stored at
−20 °C.
2.3. Sample preparation
2.3.1. Parabens extraction
One gram of the breast tissue was homogenized with 3 g of
anhydrous sodium sulfate and 15 mL of acetone:n-hexane (1:1 v/v),
and transferred to a conical flask for extraction by mechanical shaking
for 12 h. After extraction, the extract was transferred to a centrifuge
tube and centrifuged (3000 rpm; 10 min) at room temperature. The
supernatant was collected in a glass tube, the pellet was again
extracted by shaking manually with 5 mL of acetone–hexane mixture
and the supernatant was collected. The extracts were pooled and 3 g
of anhydrous sodium sulfate was added to it, shaken well to remove
moisture. Then the extract was condensed (dried) using a vacuum
rotavapour (Buchi R 210, Switzerland) at 35 °C and reconstituted in
1 mL of ethyl acetate.
2.3.2. Silica gel cleanup
The extract cleanup was done with solid phase extraction (SPE)
using silica gel. Three grams of silica gel (60–120 mesh, baked at
200 °C for 12 h) was stirred with ethyl acetate to form slurry and
poured into a glass column (16 × 1.5 cm), then Na2SO4 was layered
(~1 cm) above the silica gel and finally, conditioned with 15 mL of
ethyl acetate. The concentrated extract was transferred to the column
and eluted with 15 mL of ethyl acetate. The eluate was collected and
condensed (0.5 mL) with a rotavapour and by gentle purging of N2
and then transferred to amber glass GC vial for derivatization.
2.3.3. Derivatization
Derivatization is a very useful process for detecting compounds in
complex samples, and applied widely in forensic, medical and
environmental chemistry [16]. Silylation is the most widely used
derivatization technique and normally it does not require a purification
step, and the derivatives can be injected directly into the GC [17]. The
introduction of a silyl group(s) can also serve to enhance mass
spectrometric properties of the derivatives, by producing either more
favorable diagnostic fragmentation patterns for use in structure
investigations, or the characteristic ions for use in trace analyses in
GC–MS under selected ion monitoring mode.
In this study, we aimed to detect and quantify parabens that are
highly polar and thermally fragile. In order to make the parabens
suitable for GC analysis, they require transformation into more volatile
and thermally stable compounds. For this purpose, we used derivatizing
reagent MSTFA. The MSTFA derivatization achieved complete derivati-
zation and permitted the detection of compounds containing polar
function groups with adequate signal-to-noise (S/N) ratio. Derivatiza-
tion decreases the LODs of GC–MS methods and therefore achieves
sensitivity comparable to that of LC–MS, and GC–MS analysis after
derivatization is an efficient alternative to LC–MS [18].
For derivatization of parabens, 100 ng of working standards was
transferred to amber glass GC vials containing 500 μL of ethyl acetate.
Then derivatizing reagent (MSTFA) was added. The conditions for
derivatization were determined through investigating the effects of
various experimental parameters including the reaction time and the
volume of reagent on the analytical response of the compounds.
Silylation was optimized with different volumes (ie. 10, 20, 30, 40 and
50 μL) of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamide)
and different incubation times (ie. 15, 30, 45, 60, 75 and 90 min) at
70 °C. After silylation, 1 μL of the derivatized solution was analyzed by
GC–MS.
2.3.4. Quality control
One gram of non-cancerous breast tissue was spiked individually
with 50, 100, 200 and 300 ng mL− 1 parabens (methyl-, ethyl-, propyl-
and butyl paraben) and extracted, followed by SPE cleanup and
derivatized as mentioned in earlier sections, and the recovery and
linearity were measured. Precision was also calculated as relative
standard deviation (RSD) of the experimental concentrations.
2.4. GC–MS analysis
The detection and quantification of parabens were performed
using a Shimadzu (Japan) QP-2010 GC–MS system equipped with a
Shimadzu AOC-20i auto sampler. Chromatographic separation of
parabens was achieved with DB-1 fused silica capillary column
(30 m × 0.32 mm i.d., 0.25 μm film thickness, J&W Scientific, Folsom,
CA, USA). Helium with a purity of 99.999% was used as the carrier gas
at a flow rate of 2.25 mL min− 1. One μL of derivatized extract was
injected in splitless mode using an auto sampler. The injector port,
interface and ion source temperatures were set at 250, 270 and
230 °C, respectively. The GC temperature was programmed as
follows: 50 °C (1 min), 30 °C min− 1 ramp to 220 °C, 2 °C min− 1
ramp to 230 °C, and 15 °C min− 1 ramp to 320 °C (10 min hold). The
mass spectrometer was operated in electron ionization (EI) mode at
70 eV and at an emission current of 60 μA. Full scan data was obtained
in a mass range of m/z 35–500. Scanning interval and SIM sampling
rate were 0.5 and 0.2 s, respectively. The mass selective detector was
operated in selected ion monitoring (SIM) mode and mass ions for
each compound are given in Table 1.
 G. Shanmugam et al. / Microchemical Journal 96 (2010) 391–396 393

Table 1 3.2. Effect of reaction time on derivatization

Summary of the GC–MS method showing correlation coefficient (R2), precision, LODs,
LOQs and recovery of tissue spiked parabens.
The reaction time is also crucial in derivatization, and so we
Analyte Mass ions R2a Precision LOD LOQ Recovery (%) investigated by spiking 100 ng mL− 1 concentration of parabens and
(% RSD; n = 3) ng g− 1 ng g− 1 (n = 3) checked the extent of derivatization at 15, 30, 45, 60, 75, and 90 min
MP 209, 224, 135 0.989 5.1 2.02 6.75 99.8 ± 5.1 with 20 μL MSTFA at 70 °C. The findings (Fig. 2) revealed that the
EP 223, 193, 238 0.998 4.6 1.05 3.49 96 ± 4.4 lowest reaction time (15 min) gave less recovery (from 96 ± 2.6 to
PP 193, 210, 195 0.998 15.6 1.71 5.68 107 ± 17
99.6 ± 3.5 ng mL− 1) than the higher reaction times (i.e. 30 to
BP 210, 195, 193 0.996 13 3.75 12.5 113 ± 13
90 min). However, at 30 and 45 min reaction times the recoveries
a
Linear range 50–300 ng mL− 1. were almost same at around 100% (102 ± 8–106 ± 6.7 ng mL− 1 and
101 ± 9.4–106 ± 6 ng mL− 1, respectively). However, higher reaction
times (60, 75 and 90 min) gave greater recoveries with greater
2.5. Preparation of human tissue samples deviations, (ranged between 107 ± 11 and 136 ± 48 ng mL− 1). Since
there was not much variation between 30 and 45 min, the lower
Human breast tissue samples (cancerous: n = 9 and non-cancerous: reaction time of 30 min was chosen as an optimal reaction time for
n = 1) were obtained from the archives of Government Hospitals at sufficient recovery. The reaction temperature was maintained at
Tiruchirappalli and Thanjavur, Tamil Nadu state, India. Tissue was sliced 70 °C, since MSTFA's boiling point is 70 °C and at this temperature it
finely using a sterile blade and ground well. One gram of the tissue was reacts with compounds to replace labile hydrogen with a –Si(CH3)3
homogenized and treated as mentioned in earlier sections for group. Bowden et al. [15] stated that steriod derivatization beyond
extraction, clean up and derivatization in the optimized conditions of 60 min and above 70 °C do not provide any additional benefit in the
20 μL MSTFA, 30 min reaction time at 70 °C. RRFs (relative response factors) obtained. In addition, they reported
that temperatures below 40 °C are not adequate for efficient
derivatization with the reaction times tested in their study. Basheer
3. Result and discussion et al. [19] reported complete derivatization of estrogens in water
samples at a derivatization time of 30 min at 60 °C with 50 μL of
The extraction with n-hexane:acetone (1:1 v/v) solvent mixture MSTFA.
and clean up using silica gel (60–120 mesh) were good (data not Fig. 3 shows the effect of derivatization on resolution (in terms of
shown). Ethyl acetate was used for both column conditioning and peak intensity and peak tailing) of paraben standards. From
elution. comparing the representative chromatograms of paraben standards
in scan mode without (Fig. 3a) and with (Fig. 3b) MSTFA derivati-
zation, it is obvious that prior to derivatization (Fig. 3a) the peak
3.1. Effect of MSTFA volume on derivatization intensity was very low and moreover not sharp but tailing. However,
after derivatization (Fig. 3b) the peaks were well resolved (i.e. sharp
In order to know the influence of parameters on derivatization peaks with many fold greater intensity and without tailing).
process, the effect of the changes in the volume of MSTFA and the
derivatization reaction time were studied. Fig. 1 shows that
derivatization of spiked parabens with 10–50 μL of MSTFA, in which 3.3. Quality control (recovery and detection limit)
20 μL (at 30 min for 70 °C) gave better derivatization and good
recovery (n = 3), ranged between 92 ± 0.7 and 101 ± 6.3 ng mL− 1 for Recoveries of parabens were investigated in triplicate using non-
all compounds. Low recovery (79 ± 16–90 ± 13 ng mL− 1) was cancerous tissue sample spiked with known standards. The mean
attained with 10 μL, but higher amount of MSTFA (i.e. 30, 40 and recoveries of MeP, EtP, PrP and BuP were 99.8 ± 5.1, 96 ± 4.4, 107 ± 17
50 μL) showed more than 100% recovery with wide deviations (110 ± and 113 ± 13%, respectively. Quantitative linearity was investigated
3.9–148 ± 13.4 ng mL− 1). Therefore, 20 μL could be considered by elucidating the calibration curves for the four parabens over the
sufficient for complete derivatization of parabens (~ 100%). This is in concentration range 50–300 ng mL− 1. The line of best fit for the
contrast to Basheer et al. [19] who reported that the amount of MSTFA relationship between the peak intensity (area) and concentration of
did not have any impact on derivatization of estrogens in water each analyte in the standard solution was determined by linear
samples. Canosa et al. [8] used 20 μL of MTBSTFA for 15 min at room regression. The relationship is acceptably linear for all the four
temperature for on-fiber derivatization of parabens from surface and analytes over the full range of concentrations. The calibration curves
sewage water samples. are shown in Fig. 4 and correlation coefficients are presented in
Table 1, together with the method precision, quantification, and
detection limits. The limits of detection and limits of quantification

Fig. 1. Effect of MSTFA volume in the derivatization efficiency of parabens. Fig. 2. Effect of reaction time in MSTFA derivatization efficiency of parabens.
394 G. Shanmugam et al. / Microchemical Journal 96 (2010) 391–396

Fig. 3. GC–MS chromatogram of target compounds in scan mode (a) without derivatization and (b) with derivatization.

were estimated from the signal-to-noise ratio of 3 and 10, respectively
(Table 1).
3.4. Analysis of real samples (human breast cancer tissues)
Analytical concentrations of paraben compounds in both cancer-
ous (n = 9) and non-cancerous breast tissue (n = 1) samples were
quantified (Table 2). All the four parabens were detected in cancerous
tissues, the detection frequency of MeP and BuP is 100%, and 67% for
EtP and 78% for PrP. These compounds were detected at concentra-
tions ranging from 40 to 1719, 535 to 26,469, ND (not detected) to
10,608, and ND to 4490 ng g− 1, respectively. The chromatograms of
the target compounds in cancerous breast tissue by SIM mode before
and after derivatization were shown in Fig. 5. Well-resolved peaks
with good separation of analytes were seen after derivatization
(Fig.5b). Darbre et al. [1] quantified the parabens in breast tissues, and
found methyl paraben at higher concentrations (62% of total
parabens), which is quite opposite to the present investigation
where methyl paraben is the least quantified. The mean concentration
obtained in the present study (Table 2) is two to three orders of
magnitude higher than the levels reported by Darbre et al. [1].
Probably, derivatization might have enhanced compounds' detect-
ability and peak area intensity. Further, we failed to detect propyl- and
butyl parabens in the tissue samples without derivatization (Fig. 5a).
Fig. 6 shows the chromatogram of target compounds in non-
cancerous tissue after derivatization with and without standard
spike. In non-cancerous tissue only methyl paraben was detected
(26.6 ng g− 1), which is an order of magnitude lower than the mean
concentration of methyl paraben from nine cancerous tissues, where
all the four parabens were found, with 100% frequency of butyl
paraben. Estrogenic activity of parabens is known to increase with
increasing length of the linear alkyl chain from methyl paraben to n-

Table 2
Concentrations (ng g− 1 wet wt.) of parabens in human breast tissue.

Sample id MeP EtP PrP BuP

a
ncb 1 26.5 – –
cb 1 103 1431 391 2640
cb 2 102 – 506 1052
cb 3 171 – 273 734
cb 4 989 10,608 4490 26,469
cb 5 260 5436 1515 6913
cb 6 1239 3094 2725 3797
cb 7 1719 3101 – 3933
cb 8 1678 243 – 723
cb 9 40.1 – 321 535
Mean 802 2657 1136 5199

Fig. 4. Calibration curves for MeP, EtP, PrP and BuP by GC–MS. ncb—non-cancerous breast tissue, cb—cancerous breast tissue, a—not detected.
 G. Shanmugam et al. / Microchemical Journal 96 (2010) 391–396 395

Fig. 5. Selected ion monitoring (SIM) chromatogram of target compounds in breast cancerous tissue (Cb3) (a) without derivatization and (b) with derivatization.

Fig. 6. Selected ion monitoring (SIM) chromatogram of target compounds after MSTFA derivatization (a) non-cancerous breast tissue and (b) non-cancerous breast tissue spiked
with 100 ng mL− 1 paraben standards.
396 G. Shanmugam et al. / Microchemical Journal 96 (2010) 391–396
Table 3
Levels (mean or range) of parabens (ng g− 1) in human samples.
Sample (n) MeP EtP PrP
CBT (09) 802 2657 1136
BT (20) 12.8 2.0 2.6
Milk (04) 1.24 NR 0.08
Serum (11) NR NR NR
Serum (16) 42.6 NR 7.4
Urine (100) 0.8 NR BDL
CBT—cancerous breast tissue, BT—breast tumor, NR—not reported, BDL—below detectable level, a—ng mL− 1.
butyl paraben [6], which is in agreement with elevated levels of butyl
parabens in cancerous tissues in this study.
The mean concentrations of MeP, EtP, PrP and BuP in cancerous
breast tissues were 802, 2657, 1136 and 5199 ng g− 1, respectively
(Table 2) and the mean levels were decreased in the order
BuP N EtP N PrP N MeP. This accumulation order is quite opposite to
the parabens quantified in indoor dust samples by Canosa et al. [20].
Higher concentration of BuP encountered may be due to the fact that
it is more lipophilic (log Kow = 3.5) than other parabens analyzed
and/or less metabolizable than MeP (log Kow = 1.91) in humans.
Even Janjua et al. [11] have observed a systematic absorption of butyl
paraben (ie. 135 ng mL− 1 in serum) in man after the first topical
application of basic cream with 2% (w/w) butyl paraben. Soni et al.
[21] reported that there was no evidence of accumulation of methyl
paraben in animals. Table 3 lists the comparison of analytical
methods for parabens in human samples. So far no report was
made on GC–MS determination of paraben compounds in human
samples, but this study showed a good recovery (from 96 ± 4.4 to
113 ± 13%) with low detection limits compared to the earlier method
of Darbre et al. [1] which was done using HPLC–MS/MS with the
mean recovery of 48.5 ± 4.8%. Our method is comparable with other
HPLC–MS/MS and LC–MS/MS techniques (Table 3) used for human
body fluids [10–13].
4. Conclusion
In this study we have developed a sensitive and reliable analytical
method for the successful determination and quantification of MeP,
EtP, PrP and BuP in human tissue sample using GC–MS. The method
involves tissue extraction with acetone:n-hexane mixture, silica gel
cleanup and complete derivatization using 20 μL of MSTFA at 70 °C for
30 min. This method is very attractive for routine applications because
it is acceptably reproducible (RSD: 4.6–15.6%), linear (R2: 0.996–0.998
for 50–300 ng mL− 1 levels) and sensitive (i.e., with low LOD 1.05–
3.75 ng g− 1 wet wt.). This method can be used for large scale
screening of cancerous and non-cancerous breast tissues for the
presence of suspected paraben preservative residues of personal care
products/food commodities. In addition, it can be applied for the
determination of parabens in organisms used in monitoring studies, to
know the occurrence and fate of parabens in the environment, and to
generate systematic data on ecotoxicological risk assessment of
PPCPs.
Acknowledgements
We wish to thank Dr. D.G. Joakim Larsson, Department of
Neuroscience and Physiology, The Sahlgrenska Academy at the
University of Gothenburg, Sweden for his motivation to initiate this
PPCPs research in our laboratory. We are grateful to the United
Nations University, Japan and Shimadzu Corporation, Japan for the
GC–MS facility extended through “POPs Monitoring in Asian Coastal
Hydrosphere” project to Bharathidasan University by which the
analyses were made. First author (G.S.) thanks the University Grants
Commission, New Delhi, India for the award of Junior Research
Fellowship through non-SAP grant to the Department of Environ-
BuP Method Rec.% LOD (ng g− 1) Reference
5199 GC–MS 96–113 1–3.75 This study
2.3 HPLC–MS/MS 48.5 NA [1]
NR HPLC–MS/MS 89–103 0.1a [10]
135 LC–MS/MS 108 0.3a [11]
NR HPLC–MS/MS NR 0.2–1a [12]
NR HPLC–MS/MS NR 0.1–0.18a [13]
mental Biotechnology, Bharathidasan University, India. We thank Dr.
A.N. Subramanian and Dr. A.U. Thangavelu of Ehime University, Japan
for critical reading of the manuscript.
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