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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
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
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
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.
Table 1
Elution gradient program used in the liquid chromatography analysis.
t (min)
0
4
10
11
11.1
14
60
60
100
100
60
60
40
40
0
0
40
40
Absorbance (mAU)
Time (min)
Relative stability
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).
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
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
Table 3
Obtained concentrations (%, w/w) of the target compounds in the analyzed cosmetic samples.
Samplea
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
a)
Absorbance (mAU)
b)
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.
Table 4
Recovery values (%) obtained by applying the proposed method to four samples spiked with known amounts of the analytes.
Analyte
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.
[1] Regulation (EC) No. 1233/2009 of the European Parliament and of the Council
of 30 November 2009 on cosmetic products. http://eur-lex.europa.eu/legal-