Microchemical Journal 96 (2010) 391–396

Contents lists available at ScienceDirect

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

their potency to induce allergic contact dermatitis. Based on in vitro
The esters of p-hydroxybenzoic acid are commonly known as assays and in vivo animal models, it was shown that parabens possess
parabens including methyl paraben, ethyl paraben, propyl paraben estrogenic activity [6]. Since estrogen is a major etiological factor in
and butyl paraben. They are widely used as antibacterial, antifungal the growth and development of human breast cancer in majority
and preservative agents in food and beverages, and in pharmaceutical cases, it has been proposed that the use of parabens in cosmetics,
and personal care products (PPCPs) [1,2]. Parabens are extensively particularly underarm deodorants and antiperspirants may contribute
used in PPCP formulations because they have no perceptible odor or to the rising incidence of breast cancer and they were detected in
taste, have neutral pH, and do not produce discoloration and cause human breast tumor tissues at low ng g− 1 levels [1].
hardening [3]. Individually or in combination, they are used in over Parabens have been widely detected in food and cosmetic
13,200 formulations in nearly all types of cosmetics [4]. In a survey of products [7], waters and wastewaters [2,8], and indoor dust [9]
215 cosmetic products, 77% of the rinse-off cosmetics and 99% of using HPLC–CL, HPLC–MS/MS, LC–MS/MS, GC–MS, and Micellar
leave-on products contained parabens [5]. The products containing Electrokinetic Chromatography (MEKC); they were scarcely ana-
parabens are applied on the skin, hair, scalp, lips, mucosae (oral, lyzed in human breast tissue [1], breast milk [10], serum[11,12] and
ocular and vaginal) and nails, either occasionally or on a daily basis, urine [13] by TLC, HPLC–MS/MS and LC–MS/MS techniques. Darbre
and their use may be continuing over a period of few decades. Thus, and Harvey [14] raised the issue of breast cancer caused by the
the frequency and duration of paraben application by both oral and intentional use of under arm cosmetics, and warranted further
topical ways may lead to continuous exposure of humans [4,6]. 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].
⁎ Corresponding author. Center for Marine Environmental Studies (CMES), Ehime
University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan. Tel./fax: + 81 89 927 8171.
Further, Ye et al. [13] pointed out that high frequency of detection of
E-mail addresses: [email protected], [email protected] free and conjugated methyl, ethyl, propyl and butyl parabens could
(B.R. Ramaswamy). 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 condensed (0.5 mL) with a rotavapour and by gentle purging of N2
skin (not only underarm cosmetics) can be a source of local and then transferred to amber glass GC vial for derivatization.
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 2.3.3. Derivatization
number of potential steroids and metabolites due to its high Derivatization is a very useful process for detecting compounds in
chromatographic resolution capacity and reproducible ionization complex samples, and applied widely in forensic, medical and
efficiency [15]. This study was aimed to develop a simple and suitable environmental chemistry [16]. Silylation is the most widely used
method for the detection and quantification of paraben preservatives derivatization technique and normally it does not require a purification
in human tissue using GC–MS. Silylation of parabens with MSTFA step, and the derivatives can be injected directly into the GC [17]. The
was adopted in the analysis to improve the performance of GC–MS introduction of a silyl group(s) can also serve to enhance mass
determination. spectrometric properties of the derivatives, by producing either more
favorable diagnostic fragmentation patterns for use in structure
2. Experimental investigations, or the characteristic ions for use in trace analyses in
GC–MS under selected ion monitoring mode.
2.1. Reagents and chemicals In this study, we aimed to detect and quantify parabens that are
highly polar and thermally fragile. In order to make the parabens
The reference standards of MeP, EtP, PrP and BuP, and the suitable for GC analysis, they require transformation into more volatile
derivatizing reagent, N-Methyl-N-(trimethylsilyl) trifluoroacetamide and thermally stable compounds. For this purpose, we used derivatizing
(MSTFA) were purchased from Sigma-Aldrich (USA). Acetone and reagent MSTFA. The MSTFA derivatization achieved complete derivati-
ethyl acetate of HPLC grade were procured from Qualigens Fine zation and permitted the detection of compounds containing polar
Chemicals (Mumbai, India). Sodium sulfate (anhydrous) and glass function groups with adequate signal-to-noise (S/N) ratio. Derivatiza-
wool were obtained from HiMedia Laboratory Pvt. Ltd., (Mumbai, tion decreases the LODs of GC–MS methods and therefore achieves
India). Silica gel and florisil (60–120 mesh) were procured from sensitivity comparable to that of LC–MS, and GC–MS analysis after
Merck Specialties Private Limited (Mumbai, India). The laboratory- derivatization is an efficient alternative to LC–MS [18].
purified water was obtained from a water purification system (ELGA, For derivatization of parabens, 100 ng of working standards was
UK). To avoid potential contamination with parabens, the glassware transferred to amber glass GC vials containing 500 μL of ethyl acetate.
were sequentially washed with 10% soap solution (Labolene), tap Then derivatizing reagent (MSTFA) was added. The conditions for
water and 50% hydrochloric acid (HCl), ultrapure water and acetone. derivatization were determined through investigating the effects of
Then they were air dried and covered with aluminium foil and various experimental parameters including the reaction time and the
sterilized in hot air oven (Heco, Chennai, India) at 200 °C for 12 h. volume of reagent on the analytical response of the compounds.
Silylation was optimized with different volumes (ie. 10, 20, 30, 40 and
2.2. Standard solutions 50 μL) of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamide)
and different incubation times (ie. 15, 30, 45, 60, 75 and 90 min) at
The individual standard stock solutions were prepared by 70 °C. After silylation, 1 μL of the derivatized solution was analyzed by
accurately weighing 10 mg of methyl, ethyl, propyl and butyl GC–MS.
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 2.3.4. Quality control
calibration and for recovery spike were prepared at required One gram of non-cancerous breast tissue was spiked individually
concentrations from the stock standard solutions, and stored at with 50, 100, 200 and 300 ng mL− 1 parabens (methyl-, ethyl-, propyl-
−20 °C. and butyl paraben) and extracted, followed by SPE cleanup and
derivatized as mentioned in earlier sections, and the recovery and
2.3. Sample preparation linearity were measured. Precision was also calculated as relative
standard deviation (RSD) of the experimental concentrations.
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), 2.4. GC–MS analysis
and transferred to a conical flask for extraction by mechanical shaking
for 12 h. After extraction, the extract was transferred to a centrifuge The detection and quantification of parabens were performed
tube and centrifuged (3000 rpm; 10 min) at room temperature. The using a Shimadzu (Japan) QP-2010 GC–MS system equipped with a
supernatant was collected in a glass tube, the pellet was again Shimadzu AOC-20i auto sampler. Chromatographic separation of
extracted by shaking manually with 5 mL of acetone–hexane mixture parabens was achieved with DB-1 fused silica capillary column
and the supernatant was collected. The extracts were pooled and 3 g (30 m × 0.32 mm i.d., 0.25 μm film thickness, J&W Scientific, Folsom,
of anhydrous sodium sulfate was added to it, shaken well to remove CA, USA). Helium with a purity of 99.999% was used as the carrier gas
moisture. Then the extract was condensed (dried) using a vacuum at a flow rate of 2.25 mL min− 1. One μL of derivatized extract was
rotavapour (Buchi R 210, Switzerland) at 35 °C and reconstituted in injected in splitless mode using an auto sampler. The injector port,
1 mL of ethyl acetate. interface and ion source temperatures were set at 250, 270 and
230 °C, respectively. The GC temperature was programmed as
2.3.2. Silica gel cleanup follows: 50 °C (1 min), 30 °C min− 1 ramp to 220 °C, 2 °C min− 1
The extract cleanup was done with solid phase extraction (SPE) ramp to 230 °C, and 15 °C min− 1 ramp to 320 °C (10 min hold). The
using silica gel. Three grams of silica gel (60–120 mesh, baked at mass spectrometer was operated in electron ionization (EI) mode at
200 °C for 12 h) was stirred with ethyl acetate to form slurry and 70 eV and at an emission current of 60 μA. Full scan data was obtained
poured into a glass column (16 × 1.5 cm), then Na2SO4 was layered in a mass range of m/z 35–500. Scanning interval and SIM sampling
(~1 cm) above the silica gel and finally, conditioned with 15 mL of rate were 0.5 and 0.2 s, respectively. The mass selective detector was
ethyl acetate. The concentrated extract was transferred to the column operated in selected ion monitoring (SIM) mode and mass ions for
and eluted with 15 mL of ethyl acetate. The eluate was collected and 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 found methyl paraben at higher concentrations (62% of total
(Table 1). parabens), which is quite opposite to the present investigation
where methyl paraben is the least quantified. The mean concentration
3.4. Analysis of real samples (human breast cancer tissues) obtained in the present study (Table 2) is two to three orders of
magnitude higher than the levels reported by Darbre et al. [1].
Analytical concentrations of paraben compounds in both cancer- Probably, derivatization might have enhanced compounds' detect-
ous (n = 9) and non-cancerous breast tissue (n = 1) samples were ability and peak area intensity. Further, we failed to detect propyl- and
quantified (Table 2). All the four parabens were detected in cancerous butyl parabens in the tissue samples without derivatization (Fig. 5a).
tissues, the detection frequency of MeP and BuP is 100%, and 67% for Fig. 6 shows the chromatogram of target compounds in non-
EtP and 78% for PrP. These compounds were detected at concentra- cancerous tissue after derivatization with and without standard
tions ranging from 40 to 1719, 535 to 26,469, ND (not detected) to spike. In non-cancerous tissue only methyl paraben was detected
10,608, and ND to 4490 ng g− 1, respectively. The chromatograms of (26.6 ng g− 1), which is an order of magnitude lower than the mean
the target compounds in cancerous breast tissue by SIM mode before concentration of methyl paraben from nine cancerous tissues, where
and after derivatization were shown in Fig. 5. Well-resolved peaks all the four parabens were found, with 100% frequency of butyl
with good separation of analytes were seen after derivatization paraben. Estrogenic activity of parabens is known to increase with
(Fig.5b). Darbre et al. [1] quantified the parabens in breast tissues, and 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 BuP Method Rec.% LOD (ng g− 1) Reference

CBT (09) 802 2657 1136 5199 GC–MS 96–113 1–3.75 This study
BT (20) 12.8 2.0 2.6 2.3 HPLC–MS/MS 48.5 NA [1]
Milk (04) 1.24 NR 0.08 NR HPLC–MS/MS 89–103 0.1a [10]
Serum (11) NR NR NR 135 LC–MS/MS 108 0.3a [11]
Serum (16) 42.6 NR 7.4 NR HPLC–MS/MS NR 0.2–1a [12]
Urine (100) 0.8 NR BDL NR HPLC–MS/MS NR 0.1–0.18a [13]

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 mental Biotechnology, Bharathidasan University, India. We thank Dr.
parabens in cancerous tissues in this study. A.N. Subramanian and Dr. A.U. Thangavelu of Ehime University, Japan
The mean concentrations of MeP, EtP, PrP and BuP in cancerous for critical reading of the manuscript.
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 References
the parabens quantified in indoor dust samples by Canosa et al. [20].
[1] P.D. Darbre, A. Aljarrah, W.R. Miller, N.G. Coldham, M.J. Sauer, Concentrations of
Higher concentration of BuP encountered may be due to the fact that parabens in human breast tumours, J. Appl. Toxicol. 24 (2004) 5–13.
it is more lipophilic (log Kow = 3.5) than other parabens analyzed [2] E. Blanco, C.C. Maria del, C.M. Maria del, R. Cela, Combination of off-line solid-
and/or less metabolizable than MeP (log Kow = 1.91) in humans. phase extraction and on-column sample stacking for sensitive determination of
parabens and p-hydroxybenzoic acid in waters by non-aqueous capillary
Even Janjua et al. [11] have observed a systematic absorption of butyl electrophoresis, Anal. Chim. Acta 647 (2009) 104–111.
paraben (ie. 135 ng mL− 1 in serum) in man after the first topical [3] M.R. Lee, C.Y. Lin, Z.G. Li, T.F. Tsai, Simultaneous analysis of antioxidants and
application of basic cream with 2% (w/w) butyl paraben. Soni et al. preservatives in cosmetics by supercritical fluid extraction combined with liquid
chromatography–mass spectrometry, J. Chromatogr. A 1120 (2006) 244–251.
[21] reported that there was no evidence of accumulation of methyl
[4] R.L. Elder, Final report on the safety assessment of methyl paraben, ethylparaben,
paraben in animals. Table 3 lists the comparison of analytical propylparaben and butylparaben, Inter. J. Toxicol. 3 (1984) 147–209.
methods for parabens in human samples. So far no report was [5] S.C. Rastogi, A. Schouten, N. De Kruijf, J.W. Weijland, Contents of methyl-, ethyl-,
propyl-, butyl- and benzylparaben in cosmetic products, Contact Dermat. 32 (1995)
made on GC–MS determination of paraben compounds in human
28–30.
samples, but this study showed a good recovery (from 96 ± 4.4 to [6] J.E. Routledge, J. Parker, J. Odum, J. Ashby, J.P. Sumpter, Some alkyl hydroxy
113 ± 13%) with low detection limits compared to the earlier method benzoate preservatives (parabens) are estrogenic, Toxicol. Appl. Pharmacol. 153
of Darbre et al. [1] which was done using HPLC–MS/MS with the (1998) 12–19.
[7] Q. Zhang, M. Lian, L. Liu, H. Cui, High-performance liquid chromatographic assay of
mean recovery of 48.5 ± 4.8%. Our method is comparable with other parabens in wash-off cosmetic products and foods using chemiluminescence
HPLC–MS/MS and LC–MS/MS techniques (Table 3) used for human detection, Anal. Chim. Acta 537 (2005) 31–39.
body fluids [10–13]. [8] P. Canosa, I. Rodriguez, E. Rubi, M.H. Bollain, R. Cela, Optimisation of a solid-phase
microextraction method for the determination of parabens in water samples at
the low ng per litre level, J. Chromatogr. A 1124 (2006) 3–10.
4. Conclusion [9] P. Canosa, I. Rodriguez, E. Rubi, R. Cela, Determination of parabens and triclosan in
indoor dust using matrix solid-phase dispersion and gas chromatography with
tandem mass spectrometry, Anal. Chem. 79 (2007) 1675–1681.
In this study we have developed a sensitive and reliable analytical [10] X. Ye, A.M. Bishop, L.L. Needham, A.M. Calafat, Automated on-line column-
method for the successful determination and quantification of MeP, switching HPLC–MS/MS method with peak focusing for measuring parabens,
EtP, PrP and BuP in human tissue sample using GC–MS. The method triclosan, and other environmental phenols in human milk, Anal. Chim. Acta 622
(2008) 150–156.
involves tissue extraction with acetone:n-hexane mixture, silica gel [11] N.R. Janjua, G.K. Mortensen, A.M. Andersson, B. Kongshoj, N.E. Skakkebaek, H.C.
cleanup and complete derivatization using 20 μL of MSTFA at 70 °C for Wulf, Systemic uptake of diethyl phthalate, dibutyl phthalate, and butyl paraben
30 min. This method is very attractive for routine applications because following whole-body topical application and reproductive and thyroid hormone
levels in humans, Environ. Sci. Technol. 41 (2007) 5564–5570.
it is acceptably reproducible (RSD: 4.6–15.6%), linear (R2: 0.996–0.998
[12] X. Ye, L. Wong, L.T. Jia, L.L. Needham, A.M. Calafat, Stability of the conjugated
for 50–300 ng mL− 1 levels) and sensitive (i.e., with low LOD 1.05– species of environmental phenols and parabens in human serum, Environ.
3.75 ng g− 1 wet wt.). This method can be used for large scale Internat. 35 (2009) 1160–1163.
screening of cancerous and non-cancerous breast tissues for the [13] X. Ye, A.M. Bishop, J.A. Reidy, L.L. Needham, A.M. Calafat, Parabens as urinary
biomarkers of exposure in humans, Environ. Health Perspect. 114 (2006) 1843–1846.
presence of suspected paraben preservative residues of personal care [14] P. Darbre, P.W. Harvey, Paraben esters: review of recent studies of endocrine
products/food commodities. In addition, it can be applied for the toxicity, absorption, esterase and human exposure, and discussion of potential
determination of parabens in organisms used in monitoring studies, to human health risks, J. Appl. Toxicol. 28 (2008) 561–578.
[15] J.A. Bowden, D.M. Colosi, D.C. Mora-Montero, T.J. Garrett, R.A. Yost, Enhancement
know the occurrence and fate of parabens in the environment, and to of chemical derivatization of steroids by gas chromatography/mass spectrometry
generate systematic data on ecotoxicological risk assessment of (GC/MS), J. Chromatogr. B 877 (2009) 3237–3242.
PPCPs. [16] J.M. Halket, V.G. Zaikin, Review: derivatization in mass spectrometry—1. Silylation,
Eur. Mass Spectrom. 9 (2003) 1–21.
[17] C. Schummer, O. Delhomme, B.M.R. Appenzeller, R. Wennig, M. Millet,
Acknowledgements Comparison of MTBSTFA and BSTFA in derivatization reactions of polar
compounds prior to GC/MS analysis, Talanta 77 (2009) 1473–1482.
[18] M.C. Pietrogrande, G. Basaglia, GC–MS analytical methods for the determination of
We wish to thank Dr. D.G. Joakim Larsson, Department of personal-care products in water matrices, Trends Anal. Chem. 26 (2007) 1086–1094.
Neuroscience and Physiology, The Sahlgrenska Academy at the [19] C. Basheer, A. Jayaraman, M.K. Kee, S. Valiyaveettil, H.K. Lee, Polymer-coated
University of Gothenburg, Sweden for his motivation to initiate this hallow-fiber microextraction of estrogens in water samples with analysis by gas
chromatography–mass spectrometry, J. Chromatogr. A 1100 (2005) 137–143.
PPCPs research in our laboratory. We are grateful to the United
[20] P. Canosa, D. Perez-Palacios, A. Garrido-Lopez, M.T. Tena, I. Rodriguez, E. Rubi, R.
Nations University, Japan and Shimadzu Corporation, Japan for the Cela, Pressurized liquid extraction with in-cell clean-up followed by gas
GC–MS facility extended through “POPs Monitoring in Asian Coastal chromatography–tandem mass spectrometry for the selective determination of
Hydrosphere” project to Bharathidasan University by which the parabens and triclosan in indoor dust, J. Chromatogr. A 1161 (2007) 105–112.
[21] M.G. Soni, S.L. Taylor, N.A. Greenberg, G.A. Burdock, Evaluation of the health
analyses were made. First author (G.S.) thanks the University Grants aspects of methyl paraben: a review of the published literature, Food Chem.
Commission, New Delhi, India for the award of Junior Research Toxicol. 40 (2002) 1335–1373.
Fellowship through non-SAP grant to the Department of Environ-