Ionic Liquid-Based Ultrasound-Assisted Emulsification Microextraction Coupled with HPLC for Simultaneous Determination of Glucocorticoids and Sex Hormones in Cosmetics

An effective and environmentally friendly method based on ionic liquid-based ultrasound-assisted emulsification microextraction (IL-USAEME) and high performance liquid chromatography (HPLC) has been developed for the determination of nine hormones including three glucocorticoids, one androgen and five progestogens in cosmetics. Several factors that affect the extraction efficiency, such as the kinds and volume of ionic liquid, salt concentration, ultrasonic power and time, and centrifugation time were investigated and optimized. Under the optimum extraction condition, the recoveries of analytes ranged from 85.97% to 108.84% except prednisolone (62.30%). The intraday and interday precision was below 2.51% and 3.30%, respectively.


Introduction
Cosmetics have become more and more important in our daily life for skin whitening, anti-wrinkle, removing freckle, moisture, and so on [1].In order to achieve these effects remarkably, illicit chemicals such as hormones are added to the cosmetics.And the potential accumulation hazard of these additives is terrible [2].Although the Hygienic Standard for Cosmetic of China and European Union Directive 76/768/EEC has banned the addition of hormones to commercial cosmetics, cosmetics containing hormones are still in the market.
At present, the high performance liquid chromatography and its combination with mass spectrometry are the most common methods for determination of trace hormones in the complex matrices.For these analytic techniques, in order to decrease the interference of matrix and concentrate the targeted compounds, a sample pretreatment is indispensable, which is often laborious and consumes a large amount of organic solvents [3][4][5].
Ionic liquids (ILs) composed of organic cations and various types of anions have been successfully applied to various areas of analytical chemistry as environmental benign solvents [6][7][8][9][10].Ionic liquid-based ultrasound-assisted emulsification microextraction (IL-USAEME) is an efficient, simple, rapid and cheap extraction technique, in which applying ultrasound radiation to facilitate the emulsification of a microvolume of ionic liquid extractant in an aqueous sample, and subsequent separation of the two immiscible liquid phases by centrifugation [11,12].This method has been used for the extraction of organic compounds in environmental samples.However, to the best of our knowledge, IL-USAEME has not been applied for the extraction of hormones from cosmetics.
In this paper, an ionic liquid-based ultrasound-assisted emulsification microextraction (IL-USAEME) combined with HPLC method for simultaneous determination of glucocorticoids and sex hormones in cosmetics will be investigated.

Reagents and Chemicals
The pharmaceutical standards (purity, 97

Ionic Liquid-Based Ultrasound-Assisted Emulsification Microextraction
Ionic liquid-based ultrasound-assisted emulsification microextraction was performed in KQ-100 DA and KQ-500 DA ultrasonic water baths (Kunshan, Jiangsu, China).A 0.2 g cosmetic sample, 125 µl [C 7 MIM][PF 6 ], 62.5 µl saturated NaCl aqueous solution and 187.5 µl water were introduced to a 1.5 ml eppendorf centrifuge tube.And the mixed solution was ultrasonically extracted at ultrasonic power of 100 W for 15 min.The emulsification solution obtained after ultrasonication was centrifuged at 10000 rpm for 12 min by the Centrifuge 5804 (Eppendorf AG, Hamburg, Germany).After centrifugation, the IL extraction solution (~100 µl) which was deposited at the bottom of the centrifuge tube was collected using a 200 µl pipette and then diluted by methanol to 1ml.Finally, the solution was filtered through 0.22 µm nylon filter membrance.

HPLC Analyses
The HPLC analysis was performed using a Waters 2695 HPLC system (Waters, Milford, MA, USA) equipped with a quaternary solvent delivery system, an auto-sampler with a 100 µl sample loop, a PDA detector, a column oven, and a data station running the Empower data software.A CAPCELL PAK C18 column (150 mm × 4.6 mm, 3 µm, Chuo-ku, Tokyo, Japan) was used for separation and was maintained at 25˚C.The mobile phase was composed of water (eluent A) and acetonitrile (eluent B), and the elution was carried out in gradient mode at a flow rate of 0.

Optimization of Extraction Conditions
To acquire the high recoveries and enrichment efficiency of the studied hormones (shown as Figure 1), the effects of several factors, such as kinds of ionic liquid, volume of [C 7 MIM][PF 6 ], salt concentration, ultrasonic power and time, and centrifugation time, were examined and optimized.

Screening of the IL-Based Extraction Solvent
In the case of USAEME, the suitable extraction solvent should have density greater than that of water and also a low solubility in water [14].And the structures of ILs have a significant effect on its physicochemical proper-   ] increased, the hormones could not be transferred into the IL phase sufficiently for its high viscosity and density [12].Therefore, 125 µl of [C 7 MIM][PF 6 ] was selected for the further study.

Effect of Salt Addition
The influence of salt addition on the performance of IL-USAEME was investigated by adding different amount of NaCl (0 to 9%, w/v) to solution.The results demonstrated that the salt addition had neglectful effect on the extraction efficiency of analytes.On the other hand, the adequate salt addition is helpful to the formation of IL extraction phase.Consequently, the 9% NaCl was used in subsequent experiments.

Effect of Ultrasonic Power and Time
In this work, the emulsification of the ionic liquid into sample solution was facilitated by ultrasound radiation.The ultrasonic power can affect the mass transfer rate and the contact area between the sample solution and the ionic liquid extractant.When the ultrasonic power was tested at two different levels: 100 W, 500 W, it was obvious that the suitable ultrasonic power should be 100 W.
Additionally, the effect of ultrasonic time on extraction efficiency was evaluated in the range of 5 min -25 min.The results shown in Figure 4 indicated that the extraction efficiency achieved the maximum at the ultrasonic time of 15 min.The main reason may be that adequate ultrasonic time is needed to ensure the equilibrium of extraction.However, prolonging the ultrasonic time induced the descent of extraction efficiency because of the dissolution of IL in sample solution.Consequently, the ultrasonic time of 15 min was enough.

Effect of Centrifugation Time
The centrifugation plays an important role on the sedimentation of IL phase.The effect of centrifugation time ranging from 9 to 21 min was tested.As observed in Figure 5, the extraction efficiency of the most analytes increased with the increase of the centrifugation time from 9 to 12 min.When the centrifugation time was longer than 12 min, the extraction efficiency of dexamethasone, hydroxyprogesterone acetate and beclometasone dipropionate decreased and no significant change was observed for others.Therefore, 12 min was chosen as the centrifugation time.

Evaluation of the method
The chromatogram of a spiked sample obtained by IL-USAEME under the optimum conditions was shown in Figure 6.Under the optimum conditions, linear range, limits of detection (LODs), recovery and precision were determined and shown in Tables 1 and 2. As can be seen, good linearities were observed for all analytes with the coefficient of correlation (r 2 ) ranging from 0.9980 to 0.9994.The LODs were calculated at a signal-to-noise (S/N) ratio of 3. The mean recoveries were 62.30% -108.84% and the RSDs were 0.26% -4.69%.The intraday and interday precisions varied from 0.10% to 2.51 % and from 0.33% to 3.30 %, respectively.The results demonstrated that the method is potential for the detection of hormones fortified in cosmetics.

Conclusion
In this study, a simple, cheap and green IL-USAEME combined with HPLC method was developed and successfully applied to simultaneously determine three glucocorticoids, one androgen and five progestogens in cosmetics, in which using ultrasound radiation to facilitate    the emulsification of a microvolume of ionic liquid instead of organic solvent into the sample solution.The experimental conditions for the extraction of these analytes have been investigated.Under the optimized conditions, satisfactory extraction efficiency of the analytes was obtained.

Figure 1 .
Figure 1.The chemical structures of the studied hormones.

Table 2 . Intraday and interday precision.
The intraday precision was gained by analyzing a sample three times in one day.bTheinterday precision was received by analyzing a sample once a day over three consecutive day. a