Talanta 154 (2016) 16

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Determination of alternative preservatives in cosmetic products by

chromophoric derivatization followed by vortex-assisted liquidliquid
semimicroextraction and liquid chromatography
Pablo Miralles, Ilianna Vrouvaki, Alberto Chisvert, Amparo Salvador n
Departamento de Qumica Analtica, Facultad de Qumica, Universitat de Valncia, 46100 Burjassot, Valencia, Spain

art ic l e i nf o

a b s t r a c t

Article history:
Received 12 February 2016
Received in revised form
10 March 2016
Accepted 11 March 2016
Available online 12 March 2016

An analytical method for the simultaneous determination of phenethyl alcohol, methylpropanediol,

phenylpropanol, caprylyl glycol, and ethylhexylglycerin, which are used as alternative preservatives in
cosmetic products, has been developed. The method is based on liquid chromatography with UV spectrophotometric detection after chromophoric derivatization with benzoyl chloride and vortex-assisted
liquidliquid semimicroextraction. Different chromatographic parameters, derivatization conditions, and
sample preparation variables were studied. Under optimized conditions, the limits of detection values for
the analytes ranged from 0.02 to 0.06 mg mL
1. The method was validated with good recovery values
(84118%) and precision values (3.99.5%). It was successfully applied to 10 commercially available
cosmetic samples. The good analytical features of the proposed method besides of its environmentallyfriendly characteristics, make it useful to carry out the quality control of cosmetic products containing
the target compounds as preservative agents.
& 2016 Elsevier B.V. All rights reserved.

Keywords:
Cosmetic products
Alternative preservatives
Liquid chromatography

1. Introduction
According to the European Regulation of Cosmetic Products (EC
Regulation) [1], cosmetic product means any substance or mixture
intended to be placed in contact with the external parts of the human
body (epidermis, hair system, nails, lips and external genital organs)
or with the teeth and the mucous membranes of the oral cavity with a
view exclusively or mainly to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odours. Typical cosmetic formulation includes active principles, excipients, and additives (colorants, perfumes, preservatives, etc.). The Annex V of this regulation contains
the list of the currently allowed preservative agents in cosmetics
and the restrictions to be used, including their maximum concentration, in order to ensure the safety of consumers.
After the recent prohibition of some parabens by the European
Commission [2], the cosmetic industries are continuously looking
for new compounds that can perform the preservative function
effectively and safely. In fact, it is well-known that some cosmetic
ingredients can play more than one role in a cosmetic formulation.
In this sense, according to the Inventory of Cosmetic Ingredients (2006/257/EC) [3], phenethyl alcohol (2-phenylethanol,
n

Corresponding author.
E-mail address: amparo.salvador@uv.es (A. Salvador).

http://dx.doi.org/10.1016/j.talanta.2016.03.033
0039-9140/& 2016 Elsevier B.V. All rights reserved.

PA) is used in cosmetics as deodorant; methylpropanediol (2methyl-1,3-propanediol, MP) and phenylpropanol (3-phenyl-1propanol, PP) are commonly employed as solvents; caprylyl glycol
(1,2-octanediol, CG) acts as emollient, humectant and hair conditioning; ethylhexylglycerin (3-[(2-ethylhexyl)oxy]-1,2-propanediol, EG) is used as skin conditioning. However, all of them show
an important antimicrobial activity [413] and their use in the
cosmetic industries is widespread due to their antimicrobial
properties, being the subject of several patents [6,1417].
Despite their preservative features, these compounds, whose
chemical structures are shown in Fig. 1, are not listed as preservatives in the above mentioned Annex V of the European
regulation.
It is therefore necessary that the cosmetic companies have
procedures to perform the analytical control of these alternative
preservatives and to assure the quality of the nal products containing them. However, there are not ofcial methods to quantify
the target compounds in cosmetic samples. Besides, to the best of
our knowledge, there are not published analytical methods regarding to their determination. Only a few articles in which PA was
determined in alcoholic beverages, such as wine [1820], beer
[21,22] and alcoholic distillates [23] by chromatographic techniques have been published.
Vortex-assisted extraction procedures have been successfully
applied for sample preparation in the analysis of water [2426],
food and drinks [2729], and also cosmetic products [30,31] with

P. Miralles et al. / Talanta 154 (2016) 16

Phenethyl allcohol (PA)

Me
ethylpropane
ediol (MP)

Ph
henylpropan
nol (PP)

(CAS 60
0-12-8)

(CAS 2163
3-42-0)

((CAS 1335--12-2)

Cap
prylyl glycol (CG)

ylhexylglyce
erin (EG)
Ethy

(CAS 1117-86
6-8)

CAS 70445
5-33-9)
(C

Fig. 1. Chemical structures of the target compounds.

good analytical features. Other applications of vortex-assisted

techniques can be found in some recent reviews [3235].
For the rst time, a liquid chromatography (LC) method with
UV/Vis spectrophotometric detection is proposed here for the
determination of these alternative preservatives, i.e., PA, MP, PP,
CG, and EG, in cosmetic samples.
Owing to the low UV/Vis absorbance of the target compounds,
a derivatization step is proposed to convert them to more absorptive derivatives, in order to enhance their analytical response
during the LC analysis. Benzoyl chloride is a common derivatizing
agent used to perform chromophoric derivatizations through a
simple and effective esterication reaction under soft conditions
in basic medium [3641]. In this sense, the chromophoric derivatization combined with vortex-assisted liquidliquid semimicroextraction (VALLsME) make possible their simultaneous determination with good sensitivity and low reagents and solvents
consumption.

LC-grade n-hexane 96% from Scharlab Chemie (Barcelona,

Spain) was used as extraction solvent in the VALLsME procedure.
LC-grade acetonitrile from VWR International (Pennsylvania,
USA) was used as solvent to reconstitute the extracts after the
VALLsME and as organic modier of the mobile phase.
Extra-pure glacial acetic acid from Scharlab Chemie (Barcelona,
Spain) was used to prepare the acetic acid solution used as aqueous mobile phase.
LC-grade absolute ethanol from Scharlab Chemie (Barcelona,
Spain) was also tested as organic modier of the mobile phase.
Benzoyl chloride 99% from Aldrich (Steinheim, Germany) was
used as chromophoric derivatizing agent and reagent-grade sodium hydroxide (NaOH) from Scharlab Chemie (Barcelona, Spain)
was used to prepare the basic solutions needed to carry out the
derivatization reaction.
Ten commercial cosmetic samples with different formulations,
including creams (samples AD, moisturizing creams; samples E
and F, sunscreen creams) and gels (samples GJ, bath gels), were
purchased at local stores.

2. Experimental
2.3. Proposed method
2.1. Apparatus
An Agilent 1220 Innity LC system including a degasser, a
binary pump, an autosampler with up to 100 mL injection volume,
a thermostated column oven, and a UV/Vis detector was employed. The column was a Purosphers STAR RP-18 endcapped
(12.5 cm length, 4 mm I.D., 5 mm particle size) from Merck
(Darmstadt, Germany).
A ZX3 vortex mixer from VELP Scientica (Usmate Velate, Italy)
was used to ease the vortex-assisted liquidliquid extraction in the
preparation of cosmetic samples and an EBA 21 centrifuge from
Hettich (Tuttlingem, Germany) was used for phase separation. A
hotplate from Stuart Scientic (Staffordshire, United Kingdom)
was used for the evaporation of the organic solvent prior the reconstitution of the extract.
2.2. Reagents and samples
PP (3-phenyl-1-propanol) 98%, CG (1,2-octanediol) 98%, PA (2phenylethanol) 99%, MP (2-methyl-1,3-propanediol) 99%, all from
Aldrich (Steinheim, Germany), and EG (3-[(2-ethylhexyl)oxy]-1,2propanediol) 98% from Schlke&Mayr GmbH (Norderstedt, Germany) were used as standards.
Deionized water obtained using a NANOpure II ultrapure water
system from Barnstead (Boston, USA) was used as solvent to prepare the sample and standard solutions and the aqueous mobile
phase.

2.3.1. Preparation of sample solutions and standards

Samples were prepared by weighing approximately 0.1 g into a
5 mL volumetric ask and diluted up to the line with deionized
water. The solution was centrifuged and/or ltered when needed
to remove the non-soluble compounds and an aliquot of 0.5 mL
was placed into a 5 mL volumetric ask. Then, 1 mL of NaOH 5 M
and 20 mL of benzoyl chloride were added and the ask was stirred
vigorously for 3 min using a vortex mixer (40 Hz) in order to
perform the chromophoric derivatization. Then, deionized water
was used to complete the total volume of the volumetric ask.
After that, the solution was transferred into a polypropylene
conical-bottom tube and 1 mL of n-hexane was added to carry out
the VALLsME by vortex mixing for 30 s. Then, both phases were
separated by centrifugation (6000 rpm, 2 min). The upper organic
extract was collected and transferred into a LC injection vial and it
was then evaporated to dryness under a fume hood using a hotplate set approximately at 60 C. Finally, the extract was reconstituted adding 400 mL of acetonitrile.
Regarding with the standard solutions, a stock solution containing all the analytes at 500 mg mL
1 was prepared using deionized water as solvent. From this solution, the calibration solutions
(125 mg mL
1) were prepared daily and were subjected to the
same derivatization and extraction processes than samples.
2.3.2. Chromatographic analysis
Twenty microliters of the standard or sample solution were

P. Miralles et al. / Talanta 154 (2016) 16

Table 1
Elution gradient program used in the liquid chromatography analysis.
t (min)

Acetonitrile (%, v/v)

Aqueous 0.5% acetic acid (%, v/v)

0
4
10
11
11.1
14

60
60
100
100
60
60

40
40
0
0
40
40

injected into the column set at 30 C. The elution was performed at

1 mL min
1 ow rate with a mixture of acetonitrile:aqueous 0.5%
acetic acid solution as mobile phase, following the elution gradient
program shown in Table 1. The detection wavelength for signal
monitoring was xed at 235 nm and the runtime was completed
with 14 min.

3. Results and discussion

Absorbance (mAU)

Experimental conditions: injection volume 20 mL; ow rate 1 mL min
1; oven

temperature 30 C; 235 nm.

Time (min)

Relative stability

Fig. 2. Chromatographic separation obtained applying the proposed method to a

standard solution containing the derivatized analytes at 10 mg mL
1.

Time (min)
Fig. 3. Relative stability (analytical signal/initial analytical signal) of phenethyl alcohol performing the VALLsME (dotted line) and without performing the VALLsME
(continuous line).

3.1. Study of the chromatographic variables

Different variables may affect the chromatographic determination, such as the mobile phase composition and the column
temperature.
With the purpose of obtaining a good chromatographic resolution in a time as short as possible, the effect of the mobile
phase composition was studied. A solution of acetic acid in deionized water (0.5%, v/v) was used as aqueous mobile phase. Although the analytes are in their neutral form in the whole range of
column pH compatibility, according to their predicted pKa values
(ranged from 13.6 to 15.2), the use of an aqueous acetic acid solution as mobile phase allowed us to improve the chromatographic
peak prole, increasing the symmetry and reducing the tailing of
the chromatographic peaks.
Moreover, acetonitrile and ethanol were tested as organic
modiers, obtaining the best results when acetonitrile was used,
due to its lower absorbance at the selected wavelength for signal
monitoring (235 nm).
Different programs of gradient elution were tested using acetonitrile and aqueous 0.5% acetic acid solution as mobile phases,
obtaining the best results when using the elution gradient program shown in Table 1.
The effect of the column temperature was also studied. Oven
temperatures from 30 C to 50 C were tested. No signicant differences were observed, so 30 C was selected for further analysis in
order to set the system at xed conditions, although the analysis can
be performed at room temperature without temperature control.
With those conditions, a good chromatographic separation of
the ve derivatives was achieved in less than 14 min, as can be
seen in Fig. 2.
3.2. Study of the sample preparation variables
3.2.1. Study of the chromophoric derivatization variables
A chromophoric derivatization was carried out in order to

Table 2
Analytical gures of merit of the proposed method.
Analyte Slope
(mAU min mL lg
1)a

Intercept
(mAU min)a

R2b

sy/xc LOD
(lg mL
1)d

LOQ
(lg mL
1)e

Precision (RSD, %)f

EFg

Intra-day
1 lg mL
PA
MP
PP
CG
EG

7107 10
20107 40
750 7 20
1650 7 80
1250 7 20

307 80
2407 200

607 100
3507 60
1707 50

0.9995
8
0.9990 40
0.9995
5
0.9998 30
0.9994
9

0.03
0.06
0.02
0.06
0.02

0.11
0.19
0.06
0.18
0.07

3.9
6.2
6.9
6.0
6.8

1

Inter-day
10 lg mL
4.7
6.5
6.7
6.2
7.0

1

1 lg mL
1 10 lg mL
1
6.7
8.7
9.5
8.6
9.2

6.9
8.4
9.1
8.5
9.3

13.17 0.9
13.6 7 1.2
12.6 7 0.7
11.5 7 0.4
10.7 7 0.6

Parameters of the calibration curve obtained by simple linear regression. Working range: 125 mg mL
1, n6. Expressed as the value 7 standard deviation.
Regression coefcient (R2) of the calibration curve.
c
Residual standard deviation (sy/x) of the calibration curve.
d
Limit of detection (LOD) estimated as 3 sy/x/b, being sy/x the residual standard deviation and b the slope of the calibration curve.
e
Limit of quantication (LOQ) estimated as 10 sy/x/b, being sy/x the residual standard deviation and b the slope of the calibration curve.
f
Precision expressed as relative standard deviation (RSD, %), n10.
g
Enrichment factor (EF). The values are expressed as the mean of three replicates7 standard deviation.
a

P. Miralles et al. / Talanta 154 (2016) 16

Table 3
Obtained concentrations (%, w/w) of the target compounds in the analyzed cosmetic samples.
Samplea

Concentration (%, w/w)b

A
B
C
D
E
F
G
H
I
J

PA

MP

PP

CG

EG

n.d.
n.d.
n.d.
n.d.
0.767 0.07
0.79 7 0.03
n.d.
n.d.
n.d.
n.d.

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.88 7 0.07
1.95 7 0.05
1.72 70.09
1.84 7 0.05

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.07870.005
0.084 7 0.006
0.087 7 0.006
0.076 70.004

n.d.
n.d.
0.187 0.01
0.0657 0.005
n.d.
n.d.
0.517 0.07
0.62 7 0.04
0.59 7 0.02
0.55 7 0.03

n.d.
n.d.
n.d.
0.0477 0.003
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

n.d.: Not detected, analytes were not included in sample formulation. See text for experimental details.
a
b

Samples AF: creams; Samples GJ: gels.

The values are expressed as the mean of three replicates 7standard deviation.

enhance the analytical response of the target compounds. In the

preparation of standard and sample solutions, benzoyl chloride
was chosen as derivatizing reagent and a solution of NaOH in
deionized water (5 M) was chosen as solvent to get the basic
medium needed to perform the derivatization. Different parameters such as the volumes of the reagents and the reaction time
were studied. The following tests were performed employing
standard solutions containing the target analytes at 25 mg mL
1.
10, 20, and 30 mL of benzoyl chloride combined with 500 and
1000 mL of NaOH 5 M were tested, obtaining the best analytical
signal when 20 mL of benzoyl chloride and 1000 mL of NaOH 5 M
were used.
In order to ease the derivatization, the reagents were shaken
using a vortex-mixer and the effect of the reaction time was studied. 0.5, 1, 2, 3, and 5 min were tested as reaction time, obtaining
the best results with 3 min of vortex mixing.

a)

Absorbance (mAU)

b)

3.2.2. Study of the vortex-assisted liquidliquid semimicroextraction

(VALLsME) variables
After the derivatization reaction, VALLsME, using n-hexane as
extraction solvent and followed by reconstitution with acetonitrile, was performed in order to preconcentrate the target compounds, to avoid possible matrix interferences and to prevent the
degradation of the derivatized analytes (see Section 3.2.3). The
effect of the extraction volume, the extraction time, and the reconstitution volume were studied.
As extraction volume, 0.5, 1, and 2 mL of n-hexane were tested.
No signicant differences were observed, so 1 mL of n-hexane was
chosen for further analysis in order to reduce the consumption of
organic solvent and to ease the subsequent separation of the extract (upper phase).
In order to increase the contact surface between phases, improving the extraction effectiveness, the mixture was shaken using
a vortex-mixer. 10, 30, 60, and 90 s were tested as extraction time,
obtaining the best results with 30 seconds of vortex mixing.
Regarding to the reconstitution volume, 100, 250, 400, and
500 mL of acetonitrile were tested to reconstitute the extract after
the evaporation to dryness. Since all the tested volumes provided
the required sensitivity, a 400 mL volume was nally chosen because it provided the best compromise between sensitivity and
precision.

c)

d)

Time (min)
Fig. 4. Chromatographic separation obtained for four examples of the cosmetic
samples analyzed: (a) sample C, (b) sample D, (c) sample E and (d) sample G.

3.2.3. Study of the stability of derivatives

In an early stage of the method development, the sample and
standard solutions were measured after the derivatization reaction
without performing the VALLsME. Although the concentrations of
the analytes in the solution after the derivatization were enough to

P. Miralles et al. / Talanta 154 (2016) 16

Table 4
Recovery values (%) obtained by applying the proposed method to four samples spiked with known amounts of the analytes.
Analyte

Recovery values (%)a

Sample Ab

PA
MP
PP
CG
EG

Sample Bb

Sample Cb

Sample Gb

1 lg mL
1

10 lg mL
1

1 lg mL
1

10 lg mL
1

1 lg mL
1

10 lg mL
1

1 lg mL
1

10 lg mL
1

102 75
87 77
97 75
85 79
104 74

1057 8
847 3
1077 5
106 7 3
1157 7

997 4
907 6
917 3
1187 8
1057 8

867 3
947 4
106 7 3
977 6
927 5

947 6
927 3
977 4
1087 9
987 5

1107 9
847 7
1087 7
106 7 5
1007 6

907 7
n.c.
897 3
987 4
947 6

937 6
927 5
1127 8
1007 5
1037 3

n.c.: Not calculated, the analyte was present in sample G at high concentration.
a
b

The values are expressed as the mean of three replicates7 standard deviation.
Samples AC: creams; Sample G: gel.

quantify them, it was observed that their stability was low in the
basic aqueous medium, due to their chemical structure as carboxylate esters.
The VALLsME allowed us to increase the sensitivity of the
proposed method but also it was a way to stabilize the derivatized
analytes. As an example, the relative stability (analytical signal/
initial analytical signal) of PA is shown in Fig. 3, without performing the VALLsME and performing it. Similar results were observed with the other target compounds.
3.3. Analytical gures of merit of the proposed method
Quality parameters of the proposed method were evaluated
under the selected conditions (see Section 2.3) with standard solutions containing the target compounds. These results are shown
in Table 2.
The linearity studied reached at least to 25 mg mL
1 for all the
analytes, obtaining a high level of linearity (R2 40.9990).
The instrumental limits of detection values ranged from 0.02 to
0.06 mg mL
1 and the limits of quantication values ranged from
0.06 to 0.19 mg mL
1 (3 sy/x/b and 10 sy/x/b criteria respectively,
being sy/x the residual standard deviation and b the slope of the
calibration line). These low limits of detection and quantication
in addition to the linear range observed, allowed us to determine
the target compounds in a wide range of concentrations.
The precision, expressed as relative standard deviation (RSD),
was evaluated by applying the entire proposed method to ten
replicates of standard solutions containing the analytes at 1 and
10 mg mL
1 in the same day (intra-day precision) and in different
days (inter-day precision). The intra-day precision values ranged
from 3.9% to 6.9% at 1 mg mL
1, and from 4.7% to 7.0% at
10 mg mL
1. The inter-day precision values ranged from 6.7% to
9.5% at 1 mg mL
1, and from 6.9% to 9.3% at 10 mg mL
1. These
results reveal that good precision was achieved.
In order to evaluate both the enrichment factor and the extraction yield, standard solutions containing the analytes
(10 mg mL
1) were analyzed both directly and after performing the
VALLsME procedure under the selected conditions (see Section
2.3.1). The analytical signals were compared and the obtained
values for the enrichment factor (analytical signal with VALLsME/
analytical signal without VALLsME) ranged from 10.7 to 13.6 (see
Table 2). Considering the experimental procedure, these results
shown that the extraction yield achieved for the target compounds
could be considered quantitative in all the cases.

chromatographic resolution was obtained for all the tested samples. The results obtained for the 10 analyzed cosmetic samples
are shown in Table 3. Fig. 4 shows, as an example, the chromatograms obtained for some of them.
In order to study the matrix effects, four cosmetic samples were
prepared by triplicate according to the proposed method, and then
spiked with the target analytes at two concentration levels (1 and
10 mg mL
1). Finally, the recoveries were evaluated by determining the concentration of the analytes in both spiked and nonspiked samples, applying the proposed method. The obtained recoveries (Table 4) ranged from 84% to 118%, thus showing that
matrix effect was negligible and conventional calibration can be
used as described in the proposed method.

4. Conclusions
A new LC method with chromophoric derivatization followed
by vortex-assisted liquidliquid semimicroextration (VALLsME) is
proposed for the determination of ve alternative preservatives in
cosmetics products: phenethyl alcohol, methylpropanediol, phenylpropanol, caprylyl glycol and ethylhexylglycerin. These alternative preservatives are not yet included in the current European
Regulation on cosmetic products.
The method allows the quantication of the target compounds
in both fat- and water-soluble cosmetic samples with good analytical features, such as accuracy and precision, as well as secondary gures of merit such as simplicity and affordable procedure, making the proposed method useful for the quality control of
cosmetic products containing the target compounds as preservative agents.
In addition, the use of small volumes of organic solvents, such
as acetonitrile or n-hexane, makes the proposed method safe for
both the operator and the environment, according to the principles of the so-called Green Analytical Chemistry.

Acknowledgements
Authors wish to acknowledge the Spanish Ministerio de
Economa y Competitividad for the nancial support (Project CTQ70301-R). P.M. also would like to thank the Spanish Ministerio de
Educacin, Cultura y Deporte for his predoctoral grant.

3.4. Analysis of commercial samples

References
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