A green and simple procedure based on deep eutectic solvents for the extraction of phthalates from beverages

Journal Pre-proofs A green and simple procedure based on deep eutectic solvents for the extraction of phthalates from beverages Álvaro Santana-Mayor, Bárbara Socas-Rodríguez, Ruth Rodríguez-Ramos, Miguel Ángel Rodríguez-Delgado PII: DOI: Reference:

S0308-8146(19)31931-4 https://doi.org/10.1016/j.foodchem.2019.125798 FOCH 125798

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

7 June 2019 23 October 2019 24 October 2019

Please cite this article as: Santana-Mayor, A., Socas-Rodríguez, B., Rodríguez-Ramos, R., Ángel RodríguezDelgado, M., A green and simple procedure based on deep eutectic solvents for the extraction of phthalates from beverages, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125798

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A green and simple procedure based on deep eutectic solvents for the extraction of phthalates from beverages

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Álvaro Santana-Mayor1, Bárbara Socas-Rodríguez1, **, Ruth Rodríguez-Ramos1, Miguel

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Ángel Rodríguez-Delgado1, *

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1Departamento

de Química, Unidad Departamental de Química Analítica, Facultad

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de Ciencias, Universidad de La Laguna (ULL). Avda. Astrofísico Fco. Sánchez, s/nº. 38206

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San Cristóbal de La Laguna, España.

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*Corresponding author: Dr. Miguel Ángel Rodríguez Delgado

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Tel: + 34 922 31 80 46

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Email: [email protected]

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**Co-corresponding author: Dra. Bárbara Socas Rodríguez

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Tel: +34 922 31 80 50

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Email: [email protected]

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Abstract

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In this work, a green, inexpensive, simple and fast deep eutectic solvent (DES)-based

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dispersive liquid-liquid microextraction was evaluated, for the first time, for the extraction of

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phthalates (i.e. benzylbutyl phthalate, diisobutyl phthalate, diisopentyl phthalate, di-n-pentyl

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phthalate, di-(2-ethylhexyl) phthalate, di-n-octyl phthalate, diisononyl phthalate, diisodecyl

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phthalate) from different beverages. Separation and determination were achieved by high

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performance liquid chromatography-diode-array detection while confirmation was carried out

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by tandem mass spectrometry. The main factors affecting the extraction such as type and

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volume of DES and emulsifier, pH and ionic strength, were optimised. Choline

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chloride:phenol-based DES showed the best results. The methodology was validated for tea,

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apple-based beverage and pineapple juice. Recovery values ranged from 84 to 120 % with

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relative standard deviation values lower than 11 %. Limits of detection of the method were in

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the range µg L-1 for tea, 5.3-17.8 µg L-1 for apple beverages and 5.9-15.6 µg L-1 for pineapple

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juices.

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Keywords: plastic migrants; green solvent; dispersive liquid-liquid microextraction; food

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samples; drinks; high performance liquid chromatography.

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List of abbreviations

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ACN: acetonitrile; BBP: benzylbutyl phthalate; CCD: central composite design; ChCl:

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choline chloride; DAD: diode-array detection; DEHP: di-(2-ethylhexyl) phthalate; DES: deep

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eutectic solvent; DHP-d4: dihexyl phthalate-3,4,5,6-d4; DIBP: diisobutyl phthalate; DIDP:

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diisodecyl phthalate; DINP: diisononyl phthalate; DIPP: diisopentyl phthalate; DLLME:

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dispersive liquid-liquid microextraction; DNOP: di-n-octyl phthalate; DNPP: di-n-pentyl

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phthalate; EPA: Environmental Protection Agency; EtGly: ethylene glycol; EU: European

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Union; FT-IR: Fourier transform infrared; Gly: glycerol; HBA: hydrogen bond acceptor;

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HBD: hydrogen bond donor; HPLC: high performance liquid chromatography; IL: ionic

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liquid; IS: internal standard; LC: liquid chromatography; LOD: limit of detection; LOQ: limit

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of quantification; ME: matrix effect; MRM: multiple reaction monitoring; MS/MS: tandem

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mass spectrometry; MS: mass spectrometry; PAE: phthalic acid ester; R2: determination

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coefficient; RSD: relative standard deviation; THF: tetrahydrofuran; UHPLC: ultra-high

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performance liquid chromatography; VA-EDLLME: vortex-assisted-emulsification dispersive

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liquid-liquid microextraction.

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1.- Introduction

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In the last years, deep eutectic solvents (DESs), introduced by Abbott et al. in 2003,

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(Abbott, Capper, Davies, Rasheed & Tambyrajah, 2003), have emerged as a new type of

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green extraction solvents due to their excellent physicochemical properties. DESs are

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considered as a new generation of ionic liquids (ILs), however, they have some advantages in

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terms of low cost and greater availability of the components, ease to be prepared and they are

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also more environmentally friendly than ILs (Aydin, Yilmaz & Soylak, 2018). DESs have

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been employed in different fields and applications, especially as extraction solvents in sample

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preparation for a wide variety of analytes and matrices (Płotka-Wasylka, Rutkowska,

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Owczarek, Tobiszewski & Namiśenik, 2017). Generally, DESs are obtained by the

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complexation of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA) that are

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capable to interact with each other through strong hydrogen bond interactions, occasional

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electrostatic and van der Waals interactions, resulting in the formation of a liquid with lower

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melting point than those of any of its components (Aydin et al., 2018).

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Choline chloride (ChCl) is the most common HBA. DESs based on ChCl present

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several benefits such as low cost, simple synthesis, biodegradability and non-toxicity, among

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others. ChCl has been combined with a large number of HBDs, among which phenol is

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included since it allows not only the interaction with both organic compounds and inorganic

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species, but also lets easy phases separation using an aprotic solvent, unlike other ChCl-based

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DESs that are water-miscible (Khezeli, Daneshfar & Sahraei, 2015; Moghadam, Rajabi &

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Asghari, 2018).

80

Phthalic acid esters (PAEs) are a group of additives widely used in the plastic industry

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to improve the properties of this kind of materials. In recent years, these compounds have

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caused great concern due to their ubiquitous presence in the environment since they are not

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linked to the polymer matrix of plastics and, therefore, can migrate from the material to the

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environment, as well as their harmful effect from an ecological and health point of view (Lü

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et al., 2018). As a result of the negative effects, due to their known endocrine-disrupting

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activity that these analytes produce on the human’s health, the European Parliament in the

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resolution of March 14th, 2013 on the protection of public health from endocrine disrupters

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(European Parliament resolution, 2013) pointed out that even at very low levels of

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concentration, any exposure, even at very low levels of concentration, to endocrine disrupters,

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such as PAEs, may entail a risk and, therefore, such substances lack a limit value below

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which adverse effects do not occur. For this reason, the development of highly sensitive and

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selective analytical methodologies capable of determining this type of analytes at low levels

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of concentration is of special interest for the scientific community and regulatory

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organisations.

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Different extraction procedures have been used to analyse phthalates in beverages

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including solid-phase extraction and solid-phase microextraction procedures, as well as liquid

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phase microextraction techniques in their different approaches (González-Sálamo, Socas-

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Rodríguez & Hernández-Borges, 2018). Among the latter, the use of dispersive liquid-liquid

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microextraction (DLLME) should be highlighted. However, this extraction technique, which

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offers high speed and operational simplicity, as well as large preconcentration factors, has

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been mainly used mainly employing organic solvents (Rezaee, Yamini & Faraji, 2010). Since

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the first publication by Rezaee et al. (Rezaee, Assadi, Milani-Hosseini, Aghaee, Ahmadi &

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Berijani, 2006), and in order to eliminate this type of solvents due to its high environmental

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damage and toxicity, other extraction agents such ILs have been tested (Fan, Liu & Xie,

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2014). Nevertheless, and despite the above-mentioned advantages that DESs offer as

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extraction solvents, no publications have been reported that use DESs for the extraction of

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PAEs.

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The aim of this work is the application, for the first time, of a laboratory synthesised

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DES as solvent for the vortex-assisted-emulsification DLLME (VA-EDLLME) of a group of

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eight PAEs (i.e. benzylbutyl phthalate (BBP), diisobutyl phthalate (DIBP), diisopentyl

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phthalate (DIPP), di-n-pentyl phthalate (DNPP), di-(2-ethylhexyl) phthalate (DEHP), di-n-

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octyl phthalate (DNOP), diisononyl phthalate, (DINP),diisodecyl phthalate (DIDP)) from

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different food samples prior to their separation and determination by liquid chromatography

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(LC)-diode-array (DAD)/tandem mass spectrometry (MS/MS). To the best of our knowledge,

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this is the first time in which this type of solvent has been applied for the extraction of PAEs

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from any type of samples, including widely consumed beverages such as the ones analysed in

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this work such as tea, soft drinks and fruit juices.

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2.- Experimental

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2.1.- Chemicals and materials

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Analytical standards of DEHP (CAS 117-81-7), DNOP (CAS 117-84-0), DINP (CAS

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28553-12-0) and DIDP (CAS 26761-40-0) from Sigma-Aldrich Chemie (Madrid, Spain), and

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BBP (CAS 85-68-7), DIBP (CAS 84-69-5), DIPP (CAS 605-50-5), DNPP (CAS 131-18-0)

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and dihexyl phthalate-3,4,5,6-d4 (DHP-d4, CAS 1015854-55-3) from Dr. Ehrenstorfer GmbH

125

(Augsburg, Germany) were used without further purification (purity ≥ 97 %).

126

Individual stock solutions of each analyte were prepared in acetonitrile (ACN) of LC-

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mass spectrometry (MS) grade at 70 mg·L-1 for DHP-d4, 100 mg·L-1 for DINP, 500 mg·L-1

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for BBP, DIBP, DNPP, DNOP and DIDP, and 1000 mg·L-1 for DEHP and DIPP and stored in

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the darkness at -18 ºC. Working analyte mixtures were daily obtained by dilution with the

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appropriate volume of initial mobile phase. Range of working concentrations were 15.86-

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17.14 mg·L-1 for BBP; 15.84 mg·L-1 for DIBP, 19.26-23.56 mg·L-1 for DIPP, 7.28 mg·L-1 for

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DNPP, 7.29 mg·L-1 for DEHP, 7.27 mg·L-1 for DNOP, 11.57 mg·L-1 for DINP, and 11.56-

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15.85 mg·L-1 for DIDP, while the internal standard (IS) concentration was 10 mg·L-1.

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All chemicals were of analytical reagent grade (unless otherwise indicated) and used

135

as received. ACN, acetone of LC grade, 1,4-dioxane ( 99.5 %), acetic acid (99-100 %), urea

136

(99.0-100.5 %) and hydrochloric acid (25 %, w/w) were from Merck (Darmstadt, Germany).

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Tetrahydrofuran (THF) of high performance liquid chromatography (HPLC) grade was from

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from Scharlau Chemie S.A. (Barcelona, Spain). ChCl ( 98 %), phenol (99.0-100.5 %),

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glycerol (Gly,  99.5 %), sodium hydroxide ( 98 %) and sodium chloride ( 99.5 %) were

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from Sigma-Aldrich Chemie (Madrid, Spain). Ethylene glycol (EtGly,  99 %) was from

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Honeywell (New Jersey, USA). Water was deionised by Milli-Q gradient system A10 from

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Millipore (Massachusetts, USA).

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Based on the ubiquitous presence of PAEs in the environment, special effort was

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carried out to avoid the possible contamination in the laboratory and guarantee the absence of

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PAEs in the laboratory material. With this objective, Nochromix® cleaning mixture (prepared

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as indicated by the manufacturer) from Godax Laboratories (Maryland, USA) was used for

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the volumetric glassware, whereas non-volumetric glassware was calcinated at 550 ºC during

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4 h. In addition, PAEs-free gloves and pipette tips were used in all cases.

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2.2.- Apparatus and software

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The Fourier transform infrared (FT-IR) spectrums were recorded on the range 4000-

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600 cm-1 at room temperature using a Thermo Nicolet Avatar 360 FT-IR E.S.P spectrometer

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(Thermo Fisher Scientific, Massachusetts, USA).

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Analyses of PAEs were carried out in a HPLC system equipped with a binary pump

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(model 1525), an autosampler (model 717 plus) and a column heater (Model 5CH 1500

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series), working with Empower 2 software from Waters. The HPLC system was coupled to a

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DAD (model 2998) all of them from Waters Chromatography (Milford, MA, USA).

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Separation was carried out at 45 ºC in an X-Bridge C18 column (100 mm × 4.6 mm, 3.5 µm)

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with a pre-column with the same stationary phase (20 mm × 4.6 mm, 3.5 µm), both from

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Waters Chromatography.

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The mobile phase used for the analysis consisted of ACN (as solvent A) and water (as

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solvent B). The initial composition of the mobile phase was 50/50 (v/v) A/B with a flow of

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1.0 mL min-1. It was changed to 70/30 (v/v) A/B in 12.0 min. Then, the composition changed

164

to 100% of A in 1.0 min which was maintained during 7.0 min. The injection volume was 20

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µL and the wavelength of the detector was set at 225 nm.

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Waters Acquity UPLC® H-Class (Milford, MA, USA), equipped with a quaternary

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solvent manager and a sample manager with flow-through needle, controlled with

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MasslynxTM software from Waters Chromatography and coupled to a MS Xevo TQD detector

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(Waters Chromatography) using electrospray ionisation in positive mode were employed to

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confirm the presence of PAEs in real samples. Control of MS parameters and collection and

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processing of spectrum data was performed with MasslynxTM V4.1 software from Waters

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Chromatography. Separations were carried out in an Acquity UPLC® BEH C18 column (50

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mm x 2.1 mm, 1.7 µm) using an Acquity UPLC® BEH C18 pre-column (5 mm x 2.1 mm, 1.7

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µm), both from Waters Chromatography.

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Chromatographic and MS conditions used for confirmation analysis were applied as

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indicated in the previous work developed by Santana-Mayor et al. (Santana-Mayor, Socas-

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Rodríguez, Afonso, Palenzuela-López & Rodríguez-Delgado, 2018). Source conditions,

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which provided the highest intensity of precursor ions, were as follows: capillary voltage 3.5

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kV, source temperature 150 ºC, desolvation temperature 500 ◦oC, cone gas (N2) flow rate 50 L

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h-1, desolvation gas (N2) flow 900 L h-1, collision gas (Ar) pressure 0.5 bar. MS/MS

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experiments were performed by fragmentation of the protonated molecule [M−H]+ that was

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selected as the precursor ion. Multiple reaction monitoring (MRM) transitions as well as the

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values of cone voltage and collision energy for each analyte were optimised by direct infusion

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of individual standards of phthalates at 2 mg·L-1 in a mixture of A/B (50/50, v/v).

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2.3.- Samples selection

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Three different beverages, peach tea drink (stored in an aluminium bottle), apple soft

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drink (contained in a glass bottle) and pineapple juice (storage in a glass bottle), were selected

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as matrices to carry out the validation of the developed methodology. pH values at 25 ºC for

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tea, apple soft drink and pineapple juice were 2.76, 3.52 and 3.34, respectively.

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Apart from that, fourteen more samples were analysed in order to evaluate the

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presence of selected PAEs in real beverage samples. Among them, four tea samples including

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peach, mango and green tea, all stored in plastic bottles (pH of 2.71, 3.44 and 2.96 at 25 ºC,

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respectively), and peach tea drink contained in a glass bottle (pH of 3.16 at 25 ºC); five apple-

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based beverages including apple soft drink, storage in aluminium bottle (pH of 2.57 at 25 ºC),

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and plastic bottle (pH of 2.56 at 25 ºC), apple non-carbonated soft drink contained in a plastic

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bottle (pH of 2.82 at 25 ºC) and two apple juices, one storage in a glass bottle (pH of 3.40 at

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25 ºC) and the other one contained in a Tetra Brik® (pH of 3.62 at 25 ºC); and five pineapple-

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based juices including, three pineapple juices stored in plastic containers from different

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brands (pH of 3.53, 3.41 and 3.27 at 25 ºC), pineapple juice (pH of 3.27 at 25 ºC) and

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pineapple-coconut juice (pH of 3.37 at 25 ºC) both contained in a Tetra Brik®. All beverage

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samples were purchased in local supermarkets of Tenerife (Canary Islands, Spain).

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2.4.- Synthesis of DESs

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In this work, several DESs were prepared using ChCl as HBA and Gly, EtGly, urea,

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phenol or acetic acid as HBDs at molar ratio of 1:2, according to the literature (Khezeli et al.,

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2015; Moghadam et al., 2018). Then, after selecting phenol as the most suitable HBD,

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different molar ratios (i.e. 1:1, 1:3 and 1:4) of ChCl:phenol were also tested, in order to find

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out the most suitable composition. For synthesis, the components of DESs were placed in 50

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mL screw-capped glass tubes and then magnetically stirred, at room temperature for

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ChCl:phenol eutectic mixtures and at 80 ºC for the others, until obtaining colourless obtaining

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homogeneous liquids within 10 min in all cases. Finally, the products were cooled to room

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temperature in a vacuum desiccator to avoid moisture absorption.

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2.5.- Sample pre-treatment

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After pH and ionic strength adjustment to 6 and 16 % of NaCl (w/v), respectively, ; tea

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samples were centrifuged at 3000 r.p.m. for 5 min and the supernatant was subsequently

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filtered through a CHROMAFIL® PET-20/15 MS syringe filter with 0.20 µm pore size in

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order to remove any solid particle. In the case of the tea drink stored in glass and pineapple

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juice samples, pH was previously adjusted to pH 2 (with HCl conc.) and pH 12, respectively

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so that the centrifugation stage would be effective and before carrying out the extraction

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procedure, the pH was re-adjusted to the optimum value using NaOH 10 M or HCl conc.

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Apple soft drinks were initially sonicated for 30 min and then the procedure was applied as

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previously indicated for tea samples.

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2.6.- VA-EDLLME procedure

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VA-EDLLME method was applied for the extraction of eight PAEs from different

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beverages. Ten mL of spiked or non-spiked sample (containing 16 % NaCl (w/v)) was

229

adjusted to pH 6, using NaOH 10 M or HCl conc., introduced in a 15 mL screw-capped glass

230

centrifuge tube and 440 µL of DES ChCl:phenol 1:2 (as water-miscible extraction solvent)

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was added to the sample and vortexed for 1 min to obtain a homogeneous solution. Then, 440

232

µL of THF (as emulsifier agent) was added and followed by vortex for 1 min. At this stage,

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PAEs were quickly and easily extracted by DES due to the formation of a cloudy solution of

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micro-droplets with huge effective surface areatook places as a consequence of the self-

235

aggregation process of DES, and PAEs were quickly and easily extracted by DES.

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Afterwards, the mixture was centrifuged at 3000 r.p.m. for 7 min in a 5810 R centrifuge from

237

Eppendorf (Hamburg, Germany) to achieve phases separation and 300 µL of the DES (upper

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phase), containing the target analytes, was collected with a micropipette and transferred into

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an HPLC vial. Finally, it was diluted with 128.6 µL of ACN, and 20 µL were injected into the

240

HPLC system. Due to the ease, simplicity of the synthesis as well as the low cost of the raw

241

materials, DES was only used once, trying to avoid the possible carry-over effects as well

242

asand the excessive solvent consumption of the washing procedure.

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In the case of apple-based beverages, the DES-enriched phase was previously filtered

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through a 0.22 µm Corning® Costar® Spin-X® cellulose acetate centrifuge tube filter from

245

Sigma-Aldrich Chemie (Madrid, Spain) before the corresponding dilution in order to remove

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some solid co-extracted components.

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2.7.- Experimental Design

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In the present work, a response surface methodology was performed for the partial

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optimisation of the VA-EDLLME procedure using StatGraphics Centurion XVI software,

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16.2.04 version. In this case, a central composite design (CCD), consisting of three blocks,

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five levels (edge points (- 1 and + 1), the centre point and the star points (- α and + α)) and

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three factors (salt amount, volume of DES and volume of the emulsifier agent), with three

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replicates of the central point (15 % NaCl (w/v), 450 µL of DES and 450 µL of emulsifier)

255

and an axial distance of 1.67 (orthogonal assay), was applied in order to, simultaneously,

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evaluate the effects of these experimental parameters that affect the VA-EDLLME procedure.

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The design was carried out randomly in order to minimise the effect of non-controlled

258

variables (Khezeli et al., 2015). The different factor levels were selected according to the

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results obtained in previous studies. Salt amount was varied between 0 and 30 % (w/v) and

260

DES volume, as well as THF volume, between 100 and 800 µL as it is indicated in Table S1

261

of the Supplementary Material.

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The design involved 17 experiments using spiked Milli-Q water at 1.45 mg L-1 for

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BBP and DIBP, 2.60 mg L-1 for DIPP, 1.70 mg L-1 for DNPP and DINP, 0.95 mg L-1 for

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DEHP and DNOP, and 2.25 mg L-1 for DIDP.

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3.- Results and discussion

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3.1.- Evaluation of the synthesised DESs for the extraction of PAEs

268

DESs based on ChCl are considered excellent solvents due to their low toxicity and

269

biodegradability (Moghadam et al., 2018). However, the structure of the different HBDs is

270

also determinant in the physicochemical properties and, therefore, in the extraction efficiency

271

of the target analytes (Liu, Zhang, Qin & Yu, 2017). In order to select the most suitable

272

extraction solvent for the extraction of PAEs, five DESs based on ChCl as HBA and phenol,

273

urea, Gly, EtGly or acetic acid, as HBDs in molar ratio 1:2, were tested. The salt content of

274

the sample solution and/or the volume of THF were modified to obtain a quantitative volume

275

of extractant phase in all experiments.

276

With this aim, in the case of ChCl:phenol DES, 10 mL of Milli-Q water containing 15

277

% NaCl (w/v) at pH 6, 500 µL of DES, as extraction solvent, and 500 µL of THF as

278

emulsifier solvent were used. For acetic acid-based DES, was necessary to increase the ionic

279

strength to 30 % NaCl (w/v) and use 750 µL of THF to obtain a collectable DES phase

280

volume. In contrast, using the same conditions, or even employing higher volumes of

281

emulsifier agent, for ChCl:urea, ChCl:Gly and ChCl:EtGly, DESs, no phase separation

282

occurred hidden the successful extraction of the analytes from the aqueous media, which can

283

be probably justified with the high miscibility of these DESs in aqueous systems, making it

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284

impossible to carry out the phase separation.

285

In this way, even though the results obtained for the DES based on phenol and acetic

286

acid were similar, the extraction procedure for ChCl:phenol DES was carried out under milder

287

conditions and, in addition, a greater volume of enriched extractant phase was obtained,

288

improving the efficiency of the procedure. PAEs have both ester groups and aromatic rings,

289

which allows the phenol-based DESs to establish hydrogen bond as well as π-π interactions

290

with this type of compounds. Besides, the logarithm of the partition coefficients of the eight

291

PAEs studied ranged 4.11 to 10.36, which implies that these compounds have a high or very

292

high hydrophobicity, which is reflected in the good recovery values obtained when the

293

phenol-based DES was used. Thus, ChCl:phenol DES was selected as extraction solvent in

294

this study.

295 296

3.2.- DES characterisation

297

Prior to the application of the laboratory synthesised ChCl:phenol DES for the

298

extraction of the selected PAEs, this was characterised in order to confirm the formation of

299

hydrogen bond interactions between chloride anion of ChCl and hydroxyl group of phenol,

300

which is the main driving force for the DES formation. FT-IR spectra of pure ChCl, phenol,

301

and DES were recorded in order to confirm the formation of DES. Results are shown in

302

Figure S1. As can be seen in the Figure S1a, a peak positioned at 3415 cm-1 belongs to the

303

hydroxyl group of ChCl. The peak belonging to C–N vibration of pure ChCl was observed at

304

957 cm-1 (Mulia, Krisanti, Terahadi & Putri, 2015). Figure S1b shows the FT-IR spectrum of

305

pure phenol. Peaks at 3398 cm-1 and 1375 cm-1 are associated with the stretching and bending

306

vibrations of O–H functional group, respectively, while the peak observed at 1236 cm-1

307

belongs to the C–O stretching vibration (Smith, 2017). In Figure S1c, the O–H vibration of

308

pure phenol at 3398 cm-1 shifted to 3226 cm-1 suggesting the formation of hydrogen bonding

13

309

between phenol and ChCl when DES is formed.

310 311

3.3.- HPLC-DAD method

312

In this work, a group of eight PAEs was analysed using an X-Bridge C18 column.

313

Based on the previous experience in separation of phthalates, different gradients constituted

314

of mixtures of ACN/H2O (v/v) were tested. The best results were obtained applying the

315

gradient described in Section 2.2. In order to get the maximum absorption wavelength of each

316

analyte, a study was conducted in which a wavelength scan was carried out by the injection of

317

individual standards. 225 nm was selected as the most appropriate wavelength for all analytes.

318

In order to evaluate the linearity of the developed separation, instrumental calibration curves

319

based on the ratio of each analyte and the internal standard (IS), dihexyl phthalate-3,4,5,6-d4,

320

were carried out. Peak areas for each compound were obtained by injecting seven

321

concentration levels (n = 7; concentrations in the range 0.37–4.44 mg·L-1 for BBP; 0.37–3.70

322

mg·L-1 for DIBP; 0.457–5.39 mg·L-1 for DIPP; 0.17–1.70 mg·L-1 for DNPP, DEHP and

323

DNOP; 0.27–2.70 mg·L-1 for DINP; 0.27–3.78 mg·L-1 for DIDP) in triplicate. Determination

324

coefficients (R2) higher than 0.9954 in all cases (Table S2) and an analysis time of 20 min

325

were achieved.

326 327

3.4.- DES-VA-EDLLME optimisation

328

As it was indicated above, this work constitutes the first application of DESs for the

329

extraction of the selected group of PAEs. For this reason, a step by step study was initially

330

carried out to evaluate the extraction efficiency of different DESs. In addition, different molar

331

ratios of HBA:HBD were also tested. Likewise, the pH of the aqueous phase as well as

332

different emulsifier agents were studied with this aim. Once established these parameters,

333

NaCl % (w/v), amount of DES and volume of emulsifier agent were optimised using a CCD

14

334

model. All experiments were carried out in duplicate using Milli-Q water in order to avoid the

335

possible influence of matrix effects (MEs). In this way, 10 mL of spiked Milli-Q water

336

containing an injected concentration of twice and eight times the limit of quantification

337

(LOQ) of each analyte was employed: 1.45 mg·L-1 for BBP and DIBP, 2.60 mg·L-1 for DIPP,

338

1.70 mg·L-1 for DNPP and DINP, 0.95 mg·L-1 for DEHP and DNOP, and 2.25 mg·L-1 for

339

DIDP.

340 341

3.4.1.- Selection of DES molar ratio

342

The molar ratio of HBA to HBD plays an important role in the physicochemical

343

properties of DES and, consequently, on its extraction capacity (Lu, Cao, Wang & Su, 2016).

344

Therefore, different molar ratios ChCl:phenol from 1:1 to 1:4 were tested maintaining the rest

345

of conditions without changes (10 mL of Milli-Q water containing 15 % NaCl (w/v) at pH 6,

346

500 µL of ChCl:phenol, as extraction solvent, and 500 µL of THF. As can be seen in Figure

347

1, although 1:1 and 1:2 molar ratio provided similar results for almost all analytes, the best

348

results were obtained when molar ratio HBA to HBD 1:2 were used whereas increasing the

349

ratio above 1:2 produced a decrease in the recovery values for the longest chain PAEs

350

(DEHP, DNOP, DINP and DIDP). This fact could be associated with the reduction of the

351

proportion of hydrogen bond receptors in the DES as a consequence of the decrease in the

352

amount of ChCl (Li, Han, Zou & Yu, 2015). Thus, ChCl:phenol molar ratio of 1:2 was

353

selected for further experiments.

354 355

3.4.2.- Selection of emulsifier solvent

356

Because most ChCl-based DESs are highly water-miscible, phases separation after

357

carrying out the extraction process can be complicated (Shishov, Bulatov, Locatelli, Carradori

358

& Andruch, 2017). By the use of aprotic solvents such as THF, acetone or 1,4-dioxane,

15

359

among others, the phenomenon of self-aggregation takes places and DESs molecules are

360

easily separated from the aqueous phase. The main reason for of this process is that when an

361

aprotic solvent is adding to a homogeneous aqueous phase/DES mixture, the tendency of

362

water molecules to interact with the aprotic solvent will be greater, than with DES molecules

363

and, therefore, their interaction with the latter decreases and self-aggregation is favoured

364

(Khezeli et al., 2015). In order to achieve an adequate emulsifier agent, acetone, 1,4-dioxane

365

and THF were evaluated while the rest of conditions were maintained as follows: 10 mL of

366

Milli-Q water containing 15 % NaCl (w/v) at pH 6, 500 µL of ChCl:phenol 1:2, as extraction

367

solvent, and 500 µL of emulsifier solvent. The results showed recovery values in the range

368

51-56 % when acetone was applied and 63-71 % for almost all analytes in the case of 1,4

369

dioxane. However, a significant improvement was found when THF was employed, reaching

370

recovery values in the range 81-86 %. Therefore, THF was selected as emulsifier agent for

371

further experiments in the extraction procedure.

372 373

3.4.3.- Selection of sample pH

374

Changes in pH values can play an important role in the transfer of target analytes from

375

the aqueous phase to the extractant organic phase (Aydin et al., 2018). To study this fact, the

376

effect of the pH of the aqueous phase on the extraction efficiency of PAEs was investigated in

377

the range 2–10 and keeping the rest of experimental parameters unchanged: 10 mL of Milli-Q

378

water containing 15 % NaCl (w/v) at pH 6, 500 µL of ChCl:phenol 1:2, as extraction solvent,

379

and 500 µL of THF as emulsifier solvent. As can be seen in Figure S2, no significant

380

differences were found. This can be explained since PAEs are analytes that have no

381

established pKa values and, therefore, they are very little influenced by changes in pH.

382

However, in order to apply intermediate extraction conditions and avoid possible

383

irreproducibility during the process, pH 6 was established in all experiments.

16

384 385

3.4.4.- Experimental design

386

As can be shown above, the factors influencing the extraction process, i.e. ionic

387

strength, DES volume and THF volume, were optimised by employing a CCD, which consists

388

on the combination of a full factorial design and additional points (star points) (Hajji, Turki,

389

Hajji & Mzoughi, 2018). The response was based on the relative recovery values obtained by

390

comparing the area of peaks of the analytes in the samples enriched at the beginning and the

391

end of the methodology, using DHP-d4 as IS.

392

Figure S3 shows the Pareto chart of the standardised effects of ionic strength

393

expressed as % (w/v) of NaCl (A), volume of DES (µL) (B) and volume of THF (µL) (C)

394

with a confidence level of 95 % and indicates the significance of these last two on the

395

response surface of the study. All the variables studied have a positive influence in on

396

recovery values for all PAEs analysed. The interactions AB and AC have a negative influence

397

on recoveries recovery values for BBP, DIBP, DIPP and DNPP, and positive for DEHP,

398

DNOP, DINP and DIDP, while the interaction BC only influence negatively the results of

399

recoveries recovery for DNOP. The interaction of each variable with itself has a negative

400

influence on recoveries recovery in all cases. These results suggest that it is not necessary to

401

generate the salting-out effect to carry out the extraction procedure and, in addition, PAEs

402

have a great affinity for the extraction solvent. However, it was necessary to add NaCl to

403

ensure proper phase separation and to adequately collect adequately the extracting phase.

404

As can be seen in Figure 2a, in which the individual effects on the response of the

405

factors studied for DNPP, selected as representative analyte of the rest of PAEs, are shown;

406

the increase of the amount of salt as well as, the DES and THF volumes led to an increase in

407

the extraction efficiency, especially in the case of the volume of DES and emulsifier as it was

408

also indicated by the Pareto chart. The obtained response surface, considering a percentage of

17

409

NaCl of 15 % (w/v) is shown in Figure 2b. As can be seen, the values of relative recovery are

410

higher, which implies the maximization maximisation of the extraction efficiency of the

411

developed procedure, when both the volume of DES and THF are increased to intermediate

412

values. Furthermore, these results are consistent with those obtained from the evaluation of

413

individual effects.

414

The optimum values predicted by the CCD experimental design were an ionic strength

415

of 16 % NaCl (w/v), 440 µL of DES ChCl:phenol 1:2, as extraction solvent, and 440 µL of

416

THF as emulsifier agent. Then, three extractions, using the predicted optimal conditions, were

417

carried out consecutively, using the predicted optimal conditions confirming the obtained

418

results.

419 420

3.5.- VA-EDLLME-HPLC-DAD validation

421

Firstly, and once optimised the parameters that affect the VA-EDLLME procedure, a

422

thorough study of the repeatability between batches of the laboratory synthesised extraction

423

solvent was developed. With this regards, extractions using four different batches (n = 4) of

424

prepared DES were carried out. As can be seen in Table S3 of the Supplementary Material,

425

relative standard deviation (RSD) values were lower than 6 % in all cases, which

426

demonstrates the high repeatability of the synthetic procedure.

427

The ubiquitous presence of PAEs in the environment has been widely reported,

428

finding this type of compounds in various materials usually commonly used in the laboratory,

429

among which are high purity reagents and solvents (Guo & Kannan, 2012). For this reason,

430

the daily analysis of laboratory blanks was carried out in Milli-Q water, finding the presence

431

of some of the phthalates selected. In this way, in order to ensure the correct validation of the

432

developed methodology, as well as the adequate reliable analysis of the real samples, peak

433

areas of these analytes were subtracted when necessary.

18

434

Taking into account that it is the first time that a methodology developed for the

435

extraction of PAEs has been applied in to this type of samples, an exhaustive validation of the

436

procedure was carried out. Due to the large complexity and variability of the different

437

matrices selected (Meerpoel et al., 2018), a ME study was carried out in each case. In this

438

way, five replicates of each kind of matrix were spiked at two levels of concentration (twice

439

and eight times the LOQ of the method) and extracted using the developed VA-EDLLME

440

procedure. ME was calculated, following the Matuszewski method (Matuszewski, Constanzer

441

& Chavez-Eng, 2003), as the percentage of the ratio between the peak areas of a spiked

442

sample and the peak areas of a standard solvent at the same concentration level. As can be

443

seen in Table 1, no MEs were observed with values in the range 94-120 %.

444

Considering the aforementioned presence of this type of compounds in the matrices

445

analysed, matrix-matched calibration curves were prepared for each matrix in order to study

446

the linearity of the developed method, by injecting seven different levels of concentration (n =

447

7) in triplicate (see Figure S4). In this case, DHP-d4 was used as IS for all phthalates and

448

added at the beginning of the extraction procedure to correct the possible errors during sample

449

preparation (Carrillo, Martínez & Tena, 2008). As shown in Table 2, which also includes the

450

studied linear range and the LOQs, the values of R2 were higher than 0.9994 for all phthalates

451

and matrices.

452

After that, with the aim of studying the extraction efficiency of the method, a recovery

453

study was performed for all samples spiked at two levels of concentration at the beginning

454

and at end of the procedure (twice and eight times the LOQ of the method) by the extraction

455

of five replicates at each level and comparing concentrations (Alcântara et al., 2018). Figure

456

3a and Figure 3b show the chromatograms of an apple soft drink spiked and a blank,

457

respectively. Similar results were obtained for the other matrices. The obtained results, which

458

are shown in Table 3, showed a good efficiency of the extraction methodology with relative

19

459

recovery values in the range 95-118 % for tea drink, 93-110 % for apple soft drink and 84-120

460

% for pineapple juice and its excellent ruggedness with RSDs values lower than 11 % for all

461

samples and target analytes. Limits of detection (LODs) of the method defined as the

462

concentration which provided a signal-to-noise ratio of 3, shown in Table 3, ranged between

463

5.1-14.2 µg L-1 for tea drink, 5.3-17.8 µg L-1 for apple-based beverage and 5.9-15.6 µg L-1 for

464

pineapple juice, while LOQs of the method were found in the ranges 17.2-47.2 µg L-1 for tea

465

drink, 17.7-59.4 µg L-1 for apple-based beverage and 19.6-52.0 µg L-1 for pineapple juice.

466

LOQ values, defined as the concentration which provided a signal-to-noise ratio of 10, were

467

experimentally checked by the analysis of samples spiked at this concentration level.

468

Although other green solvents such as ILs have been applied for the DLLME of

469

phthalates in several matrices (Cacho, Campillo, Viñas & Hernández-Córdoba, 2017; Fan et

470

al., 2014; Hu et al., 2016; Sun, Shi & Chen, 2013; Zhou, Zhang & Xie, 2011), DESs, which

471

offer numerous advantages in termssuch as of high biodegradability and low toxicity, as well

472

as low costs and, easy preparation, and its great potential in sample preparation (Płotka-

473

Wasylka et al., 2017) have not been used so far on the extraction of these this kind of

474

compounds. The LOD values are in the same range than the obtained in previous publications

475

in which ILs have been used for the extraction of PAEs (Wang, Su & Yang, 2013; Zhang,

476

Chen & Jiang, 2011; Zhou et al., 2011). Only in the case of the work developed by Tan et al.

477

(Tan, Lu, Gao, Wang, Zhao & Liang, 2018) and the one carried out by Cacho et al. (Cacho et

478

al., 2017), in which MS was used as a detection system, improving thebetter sensitivity of the

479

technique employed respect the results obtained in the presented study was observed.

480

However, the detector used in the development of this work is available to a greater number

481

of laboratories due to its cost and ease of acquisition. Apart from that, the recovery values and

482

precision obtained in the present work are comparable or better than those of previous works.

483

Zhou et al. (Zhou et al., 2011) obtained recovery values in the range 85.5-102.5 % with RSDs

20

484

lower than 5.1 % for two PAEs, while in the work developed by Wang et al. (Wang et al,

485

2013) recovery values between 85.2 % and 103.3 %, with RSDs lower than 5.9 % were

486

obtained for five PAEs. Zhang et al. (Zhang et al., 2011) reached relative recovery values in

487

the range of 90.1-99.2 %, with RSDs between 2.2 % and 3.7 %, for three PAEs. Only the

488

work carried out by Tan et al. (Tan et al., 2018) determined a similar number of compounds

489

and obtained recovery values of 92.3-105.3 % with RSDs lower than 6.7 % in all cases.

490

Likewise, the extraction procedure developed, compared with the previous bibliography,

491

stands out for its great simplicity and an operational time of a few minutes to perform the

492

extraction of the compounds of interest (Cacho et al., 2017; Hu et al., 2016; Sun et al., 2013;

493

Wang et al., 2013; Zhang et al., 2011; Zhou et al., 2011), to carry out the separation of the

494

phases and/or to improve the extraction efficiency. Besides, in other cases it was necessary to

495

use the IL combined with another type of sorbents to improve the sensitivity of the method

496

(Tan et al., 2018; Wang, Yang, Liu, Cheng & Yang, 2017). The method developed not only

497

has greater simplicity, lower cost and is respectful with the environment, but also allows

498

evaluating simultaneously a higher number of analytes and reaching levels of concentration

499

similar to those of previous works, requiring lower procedure time.The method developed not

500

only has greater simplicity and cost, as well as being respectful with the environment, but also

501

allows evaluating simultaneously a higher number of analytes and reaching levels of

502

concentration similar to those of previous works requiring lower procedure time.

503 504

3.6.- Analysis of real samples

505

To evaluate the performance of the validated methodology, the optimised VA-

506

EDLLME-DAD procedure was applied to the analysis of a group of seventeen samples of

507

different nature and brands, commercialised in diverse storage materials. In this sense, five tea

508

drinks samples (three of them in plastic and the others in glass and aluminium bottles), six

21

509

apple-based beverages commercialised in aluminium, plastic, glass and Tetra Brik®

510

containers, and pineapple juices one of them collected in a glass bottle, two in Tetra Brik®

511

and three in plastic containers were selected for the present study.

512

It should be noted that DEHP was detected in some of the analysed samples, including

513

peach tea drink stored in glass bottle, apple soft drink and pineapple juice contained in plastic

514

bottle, and pineapple juice storage in Tetra Brik®, however not quantification could be

515

carried since the signals were lower than the respective LOQ of the method. In these cases, in

516

order to ensure the unambiguous determination of the analytes of interest and guarantee the

517

reliability of the results obtained, the samples were injected in an ultra-high performance

518

liquid chromatography (UHPLC)-MS/MS system for confirmation analysis. It was operated

519

in operating in MRM mode using 1 precursor and 2 product ions as well as the retention time

520

as identification points and establishing a maximum tolerance of ± 20% for the relative ion

521

intensities of the product and precursor ions (Commission Decision 2002/657/EC). Figure S6

522

S5 shows the chromatogram and mass spectrum of the confirmation analysis for DEHP. It is

523

specially remarkable the presence of DEHP since this compound has been included by both

524

the European Union (EU) (Commission Regulation (EU) No. 10/2011) and US

525

Environmental Protection Agency (EPA) (US EPA, 2012) in the group of substances with a

526

restricted use in the preparation and production of plastic material intended to come into

527

contact with food, due to their toxic effect on the reproductive system. This phthalate has

528

been found in previous studies in a wide range of concentrations. Regarding pineapple juice

529

samples, no data has been found on analysis of PAEs in this matrix. However, there are

530

researches based on the analysis of these compounds in other fruit juices, such as lemon

531

(Farajzadeh, Khorram & Nabil, 2014; Yılmaz, Ertaş & Kolak, 2014), turnip, cherry (Yılmaz

532

et al., 2014) and apple (Vidal, Ibañez & Escandar, 2016), as well as other unspecified

533

varieties (Luo, Yu, Yuan & Feng, 2012; Wu, Pa, Ma, Wang & Zhang, 2014), where DEHP

22

534

concentration values were found in the range 0.09-126 µg L-1. With respect to tea beverages,

535

Wu et al. (Wu et al., 2014) found concentration values of DEHP ranged between 16-123 µg L-

536

1

537

soft drinks, no studies have been published so far in which DEHP was determined in this

538

specific matrix, although it was analysed in other types of soft drinks. In this sense, Vidal et

539

al. (Vidal et al., 2016) found this compound in lime soda in ranges 6.80-7.23 µg L-1 while Luo

540

et al. (Luo et al., 2012) detected it in not specified carbonated drinks at levels between 3.4-

541

16.3 µg L-1. These results are in concordance with the ones obtained in the present work,

542

except in the case of Farajzadeh et al. (Farajzadeh et al., 2014) whose results involve

543

concentrations in cola soda at 76 µg L-1, which are considerably higher.

which are similar or higher than the ones obtained in the present work. As for apple-based

544 545

4.- Conclusions

546

In this work, for the first time, a methodology based on the application of a VA-

547

DLLME using a laboratory synthesised ChCl:phenol DES, combined with HPLC-DAD has

548

been successfully applied for the determination of eight phthalates of interest in different

549

beverages including tea, soft drinks and fruit juices. The whole methodology was

550

exhaustively validated in terms of matrix effect, linearity, extraction efficiency and sensitivity

551

with good results. LODs of the method ranged between 5.1 and 17.8 µg L-1 while LOQs of

552

the method were found in the range 17.2-59.4 µg L-1 for all studied matrices. Recovery

553

values were between 84 and 120 % with RSDs below 11 % for all samples and analytes.

554

The proposed methodology constitutes a very simple, fast, cheap, efficient and green

555

alternative to conventional extraction techniques previously developed for the extraction of

556

PAEs using conventional solvents allowing the use of low volumes of biodegradable and

557

environmentally friendly extraction agents, following the principles of Green Analytical

558

Chemistry. The optimised method was applied for the evaluation of real samples finding the

23

559

presence of DEHP at levels lower than 17.2 µg L-1 which was confirmed by MS/MS. The

560

proposed method is a green alternative for the analysis of phthalates in complex samples

561

matrices such as beverage samples.

562 563 564 565

Acknowledgements This work has been supported by the Spanish Ministry of Economy, Industry and Competitiveness (project AGL2017-89257-P).

566 567

Authors declare no conflict of interest.

568

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658

deep eutectic solvents in analytical chemistry. A review. Microchemical Journal, 135, 33–38.

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Smith, B. C. (2017). Infrared Spectral Interpretation: A Systematic Approach. (2nd ed.). Boca

660

Ratón Florida: Taylor & Francis (Chapter 3).

661

Sun, J.- N., Shi, Y.- P., & Chen, J. (2013). Simultaneous determination of plasticizer di(2-

662

ethylhexyl)phthalate and its metabolite in human urine by temperature controlled ionic liquid

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dispersive

664

chromatography. Analytical Methods, 5, 1427-1434.

665

Tan, Y., Lu, Y., Gao, Y., Wang, B., Zhao, L., & Liang, H. (2018). Facile Preparation of

666

Hydrophilic-Bifunctional-Groups Modified Magnetic Microspheres as a Novel Matrix for

667

Detection of Phthalate Esters from Human Plasma Samples. ChemistrySelect, 3, 9526 –9532

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US

669

https://www.epa.gov/assessing-and-managing-chemicals-under-tsca/phthalates/ Accessed 01

670

May 2019.

liquid–liquid

Environmental

microextraction

Protection

combined

Agency.

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with

Phthalates

high

performance

Action

Plan.

liquid

(2012).

671

Vidal, R. B. P., Ibañez, G. A., & Escandar, G. M. (2016). A green method for the

672

quantification of plastics-derived endocrine disruptors in beverages by chemometrics-assisted

673

liquid chromatography with simultaneous diode array and fluorescent detection. Talanta, 159,

674

336–343.

675

Wang, R., Su, P., & Yang, Y. (2013). Optimization of ionic liquid-based microwave-assisted

676

dispersive liquid–liquid microextraction for the determination of plasticizers in water by

677

response surface methodology. Analytical Methods, 5, 1033–1039.

678

Wang, M., Yang, F., Liu, L., Cheng, C., & Yang, Y. (2017). Ionic Liquid-Based Surfactant

679

Extraction Coupled with Magnetic Dispersive μ-Solid Phase Extraction for the Determination

680

of Phthalate Esters in Packaging Milk Samples by HPLC. Food Analytical Methods, 10,

681

1745–1754.

682

Wu, P.- G., Pa, X.- D., Ma, B.- J., Wang, L.- Y., & Zhang, J. (2014). Determination of

683

phthalate esters in non‑alcoholic beverages by GC–MS and optimization of the extraction

684

conditions. European Food Research and Technology, 238, 607–612.

685

Yılmaz, P. K., Ertaş, A., & Kolak, U. (2014). Simultaneous determination of seven phthalic

686

acid esters in beverages using ultrasound and vortex-assisted dispersive liquid–liquid

687

microextraction followed by high-performance liquid chromatography. Journal of Separation

688

Science, 37, 2111–2117.

689

Zhang, H., Chen, X., & Jiang, X. (2011). Determination of phthalate esters in water samples

690

by ionic liquid cold-induced aggregation dispersive liquid–liquid microextraction coupled

691

with high-performance liquid chromatography. Analytica Chimica Acta, 689, 137–142.

692

Zhou, Q., Zhang, X., & Xie, G. (2011). Simultaneous analysis of phthalate esters and

693

pyrethroid insecticides in water samples by temperature-controlled ionic liquid dispersive

29

694

liquid-phase microextraction combined with high-performance liquid chromatography.

695

Analytical Methods, 3, 1815-1820..

30

697

Figure captions

698

Figure 1.- Effect of ChCl:phenol DES composition on the extraction efficiency of the target

699

analytes after the application of the VA-EDLLME procedure. Extraction conditions: 10 mL of

700

spiked Milli-Q water containing 15 % NaCl (w/v) at pH 6, 500 µL of ChCl:phenol, as

701

extraction solvent, and 500 µL of THF as emulsifier solvent. Concentration of target analytes:

702

1.45 mg·L-1 for BBP and DIBP, 2.60 mg·L-1 for DIPP, 1.70 mg·L-1 for DNPP and DINP, 0.95

703

mg·L-1 for DEHP and DNOP, and 2.25 mg·L-1 for DIDP. The horizontal line indicates 100 %

704

of recovery.

705

Figure 2.- (a) Main effect plots of the evaluated factors for DNPP response. (b) Estimated

706

response surfaces for the CCD. Relative recovery values versus DES and THF volume

707

(considering an ionic strength of 15 % NaCl (w/v)).

708

Figure 3.- (a) HPLC-DAD chromatogram of selected PAEs and DHP-d4 (IS) of a spiked

709

apple-based soft drink. Injection volume: 20 µL. Detection wavelength: 225 nm. Mobile

710

phase flow: 1 mL/min. Column temperature: 45 ºC. Analytes identification and concentration

711

in the sample: (1) BBP (0.12 mg·L-1), (2) DIBP (0.11 mg·L-1), (3) DIPP (0.17 mg·L-1), (4)

712

DNPP (0.05 mg·L-1), (5) DEHP (0.05 mg·L-1), (6) DNOP (0.05 mg·L-1), (7) DINP (0.08

713

mg·L-1) and (8) DIDP (0.11 mg·L-1). (b) HPLC-DAD chromatogram of non-spiked apple-

714

based soft drink. Injection volume: 20 µL. Detection wavelength: 225 nm. Mobile phase flow:

715

1 mL/min. Column temperature: 45 ºC.

31

717

Figure captions of the Supplementary Material

718

Figure S1.- FT-IR spectra of: (a).ChCl; (b) phenol; (c) ChCl:phenol DES within the range of

719

400–4000 cm−1 at room temperature.

720

Figure S2.- Effect of pH media on the extraction efficiency of the target analytes after the

721

application of the VA-EDLLME procedure (n = 2). Extraction conditions: 10 mL of spiked

722

Milli-Q water containing 15 % NaCl (w/v) at pH 6, 500 µL of ChCl:phenol 1:2, as extraction

723

solvent, and 500 µL of THF as emulsifier solvent. Concentration of target analytes: 1.45

724

mg·L-1 for BBP and DIBP, 2.60 mg·L-1 for DIPP, 1.70 mg·L-1 for DNPP and DINP, 0.95

725

mg·L-1 for DEHP and DNOP, and 2.25 mg·L-1 for DIDP.

726

Figure S3.- Pareto chart of standardised effects of the CCD for DNPP, selected as

727

representative analyte of the rest of PAEs, for the analysis of the variables: % (w/v) of NaCl

728

(A), volume of DES (µL) (B) and the volume of THF (µL) (C), in the relative recovery

729

values. The blue vertical line in the chart defines the 95 % confidence level, while bars in gray

730

and blue show whether the variables, and the interactions between them, affect positively or

731

negatively to the recoveries, respectively.

732

Figure S4.- Matrix-matched calibration curve in an apple-based soft drink of DNPP, as a

733

representative analyte of the rest of PAEs.

734

Figure S5.- HPLC-DAD chromatogram of non-spiked apple-based soft drink. Injection

735

volume: 20 µL. Detection wavelength: 225 nm. Mobile phase flow: 1 mL/min. Column

736

temperature: 45 ºC.

737

Figure S6S5.- UHPLC-MS/MS chromatogram and MS/MS spectrum of DEHP found in an

738

analysed pineapple juice stored in plastic bottle, using the developed procedure.

739

Table 1.- Matrix effect study (n = 10) of the VA-EDLLME-HPLC-DAD method in the different matrices. Analyte

Type of sample

BBP

Peach tea drink Apple soft drink

MEa), b) (%)

RSD (%)

100

6

Analyte

Type of soil

MEa), b) (%)

RSD (%)

Peach tea drink

99

9

Apple soft drink

115

14

DEHP 107

4

32

DIBP

DIPP

DNPP

740 741

Pineapple juice Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice

101

5

Pineapple juice

103

9

101

2

Peach tea drink

102

6

106

5

Apple soft drink

110

5

97

3

Pineapple juice

105

11

97

6

Peach tea drink

104

6

102

3

Apple soft drink

111

9

98

7

Pineapple juice

111

14

101

5

Peach tea drink

105

6

98

2

Apple soft drink

113

7

94

15

Pineapple juice

120

17

DNOP

DINP

DIDP

a) Results obtained as an average (n = 10) of each analyte at two levels of concentration (low level: twice the LOQ; high level: eight times the LOQ. b) Calculated following the Matuszewski method (Matuszewski et al., 2003).

33

743 744

Table 2.- Matrix-matched calibration data of the selected compounds in the different matrices. Calibration data (n = 7)

Analyt e

BBP

DIBP

DIPP

DNPP

745

Type of sample

Calibration data (n = 7)

Range of concentratio n studied (mg L-1)

Slope

Intercept

R2

Peach tea drink

0.37-3.33

1.02·100 ± 2.07·10-2

-1.14·10-1 ± 3.98·10-2

0.999 7

Apple soft drink

0.40-4.00

1.11·100 ± 1.26·10-2

-9.29·10-2 ± 3.10·10-2

0.999 9

Pineappl e juice

0.37-3.70

1.01·100 ± 1.65·10-2

-1.09·10-2 ± 3.78·10-2

Peach tea drink

0.37-3.33

9.87·10-1 ± 2.27·10-2

Apple soft drink

0.37-3.70

Pineappl e juice

Analyt e

Type of sample

Range of concentratio n studied (mg L-1)

Peach tea drink

0.17-1.70

Apple soft drink

0.17-1.70

0.999 8

Pineappl e juice

0.17-1.70

-9.40·10-2 ± 4.44·10-2

0.999 6

Peach tea drink

0.17-1.70

1.05·100 ± 7.74·10-3

-7.99·10-2 ± 1.61·10-2

0.999 9

Apple soft drink

0.17-1.70

0.37-3.70

9.53·10-1 ± 2.23·10-2

-9.98·10-2 ± 2.46·10-2

0.999 6

Pineappl e juice

0.17-1.70

Peach tea drink

0.45-4.49

4.99·10-1 ± 1.12·10-2

1.83·10-2 ± 4.89·10-

0.999 6

Peach tea drink

0.27-2.70

Apple soft drink

0.55-5.50

5.13·10-1 ± 6.04·10-3

-1.13·10-1 ± 1.86·10-2

0.999 9

Apple soft drink

0.27-2.70

Pineappl e juice

0.45-4.49

4.88·10-1 ± 4.26·10-3

-6.26·10-2 ± 1.13·10-2

0.999 9

Pineappl e juice

0.27-2.70

Peach tea drink

0.17-1.70

8.73·10-1 ± 1.76·10-2

-2.92·10-2 ± 1.62·10-2

0.999 7

Peach tea drink

0.27-2.70

Apple soft drink

0.17-1.70

8.92·10-1 ± 1.09·10-2

-3.58·10-2 ± 1.05·10-2

0.999 9

Apple soft drink

0.37-3.70

Pineappl e juice

0.17-1.70

8.43·10-1 ± 2.00·10-2

3.50·10-2 ± 2.01·10-

0.999 6

Pineappl e juice

0.27-2.70

2

2

DEHP

DNOP

DINP

DIDP

R2: determination coefficient. DHP-d4 was used as internal standard in all cases.

34

Slope 6.81·1 0-1 ± 1.96·1 0-2 6.92·1 0-1 ± 8.72·1 0-3 6.66·1 0-1 ± 6.74·1 0-3 7.10·1 0-1 ± 1.27·1 0-2 6.91·1 0-1 ± 1.19·1 0-2 6.83·1 0-1 ± 7.01·1 0-3 6.14·1 0-1 ± 1.57·1 0-2 6.26·1 0-1 ± 8.01·1 0-3 5.86·1 0-1 ± 7.30·1 0-3 6.36·1 0-1 ± 1.47·1 0-2 6.39·1 0-1 ± 7.73·1 0-3 6.05·1 0-1 ± 8.24·1 0-3

Intercep t

R2

-2.23·102 ± 1.73·10-2

0.999 4

-1.37·102 ± 8.78·10-3

0.999 9

-1.33·102 ± 6.79·10-3

0.999 9

-5.02·102 ± 1.06·10-2

0.999 8

-8.93·103 ± 1.20·10-2

0.999 8

-1.59·102 ± 7.04·10-3

0.999 9

-5.94·102 ± 2.07·10-2

0.999 5

-3.14·102 ± 1.40·10-2

0.999 9

-8.07·103 ± 1.17·10-2

0.999 9

-5.83·102 ± 1.94·10-2

0.999 6

-4.98·102 ± 1.70·10-2

0.999 9

8.01·10-2 ± 1.32·10-2

0.999 9

747 748

Table 3.- Results of the recovery study (n = 5) of the VA-EDLLME-HPLC-DAD method for the selected compounds in the different beverages at two levels of concentration.

Analyte

BBP

DIBP

DIPP

DNPP

749 750 751

Type of sample

Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice

Relative recovery % Level 2b) (n Level 1a) (n = 5) = 5) (RSD, %) (RSD, %)

LODmethodc) LOQmethodd) Analyte (µg L-1) (µg L-1)

105 (2)

99 (3)

11.2

37.3

101 (4)

101 (2)

12.5

41.6

107 (6)

111 (6)

12.0

40.1

118 (9)

102 (3)

10.2

34.0

110 (7)

103 (2)

11.0

36.6

117 (7)

120 (8)

10.9

36.3

98 (2)

97 (3)

14.2

47.2

93 (4)

102 (2)

17.8

59.4

101 (4)

106 (5)

15.6

52.0

99 (11)

98 (2)

5.3

17.8

94 (3)

100 (1)

5.5

18.4

93 (3)

108 (3)

5.9

19.6

DEHP

DNOP

DINP

DIDP

Type of sample

Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice Peach tea drink Apple soft drink Pineapple juice

Relative recovery % Level 1a) (n = Level 2b) (n 5) = 5) (RSD, (RSD, %) %)

LODmethodc) LOQmethodd) (µg L-1) (µg L-1)

104 (4)

100 (2)

5.1

17.2

94 (3)

104 (1)

5.4

18.1

98 (1)

104 (3)

6.0

20.2

100 (4)

96 (3)

5.3

17.8

101 (3)

101 (1)

5.3

17.7

85 (7)

101 (3)

6.1

20.3

96 (2)

95 (3)

8.7

28.9

95 (3)

102 (1)

8.6

28.7

84 (6)

100 (3)

9.4

31.4

98 (6)

97 (3)

8.5

28.3

94 (3)

99 (2)

12.0

40.1

110 (5)

103 (2)

9.1

30.3

a) Concentration of target analytes was twice the LOQ. b) Concentration of analytes was eight times the LOQ. c) Defined as the concentration which provides a signal-to-noise ratio of 3. d) Defined as the concentration which provides a signal-tonoise ratio of 10.

752 753

Santana-Mayor et al. Highlights

754 755

- A ChCl:phenol based deep eutectic solvent was applied for the DLLME of phthalates.

756

- Parameters affecting the extraction efficiency of DLLME were studied and optimised.

757

- DLLME-HPLC-DAD method was validated for different kinds of beverages.

758

- Commercially available products were analysed using the developed methodology.

759

- Positive samples were confirmed by UHPLC-MS/MS analysis.

760 761

Declaration of interests

762 763 764

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

35

765 766 767 768

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

769 770 771 772 773

36