Determination of oxidative hair dyes using miniaturized extraction techniques and gas chromatography-tandem mass spectrometry

Accepted Manuscript Determination of oxidative hair dyes using miniaturized extraction techniques and gas chromatography-tandem mass spectrometry

Eugenia Guerra, J. Pablo Lamas, Maria Llompart, Carmen Garcia-Jares PII: DOI: Reference:

S0026-265X(16)30649-X doi: 10.1016/j.microc.2017.02.017 MICROC 2703

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

22 November 2016 14 February 2017 14 February 2017

Please cite this article as: Eugenia Guerra, J. Pablo Lamas, Maria Llompart, Carmen Garcia-Jares , Determination of oxidative hair dyes using miniaturized extraction techniques and gas chromatography-tandem mass spectrometry. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Microc(2016), doi: 10.1016/j.microc.2017.02.017

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ACCEPTED MANUSCRIPT Determination of oxidative hair dyes using miniaturized extraction techniques and gas chromatography-tandem mass spectrometry Eugenia Guerra, J. Pablo Lamas, Maria Llompart, Carmen Garcia-Jares* Laboratory of Research and Development of Analytical Solutions (LIDSA), Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, Universidade de Santiago de Compostela, E-15782, Santiago de Compostela, Spain.

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* Corresponding author. Phone: 34-881814394

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E-mail address: [email protected]

Keywords: hair dyes; cosmetics; vortex extraction; ultrasound-assisted extraction; matrix solid-

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phase dispersion; gas chromatography-tandem mass spectrometry

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ABSTRACT

Three methodologies based on miniaturized solvent extraction: vortex extraction (VE), ultrasound-assisted extraction (UAE), and matrix solid-phase dispersion (MSPD), were

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optimized for the gas chromatography-tandem mass spectrometry analysis of seven

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regulated oxidative hair dyes, including some of the most frequently used sensitizers in hair products (resorcinol, RL; 1-naphthol, 1NL), as well as their banned isomers (hydroquinone, HQ; 2-naphthol, 2NL). The use of MS/MS in combination with GC is a

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valuable approach that had not been applied before in the analysis of the considered compounds in hair dyes formulations. Parameters influencing the extraction efficiency were optimized for the three sample preparation techniques employing real sample. 0.1

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g of sample and 1 mL of organic solvent were used in all cases, which reduced the solvent consumption compared to other methods proposed in literature. Under the optimal selected conditions, three methods were validated. Good linearity (R2≥ 0.991, %RSD ≤ 10) was achieved in a broad range of concentrations. In general, quantitative recoveries were obtained in real samples spiked at low, medium and high concentration levels using the three extraction techniques (ranged from 71.4 to 118% with exceptions). Good method precision (%RSD ≤ 14) and LOQ ≤ 0.13 µg g-1 were achieved in all cases. Results demonstrated that the three methods could be implemented indistinctly. In the commercial hair dye formulations analyzed, all

ACCEPTED MANUSCRIPT investigated compounds but 2-naphtol were found. Thus, hydroquinone, one of the banned hair dyes according to EU Cosmetic Directive, was evidenced in at least one formulation.

1. Introduction Hair plays a significant role in body image and people have applied dyes for ages.

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Among the different types of products for changing the hair color, oxidative hair dyes are by far the most frequently used and they have held the dominant share of the market

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for decades. Oxidative (also called permanent) hair dyes are uncolored or faintly

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colored compounds but when they are mixed in presence of an oxidant, usually a solution of hydrogen peroxide, through a process of oxidative condensation between an

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intermediate and a coupler, they produce colored complexes that penetrate the cortex of the hair providing the desired shade [1]. These precursors are essentially aromatic ring derivatives such as diamines, aminophenols, phenols or naphthols. Although many

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studies have reported their potentially allergenic effects [2], these are focused on a few substances. Despite hair dyes that are strong or extreme sensitizers are very common in

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consumer products on the European market, dermatitis patients are generally patch tested with one substance only, p-phenylenediamine [3]. Hence, toxicity of hair dyes is

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still a current issue of concern and subject to assessment. The EU Cosmetic Regulation (EC) No 1223/2009 [4] establishes the rules to be complied with by all cosmetic products available on the market in order to ensure a high level of protection of human

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health. Restrictions of use and maximum concentration permitted (MCP) according to annexes II and III of the Regulation for the compounds considered in this work are

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presented in Table 1. Since the limited data on the allergenicity of many hair dyes, those rules of use are amended too frequently according to new risk assessments and hence there are no specific positive lists established for the time being [5]. An example is hydroquinone which was restricted in hair products when the regulation was published but at present is prohibited due to their high toxicity. However, its isomer, resorcinol, is one of the most common ingredients in hair dyes and is also present in high concentrations (generally higher than 0.1% (w/w)). All hair dye formulations that contain any selected compound must include an advert “hair colorants can cause severe allergic reactions” in the packing. In some cases, such as resorcinol, due to its potentially allergenic effects, label must also warn that the hair product contains that

ACCEPTED MANUSCRIPT particular ingredient. For this reason, some manufacturers show the label “free of resorcinol” in the packing to attract consumers. Therefore, the development of selective and sensitive analytical methods for the control of these hair dyes is advisable in order to safeguard consumer safety. However, there is a lack of analytical methodologies for the determination of hair dyes in cosmetics. One evidence of this is that there is not any method validated for the determination of 2-

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naphthol in hair dyes, included in this study, even though its use is prohibited. Table 2 shows the analytical methods reported in last decades in international scientific journals

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for the determination of the hair dyes considered in this work [6-16]. The most employed widely analytical technique is high performance liquid chromatography

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(HPLC) with absorbance detectors, UV or DAD [7, 9, 13-16]. Gas chromatography (GC) is an approach scarcely reported in the international scientific literature for hair

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dyes determination [17-20] and the proposed methodologies are focused on the analysis of amino-containing compounds. This lack may be attributed to the fact that

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derivatization previous to the analysis is highly recommendable due to the high polarity and low volatility of the analytes. However, acetylation is a low-cost, simple and efficient derivatization procedure that allows easily overcoming this limitation, and it

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has been successfully applied in the cosmetic analysis [21-23]. Furthermore, tandem

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mass spectrometry combined with GC offers improved analytical sensitivity and selectivity, which is particularly advisable in case of hair dyes since there are isomeric compounds with very different legal regulation. GC-MS has been used for the

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determination of resorcinol and hydroquinone in skin-whitening and anti-agents in products [24] and GC-MS/MS has been recently applied to the analysis of cosmetic ingredients [21-23, 25-27]. So far, GC-MS/MS has not been reported in the

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international scientific literature for the determination of the target hair dyes (hydroxylated and phenyl methyl pyrazolone) in hair formulations. An appropriate sample pre-treatment is an essential step before the chromatographic analysis. Procedures based on extraction with solvents or assisted by ultrasound are the most commonly employed in the literature for the analysis of hair dyes (see Table 2). In recent years, there is an increasing trend towards extraction methods with lower sample and reagent consumption, as well as simpler in equipment and handling requirements. Following that trend, this study proposes rapid and eco-friendly extraction techniques (VE, UAE and MSPD) for the analysis of oxidative hair dyes.

ACCEPTED MANUSCRIPT Vortex extraction (VE) is an easy and reliable approach for the extraction of matrices with relative complexity as hair dye color creams. It leads to extraction in a single step with very simple equipment. Ultrasound-assisted extraction (UAE) is an effective technique that has attracted growing interest in recent years and is also becoming a matter of routine practice in analytical laboratories. The use of ultrasound energy provides an enhanced mass transfer and solvent penetration, leading to an efficient extraction with a great reduction in time and solvent consumption [28]. Sometimes the

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use of VE as first step to disperse cosmetic samples before UAE has also been used in cosmetic analysis [29]. Matrix solid-phase dispersion (MSPD) is recognized as a

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valuable strategy for the extraction of solid, semisolid and viscous samples. It enables

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performing the extraction and clean-up at the same time to obtain a cleaner extract. A miniaturized procedure based on MSPD has been applied to the analysis of cosmetic

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ingredients such as water-soluble dyes [30], preservatives and its unwanted products [31], synthetic musks and plasticizers [32], in a broad range of cosmetic matrices. Miniaturization of MSPD allows reducing sample preparation time and solvent

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consumption, and does not require special equipment since the extraction is performed in glass Pasteur pipettes with glass wool plugs, and thus, it can be easily implemented in

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any laboratory at negligible costs.

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This work aims to optimize, validate, and apply methodologies based on miniaturized VE, UAE and MSPD, followed by GC-MS/MS, for the simultaneous determination of several oxidative hair dyes, including some of the most frequently sensitizers used in

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domestic hair dye products. To the best of our knowledge, this is the first time that GCMS/MS has been proposed for the analysis in hair cosmetics of hydroxylated dyes including dihydroxybenzenes and derivatives, as well as naphthols and phenyl methyl

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pyrazolone, for which the analytical methodology published is scarce or nonexistent.

2. Experimental 2.1 Chemicals, materials and samples All solvents and reagents were of analytical grade. Resorcinol (RL; 99.8%), 2methylresorcinol (2MR; 99.3%), 4-chlororesorcinol (4CLR; 98.8%), 1-naphthol (1NL; 99.9%) and phenyl methyl pyrazolone (PMP; 99.8%) were supplied by AlfaAesar (Karlsruhe, Germany). Hydroquinone (HQ; 99.7%) was acquired from Sigma and 2naphthol (2NL; 99.9%) from Cymit, TCI (Zwijndrecht, Belgium).

ACCEPTED MANUSCRIPT Methanol, ethyl acetate, pyridine, and N,O-bis (trimethylsilyl) trifluoro-acetamide + trimethylchlorosilane (BSTFA + TMCS, 99:1) were provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Acetonitrile and acetic anhydride were supplied by Merck (Darmstadt, Germany). Sweet almond oil (cosmetic oil) was supplied by Acofarma (Barcelona, Spain). Ascorbic acid was purchased by Merck (Darmstadt, Germany) and disodium

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metabisulphite and disodium tiosulphate were supplied by Panreac (Barcelona, Spain). Florisil (60–100 mesh) was purchased from Supelco Analytical (Bellefonte, PA, USA).

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C18 (50–70 mesh), aluminum oxide activated neutral and diatomaceous earth Hyflo Super Cel were obtained from Sigma-Aldrich (Steinheim, Germany). Sand (200 mesh

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particle size) was provided by Scharlau (Barcelona, Spain). Silica gel (60 mesh particle size) was supplied by Merck (Darmstadt, Germany). Anhydrous sodium sulphate (99%)

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was purchased from Panreac (Barcelona, Spain). Before being used, Florisil, alumina and silica were activated at 230 ⁰C for 15 h and anhydrous sodium sulphate was kept at

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the same temperature for 2 h to prevent its hydration. Both of them were allowed to cool down in a desiccator.

Individual stock solutions of each compound were prepared in methanol at

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concentrations ranging from 9 to 13 mg mL-1. Further dilutions and mixtures in ethyl

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acetate were prepared by convenient dilution of the stock solution to spike cosmetic samples (when needed). All solutions were stored in glass vials protected from light exposure with aluminum foil, and kept in a freezer at -20ºC.

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Hair dyes formulations for women and men from national and international brands were purchased from local sources. Samples were kept in their original containers at room

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temperature until their analysis. For validation purposes three real samples (T1, T2 and T3) free of all target compounds were selected. Other samples analyzed are described in detail in Section 3.6 and Table 6 and 7.

2.2 Sample extraction procedures In all cases, approximately, the first portion (2-3 cm) of hair coloring cream from the tube was discarded due to the possible oxidation of the sample in contact with the oxygen of the room air.

ACCEPTED MANUSCRIPT 2.2.1 Vortex extraction 0.1 g of hair dye product was exactly weighted into a 1.5 mL Eppendorf tube. Then, the sample was spiked with 20 µL of an ascorbic acid solution in methanol at 10 mg mL-1 in order to prevent the hair dye from oxidizing, and for optimization and recovery experiments, 20 µL of the corresponding working solution of the compounds were also added to get the desired final concentration. One mL of acetonitrile was used as

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extracting solvent. The tube was stirred for 5 min using a vortex agitator from VELP Scientifica (Usmate, Italy) at 2400 rpm (maximum setting). Finally, 800 µL of the

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supernatant were filtered through a 0.22 µm PTFE syringe filter for its subsequent

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dilution and derivatization.

2.2.2 Ultrasound-assisted extraction

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Sample was prepared as described before in Section 2.2.1 for VE. The tube was then immersed for 5 min into an ultrasonic water bath Raypa® model UCI 150 (Barcelona,

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Spain), and extractions were performed at 35 kHz of ultrasound frequency and 400 W for 5 min at 25°C at the beginning of every experiment. Finally, 800 µL of the

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dilution and derivatization.

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supernatant were filtered through a 0.22 µm PTFE syringe filter for its subsequent

2.2.3 Matrix solid-phase dispersion extraction

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0.1 g of hair dye product was placed into a 10 mL glass vial, and spiked with 20 µL of an ascorbic acid solution in methanol at 10 mg mL-1. When necessary, 20 µL of the

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corresponding solution of the compounds was added to get the desired concentration. Then, the sample was gently blended with 0.3 g of a drying agent (anhydrous Na2SO4), and 0.4 g of the dispersing agent (Florisil) into the vial using a glass rod until a homogeneous mixture was obtained. The mixture was transferred into a glass Pasteur pipette (approximately 150 mm), with a small amount of glass wool placed at the bottom, containing 0.1 g of Florisil (to obtain a further degree of fractionation and sample clean-up). A small amount of glass wool was placed on top of the sample before compression with a spatula. Elution with acetonitrile was made by gravity flow, collecting 1 mL of extract into a volumetric flask. Finally, it was filtered through a 0.22 µm PTFE syringe filter for its subsequent dilution and derivatization.

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2.3 Derivatization procedure Acetylation was carried out by adding 200 µL of acetic anhydride containing 2.5% of pyridine and 10 µL of sweet almonds oil solution in ethyl acetate (10% v/v) to 1 mL of the standard or extract solution. The mixture was vortexed, heated at 80ºC for 20 min, and then allowed to cool down before GC-MS/MS analysis. To prove that there were no

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losses by keeping the solution at high temperature, the vials containing the solution were weighed before and after heating. The same mass was obtained in both cases,

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concluding that no loss had occurred.

2.4 Gas chromatography-tandem mass spectrometry

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The GC–MS/MS analysis was performed using a Thermo Scientific Trace 1310 gas chromatograph coupled to a triple quadrupole mass spectrometer (TSQ 8000) and an IL

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1310 autosampler from Thermo Scientific (San Jose, CA, USA). Separation was carried out on a 5% phenyl-arylene/95%- dimethylpolysiloxane Zebron ZB-SemiVolatiles capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness)

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supplied by Phenomenex (Torrance, CA, USA). Helium (purity 99.999%) was employed as carrier gas at a constant column flow of 1.0 mL min -1. The GC oven

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temperature was programmed from 60°C (held 1 min) to 130°C at 25°C min-1 (held 1 min); to 160ºC at 5ºC min-1 (held 3 min); to 200ºC at 15ºC min-1 and 5ºC min-1 to 240ºC

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(held 3 min). The injector temperature was 260°C. Sample (1 µL) was injected in the splitless mode (1 min).

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The mass spectrometer was operated in the electron impact (EI) ionization positive mode (+70 eV). The temperatures of the transfer line and the ion source were set at 290 and 350°C, respectively. Selected reaction monitoring (SRM) acquisition mode was implemented. Quantification was performed using the Trace Finder software (Manugistics, Rockville, MD, USA).

3. Results and discussion 3.1 GC-MS/MS analysis The use of tandem mass spectrometry offers an increased analytical selectivity and sensitivity providing lower limits of detection compared to other detectors, which is a

ACCEPTED MANUSCRIPT valuable characteristic in the control of prohibited substances. MS/MS transitions allow a distinct identification of a target compound in the presence of other interfering matrix components. Nevertheless, since the ionization and fragmentation patterns of isomeric compounds are highly similar, mostly identical, spectral data generated are not enough for the reliable quantification and confirmation of these substances, and thus, an adequate chromatographic separation is needed. Regarding oxidative hair dyes analysis, the relevance of the separation of isomers is still greater since cosmetic legislation, as

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previously mentioned in the introduction, prohibits the use of some of them due to their toxicity. The GC-MS/MS method optimized in this study provides a satisfactory

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chromatographic resolution between these isomers (1.2 for RL and HQ and 2.1 for 1NL

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and 2NL).

MS/MS working conditions were optimized using selected reaction monitoring

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optimization, implying the selection of a precursor ion, the product ions study and optimization. Thus, working in electron ionization (EI) mode, the precursor ion was identified by full scan mode for each compound. In a second step, the mass

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spectrometer was set to product-ion-scan mode and the most intense precursor ions were selected for subsequent fragmentation with different collision energies. Three

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transitions were selected for each target compound according to ion abundance. The most intense transition was used for quantification purpose, whereas the second and the

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third ones were employed for identification/confirmation purposes. Chromatographic parameters, such as the oven temperature program, were optimized using a derivatized standard mixture solution of the target analytes in ethyl acetate (see

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Section 3.2). Retention times and selected SRM transitions of the derivatized

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compounds are shown in Table 3.

3.2 Derivatization optimization Preliminary analyses showed that a previous derivatization step would improve the peak shape and subsequently increase the analytical sensitivity. Acetylation with acetic anhydride is one of the simplest and cheapest derivatization procedures for phenolic compounds [33] and it has already been successfully applied in the analysis of several families of ingredients in cosmetic samples [21, 23]. Consequently, we investigated an acetylation procedure based on the conditions proposed in this study. In general, a substantial improvement in terms of analytical signal and peak shape was obtained. As expected, no differences in the chromatographic response were observed for PMP since

ACCEPTED MANUSCRIPT it does not have chemical groups susceptible to acetylation. In some cases, especially for dihydroxybenzenes, the full-scan analysis revealed several peaks corresponding with the mono and diacetyl derivatives as well as with a small moiety of underivatized compound, being the diacetylated compound the most abundant. Considering a previous work on phenolic dyes [34], the efficiency of a silylation reagent was also tested by derivatizing a standard mixture solution in ethyl acetate with 40 µL of N,O-bis (trimethylsilyl) trifluoro-acetamide (BSTFA) catalyzed by

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trimethylchlorosilane (TMCS) (99:1) under the conditions described in that paper (60ºC, 1 h). BSTFA is very versatile, reacting with a range of polar organic compounds

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and its by-products (trimethylsilyltrifluoroacetamide and trifluoroacetamide) are more

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volatile than many other silylating reagents, causing less chromatographic interference. However, poor chromatographic behavior of the silylated derivatives was found in

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comparison with acetylated ones, and hence, efforts in the optimization of derivatization were focused in the use of acetic anhydride and pyridine as catalyzer. Different conditions of time and temperature of acetylation (10, 20 and 30 min, and 80

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and 100ºC) were evaluated finding comparable results, thus moderate conditions (80ºC and 20 min) were selected to achieve an as complete chemical derivatization of dye

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components as possible. Concerning dihydroxybenzenes and naphthols, the diacetylated derivatives demonstrated to be stable and the most abundant, so they were selected to

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determine those compounds.

3.3 Influence of antioxidants

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Oxidative hair dyes are highly sensitive to UV light, air and other oxidizing chemicals. A noticeable change of color can be rapidly observed in the hair coloring cream exposed

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to room air. So, the role of an antioxidant addition during the sample preparation in the analysis of hair dyes had been reported in the literature [7, 35]. Different antioxidants were tested using a real sample. Ascorbic acid, disodium metabisulphite, and disodium tiosulphate solutions at 10 mg mL-1 in methanol were added to 0.1 g sample aliquots. Total ion chromatograms (TIC) were compared, and no significant differences were observed (data not shown). Since ascorbic acid is frequently used in hair dye formulations, it was selected to prevent the sample from oxidation during handling. Different ways of adding the ascorbic acid were also evaluated using a real sample spiked with the compounds at 100 µg g-1. Results obtained without ascorbic acid, with

ACCEPTED MANUSCRIPT 10 mg ascorbic acid directly mixed with the sample, and a sample spiked with 20 µL of a 10 mg mL-1 solution of ascorbic acid in methanol, showed remarkable differences of the analytical response (see Figure 1). For all analytes, except for PMP, the sample spiked with 20 µL of a solution of ascorbic acid led to higher responses, highlighting the case of HQ, whose signal was increased more than 10 times compared to the sample without ascorbic acid. In view of these results, it was clear that the presence of ascorbic acid in the sample improved the responses, except for PMP. Then, different

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concentrations of ascorbic acid solutions were considered: 1, 10 and 100 mg mL-1. Since no significant differences were found, the medium concentration was selected.

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In order to evaluate the stability of the extracts adding the considered amount of

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ascorbic acid to the sample prior the extraction, analytical responses within 72 hours

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were compared in terms of RSD (%) obtaining values between 6.5 and 13.

3.4 Extraction optimization

The experiments to evaluate the influence of main variables potentially affecting each

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technique were carried out with 0.1 g of the same sample spiked at 100 µg g-1 with the analytes in order to compare the results among the different procedures. The extracts

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obtained were diluted (1:10, v/v) with ethyl acetate due to its suitable volatility for the GC analysis.

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In case of VE and UAE, a few experimental parameters need to be evaluated in comparison with other techniques. For VE, the effect of the extracting solvent was studied. Methanol, acetonitrile and ethyl acetate were chosen in accordance with the

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principles of sustainability and green chemistry. For extraction, 1 mL of solvent was added to 0.1 g of sample in an Eppendorf tube and the mixture was vigorously shaken

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using a vortex agitator for 5 min as described in Section 2.2. As shown in Figure 2, greater analytical responses were obtained using acetonitrile and ethyl acetate with noticeable differences for some analytes compared to methanol. Employing acetonitrile, RL and 2MR showed an increased signal almost two and five times, respectively, compared to the use of methanol. This fact might be explained considering that methanol contained in the extract (10% v/v) may react with the derivatization agent, acetic anhydride. Since no significant differences between acetonitrile and ethyl acetate were found, the former was selected as extracting solvent in such a way that the proposed procedure would be compatible with LC.

ACCEPTED MANUSCRIPT For the optimization of UAE, extracting solvent and sonication time were studied simultaneously at three levels. The same solvents as in the previous optimization were selected and the mixtures were sonicated for 1, 5 and 10 min (Figure 3a). Analytical responses obtained with the three solvents (5 min of sonication time) were compared and, as expected according to the results achieved in the VE, acetonitrile and ethyl acetate provided the highest efficiencies (Figure 3b). Fixing acetonitrile as extracting solvent, the application of ultrasound for 5 min led to the best results for the extraction

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of all compounds.

The possibility of simultaneously performing a clean-up step was considered in VE and

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UAE. Experiments were carried out in optimal conditions. Florisil (0.1 g) was added to

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the mixture before shaking. After extraction, 800 µL of the supernatant were collected and filtered for subsequent dilution and derivatization. Results showed no significant

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differences between using Florisil or not (see Figure 4). Thus, the addition of sorbent was discarded.

In the optimization of MSPD, three factors (dispersant, solvent, and elution volume)

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were evaluated. Preliminary experiments were carried out with a non-spiked real sample containing some target compounds (RL, 2MR and PMP). Dispersants with intermediate

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and upper-intermediate polarities (sand, C18, diatomaceous earth, alumina, Florisil and silica) were checked. Results allowed discarding sand, C18, and diatomaceous earth,

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since they led to an improper elution of the analytes. Alumina, silica and Florisil were kept for further studies using a spiked sample. As can be seen in Figure 5a, comparable results were generally obtained using the three dispersants. Given the suitability of

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Florisil for the MSPD extraction of other cosmetic ingredients, as demonstrated in previous studies [21, 30, 32], it was selected for the extraction of hair dyes. Regarding

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the solvent, the same trend as in VE and UAE was observed for MSPD, thus acetonitrile was selected. The elution volume was evaluated by collecting 2 mL in two 1 mL volumetric flasks and analyzing each fraction separately. Recoveries lower than 12% were found in the second fraction, so 1 mL was chosen as elution volume.

3.5 Method performance GC-MS/MS quality parameters were evaluated in terms of linearity and intra- and interday precision (see Table 4). First experiments showed that the analytical signal obtained for some compounds in the extracts (samples T1, T2 and T3 spiked at 1 µg g-1) was higher than the corresponding

ACCEPTED MANUSCRIPT standard solutions. This could suggest a likely positive matrix effect occurring either in the MS ion source or/and in the GC inlet. In an attempt to minimize this problem, cosmetic oil was added to the standard solutions to simulate a general cosmetic sample matrix. To check the suitability of this procedure, the analytical responses obtained for a standard solution of 0.1 µg mL-1, a standard solution spiked with cosmetic oil (10 µL of a 10% v/v in ethyl acetate), and a standard solution spiked with 100 µL of a blank sample extract (see Section 2.2), were compared. Results are shown in Figure 6. Since

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differences between responses obtained for matrix spiked standards were similar for most compounds, for the sake of simplicity, the “pseudo matrix matched” procedure

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using oil was selected to assess the performance of the proposed method.

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Regarding the instrumental linearity, calibration curves were obtained in a range of 1 to 2000 ng mL-1 with standards prepared in ethyl acetate containing cosmetic oil.

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Determination coefficients (R2 ≥ 0.991) demonstrated a directly proportional relationship between the amount of each analyte and the chromatographic response. Instrumental precision was evaluated at three concentration levels (10, 100 and 1000 ng

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mL-1) and expressed in terms of relative standard deviation (RSD%). In general, repeatability (intra-day, n=6) as well as reproducibility (inter-day, n=6) showed

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satisfactory values lower than 10% in all cases and mean RSD values lower than 7%. To evaluate accuracy, a recovery study was carried out for the three extraction methods, 1

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using three samples spiked at low, medium and high concentrations (1, 10 and 100 µg g). Recovery values were calculated as the ratio of the response obtained for spiked

samples, adequately diluted (see Section 2.3), to the response measured for standards (at

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the same concentration level) prepared in a pseudo matrix matched solution, and expressed as percentage (Table 5). Applying VE and UAE procedures, satisfactory

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recoveries were achieved for all compounds at any studied concentration level in the three samples except for PMP. Mean recovery values ranged from 80.6 to 101%. Therefore, they were quantitative. In the case of MSPD, quantitative recoveries were also found for all target dyes except for PMP in samples T2 and T3 at any studied level, achieving mean recoveries between 80.5 and 108%. For sample T1 spiked at 1 and 10 µg g-1, lower recoveries were obtained for RL and 4CLR. This sample is used as a lightening cream, so particular composition may explain the lower recoveries of these two dyes. Regarding PMP, it could not be determined in this matrix by any of the methods. In samples T2 and T3, recoveries values were 55.6 and 75.2 % using VE and 58.6 and 79.1% using UAE, being slightly lower for MSPD.

ACCEPTED MANUSCRIPT The limits of quantification (LOQs) for each method were calculated as the compound concentration giving a signal-to-noise ratio of ten (S/N = 10), taking into account the dilution factors, and they are shown in table 6. LOQs were in all cases lower than 0.13 µg g-1, which provide improved sensitivity compared to the methodologies reported in the literature (Table 2) to detect and determine compounds prohibited according the EU Regulation that may be present at trace levels in the commercial samples. Finally, the three methodologies were applied to the analysis of three non-spiked

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commercial samples (HD1, HD2 and HD3). As can be seen in Table 6, comparable

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results were achieved with the three approaches.

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3.6 Analysis of commercial samples

Validation results evidenced the suitability of all the three extraction techniques (VE,

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UAE and MSPD) for the treatment of hair dyes samples. Regarding UAE, some authors pointed out that, even at low frequencies and relatively short application times, analyte degradation may occur through large pressure and temperature gradients, high shear

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forces or even by free radicals generation [36, 37]. On the other hand, MSPD gave lower recovery for PMP. Vortex mixing was selected for application to commercial

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products analysis due to its procedural simplicity. The validated method was applied to eight permanent hair dye formulations (HD4-

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HD11) in addition to HD1-HD3 previously used for methodologies comparison (Table 6). Hair dyeing products included some intended for women but also for men. Products in form of color cream including dark, brown and blonde shades have been analyzed.

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Results are shown in Table 7. Since the target compounds can be present from trace concentrations at the sub µg g-1 up to thousands of µg g-1 (several orders of magnitude

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within a sample), adequate dilutions of the derivatized extracts with ethyl acetate were performed to get concentrations within the calibration range. To avoid possible carryover, two blanks (ethyl acetate) were injected after every set of samples. In the selection of hair color creams, three blonde shades, one coppery red and several dark and light brown shades, purchased in local markets and professional stores, were selected. Blonde shades tend to show lower content of the oxidative dyes studied (for example HD2 and HD9). Regarding concentrations of the studied compounds, comparable results were found in products acquired in local markets and in stores specialized in hair care.

ACCEPTED MANUSCRIPT Six of the studied compounds were found in the analyzed samples. The target hair dyes found most often and in the greatest quantities were RL and 2MR. They were detected in all products at concentrations ranging from 14 to 8675 µg g-1, and from 0.119 to 2644 µg g-1, respectively. In the case of RL, the highest content was found in a hair dye formulation for men. Regarding PMP, it was identified in five of the analyzed samples. Due to the lower recoveries obtained for this compound, results of its analysis should be considered as semi-quantitative. Concentrations found were between 0.214 µg g-1 and

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255 µg g-1.

Concerning the number of compounds per sample, in general, four analytes were found.

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A remarkable point is that all samples contained at least one compound not declared in

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the label at concentrations up to 2 µg g-1. It is worth mentioning the presence of RL (at 14 µg g-1) in a product that expressly included in its packing “free of resorcinol” as

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commercial claim for the consumers. In addition, HQ, prohibited in cosmetics by the current European Regulation, was also detected in this sample at a concentration higher than 1 µg g-1. In the rest of products analyzed, EU Regulation restrictions were fulfilled.

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The SRM chromatogram of a sample containing five dyes (HD1) is displayed in Figure

D

7.

4. Conclusions

PT E

Three simple, eco-friendly and low time consumption sample preparation methods based on VE, UAE and MSPD, were optimized for the GC-MS/MS analysis of some of the most frequently oxidative hair dyes. Selected conditions led in all cases to low

CE

extraction times and solvent consumption. GC-MS/MS was for the first time applied to the analysis of dihydroxybenzenes and their derivatives, naphthols and PMP in hair dye

AC

products allowing separation for the identification of isomeric compounds with very different legal regulation. The validation of the optimized methods was performed using real samples. Good linearity (R2 ≥ 0.991) was obtained in a wide concentration range. Recovery of the compounds was quantitative at low, medium and high spiked concentrations with good method precision (% RSD ≤ 14) using all the extraction methods (VE, UAE and MSPD). LOQ values ≤ 0.13 µg g-1, provide an improved sensitivity compared to the reported methodologies. The three methods showed similar results when applied to real non-spiked samples. In this way, VE was chosen for the analysis of commercial hair dye formulations. All samples, available at markets and professional stores, showed the presence of RL and 2MR at concentrations up to 0.87

ACCEPTED MANUSCRIPT and 0.26% (w/w), respectively, as well as of various compounds not mentioned on the list of ingredients (concentrations up to 0.0014%, w/w). Hydroquinone, a prohibited compound, was found in one sample (1.19 µg g-1). These findings highlight the need of sensitive and selective methodologies for implementation in the required quality control of hair dyeing.

Acknowledgements

PT

This research was supported by European Regional Development Fund 2007–2013 (FEDER) and projects CTQ2013-46545-P (Ministry of Economy and Competitiveness,

RI

Spain), UNST10-1E-491 (Infrastructure Program, Ministry of Science and Innovation,

SC

Spain) and GPC2014/035 (Consolidated Research Groups Program, Xunta de Galicia). E.G. acknowledges Xunta de Galicia for her predoctoral contract and the Ministry of

NU

Education, Culture and Sport for a FPU grant.

MA

References

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[1] P. Ghosh, A.K. Sinha, Hair Colors: Classification, Chemistry and a Review of Chromatographic and Electrophoretic Methods for Analysis, Anal. Lett., 41 (2008) 2291-2321. [2] G.J. Nohynek, E. Antignac, T. Re, H. Toutain, Safety assessment of personal care products/cosmetics and their ingredients, Toxicol. Appl. Pharm., 243 (2010) 239-259. [3] C. Lidén, K. Yazar, J.D. Johansen, A.T. Karlberg, W. Uter, I.R. White, Comparative sensitizing potencies of fragrances, preservatives, and hair dyes, Contact Dermatitis, 75 (2016) 265-275. [4] Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (recast), in: Off. J. Union, 2009. [5] A. Salvador, A. Chisvert, Hair dyes. Regulatory aspects and analytical methods, in: A. Salvador, A. Chisvert (Eds.) Analysis of Cosmetic Products, Elsevier Amsterdam, 2007, pp. 190-209. [6] J.L. Nizzia, A.E. O'Leary, A.T. Ton, C.C. Mulligan, Screening of cosmetic ingredients from authentic formulations and environmental samples with desorption electrospray ionization mass spectrometry, Anal. Methods, 5 (2013) 394-401. [7] Y. Lai, H. Wang, Q. Dong, H. Chen, R. Lin, Y. Cai, Simultaneous determination of 13 components in oxidative hair dyes by high-performance chromatography using an ion-pair reagent, J. Cosmet. Sci., 63 (2012) 321-331. [8] J.-F. He, F.-J. Yao, H. Cui, X.-J. Li, Z.-B. Yuan, Simultaneous determination of dihydroxybenzene positional isomers by capillary electrochromatography using gold nanoparticles as stationary phase, J. Sep. Sci., 35 (2012) 1003-1009.

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[9] Z. Zhong, G. Li, Y. Wu, Z. Luo, B. Zhu, Ultrasound-assisted matrix solid-phase dispersive liquid extraction for the determination of intermediates in hair dyes with ion chromatography, Anal. Chim. Acta, 752 (2012) 53-61. [10] S. Dong, L. Chi, S. Zhang, P. He, Q. Wang, Y. Fang, Simultaneous determination of phenylenediamine isomers and dihydroxybenzene isomers in hair dyes by capillary zone electrophoresis coupled with amperometric detection, Anal. Bioanal. Chem., 391 (2008) 653-659. [11] M. Narita, K. Murakami, J.-M. Kauffmann, Determination of dye precursors in hair coloring products by liquid chromatography with electrochemical detection, Anal. Chim. Acta, 588 (2007) 316-320. [12] J. Zhou, H. Xu, G.-H. Wan, C.-F. Duan, H. Cui, Enhancing and inhibiting effects of aromatic compounds on luminol-dimethylsulfoxide-OH- chemiluminescence and determination of intermediates in oxidative hair dyes by HPLC with chemiluminescence detection, Talanta, 64 (2004) 467-477. [13] U. Vincent, G. Bordin, A.R. Rodriguez, Optimization and validation of an analytical procedure for the determination of oxidative hair dyes in commercial cosmetic formulations, J. Cosmet. Sci., 53 (2002) 101-119. [14] U. Vincent, G. Bordin, A.R. Rodriguez, Validation of an analytical procedure for the determination of oxidative hair dyes in cosmetic formulations, J. Cosmet. Sci., 53 (2002) 43-58. [15] S.C. Rastogi, A method for the analysis of intermediates of oxidative hair dyes in cosmetic products, J. Sep. Sci., 24 (2001) 173-178. [16] B.H. Shao, X.Z. Xu, J.W. Yan, X.Y. Fu, Quantitative determination of commercial oxidation hair dyes by reversed-phase HPLC, J. Liq. Chromatogr. Relat. Technol., 24 (2001) 241-249. [17] M. Akyuz, S. Ata, Determination of aromatic amines in hair dye and henna samples by ion-pair extraction and gas chromatography-mass spectrometry, J. Pharm. Biomed. Anal., 47 (2008) 68-80.

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[18] M.L. Di Gioia, A. Leggio, A. Le Pera, A. Liguori, A. Napoli, F. Perri, C. Siciliano, Determination by gas chromatography/mass spectrometry of p-phenylenediamine in hair dyes after conversion to an imine derivative, J. Chromatogr. A, 1066 (2005) 143148. [19] N. Tanada, S. Kashimura, M. Kageura, K. Hara, Practical GC/MS analysis of oxidation dye components in hair fiber as a forensic investigative procedure, J. Forensic Sci., 44 (1999) 292-296. [20] R.M. Facino, M. Carini, G. Aldini, C. Marinello, P. Traldi, R. Seraglia, Analysis of cosmetic products by gas chromatography/mass spectrometry and fast-atom bombardment mass spectrometry: when a combined approach is successful, Rapid Commun. Mass Spectrom., 11 (1997) 1329-1334. [21] M. Celeiro, E. Guerra, J.P. Lamas, M. Lores, C. Garcia-Jares, M. Llompart, Development of a multianalyte method based on micro-matrix-solid-phase dispersion for the analysis of fragrance allergens and preservatives in personal care products, J. Chromatogr. A, 1344 (2014) 1-14.

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[22] G. Alvarez-Rivera, M. Vila, M. Lores, C. Garcia-Jares, M. Llompart, Development of a multi-preservative method based on solid-phase microextraction–gas chromatography–tandem mass spectrometry for cosmetic analysis, J. Chromatogr. A, 1339 (2014) 13-25. [23] M. Vila, J.P. Lamas, C. Garcia-Jares, T. Dagnac, M. Llompart, Optimization of an analytical methodology for the simultaneous determination of different classes of ultraviolet filters in cosmetics by pressurized liquid extraction–gas chromatography tandem mass spectrometry, J. Chromatogr. A, 1405 (2015) 12-22. [24] A. Chisvert, J. Sisternes, Á. Balaguer, A. Salvador, A gas chromatography-mass spectrometric method to determine skin-whitening agents in cosmetic products, Talanta, 81 (2010) 530-536. [25] W. Zhou, P.G. Wang, J.B. Wittenberg, D. Rua, A.J. Krynitsky, Simultaneous determination of cosmetics ingredients in nail products by fast gas chromatography with tandem mass spectrometry, J. Chromatogr. A, 1446 (2016) 134-140. [26] M. Celeiro, J.P. Lamas, C. Garcia-Jares, M. Llompart, Pressurized liquid extraction-gas chromatography-mass spectrometry analysis of fragrance allergens, musks, phthalates and preservatives in baby wipes, J. Chromatogr. A, 1384 (2015) 9-21. [27] R.J. Turesky, J.P. Freeman, R.D. Holland, D.M. Nestorick, D.W. Miller, D.L. Ratnasinghe, F.F. Kadlubar, Identification of Aminobiphenyl Derivatives in Commercial Hair Dyes, Chem. Res. Toxicol., 16 (2003) 1162-1173. [28] Y. Picó, Ultrasound-assisted extraction for food and environmental samples, TrACTrend Anal. Chem., 43 (2013) 84-99. [29] P.G. Wang, W. Zhou, Rapid determination of parabens in personal care products by stable isotope GC-MS/MS with dynamic selected reaction monitoring, J. Sep. Sci., 36 (2013) 1781-1787. [30] E. Guerra, M. Celeiro, J.P. Lamas, M. Llompart, C. Garcia-Jares, Determination of dyes in cosmetic products by micro-matrix solid phase dispersion and liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. A, 1415 (2015) 27-37. [31] G. Alvarez-Rivera, M. Llompart, C. Garcia-Jares, M. Lores, Identification of unwanted photoproducts of cosmetic preservatives in personal care products under ultraviolet-light using solid-phase microextraction and micro-matrix solid-phase dispersion, J. Chromatogr. A, 1390 (2015) 1-12. [32] M. Llompart, M. Celeiro, J. P. Lamas, L. Sanchez-Prado, M. Lores, C. GarciaJares, Analysis of plasticizers and synthetic musks in cosmetic and personal care products by matrix solid-phase dispersion gas chromatography–mass spectrometry, J. Chromatogr. A, 1293 (2013) 10-19. [33] L. Sanchez-Prado, G. Alvarez-Rivera, J.P. Lamas, M. Lores, C. Garcia-Jares, M. Llompart, Analysis of multi-class preservatives in leave-on and rinse-off cosmetics by matrix solid-phase dispersion, Anal. Bioanal. Chem., 401 (2011) 3293-3304. [34] L. He, Y. Su, B. Fang, X. Shen, Z. Zeng, Y. Liu, Determination of Sudan dye residues in eggs by liquid chromatography and gas chromatography–mass spectrometry, Anal. Chim. Acta, 594 (2007) 139-146.

ACCEPTED MANUSCRIPT

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[35] E. Pel, G. Bordin, A.R. Rodriguez, HPLC Candidate Reference Method for Oxidative Hair Dye Analysis. I. Separation and Stability Testing, J. Liq. Chromatogr. Relat. Technol, 21 (1998) 883-901. [36] L. Sanchez-Prado, R. Barro, C. Garcia-Jares, M. Llompart, M. Lores, C. Petrakis, N. Kalogerakis, D. Mantzavinos, E. Psillakis, Sonochemical degradation of triclosan in water and wastewater, Ultrason. Sonochem., 15 (2008) 689-694. [37] E. Yiantzi, E. Psillakis, K. Tyrovola, N. Kalogerakis, Vortex-assisted liquid-liquid microextraction of octylphenol, nonylphenol and bisphenol-A, Talanta, 80 (2010) 20572062.

ACCEPTED MANUSCRIPT Figure captions Figure 1. Influence of the ascorbic acid addition (0, 10 mg solid and 0.2 mg in solution) on the standard response Figure 2. Influence of the solvent in vortex extraction Figure 3. Influence of the sonication time (A) and solvent type (B) in ultrasoundassisted extraction.

PT

Figure 4. Influence of clean-up in vortex extraction (A) and ultrasound-assisted extraction (B)

RI

Figure 5. Influence of the dispersant (A) and solvent type (B) in MSPD.

SC

Figure 6. Effect of matrix addition on the GC/MS-MS response of a 0.1 µg mL-1 standard solution of the compounds

AC

CE

PT E

D

MA

NU

Figure 7. SRM chromatogram of a sample (HD1) containing five target compounds (see concentrations in Table 6)

ACCEPTED MANUSCRIPT Figure 1. Influence of the ascorbic acid addition (0, 10 mg solid and 0.2 mg in solution) 3.00E+07

0

10

0.2

PT

2.50E+07

RI NU

SC

1.50E+07

MA

1.00E+07

0.00E+00

HQ

CE

PT E

RL

D

5.00E+06

AC

Abundance

2.00E+07

2MR

4CLR

1NL

2NL

PMP

ACCEPTED MANUSCRIPT Figure 2. Influence of the solvent in vortex extraction 4.50E+07 MeOH 4.00E+07 ACN 3.50E+07

EA

PT RI

2.50E+07

SC

2.00E+07

NU

1.50E+07

5.00E+06

0.00E+00 HQ

2MR

CE

PT E

D

RL

MA

1.00E+07

AC

Abundance

3.00E+07

4CLR

1NL

2NL

PMP

ACCEPTED MANUSCRIPT Figure 3. Influence of the sonication time (A) and solvent type (B) in ultrasoundassisted extraction.

A

1 min

3.00E+07

5 min

2.50E+07

10 min

2.00E+07

PT

Abundance

3.50E+07

1.50E+07

RI

1.00E+07

0.00E+00

3.50E+07

HQ

4CLR

1NL

2NL

2.50E+07

D

2.00E+07

PMP

MeOH ACN EA

MA

B

3.00E+07

PT E

1.50E+07 1.00E+07 5.00E+06

CE

0.00E+00

RL

AC

Abundance

2MR

NU

RL

SC

5.00E+06

HQ

2MR

4CLR

1NL

2NL

PMP

ACCEPTED MANUSCRIPT Figure 4. Influence of clean-up in vortex extraction (A) and ultrasound-assisted extraction (B)

3.00E+07

A

VE clean-up

VE

2.50E+07

1.50E+07

PT

Abundance

2.00E+07

RI

1.00E+07

0.00E+00

3.50E+07

HQ

B

UAE clean-up

MA

3.00E+07 2.50E+07 2.00E+07

4CLR

1NL

2NL

PMP

1NL

2NL

PMP

UAE

D

1.50E+07

PT E

1.00E+07 5.00E+06 0.00E+00

CE

RL

AC

Abundance

2MR

NU

RL

SC

5.00E+06

HQ

2MR

4CLR

ACCEPTED MANUSCRIPT Figure 5. Influence of the dispersant (A) and solvent type (B) in MSPD

3.00E+07

A

FLORISIL ALUMINA

2.50E+07

SILICA

PT

Abundance

2.00E+07 1.50E+07

RI

1.00E+07

0.00E+00

B

3.00E+07

4CLR

1NL

2NL

PMP

MeOH ACN EA

PT E

D

2.00E+07 1.50E+07 1.00E+07

CE

5.00E+06

RL

AC

Abundance

2.50E+07

0.00E+00

2MR

MA

3.50E+07

HQ

NU

RL

SC

5.00E+06

HQ

2MR

4CLR

1NL

2NL

PMP

ACCEPTED MANUSCRIPT -1

Figure 6. Effect of matrix addition on the GC/MS-MS response of a 0.1 µg mL standard solution of the compounds

6.00E+06

No spiked Spiked with matrix

Spiked with oil

PT

5.00E+06

RI SC

3.00E+06

NU

2.00E+06

0.00E+00 HQ

CE

PT E

D

RL

MA

1.00E+06

AC

Abundance

4.00E+06

2MR

4CLR

1NL

2NL

PMP

ACCEPTED MANUSCRIPT Figure 7. SRM chromatogram of a sample (HD1) containing five target compounds (see concentrations in Table 6) RT: 7.92 - 14.39 8.85

100

RL 152.0-110.1 (10 eV)

50

0 100

9.70

11.11

0 100

11.39

1NL 144.0-115.1 (20 eV)

NU

50

50

8.5

9.0

9.5

10.0

10.5

11.0

11.5

PT E

D

Time (min)

CE

8.0

MA

0 100

0

SC

RI

4CLR 144.0-80.8 (15 eV)

50

AC

Relative Abundance

0 100

PT

2MR 124.0-123.1 (10 eV)

50

12.0

13.59

PMP 174.0-129 (10 eV)

12.5

13.0

13.5

14.0

ACCEPTED MANUSCRIPT Table 1. Target compounds, use restrictions, and maximum concentration permitted (MCP) according to the Regulation (EC) No 1223/2009 and following amendments. Compound

Chemical name

1,3-benzenediol

Use restrictions

108-46-3

Hair dye substance in oxidative hair dye products Products intended for colouring eyelashes (for professional use only) Hair lotions and shampoos

RI

PT

Resorcinol (RL)

CAS number

4-chlorobenzenediol

1-naphthol (1NL)

1-naphthalenol

CE

PT E

4-chlororesorcinol (4CLR)

SC

608-25-3

2-naphthalenol

95-88-5

90-15-3 135-19-3

1.25%a

1.25%a

0.5%

Prohibited

1.8% a

1.8%

2.5% a

2.0% a

Any type of product Prohibited Hair dye substance in Phenyl methyl 3-methyl-1-phenyl89-25-8 oxidative hair dye 0.25% a pyrazolone (PMP) 5-pyrazolone products a After mixing under oxidative conditions, in ready product to apply to hair or eyelashes

AC

2-naphthol (2NL)

NU

2-methyl-1,3benzenediol

123-31-9

MA

2-methylresorcinol (2MR)

1,4-benzenediol

D

Hydroquinone (HQ)

Any type of products (except artificial nail systems for professional use only) Hair dye substance in oxidative hair dye products Hair dye substance in non-oxidative hair dye products Hair dye substance in oxidative hair dye products Hair dye substance in oxidative hair dye products

MCP

ACCEPTED MANUSCRIPT Table 2. Analytical methodologies proposed for the target oxidative hair dyes

Analytes

Sample preparation (extraction method)

Analysis

RL

Without sample preparation

DESI-MS

RL, HQ

UAE: 0.1 g sample dissolved in 15 mL 50% (v/v) ethanol, 1 mL 1% sodium dithionite added as antioxidant, before 15 min ultrasonic extraction

IPC-DAD

RL, HQ

UAE: sample dissolved in 25 mL ethanol, before 60 min ultrasonic extraction

CEC

RL, HQ, 2MR, 4CLR,

UA-MSPDLE: 0.2 g sample weighted, followed by adding 5% (w/w) sodium sulphite as antioxidant, 25 mL scale with 0.1 mol L-1 methanesulphonic acid, 0.4 g of neutral alumina, and 0.5 mL of nhexane. Stirring and sonication at

Recovery (%)

84.8-97.8 (RL); 81.787.2 (HQ)

LOD/LOQa (%w/w) x 104

RSD (%)

Ref.

1.0

9-27

Nizzia et al, 2013

0.3 (RL); 0.2 (HQ)

0.055 (RL); 0.011 (HQ) / 0.61 (RL); 0.56 (HQ)

D E

I R

C S U

< 3.2 (RL); < 3.0 (HQ)

Lai et al, 2012

N A

M

< 3.6 (RL); < 2.4 (HQ)

He et al, 2012

< 2.9 (RL); < 2.8 (HQ); < 2.6 (2MR); < 3.7 (4CLR)

Zhong et al, 2012

T P E

A

C C

IPC-UV

92.0-99.4 (RL); 90.5103.5 (HQ); 85.7-98.3 (2MR); 93.699.2 (4CLR)

0.019 (RL); 0.035 (HQ); 0.026 (2MR); 0.044 (4CLR) / 0.06 (RL); 0.12 (HQ); 0.09 (2MR); 0.15 (4CLR)

T P

ACCEPTED MANUSCRIPT 60ºC and centrifuging.

RL

UAE: sample dissolved in 10 mL ethanol, before 25 min ultrasonic extraction.

CZE-AD

RL

UAE: 0.1 g sample dissolved in 10 mL methanol, before 1 min ultrasonic extraction

HPLC-ED

RL, HQ

UAE: 0.05 g sample dissolved in a mixture solution (20 mL methanol and 30 mL mobile phase) containing 0.5 g sodium sulphite, before 10 min ultrasonic extraction

RL, HQ, 1NL

LLE: sample dissolved in methanoltetraborate buffer pH 8 containing sodium ascorbate as antioxidant 15 times or 10 times (w/w); three times extracted with nheptane

RL, HQ, 1NL

UAE: 1 g sample dissolved in 25

96.4-107

0.068

< 4.9

Dong et al, 2008

T P

I R

Narita et al, 2007

C S U

90.6-105.2 (RL)

HPLC-CL

0.032 (RL); 0.003 (HQ)

D E

N A

M

< 2.6 (RL); < 2.2 (HQ)

Zhou et al, 2004

T P E

C C

A

HPLC-DAD

IPC-DAD

94 (RL), 98 (HQ), n.c. (1NL)

94 (RL), 98 (HQ), 95

Vincent et al, 2002a, 2002b

≤ 5 for all compounds

<5

Rastogi, 2001

ACCEPTED MANUSCRIPT mM phosphate buffer (pH 6.0, containing 0.1% 1heptanesulphonic acid sodium salt and 0.05% sodium ascorbate) and 15 mL of acetonitrile before heating at 60ºC for 5 min and sonication for 5 min

RL

UAE: 0.1 g sample dissolved in 5 mL of ethanol before ultrasonic extraction

(1NL)

T P

I R

HPLC-DAD

93.3

-/ 0.72

1.3

N A

C S U

Shao et al, 2001

IPC: ion-pair chromatography; CEC: capillary electrochromatography; UA-MSPDLE: ultrasound-assisted matrix solid-phase dispersive liquid extraction; CZE-AD: capillary zone electrophoresis-amperometric detection; ED: electrochemical detection; CL: chemiluminescence; DAD: diode array detector; n.c.: not calculated a

Equivalent to µg g

D E

-1

T P E

A

C C

M

ACCEPTED MANUSCRIPT

RT

SRM1

(min

(quantification

)

)

RL

8.81

152> 110.1

10

110> 82.1

10

194> 110.1

10

HQ

8.95

152.1> 110

10

110> 82.1

10

194> 110.1

10

2MR

9.68

124> 123.1

10

124> 95

15

166.1> 124

10

4CLR

11.09

144> 80.8

15

144> 51.7

25

186> 144

10

1NL

11.39

144> 115.1

20

144> 89

35

186.1> 144.1

10

2NL

11.85

144> 115

20

144.1> 89

35

186.1> 144.1

10

PMP

13.60

174> 129

10

174> 77

RI

Table 3. Experimental GC-MS/MS parameters

174> 145.3

15

d

CE1 a

SRM3

(confirmation

CE2 a

(confirmation

)

a

)

30

CE

PT E

D

MA

NU

SC

Collision energy (eV)

CE3

AC

a

SRM2

PT

Compoun

31

ACCEPTED MANUSCRIPT Table 4. GC-MS/MS performance: linearity and precision at three concentrations (10, 100, and 1000 ng mL-1). Linearity Compound

Range -1

(ng mL )

Precision RSD (%) R2

Intra-day (n=6)

Inter-day (n=6)

10

100

1000

10

100

1000

1-2000

0.9999

6.0

3.1

2.5

7.4

1.8

4.0

HQ

1-2000

0.9998

4.6

3.1

2.4

9.2

6.5

5.6

2MR

1-2000

0.9908

5.5

9.3

2.7

10

10

8.6

4CLR

1-2000

0.9998

6.3

5.2

4.5

6.3

6.4

3.3

1NL

1-2000

0.9998

8.5

3.3

1.9

8.7

8.7

3.6

2NL

1-2000

0.9995

9.8

4.6

1.7

10

6.4

4.2

PMP

1-2000

0.9990

5.6

8.5

9.7

7.7

8.6

2.6

AC

CE

PT E

D

MA

NU

SC

RI

PT

RL

32

ACCEPTED MANUSCRIPT Table 5. Recovery study (%RSD, n=4) performed with three samples (T1, T2 and T3) spiked at 1, 10 and 100 µg g-1, extracted by the three procedures (VE, UAE, and MSPD).

T1 VE

UAE

T2 MSPD

VE

T3

UAE

MSPD

VE

UAE

MSPD

-1

96.2 (9.0)

2MR

78.8 (6.4)

61.4 (2.7)

79.2 (3.2)

47.1(6.2)

84.7 (10)

2NL

82.4 (0.7)

85.3 (10)

1NL

77.1 (1.5)

88.6 (9.9)

4CLR

51.5 (1.2)

82.0 (2.6)

89.9 (10)

102 (11)

99.5

94.1

94.8

74.7

(11)

(9.7)

(8.4)

(1.7)

(3.0)

(2.8)

87.1

79.9

97.7

82.8

94.0

94.7

(10)

(9.6)

(4.8)

(0.6)

(5.6)

(11)

80.3

118

PT

HQ

74.8 (1.6)

89.2

92.7

112

81.0

(12)

(5.5)

110 (14)

(3.2)

(7.2)

(4.7)

87.5

86.2

118

87.4

95.8

71.8

(7.0)

(7.2)

(5.3)

(3.2)

(8.3)

(6.8)

88.9

93.1

115

94.6

104

85.4

(8.4)

(6.8)

(2.5)

(2.1)

(6.1)

(8.9)

96.2

113

101

103

87.0

(8.4)

(3.7)

(1.1)

(0.1)

(6.7)

94.8 91.6 (6.4)

83.9 (6.6)

(12)

RI

(7.6)

86.8

SC

86.3

MA

RL

NU

1 µg g

-1

10 µg g

72.8

75.2 (4.4)

2MR

81.8

4CLR

67.1 (6.6)

79.3 (5.5)

78.1 (5.8)

84.7 (5.8)

101 (8.9)

93.9 (11)

91.1

98.8

83.6

85.6

74.5

(7.1)

(3.2)

(2.1)

(1.0)

(3.5)

(0.9)

82.7

90.3

79.9

78.1

79.5

71.4

(9.5)

(3.6)

(5.3)

(2.3)

(5.9)

(8.5)

110

117

94.4

104

101

87.6

(6.5)

(5.2)

(10)

(2.9)

(5.9)

(7.6)

78.4

85.8

90.5

77.9

77.1

76.3

(10)

(4.6)

(11)

(1.7)

(6.1)

(2.3)

76.4

85.3

92.4

74.4

72.8

82.6

(11)

(4.8)

(13)

(0.5)

(11)

(1.3)

79.1

87.9

98.3

74.9

78.3

84.6

(12)

(6.3)

(14)

(2.5)

(10)

(14)

100 µg g-1

73.0 (1.3)

88.5 (9.5)

2MR

73.0 (5.5)

100 (7.0)

HQ

50.3 (5.4)

90.5 (11)

RL

96.5 (6.6)

AC

(10) 2NL

93.5 (6.9)

80.3 (11)

1NL

83.7 (11)

90.2 (6.0)

4CLR

73.7 (4.9)

PT E

HQ

74.0 (9.8)

82.9

D

(0.5)

56.3 (7.7)

CE

RL

80.6 (0.4)

106

98.4

95.3

102

96.5

85.8

80.2

(2.4)

(6.0)

(2.3)

(2.4)

(7.8)

(11)

95.4

96.9

89.3

98.7

85.2

71.9

(2.6)

(1.8)

(1.6)

(1.6)

(8.3)

(9.8)

81.8

105

91.1

98.5

89.5

79.9

(5.6)

109 (9.5)

95.8 (0.3)

(4.5)

(7.1)

(5.2)

(2.3)

(6.8)

(8.7)

98.3

104 (11)

70.0 (5.1)

88.2

99.4

87.5

92.8

88.2

76.5

33

ACCEPTED MANUSCRIPT

1NL

(5.8)

(1.7)

(6.1)

(5.2)

(3.0)

(6.9)

(13)

99.0

89.6

102

87.1

97.6

87.8

84.4

(2.5)

(9.7)

(3.9)

(3.1)

(4.0)

(10)

90.8

82.9

89.3

104

86.3

87.4

(3.5)

(6.0)

(6.3)

(1.8)

(3.5)

(10)

(7.6)

89.9 (3.5)

105 97.8 (11)

87.4 (3.7)

CE

PT E

D

MA

NU

SC

RI

PT

(3.7)

AC

2NL

100 (12)

34

ACCEPTED MANUSCRIPT Table 6. Limits of quantification and application of the methods to three commercial samples (HD1, HD2 and HD3, concentrations in µg g-1). LOQ MSP

VE

D 0.06

1

3

0.06

0.11

0

6

0.09

0.12

7

5

4CL

0.08

0.07

R

6

9

1NL

0.09

0.13

8

1

0.08

0.06

3

7

2NL

D

UA

MSP

E

D

396 1

3863

3803

491

452

0.074

0.086

UA

MSP

E

D

3974

3721

9

1715

1294

1.86

1.55

1.20

VE

414

264

2.1

4

2455

2608

5

2.08

1.71

1.65

SC

2MR

E

VE

418

389

390

0.108

0.125

0.066

531

5

1.83

1.99

150

MA

HQ

0.080

MSP

PT

0.06

RL

UA

HD3

RI

UAE

HD2

NU

VE

HD1

Values given in italics and underlined correspond with compounds found in the sample but not declared

AC

CE

PT E

D

in the label.

35

ACCEPTED MANUSCRIPT Table 7. Concentrations (%, w/w x 104)a of the target compounds in commercial hair dying productsb.

RL

HD4

HD5

HD6

HD7

HD8

HD9

HD10

HD11

3467

4483

7264

5315

8675

14.0

2560

190

249

27.8

0.119

764

0.107

HQ

1.19

2MR

498

0.498

473

2486

4CLR

0.147

0.186

0.571

0.147

0.751

1NL

0.169

PT

2NL Blank spaces: below LOQ.

b

By VE-GC-MS/MS

SC

Equivalent to µg g-1

AC

CE

PT E

D

MA

NU

a

RI

Values given in italics and underlined correspond with compounds found in the sample but not declared in the label.

36

ACCEPTED MANUSCRIPT

Highlights

PT

RI SC NU MA D PT E

 

CE



Miniaturized VE, UAE, and MSPD were optimized for hair dyes analysis. GC-MS/MS allowed sensitive and selective detection of regulated and banned isomers. Three validated methods performed successfully in a broad range of dyeing products. Most dyes found in real samples, including banned hydroquinone at the low ppb. Methods allowed detecting dyes in products that did not comply with EU regulation.

AC

 

37