Accepted Manuscript Title: Determination of atranol and chloroatranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry Author: Marina L´opez-Nogueroles Alberto Chisvert Amparo Salvador PII: DOI: Reference:
S0003-2670(14)00378-X http://dx.doi.org/doi:10.1016/j.aca.2014.03.042 ACA 233179
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
16-9-2013 21-3-2014 30-3-2014
Please cite this article as: Marina L´opez-Nogueroles, Alberto Chisvert, Amparo Salvador, Determination of atranol and chloroatranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Determination of atranol and chloroatranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry.
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Marina López-Nogueroles, Alberto Chisvert*, Amparo Salvador
6 Departamento de Química Analítica, Facultad de Química, Universitat de València, 46100 Burjassot, Valencia, Spain
9 * Corresponding author: Tel.: +34 96 354 49 00; Fax: + 34 96 354 44 36
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e-mail address: [email protected] (A. Chisvert)
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Graphical abstract Abstract
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► The potent allergens atranol and chloroatranol are determined in perfumes by GCMS ► LLE is used to clean up the sample solution from lipophilic substances ► In situ derivatization and DLLME is used to remove polar compounds and concentrate ► The method presents good analytical features ► The method has been assayed in four real perfume samples
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26 27 28 29 30 31 32 33 34 35 36 37 38 39
Highlights
A new analytical method based on simultaneous derivatization and dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS), for the determination of the allergenic compounds atranol and chloroatranol in perfumes, is presented. Derivatization of the target analytes by means of acetylation with anhydride acetic in carbonate buffer was carried out. Thereby volatility and detectability were increased for improved GC-MS
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sensitivity. In addition, extractability by DLLME was also enhanced due to a less polar character of the solutes. A liquid-liquid extraction was performed before DLLME to clean up the sample and to obtain an aqueous sample solution, free of the low polar matrix from the essential oils, as donor phase. Different parameters, such as the nature and volume of both the extraction and disperser solvents, the ionic strength of the aqueous donor phase or the effect of the derivatization reagent volume, were optimized. Under the selected conditions (injection of a mixture of 750 µL of acetone as disperser solvent, 100 µL of chloroform as extraction solvent and 100 µL of anhydride acetic as derivatization reagent) the figures of merit of the proposed method were evaluated. Limits of detection in the low ng mL
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range were obtained. Matrix
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effect was observed in real perfume samples and thus, standard addition calibration is recommended.
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Keywords:
Dispersive
liquid-liquid
microextraction;
Simultaneous
derivatization;
Gas
chromatography-mass
spectrometry; Atranol; Chloroatranol; Allergens.
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46 1. Introduction
A list of 26 fragrance chemicals classified as potentially allergenic substances (PASs)
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was first included in the European Union (EU) Directive on cosmetic products in 2003 [1] and
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they are also compiled in the current EU Regulation [2]. It was established that, unlike other
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fragrance chemicals, their presence in any cosmetic product has to be declared on the label
52
when present at a higher concentration than 0.001 % in those products to remain on the skin or
53
0.01 % in those intended to be rinsed off. 24 of these PASs are chemically defined volatile
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compounds while the other two are natural moss extracts, i.e. oakmoss extract (Evernia
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prunastri extract) and treemoss extract (Evernia furfuracea extract), which are not a defined
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compound, but a complex mixture of natural products.
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Since then, many works dealing with the determination of these skin-sensitizing
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ingredients can be found in the literature, especially regarding those 24 chemically defined
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PASs [3-8]. However, the number of publications regarding the determination of the two natural
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moss extracts is rather limited, and surely it presents an important challenge. Indeed, Joulain et
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al. published two comprehensive reviews on the composition of these two extracts identifying
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170 different constituents in oakmoss extracts and 90 in the case of treemoss [9,10]. It should
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be noted that their natural conditions and a not standardized industrial processing makes the
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composition of these extracts variable. The industrial processing of these two natural extracts
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usually consists on the extraction with hexane or other solvents of the harvested lichens
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followed by dilution with ethanol and submission to physical treatments to remove their original
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colour.
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From all the different compounds present in oakmoss and treemoss extracts, atranol
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and chloroatranol are very potent allergens [11-13]. These two compounds are degradation
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products, formed after transesterification and decarboxylation of the lichen depsides atranorin
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and chloroatranorin, and are formed in the moss absolute production [14]. It is worth mentioning
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that the EU Regulation, which only restricts total concentration of oakmoss and treemoss
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extracts, does not list these individually allergenic components of mosses and their
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quantification is not indicative of the total amount of the regulated moss. However the EU
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Scientific Committee on Consumer Products (SCCP) stated that these compounds are such
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potent allergens they should not be present at all in cosmetic products [15] so it can be
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expected they will be banned or restricted in the coming years. Therefore, works dealing with
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their determination could be useful in the future, specially taking into account the lack of
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analytical methods available for this purpose.
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In this sense, Bernard et al. [11] searched for allergenic molecules in oakmoss extract
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using GC-MS after chemical fractionation of the extract by gel permeation chromatography.
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David et al. [16] semiquantitatively determined chloroatranol in moss extracts by GC-MS using a
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programmed temperature vaporizing inlet with an automated liner exchange. The liner was
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packed with polydimethylsiloxane that retained non-volatile material. Hiserodt et al. [14]
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developed a qualitative method using liquid chromatography-tandem mass spectrometry (LC-
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MS/MS) to identify atranorin and some related potential allergens, including atranol and
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chloroatranol, in oakmoss absolute. Bossi et al. [17] further developed this method to quantify
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atranol and chloroatranol in perfumes. The method was based on direct injection of the sample
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and limits of detection (LOD) of 5 ng mL
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respectively, were obtained. The recovery of chloratranol from spiked perfumes was 89±10%.
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Low recoveries (49±6%) were observed for atranol in spiked perfumes, indicating ion
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suppression caused by matrix components. Same authors also applied this method to quantify
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the presence of these two allergenic compounds in different commercial perfumes [18, 19].
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and 2.4 ng mL
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for atranol and chloroatranol
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It is worth mentioning that perfumes are elaborated by mixing different natural raw
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materials or complex synthetic formulations that commonly contain hundreds or thousands of
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different compounds. This fact complicates the determination of compounds in this matrix. Thus,
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an effective sample preparation, that enables a significant clean-up, is crucial to obtain a good
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accuracy. The aim of this work is to develop a method to determine atranol and chloroatranol in
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perfumes with a good accuracy.
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In this work, liquid-liquid extraction (LLE) is used to clean up the sample solution from
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lipophilic substances. This step is followed by a simultaneous derivatization and dispersive
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liquid-liquid microextraction (DLLME) step that enables to change the solvent (for further GC-
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MS analysis) and to concentrate the target compounds. Moreover it provides a second, less
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crucial, clean up from possible components that would have remained in the polar phase with
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the analytes after the LLE process.
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It should be pointed out that the derivatization step is proposed since atranol and
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chloroatranol are relatively polar compounds, and without it, a poor extraction efficiency would
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be achieved in the DLLME process. Moreover, the derivatization allows volatility and thus
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increases sensitivity in GC-MS. The chosen approach was an in situ derivatization via
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acetylation of the hydroxyl groups under basic aqueous conditions, using acetic anhydride as
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acetylating agent. The derivatization is performed at the same time as DLLME, allowing the
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simplification of the procedure and a decrease in the time of analysis. This one step
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derivatization and DLLME was first introduced by Fattahi et al. to determine clorophenols in
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waters [20] and has been later used by other authors to determine phenols in a similar
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approach although also performing other types of derivatization reactions such as silylation [21,
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22] and O-alkoxycarbonylation [23, 24].
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2. Experimental
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2.1 Reagents and samples
Atranol (97%) and chloroatranol (98.9%), purchased from Ambinter (Orleans, France),
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were used as standards. O-cresol, purchased from Aldrich, was used as surrogate. Figure 1
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shows the chemical structure of the three of them.
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Liquid chromatography grade absolute ethanol from Scharlau (Barcelona, Spain), was
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used as solvent to prepare dilute solutions of the standards. Deionised water (resistivity 18
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M cm) obtained by means of a Nanopure II water purification system from Barnstead (Boston,
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MA, USA) was used to prepare the working standard solutions.
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Analytical reagent grade chloroform and dichloromethane, from Scharlau Chemie
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(Barcelona, Spain), were tested as extraction solvents. Ethanol, ultrapure acetone and liquid
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chromatography grade acetonitrile, all from Scharlau Chemie, were tested as disperser
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solvents. Analytical reagent grade sodium chloride 99% from Scharlau Chemie was used to
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adjust the ionic strength. Acetic anhydride extra pure and anhydrous diethyl ether from Scharlau
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Chemie, and potassium carbonate analysis grade from Panreac (Barcelona, Spain) were used
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in the derivatization of the compounds.
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High purity helium (99.9999%) from Carburos Metalicos S.A. (Paterna, Spain) was used as carrier gas in the GC-MS system.
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Four different commercial perfumes from different brands (a women eau de toilette
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and eau de parfum, a man eau de parfum and a children eau de cologne) were purchased and
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stored at room temperature until analysis. For reasons of confidentiality, the manufacturers are
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not shown.
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141 2.2 Apparatus
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The GC-MS system used consisted of a Focus GC gas chromatograph coupled to a
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DSQII mass spectrometry detector equipped with an AI3000 autosampler, all from Thermo
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Fisher Scientific (Austin, TX, USA). The chromatographic separations were made using an HP-
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5MS Ultra Inert (95% dimethyl-5% diphenylpolysiloxane, 30 m length, 0.25 mm i.d., 0.25 µm film
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thickness) analytical fused-silica capillary column from Agilent Technologies (Santa Clara, CA,
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USA).
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An 8452A Hewlett Packard diode array UV/Vis spectrophotometer with a 1 cm path length quartz cell was used to determine the losses in the LLE process.
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An Avance 300 spectrometer from Bruker (Madrid, Spain) was employed to run H
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nuclear magnetic resonance (NMR) spectra at 300 MHz, using residual non-deuterated
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chloroform as internal standard ( 7.26 ppm).
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An EBA 21 centrifuge from Hettich (Tuttlingem, Germany) was also used.
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2.3 Method
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2.3.1. Internal standard calibration
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Standards preparation: 1 mL of each one of the ethanolic standard solutions
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containing the analytes (atranol and chloroatranol) in a concentration between 20 and 100 ng
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mL and the surrogate (o-cresol) in 40 ng mL were transferred to different 15 mL polyethylene
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centrifuge tubes. 1.5 mL of water and 1.5 mL of hexane were added and the mixture vortexed
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during 1 min. Then, the tubes were centrifuged for 3 min at 3000 rpm and the upper layers of
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hexane were collected with a syringe and discarded. The liquid-liquid extraction (LLE) process
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and subsequent discard of hexane was repeated two more times. After this, 2 mL of the
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remaining aqueous solutions were taken and transferred to other polyethylene centrifuge tubes.
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8 mL of water and 1 mL of a 1M potassium carbonate solution were added. A simultaneous
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derivatization and DLLME process was then performed injecting to these solutions different
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previously mixed solutions of 100 µL of chloroform (extraction solvent), 100 µL of acetic
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anhydride (derivatization reagent) and 750 µL of acetone (disperser solvent). The tubes were
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then centrifuged for 5 min at 6000 rpm. The sedimented phases were collected with a 100 μL
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Hamilton syringe and transferred into 100 µL inserts placed inside different injection vials, which
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were then ready to be injected into the chromatographic system.
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Samples preparation: 3 replicates of 1 mL of perfume sample were submitted to the same procedure.
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2.3.2. Standard addition combined with internal standard calibration -1
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Several aliquots of 1 mL of perfume sample were spiked, with a 2 µg mL ethanolic
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solution of the target analytes, in a concentration between 20 and 100 ng mL , and with a 2 µg
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mL
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procedure described in 2.3.1.
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ethanolic solution of the surrogate, in 40 ng mL , and they were submitted to the
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(As will be seen later, the results showed that standard addition calibration is
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recommended).
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2.3.3. GC-MS analysis.
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1 µL of each one of the aforementioned sedimented phases was injected into the GC
185
system coupled to a mass spectrometry detector operated in positive electron ionisation mode
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at ionisation energy of 70 eV and with a multiplier voltage set at 1300 V. The inlet temperature
187
was 280 ºC and the injection was accomplished in splitless mode (splitless time: 1 min). The
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−1
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separation was run at a 1 mL min
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was: from 80 ºC (1 min) to 200 ºC at 4 ºC min
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transfer line and ion source temperatures were set at 280 and 250 ºC, respectively. The
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chromatograms were recorded in selected ion monitoring (SIM) mode at the following
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mass/charge (m/z) ratios: m/z 108 from minute 5.0 to 11.0 for o-cresol, m/z 152 from minute
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20.0 to 28.0 for atranol and m/z 186 from minute 28.0 to 32.0 for chloroatranol. A full scan mode
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(m/z from 50 to 350) was simultaneously recorded from minute 5.0 to the end of the analysis
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time to confirm the identification of the analytes in real samples.
helium constant flow rate. The oven temperature program -1
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and finally to 280 ºC (5 min) at 20 ºC min . The
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Calibration was perfomed by plotting Ai/Asur (where Ai is the peak area of the target
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analyte and Asur that of the surrogate (i.e. o-cresol), each one obtained by its quantifier ion)
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versus target analyte concentration.
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2.4. Synthesis of the diacetylated compounds
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The diacetylated compounds were not commercially available and thus, they were
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synthesised in the laboratory in order to calculated the enrichment factor, based on a synthesis
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of a similar product already published [25]. Briefly, a flask equipped with a stir bar and a
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nitrogen line containing the target compound (atranol or chloroatranol), 3 equivalents of
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potassium carbonate, 6 equivalents of anhydride acetic and 4 mL of dry diethyl ether was stirred
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overnight. The product reaction was filtered and evaporated under reduced pressure. Then, the
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solid was rinsed thrice with hexane and evaporated again.
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3. Results and discussion
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3.1. Liquid-liquid extraction Ethanol content in perfumes is around 80%. The other remaining 20% mostly
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corresponds to the essential oils, water and keepers. Terpenes are oily, non-polar primary
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constituents of essential oils. Thus, a good clean-up is important to avoid interfering compounds
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in the subsequent chromatography. In this sense, if the perfume is directly injected, the injector
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port and the column head could suffer from dirtiness and thus jeopardize the results [5].
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Moreover, as the DLLME used to preconcentrate de target analytes requires an aqueous donor
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phase, an increase of the water content was needed in the sample solution. The direct dilution
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of the perfume with water is not possible since a slight increase in the percentage of water is
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enough to make the solution an opaque white dispersion, due to the presence of the non-polar
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components of the perfumes. However, a previous liquid-liquid extraction provides a good
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clean-up and enables the required water dilution before DLLME. Taking advantage of the relatively high polarity of the compounds of interest (with a
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log P, where P is the partition coefficient, of 1.99 and 2.99 for atranol and chloroatranol
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respectively), conventional liquid-liquid extraction (LLE) could be a useful technique in the case
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of having two immiscible phases (an organic and an hydro-alcoholic phase). The analytes could
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theoretically stay in the hydro-alcoholic phase unlike most of fragrance compounds, which are
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highly non-polar, and therefore will migrate to the organic phase. Indeed, preliminary tests
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showed that atranol and chloroatranol remained in the hydro-alcoholic phase and moreover, the
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injection of the rejected phases in the GC-MS showed that an important clean-up was
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performed. The clean-up was evidenced when even after adding 8 mL of water to 2 mL of the
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remaining hydro-alcoholic phase it still remained a completely transparent solution, that allowed
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the subsequent DLLME.
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Three extractions with aliquots of 1.5 mL of hexane were observed to be enough to
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eliminate most of these fat-soluble interferences of the perfume that made the solution used for
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DLLME not clear. Chromatograms in both full scan and SIM modes (i.e., at m/z 152 and 186) of
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the hexane rejected phases of Perfume 2 and Perfume 3 are shown, as example, as
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Supplementary Data to prove the clean-up performed.
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The recovery of atranol and chloroatranol in the LLE step was then quantified. To do
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so, the LLE process was performed to a hydroethanolic standard solution of each one of the
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separate compounds. The compounds were measured in the hydroethanolic standard solution
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with an UV spectrophotometer after 1, 2 and 3 repetitions of the LLE (all in triplicate) and
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compared to that of the hydroethanolic solution before performing the clean-up process. Its
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spectrum was recorded and the absorbance at λmax (i.e., 280 nm) was compared to that of the
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hydroethanolic solution before performing the LLE process to study how much target analyte
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was lost to the rejected phase in the process. In the case of atranol a total of 17.9±0.4 % is lost
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after the 3 extractions (12 % in the first one and 3 % in each of the other 2). Therefore, the
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recovery would be of 82.1±0.4 %. In the case of chloroatranol a total of 31.3±0.8 % is lost after
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the 3 extractions (13 % in the first and second one and 5 % in the third one). Therefore, the
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recovery is of 68.7±0.8 %. Although this may seem excessive, it is important to keep in mind
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that it is worthy compared to the clean-up performed.
251 3.2. Simultaneous dispersive liquid-liquid microextraction and derivatization
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order to achieve the highest extractability as possible and thus, the highest sensitivity.
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The simultaneous DLLME and derivatization parameters needed to be optimized in
Thus, to study the different parameters, the DLLME procedure was carried out to 1 mL
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of a 50 ng mL ethanolic solution of the two target analytes, to which 9 mL of water and 1 mL of
257
a 1M solution of potassium carbonate had been previously added (standard hydro-ethanolic
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solution).
The peak area of each analyte was plotted (Fig 2-7) as the average of 3 replicates with the error bars showing the standard deviation.
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3.2.1 Selection of the kind of extraction and disperser solvents
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In order to select the optimum extraction and disperser solvents it is important to take
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into account those features they need to have. The disperser solvent has to form the cloudy
265
solution and has to be miscible in both the aqueous donor phase and the extraction solvent.
266
The extraction solvent has to be immiscible in the aqueous donor phase and should also extract
267
the compounds of interest and have a good behaviour for the further analytical technique going
268
to be used (in this case GC).
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Regarding the extraction solvent, dichloromethane and chloroform were studied and in
270
the case of the disperser solvents, acetone, acetonitrile and ethanol. One mL of each disperser
271
solvent was mixed with 100 µL of each extraction solvent and 100 µL of acetic anhydride. Then,
272
each mixture was rapidly injected into 10 mL of the standard hydro-ethanolic solution. No phase
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separation after centrifugation was observed in any of the combinations that used
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dichloromethane as extraction solvent. Results using chloroform showed that the volume of the
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sedimented phase varied a lot depending on the disperser solvent. Although higher areas were
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achieved using ethanol as disperser solvent, it was not chosen as the volume was very low to
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handle easily. Therefore, chloroform and acetone were chosen as extraction and disperser
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solvent for further experiments.
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3.2.2. Effect of the extraction solvent volume In order to study the effect of the extraction solvent volume, different volumes of
282
chloroform, ranging from 50 to 125 µL, were mixed with 1 mL of acetone and 100 µL of acetic
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anhydride and injected into 10 mL of the standard hydro-ethanolic solution (Figure 2). When 50
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µL of chloroform were used, the volume of the obtained sedimented phase was very low to
285
inject it in the GC-MS and when 75 µL were used the total volume of the sedimented phase was
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only 13±5 µL, very low to handle, what caused a poor repeatibiliy in the results. Thus, although
287
less sensitivity is obtained, the subsequent studies were carried out using 100 µL of extraction
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solvent.
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3.2.3. Effect of the disperser solvent volume
To study this effect, different volumes of acetone, ranging from 250 to 1000 µL, were
292
tested. Figure 3 shows that the signal increases with the disperser solvent volume and tends to
293
stabilize after 750 µL. Low volumes of disperser solvent could mean poor extraction efficiencies,
294
as the extraction solvent needs to be correctly dispersed (cloudy solution). Thus 750 µL of
295
disperser solvent were selected for further experiments.
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3.2.4. Effect of the potassium carbonate amount An aqueous solution 1M of potassium carbonate was used to perform the
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derivatization reaction in a basic aqueous media. Different volumes, ranging from 0.1 to 2 mL,
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of this solution were tested to study its effect on the simultaneous derivatization and DLLME
301
process. Figure 4 shows that the best results were obtained when 1 mL of the solution was
302
added. These results could be explained by the fact that there are two effects involved. On one
303
hand, the alkaline pH is needed to perform the reaction in a reasonable yield. On the other
304
hand, potassium carbonate is a salt and it increases the ionic strength of the solution what could
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affect the results (as described in 3.2.6). In this sense, 1 mL of 1M potassium carbonate
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solution is chosen for further experiments.
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3.2.5. Effect of the acetic anhydride volume Acetic anhydride was mixed with the extraction (chloroform) and disperser (acetone)
310
solvents in order to inject the mixture and perform the simultaneous derivatization and DLLME
311
process. Different volumes, ranging from 50 to 150 µL, of acetic anhydride were tested while all
312
other parameters were kept constant. Figure 5 shows that, when comparing 50 and 100 µL,
313
similar results were obtained for atranol. However, in the case of chloroatranol the best results
314
were obtained when 100 µL were used. Thus, 100 µL of derivatizating agent were chosen for
315
further experiments. The signal was expected to increase with the volume of anhydride acetic,
316
as it would be desirable that it was in excess. However, the slight decrease of signal when 150
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μL are used could be explained as a raise in the acidity.
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Both, potassium carbonate and acetic anhydride amount, are in agreement with other
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published works where an acetylation of phenols in basic aqueous media is performed and
320
similar proportions are used [26].
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3.2.6. Effect of the ionic strength of the donor phase
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With the purpose of studying this effect, the simultaneous derivatization and DLLME
324
procedure was performed to different hydro-ethanolic standard solutions containing different
325
concentrations of NaCl ranging from 0 to 9% (m/v). It should be noted that as 1 mL of a 1M
326
potassium carbonate solution was previously added, there were other salts that already
327
contributed to the ionic strength of the donor phase.
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On one hand, the increase of the ionic strength decreases the solubility of the
329
extraction solvent (chloroform) in the aqueous phase, due to the salting out effect, and
330
therefore, the volume of the sedimented phase increases. On the other hand, the salting out
331
effect is expected to favour the extraction of the target compounds from the aqueous phase to
332
the organic phase. Figure 6 shows that the best results in the case of atranol were obtained
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when no NaCl was added, whereas no significant differences were obtained in the case of
334
chloroatranol. Therefore, in subsequent experiments, no NaCl was added to the donor phase.
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3.2.7. Effect of the pH of the donor phase The extraction of compounds can be considerably affected depending on the pH of the
338
donor phase. In this case, the donor phase needed to present an alkaline pH in order to perform
339
the derivatization reaction (potassium carbonate is added just before, resulting on a pH of 11.5
340
0.2). Thus, different pH values were not studied.
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3.2.8. Effect of the extraction and derivatization time
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The extraction and derivatization time is defined as the period between the injection of
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the mixture (i.e., the derivatization reagent and both the disperser and the extraction solvents)
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and the centrifugation step.
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Different times, ranging from 0 to 60 min, were studied. As expected, extraction and
347
derivatization time does not affect the results obtained in the DLLME. As already stated for this
348
technique, the cloudy solution generates a very large surface area between the sample, the
349
extraction solvent and the derivatization reagent, what enables the achievement of the
350
equilibrium state almost instantaneously.
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3.2.9. Enrichment factor of the DLLME process The enrichment factor (EF) is defined as the ratio between the concentration of the
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compound in the extraction solvent phase and the initial concentration in the aqueous donor
355
phase. The maximum EF value, which would correspond to the total transfer of the target
356
analytes to this sedimented phase, can be calculated as V 0/Vsed, where V0 is the sample volume
357
and Vsed the sedimented phase volume. In this case, the final volume of sedimented phase after
358
the optimization is approximately 20 µL and the initial volume of sample is 1 mL. Thus, the
359
maximum EF possible would be 50.
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Taking into account that analytes are derivatized simultaneously to DLLME, to calculate
361
the real EF of the DLLME process the pure derivatized compounds were needed. Unfortunately,
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these compounds were not commercially available and thus, they were synthesised in the
363
laboratory as described in Section 2.4. A H NMR of the product showed that the dyacetilated
364
product had been successfully synthesised and no other significant side products or reagents
365
were left.
1
To calculate them the area of a standard solution of atranol and chloroatranol submitted
367
to the DLLME process and that of a solution of derivatized atranol and chloroatranol in the same
368
concentration in chloroform (not extracted) were compared. An EF of 19 and 22 were obtained
369
for atranol and chloroatranol respectively.
cr
ip t
366
370 3.3. Use of surrogate
us
371
O-cresol was chosen as surrogate in order to increase the repeatability, not only of the
373
GC injections but of the whole method procedure (i.e., LLE, DLLME, derivatization and
374
injection). Thus, Ai/Asur (where Ai is the peak area of the target analyte and Asur that of the
375
surrogate) was used as response function for quantification purposes.
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an
372
O-cresol was selected as it presented a very similar log P than atranol so its solubility
377
features were expected to be similar to those of the analyte. Furthermore it is, as the target
378
compounds, a phenol, what is important when looking for a compound that could act as
379
surrogate not only of the extraction processes but also of the derivatization process.
ce pt
ed
376
380
382 383 384
3.4. Analytical figures of merit of the proposed method Once the simultaneous derivatization and DLLME was optimized, different quality
Ac
381
parameters were evaluated using standard solutions. An internal standard calibration curve was constructed and linearity reached at least -1
-1
385
1000 ng mL . The working range employed was set from 20 to 100 ng mL . All the solutions
386
contained 40 ng mL of o-cresol as surrogate. Figure 7 shows the chromatogram obtained for
387
an ethanolic standard solution containing the target analytes at 60 ng mL
388
submitted to the proposed LLE followed by derivatization and DLLME and GC-MS method.
-1
−1
and the surrogate,
389
The repeatability, expressed as relative standard deviation (RSD), was evaluated by
390
applying the proposed LLE followed by derivatization and DLLME and GC-MS method to five
13
Page 13 of 23
-1
391
replicates of a standard solution containing 40 ng mL
392
surrogate. RSD of 7 and 10 % were obtained for atranol and chloratranol respectively. The EFs
393
of the whole method were also calculated, taking into account the loss in the LLE process and
394
the EFs of the DLLME process, resulting in a value of 15 for both, atranol and chloroatranol.
of the target compounds and the
In order to evaluate matrix effects when internal standard calibration is used, the
396
slopes of the calibration curves obtained by this method and those obtained by standard
397
addition calibration combined with internal standard of four commercial perfumes were
398
compared. In this sense, the proposed LLE followed by derivatization and DLLME and GC-MS
399
method was applied to four perfume samples that had been previously spiked with the target
400
analytes at 20 - 100 ng mL (working range). The characteristics of the calibration equations
401
obtained for each standard addition are shown in Table 1. This table also shows the study of
402
matrix effect obtained in percentage calculated with the ratio of the slope of the standard
403
addition calibration of each sample to that of an internal standard calibration performed the
404
same day. Matrix effects can be observed, especially in the case of chloroatranol. Thus,
405
standard addition is recommended. It is worth mentioning that even though matrix effects were
406
observed in some previous published articles [12,13], no correction of the data with standard
407
addition was done. Table 2 shows the recoveries obtained after spiking blank perfume samples
408
with 20 and 100 ng mL of the target compounds and performing the proposed method. Good
409
recovery values are obtained when using standard addition calibration.
cr
ip t
395
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-1
ed
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an
us
-1
410
As standard addition is proposed, limits of detection were calculated as 3 times the
411
signal-to-noise ratio of a sample spiked with 20 ng mL of the analytes. They were of the order
412
of 3 and 2 ng mL for atranol and chloratranol respectively in the initial perfume samples. Thus,
413
the limits of quantification obtained were of the order of 9 and 6 ng mL
414
chloratranol respectively. Repeatability was re-evaluated using the same experiment but this
415
time to the four perfumes spiked instead of the standard solution. The mean of the four RSD
416
resulted in a total RSD of 9 % for both atranol and chloroatranol. This shows that there is no
417
significant difference between the internal standard and the standard addition method on
418
repeatability.
-1
Ac
-1
-1
for atranol and
419 420
14
Page 14 of 23
421
3.5. Assay of the proposed method to the analysis of real perfume samples The target analytes were determined in four different perfume samples. The results
423
indicated that both analytes were not present in the analyzed samples or that they were below
424
the limits of detection of the method. As the number of samples is rather limited these results
425
could be taken as satisfactory preliminary results and a higher number should be analysed to
426
assure the method is applicable to all market perfumes.
ip t
422
427 4. Conclusions
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428
Quality control of commercial perfumes and raw materials is necessary. Analytical
430
methods are required to guarantee there are no undesired compounds in the finished product.
431
Little has been done in this field regarding the potent allergens atranol and chloroatranol.
an
us
429
432
DLLME and GC-MS is presented to determine atranol and chloroatranol in perfumes.
M
433
In this work a method based on LLE followed by simultaneous derivatization and
434
Since matrix effects were observed when internal standardization is used, standard
435
addition calibration is recommended. In this sense, the method allows the determination of
436
atranol and chloroatranol successfully in the ng mL range with good accuracy and precision.
437 Acknowledgements
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438
ed
-1
The authors acknowledge the financial support of the Spanish Ministry of Science and
440
Innovation (Project CTQ2009-12709), especially Marina López-Nogueroles for her FPI
441
predoctoral grant, and the financial support of University of Valencia (Project UV-INV-AE13-
442
137567).
443
Ac
439
Authors also acknowledge the advice given concerning the derivatization reaction, by
444
Prof. Gonzalo Blay, Prof. Isabel Fernandez and Ms. Amparo Sanz from the Department of
445
Organic Chemistry of the University of Valencia.
15
Page 15 of 23
References
[1] Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending council directive 76/768/EEC on the approximation of the laws of the member states relating to cosmetic products
ip t
[2] Regulation (EC) no 1223/2009 of the European Parliament and of the council of 30 November 2009 on cosmetic products
cr
[3] A. Chisvert, M. López-Nogueroles, A. Salvador, Essential oils: Analytical methods to control
us
the quality of perfumes, in: M.J. Ramawat K. (Ed.), Handbook of Natural Products, SpringerVerlag, Berlin Heidelberg, 2013, pp. 3287-3310.
an
[4] A. Chaintreau, Analytical methods to determine potentially allergenic fragrance-related substances in cosmetics, in: A. Salvador, A. Chisvert (Eds.), Analysis of cosmetic products,
M
Elsevier, Amsterdam, 2007, pp 257-275.
[5] A. Chaintreau, D. Joulain, C. Marin, C. Schmidt, M. Vey, J. Agric. Food Chem. 51 (2003)
ed
6398-6403.
[6] H. Leijs, J. Broekhans, L. van Pelt, C. Mussinan, J. Agric. Food Chem. 53 (2005) 5487-5491 [7] M. Bassereau, A. Chaintreau, S. Duperrex, D. Joulain, H. Leijs, G. Loesing, N. Owen, A.
ce pt
Sherlock, C. Schippa, P. Thorel, M. Vey, J. Agric. Food Chem. 55 (2007) 25-31. [8] A. Chaintreau, E. Cicchetti, N. David, A. Earls, P. Gimeno, B. Grimaud, D. Joulain, N. Kupfermann, G. Kuropka, F. Saltron, C. Schippa, Journal of Chromatography A 1218 (2011)
Ac
7869-7877
[9] D. Joulain, R. Tabacchi, Flavour Frag. J. 24 (2009) 49-61. [10] D. Joulain, R. Tabacchi, Flavour Frag. J. 24 (2009) 105-116. [11] G. Bernard, E. Gimenez-Arnau, S. Rastogi, S. Heydorn, J. Johansen, T. Menne, A. Goossens, K. Andersen, J. Lepoittevin, Arch. Dermatol. Res. 295 (2003) 229-235. [12] J. Johansen, G. Bernard, E. Gimenez-Arnau, J. Lepoittevin, M. Bruze, K. Andersen, Contact Dermatitis 54 (2006) 192-195.
16
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[13] J. Johansen, K. Andersen, C. Svedman, M. Bruze, G. Bernard, E. Gimenez-Arnau, S. Rastogi, J. Lepoittevin, T. Menne, Contact Dermatitis 49 (2003) 180-184. [14] R. Hiserodt, D. Swijter, C. Mussinan, J. Chromatogr. A 888 (2000) 103-111. [15] Scientific Committee on Consumer Products. SCCP Opinion on Atranol and Chloroatranol Present in Natural Extracts (e.g. Oak Moss and Tree Moss Extracts). SCCP/00847/04 adopted
ip t
by the SCCP on 7 December 2004.
[16] F. David, C. Devos, P. Sandra, LC GC Europe 19 (November 2006) 602-617
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[17] R. Bossi, S. Rastogi, G. Bernard, E. Gimenez-Arnau, J. Johansen, J. Lepoittevin, T.
us
Menne, J. Sep. Sci. 27 (2004) 537-540.
[18] S. Rastogi, R. Bossi, J. Johansen, T. Menne, G. Bernard, E. Gimenez-Arnau, J.
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Lepoittevin, Contact Dermatitis 50 (2004) 367-370
[19] S.C. Rastogi, J.D. Johansen, R. Bossi, Contact Dermatits 56 (2007) 201-204.
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[20] N. Fattahi, Y. Assadi, M.R.M. Hosseini, E.Z. Jahromi, J. Chromatogr. A. 1157 (2007) 23-29. [21] R. Montes, I. Rodriguez, E. Rubi, R. Cela, J. Chromatogr. A. 1216 (2009) 205-210.
ed
[22] J. Wu, H. Chen, W. Ding, J Chromatogr. A. 1302 (2013) 20-27 [23] S. Luo, L. Fang, X. Wang, H. Liu, G. Ouyang, C. Lan, T. Luan, J. Chromatogr. A. 1217
ce pt
(2010) 6762-6768.
[24] M.K.R. Mudiam, C. Ratnasekhar, J. Chromatogr. A 1291 (2013) 10-18. [25] C. Selenski, T. R.R. Pettus, Tetrahedron 62 (2006) 5298-5307.
54.
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[26] F. Galán-Cano, R. Lucena, S. Cárdenas, M. Valcárcel, J. Chromatogr. A 1229 (2012) 48-
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Page 17 of 23
Figure captions
Fig. 1 Chemical structure of (a) atranol (b) chloroatranol and (c) o-cresol Fig. 2 Effect of the volume of extraction solvent. Extraction conditions: standard solution volume: 10 mL; disperser solvent volume: 1000 µL; K2CO3 1M volume: 1 mL; acetic anhydride
ip t
volume: 100 µL.
Fig. 3 Effect of the volume of disperser solvent. Extraction conditions: standard solution volume:
cr
10 mL; extraction solvent volume: 100 µL; K2CO3 1M volume: 1 mL; acetic anhydride volume:
us
100 µL.
Fig. 4 Effect of the volume of K2CO3. Extraction conditions: standard solution volume: 10 mL;
an
disperser solvent volume: 1000 µL; extraction solvent volume: 100 µL; acetic anhydride volume: 100 µL.
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Fig. 5 Effect of the volume of acetic anhydride. Extraction conditions: standard solution volume: 10 mL; disperser solvent volume: 1000 µL; extraction solvent volume: 100 µL; K2CO3 1M
ed
volume: 1 mL.
Fig. 6 Effect of the ionic strength. Extraction conditions: standard solution volume: 10 mL;
ce pt
disperser solvent volume: 1000 µL; extraction solvent volume: 100 µL; K2CO3 1M volume: 1 mL; acetic anhydride volume: 100 µL.
Fig. 7 A chromatogram obtained applying the proposed method to an ethanolic standard -1
-1
Ac
solution containing the target compounds at 60 ng mL and o-cresol as surrogate at 40 ng mL .
18
Page 18 of 23
Figure 1
(a)
OH
Cl
(b)
(c)
OH
OH
O
O
OH
us
cr
ip t
OH
75 µL 100 µL 125 µL
150
M
100
0
Chloroatranol
ce pt
Atranol
ed
50
Ac
Area (counts*min/105)
200
an
Figure 2
Page 19 of 23
Figure 3
250 µL
500 µL
750 µL
1000 µL
150
100
50
ip t
Area (counts*min/105)
200
0
Chloroatranol
cr
Atranol
0,5 mL
1 mL
2 mL
an
200
0.1 mL
150
M
100 50 0
ed
Chloroatranol
ce pt
Atranol
Ac
Area (counts*min/105)
250
us
Figure 4
Page 20 of 23
Figure 5 50 µL 100 µL 150 µL
150
100
50
ip t
Area (counts*min/105)
200
0 Chloroatranol
us
cr
Atranol
Figure 6
4 % NaCl
9 % NaCl
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an
2.5 % NaCl
100
50
0
Figure 7 100
Chloroatranol
ce pt
Atranol
O-cresol (m/z = 108)
50 0 100
Ac
Relative abundance (%)
0 % NaCl
ed
Area (counts*min/105)
150
Atranol (m/z = 152)
50
0 100
Chloroatranol (m/z = 186)
50 0 0
5
10
15
20
25
30
Time (min)
Page 21 of 23
ip t cr Atranol
Perfume 1 Perfume 2 Perfume 3 Perfume 4
(280±9)·10
-4
(297±7)·10
-4
(315±12)·10
-4
(336±15)·10
-4
a
-0.05±0.05 -0.04±0.03 -0.10±0.07 0.13±0.09
Regression a coefficient
Calibration b slopes ratio (%)
0.996
116 ± 5
0.998 0.994 0.992
112 ± 6 120 ± 7 146 ± 9
Slope -1 -1 (ng mL )
Intercept
a
Regression a coefficient
Calibration b slopes ratio (%)
-4
-0.0002±0.008
0.997
72 ± 6
-4
-0.006±0.02
0.990
89 ± 6
-4
0.003±0.003
0.998
71 ± 3
-4
0.03±0.02
0.98
62 ± 8
(56±2)·10 (65±4)·10 (52±1)·10 (41±4)·10
*
-1
d
Obtained by means of the internal standardization calibration. Working range: 20 to 100 ng mL . Number of calibration points: 6 (Standard addition calibration slope / Internal standard calibration slope) x 100
ep te
b
Intercept
Ac c
a
Slope -1 -1 (ng mL )
Chloroatranol
a
an
Analyte
M
a
us
Table1. Study of matrix effects
Page 22 of 23
Table 2 Study of the recovery of the proposed method (standard addition calibration) Recovery * (%) Atranol 20 ng mL
-1
Chloroatranol
100 ng mL-1
20 ng mL-1
100 ng mL
Perfume 1
94
103
95
98
Perfume 2
96
101
79
97
Perfume 3
86
103
104
100
Perfume 4
106
98
110
98
-1
* Recovery obtained after spiking free-analytes samples with 20 and 100 ng mL-1, estimated as
Ac
ce pt
ed
M
an
us
cr
((Obtained concentration – Found concentration) / Spiked concentration)) x 100
ip t
Real samples
Page 23 of 23
S0003-2670(14)00378-X http://dx.doi.org/doi:10.1016/j.aca.2014.03.042 ACA 233179
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
16-9-2013 21-3-2014 30-3-2014
Please cite this article as: Marina L´opez-Nogueroles, Alberto Chisvert, Amparo Salvador, Determination of atranol and chloroatranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3
Determination of atranol and chloroatranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry.
4 5
Marina López-Nogueroles, Alberto Chisvert*, Amparo Salvador
6 Departamento de Química Analítica, Facultad de Química, Universitat de València, 46100 Burjassot, Valencia, Spain
9 * Corresponding author: Tel.: +34 96 354 49 00; Fax: + 34 96 354 44 36
11
e-mail address: [email protected] (A. Chisvert)
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10
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Graphical abstract Abstract
ed
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► The potent allergens atranol and chloroatranol are determined in perfumes by GCMS ► LLE is used to clean up the sample solution from lipophilic substances ► In situ derivatization and DLLME is used to remove polar compounds and concentrate ► The method presents good analytical features ► The method has been assayed in four real perfume samples
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26 27 28 29 30 31 32 33 34 35 36 37 38 39
Highlights
A new analytical method based on simultaneous derivatization and dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS), for the determination of the allergenic compounds atranol and chloroatranol in perfumes, is presented. Derivatization of the target analytes by means of acetylation with anhydride acetic in carbonate buffer was carried out. Thereby volatility and detectability were increased for improved GC-MS
Ac
24 25
us
12 13 14 15 16 17 18 19 20 21 22 23
ip t
7 8
sensitivity. In addition, extractability by DLLME was also enhanced due to a less polar character of the solutes. A liquid-liquid extraction was performed before DLLME to clean up the sample and to obtain an aqueous sample solution, free of the low polar matrix from the essential oils, as donor phase. Different parameters, such as the nature and volume of both the extraction and disperser solvents, the ionic strength of the aqueous donor phase or the effect of the derivatization reagent volume, were optimized. Under the selected conditions (injection of a mixture of 750 µL of acetone as disperser solvent, 100 µL of chloroform as extraction solvent and 100 µL of anhydride acetic as derivatization reagent) the figures of merit of the proposed method were evaluated. Limits of detection in the low ng mL
-1
range were obtained. Matrix
1
Page 1 of 23
40 41
effect was observed in real perfume samples and thus, standard addition calibration is recommended.
42 43 44 45
Keywords:
Dispersive
liquid-liquid
microextraction;
Simultaneous
derivatization;
Gas
chromatography-mass
spectrometry; Atranol; Chloroatranol; Allergens.
47
ip t
46 1. Introduction
A list of 26 fragrance chemicals classified as potentially allergenic substances (PASs)
49
was first included in the European Union (EU) Directive on cosmetic products in 2003 [1] and
50
they are also compiled in the current EU Regulation [2]. It was established that, unlike other
51
fragrance chemicals, their presence in any cosmetic product has to be declared on the label
52
when present at a higher concentration than 0.001 % in those products to remain on the skin or
53
0.01 % in those intended to be rinsed off. 24 of these PASs are chemically defined volatile
54
compounds while the other two are natural moss extracts, i.e. oakmoss extract (Evernia
55
prunastri extract) and treemoss extract (Evernia furfuracea extract), which are not a defined
56
compound, but a complex mixture of natural products.
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48
Since then, many works dealing with the determination of these skin-sensitizing
58
ingredients can be found in the literature, especially regarding those 24 chemically defined
59
PASs [3-8]. However, the number of publications regarding the determination of the two natural
60
moss extracts is rather limited, and surely it presents an important challenge. Indeed, Joulain et
61
al. published two comprehensive reviews on the composition of these two extracts identifying
62
170 different constituents in oakmoss extracts and 90 in the case of treemoss [9,10]. It should
63
be noted that their natural conditions and a not standardized industrial processing makes the
64
composition of these extracts variable. The industrial processing of these two natural extracts
65
usually consists on the extraction with hexane or other solvents of the harvested lichens
66
followed by dilution with ethanol and submission to physical treatments to remove their original
67
colour.
Ac
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57
68
From all the different compounds present in oakmoss and treemoss extracts, atranol
69
and chloroatranol are very potent allergens [11-13]. These two compounds are degradation
70
products, formed after transesterification and decarboxylation of the lichen depsides atranorin
2
Page 2 of 23
and chloroatranorin, and are formed in the moss absolute production [14]. It is worth mentioning
72
that the EU Regulation, which only restricts total concentration of oakmoss and treemoss
73
extracts, does not list these individually allergenic components of mosses and their
74
quantification is not indicative of the total amount of the regulated moss. However the EU
75
Scientific Committee on Consumer Products (SCCP) stated that these compounds are such
76
potent allergens they should not be present at all in cosmetic products [15] so it can be
77
expected they will be banned or restricted in the coming years. Therefore, works dealing with
78
their determination could be useful in the future, specially taking into account the lack of
79
analytical methods available for this purpose.
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71
In this sense, Bernard et al. [11] searched for allergenic molecules in oakmoss extract
81
using GC-MS after chemical fractionation of the extract by gel permeation chromatography.
82
David et al. [16] semiquantitatively determined chloroatranol in moss extracts by GC-MS using a
83
programmed temperature vaporizing inlet with an automated liner exchange. The liner was
84
packed with polydimethylsiloxane that retained non-volatile material. Hiserodt et al. [14]
85
developed a qualitative method using liquid chromatography-tandem mass spectrometry (LC-
86
MS/MS) to identify atranorin and some related potential allergens, including atranol and
87
chloroatranol, in oakmoss absolute. Bossi et al. [17] further developed this method to quantify
88
atranol and chloroatranol in perfumes. The method was based on direct injection of the sample
89
and limits of detection (LOD) of 5 ng mL
90
respectively, were obtained. The recovery of chloratranol from spiked perfumes was 89±10%.
91
Low recoveries (49±6%) were observed for atranol in spiked perfumes, indicating ion
92
suppression caused by matrix components. Same authors also applied this method to quantify
93
the presence of these two allergenic compounds in different commercial perfumes [18, 19].
an
M
ed
ce pt
-1
and 2.4 ng mL
-1
for atranol and chloroatranol
Ac
94
us
80
It is worth mentioning that perfumes are elaborated by mixing different natural raw
95
materials or complex synthetic formulations that commonly contain hundreds or thousands of
96
different compounds. This fact complicates the determination of compounds in this matrix. Thus,
97
an effective sample preparation, that enables a significant clean-up, is crucial to obtain a good
98
accuracy. The aim of this work is to develop a method to determine atranol and chloroatranol in
99
perfumes with a good accuracy.
3
Page 3 of 23
In this work, liquid-liquid extraction (LLE) is used to clean up the sample solution from
101
lipophilic substances. This step is followed by a simultaneous derivatization and dispersive
102
liquid-liquid microextraction (DLLME) step that enables to change the solvent (for further GC-
103
MS analysis) and to concentrate the target compounds. Moreover it provides a second, less
104
crucial, clean up from possible components that would have remained in the polar phase with
105
the analytes after the LLE process.
ip t
100
It should be pointed out that the derivatization step is proposed since atranol and
107
chloroatranol are relatively polar compounds, and without it, a poor extraction efficiency would
108
be achieved in the DLLME process. Moreover, the derivatization allows volatility and thus
109
increases sensitivity in GC-MS. The chosen approach was an in situ derivatization via
110
acetylation of the hydroxyl groups under basic aqueous conditions, using acetic anhydride as
111
acetylating agent. The derivatization is performed at the same time as DLLME, allowing the
112
simplification of the procedure and a decrease in the time of analysis. This one step
113
derivatization and DLLME was first introduced by Fattahi et al. to determine clorophenols in
114
waters [20] and has been later used by other authors to determine phenols in a similar
115
approach although also performing other types of derivatization reactions such as silylation [21,
116
22] and O-alkoxycarbonylation [23, 24].
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106
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117 118
2. Experimental
119
2.1 Reagents and samples
Atranol (97%) and chloroatranol (98.9%), purchased from Ambinter (Orleans, France),
121
were used as standards. O-cresol, purchased from Aldrich, was used as surrogate. Figure 1
122
shows the chemical structure of the three of them.
123
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120
Liquid chromatography grade absolute ethanol from Scharlau (Barcelona, Spain), was
124
used as solvent to prepare dilute solutions of the standards. Deionised water (resistivity 18
125
M cm) obtained by means of a Nanopure II water purification system from Barnstead (Boston,
126
MA, USA) was used to prepare the working standard solutions.
127
Analytical reagent grade chloroform and dichloromethane, from Scharlau Chemie
128
(Barcelona, Spain), were tested as extraction solvents. Ethanol, ultrapure acetone and liquid
4
Page 4 of 23
129
chromatography grade acetonitrile, all from Scharlau Chemie, were tested as disperser
130
solvents. Analytical reagent grade sodium chloride 99% from Scharlau Chemie was used to
132
adjust the ionic strength. Acetic anhydride extra pure and anhydrous diethyl ether from Scharlau
133
Chemie, and potassium carbonate analysis grade from Panreac (Barcelona, Spain) were used
134
in the derivatization of the compounds.
135
High purity helium (99.9999%) from Carburos Metalicos S.A. (Paterna, Spain) was used as carrier gas in the GC-MS system.
cr
136
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131
Four different commercial perfumes from different brands (a women eau de toilette
138
and eau de parfum, a man eau de parfum and a children eau de cologne) were purchased and
139
stored at room temperature until analysis. For reasons of confidentiality, the manufacturers are
140
not shown.
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us
137
141 2.2 Apparatus
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142
The GC-MS system used consisted of a Focus GC gas chromatograph coupled to a
144
DSQII mass spectrometry detector equipped with an AI3000 autosampler, all from Thermo
145
Fisher Scientific (Austin, TX, USA). The chromatographic separations were made using an HP-
146
5MS Ultra Inert (95% dimethyl-5% diphenylpolysiloxane, 30 m length, 0.25 mm i.d., 0.25 µm film
147
thickness) analytical fused-silica capillary column from Agilent Technologies (Santa Clara, CA,
148
USA).
151
ce pt
150
An 8452A Hewlett Packard diode array UV/Vis spectrophotometer with a 1 cm path length quartz cell was used to determine the losses in the LLE process.
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149
ed
143
1
An Avance 300 spectrometer from Bruker (Madrid, Spain) was employed to run H
152
nuclear magnetic resonance (NMR) spectra at 300 MHz, using residual non-deuterated
153
chloroform as internal standard ( 7.26 ppm).
154
An EBA 21 centrifuge from Hettich (Tuttlingem, Germany) was also used.
155 156
2.3 Method
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2.3.1. Internal standard calibration
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Standards preparation: 1 mL of each one of the ethanolic standard solutions
159
containing the analytes (atranol and chloroatranol) in a concentration between 20 and 100 ng
160
mL and the surrogate (o-cresol) in 40 ng mL were transferred to different 15 mL polyethylene
161
centrifuge tubes. 1.5 mL of water and 1.5 mL of hexane were added and the mixture vortexed
162
during 1 min. Then, the tubes were centrifuged for 3 min at 3000 rpm and the upper layers of
163
hexane were collected with a syringe and discarded. The liquid-liquid extraction (LLE) process
164
and subsequent discard of hexane was repeated two more times. After this, 2 mL of the
165
remaining aqueous solutions were taken and transferred to other polyethylene centrifuge tubes.
166
8 mL of water and 1 mL of a 1M potassium carbonate solution were added. A simultaneous
167
derivatization and DLLME process was then performed injecting to these solutions different
168
previously mixed solutions of 100 µL of chloroform (extraction solvent), 100 µL of acetic
169
anhydride (derivatization reagent) and 750 µL of acetone (disperser solvent). The tubes were
170
then centrifuged for 5 min at 6000 rpm. The sedimented phases were collected with a 100 μL
171
Hamilton syringe and transferred into 100 µL inserts placed inside different injection vials, which
172
were then ready to be injected into the chromatographic system.
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Samples preparation: 3 replicates of 1 mL of perfume sample were submitted to the same procedure.
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2.3.2. Standard addition combined with internal standard calibration -1
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Several aliquots of 1 mL of perfume sample were spiked, with a 2 µg mL ethanolic
178
solution of the target analytes, in a concentration between 20 and 100 ng mL , and with a 2 µg
179
mL
180
procedure described in 2.3.1.
-1
ethanolic solution of the surrogate, in 40 ng mL , and they were submitted to the
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-1
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(As will be seen later, the results showed that standard addition calibration is
182
recommended).
183
2.3.3. GC-MS analysis.
184
1 µL of each one of the aforementioned sedimented phases was injected into the GC
185
system coupled to a mass spectrometry detector operated in positive electron ionisation mode
186
at ionisation energy of 70 eV and with a multiplier voltage set at 1300 V. The inlet temperature
187
was 280 ºC and the injection was accomplished in splitless mode (splitless time: 1 min). The
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−1
188
separation was run at a 1 mL min
189
was: from 80 ºC (1 min) to 200 ºC at 4 ºC min
190
transfer line and ion source temperatures were set at 280 and 250 ºC, respectively. The
191
chromatograms were recorded in selected ion monitoring (SIM) mode at the following
192
mass/charge (m/z) ratios: m/z 108 from minute 5.0 to 11.0 for o-cresol, m/z 152 from minute
193
20.0 to 28.0 for atranol and m/z 186 from minute 28.0 to 32.0 for chloroatranol. A full scan mode
194
(m/z from 50 to 350) was simultaneously recorded from minute 5.0 to the end of the analysis
195
time to confirm the identification of the analytes in real samples.
helium constant flow rate. The oven temperature program -1
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and finally to 280 ºC (5 min) at 20 ºC min . The
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Calibration was perfomed by plotting Ai/Asur (where Ai is the peak area of the target
197
analyte and Asur that of the surrogate (i.e. o-cresol), each one obtained by its quantifier ion)
198
versus target analyte concentration.
199 200
2.4. Synthesis of the diacetylated compounds
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The diacetylated compounds were not commercially available and thus, they were
202
synthesised in the laboratory in order to calculated the enrichment factor, based on a synthesis
203
of a similar product already published [25]. Briefly, a flask equipped with a stir bar and a
204
nitrogen line containing the target compound (atranol or chloroatranol), 3 equivalents of
205
potassium carbonate, 6 equivalents of anhydride acetic and 4 mL of dry diethyl ether was stirred
206
overnight. The product reaction was filtered and evaporated under reduced pressure. Then, the
207
solid was rinsed thrice with hexane and evaporated again.
210 211
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3. Results and discussion
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3.1. Liquid-liquid extraction Ethanol content in perfumes is around 80%. The other remaining 20% mostly
212
corresponds to the essential oils, water and keepers. Terpenes are oily, non-polar primary
213
constituents of essential oils. Thus, a good clean-up is important to avoid interfering compounds
214
in the subsequent chromatography. In this sense, if the perfume is directly injected, the injector
215
port and the column head could suffer from dirtiness and thus jeopardize the results [5].
216
Moreover, as the DLLME used to preconcentrate de target analytes requires an aqueous donor
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217
phase, an increase of the water content was needed in the sample solution. The direct dilution
218
of the perfume with water is not possible since a slight increase in the percentage of water is
219
enough to make the solution an opaque white dispersion, due to the presence of the non-polar
220
components of the perfumes. However, a previous liquid-liquid extraction provides a good
221
clean-up and enables the required water dilution before DLLME. Taking advantage of the relatively high polarity of the compounds of interest (with a
223
log P, where P is the partition coefficient, of 1.99 and 2.99 for atranol and chloroatranol
224
respectively), conventional liquid-liquid extraction (LLE) could be a useful technique in the case
225
of having two immiscible phases (an organic and an hydro-alcoholic phase). The analytes could
226
theoretically stay in the hydro-alcoholic phase unlike most of fragrance compounds, which are
227
highly non-polar, and therefore will migrate to the organic phase. Indeed, preliminary tests
228
showed that atranol and chloroatranol remained in the hydro-alcoholic phase and moreover, the
229
injection of the rejected phases in the GC-MS showed that an important clean-up was
230
performed. The clean-up was evidenced when even after adding 8 mL of water to 2 mL of the
231
remaining hydro-alcoholic phase it still remained a completely transparent solution, that allowed
232
the subsequent DLLME.
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Three extractions with aliquots of 1.5 mL of hexane were observed to be enough to
234
eliminate most of these fat-soluble interferences of the perfume that made the solution used for
235
DLLME not clear. Chromatograms in both full scan and SIM modes (i.e., at m/z 152 and 186) of
236
the hexane rejected phases of Perfume 2 and Perfume 3 are shown, as example, as
237
Supplementary Data to prove the clean-up performed.
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The recovery of atranol and chloroatranol in the LLE step was then quantified. To do
239
so, the LLE process was performed to a hydroethanolic standard solution of each one of the
240
separate compounds. The compounds were measured in the hydroethanolic standard solution
241
with an UV spectrophotometer after 1, 2 and 3 repetitions of the LLE (all in triplicate) and
242
compared to that of the hydroethanolic solution before performing the clean-up process. Its
243
spectrum was recorded and the absorbance at λmax (i.e., 280 nm) was compared to that of the
244
hydroethanolic solution before performing the LLE process to study how much target analyte
245
was lost to the rejected phase in the process. In the case of atranol a total of 17.9±0.4 % is lost
246
after the 3 extractions (12 % in the first one and 3 % in each of the other 2). Therefore, the
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247
recovery would be of 82.1±0.4 %. In the case of chloroatranol a total of 31.3±0.8 % is lost after
248
the 3 extractions (13 % in the first and second one and 5 % in the third one). Therefore, the
249
recovery is of 68.7±0.8 %. Although this may seem excessive, it is important to keep in mind
250
that it is worthy compared to the clean-up performed.
251 3.2. Simultaneous dispersive liquid-liquid microextraction and derivatization
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252 253
order to achieve the highest extractability as possible and thus, the highest sensitivity.
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The simultaneous DLLME and derivatization parameters needed to be optimized in
Thus, to study the different parameters, the DLLME procedure was carried out to 1 mL
256
of a 50 ng mL ethanolic solution of the two target analytes, to which 9 mL of water and 1 mL of
257
a 1M solution of potassium carbonate had been previously added (standard hydro-ethanolic
258
solution).
The peak area of each analyte was plotted (Fig 2-7) as the average of 3 replicates with the error bars showing the standard deviation.
261
3.2.1 Selection of the kind of extraction and disperser solvents
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In order to select the optimum extraction and disperser solvents it is important to take
264
into account those features they need to have. The disperser solvent has to form the cloudy
265
solution and has to be miscible in both the aqueous donor phase and the extraction solvent.
266
The extraction solvent has to be immiscible in the aqueous donor phase and should also extract
267
the compounds of interest and have a good behaviour for the further analytical technique going
268
to be used (in this case GC).
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Regarding the extraction solvent, dichloromethane and chloroform were studied and in
270
the case of the disperser solvents, acetone, acetonitrile and ethanol. One mL of each disperser
271
solvent was mixed with 100 µL of each extraction solvent and 100 µL of acetic anhydride. Then,
272
each mixture was rapidly injected into 10 mL of the standard hydro-ethanolic solution. No phase
273
separation after centrifugation was observed in any of the combinations that used
274
dichloromethane as extraction solvent. Results using chloroform showed that the volume of the
275
sedimented phase varied a lot depending on the disperser solvent. Although higher areas were
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276
achieved using ethanol as disperser solvent, it was not chosen as the volume was very low to
277
handle easily. Therefore, chloroform and acetone were chosen as extraction and disperser
278
solvent for further experiments.
279 280
3.2.2. Effect of the extraction solvent volume In order to study the effect of the extraction solvent volume, different volumes of
282
chloroform, ranging from 50 to 125 µL, were mixed with 1 mL of acetone and 100 µL of acetic
283
anhydride and injected into 10 mL of the standard hydro-ethanolic solution (Figure 2). When 50
284
µL of chloroform were used, the volume of the obtained sedimented phase was very low to
285
inject it in the GC-MS and when 75 µL were used the total volume of the sedimented phase was
286
only 13±5 µL, very low to handle, what caused a poor repeatibiliy in the results. Thus, although
287
less sensitivity is obtained, the subsequent studies were carried out using 100 µL of extraction
288
solvent.
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290
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3.2.3. Effect of the disperser solvent volume
To study this effect, different volumes of acetone, ranging from 250 to 1000 µL, were
292
tested. Figure 3 shows that the signal increases with the disperser solvent volume and tends to
293
stabilize after 750 µL. Low volumes of disperser solvent could mean poor extraction efficiencies,
294
as the extraction solvent needs to be correctly dispersed (cloudy solution). Thus 750 µL of
295
disperser solvent were selected for further experiments.
297 298
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3.2.4. Effect of the potassium carbonate amount An aqueous solution 1M of potassium carbonate was used to perform the
299
derivatization reaction in a basic aqueous media. Different volumes, ranging from 0.1 to 2 mL,
300
of this solution were tested to study its effect on the simultaneous derivatization and DLLME
301
process. Figure 4 shows that the best results were obtained when 1 mL of the solution was
302
added. These results could be explained by the fact that there are two effects involved. On one
303
hand, the alkaline pH is needed to perform the reaction in a reasonable yield. On the other
304
hand, potassium carbonate is a salt and it increases the ionic strength of the solution what could
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305
affect the results (as described in 3.2.6). In this sense, 1 mL of 1M potassium carbonate
306
solution is chosen for further experiments.
307 308
3.2.5. Effect of the acetic anhydride volume Acetic anhydride was mixed with the extraction (chloroform) and disperser (acetone)
310
solvents in order to inject the mixture and perform the simultaneous derivatization and DLLME
311
process. Different volumes, ranging from 50 to 150 µL, of acetic anhydride were tested while all
312
other parameters were kept constant. Figure 5 shows that, when comparing 50 and 100 µL,
313
similar results were obtained for atranol. However, in the case of chloroatranol the best results
314
were obtained when 100 µL were used. Thus, 100 µL of derivatizating agent were chosen for
315
further experiments. The signal was expected to increase with the volume of anhydride acetic,
316
as it would be desirable that it was in excess. However, the slight decrease of signal when 150
317
μL are used could be explained as a raise in the acidity.
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309
Both, potassium carbonate and acetic anhydride amount, are in agreement with other
319
published works where an acetylation of phenols in basic aqueous media is performed and
320
similar proportions are used [26].
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321
3.2.6. Effect of the ionic strength of the donor phase
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322
With the purpose of studying this effect, the simultaneous derivatization and DLLME
324
procedure was performed to different hydro-ethanolic standard solutions containing different
325
concentrations of NaCl ranging from 0 to 9% (m/v). It should be noted that as 1 mL of a 1M
326
potassium carbonate solution was previously added, there were other salts that already
327
contributed to the ionic strength of the donor phase.
328
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323
On one hand, the increase of the ionic strength decreases the solubility of the
329
extraction solvent (chloroform) in the aqueous phase, due to the salting out effect, and
330
therefore, the volume of the sedimented phase increases. On the other hand, the salting out
331
effect is expected to favour the extraction of the target compounds from the aqueous phase to
332
the organic phase. Figure 6 shows that the best results in the case of atranol were obtained
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333
when no NaCl was added, whereas no significant differences were obtained in the case of
334
chloroatranol. Therefore, in subsequent experiments, no NaCl was added to the donor phase.
335 336
3.2.7. Effect of the pH of the donor phase The extraction of compounds can be considerably affected depending on the pH of the
338
donor phase. In this case, the donor phase needed to present an alkaline pH in order to perform
339
the derivatization reaction (potassium carbonate is added just before, resulting on a pH of 11.5
340
0.2). Thus, different pH values were not studied.
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342
3.2.8. Effect of the extraction and derivatization time
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341
The extraction and derivatization time is defined as the period between the injection of
344
the mixture (i.e., the derivatization reagent and both the disperser and the extraction solvents)
345
and the centrifugation step.
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Different times, ranging from 0 to 60 min, were studied. As expected, extraction and
347
derivatization time does not affect the results obtained in the DLLME. As already stated for this
348
technique, the cloudy solution generates a very large surface area between the sample, the
349
extraction solvent and the derivatization reagent, what enables the achievement of the
350
equilibrium state almost instantaneously.
353
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352
3.2.9. Enrichment factor of the DLLME process The enrichment factor (EF) is defined as the ratio between the concentration of the
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346
354
compound in the extraction solvent phase and the initial concentration in the aqueous donor
355
phase. The maximum EF value, which would correspond to the total transfer of the target
356
analytes to this sedimented phase, can be calculated as V 0/Vsed, where V0 is the sample volume
357
and Vsed the sedimented phase volume. In this case, the final volume of sedimented phase after
358
the optimization is approximately 20 µL and the initial volume of sample is 1 mL. Thus, the
359
maximum EF possible would be 50.
360
Taking into account that analytes are derivatized simultaneously to DLLME, to calculate
361
the real EF of the DLLME process the pure derivatized compounds were needed. Unfortunately,
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Page 12 of 23
362
these compounds were not commercially available and thus, they were synthesised in the
363
laboratory as described in Section 2.4. A H NMR of the product showed that the dyacetilated
364
product had been successfully synthesised and no other significant side products or reagents
365
were left.
1
To calculate them the area of a standard solution of atranol and chloroatranol submitted
367
to the DLLME process and that of a solution of derivatized atranol and chloroatranol in the same
368
concentration in chloroform (not extracted) were compared. An EF of 19 and 22 were obtained
369
for atranol and chloroatranol respectively.
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370 3.3. Use of surrogate
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371
O-cresol was chosen as surrogate in order to increase the repeatability, not only of the
373
GC injections but of the whole method procedure (i.e., LLE, DLLME, derivatization and
374
injection). Thus, Ai/Asur (where Ai is the peak area of the target analyte and Asur that of the
375
surrogate) was used as response function for quantification purposes.
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372
O-cresol was selected as it presented a very similar log P than atranol so its solubility
377
features were expected to be similar to those of the analyte. Furthermore it is, as the target
378
compounds, a phenol, what is important when looking for a compound that could act as
379
surrogate not only of the extraction processes but also of the derivatization process.
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376
380
382 383 384
3.4. Analytical figures of merit of the proposed method Once the simultaneous derivatization and DLLME was optimized, different quality
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381
parameters were evaluated using standard solutions. An internal standard calibration curve was constructed and linearity reached at least -1
-1
385
1000 ng mL . The working range employed was set from 20 to 100 ng mL . All the solutions
386
contained 40 ng mL of o-cresol as surrogate. Figure 7 shows the chromatogram obtained for
387
an ethanolic standard solution containing the target analytes at 60 ng mL
388
submitted to the proposed LLE followed by derivatization and DLLME and GC-MS method.
-1
−1
and the surrogate,
389
The repeatability, expressed as relative standard deviation (RSD), was evaluated by
390
applying the proposed LLE followed by derivatization and DLLME and GC-MS method to five
13
Page 13 of 23
-1
391
replicates of a standard solution containing 40 ng mL
392
surrogate. RSD of 7 and 10 % were obtained for atranol and chloratranol respectively. The EFs
393
of the whole method were also calculated, taking into account the loss in the LLE process and
394
the EFs of the DLLME process, resulting in a value of 15 for both, atranol and chloroatranol.
of the target compounds and the
In order to evaluate matrix effects when internal standard calibration is used, the
396
slopes of the calibration curves obtained by this method and those obtained by standard
397
addition calibration combined with internal standard of four commercial perfumes were
398
compared. In this sense, the proposed LLE followed by derivatization and DLLME and GC-MS
399
method was applied to four perfume samples that had been previously spiked with the target
400
analytes at 20 - 100 ng mL (working range). The characteristics of the calibration equations
401
obtained for each standard addition are shown in Table 1. This table also shows the study of
402
matrix effect obtained in percentage calculated with the ratio of the slope of the standard
403
addition calibration of each sample to that of an internal standard calibration performed the
404
same day. Matrix effects can be observed, especially in the case of chloroatranol. Thus,
405
standard addition is recommended. It is worth mentioning that even though matrix effects were
406
observed in some previous published articles [12,13], no correction of the data with standard
407
addition was done. Table 2 shows the recoveries obtained after spiking blank perfume samples
408
with 20 and 100 ng mL of the target compounds and performing the proposed method. Good
409
recovery values are obtained when using standard addition calibration.
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395
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410
As standard addition is proposed, limits of detection were calculated as 3 times the
411
signal-to-noise ratio of a sample spiked with 20 ng mL of the analytes. They were of the order
412
of 3 and 2 ng mL for atranol and chloratranol respectively in the initial perfume samples. Thus,
413
the limits of quantification obtained were of the order of 9 and 6 ng mL
414
chloratranol respectively. Repeatability was re-evaluated using the same experiment but this
415
time to the four perfumes spiked instead of the standard solution. The mean of the four RSD
416
resulted in a total RSD of 9 % for both atranol and chloroatranol. This shows that there is no
417
significant difference between the internal standard and the standard addition method on
418
repeatability.
-1
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-1
-1
for atranol and
419 420
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Page 14 of 23
421
3.5. Assay of the proposed method to the analysis of real perfume samples The target analytes were determined in four different perfume samples. The results
423
indicated that both analytes were not present in the analyzed samples or that they were below
424
the limits of detection of the method. As the number of samples is rather limited these results
425
could be taken as satisfactory preliminary results and a higher number should be analysed to
426
assure the method is applicable to all market perfumes.
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422
427 4. Conclusions
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428
Quality control of commercial perfumes and raw materials is necessary. Analytical
430
methods are required to guarantee there are no undesired compounds in the finished product.
431
Little has been done in this field regarding the potent allergens atranol and chloroatranol.
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429
432
DLLME and GC-MS is presented to determine atranol and chloroatranol in perfumes.
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433
In this work a method based on LLE followed by simultaneous derivatization and
434
Since matrix effects were observed when internal standardization is used, standard
435
addition calibration is recommended. In this sense, the method allows the determination of
436
atranol and chloroatranol successfully in the ng mL range with good accuracy and precision.
437 Acknowledgements
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The authors acknowledge the financial support of the Spanish Ministry of Science and
440
Innovation (Project CTQ2009-12709), especially Marina López-Nogueroles for her FPI
441
predoctoral grant, and the financial support of University of Valencia (Project UV-INV-AE13-
442
137567).
443
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439
Authors also acknowledge the advice given concerning the derivatization reaction, by
444
Prof. Gonzalo Blay, Prof. Isabel Fernandez and Ms. Amparo Sanz from the Department of
445
Organic Chemistry of the University of Valencia.
15
Page 15 of 23
References
[1] Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending council directive 76/768/EEC on the approximation of the laws of the member states relating to cosmetic products
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[2] Regulation (EC) no 1223/2009 of the European Parliament and of the council of 30 November 2009 on cosmetic products
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[3] A. Chisvert, M. López-Nogueroles, A. Salvador, Essential oils: Analytical methods to control
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the quality of perfumes, in: M.J. Ramawat K. (Ed.), Handbook of Natural Products, SpringerVerlag, Berlin Heidelberg, 2013, pp. 3287-3310.
an
[4] A. Chaintreau, Analytical methods to determine potentially allergenic fragrance-related substances in cosmetics, in: A. Salvador, A. Chisvert (Eds.), Analysis of cosmetic products,
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Elsevier, Amsterdam, 2007, pp 257-275.
[5] A. Chaintreau, D. Joulain, C. Marin, C. Schmidt, M. Vey, J. Agric. Food Chem. 51 (2003)
ed
6398-6403.
[6] H. Leijs, J. Broekhans, L. van Pelt, C. Mussinan, J. Agric. Food Chem. 53 (2005) 5487-5491 [7] M. Bassereau, A. Chaintreau, S. Duperrex, D. Joulain, H. Leijs, G. Loesing, N. Owen, A.
ce pt
Sherlock, C. Schippa, P. Thorel, M. Vey, J. Agric. Food Chem. 55 (2007) 25-31. [8] A. Chaintreau, E. Cicchetti, N. David, A. Earls, P. Gimeno, B. Grimaud, D. Joulain, N. Kupfermann, G. Kuropka, F. Saltron, C. Schippa, Journal of Chromatography A 1218 (2011)
Ac
7869-7877
[9] D. Joulain, R. Tabacchi, Flavour Frag. J. 24 (2009) 49-61. [10] D. Joulain, R. Tabacchi, Flavour Frag. J. 24 (2009) 105-116. [11] G. Bernard, E. Gimenez-Arnau, S. Rastogi, S. Heydorn, J. Johansen, T. Menne, A. Goossens, K. Andersen, J. Lepoittevin, Arch. Dermatol. Res. 295 (2003) 229-235. [12] J. Johansen, G. Bernard, E. Gimenez-Arnau, J. Lepoittevin, M. Bruze, K. Andersen, Contact Dermatitis 54 (2006) 192-195.
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[13] J. Johansen, K. Andersen, C. Svedman, M. Bruze, G. Bernard, E. Gimenez-Arnau, S. Rastogi, J. Lepoittevin, T. Menne, Contact Dermatitis 49 (2003) 180-184. [14] R. Hiserodt, D. Swijter, C. Mussinan, J. Chromatogr. A 888 (2000) 103-111. [15] Scientific Committee on Consumer Products. SCCP Opinion on Atranol and Chloroatranol Present in Natural Extracts (e.g. Oak Moss and Tree Moss Extracts). SCCP/00847/04 adopted
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by the SCCP on 7 December 2004.
[16] F. David, C. Devos, P. Sandra, LC GC Europe 19 (November 2006) 602-617
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[17] R. Bossi, S. Rastogi, G. Bernard, E. Gimenez-Arnau, J. Johansen, J. Lepoittevin, T.
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Menne, J. Sep. Sci. 27 (2004) 537-540.
[18] S. Rastogi, R. Bossi, J. Johansen, T. Menne, G. Bernard, E. Gimenez-Arnau, J.
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Lepoittevin, Contact Dermatitis 50 (2004) 367-370
[19] S.C. Rastogi, J.D. Johansen, R. Bossi, Contact Dermatits 56 (2007) 201-204.
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[20] N. Fattahi, Y. Assadi, M.R.M. Hosseini, E.Z. Jahromi, J. Chromatogr. A. 1157 (2007) 23-29. [21] R. Montes, I. Rodriguez, E. Rubi, R. Cela, J. Chromatogr. A. 1216 (2009) 205-210.
ed
[22] J. Wu, H. Chen, W. Ding, J Chromatogr. A. 1302 (2013) 20-27 [23] S. Luo, L. Fang, X. Wang, H. Liu, G. Ouyang, C. Lan, T. Luan, J. Chromatogr. A. 1217
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(2010) 6762-6768.
[24] M.K.R. Mudiam, C. Ratnasekhar, J. Chromatogr. A 1291 (2013) 10-18. [25] C. Selenski, T. R.R. Pettus, Tetrahedron 62 (2006) 5298-5307.
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[26] F. Galán-Cano, R. Lucena, S. Cárdenas, M. Valcárcel, J. Chromatogr. A 1229 (2012) 48-
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Figure captions
Fig. 1 Chemical structure of (a) atranol (b) chloroatranol and (c) o-cresol Fig. 2 Effect of the volume of extraction solvent. Extraction conditions: standard solution volume: 10 mL; disperser solvent volume: 1000 µL; K2CO3 1M volume: 1 mL; acetic anhydride
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volume: 100 µL.
Fig. 3 Effect of the volume of disperser solvent. Extraction conditions: standard solution volume:
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10 mL; extraction solvent volume: 100 µL; K2CO3 1M volume: 1 mL; acetic anhydride volume:
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100 µL.
Fig. 4 Effect of the volume of K2CO3. Extraction conditions: standard solution volume: 10 mL;
an
disperser solvent volume: 1000 µL; extraction solvent volume: 100 µL; acetic anhydride volume: 100 µL.
M
Fig. 5 Effect of the volume of acetic anhydride. Extraction conditions: standard solution volume: 10 mL; disperser solvent volume: 1000 µL; extraction solvent volume: 100 µL; K2CO3 1M
ed
volume: 1 mL.
Fig. 6 Effect of the ionic strength. Extraction conditions: standard solution volume: 10 mL;
ce pt
disperser solvent volume: 1000 µL; extraction solvent volume: 100 µL; K2CO3 1M volume: 1 mL; acetic anhydride volume: 100 µL.
Fig. 7 A chromatogram obtained applying the proposed method to an ethanolic standard -1
-1
Ac
solution containing the target compounds at 60 ng mL and o-cresol as surrogate at 40 ng mL .
18
Page 18 of 23
Figure 1
(a)
OH
Cl
(b)
(c)
OH
OH
O
O
OH
us
cr
ip t
OH
75 µL 100 µL 125 µL
150
M
100
0
Chloroatranol
ce pt
Atranol
ed
50
Ac
Area (counts*min/105)
200
an
Figure 2
Page 19 of 23
Figure 3
250 µL
500 µL
750 µL
1000 µL
150
100
50
ip t
Area (counts*min/105)
200
0
Chloroatranol
cr
Atranol
0,5 mL
1 mL
2 mL
an
200
0.1 mL
150
M
100 50 0
ed
Chloroatranol
ce pt
Atranol
Ac
Area (counts*min/105)
250
us
Figure 4
Page 20 of 23
Figure 5 50 µL 100 µL 150 µL
150
100
50
ip t
Area (counts*min/105)
200
0 Chloroatranol
us
cr
Atranol
Figure 6
4 % NaCl
9 % NaCl
M
an
2.5 % NaCl
100
50
0
Figure 7 100
Chloroatranol
ce pt
Atranol
O-cresol (m/z = 108)
50 0 100
Ac
Relative abundance (%)
0 % NaCl
ed
Area (counts*min/105)
150
Atranol (m/z = 152)
50
0 100
Chloroatranol (m/z = 186)
50 0 0
5
10
15
20
25
30
Time (min)
Page 21 of 23
ip t cr Atranol
Perfume 1 Perfume 2 Perfume 3 Perfume 4
(280±9)·10
-4
(297±7)·10
-4
(315±12)·10
-4
(336±15)·10
-4
a
-0.05±0.05 -0.04±0.03 -0.10±0.07 0.13±0.09
Regression a coefficient
Calibration b slopes ratio (%)
0.996
116 ± 5
0.998 0.994 0.992
112 ± 6 120 ± 7 146 ± 9
Slope -1 -1 (ng mL )
Intercept
a
Regression a coefficient
Calibration b slopes ratio (%)
-4
-0.0002±0.008
0.997
72 ± 6
-4
-0.006±0.02
0.990
89 ± 6
-4
0.003±0.003
0.998
71 ± 3
-4
0.03±0.02
0.98
62 ± 8
(56±2)·10 (65±4)·10 (52±1)·10 (41±4)·10
*
-1
d
Obtained by means of the internal standardization calibration. Working range: 20 to 100 ng mL . Number of calibration points: 6 (Standard addition calibration slope / Internal standard calibration slope) x 100
ep te
b
Intercept
Ac c
a
Slope -1 -1 (ng mL )
Chloroatranol
a
an
Analyte
M
a
us
Table1. Study of matrix effects
Page 22 of 23
Table 2 Study of the recovery of the proposed method (standard addition calibration) Recovery * (%) Atranol 20 ng mL
-1
Chloroatranol
100 ng mL-1
20 ng mL-1
100 ng mL
Perfume 1
94
103
95
98
Perfume 2
96
101
79
97
Perfume 3
86
103
104
100
Perfume 4
106
98
110
98
-1
* Recovery obtained after spiking free-analytes samples with 20 and 100 ng mL-1, estimated as
Ac
ce pt
ed
M
an
us
cr
((Obtained concentration – Found concentration) / Spiked concentration)) x 100
ip t
Real samples
Page 23 of 23
