Simultaneous in-cell derivatization pressurized liquid extraction for the determination of multiclass preservatives in leave-on cosmetics

Article abstract of DOI:10.1021/ac101985h

An effective one-step sample preparation methodology for the determination of multiclass preservatives in cosmetics has been developed, applying, for the first time to this kind of matrix, pressurized liquid extraction (PLE) and a very simple, cheap, and fast derivatization procedure: acetylation with acetic anhydride and pyridine. A multifactorial experimental design has been used to evaluate and optimize the main experimental parameters potentially affecting the extraction process. In the final conditions the sample was mixed with Florisil as the dispersing sorbent and extracted with ethyl acetate for 15 min at 120 °C. One of the main goals of this work was to demonstrate the possibility of carrying out direct cosmetic preservative acetylation by simply adding the derivatization reagents into the PLE cell. The extract was then analyzed by GC/MS without any further cleanup or concentration step. The accuracy, precision, linearity, and detection limits (LODs) were evaluated to assess the performance of the proposed method. Quantitative recoveries were obtained, and relative standard deviation values were lower than 10% in all cases. The obtained LODs ranged from 0.000004% to 0.0001% (w/w), values far below the established restrictions in the European Cosmetics Regulation, making this multicomponent analytical method suitable for routine control. Finally, several cosmetic products such as moisturizing and antiwrinkle creams and lotions, hand creams, sunscreen and after-sun creams, baby lotions, and hair care products were analyzed. All the samples contained several of the target cosmetic ingredients, in some cases at quite high concentrations, although the actual European Cosmetics Regulation was fulfilled in all cases.

Full text of DOI:10.1021/ac101985h

Anal. Chem. 2010, 82, 9384–9392
Simultaneous In-Cell Derivatization Pressurized
Liquid Extraction for the Determination of
Multiclass Preservatives in Leave-On Cosmetics
Lucia Sanchez-Prado, J. Pablo Lamas, Marta Lores, Carmen Garcia-Jares, and Maria Llompart*
Departamento de Quimica Analitica, Nutricion y Bromatologia, Campus Sur, Universidad de Santiago de Compostela,
E-15782 Santiago de Compostela, Spain
An effective one-step sample preparation methodology for
the determination of multiclass preservatives in cosmetics
has been developed, applying, for the first time to this
kind of matrix, pressurized liquid extraction (PLE) and a
very simple, cheap, and fast derivatization procedure:
acetylation with acetic anhydride and pyridine. A multi-
factorial experimental design has been used to evaluate
and optimize the main experimental parameters poten-
tially affecting the extraction process. In the final condi-
tions the sample was mixed with Florisil as the dispersing
sorbent and extracted with ethyl acetate for 15 min at 120
°C. One of the main goals of this work was to demonstrate
the possibility of carrying out direct cosmetic preservative
acetylation by simply adding the derivatization reagents
into the PLE cell. The extract was then analyzed by GC/
MS without any further cleanup or concentration step. The
accuracy, precision, linearity, and detection limits (LODs)
were evaluated to assess the performance of the proposed
method. Quantitative recoveries were obtained, and rela-
tive standard deviation values were lower than 10% in all
cases. The obtained LODs ranged from 0.000004% to
0.0001% (w/w), values far below the established restric-
tions in the European Cosmetics Regulation, making this
multicomponent analytical method suitable for routine
control. Finally, several cosmetic products such as mois-
turizing and antiwrinkle creams and lotions, hand creams,
sunscreen and after-sun creams, baby lotions, and hair
care products were analyzed. All the samples contained
several of the target cosmetic ingredients, in some cases
at quite high concentrations, although the actual Euro-
pean Cosmetics Regulation was fulfilled in all cases.
and personal-care products to prevent or retard bacterial growth.
Parabens are the most widely used antimicrobial preservatives in
cosmetic products. Their antimicrobial activity is generally selec-
tive, so their mixtures or mixtures with other classes of preserva-
tives offer powerful antimicrobial activity against an extremely
broad spectrum of microorganisms.1
2-tert-Butyl-4-methoxyphenol (BHA) and 2,6-bis(1,1-dimethyl-
ethyl)-4-methylphenol (BHT) are antioxidant preservatives fre-
quently used to prevent oxidation in foods and cosmetics. The
use of mixtures of both of them is very common since there is a
synergic increase of their antioxidant power.2
Together with the positive protective effects of cosmetic
preservatives, unintended possible side effects of these ingredients
are a matter of concern, because exposure to some of these com-
pounds could have harmful effects on human health. Some of
these ingredients, such as parabens and BHA, may modulate and
disrupt the endocrine system,3 IPBC could cause acute inhalation
toxicity,4 and some compounds such as BHA or some transforma-
tion products of triclosan, Bronidox, and Bronopol are even
suspected carcinogenics.5-7 There is also current scientific
evidence that indicates that the use or misuse of biocidal products
may contribute to the increased occurrence of antibiotic-resistant
bacteria, both in humans and in the environment.8
To ensure a high level of protection of human health, cosmetic
products are regulated and controlled worldwide. The new
European Union (EU) Cosmetic Products Regulation9 (which is,
(1) Ingredients Prohibited & Restricted by FDA Regulations. http://www.fda.gov/
Cosmetics/ProductandIngredientSafety/SelectedCosmeticIngredients/
ucm127406.htm (accessed July 2010).
(2) Polati, S.; Gosetti, F.; Gennaro, M. C. In Analysis of Cosmetic Products;
Salvador, A., Chisvert, A., Eds.; Elsevier: New York, 2007, p 211.
(3) DHI Water and Environment. Study on Enhancing the Endocrine Disrupter
Priority List with a Focus on Low Production Volume Chemicals; Revised
Report to DG Environment; DHI: Hersholm, Denmark, 2007. http://
ec.europa.eu/environment/endocrine/documents/final_report_2007.pdf.
Preservatives are substances added to cosmetics for the
primary purpose of inhibiting the development of microorganisms
(antimicrobial function), but may also be added to protect such
products against damage and degradation caused by the exposure
to oxygen (antioxidant function).
The esters of p-hydroxybenzoic acid (parabens), iodopropynyl
butylcarbamate (IPBC), 2,4,4′-trichloro-2′-hydroxydiphenyl ether
(triclosan, TCS), and bromine-containing preservatives such as
5-bromo-5-nitro-1,3-dioxane (Bronidox) and 2-bromo-2-nitropro-
pane-1,3-diol (Bronopol) are included in a wide variety of cosmetics
(4) Lanigan, R. S. Int. J. Toxicol. 1998, 17 (Suppl. 5), 1–37
(5) Matyska, M. T.; Pesek, J. J.; Yang, L. J. Chromatogr., A 2000, 887, 497–
503
.
.
(6) International Agency for Research on Cancer. IARC Monographs on the
Evaluation of Carcinogenic Risks to Humans; World Health Organization:
Geneva, Switzerland, 1996; Vol. 40. http://monographs.iarc.fr/ENG/
Monographs/vol40/volume40.pdf#12.
(7) Cosmetic Ingredient Review. Safety of Triclosan as a Preservative in
Cosmetics. http://www.cir-safety.org/newreports.shtml (accessed July
2010).
(8) Scientific Committee on Emerging and Newly Identified Health Risks
(SCENIHR). Assessment of the Antibiotic Resistance Effects of Biocides;
European Commission: Brussels, Belgium, 2009.
* Corresponding author. Phone: 34-981563100, ext. 14225. E-mail:
maria.llompart@usc.es.
(9) Off. J. Eur. Union 2009, L342, 59-209.
9384 Analytical Chemistry, Vol. 82, No. 22, November 15, 2010
10.1021/ac101985h  2010 American Chemical Society
Published on Web 10/27/2010

to a great extent, a recast of the previous Cosmetics Directive10
and its successive amendments and adaptations), the federal Food,
Drug and Cosmetic Act (FD&C Act) and the Fair Packaging and
Labeling Act (FPLA) drawn up by the Food and Drug Administra-
tion (FDA) in the United States, and, finally, the Pharmaceutical
Affairs Law (PAL) adopted in Japan constitute the three main
regulatory systems on cosmetic products. The preservatives
allowed in the EU context are listed in Annex VI of the EU
Cosmetics Regulation,9 where limitations, requirements, label
warnings, and the maximum permissible concentrations are
indicated (see Table S-1 (Supporting Information) for the target
preservatives of this study). In Japanese legislation there is also
a positive list of preservatives, but the allowed substances and
authorized contents are quite different.11 In the U.S. framework
there is not a positive list of preservatives, although there is a
short list of substances, published by the FDA, banned or
restricted in cosmetics, including different compounds formerly
used as preservatives.1
Thus, to protect consumer health and ensure compliance to
existing government regulations, there is a need for the develop-
ment of effective and convenient methodologies to identify and
determine preservatives in cosmetics both accurately and sensitively.
A great part of the analytical effort has been focused on paraben
determination,12-15 while methods for the determination of other
preservatives in cosmetic formulations are very limited or inex-
istent. However, multicomponent analytical methods are required
since cosmetic products very often contain mixtures of preserva-
tives belonging to different chemical classes. Simultaneous
analysis of more than one class of preservatives is scarce and
mainly based on liquid chromatography (LC)16-18 and capillary
electrophoresis (CE).19,20 Flow injection analysis (FIA) has also
been employed, enhancing sample throughput.21
croextraction (SPME)23 have been recently applied for the
determination of different additives in cosmetics.
Pressurized liquid extraction (PLE) has been applied for the
analysis of cosmetic ingredients (parabens and TCS, among them)
in environmental matrixes, such as sewage sludge.24,25 PLE is fast,
increases automation, decreases the amount of organic solvents,
and offers the possibility of controlling the selectivity of the
extraction by loading different sorbents instead of inert materials
into the extraction cell.
Due to the polar nature of most preservatives, a derivatization
step previous to gas chromatography (GC) analysis is highly
recommended to reduce adsorption in the chromatographic
system and improve sensitivity, peak separation, and peak
symmetry.14,22 Acetylation is one of the most common derivati-
zation procedures for phenolic compounds,26,27 and it has been
applied for the determination of parabens and triclosan in
water,27,28 but to our knowledge, this derivatization procedure has
never before been employed for cosmetic samples. The advan-
tages of acetylation are the high efficiency obtained using low-
cost reagents, especially compared with silylation agents.
The aim of this work is to develop a method based on PLE
with acetylation followed by gas chromatography/mass spectrom-
etry (GC/MS) for the simultaneous determination of different
classes of preservatives including two bromine-containing preser-
vatives, seven parabens, IPBC, TCS, and the antioxidant preserva-
tives BHA and BHT in multimatrix cosmetic samples. The
possibility of performing simultaneous derivatization and extrac-
tion by adding the acetylation reagents into the PLE cell will be
evaluated. To our knowledge, both acetylation and PLE are applied
for the first time to the analysis of cosmetics.
MATERIALS AND METHODS
In most of these procedures, sample preparation is usually
performed through several steps which can include solvent
extraction or dilution, mixing, sonication, heating, addition of acids
or bases, centrifugation, and filtration. These procedures are
frequently tedious and time-consuming, and the use of hazardous
solvents is usually required. In addition, the possible presence of
interferences that could distort the results is not rejectable. To
overcome some of these drawbacks, supercritical fluid extraction
(SFE),16,22 solid-phase extraction (SPE),12 and solid-phase mi-
Chemicals. Bronidox (g99.0%) was acquired from Fluka
(Buchs, Switzerland). Bronopol (98%), methylparaben (99%, MeP),
ethylparaben (99%, EtP), propylparaben (99%, PrP), butylparaben
(99%, BuP), benzylparaben (99%, BzP), butylated hydroxyanisole
(g98.5%, BHA), butylated hydroxytoluene (99%, BHT), IPBC
(97%), and triclosan (g97.0%, TCS) were purchased from Aldrich
(Milwaukee, WI). Isopropylparaben (g99%, iPrP) and isobutylpa-
raben (g97%, iBuP) were purchased from TCI Europe (Belgium).
Table S-1 (Supporting Information) shows the IUPAC names and
chemical structures of the studied compounds.
Deuterated methyl 4-hydroxybenzoate-2,3,5,6-d4 (MePd4, 98.3
atom % D) was obtained from C/D/N Isotopes (Quebec,
Canada). The internal standard PCB-30 (2,4,6-trichlorobiphenyl)
was purchased from Dr. Ehrenstorfer (Augsburg, Germany).
Acetone, ethyl acetate, n-hexane, pyridine, and acetic anhydride
(Ac2O) were provided by Merck (Darmstadt, Germany). Florisil
(60-100 mesh) and C18 (70-230 mesh) were obtained from
(10) Council Directive 76/768/EEC of 27 July 1976 on the Approximation of the
Laws of the Member States Relating to Cosmetic Products; European Union:
Brussels, Belgium.
(11) Ministry of Health and Welfare. Standards for Cosmetics. http://www.
mhlw.go.jp/english/topics/cosmetics/index.html (accessed July 2010).
(12) Ma´rquez-Sillero, I.; Aguilera-Herrador, E.; Ca´rdenas, S.; Valca´rcel, M.
J. Chromatogr., A 2010, 1217, 1–6
(13) Wang, S.-P.; Chang, C.-L. Anal. Chim. Acta 1998, 377, 85–93
(14) Saraji, M.; Mirmahdieh, S. J. Sep. Sci. 2009, 32, 988–995
(15) Melo, L. P.; Queiroz, M. E. C. J. Sep. Sci. 2010, 33, 1849–1855
(16) Lee, M.-R.; Lin, C.-Y.; Li, Z.-G.; Tsai, T.-F. J. Chromatogr., A 2006, 1120,
244–251
(17) Wu, T.; Wang, C.; Wang, X.; Ma, Q. Int. J. Cosmet. Sci. 2008, 30, 367–372
(18) Gagliardi, L.; Cavazzutti, G.; Turchetto, L.; Manna, F.; Tonelli, D. J. Chro-
matogr., A 1990, 508, 252–258
(19) Wang, J.; Zhang, D.; Chu, Q.; Ye, J. Chin. J. Chem. 2010, 28, 313–319
(20) Huang, H.-Y.; Lai, Y.-C.; Chiu, C.-W.; Yeh, J.-M. J. Chromatogr., A 2003,
993, 153–164
(21) Garc´ıa-Jime´nez, J. F.; Valencia, M. C.; Capita´n-Vallvey, L. F. Anal. Chim.
Acta 2007, 594, 226–233
(22) Yang, T.-J.; Tsai, F.-J.; Chen, C.-Y.; Yang, T. C.-C.; Lee, M.-R. Anal. Chim.
Acta 2010, 668, 188–194
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(23) Tsai, T.-F.; Lee, M.-R. Chromatographia 2008, 67, 425–431
(24) Nieto, A.; Borrull, F.; Marce´, R. M.; Pocurull, E. J. Chromatogr., A 2009,
1216, 5619–5625
(25) Nieto, A.; Borrull, F.; Pocurull, E.; Marce´, R. M. TrAC, Trends Anal. Chem.
2010, 29, 752–764
(26) Llompart, M. P.; Lorenzo, R. A.; Cela, R.; Pare´, J. R. J.; Be´langer, J. M. R.;
Li, K. J. Chromatogr., A 1997, 757, 153–164
(27) Regueiro, J.; Becerril, E.; Garcia-Jares, C.; Llompart, M. J. Chromatogr., A
2009, 1216, 4693–4702
(28) Regueiro, J.; Llompart, M.; Psillakis, E.; Garcia-Monteagudo, J. C.; Garcia-
Jares, C. Talanta 2009, 79, 1387–1397
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Analytical Chemistry, Vol. 82, No. 22, November 15, 2010 9385

Aldrich (Milwaukee, WI). Before being used, Florisil was
activated at 130 °C for 12 h and then allowed to cool in a
desiccator. Sodium sulfate anhydrous (99%) was purchased by
Panreac (Barcelona, Spain).
Individual stock solutions of each compound were prepared
in acetone. Further dilutions and mixtures were prepared in
acetone, hexane, hexane/acetone (1:1, v/v), and ethyl acetate.
All solutions were stored in amber glass vials at -20 °C. All
solvents and reagents were of analytical grade.
matography Systems). Separation was carried out on an HP5
capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness)
from Agilent Technologies (Palo Alto, CA). Helium (purity
99.999%) was employed as the carrier gas at a constant column
flow of 0.8 mL min-1. Two different GC oven temperature
programs were tested. The first was used for the derivatization
studies, and it consisted of the following: 45 °C (held 2 min)
to 100 °C at 8 °C min-1, to 150 °C at 20 °C min-1, to 200 °C at
25 °C min-1 (held 5 min), to 220 °C (held 1 min) at 8 °C min-1
,
and a final ramp to 260 °C (held 7 min) at 30 °C min-1. The
second program was optimized to keep good resolution of the
target compounds, increasing the sample throughput: 60 °C
(held 2 min) to 200 °C at 30 °C min-1 and a final ramp to 260
°C (held 4 min) at 40 °C min-1 (total analysis time 15 min).
The injector was programmed to return to the split mode after
2 min from the beginning of a run. The split flow was set at 20
mL min-1. The injector temperature was held constant at 260
°C. The trap, manifold, and transfer-line temperatures were 220,
120, and 280 °C, respectively.
The GC/MS system was operated by Saturn GC/MS Worksta-
tion v5.52 software. In the full scan mode the mass range was
varied from 50 to 320 m/z at 0.6 s scan-1, starting at 4 min and
ending at 15 min. The filament emission current was 15 µA.
The analytes were positively identified by comparison of their
mass spectra and retention times to those of the standards.
Statistical Analysis. Basic and descriptive statistics and
experimental design analysis were performed using Statgraphics-
Plus v5.1 (Manugistics, Rockville, MD) as the software package.
The experimental design was applied in the optimization of the
extraction method to analyze the simultaneous effect of the main
parameters affecting PLE.
Acetylation was carried out by adding 100 µL of acetic
anhydride containing 2.5% pyridine to 1 mL of the standard or
extract solutions. The mixture was then maintained at 80 °C for
30 min and then allowed to cool to room temperature.
Cosmetic Samples. Different cosmetics from national and
global companies were purchased from local stores. They included
moisturizing and antiwrinkle creams and lotions, hand creams,
sunscreen and after-sun creams, and baby lotions. Two products
for hair care were also considered. Samples were kept in their
original containers at room temperature until their analysis.
A 0.5 g portion of cosmetic sample was weighed exactly into
a 10 mL glass vial. When it was necessary, the sample was spiked
with 50 µL of the corresponding acetone solution of the target
compounds to get the desired final concentration in the cosmetic
sample. The sample was then mixed with 1 g of a drying agent
(anhydrous Na2SO4) and 1 g of dispersing sorbent (C18 or
Florisil).
PLE and Derivatization Procedures. Extractions were
performed on an ASE 200 system (Dionex Co., Sunnyvale, CA)
equipped with a 24-sample carousel, 11 mL stainless steel cells,
and 40 mL collection vials. Two cellulose filters (Dionex) were
placed at each end of the PLE cell. The sample, mixed with the
drying agent and the dispersing sorbent, was introduced into the
cell, where previously 1 g of clean sand (50-70 mesh particle
size, Sigma-Aldrich) was placed. In all experiments, 20 µL of
MePd4 surrogate solution (2500 µg mL-1) was added to each
sample before extraction. Finally, the dead volume of the cell
was filled with sand. The cell was tightly closed and placed
into the carousel of the ASE system. Extractions were per-
formed by preheating the cell before filling with solvent
(preheat method). The extraction pressure was set to 1500 psi,
the flush volume was 60%, and the purge time was set to 60 s.
Hexane/acetone (1:1, v/v) or ethyl acetate was employed as
the extraction solvent, depending on the experiment. The
extraction temperature and extraction time varied during the
optimization of the method. After extraction, 20 µL of PCB 30
(100 µg mL-1) was added to the final extract (∼15 mL) to
correct possible variations of the extract volume. Then PLE
extracts were derivatized and analyzed by GC/MS.
RESULTS AND DISCUSSION
Derivatization and GC/MS Analysis. Optimization of the
chromatographic conditions was accomplished using a standard
mixture solution of all target compounds in n-hexane. Direct
analysis produced peaks with appreciable tailing for most com-
pounds due to the interaction of hydroxyl groups with the
chromatographic system. Therefore, a derivatization step was
introduced prior to GC determination to improve the chromato-
graphic analysis. Acetylation with acetic anhydride is one of the
most simple and cheap derivatization procedures for phenolic
compounds. The procedure to obtain standard solutions of the
corresponding acetylated compounds was based on a previous
work dealing with the acetylation of other phenolic species26 and
a recent study including some of the target compounds,28 and it
is described in the Materials and Methods.
Different families of preservatives are studied in this work
(Table S-1, Supporting Information), and for some of these
compounds no previous studies on their acetylation reaction were
found (e.g., Bronopol). It is necessary to ensure which compounds
undergo derivatization and to demonstrate the chromatographic
benefits of this reaction. Figure 1 shows the extracted ion
chromatograms before (A) and after (B) acetylation, and Figure
S-1 (Supporting Information) compares the chromatographic
responses obtained. The retention times and the quantification
and identification ions for the nonderivatized and derivatized
analytes are included in Table S-2 (Supporting Information).
In the simultaneous derivatization-extraction experiments, 100
µL of acetic anhydride containing 2.5% pyridine was added to the
cosmetic sample before the addition of the drying agent and the
dispersing sorbent. Then the PLE procedure previously described
was carried out. Finally, the extracts were directly analyzed since
in-cell derivatization was accomplished during extraction.
GC/MS Analysis. Analyses were performed on a Varian CP
3900 gas chromatograph (Varian Chromatography Systems,
Walnut Creek, CA) equipped with a 1079 split/splitless injector
and an ion trap spectrometer, Varian Saturn 2100 (Varian Chro-
9386 Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

Figure 1. Extracted ion chromatograms corresponding to a 10 µg mL-1 solution of the target analytes before (A) and after (B) derivatization.
Bronidox and IPBC do not undergo derivatization since these
compounds do not have chemical groups susceptible to acetyla-
tion; the retention times are not modified, and neither are their
chromatographic responses (Figures 1 and S-1, Supporting
Information).
On the contrary, parabens and triclosan are acetylated under
selected conditions. This fact is confirmed because of the
displacement of the retention times (see Figure 1), as well as the
improvement in the peak shapes, since the tailing observed in
the nonderivatized species disappears and peaks completely
symmetric are obtained. This improvement is especially noticeable
for MeP, BzP, and TCS; for these compounds, responses are also
significantly higher (Figure S-1, Supporting Information). Ad-
ditionally, small differences can also be observed in the obtained
mass spectra. The ratio of ion intensities is slightly modified when
the derivatization takes place (see as an example MeP in Figure
S-2, Supporting Information). The molecular ions corresponding
to the acetylated derivatives were not present in the mass spectra
Analytical Chemistry, Vol. 82, No. 22, November 15, 2010 9387

in most cases. This absence has been previously reported as a
result of the loss of the acetyl group upon ionization.28,29 Complete
acetylation can be assured since nonderivatized species were not
detected.
extractions.33 The flush volume and purge time were set at 60%
and 60 s, respectively. The influence of the remaining variables
was studied using a multifactor strategy. The study consisted of
a complete factorial 2∧4 design, involving 16 randomized
experiments and allowing 5 degrees of freedom to estimate
the experimental error. This design has resolution V, which
means that it is capable of evaluating all main effects and all
two-factor interactions. Numerical analysis of data resulting
from the experimental design was made with the statistical
software package Statgraphics-Plus v5.1. The experiments were
performed using 0.5 g of a real moisturizing cream sample
initially labeled as containing some of the target compounds
(Bronopol, MeP, BHT, and PrP) and fortified with all com-
pounds at 100 µg g-1. Since drying of the sample is essential
for an efficient PLE, in all experiments 1 g of anhydrous sodium
sulfate was added. Sand was employed to avoid dead volume.
The studied factors were the extraction temperature (A),
extraction solvent (B), dispersing sorbent (C), and extraction
time (D).
The temperature factor (A) was studied at 80 and 120 °C. The
choice of an appropriate solvent is another essential aspect in the
development of extraction methods. For an efficient extraction,
the solvent must solubilize the target compounds while leaving
the sample matrix as intact as possible.33 Two solvents (factor B)
were investigated: hexane/acetone (1:1, v/v) and ethyl acetate.
The inclusion of an in situ cleanup step by adding certain
sorbents to the PLE cell contributes to obtaining clean extracts.
In this way, lipids and other coextractable matrix materials are
prevented from coming out to the extract. In addition, these
sorbents can act as a dispersing phase, contributing to the
execution of a more efficient extraction. Thus, 1 g of dispersing
sorbent (factor C), C18 or Florisil, was mixed with the sample
and packed in the PLE cell. The last factor considered was the
extraction time (factor D), and it was assessed at 5 and 15 min.
The 16 experiments were carried out; after extraction, the
extracts were acetylated at 80 °C for 30 min before GC/MS
analysis (see the Materials and Methods). Numerical analysis of
the results obtained leads to the analysis of variance (ANOVA)
results shown in Table 1. As can be seen, the most important
factor, with statistical significance for most of the target com-
pounds, is the extraction solvent. The extraction time was also
significant for many analytes, whereas the temperature and the
dispersing sorbent were each significant for five compounds.
Some second-order effects are also important, especially interac-
tions AB (temperature-solvent) and BD (solvent-time).
The information included in the ANOVA can be graphically
plotted by means of the Pareto charts. In Figure S-3 (Supporting
Information), some representative graphics are shown. The length
of each bar is proportional to the absolute value of its associated
standardized effect. The vertical line in the graphs represents the
statistically significant bound at the 95% confidence level.
Figure 2 shows the main effects diagrams for several repre-
sentative compounds. This kind of plot shows the main effects
with a line drawn between the low and the high levels of the
Regarding Bronopol, the effect of the derivatization on the peak
shape and chromatographic response is much more evident as
can be seen in Figures 1 and S-1 (Supporting Information). The
retention time was also considerably modified (more than 1 min).
In addition, the mass spectrum of the acetylated derivative differs
significantly from the spectrum of Bronopol (see both spectra in
Figure S-2). In this compound two hydroxyl groups are present
(Table S-1), which means that the acetylation can take place in
two reaction centers. In fact, in this case, the molecular ion
corresponding to the doubly acetylated compound was identified
in the mass spectrum (m/z 283), confirming the above hypothesis.
In addition, a cluster of ions, typical of bromine-containing
compounds, around m/z 195 and 197 corresponding to the loss
of two CH3CO groups is present. The base peak (m/z 115) was
formed by the subsequent loss of the bromine atom.
BHA also undergoes derivatization (see Figure 1). The most
intense fragment ions for the acetylated derivative (see Figure
S-2, Supporting Information) were also formed by the loss of
CH3CO, in such a way that the mass spectrum of the derivative
was similar to that of the nonderivatized compound, with the
exception of the ratio of ion intensities and the presence of
the acetylated BHA molecular ion at m/z 222.
In the case of BHT, the acetylation could not be demonstrated
since the retention time, peak shape, chromatographic response
(Figure 1), and mass spectra were equivalent before and after
the addition of the acetylation reagents. The highly hindered
hydroxyl group with poor nucleophilicity (see the structure in
Table S-1, Supporting Information) may prevent the acetylation
under the studied conditions. This is in agreement with the study
of Monsef-Mirzai,30 who demonstrated that very hindered phenols,
such as BHT, remain unacetylated. Anyway, the underivatized
BHT peak shape and chromatographic response are both
satisfactory.
In summary, three of the compounds (Bronidox, IPBC, and
BHT) did not undergo derivatization. For the other compounds,
the reaction yield was quantitative, since we could not find any
trace of the underivatized analytes, and satisfactory, improving
significantly the chromatographic analysis of the target com-
pounds both qualitatively and quantitatively. The reaction was also
carried out with standard solutions in ethyl acetate and hexane/
acetone (1:1, v/v), demonstrating the suitability of these solvents
to accomplish derivatization. The acetylated derivatives were stable
for at least several weeks.
PLE Optimization. The influence of the main variables
potentially affecting the PLE process must be evaluated to obtain
an efficient extraction. In the usual working range for this
technique, the pressure generally has a negligible effect on the
extraction yield,31,32 so we decided to conduct the experiments
at 1500 psi, which is the standard operating pressure in PLE
(29) Croley, T. R.; Lynn, B. C., Jr. Rapid Commun. Mass Spectrom. 1998, 12,
(32) EPA Method 3545, Pressurised Fluid Extraction. Test Methods for Evaluating
Solid Waste, Physical/Chemical Methods, 3rd ed.; Final Update IV; EPA SW-
846; U.S. Government Printing Office: Washington, DC, 2008.
(33) Methods Optimization in Accelerated Solvent Extraction (ASE); Technical
Note 208; Dionex Corp.: Sunnyvale, CA, 2004.
171–175
(30) Monsef-Mirzai, P. Fuel 1996, 75, 1684–1687
(31) Carabias-Mart´ınez, R.; Rodr´ıguez-Gonzalo, E.; Revilla-Ruiz, P.; Herna´ndez-
Me´ndez, J. J. Chromatogr., A 2005, 1089, 1–17
.
.
.
9388 Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

Table 1. F Ratios and p Valuesa Obtained in the Analysis of Variance
main effects
interactions
AD
(A)
temperature
(B)
(C)
(D)
time
solvent
dispersant
AB
AC
BC
BD
F
P
F
p
F
p
F
p
F
p
F
p
F
p
F
p
F
p
ratio value ratio value ratio value ratio value ratio value ratio value ratio value ratio value ratio value
Bronidox
Bronopol
MeP
BHA
BHT
EtP
20
5
+
11
15
19
28
1
19
10
31
40
54
36
+
+
+
+
8
+
1
4
14
40
2
28
17
6
19
35
41
11
3
18
6
+
8
+
22
1
+
3
21
6
+
0.3
0.1
2
5
0
5
+
+
5
4
9
2
2
5
16
1
+
10
5
+
+
0.01
1
3
13
1
+
0
0.2
3
1
12
7
+
+
+
+
+
+
+
+
2
+
+
13
7
+
+
6
0.01
0.01
0.1
0.01
0.1
0.3
0.2
0.4
9
+
+
iPrP
1
2
3
8
PrP
0
4
1
0.2
4
1
1
IPBC
iBuP
BuP
8
+
37
+
+
+
+
+
+
+
+
12
11
12
1
+
+
+
5
10
5
+
5
7
9
7
6
3
6
0.1
2
6
7
+
+
+
BzP
TCS
1
0.5
1
0.3
1
0.2
1
11
12
1
0
a Key: + cell, p value <0.05; empty cell, p value >0.05.
Figure 2. Main effects plots for some representative compounds (EtAc ) ethyl acetate; Hex/Acet ) hexane/acetone).
corresponding factors. The length of the lines is proportional to
the effect magnitude of each factor in the extraction process,
and the sign of the slope indicates the level of the factor that
produces the highest response. Regarding the significant factors
B and D (see the ANOVA in Table 1), the best extractions were
obtained at the high level of the factors for all compounds, which
means ethyl acetate and 15 min. The other two main factors A
and C were significant for less compounds (Table 1) but, in those
cases, were also characterized by a positive slope, so better
extractions were also achieved at the high level of the factors,
120 °C and Florisil.
Before a general method for the simultaneous extraction of
the 13 target compounds is proposed, it is necessary to examine
the interaction effects, since some of them, especially AB and BD
(see Table 1), were significant for several analytes. These second-
order effects are shown in Figure 3 for some analytes, as an
example, since the trends were equivalent in all cases. Although
the slopes of the lines are quite different, the lines do not intercept,
so the general conditions established after analysis of the main
effects do not change. Interaction AB shows again as the most
favorable conditions the extraction at 120 °C using ethyl acetate.
Regarding the BD effect, the most favorable conditions were ethyl
acetate and 15 min, although it is interesting to notice that in
general the time is only significant when hexane/acetone is used.
An exception to this behavior was BzP and TCS (see the BzP
plot in Figure 3). For these two compounds, the most favorable
conditions would involve the extraction with hexane/acetone for
15 min.
In view of the results of the experimental design, the selected
general conditions for the simultaneous extraction of the target
preservatives and antioxidants were established as follows: extrac-
tion temperature of 120 °C, ethyl acetate as solvent, Florisil as
dispersing sorbent, and extraction time of 15 min.
Analytical Chemistry, Vol. 82, No. 22, November 15, 2010 9389

Figure 3. Interaction effects plots: AB (temperature-solvent) and BD (solvent-time).
Table 2. Quality Parameters of the Proposed Methoda
recoveryb (RSD) (%) (n ) 3)
instrumental parameters
derivatization after extraction
in-cell derivatization
compd
R2
IDL (ng mL-1
)
20 µg g-1
100 µg g-1
20 µg g-1
100 µg g-1
LOD (%, w/w)
LOQ (%, w/w)
Bronidox
Bronopol
MeP
BHA
BHT
EtP
0.9971
1.0000
0.9991
0.9996
0.9994
1.0000
0.9992
0.9965
0.9946
0.9971
0.9988
0.9998
0.9977
5.6
18
73.7 (1.5)
nc
98.3 (2.7)
98.2 (7.3)
94.8 (9.5)
93.0 (2.3)
98.1 (0.5)
101 (0.4)
101 (0.8)
107 (0.1)
90.9 (5.2)
96.5 (1.6)
97.2 (2.3)
99.1 (9.1)
110 (5.3)
97.9 (1.5)
83.5 (3.7)
nc
87.9 (8.0)
107 (4.0)
109 (8.7)
95.4 (8.1)
nc
90.6 (7.9)
102 (6.4)
108 (1.1)
88.9 (4.1)
93.6 (8.4)
85.7 (9.0)
88.4 (1.9)
113 (4.1)
90.1 (4.1)
105 (0.3)
111 (0.9)
104 (1.8)
89.7 (6.8)
100 (2.7)
97.0 (4.8)
106 (5.1)
104 (1.3)
111 (2.9)
0.000094
0.00015
0.00031
0.00051
0.000018
0.000027
0.000013
0.000027
0.000033
0.000019
0.00028
0.000022
0.000020
0.00023
0.00013
1.0
nc
0.0000053
0.0000081
0.0000041
0.0000080
0.0000098
0.0000058
0.000085
0.0000065
0.0000060
0.000068
0.000040
1.2
110 (0.6)
91.0 (5.8)
95.5 (2.2)
100 (7.0)
99.3 (8.4)
94.5 (5.3)
104 (6.0)
101 (4.2)
95.5 (7.2)
109 (4.5)
0.41
1.4
iPrP
1.7
PrP
1.0
IPBC
iBuP
BuP
2.3
0.86
0.64
2.0
BzP
TCS
0.73
a nc ) not calculated since the concentrations in the sample were much higher than the added concentration. b Real sample MC1 (Table 3) was
employed in the recovery studies.
Experiments were also run with the objective of studying the
possibility of performing in-cell derivatization of the target
compounds in the PLE cell. In the simultaneous derivatization-
extraction experiments, 100 µL of acetic anhydride containing 2.5%
pyridine was added to the cosmetic sample and the PLE procedure
was carried out in the selected conditions indicated above. The
initial results were fully satisfactory, obtaining equivalent extracts,
and as a consequence, both processes, PLE followed by deriva-
tization, as well as the simultaneous pressurized liquid deriva-
tization-extraction, were considered for method validation.
Method Performance. Application to Real Samples.
Method quality parameters were evaluated (Table 2). The
instrumental linearity was proved at a concentration range
between 0.05 and 10 µg mL-1 (including six concentration
levels) using derivatized standard solutions prepared in ethyl
acetate (see the Materials and Methods). Each concentration
level was injected in triplicate, and the response function was
found to be linear with determination coefficients (R2) higher
than 0.9946.
Instrumental detection limits (IDLs) were calculated as the
concentration giving a signal-to-noise ratio of 3 (S/N ) 3). Values
ranged from 0.41 to 18 ng mL-1, as can be seen in Table 2
.
The other figures of merit were calculated using real cosmetic
samples.
Recovery studies were carried out by applying the optimized
PLE method to the extraction of a real cream sample spiked at
two different concentrations, 20 and 100 µg g-1. Previous analyses
of this sample showed the presence of some of the target
compounds (see sample MC1 in Table 3), and these initial
concentrations were taken into account to calculate the recoveries.
9390 Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

As can be seen in Table 2, the recoveries were between 74% and
110% in all cases. The precision was also evaluated, and the relative
standard deviation (RSD) values were lower than 10% with an
average value of 4.2%.
As was commented, the possibility of performing simultaneous
derivatization-extraction by adding the acetylation reagents in
the PLE cell was also evaluated. Recoveries are also given in Table
2. As can be seen, the recoveries were satisfactory, with values
ranging from 84% to 111%. The precision of the method expressed
as the RSD was between 1% and 9%. Performing the combined
derivatization-extraction process, the method quality parameters
are equivalent and the method is even more simple and time
saving.
The limits of detection (LODs) and quantification (LOQs) of
the overall method were calculated as the compound concentration
giving a signal-to-noise ratio of 3 (S/N ) 3) and 10 (S/N ) 10),
respectively. These values are shown in Table 2, expressed as a
percentage (w/w) to be equivalent with the units used in the
European Cosmetics Regulation.9 The obtained limits are much
lower than the established restrictions (see Table S-1, Supporting
Information), and it is important to emphasize that, if necessary,
these limits could be easily reduced (by at least 1 order of
magnitude) by concentrating the PLE extract (∼15 mL).
Finally, the method was applied to the analysis of real cosmetic
samples including moisturizing creams (MCs) and lotions (MLs),
antiwrinkle (AW) creams, hand creams (HCs), sunscreen creams
(SCs), after-sun (AS) creams, baby lotions (BLs), and hair
conditioning (CO) and shampoo (SH) products. The results are
shown in Table 3. The extracted ion chromatogram of sample HC1
is shown in Figure S-4 (Supporting Information). For all the
samples, the recoveries of MePd4 (surrogate standard) were
satisfactory, with values ranging from 83.7 to 115 (see the first
row, Table 3). As commented in the first section of this paper,
the presence of these ingredients must be included in the cosmetic
label, and these levels cannot exceed the regulated limit in each
case. Regarding parabens, the compounds mainly found were MeP
and PrP; both compounds are usually associated with an increase
in the preservative activity. EtP, iBuP, and BuP were also found
in the samples but much less frequently. The maximum allowed
concentration of parabens in ready for use preparations is 0.4%
for a single ester and 0.8% for mixtures of esters, expressed as
acid (see the European Cosmetics Regulation limits in Table S-1).
For this reason, the total content of parabens in the samples was
determined and expressed as a percentage (w/w) of acid, being
included in the last row of Table 3. All samples presented paraben
concentrations below the legal limits, although one of the samples,
a baby moisturizing lotion (BL2), was close to the total paraben
maximum concentration limit. Most of the samples were correctly
labeled with the exception of EtP, iBuP, and BuP in HC2, PrP in
AW1 and MC5, and iPrP in CO, which were not included in the
label. The antioxidant BHT was found in most of the samples,
whereas BHA was found in four samples, in two of them associated
with BHT, which increases the antioxidant power due to the
synergism. Although there is some concern about the safety of
both compounds, there are no restrictions about their use in
cosmetic formulations. The presence of BHT and BHA was not
indicated in the label with the exception of HC2 and BL1. IPBC
was found in one rinse-off product (SH), and it was included in
Analytical Chemistry, Vol. 82, No. 22, November 15, 2010 9391

the product label. Finally, Bronopol was detected in one leave-on
cosmetic (MC1), and in this case, it was also listed as an
ingredient.
control. The reliability of the method was demonstrated through
a broad range of cosmetic products showing compliance with the
actual European Cosmetics Regulation.
CONCLUSIONS
ACKNOWLEDGMENT
A method based on acetylation PLE followed by GC/MS for
the simultaneous determination of different classes of preserva-
tives, including two bromine-containing preservatives, seven
parabens, IPBC, TCS, and the antioxidant preservatives BHA and
BHT, in multimatrix cosmetic samples has been developed. To
our knowledge, both acetylation and PLE are applied for the first
time to the analysis of cosmetics. We have demonstrated the
possibility of performing simultaneous in situ derivatization by
adding the acetylation reagents directly on the cosmetic sample
into the PLE cell, making possible the GC/MS analysis of the
extract without any further step. The obtained LODs are far below
the established restrictions in the European Cosmetics Regulation,
making this multicomponent analytical method suitable for routine
This research was supported by FEDER funds and Project
CTQ2010-19831 (Ministerio de Ciencia e Innovacion, Spain).
L.S.-P. and J.P.L. acknowledge Xunta de Galicia for postdoctoral
Angeles Alvarin˜o and Isabel Barreto contracts, respectively.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in the text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review July 30, 2010. Accepted October 12,
2010.
AC101985H
9392 Analytical Chemistry, Vol. 82, No. 22, November 15, 2010