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Talanta journal homepage: www.elsevier.com/locate/talanta Determination of alternative preservatives in cosmetic

Determination of alternative preservatives in cosmetic products by chromophoric derivatization followed by vortex-assisted liquid liquid semimicroextraction and liquid chromatography

Pablo Miralles, Ilianna Vrouvaki, Alberto Chisvert, Amparo Salvador n

Ilianna Vrouvaki, Alberto Chisvert, Amparo Salvador n Departamento de Química Analítica, Facultad de Química,

Departamento de Química Analítica, Facultad de Química, Universitat de València, 46100 Burjassot, Valencia, Spain

article info

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

abstract

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 spec- trophotometric detection after chromophoric derivatization with benzoyl chloride and vortex-assisted liquid liquid 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 m g mL 1 . The method was validated with good recovery values (84 118%) and precision values (3.9 9.5%). It was successfully applied to 10 commercially available cosmetic samples. The good analytical features of the proposed method besides of its environmentally- friendly 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.

1. Introduction

According to the European Regulation of Cosmetic Products (EC Regulation) [1] , cosmetic productmeans 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, chan- ging their appearance, protecting them, keeping them in good con- dition or correcting body odours . Typical cosmetic formulation in- cludes active principles, excipients, and additives (colorants, per- fumes, 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 con- centration, 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 In- gredients (2006/257/EC) [3] , phenethyl alcohol (2-phenylethanol,

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

0039-9140/ & 2016 Elsevier B.V. All rights reserved.

PA) is used in cosmetics as deodorant; methylpropanediol (2- methyl-1,3-propanediol, MP) and phenylpropanol (3-phenyl-1- propanol, PP) are commonly employed as solvents; caprylyl glycol (1,2-octanediol, CG) acts as emollient, humectant and hair con- ditioning; ethylhexylglycerin (3-[(2-ethylhexyl)oxy]-1,2-propane- diol, EG) is used as skin conditioning. However, all of them show an important antimicrobial activity [4 13] and their use in the cosmetic industries is widespread due to their antimicrobial properties, being the subject of several patents [6 , 14 17] . Despite their preservative features, these compounds, whose chemical structures are shown in Fig. 1 , are not listed as pre- servatives 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 con- taining them. However, there are not of cial methods to quantify the target compounds in cosmetic samples. Besides, to the best of our knowledge, there are not published analytical methods re- garding to their determination. Only a few articles in which PA was determined in alcoholic beverages, such as wine [18 20] , beer [21 , 22] and alcoholic distillates [23] by chromatographic techni- ques have been published. Vortex-assisted extraction procedures have been successfully applied for sample preparation in the analysis of water [2426] , food and drinks [27 29] , and also cosmetic products [30 , 31] with

2

P. Miralles et al. / Talanta 154 (2016) 1 6

PPhenethyl al lcohol (PA) Me ethylpropane ediol (MP) Ph henylpropan nol (PP) (CAS 60 0-12-8)
PPhenethyl al lcohol (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)

(C AS 1117-86 6-8)

Ethy ylhexylglyce erin (EG)

CAS 70445

(C

5-33-9)

Fig. 1. Chemical structures of the target compounds.

good analytical features. Other applications of vortex-assisted techniques can be found in some recent reviews [32 35]. 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 ab-

sorptive 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 esteri cation reaction under soft conditions in basic medium [36 41] . In this sense, the chromophoric deri- vatization combined with vortex-assisted liquid liquid semi- microextraction (VALLsME) make possible their simultaneous de- termination with good sensitivity and low reagents and solvents consumption.

2. Experimental

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 modi er of the mobile phase. Extra-pure glacial acetic acid from Scharlab Chemie (Barcelona, Spain) was used to prepare the acetic acid solution used as aqu- eous mobile phase. LC-grade absolute ethanol from Scharlab Chemie (Barcelona, Spain) was also tested as organic modi er of the mobile phase. Benzoyl chloride 99% from Aldrich (Steinheim, Germany) was used as chromophoric derivatizing agent and reagent-grade so- dium 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 A D, moisturizing creams; samples E and F, sunscreen creams) and gels (samples G J, bath gels), were purchased at local stores.

 

2.3.

Proposed method

2.1.

Apparatus

 

2.3.1.

Preparation of sample solutions and standards

An Agilent 1220 In nity LC system including a degasser, a binary pump, an autosampler with up to 100 m L injection volume,

a thermostated column oven, and a UV/Vis detector was em-

ployed. The column was a Purospher s STAR RP-18 endcapped (12.5 cm length, 4 mm I.D., 5 m m particle size) from Merck (Darmstadt, Germany). A ZX3 vortex mixer from VELP Scienti ca (Usmate Velate, Italy) was used to ease the vortex-assisted liquid liquid 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 Scienti c (Staffordshire, United Kingdom) was used for the evaporation of the organic solvent prior the re- constitution of the extract.

2.2. Reagents and samples

PP (3-phenyl-1-propanol) 98%, CG (1,2-octanediol) 98%, PA (2- phenylethanol) 99%, MP (2-methyl-1,3-propanediol) 99%, all from Aldrich (Steinheim, Germany), and EG (3-[(2-ethylhexyl)oxy]-1,2- propanediol) 98% from Schülke&Mayr GmbH (Norderstedt, Ger- many) were used as standards. Deionized water obtained using a NANOpure II ultrapure water system from Barnstead (Boston, USA) was used as solvent to pre- pare the sample and standard solutions and the aqueous mobile phase.

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 m L 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 hot- plate set approximately at 60 ° C. Finally, the extract was recon- stituted adding 400 m L of acetonitrile. Regarding with the standard solutions, a stock solution con- taining all the analytes at 500 m g mL 1 was prepared using deio- nized water as solvent. From this solution, the calibration solutions (1 25 m g 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) 1 6

3

Table 1 Elution gradient program used in the liquid chromatography analysis.

t (min)

Acetonitrile (%, v/v)

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

0

6

0

4

0

4

6

0

4

0

10

100

0

11

100

0

11.1

60

40

14

60

40

Experimental conditions: injection volume ¼ 20 m L; ow rate ¼ 1 mL min 1 ; oven temperature ¼ 30 ° C; λ ¼ 235 nm.

Absorbance (mAU)
Absorbance (mAU)

Time (min)

Fig. 2. Chromatographic separation obtained applying the proposed method to a standard solution containing the derivatized analytes at 10 m g mL 1 .

Relative stability
Relative stability

Time (min)

Fig. 3. Relative stability (analytical signal/initial analytical signal) of phenethyl al- cohol performing the VALLsME (dotted line) and without performing the VALLsME (continuous line).

Table 2 Analytical gures of merit of the proposed method.

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

3.1.

Study of the chromatographic variables

Different variables may affect the chromatographic determi- nation, such as the mobile phase composition and the column temperature. With the purpose of obtaining a good chromatographic re- solution in a time as short as possible, the effect of the mobile phase composition was studied. A solution of acetic acid in deio- nized water (0.5%, v/v) was used as aqueous mobile phase. Al- though 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 so- lution as mobile phase allowed us to improve the chromatographic peak pro le, increasing the symmetry and reducing the tailing of the chromatographic peaks. Moreover, acetonitrile and ethanol were tested as organic modi ers, 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 acet- onitrile and aqueous 0.5% acetic acid solution as mobile phases, obtaining the best results when using the elution gradient pro- gram 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 differ- ences 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

Analyte Slope (mAU min mL l g 1 ) a

Intercept (mAU min) a

R 2 b

s y/x c LOD

 

LOQ

Precision (RSD, %) f

 

EF g

 

(

l g

mL 1 ) d

( l g mL 1 ) e

 
 
 

Intra-day

Inter-day

1 l g mL 1 10 l g mL 1 1 l g mL 1 10 l g mL 1

 

PA

710 7 10 2010 7 40 750 7 20 1650 7 80 1250 7 20

30 7 80 240 7 200 60 7 100 350 7 60 170 7 50

0.9995

8

0.03

 

0.11

3.9

4.7

6.7

6.9

13.1 7 0.9 13.6 7 1.2 12.6 7 0.7 11.5 7 0.4 10.7 7 0.6

MP

0.9990 40

0.06

0.19

6.2

6.5

8.7

8.4

PP

0.9995

5

0.02

0.06

6.9

6.7

9.5

9.1

CG

0.9998 30

0.06

0.18

6.0

6.2

8.6

8.5

EG

0.9994

9

0.02

0.07

6.8

7.0

9.2

9.3

a Parameters of the calibration curve obtained by simple linear regression. Working range: 1 25 m g mL 1 , n ¼ 6. Expressed as the value 7 standard deviation.

b Regression coef cient ( R 2 ) of the calibration curve.

c Residual standard deviation ( s y/x ) of the calibration curve.

d Limit of detection (LOD) estimated as 3 s y/x / b , being s y/x the residual standard deviation and b the slope of the calibration curve.

e Limit of quanti cation (LOQ) estimated as 10 s y/x / b , being s y/x the residual standard deviation and b the slope of the calibration curve.

f Precision expressed as relative standard deviation (RSD,

g Enrichment factor (EF). The values are expressed as the mean of three replicates 7 standard deviation.

%), n ¼ 10.

4

P. Miralles et al. / Talanta 154 (2016) 1 6

Absorbance (mAU)

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

Sample a

Concentration (%, w/w) b

 

PA

MP

PP

CG

EG

A

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 7 0.09 1.84 7 0.05

n.d. n.d. n.d. n.d. n.d. n.d. 0.078 7 0.005 0.084 7 0.006 0.087 7 0.006 0.076 7 0.004

n.d. n.d. 0.18 7 0.01 0.065 7 0.005 n.d. n.d. 0.51 7 0.07 0.62 7 0.04 0.59 7 0.02 0.55 7 0.03

n.d. n.d. n.d. 0.047 7 0.003 n.d. n.d. n.d. n.d. n.d. n.d.

B

n.d.

C

n.d.

D

n.d.

E

0.76 7 0.07

F

0.79 7 0.03

G

n.d.

H

n.d.

I

n.d.

J

n.d.

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

a Samples A F: creams; Samples G J: gels.

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

a)
a)
b)
b)
the mean of three replicates 7 standard deviation. a) b) c) enhance the analytical response of
c)
c)
mean of three replicates 7 standard deviation. a) b) c) enhance the analytical response of the

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 para- meters 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 m g mL 1 . 10, 20, and 30 m L of benzoyl chloride combined with 500 and

1000 m L of NaOH 5 M were tested, obtaining the best analytical signal when 20 m L of benzoyl chloride and 1000 m L 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 stu- died. 0.5, 1, 2, 3, and 5 min were tested as reaction time, obtaining the best results with 3 min of vortex mixing.

3.2.2. Study of the vortex-assisted liquid liquid semimicroextraction

(VALLsME) variables

After the derivatization reaction, VALLsME, using n-hexane as

extraction solvent and followed by reconstitution with acetoni- trile, was performed in order to preconcentrate the target com- pounds, 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 re- Section 3.2.3 ). The effect of the extraction volume, the extraction time, and the re- constitution volume were studied. As extraction volume, 0.5, 1, and 2 mL of n-hexane were tested. No signi cant 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 ex-

tract (upper phase). In order to increase the contact surface between phases, im- proving 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 m L of acetonitrile were tested to reconstitute the extract after the evaporation to dryness. m L of acetonitrile were tested to reconstitute the extract after the evaporation to dryness. Since all the tested volumes provided the required sensitivity, a 400 m L volume was nally chosen be- cause it provided the best compromise between sensitivity and

Time (min)
Time (min)

precision.the best compromise between sensitivity and Time (min) 3.2.3. Study of the stability of derivatives In

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

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.

P. Miralles et al. / Talanta 154 (2016) 1 6

5

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 A b

Sample B b

Sample C b

Sample G b

1 l g mL 1

10 l g mL 1

1 l g mL 1

10 l g mL 1

1 l g mL 1

10 l g mL 1

1 l g mL 1

10 l g mL 1

PA

102 7 5 87 7 7 97 7 5 85 7 9 104 7 4

105 7 8 8 4 7 3 107 7 5 106 7 3 115 7 7

9 9 7 4 9 0 7 6 91 7 3 118 7 8 10 5 7 8

8 6 7 3 9 4 7 4 106 7 3 97 7 6 9 2 7 5

9 4 7 6 9 2 7 3 97 7 4 10 8 7 9 9 8 7 5

110 7 9 8 4 7 7 108 7 7 106 7 5 100 7 6

9 0 7 7 n.c. 8 9 7 3 9 8 7 4 9 4 7 6

9 3 7 6 92 7 5 112 7 8 100 7 5 10 3 7 3

 

MP

PP

CG

EG

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

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

b Samples A C: 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 car- boxylate 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 per- forming the VALLsME and performing it. Similar results were ob- served 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 so- lutions containing the target compounds. These results are shown in Table 2 . The linearity studied reached at least to 25 m g mL 1 for all the analytes, obtaining a high level of linearity ( R 2 4 0.9990). The instrumental limits of detection values ranged from 0.02 to 0.06 m g mL 1 and the limits of quanti cation values ranged from 0.06 to 0.19 m g mL 1 (3 s y/x / b and 10 s y/x / b criteria respectively, being s y/x the residual standard deviation and b the slope of the calibration line). These low limits of detection and quanti cation 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 m g 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 m g mL 1 , and from 4.7% to 7.0% at 10 m g mL 1 . The inter-day precision values ranged from 6.7% to 9.5% at 1 m g mL 1 , and from 6.9% to 9.3% at 10 m g mL 1 . These results reveal that good precision was achieved. In order to evaluate both the enrichment factor and the ex- traction yield, standard solutions containing the analytes (10 m g 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.

3.4. Analysis of commercial samples

Samples were prepared by triplicate as speci ed in the pro- posed method (see Section 2.3 ) and injected into the chromato- graphic system under the selected conditions. A good

chromatographic resolution was obtained for all the tested sam- ples. The results obtained for the 10 analyzed cosmetic samples are shown in Table 3 . Fig. 4 shows, as an example, the chroma- tograms 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 m g mL 1 ). Finally, the recoveries were evaluated by determin- ing the concentration of the analytes in both spiked and non- spiked samples, applying the proposed method. The obtained re- coveries ( 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 liquid liquid semimicroextration (VALLsME) is

proposed for the determination of ve alternative preservatives in cosmetics products: phenethyl alcohol, methylpropanediol, phe- nylpropanol, caprylyl glycol and ethylhexylglycerin. These alter- native preservatives are not yet included in the current European Regulation on cosmetic products. The method allows the quanti cation of the target compounds in both fat- and water-soluble cosmetic samples with good ana- lytical features, such as accuracy and precision, as well as sec- ondary gures of merit such as simplicity and affordable proce- dure, making the proposed method useful for the quality control of cosmetic products containing the target compounds as pre- servative 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 princi- ples of the so-called Green Analytical Chemistry.

Acknowledgements

Authors wish to acknowledge the Spanish Ministerio de Economía y Competitividad for the nancial support (Project CTQ- 70301-R). P.M. also would like to thank the Spanish Ministerio de Educación, Cultura y Deporte for his predoctoral grant.

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