CHAPTER 6

Tanning and Whitening Agents

in Cosmetics: Regulatory Aspects
and Analytical Methods
Alberto Chisvert, Juan L. Benedé, Amparo Salvador
University of Valencia,Valencia, Spain

INTRODUCTION
Tanning and whitening cosmetic products are closely related products despite their
opposite effects (i.e., employed for tanning or for bleaching the skin, respectively).
Leaving aside pharmaceutical products to treat severe skin disorders, such as vitiligo
(hypopigmentation) or melasma (hyperpigmentation), tanning and whitening cosmetic
products are used to darken or to lighten the original tone of our skin for aesthetic rea-
sons closely related with cultural and socioeconomical status or simply for fashion
(Herrmann et al., 2015; Naidoo et al., 2016).
Before we describe all of these products, the natural process associated with the syn-
thesis of melanin should be described to better understand how they work. Melanin is
the natural pigment that appears as a protective measure when skin is exposed to the
sun. This process is called melanogenesis and involves enzymatically catalysed and
chemical reactions. Briefly, the biosynthesis of melanin is initiated with the first step of
tyrosine oxidation to dopaquinone, catalysed by tyrosinase. This key enzyme for the
melanogenesis process is located in the membrane of melanosomes, which are vesicles
inside specialized cells called melanocytes located at the basal epidermis layer (Gillbro
and Olsson, 2011; Burger et al., 2016). It should be emphasized that this first step is the
rate-limiting step of the overall process (Chang, 2009). From dopaquinone synthesis, the
melanin biosynthesis diverges into two different pathways depending on whether there
is cysteine or glutathione present (Burger et al., 2016).This results in two types of mela-
nin depending on the synthesis pathway, eumelanin (dark brown-black) and pheomela-
nin (light red-yellow), and the relative amounts of them are responsible for the
constitutive skin pigmentation. Once melanin is synthesized, melanocytes transport the
mature melanosomes containing melanin through their dendrites to surrounding kera-
tinocytes (Desmedt et al., 2016), where it is accumulated and protects us from the sun
(Burger et al., 2016).

Analysis of Cosmetic Products

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108 Analysis of Cosmetic Products

Tanning Products
Among the different tanning products available on the market, two groups should be
differentiated, i.e., sunless tanning products and sun tanning accelerators.
The sunless tanning products, also called self-tanners, produce a tanning effect without
sun, and thus they avoid the harmful side effects associated with solar radiation (see
Chapter 5).Their active ingredients interact with amino acids at the stratum corneum level
to form substances called melanoidins (Balogh et al., 2011). These orange to brown poly-
meric compounds artificially formed should not be confused with melanin. Despite this
simple staining reaction, this artificial tanning has been shown to provide a slight protec-
tion against UVA radiation in animals and humans (Brown, 2001). It should be noted that
the tan provided by these products is not removed by simple washing but it does not last
more than 5–7 days, as the skin cells are continuously being shed (Pantini et al., 2007).
These products can be spread by the users themselves, but usually they are sprayed by tan-
ning booths to achieve a more homogeneous tone. The most popular active ingredient is
dihydroxyacetone, commonly known by its acronym DHA. Erythrulose is a compound
chemically very similar to DHA that in fact works similar to DHA and, despite having
better tanning properties at high concentrations, it has been used as a self-tanning active
ingredient to a much lesser extent (Lenz et al., 2010). There are other active ingredients
with self-tanning properties, but they are under study and under patents.
Regarding tanning accelerators, they increase tanning when sunbathing outdoors, or
also indoors after sun exposure. Although different compounds have been shown to
accelerate skin pigmentation in vitro via different mechanisms (Brown, 2001), the mar-
keted cosmetic products are mainly based on tyrosine as the active ingredient, on the
basis that, as described before, tyrosine is involved in the rate-limiting step of melano-
genesis. Therefore, these compounds containing tyrosine (or tyrosine derivatives, e.g.,
acetyl tyrosine) are thought to penetrate to the basal epidermis layer, where melanocytes
are stimulated, producing melanin and therefore increasing the tan. Sometimes, ribofla-
vin is added to these cosmetic preparations because it is known to activate the oxidation
of tyrosine in melanogenesis, and thus it improves the efficacy of tyrosine-based prepara-
tions. Psoralens, which were incorporated several decades ago either as bergamot oil or
as purified 5-methoxypsoralen, are no longer used in the cosmetics industry because of
their prohibition motivated by their multiple side effects related to erythema and skin
cancer (Herrmann et al., 2015).
These tanning agents are listed in Table 6.1.

Whitening Products
These products are designed to produce a whitening effect on the skin. They contain
various ingredients that interfere in the melanogenesis process by means of different
mechanisms, such as tyrosinase inhibition or melanosomal transfer disturbance, among
others (Gillbro and Olsson, 2011; Burger et al., 2016; Desmedt et al., 2016).
 Tanning and Whitening Agents in Cosmetics: Regulatory Aspects and Analytical Methods 109

Table 6.1 Tanning and whitening agents considered in this chapter

INCI name Keya

Tanning agents
Dihydroxyacetone DHA
Erythrulose
Tyrosine
Psoralen
Whitening agents
Hydroquinone and its derivatives
Hydroquinone HQ
Hydroquinone monomethyl ether HQMM
Hydroquinone monoethyl ether HQME
Hydroquinone monobenzyl ether HQMB
Resorcinol RS
Arbutin ARB
Mercury derivatives
Mercury chloride
Mercury iodide
Retinoids
Retinoic acid (tretinoin) RA
Retinyl palmitate RP
Ascorbic acid and its derivatives
Ascorbic acid AA
Ascorbyl palmitate AP
Ascorbyl dipalmitate ADP
Ascorbyl stearate AS
Magnesium or sodium ascorbyl phosphate MAP, SAP
Ascorbyl glucoside AG
Others
Kojic acid KA
Kojic dipalmitate KDP
Azelaic acid AZA
Niacinamide NC
INCI, International Nomenclature of Cosmetic Ingredients.
aKey system adopted by the authors.

The most popular active ingredient traditionally used in this type of cosmetic prod-
uct is hydroquinone (HQ). However, different dermatological (dermatitis, ochronosis,
permanent depigmentation) and carcinogenic side effects have been associated with
these compounds (Couteau and Coiffard, 2016; Naidoo et al., 2016). In this sense, HQ
is no longer used in the cosmetic industry (but it is still largely used in the pharmaceuti-
cal industry), not even its derivatives traditionally used in the past, such as its mono-
methyl (HQMM), monoethyl (HQME) and monobenzyl ether (HQMB). Resorcinol
110 Analysis of Cosmetic Products

(RS), a positional isomer of HQ, was also used in the past, but it is currently not used.
However, arbutin (ARB), which is a natural HQ derivative found in different plants, has
been most popularly accepted by the cosmetics industry rather than its parent com-
pound, as it is less harmful (Gillbro et al., 2011; Naidoo et al., 2016). In fact, it is added
to cosmetics as a pure compound or through extracts from Arctostaphylos uva-ursi and
Arbutus unedo (Chisvert et al., 2007).
Some mercury derivatives, such as mercury chloride and mercury iodide, were used
in the past to bleach the skin but are no longer used in skin whitening cosmetic products
because mercury is broadly not allowed in cosmetic products (Naidoo et al., 2016), with
few exceptions like thimerosal or phenyl mercury that are used as preservatives in some
countries.
Other controversial whitening agents are those derived from vitamin A (retinoids),
especially retinoic acid (RA), also known as tretinoin, and its derivative retinyl palmitate.
However erythema, desquamation and teratogenic effects have been associated with RA,
and thus it is not currently used in cosmetic preparations (Couteau and Coiffard, 2016;
Naidoo et al., 2016; Desmedt et al., 2016).
Some corticosteroids, such as fluocinonide, betamethasone dipropionate and clobeta-
sol propionate, among others, have been also used in the past as whitening agents in
some countries.
Nowadays, other than ARB, the most popular whitening agents found in cosmetic
products are ascorbic acid (AA), kojic acid (KA), azelaic acid and niacinamide. It should
be emphasized that owing to its labile oxidative properties, KA is usually added to cos-
metics by means of its dipalmitic ester, i.e., as kojic dipalmitate. The same happens with
AA, which is usually found as ascorbyl palmitate, ascorbyl dipalmitate, ascorbyl stearate,
magnesium (or sodium) ascorbyl phosphate and ascorbyl glucoside (Chisvert et al.,
2007). Moreover, these derivatives have different solubility properties compared to the
parent compound, and thus, they are employed in the formulation of new preparations
having different properties (Chisvert et al., 2007).
All these whitening agents are listed in Table 6.1.
Usually, whitening cosmetic products will also contain peeling chemicals, such as
α-hydroxy acids (glycolic, lactic or malic acids) or β-hydroxy acids (salicylic acid), to
improve their effectiveness, because these chemicals remove the dead skin cells, making
the task of the whitening agents easier. Also sunscreen agents are added to protect users’
skin from sunlight to avoid tanning (Chisvert et al., 2007).
For all the aforementioned reasons, analytical methods are needed to control the
concentration of tanning and whitening agents and thus to ensure the efficacy and the
safety of cosmetic products that contain them as ingredients.
The aim of this chapter is to familiarize the reader with the different compounds
used as tanning and whitening agents and the legislation regulating these compounds
and especially to review the analytical methods for tanning and whitening agents
 Tanning and Whitening Agents in Cosmetics: Regulatory Aspects and Analytical Methods 111

determination in cosmetic products since 2006. Those articles published earlier (i.e.,
before 2006) were reviewed in the first edition of this book (Salvador and Chisvert,
2007).

REGULATORY ASPECTS
In contrast to UV filters, described in Chapter 5, there are no positive lists for tanning
and whitening agents. Some of these active ingredients have been banned because of
their side effects, whereas others have been subjected to restrictions. In any case, their
prohibition/restriction or not depends on the legislation in force in each country, which
differs from one part of the world to the other, as was mentioned in Chapters 1 and 2.

Tanning and Whitening Agents in the European Union

Products containing these ingredients can be considered pharmaceuticals or cosmetics
depending on how they are declared, i.e., following guidelines established in Regulation
EC 726/2004 (related to pharmaceutical products) or in Regulation EC 1223/2009
(known as the EU Cosmetics Regulation). In the first case, products are intended to
correct skin disorders in small areas and are prescribed by a doctor, whereas in the last
case products are used for aesthetic reasons on large skin areas and are freely available. In
this regard, it should be emphasized that the active agents allowed in both types of prod-
ucts could be completely different, cosmetic products being subjected to more restric-
tions than pharmaceutical products. As example, it should be said that a user could find
a pharmaceutical product containing HQ, whereas this compound is banned as a whit-
ening agent in cosmetic products. Its use in cosmetics, and that of its monomethyl ether
(HQMM), is restricted to artificial nail systems according to Annex III of the EU
Cosmetics Regulation. Its positional isomer RS is also prohibited as a skin whitening
agent and its use is restricted to oxidative hair dyes and other hair care products. In the
case of mercury derivatives, they are banned (except for thimerosal or phenyl mercury
used as preservatives), since the use of mercury-based ingredients is not allowed in cos-
metic products according to Annex II of the EU Cosmetics Regulation. Similarly, RA,
corticosteroids (e.g., fluocinonide, betamethasone dipropionate and clobetasol propio-
nate) or psoralens (e.g., 5-methoxypsoralen) are completely banned in cosmetic prod-
ucts, as is stated in Annex II of the EU Cosmetics Regulation. However, market
surveillance has shown an illegal inclusion and product mislabelling in some countries
(Naidoo et al., 2016; Desmedt et al., 2016). No prohibition or restriction has been found
for the other tanning or whitening agents described earlier.

Tanning and Whitening Agents Outside the European Union

In other countries, such as the countries of the Association of Southeast Asian Nations
and the Southern Common Market in South America, regulation on cosmetics con-
verges to the EU, and thus all the aforementioned could be applicable to these.
112 Analysis of Cosmetic Products

However, in the United States, according to the Food and Drug Administration
(FDA),Title 21 of the Code of Federal Regulations (CFR), the unique reference to tan-
ning agents is related to DHA, which is considered a colour additive exempt from cer-
tification, being allowed in cosmetics (21 CFR 73.2150). Regarding whitening agents,
as expected, the use of mercury derivatives is not allowed based on the known hazards
of mercury and its questionable efficacy as a skin bleaching agent (21 CFR 700.13). Any
other reference regarding whitening agents has not been found. However, the FDA
issued in 2006 a notice through the Federal Register (2006) in which they proposed to
withdraw the rule in force on skin bleaching drug products for over-the-counter human
use, based on new data and information on the safety of the only active ingredient that
had been proposed for inclusion in these products, i.e., HQ. This proposal intended to
consider all skin bleaching drug products to be new drugs that require approval as a new
drug application to continue on the market. In Japan, HQMB is not allowed in cosmetic
products (MHWN, 2000).

ANALYTICAL METHODS FOR TANNING AND WHITENING

AGENTS DETERMINATION IN COSMETIC PRODUCTS
All the aforementioned restrictions established for these active ingredients need to be
accompanied by efficient and reliable analytical methods to control them. This is even
more so, if it is taken into account that market inspections have shown an illegal inclu-
sion of certain compounds and product mislabelling in some countries (Naidoo et al.,
2016; Desmedt et al., 2016).

Determination of Tanning Agents

There are no official analytical methods for the determination of any of the scarce afore-
mentioned tanning agents. Moreover, as said before, DHA is the most popular one, and
no serious side effects have been associated with its use. For this same reason the analyti-
cal community has paid very little attention to developing analytical methods for its
control. A detailed review of those papers published until 2006 was carried out in the
first edition of this book, revealing very few and old publications on this matter (Salvador
and Chisvert, 2007). Because of its polar character, it was usual to derivatize DHA to a
less polar or more volatile derivative so that it could be determined by reversed-phase
liquid chromatography (LC) or gas chromatography (GC), respectively (Chisvert et al.,
2007). Since 2006, only one publication regarding the determination of DHA by LC has
been published (Biondi et al., 2007). In this paper, the cream samples are cleaned up by
liquid–liquid extraction using aqueous sodium chloride and dichloromethane, and then
the DHA is derivatized with pentafluorobenzylhydroxylamine after 5 min at room tem-
perature, which in the authors’ words is more rapid and selective than the methods
published before.
 Tanning and Whitening Agents in Cosmetics: Regulatory Aspects and Analytical Methods 113

Determination of Whitening Agents

Regarding whitening agents, there is an official analytical method published in the EU
framework under Commission Directive 95/32/EC. This method focuses on the deter-
mination of HQ, HQMM, HQME and HQMB, and it is based on their identification
by means of thin-layer chromatography (TLC), followed by their quantitative determi-
nation using LC with ultraviolet/visible (UV/VIS) detection, for which the sample is
extracted with a water/methanol mixture under heating. As of this writing, in 2017, to
update and extend this official method for the determination of HQ and its ethers, but
also for the most common corticosteroids used illegally in cosmetic products, a new
proposal is being approved as an EU standard (FprEN 16956, WI 00392023, 2017) (see
Chapter 4). This standard, based on LC–UV/VIS, also describes a screening method for
the identification of HQ, its ethers, and 38 corticosteroids, which relies on the paper
published by Gimeno et al. (2016).
As can be found in the analytical databases, there are different published papers deal-
ing with the determination of whitening agents in cosmetic products. A detailed and
exhaustive review of those methods published up to 2006 can be found in the first edi-
tion of this book (Salvador and Chisvert, 2007). On that occasion, a detailed study of
those published papers revealed that most of them were focused on HQ and its ether,
whereas other whitening agents were scarcely considered. Since 2006, the demand for
reliable and broad-spectrum analytical methods to improve and facilitate quality control
in the cosmetics industry seems to have been heard. A detailed review of those papers
published since the last edition has been carried out here. Table 6.2 summarizes the
experimental details and some interesting remarks of these published papers dealing
with the determination of whitening agents.
Skin whitening cosmetic products usually contain mixtures of whitening agents, so
their direct measurement by, for example, UV/VIS spectrometry without a previous
separation step is difficult. In this regard chromatographic techniques are usually employed
to cover the determination of a high number of these active agents. Nevertheless, some
specific colorimetric reactions or chemometric strategies have been proposed to directly
determine individual whitening agents or mixtures of these compounds. In this sense,
Uddin et al. (2011) developed a selective method to determine HQ based on its oxida-
tion to benzoquinone catalysed by metavanadate. Calaca et al. (2011) determined mix-
tures of KA and HQ by using multivariate calibration based on the reaction with Fe3+,
so that Fe3+ is complexed by KA, whereas HQ reduces Fe3+ to Fe2+ and it is further
complexed by phenanthroline. Also applying chemometrics, Elzanfaly et al. (2012) deter-
mined mixtures of RA and HQ by using the ratio difference method. Esteki et al. (2016)
used another chemometric strategy to determine HQ in the presence of the preservative
methylparaben, based on successive projections algorithm–multiple linear regression.
Attenuated total reflectance–infrared spectroscopy and subsequent chemometric data
 114
Table 6.2 Published papers from January 2006 to December 2016 concerning whitening agents determination in cosmetic products

Analysis of Cosmetic Products

(chronological order)
Target
whitening Type of
Authors agentsa matrixb Sample preparationc Analytical techniquec Remarksd
Varvaresou et al. MAP EM Sample is dispersed in LC–UV/VIS, C18 column, LOD: 0.08%
(2006) acetonitrile:water by isocratic acetonitrile: R: 99%–103%
sonication (15 min), diluted phosphate buffer containing
with water and filtered tetrabutylammonium
hydroxide as mobile phase
Wang and Wu AA, AP, MAP LO Sample is dispersed in MEKC–UV/VIS, fused- LOD: no data
(2006) water:chloroform:methanol silica capillary, borate buffer R: >95%
by sonication (10 min) and containing sodium dodecyl
centrifuged sulphate as running buffer
Thongchai et al. ARB EM Sample is dispersed in LC–UV/VIS, C18 column, LOD: 0.0001%
(2007) methanol by sonication isocratic methanol: R: 100%
(30 min), centrifuged and water:hydrochloric acid (aq)
filtered as mobile phase
Lin et al. AG, KA, NC EM, LO, Sample is dispersed in water, LC–UV/VIS, Pf column, LOD: no data
(2007a) OI vortexed, then submitted to isocratic methanol:phosphate R: 92%–106%
microdialysis and injected buffer as mobile phase
online
Lin et al. ARB, HQ, KA GE, OI Sample is dispersed in water CE–UV/VIS, uncoated LOD:
(2007b) by sonication (30 min) and fused-silica capillary, 0.07%–0.14%
centrifuged phosphate buffer as running R: >99%
buffer
Wei et al. AA, ARB EM, Sample is dispersed in LC–CL, C18 column, LOD:
(2007) GE, LO methanol, centrifuged and isocratic methanol:phosphate 0.0002%–
diluted with methanol: buffer as mobile phase 0.0003%
phosphate buffer R: 98%–106%
Shih et al. KA EM Sample is dispersed in water FI–AD, carbon-nanotube LOD: no data
(2007) and filtered modified screen-printed R: 94%–107%
electrode
Balaguer et al. KDP EM Sample is dispersed in LC–UV/VIS, PLGel LOD:
(2008a) tetrahydrofuran by Mixed-D column, 0.04%–0.15%
sonication (10 min) and tetrahydrofuran as mobile R: 99%–102%
filtered phase
Balaguer et al. AA, AG, AP, EM Sample is dispersed in LC–UV/VIS, C8 column, LOD:
(2008b) MAP ethanol:phosphate buffer by gradient ethanol:phosphate 0.003%–0.12%

Tanning and Whitening Agents in Cosmetics: Regulatory Aspects and Analytical Methods
sonication (10 min) and buffer as mobile phase R: 97%–99%
filtered
Hubinger RA, RP EM, LO Sample is mixed with LC–UV/VIS, C18 column, LOD:
(2009) diatomaceous earth, then gradient methanol: 0.00003%–
eluted with hexane: ammonium acetate 0.0001%
isopropanol:ethyl acetate buffer:dichloromethane as R: 94%–103%
mobile phase
Chisvert et al. ARB, AZA, EM Sample is dissolved in GC–MS, 95% dimethyl–5% LOD:
(2010) HQ, KA, RS dimethyl formamide, diluted diphenyl polysiloxane 0.0005%–0.24%
and derivatized with BSTFA column, helium as carrier R: 98%–103%
gas
Cheng et al. ARB, NC Sample is extracted with LC–UV/VIS, C18 column, LOD: no data
(2010) chloroform:water mixture isocratic methanol:water as R: 92%–110%
mobile phase
Calaca et al. HQ, KA Sample is solved in Clark– UV/VIS LOD: no data
(2011) Lubs buffer, then mixed (multivariate calibration R: 98%–107%
with Fe3+ and method)
phenanthroline
Uddin et al. HQ EM Sample is dispersed in UV/VIS LOD: no data
(2011) isopropanol and filtered, R: no data
then mixed with
ammonium metavanadate
Continued

115
 116
Table 6.2 Published papers from January 2006 to December 2016 concerning whitening agents determination in cosmetic products

Analysis of Cosmetic Products

(chronological order)—cont’d
Target
whitening Type of
Authors agentsa matrixb Sample preparationc Analytical techniquec Remarksd
Yang et al. NC EM Sample is dispersed in LC–UV/VIS, C18 column, LOD: no data
(2011) methanol by vortex and water as mobile phase R: 100%
filtered
Gao and HQ EM, LO Sample is dispersed with LC–UV/VIS, C18 column, LOD: 0.0005%
Legido-Quigley methanol:water by isocratic methanol: R: 103%–116%
(2011) sonication (30 min), ammonium formate buffer
centrifuged and filtered as mobile phase
Elzanfaly et al. HQ, RA EM Sample is dispersed in UV/VIS LOD: no data
(2012) methanol by sonication (ratio difference method) R: 100%
(10 min), then filtered
Desmedt et al. ARB, HQ, KA, EM, LO, Soaps are dissolved in water LC–UV/VIS, C18 column, LOD:
(2013, 2014) NC, RA (and SO and neutralized with gradient acetonitrile: 0.00005%–
some hydrochloric acid; ammonium borate buffer as 0.00075%
corticosteroids) neutralized soaps, EMs and mobile phase R: 90%–103%
LOs are dispersed in LC–MS was also
acetonitrile by sonication used for
(30 min) and filtered screening
purposes
Jin et al. (2013) ARB, HQ, KA, EM, LO Sample is dispersed in MEKC–AD, fused-silica LOD: no data
RS ethanol or ethanol:water by capillary, borate buffer R: 100%
sonication (20 min), containing sodium dodecyl
centrifuged and filtered sulphate as running buffer
Thongchai and ARB, HQ EM Sample is dispersed in LC–UV/VIS, C18 column, LOD:
Liawruangrath acetonitrile:phosphate buffer isocratic acetonitrile: 0.002%–0.003%
(2013) by sonication (30 min), phosphate buffer containing R: 96%–99%
centrifuged and filtered Brij 35 as mobile phase
Sun and Wu NC EM Sample is dispersed in MEKC–UV/VIS, fused- LOD: 0.0004%
(2013) trichloromethane:water by silica capillary, borate buffer R: 87%–108%
sonication (30 min) and the containing sodium dodecyl
water phase was collected sulphate as running buffer
Jeon et al. ARB, NC EM, LO Sample is dispersed in LC–UV/VIS, C18 column, LOD:
(2014) methanol by sonication gradient methanol:water as 0.006%–0.04%
(10 min), diluted with water mobile phase R: 101%–107%
and centrifuged
Tidjarat et al. HQ, RA EM Sample is mixed with TLC, cellulose acetate Only screening

Tanning and Whitening Agents in Cosmetics: Regulatory Aspects and Analytical Methods
(2014) ethanol and sonicated nanofibers as stationary purposes
(10 min) phase, methanol:water:acetic
acid as mobile phase
Deconinck ARB, HQ, KA, EM, Sample is directly brought ATR–IR Only screening
et al. (2014) NC, RA GE, LO, on the crystal and pressed (k-nearest neighbours purposes
OI, SO method)
Tsai et al. ARB, KA, RS EM, LO Sample is dissolved in MEKC–UV/VIS, fused- LOD:
(2014) ethanol by sonication silica capillary, borate buffer 0.005%–0.04%
(10 min), then centrifuged containing sodium dodecyl R: 85%–115%
and diluted with water sulphate as running buffer
Chen et al. AA, AG, MAP EM, Low-fat samples are LC–UV/VIS, C18 column, LOD:
(2015a) GE, LO extracted with phosphate gradient methanol:phosphate 0.004%–0.008%
buffer, whereas high-fat buffer as mobile phase R: 96%–101%
samples are dispersed in
dichloromethane and
extracted with phosphate
buffer, then centrifuged and
filtered
Chen et al. AG, ARB, HQ, EM, LO Sample is dispersed in water LC–AD, C18 column, LOD: no data
(2015b) KA, MAP by stirring, then submitted isocratic citrate R: 91%–108%
to microdialysis and injected buffer containing
online tetrabutylammonium
chloride as mobile phase
Continued

117
 118
Table 6.2 Published papers from January 2006 to December 2016 concerning whitening agents determination in cosmetic products

Analysis of Cosmetic Products

(chronological order)—cont’d
Target
whitening Type of
Authors agentsa matrixb Sample preparationc Analytical techniquec Remarksd
Jeon et al. ARB, HQ EM Sample is dispersed in LC–UV/VIS, C18 column, LOD:
(2015) methanol by sonication gradient methanol:water as 0.0004%–
(30 min), diluted with water mobile phase 0.0005%
and sonicated (30 min), then R: 90%–103%
centrifuged and filtered
Esteki et al. HQ EM, Sample is dispersed in UV/VIS LOD: no data
(2016) GE, LO methanol:water by (successive projections R: 84%–114%
sonication (30 min), algorithm method)
centrifuged and filtered
Galimany- HQ, KA EM Sample is dispersed in LC–UV/VIS, Phenyl LOD: no data
Rovira et al. acetonitrile:water by column, gradient R: 98%–101%
(2016) sonication (15 min), then acetonitrile:acetic acid (aq)
centrifuged as mobile phase
Shams et al. ARB EM Sample is dispersed in LC–UV/VIS, C18 column, LOD: 0.0006%
(2016) (and some acetonitrile:methanol:water, isocratic acetonitrile: R: 96%–99%
corticosteroids) by stirring (30 min), then methanol:water as mobile
filtered phase
Gimeno et al. HQ, HQMM, EM, OI Sample is dispersed in LC–UV/VIS, C18 column, LOD: <0.005%
(2016) HQME, methanol:water by shaking gradient acetonitrile:acetic R: no data
HQMB (and (5 min), diluted, stirred acid (aq) as mobile phase
some (30–60 min) and filtered
corticosteroids)
aSee Table 6.1 for key to abbreviations.
bEM, emulsion; GE, gel; LO, lotion; OI, oil; SO, soap.
cAD, amperometric detection; ATR–IR, attenuated total reflectance–infrared spectrometry; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; C18, octadecylsilica;
CE, capillary electrophoresis; CL, chemiluminescence; FI, flow injection; GC, gas chromatography; LC, liquid chromatography; MEKC, micellar electrokinetic
chromatography; MS, mass spectrometry; Pf, perfluorinated phenyl; TLC, thin-layer chromatography; UV/VIS, ultraviolet/visible spectrometry.
dLOD, limit of detection (referred to sample, not to sample solution); R, recovery.
 Tanning and Whitening Agents in Cosmetics: Regulatory Aspects and Analytical Methods 119

treatment based on k-nearest neighbours has also been used for screening whitening
agents in cosmetic samples (Deconinck et al., 2014).
On just one occasion, electroanalytical detection, such as amperometric detection
(AD), was used to determine an individual whitening agent (i.e., KA) without a previous
separation (Shih et al., 2007). In this case, solutions were propelled by using a flow-
injection system.
With regard to chromatographic techniques, LC is the most commonly employed
because whitening agents present relatively high boiling points for employing GC.
Usually, a UV/VIS spectrometric detector is preferred because most of them present
chromophore moieties, although chemiluminescence (Wei et al., 2007) and AD (Chen
et al., 2015b) have also been used. In 2014, mass spectrometry (MS) detection was used
for identification purposes (Desmedt et al., 2014).
GC has been used on just one occasion (Chisvert et al., 2010), on which a previous
derivatization of the whitening agents with N,O-bis(trimethylsilyl)trifluoroacetamide
was carried out to convert them to the more volatile trimethylsilyl derivatives. MS was
used as the detector to unequivocally determine HQ and RS.
Capillary electrophoresis and micellar electrokinetic chromatography have also been
employed with very good analytical features for the quantitative determination of differ-
ent whitening agents (Wang and Wu, 2006; Lin et al., 2007b; Jin et al., 2013; Sun and Wu,
2013; Tsai et al., 2014).
Finally, it should be said that TLC was used as a screening method for HQ and RA
(Tidjarat et al., 2014).
Regarding sample preparation, no complex operations are required. Most of the cases
consisted just in adding an appropriate solvent to the cosmetic sample and then sonicating
it for several minutes to dissolve the sample totally or at least to leach the target com-
pounds. Centrifugation or filtration is sometimes required to obtain clear solutions prior
to measurement.The use of sample preconcentration techniques is not really necessary in
this case, since whitening agents are major ingredients, even when used fraudulently.

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