Miniaturized matrix solid-phase dispersion for the analysis of ultraviolet filters and other cosmetic ingredients in personal care products

separations Article Miniaturized Matrix Solid-Phase Dispersion for the Analysis of Ultraviolet Filters and Other Cosmetic Ingredients in Personal Care Products Maria Celeiro , Lua Vazquez, J. Pablo Lamas , Marlene Vila, Carmen Garcia-Jares and Maria Llompart * Laboratory of Research and Development of Analytical Solutions (LIDSA), Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain; maria.celeiro.monter[email protected] (M.C.); lua.vazquez.ferreir[email protected] (L.V.); [email protected] (J.P.L.); [email protected] (M.V.); carmen.garcia.jar[email protected] (C.G.-J.) *Correspondence: [email protected]; Tel.: +34-881-814-225 Received: 5 April 2019; Accepted: 3 June 2019; Published: 10 June 2019   Abstract: A method based on micro-matrix solid-phase dispersion ( µ -MSPD) followed by gas-chromatography tandem mass spectrometry (GC–MS/MS) was developed to analyze UV filters in personal care products. It is the first time that MSPD is employed to extract UV filters from cosmetics samples. This technique provides efficient and low-cost extractions, and allows performing extraction and clean-up in one step, which is one of their main advantages. The amount of sample employed was only 0.1 g and the extraction procedure was performed preparing the sample-sorbent column in a glass Pasteur pipette instead of the classic plastic columns in order to avoid plastizicer contamination. Factors affecting the process such as type of sorbent, and amount and type of elution solvent were studied by a factorial design. The method was validated and extended to other families of cosmetic ingredients such as fragrance allergens, preservatives, plasticizers and synthetic musks, including a total of 78 target analytes. Recovery studies in real sample at several concentration levels were also performed. Finally, the green extraction methodology was applied to the analysis of real cosmetic samples of different nature. Keywords: UV filters; matrix solid-phase dispersion; µ -MSPD; miniaturized extraction technique; GC–MS/MS; cosmetic analysis; personal care products; fragrance allergens; preservatives; plasticizers; synthetic musks 1. Introduction The cosmetic industry is one of the fastest growing markets in the world, due to a high demand for cosmetics and personal care products. Manufacturers must innovate to offer attractive and safe products for consumers to stay ahead in a highly competitive sector. Cosmetic formulations usually include a large number or organic compounds, such as fragrances, preservatives, antioxidants, plasticizers, or surfactants among others. One type of these compounds are the ultraviolet filters ( UV filters ). These substances are intended to protect consumers against the harmful solar radiation and, although their presence is especially important in sunscreens, they can be found in a broad range of daily care products such as creams, hair-care products, lip protectors, make-up, and many others. The widespread inclusion of UV filters in personal care and consumer products increases the human exposure to these compounds. Some of them are considered as endocrine disruptors, with high bioaccumulative properties. In fact, some of them have been recently detected in human breast milk. Nowadays, according to the Annex VI of the Regulation EC No 1223/2009 [ 1 ], 26 organic UV filters are allowed for Separations 2019,6, 30; doi:10.3390/separations6020030 www.mdpi.com/journal/separations Separations 2019,6, 30 2 of 14 their use in the formulation of cosmetic products, being the maximum concentration permitted in the final product up to 10% (w/w). It is important to note that the Regulation regarding cosmetic products is being continually updated, with the restriction and even prohibition of several compounds each year. Therefore, the cosmetic sector demands the development of reliable, fast and easy to implement analytical methodology to analyze a broad range of cosmetics ingredients. One major drawback for the analysis of cosmetics is sample preparation, since the cosmetic matrices are complex and varied. Besides, the concentration of the different ingredients in cosmetic formulations usually ranges several orders of magnitude, from the ng g−1to thousands of µg g−1. Most of the reported methodologies for the determination of UV filters in cosmetics deal with the simultaneous analysis of few target compounds. Regarding the sample preparation, solid-liquid or liquid–liquid extraction, or simple dilution, have been the most employed procedures [ 2 – 4 ]. However, since cosmetics are complex mixtures of ingredients, the direct dilution of the samples can negatively affect the chromatographic determination and the chromatographic system, producing damage in the injector, column and detector. Therefore, the use of sample preparation techniques which imply an in-situ clean-up step is a good approach. In this way, matrix solid-phase dispersion (MSPD) has been proposed for the extraction of different families of cosmetic ingredients such as fragrances, preservatives or dyes [5–7]. New trends in sample preparation are focused on the development of miniaturized procedures which complies with the green chemistry principles [ 8 , 9 ], and techniques such as ultrasound-assisted emulsification microextraction (USAEME) or single drop microextraction have been developed [ 10 , 11 ] for the determination of parabens of phthalates. In this way, a miniaturization of the classical MSPD, micro-MSPD ( µ -MSPD), employing low-cost material, low amount of sample and organic solvent consumption, has been successfully proposed for the extraction of different compounds such as synthetic musks, preservatives, fragrance allergens, or dyes [ 12 – 15 ] in cosmetics and personal care products. However, to the best of our knowledge MSPD and µ -MSPD have never been applied for the determination of UV filters. Regarding the analytical determination of UV filters in cosmetic samples, LC-DAD has been the most employed technique [ 2 ]. However, the use of other detectors, such as MS, and especially the use of triple quadrupole working under MS/MS provides improved selectivity and sensitivity [16,17]. The main goal of this work is the development of an analytical methodology based on µ -MSPD-GC–MS/MS for the simultaneous determination of 14 multiclass UV filters in cosmetic samples. The main experimental parameters affecting extraction, such as the type of sorbent, and amount and type of extraction solvent have been optimized by means of experimental design. The method was validated and applied to a broad range of cosmetic and personal care products to quantify not only UV filters, but also other families of compounds such as fragrances, preservatives, plasticizers, and synthetic musks, allowing the simultaneous analysis of 78 compounds with very different chemical nature in a single extraction and chromatographic run. 2. Materials and Methods 2.1. Chemicals, Reagents and Materials The studied UV filters, their Chemical Abstracts Service (CAS) number, retention times, and MS/MS transitions are summarized in Table 1. Target fragrance allergens, preservatives, plasticizers and synthetic musks are shown in Table S1. Ethyl acetate, acetonitrile (ACN) and isooctane were provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany), methanol (MeOH) was supplied by Scharlab (Barcelona, Spain), and acetone was provided by Fluka Analytical (Steinheim, Germany). Florisil (60–100 µ m mesh), and glass wool were purchased from Supelco Analytical (Bellefonte, PA, USA), and sand (200–300 µ m mesh) and anhydrous sodium sulphate, Na 2 SO 4 , (99%) from Panreac (Barcelona, Spain). Individual stock solutions of all the compounds were prepared in acetone, isooctane or methanol. Further dilutions and mixtures were prepared in acetone (spike solutions) or acetonitrile Separations 2019,6, 30 3 of 14 (calibration study). Solutions were stored in amber glass vials at − 20 ◦ C. All solvents and reagents were of analytical grade. Table 1. Studied ultraviolet (UV) filters. CAS number, retention time and mass spectrometry (MS)/MS transitions. UV Filter Acronym CAS Retention Time (min) MS/MS Transition (CE a, eV) Ethylhexylsalicylate EHS 118-60-5 12.85 120.0 →92.0 (10) 138.0 →120.0 (10) 250.1 →120.0 (15) Benzyl salicylate BS 118-58-1 13.73 91.0 →39.0 (30) 91.0 →65.0 (15) 228.1 →91.1 (10) Homosalate HMS 118-56-9 13.88 120.0 →92.0 (10) 138.0 →120.0 (10) 262.2 →120.0 (15) Benzophenone-3 BP3 131-57-7 16.22 151.0 →95.0 (10) 227.1 →127.9 (35) 227.1 →184.0 (20) Isoamyl-4-methoxycinnamate IAMC 71617-10-2 16.38 161.0 →133.0 (10) 178.1 →161.1 (10) 248.1 →178.0 (10) 4-methylbenzylidene camphor 4MBC 36861-47-9 16.63 127.9 →102.0 (20) 170.6 →128.1 (15) 254.1 →239.2 (10) Methyl anthranilate MA 134-20-3 17.66 119.0 →91.8 (10) 137.0 →119.0 (10) 275.2 →137.0 (10) Etocrylene ETO 5232-99-5 18.22 231.9 →176.5 (20) 248.0 →164.9 (25) 276.9 →248.1 (10) Ethylhexyl-p-aminobenzoic acid EHPABA 21245-02-3 19.33 148.0 →104.2 (25) 165.1 →148.6 (15) 277.2 →164.9 (10) 2-ethylhexyl 4-methoxycinnamate 2EHMC 5466-77-3 19.69 161.0 →133.1 (10) 177.9 →133.1 (20) 290.2 →178.1 (10) Octocrylene OCR 6197-30-4 21.48 232.0 →203.0 (20) 248.0 →165.0 (30) 360.2 →276.1 (20) Avobenzone BMDM 70356-09-1 22.44 161.1 →118.0 (15) 295.1 →135.1 (15) 309.2 →279.1 (20) Diethylamino hydroxybenzoyl hexyl benzoate DHHB 302776-68-7 23.10 382.2 →280.2 (10) 382.2 →298.1 (10 397.2 →382.2 (10 Drometrizole trisiloxane DRT 155633-54-8 25.50 221.1 →73.1 (15) 369.1 →250.2 (10) 444.1 →296.1 (25 aCE: collision energy; underlined SRM transitions: quantification transitions. Metallic, glass materials, dispersing agents (Florisil and sand), Na 2 SO 4 and glass wool were maintained at 230 ◦C for 12 h before use to eliminate possible phthalate contamination. All materials were allowed to cool down, wrapped with aluminum foil, and Florisil, sand, and Na 2 SO 4 were kept in desiccator. 2.2. Cosmetic Samples Cosmetics and personal care products from national and international brands were obtained from local sources. They included sunscreens intended for adults and for children, hair-care products, moisturizing face creams, antiwrinkle creams, make-up, lip protectors, make-up, lipsticks, among others. The samples were kept in their original containers and protected from light at room temperature. Separations 2019,6, 30 4 of 14 2.3. µ-MSPD Procedure Cosmetic samples (0.1 g) were exactly weighed into a glass vial. Then, the sample was gently blended with 0.4 g of the drying agent anhydrous Na 2 SO 4 , and 0.4 g of the corresponding dispersing agent (Florisil or sand), into the vial, using a glass rod, until a homogeneous mixture was obtained ( ca. 5 min ). The mixture was then transferred into a glass Pasteur pipette (approximately 150 mm), with a small amount of glass wool at the bottom, containing 0.1 g of Florisil (to obtain a further degree of fractionation and an in-situ clean-up step). Finally, a small amount of glass wool was placed on top to compress the mixture. Elution with the corresponding solvent (ethyl acetate, ACN, MeOH or the mixture MeOH/acetone (1:1, v/v)) depending on the experiment was made by gravity flow, collecting the extract into a 1 mL or 2 mL volumetric flask. The obtained extracts were diluted 1:10 (v/v) and 1:100 (v/v) in ACN (or even more when necessary), and analyzed by GC–MS/MS. Fortified samples were spiked with 10 µ L of the corresponding spiking solution to get the desired final concentration of the target compounds, and submitted to the same process described above. Figure 1illustrates the described µ-MSPD procedure under the optimal conditions. Separations 2018, 5, x FOR PEER REVIEW 4 of 15 2.2. Cosmetic Samples Cosmetics and personal care products from national and international brands were obtained from local sources. They included sunscreens intended for adults and for children, hair-care products, moisturizing face creams, antiwrinkle creams, make-up, lip protectors, make-up, lipsticks, among others. The samples were kept in their original containers and protected from light at room temperature. 2.3. µ-MSPD Procedure Cosmetic samples (0.1 g) were exactly weighed into a glass vial. Then, the sample was gently blended with 0.4 g of the drying agent anhydrous Na2SO4, and 0.4 g of the corresponding dispersing agent (Florisil or sand), into the vial, using a glass rod, until a homogeneous mixture was obtained (ca. 5 min). The mixture was then transferred into a glass Pasteur pipette (approximately 150 mm), with a small amount of glass wool at the bottom, containing 0.1 g of Florisil (to obtain a further degree of fractionation and an in-situ clean-up step). Finally, a small amount of glass wool was placed on top to compress the mixture. Elution with the corresponding solvent (ethyl acetate, ACN, MeOH or the mixture MeOH/acetone (1:1, v/v)) depending on the experiment was made by gravity flow, collecting the extract into a 1 mL or 2 mL volumetric flask. The obtained extracts were diluted 1:10 (v/v) and 1:100 (v/v) in ACN (or even more when necessary), and analyzed by GC–MS/MS. Fortified samples were spiked with 10 µL of the corresponding spiking solution to get the desired final concentration of the target compounds, and submitted to the same process described above. Figure 1 illustrates the described µ-MSPD procedure under the optimal conditions. Figure 1. Schematic representation of the micro-matrix solid-phase dispersion (µ-MSPD) procedure under the optimal conditions. 2.4. GC–MS/MS Analysis The GC–MS/MS analysis was carried out employing a Thermo Scientific Trace 1310 gas chromatograph coupled to a triple quadrupole mass spectrometer (TSQ 8000) with IL 1310 autosampler from Thermo Scientific (San Jose, CA, USA). Separation was performed on a Zebron ZB- Semivolatiles (30 m × 0.25 mm i.d., 0.25 μm film thickness) obtained from 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 temperature was programmed from 60 °C (held 1 min) to 100 °C at 8 °C min−1, to 150 °C at 20 °C min−1, to 200 °C at 25 °C min−1 (held 5 min), to 220 °C at 8 °C min−1, and to 290 °C at 30 °C min−1 (held 3 min). Pulsed splitless mode (200 kPa, held 1 min) was used for injection and the injector temperature was set at 260 °C. The injection volume was 1 µL and the total run time was 23.5 min. The mass spectrometer (MSD) 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 °C and 350 °C, respectively. Selected reaction monitoring (SRM) acquisition mode was implemented monitoring Figure 1. Schematic representation of the micro-matrix solid-phase dispersion ( µ -MSPD) procedure under the optimal conditions. 2.4. GC–MS/MS Analysis The GC–MS/MS analysis was carried out employing a Thermo Scientific Trace 1310 gas chromatograph coupled to a triple quadrupole mass spectrometer (TSQ 8000) with IL 1310 autosampler from Thermo Scientific (San Jose, CA, USA). Separation was performed on a Zebron ZB-Semivolatiles (30 m × 0.25 mm i.d., 0.25 µ m film thickness) obtained from 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 temperature was programmed from 60 ◦ C (held 1 min) to 100 ◦ C at 8 ◦ C min −1 , to 150 ◦ C at 20 ◦ C min −1 , to 200 ◦ C at 25 ◦ C min −1 (held 5 min), to 220 ◦ C at 8 ◦ C min −1 , and to 290 ◦ C at 30 ◦ C min −1 (held 3 min). Pulsed splitless mode (200 kPa, held 1 min) was used for injection and the injector temperature was set at 260 ◦C. The injection volume was 1 µL and the total run time was 23.5 min. The mass spectrometer (MSD) 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 ◦ C and 350 ◦ C, respectively. Selected reaction monitoring (SRM) acquisition mode was implemented monitoring three transitions per compound (see Table 1for UV filters, and Table S1 for the other compounds). The system was operated by Xcalibur 2.2 and Trace FinderTM 3.2 software. Separations 2019,6, 30 5 of 14 2.5. Statistical Analysis Basic and descriptive statistical analysis were performed using Statgraphics Centurion XVII (Manugistics, Rockville, MD, USA) as software package. 3. Results and Discussion 3.1. Chromatographic Analysis The chromatographic GC–MS/MS method for the determination of the target UV filters was previously proposed by the authors [ 16 – 18 ], and it was extended to other compounds including 25 fragrance allergens, 13 preservatives, 15 plasticizers, and 11 synthetic musks, making a total of 78 compounds . The chromatographic conditions have been previously described in Section 2.4. SRM acquisition mode was employed monitoring two or three transitions per compound (see Table 1 and Table S1). 3.2. Optimization of the µ-MSPD Procedure The influence of the main parameters potentially affecting the µ -MSPD procedure must be evaluated to obtain an efficient extraction. Several factors, such as the amount of sample, desiccant and dispersing agents were maintained constant, based on previous studies [ 3 , 12 – 14 ]. The amount of sample was 0.1 g, which was mixed with 0.4 g of Na 2 SO 4 to remove the moisture of the samples, which could negatively affect the extraction. Regarding the dispersing agent, its amount was fixed at 0.4 g. The studied parameters were the extraction solvent (factor A), the dispersing agent (factor B), and the extraction volume (factor C), and the different levels are summarized in Table 2. The choice of an appropriate solvent is essential in the development of extraction methods. For an efficient extraction, the solvent must solubilize the target compounds while leaving the sample matrix as intact as possible. Four solvents were investigated: ACN, ethyl acetate (EtAc), methanol (MeOH), and the mixture MeOH/acetone (1:1, v/v). The dispersing agent can be also a very important factor affecting the extraction. In addition, it can contribute to obtain cleaner extracts, preventing lipids and other co-extractable matrix materials from coming out to the extract. Based on our previous works [ 6 , 12 , 14 , 15 ], this factor was considered at two levels: Florisil and sand. The solvent volume was also studied at two levels: 1 mL and 2 mL. Larger solvent volumes were not evaluated since the purpose of this study was the development of a green miniaturized extraction protocol. Lower solvent volumes were also not considered since they are not suitable for practical purposes, making necessary the use of inserts to perform further chromatographic analysis. Table 2. Experimental factors and levels included in the experimental design. Factor Code Level 1 Level 2 Level 3 Level 4 Solvent A ACN EtAc MeOH MeOH/acetone (1:1, v/v) Dispersant B Florisil Sand Volume of solvent (mL) C 1 2 The influence of the three variables was studied using a multifactor strategy. The study consisted of a multifactor 4*2 2 design, involving 16 randomized experiments and allowing three degrees of freedom to estimate the experimental error. The design has resolution V, which means that it is capable of evaluating all main effects and all two-factor interactions. Numerical analysis of data resulting from the experimental design was made employing the software package Statgraphics Centurion XVII (Manugistics, CA, USA). The experiments were performed using composite sample prepared as a mixture of four real samples including a sunscreen, a facial cream, a body lotion, and a lip protector. Since the composite sample contained six of the target compounds from the different families of the UV filters studied, it was decided to work with the sample as it, without compounds addition, to really Separations 2019,6, 30 6 of 14 evaluate the capability of the miniaturized procedure to break analyte-matrix interactions, providing efficient extractions. Besides, other compounds such as 11 fragrance allergens, seven preservatives, three plasticizers, and two synthetic musks, were detected in the composite sample. The analysis of variance, ANOVA, describes the impact of the studied factors on the obtained responses. Results for the UV filters are shown in the ANOVA table, Table 3. For the sake of simplicity, only F-ratios and p-values are given. The F-ratio measures the contribution of each factor and interaction on the variance of the response. The p-value tests the statistical significance of each factor and interaction. When p-value is lower than 0.05, the factor has a statistically significant effects at the 95% confidence level. Table 3. ANOVA summary table obtained for the micro-matrix solid-phase dispersion (µ-MSPD) procedure. Compound Solvent (A) Dispersant (B) Volume (C) AB AC BC F p F p FpFpFPFp EHS 63 0.0032 75 0.0032 47 0.0063 150 0.0009 1.3 0.4114 0.82 0.4313 BP3 157 0.0008 8.1 0.0647 49 0.0059 422 0.0002 4.5 0.1238 0.64 0.4817 IAMC 18 0.0200 663 0.0001 43 0.0072 65 0.0031 6.4 0.0802 0.01 0.9361 4MBC 13 0.0288 545 0.0002 48 0.0060 45 0.0054 6.3 0.0815 0.75 0.4490 2EHMC 2.6 0.2264 163 0.0010 9.7 0.0525 17 0.0202 2.2 0.2667 0.03 0.8792 OCR 4.0 0.1425 172 0.0010 13 0.0360 11 0.0374 2.3 0.2560 0.48 0.5392 p-values lower than 0.05 (in bold) denotes statistical significance. As can be seen, the three studied factors were significant for all the UV filters present in the sample in most cases. The interaction solvent-dispersant (AB) was significant for all the compounds. The other two second order factors (solvent-volume, AC and dispersant-volume, BC) were not significant. Figure 2shows some selected mean plot graphs, that illustrate the effect of the main factors by showing the mean values as well as the confidence intervals for each level, easily visualizing the most favorable extraction conditions. For all the UV filters, the most efficient solvent was ACN providing higher responses (see Figure 2a). Regarding the dispersing agent, Florisil gave also higher responses for all the analytes (see Figure 2b). As regards the interaction AB, some examples are included in Figure 2c. The two-factor plots display the least squared means at all combinations of two factors, which allows studying the effect of both factors simultaneously. In this case, two different behaviors can be observed. For 2-ethylhexyl 4-methoxycinnamate (2EHMC), 4-methylbenzylidene camphor (4MBC), octocrylene (OCR) and isoamyl-4-methoxycinnamate (IAMC), the use of Florisil provided the highest response regardless of the solvent used (see as example OCR graph in Figure 2c). In the case of ethylhexylsalicylate (EHS) and benzophenone-3 (BP3), the use of sand was more favorable when MeOH or the mixture MeOH/acetone (1:1, v/v) was employed but, in any case, higher responses were obtained using ACN or EtAc with Florisil (see as example BP3 graph in Figure 2c). Regarding solvent volume, 2 mL was initially more favorable, although the differences in the responses were not very high (see Figure 2d). Since Florisil was the most favorable dispersing agent for all analytes, the results were analyzed considering only the experiments carried out with this sorbent. The ANOVA results were similar for all the analytes and are graphically displayed for IAMC and EHS as example in Figure 3a. The plot shows scaled effects for each factor, so the natural variance of the points in the diagram can be comped to that of the residuals, displayed at the bottom of the plot. By comparing the variability amongst the factors to that of the residuals, it is easy to identify those factors showing differences of a greater magnitude than could be solely accounted by the experimental error. As can be observed, the solvent nature was significant, but the amount of solvent was not a significant factor. The levels of the factors at the right part of the ANOVA plot indicate the conditions that offer higher response and therefore, more efficient extraction. In the mean plot in Figure 3b the influence of the solvent is clearly appreciate. ACN and EtAc provided similar results, whereas for the other solvents the responses were clearly lower. Separations 2019,6, 30 7 of 14 Separations 2018, 5, x FOR PEER REVIEW 7 of 15 Figure 2. Mean plots (a,b,d) and interaction plots (c) of the main factors studied in the multi-factor categorical design for some representative ultraviolet (UV) filters. Since Florisil was the most favorable dispersing agent for all analytes, the results were analyzed considering only the experiments carried out with this sorbent. The ANOVA results were similar for all the analytes and are graphically displayed for IAMC and EHS as example in Figure 3a. The plot shows scaled effects for each factor, so the natural variance of the points in the diagram can be comped to that of the residuals, displayed at the bottom of the plot. By comparing the variability amongst the factors to that of the residuals, it is easy to identify those factors showing differences of a greater magnitude than could be solely accounted by the experimental error. As can be observed, the solvent nature was significant, but the amount of solvent was not a significant factor. The levels of the factors at the right part of the ANOVA plot indicate the conditions that offer higher response and therefore, more efficient extraction. In the mean plot in Figure 3b the influence of the solvent is clearly appreciate. ACN and EtAc provided similar results, whereas for the other solvents the responses were clearly lower. Figure 2. Mean plots ( a , b , d ) and interaction plots ( c ) of the main factors studied in the multi-factor categorical design for some representative ultraviolet (UV) filters. Therefore, in view of the results, the selected conditions for the analysis of UV filters comprise the use of Florisil as dispersing agent, and ACN or EtAc as eluting solvent. Under these conditions the amount of solvent was not significant and, therefore, the low solvent volume, 1 mL, was selected. Regarding the other cosmetic ingredients and additives present in the composite sample, including fragrance allergens, preservatives, plasticizers and synthetic musks, the statistical analysis showed as more favourable conditions the once previously selected for the UV filters. Therefore, a general multianalyte method for the determination of all these families of personal care products (PCPs) can be proposed. Separations 2019,6, 30 8 of 14 Separations 2018, 5, x FOR PEER REVIEW 8 of 15 Figure 3. (a,b) ANOVA plots showing the main effects for isoamyl-4-methoxycinnamate (IAMC) and ethylhexylsalicylate (EHS). Therefore, in view of the results, the selected conditions for the analysis of UV filters comprise the use of Florisil as dispersing agent, and ACN or EtAc as eluting solvent. Under these conditions the amount of solvent was not significant and, therefore, the low solvent volume, 1 mL, was selected. Regarding the other cosmetic ingredients and additives present in the composite sample, including fragrance allergens, preservatives, plasticizers and synthetic musks, the statistical analysis showed as more favourable conditions the once previously selected for the UV filters. Therefore, a general multianalyte method for the determination of all these families of personal care products (PCPs) can be proposed. 3.3. Method Performance The µ-MSPD-GC–MS/MS method was validated in terms of linearity, accuracy and precision. Method performance is summarized in Table 4 for UV filters, and Table 5 for the other compounds. The calibration study was performed employing standard solutions prepared in acetonitrile containing the 78 compounds at different levels, covering a concentration range from 0.001 to 10 mg L−1 (see specific ranges for each compound in Tables 4 and 5) with twelve levels and three replicates per level. The method exhibited a direct proportional relationship between the concentration of each analyte and the chromatographic response with determination coefficients R2 ≥ 0.9915 for all compounds. Calibration plots for some representative compounds are shown in Figure S1. Intra-day, and inter-day precision was also evaluated. The relative standard deviation (RSD) values for the inter-day are shown in Tables 4 and 5, and they were lower than 10% for all the analyzed UV filters, and lower than 14% for the other compounds. Recovery studies were carried out by implementing the optimized µ-MSPD-GC–MS/MS method to a real cosmetic sample (a moisturizing hand cream). Sample was fortified at three different concentration levels (1, 10 and 100 µg g−1) for the UV filters and the µ-MSPD-GC–MS/MS procedure was performed. Recoveries were calculated as the ratio of concentration found/added considering the responses obtained for each analyte, and they are shown in Table 4 Quantitative recoveries were Figure 3. ( a , b ) ANOVA plots showing the main effects for isoamyl-4-methoxycinnamate (IAMC) and ethylhexylsalicylate (EHS). 3.3. Method Performance The µ -MSPD-GC–MS/MS method was validated in terms of linearity, accuracy and precision. Method performance is summarized in Table 4for UV filters, and Table 5for the other compounds. Table 4. µ -MSPD-gas-chromatography tandem mass spectrometry (GC–MS/MS) performance for the UV filters. Linearity, precision, and recovery studies. UV Filters Linearity Precision aRecoveries Range (mg L−1)R2RSD, % Mean Values 100 µg g−110 µg g−11µg g−1 EHS 0.001–10 0.9999 10 109 ±11 106 ±2 111 ±4 110 ±5 BS 0.002–10 0.9999 4.8 110 ±3 111 ±2 116 ±6 103 ±1 HMS 0.002–10 0.9999 10 109 ±6 110 ±2 109 ±7 108 ±10 BP3 0.002–10 0.9980 3.9 106 ±6 103 ±3 117 ±10 98.7 ±5.3 IAMC 0.001–10 0.9992 14 98.4 ±5.8 100 ±2 102 ±6 93.3 ±9.5 4MBC 0.002–10 0.9997 8.0 97.9 ±6.7 97.9 ±2.8 99.4 ±7.2 96.6 ±10.0 MA 0.001–10 0.9994 5.8 106 ±5 104 ±2 99.4 ±8.2 114 ±4 ETO 0.001–10 0.9998 5.2 97.9 ±7.3 97.4 ±7.2 93.3 ±9.7 103 ±5 EHPABA 0.002–10 0.9997 8.6 99.0 ±4.3 101 ±2 95.2 ±6.8 101 ±4 2EHMC 0.002–10 0.9992 10 99.5 ±4.1 99.4 ±1.5 99.1 ±8.5 100 ±3 OCR 0.002–10 0.9999 9.8 104 ±4 104 ±4n.c. bn.c. b BMDM 1–1000 0.9966 6.1 111 ±2 111 ±2 n.c. cn.c. c DHHB 1–50 0.9922 10 108 ±3 108 ±3 n.c. cn.c. c DRT 0.1–100 0.9915 5.6 98.7 ±2.3 98.2 ±1.2 97.4 ±3.5 n.c c an=6; bnot calculated since the compound was detected in the sample or cbelow linear range. The calibration study was performed employing standard solutions prepared in acetonitrile containing the 78 compounds at different levels, covering a concentration range from 0.001 to 10 mg L−1 (see specific ranges for each compound in Tables 4and 5) with twelve levels and three replicates per level. The method exhibited a direct proportional relationship between the concentration of each analyte and the chromatographic response with determination coefficients R 2≥ 0.9915 for all compounds. Calibration plots for some representative compounds are shown in Figure S1. Separations 2019,6, 30 9 of 14 Table 5. µ -MSPD-GC–MS/MS performance for the fragrance allergens, preservatives, plasticizers and synthetic musks. Linearity, precision, and recovery studies. Compounds Linearity Precision aRecoveries Range (mg L−1)R2RSD, % Mean Values 100 µg g−110 µg g−1 Fragrance allergens Pinene 0.001–10 0.9994 3.5 70.2 ±6.0 77.8 ±7.8 62.6 ±4.2 Limonene 0.001–10 0.9985 7.2 85.1 ±3.7 97.1 ±3.6 73.1 ±3.8 Benzyl alcohol 0.001–10 0.9982 9.7 109 ±6 107 ±2 111 ±9 Linalool 0.005–10 0.9994 6.7 98.6 ±6.7 104 ±2 93. 2 ±11.4 Methyl-2-octynoate 0.1–10 0.9999 6.0 106 ±5 105 ±2 107 ±8 Citronellol 0.05–10 0.9999 8.8 107 ±6 107 ±2 107 ±10 Citral 0.002–10 0.9994 7.1 99.5 ±4 112 ±1 86.9 ±7.0 Geraniol 0.02–10 0.9998 6.9 106 ±3 96.4 ±0.4 116 ±6 Cinnamaldehyde 0.005–10 0.9999 6.8 101 ±7 106 ±2 95.7 ±11.3 Hydroxycitronellal 0.005–10 0.9995 6.0 108 ±2 100 ±2 116 ±3 Anise alcohol 0.01–10 0.9998 8.3 102 ±5 101 ±2 103 ±9 Cinnamyl alcohol 0.001–10 0.9996 8.7 105 ±6 105 ±4 105 ±7 Eugenol 0.005–10 0.9965 6.4 108 ±4 105 ±3 111 ±5 Methyleugenol 0.005–10 0.9981 6.8 95.6 ±3.7 102 ±2 89.2 ±5.4 Isoeugenol 0.02–10 0.9992 8.4 100 ±4 103 ±3 97.0 ±5.3 Coumarin 0.02–10 0.9980 6.6 102 ±8 104 ±3 100 ±13 α-isomethylionone 0.005–10 0.9975 8.6 99.5 ±5.6 101 ±2 98.1 ±9.3 Lilial®0.005–10 0.9995 6.6 100 ±7 106 ±2 94.2 ±12.1 Amylcinnamaldehyde 0.005–10 0.9991 8.1 106 ±4 106 ±1 106 ±7 Lyral®0.002–2 0.9971 7.2 107 ±4 108 ±1 105 ±7 Amylcinnamyl alcohol 0.005–10 0.9992 8.1 110 ±7 107 ±1 112 ±12 Farnesol 0.02–10 0.9994 12 107 ±7 106 ±4 107 ±10 Hexylcinnamaldehyde 0.01–10 0.9922 8.1 107 ±5 107 ±2 106 ±7 Benzyl benzoate 0.002–10 0.9992 6.7 102 ±6 104 ±2 99.3 ±10.2 Benzyl cinnamate 0.001–10 0.9999 5.4 103 ±4 103 ±3 102 ±5 Preservatives Bronidox 0.002–10 0.9999 4.8 103 ±6 110 ±1 95.6 ±11.1 Phenoxyethanol (PhEtOH) 0.001–10 0.9999 7.4 110 ±7 101 ±1 120 ±14 Methyl paraben (MeP) 0.001–10 0.9997 10 110 ±6 102 ±2 117 ±11 Butylhydroxyanisole (BHA) 0.0001–10 0.9990 5.5 95.6 ±4.5 103 ±3 88.1 ±5.9 Butylhydroxytoluene (BHT) 0.005–10 0.9996 4.9 95.1 ±3.3 103 ±2 87.2 ±4.7 Ethyl paraben (EtP) 0.02–10 0.9999 11 102 ±8 100 ±2 103 ±14 Isopropyl paraben (iPrP) 0.05–10 0.9995 10 103 ±4 105 ±3 99.9 ±4.6 Propyl paraben (PrP) 0.01–10 0.9998 11 98.5 ±4.2 102 ±2 94.9 ±6.4 Iodopropynylbutyl carbamate (IPBC) 0.002–10 0.9997 3.5 104 ±9 103 ±2 105 ±15 Isobutyl paraben (iBuP) 0.005–10 0.9999 8.1 103 ±2 102 ±2 104 ±2 Butyl paraben (BuP) 0.005–10 0.9999 7.6 99.6 ±1.9 101 ±1 98.3 ±2.8 Triclosan (TCS) 0.002–10 0.9983 2.3 115 ±9 113 ±3 117 ±14 Benzyl paraben (BzP) 0.05–10 0.9995 4.2 101 ±6 99.2 ±3.3 103 ±8 Plasticizers Dimethyl adipate (DMA) 0.01–10 0.9998 5.3 116 ±5 104 ±1 118 ±8 Diethyl adipate (DEA) 0.001–10 0.9989 6.5 98.5 ±7.7 103 ±2 93.9 ±13.4 Diethyl phthalate (DEP) 0.005–10 0.9984 6.2 99.9 ±4.0 102 ±1 97.8 ±7.1 Diisobutyl phthalate (DIBP) 0.001–10 0.9992 4.7 98.9 ±4.9 101 ±2 96.8 ±7.8 Dibutyl phtahalte (DBP) 0.001–10 0.9997 9.1 100 ±5 102 ±2 98.4 ±8.7 Dimethoxyethyl phthalate (DMEP) 0.005–10 0.9999 6.3 105 ±6 105 ±2 105 ±9 Diisopentyl phthalate (DIPP) 0.002–10 0.9998 8.2 99.3 ±5.5 100 ±3 98.7 ±7.9 Dipentyl phthalate (DPP) 0.001–10 0.9997 11 101 ±3 101 ±2 101 ±3 Benzylbutyl phthalate (BBP) 0.002–10 0.9997 13 99.7 ±5.5 100 ±3 99.5 ±8.0 Diethylhexyl adipate (DEHA) 0.005–10 0.9997 14 95.4 ±5.4 96.2 ±2.8 94.6 ±8.0 Diisoheptyl phthalate (DIHP) 0.002–10 0.9998 3.9 97.6 ±5.3 100 ±3 95.3 ±7.6 Dicyclohexyl phthalate (DCHP) 0.005-10 0.9996 9.5 101 ±4 101 ±3 101 ±4 Diethylhexyl phthalate (DEHP) 0.01–10 0.9997 9.2 100 ±3 100 ±4 100 ±1 Diphenyl phthalate (DPhP) 0.001–10 0.9999 11 101 ±5 98.7 ±4.5 103 ±5 Di-n-octyl phthalate (DnOP) 0.005–10 0.9998 8.0 105 ±3 107 ±5 102 ±1 Synthetic musks Cashmeran 0.001–10 0.9976 7.1 100 ±4 103 ±2 97.0 ±5.0 Celestolide 0.002–10 0.9979 5.4 100 ±8 106 ±2 94.2 ±13.8 Phantolide 0.005–10 0.9977 5.9 99.8 ±7.3 104 ±2 95.6 ±12.6 Ambrette 0.005–10 0.9998 10 97.7 ±7 104 ±1 91.4 ±13.0 Trasolide 0.1–10 0.9996 9.6 102 ±9 98.3 ±9.9 105 ±9 Galaxolide 0.001–10 0.9995 6.7 97.3 ±5.1 101 ±3 97.3 ±8.2 Tonalide 0.005–10 0.9988 7.2 96.1 ±7.2 101 ±1 91.3 ±13.5 Musk Moskene 0.002–10 0.9999 9.4 108 ±5 112 ±2 103 ±8 Musk Tibetene 0.005–10 0.9995 7.1 99.1 ±4.9 104 ±1 94.3 ±8.8 Ambrettolide 0.002–10 0.9988 7.0 105 ±3 104 ±3 106 ±2 Musk Ketone 0.001–10 0.9998 10 98.4 ±7.1 90.8 ±3.4 98.0 ±10.7 an=6.