ANALYST, MAY 1992, VOL. 117 839 Determination of Parabens in Cosmetic Products by Supercritical Fluid Extraction and High-performance Liquid Chromatography Santo Scalia" and David E. Games Mass Spectrometry Research Unit, Department of Chemistry, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK A rapid and simple supercritical fluid extraction (SFE) procedure has been developed for the isolation of paraben preservatives from cosmetic matrices. Method optimization indicates that recovery is affected most by extraction temperature and time. The parabens were assayed by high-performance liquid chromatography after extraction of the cosmetic preparations with supercritical carbon dioxide at 60°C and at a density of 0.85 g ml-I. Quantitative recoveries of parabens were obtained with two sequential 7 min extraction steps.Supercritical fluid extraction of cosmetic samples gave better recovery for para bens than conventional liquid extraction techniques within a shorter period of time. Moreover, the automated SFE system used minimized sample manipulation and allowed stand-alone operations. The SFE method is simple t o perform, accurate, reproducible and suitable for routine analyses of commercial cosmetic products. Keywords: Supercritical fluid extraction; high-performance liquid chromatography; paraben preservatives; cosmetic product Preservatives are commonly contained in cosmetic prepara- tions for the primary purpose of inhibiting the development of micro-organisms. However, all the preservatives can be harmful to the consumer by their potency to induce allergic contact dermatitis.1 The European Economic Community (EEC) Directive on cosmetics2 includes a list of preservatives authorized as cosmetic additives and their allowed maximum concentrations. Hence, the assay of these substances in cosmetic products is important for checking compliance with the EEC legislation. The p-hydroxybenzoic acid esters or parabens are the most widely used antimicrobial agents in cosmetics,3 the most important ones being the methyl, ethyl, propyl and butyl esters.' Combinations of two or more parabens are often used to increase the ability of the system to withstand microbial contaminations.4 Published methods for the determination of these preservatives in cosmetic preparations are based on gas chromatography (GC)s and high-performance liquid chroma- tography (HPLC) .fj-g The latter technique offers distinct advantages over GC such as simpler purification procedures and the lack of derivatization steps.However, solid and semi-solid samples which are often encountered in the analysis of cosmetics must first be put into a liquid form before HPLC analyses. This requires several sample manipulations7.8 (e. g., solvent extraction, mixing, sonication, heating, addition of acids and centrifugation), which represent a source of possible errors. Moreover, the organic solvents used must be pure and eventually be disposed of. Supercritical fluid extraction (SFE) is emerging as a valuable techniqueg-10 for the isolation of solutes from solid samples, using supercritical fluids as the extraction media.While supercritical fluids exhibit solvation powers approach- ing those of liquids, they have both lower viscosities and higher diffusivities," which lead to more rapid and efficient extractions of analytes. Moreover, the solvent strength of a supercritical fluid increases with increasing density, allowing modifications of the extraction selectivity simply by changing the pressure or the temperature. Finally, carbon dioxide, the supercritical fluid most frequently used in SFE, is non- flammable, non-toxic and available in a pure form at a * On leave from the Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, via Scandiana 21, 44100 Ferrara, Italy. reasonable cost. Hence, it represents an excellent alternative to the potentially hazardous solvents currently used in sample preparation.This paper describes the development of an SFE procedure, performed with a commercially available system, for the rapid and efficient purification of the complex cosmetic matrices before assay of paraben preservatives by HPLC. The applica- tion of the method to the determination of parabens in commercial cosmetic products is also reported. Experimental Materials Instrument-grade liquid carbon dioxide supplied in cylinders with a dip tube was obtained from BOC (London, UK). Methanol, acetonitrile and water of HPLC-grade were sup- plied by Fisons (Ipswich, UK). Methyl, ethyl, propyl and butyl parabens were purchased from Sigma (St. Louis, MO, USA). Their purity was checked by HPLC prior to use.All other chemicals were of analytical-reagent grade (Sigma). Commer- cial cosmetics were from retail stores. Chromatography The HPLC apparatus consisted of a Hewlett-Packard 1084B high-performance liquid chromatograph (Hewlett-Packard, Avondale, PA, USA) linked to an injection valve with a 10 PI sample loop (Rheodyne, Cotati, CA, USA). The column effluent was monitored by the built-in multiple wavelength ultraviolet/visible detector set at a wavelength of 254 nm and 0.38 a.u.f.s. Separations were performed on a Spherisorb ODS column (particle diameter 5 pm, 100 x 4.6 mm i.d.; Jones Chromatography, Hengoed, Mid-Glamorgan, UK) under gradient conditions at a flow rate of 1.0 ml min-1. Solvent A and solvent B were 20 and 80% v/v acetonitrile in water, respectively.The elution programme was as follows: isocratic elution with 40% solvent B-60% solvent A for 6.5 min, then a 1 min linear gradient to 100% solvent B. The mobile phase was filtered through HVLP-type 0.45 pm filters. Chromatography was carried out at ambient temperature. The identity of the separated compounds was assigned by co-chromatography with the authentic substances. Peak areas were used for calculations.840 ANALYST, MAY 1992, VOL. 117 Table 1 SFE parameters Table 2 Comparison of total concentrations of parabens in cosmetic products purified by SFE or liquid extraction7 Extraction fluid density Extraction fluid flow rate Extraction temperature Equilibration time Extraction time Restrictor temperature Trap temperature Rinse solvent Rinse volume Rinse rate 0.85gml-l 2 ml min-1 60 "C 2 min 7 min 60 "C extractionM0 "C rinse 30 "C extractionh0 "C rinse Methanol 1.2 ml 1.0 ml min-1 I 1 I 5 10 14 20 Fig.1 Influence of the extraction time on the average recovery of a mixture of methyl, ethyl, propyl and butyl parabens from a hand cream. Other SFE conditions as in Table 1. Values plotted are means of triplicate experiments Ti me/m i n Sample Extraction Supercritical fluid extractions were performed with a com- puter-controlled HP 7680A SFE system (Hewlett-Packard). The cosmetic product (0.2-0.3 g) was accurately weighed on a piece of filter-paper which was rolled and inserted into the extraction cell. After initiation of the extraction programme, the supercritical carbon dioxide flows through the extraction cell and then through the restrictor into the analyte trap. The sudden pressure drop at the restrictor causes the supercritical fluid to evaporate depositing the analytes on an internal trap packed with small (diameter 0.36-0.43 mm) stainless-steel balls.Finally, the trap is rinsed with methanol and the rinse solvent collected in sample vials. The contents of the vials were made up to volume (3 ml), filtered if necessary and analysed directly by HPLC. Extraction density and time, cell temperature, supercritical carbon dioxide flow rate, trap temperature and amount of rinse solvent were controlled by the software program in the personal computer. The specific extraction conditions used are reported in Table 1. Recovery and Reproducibility 'Spiked' solutions were obtained by dissolving weighed amounts of parabens in methanol. The test samples were prepared by adding 50 p.1 aliquots of the spiked solutions, corresponding to 0.04% m/m of each single paraben, to the cosmetic products (0.2 g) and mixing them thoroughly.The percentage recovery was determined by comparing the peak areas of the parabens extracted from the samples with those obtained by direct injections of the standard solutions. The intra-assay reproducibility was tested by analysing, on ten different days, 10 p1 of the same stock sample solution from a suncream. The inter-assay variability was evaluated by replicate ( n = 10) extractions of the same suncream product. Results and Discussion A hand cream product, containing no detectable parabens, was spiked with methyl, ethyl, propyl and butyl parabens at 0.04% m/m and extracted for 10 min with supercritical carbon dioxide at 40°C and at a density of 0.95 g ml-1.In order to prevent the matrix from being swept out of the extraction cell, the sample was smeared on filter-paper. In spite of the high Concentration* (% d m ) Sample SFE Liquid extraction Suncream 0.164 0.150 Moisturising lotion 0.179 0.169 Cleansing milk 0.207 0.197 Skin cream 0.237 0.219 Day cream 0.058 0.052 * Mean value of three determinations. t - Q C D 5 1 4 2 3 1 3 0 2 4 6 8 1 0 1 2 0 2 4 6 8 1 0 1 2 Ti me/m i n Fig. 2 HPLC trace of a suncream product purified by (a) SFE or (6) by the method reported in the literature.' 1, Methyl paraben; 2, ethyl paraben; 3, propyl paraben; and 4, butyl paraben. Operating conditions as described under Experimental value of the fluid density used (at the limit of the operating range of the instrument), low recoveries (19.8-22.5%) were observed for all the compounds investigated.Increasing the extraction temperature from 40 to 60°C produced higher recoveries (65.4-67.0%) even though the carbon dioxide density, and hence its solvating power, had to be reduced (0.85 g ml-1 was the maximum density achieved with the SFE system at 60 "C). A second extraction of the same sample was found to recover all the compounds quantitatively. Although the density is generally the most important parameter that influences the extraction efficiency in SFE, for the parabens the temperature has a dominant role. The improved recoveries obtained at higher temperature are due to increased solute solubility and also to sample matrix modifica- tions (such as swelling) and enhanced diffusivity.The influ- ence of the extraction time on the recovery of the parabens from the cosmetic matrix was also investigated. As illustrated in Fig. 1, the extraction is complete after 14 min. The optimized SFE procedure consists of two 7 min extraction steps performed under identical conditions (Table 1) and preceded by a static equilibration of 2 min. Three different cosmetic preparations, containing no detectable parabens, were spiked with each preservative at a concentration of 0.04% m/m and subjected to the SFE method outlined above. The average recoveries f the standard deviations for the four parabens from a hand cream, aANALYST, MAY 1992, VOL.117 84 1 1 I I I I I k 0 2 4 6 8 1 0 0 2 4 6 8 10 Time/min Fig. 3 HPLC trace of a day cream preparation purified by ( a ) SFE or ( b ) by the method reported in the literature.’ Sensitivity, 0.19 a.u.f.s. Other operating conditions as described under Experimental and peak identification as in Fig. 2 sunscreen lotion and a shampoo were 100.3 k 1.7% (n = 6), 96.7 f 1.4% (n = 6) and 99.5 -t 2.3% (n = 6), respectively. Calibration graphs were linear in the range 0.004-0.4% m/m with correlation coefficients greater than 0.998. In none of the graphs was the intercept with the y-axis significantly different from zero at the 95% confidence interval. The minimum quantifiable amounts were at least ten times below the levels normally used in the formulation of cosmetics.6.8 Applying the SFE procedure to a commercial suncream product, the total concentration of parabens (0.164% m/m) was determined with a relative standard deviation of 1.3% ( n = 10) for the intra-assay reproducibility and 1.6% ( n = 10) for the inter-assay reproducibility.The good precision achieved can be traced to the automated extraction process, which minimizes the sample handling steps. As spiked samples do not really simulate real samples, the SFE method developed in this study was further validated by comparison with the previously adopted liquid extraction procedure7 on the same cosmetic preparations known to contain parabens. Five different commercial products were assayed. The levels measured (Table 2) conform to the EEC legislation’ (limiting value, 0.8% m/m for mixtures of parabens) and indicate that the recoveries of these preserva- tives with SFE are higher than those obtained with the liquid extraction technique currently used.7.8 Moreover, the purifi- cation based on SFE is more rapid (taking less than 40 min to perform) and less labour-intensive than others reported in the literature.7.8 Faster sample preparation has been attained by other workers;h however the recoveries of the parabens were not evaluated. Further, rapid column deterioration is a disadvantage of this method as the cosmetic preparation after solubilization is injected directly onto the HPLC column without any sample clean-up.Representative chromatograms of a suncream preparation and of a day cream product, extracted by the SFE procedure described here (a) or by the method reported in the literature7 (b), are shown in Figs.2 and 3, respectively. In addition to improved recoveries (Table 2), the SFE method [Figs. 2(a) and 3(a)] affords a more effective purification of the cosmetic matrices compared with the classical liquid extraction technique [Figs. 2(b) and 3 ( b ) J , as is evident by the absence in the HPLC traces of large peaks close to the void volume. Conclusions An SFE procedure for the rapid isolation of the paraben preservatives from cosmetics has been developed. The pro- posed method is less laborious than others reported in the literature, as sample pre-treatment simply involves weighing the cosmetic product and inserting it into the extraction cell. Moreover, because SFE allows the automation of various processes, method development is faster than with the traditional manual sample preparation.The rapidity, sim- plicity, good accuracy and reproducibility of the SFE proce- dure make it suitable for routine quality control analyses of parabens in cosmetics, particularly to verify their con- formance to the EEC legislation. Work is in progress in this laboratory to investigate the effectiveness of SFE as a generally applicable procedure for extracting a variety of additives from cosmetic matrices. The authors thank the SERC for assistance in the purchase of some of the equipment used in these studies. S. S. thanks the CNR for financial support. Provision of the SFE system by Hewlett-Packard is gratefully acknowledged. 1 2 3 4 5 6 7 8 9 10 I1 References Schopf, E., and Baumgartner, A., J. Appl. Cosmetol., 2990.8, 39. European Economic Community Council Directive 76/768/ EEC, Appendix VI, 1976. Wallhausser, K . 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M., Royal Society of Chemistry, London, 1988. p. 159. 1990, pp. 313-352. Paper I /05583 F Received November 4, 1991 Accepted December 6, 1991