A gas chromatography–mass spectrometric method to determine skin-whitening agents in cosmetic products
Introduction
Currently, brown skin-spots or staining represent one of the most important aesthetic problems in humans [1]. This skin disorder, a consequence of melanin excess produced by hyperactivity of melanocytes, could have different causes, such as overexposure to solar radiation, ageing, hormonal dysfunctions during pregnancy or taking certain medicines like contraceptives, among others [2], [3], [4].
Although these spots are not normally harmful to health, many people are affected by them and seek specialist advice to try to remove these anti-aesthetic marks from their skin. Thus, apart from the most serious cases, which could require medical treatment, in most cases a cosmetic treatment can be employed, based on skin-whitening (also referred to as skin-bleaching) products. These cosmetic products contain so-called skin-whitening agents, which act by inhibiting melanin biosynthesis via different mechanisms [5], [6], [7].
Different compounds have been used as skin-whitening agents in cosmetics [8]. Among them, hydroquinone (HQ) (see Fig. 1) was one the most commonly used due to its well-known effectiveness [9], [10]. Its use as a whitening agent in cosmetics (up to 2%) was first allowed in the European Union in 1984 by the Commission Directive 84/415/EEC [11]. However, its use was restricted to hair-dye products and artificial nail systems sixteen years later by Commission Directive 2000/6/EC [12], since different safety problems were attributed to its use in skin-whitening cosmetics, such as ochronosis, irritation, allergy [10], [13], [14], and particularly due to its carcinogenic properties over long-term use [15]. Furthermore, there is evidence of its percutaneous absorption, and it has been found to be present in the body fluids of users of skin-whitening cosmetics containing HQ [16], [17], which makes its toxic potential more serious. Nevertheless, it can be used in specialised pharmaceutical products, which are not considered cosmetics, thus being regulated by the legislation concerning pharmaceutical products. Likewise, in the United States framework, HQ was allowed at concentrations up to 1.5–2% in skin-whitening products [18] (higher concentrations are only permitted in prescription drugs), which are considered over-the-counter drugs, but it has recently been banned in this type of product by the United States Food and Drug Administration [19].
Whitening properties have also been attributed to resorcinol (RS), an isomer of HQ (see Fig. 1) [20], [21], but its use as skin-whitening agent in cosmetics is not permitted in either the European Union or United States.
Arbutin (ARB), which is a glucosylic ether of HQ (see Fig. 1), has been proposed as a good alternative to HQ. Although it is less effective, it does not present the side-effects attributed to the use of HQ. Other skin-whitening agents used extensively are kojic acid (KA) and azelaic acid (AZA) (see Fig. 1), although they are also much less effective than HQ. Ascorbic acid and some of its derivatives have also been used at length as whitening agents, although their effectiveness is also lower than HQ.
Taking all this into account, skin-whitening products should be controlled for various reasons: on one hand, by the manufacturers themselves, in order to assure that they contain the correct amount of whitening agent, i.e. to assure the efficacy of these products; and, on the other hand, by safety authorities, to prevent fraudulent use of both HQ and RS in this type of product, i.e. to assure user safety.
However, no official analytical methods have been found in the literature, except those proposed by the EU Commission [22] and the United States Pharmacopeia [23]. In the former, HQ (and some of its ethers, but not including ARB) can be identified by means of thin-layer chromatography (TLC), followed by their quantitative determination using liquid chromatography (LC) with ultraviolet/visible detection. In the latter, HQ can be quantified by means of titration with cerium sulphate using diphenylamine as indicator.
Therefore, the development and validation of analytical methods to control skin-whitening cosmetic products is of great interest, as they will assure the efficacy and safety of this type of cosmetic product. A bibliographic search revealed more than 30 publications focusing on the determination of skin-whitening agents in cosmetics, which were recently critically reviewed [8]. Analytical techniques where determination is carried out in the liquid phase are preferred over others dealing with the vapour phase, owing to the low volatility of these compounds, with LC being the technique of choice. There is only one published article where skin-whitening cosmetics are analyzed by using gas chromatography (GC) [24], in which the determination of HQ is carried out.
The aim of this paper focuses on developing a reliable analytical method to determine three of the most commonly used skin-whitening agents (ARB, KA and AZA), which are often combined in modern skin-whitening cosmetics, as well as two prohibited skin-whitening agents (HQ, RS) using the potential GC offers. The method couples GC with a mass spectrometric (MS) detector, enabling forbidden skin-whitening agents to be detected at low concentrations, as well as reliably identifying them by means of their mass spectra.
Owing to the low volatility of the target compounds for GC analysis, a derivatization step was carried out by using a silylation agent to convert them to more volatile derivatives (see Fig. 1).
As was recently reviewed [8] and bibliographically search updated, there are no published papers reporting RS determination in skin-whitening cosmetics, and neither has a wide-ranging analytical method been published to determine HQ, ARB, KA and AZA together.
Section snippets
Apparatus
A Focus GC gas chromatograph, equipped with an AI 3000 autosampler and coupled to a DSQ II mass spectrometric detector, from Thermo Fisher Scientific (Austin, TX, USA) was employed.
An ultrasonic water bath from Selecta (Valencia, Spain) was used to improve sample solving process in the cases of difficulty in solubilization.
Reagents and samples
Resorcinol (RS) >99% from Fluka (Buchs, Switzerland), arbutin (ARB) >99% from Bioland Ltd. (Chungnam, Korea), and kojic acid (KA) 99.2%, hydroquinone (HQ) >99%, and azelaic
Studies on the derivatization reaction
As mentioned before, owing to the low volatility of the target analytes for GC analysis, a derivatization step was necessary to convert them to more volatile derivatives. Different derivatization strategies, such as silylation, alkylation, esterification or acylation [25], [26], [27], have been extensively used for GC analysis, and a huge number of applications in analytical chemistry can be found elsewhere. Moreover, derivatization has the added advantage in that the derivatives have the
Conclusions
A GC–MS method is proposed to determine three allowed and two forbidden skin-whitening agents in cosmetics, with good limits of detection. The mass spectra acquired in full-scan mode, jointly with the retention time, offer unequivocal proof of the identity of the target forbidden compounds.
The proposed method is accurate and precise, as can be concluded by the results obtained in the analysis of a laboratory-made skin-whitening sample.
The method can be easily applied both by the manufacturers
Acknowledgements
The authors acknowledge the financial support of the Spanish Government (Projects CTQ2006-00296 and CTQ2009-12709), especially A. Balaguer for his predoctoral grant. The collaboration of Ana Torrens (from Corporación Dermoestética S.L., Paterna, Valencia, Spain) is deeply acknowledged. The authors are also grateful to Prof. Isabel Fernández, from Department of Organic Chemistry at University of Valencia, for her assistance in the interpretation of mass spectra.
The authors would like to make
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