assessment of levels of some active skin lightening ...€¦ · jkuat, in particular mr. dennis...
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ASSESSMENT OF LEVELS OF SOME ACTIVE SKIN LIGHTENING COMPOUNDS
IN SELECTED FACIAL CREAMS AND SOAPS IN THE KENYAN MARKET
BY
Grace Kwamboka Abere (BSC.PGDE)
REG. NO: I56/CE/22232/10
A Thesis Submitted in Partial Fulfillment of the Requirement for the Award of the Degree
of Masters of Science in Applied Analytical Chemistry in the School of Pure and Applied
Sciences of Kenyatta University
March, 2015
ii
DECLARATION
I declare that this thesis is my original work and has not been previously presented for the award
of a degree in any other University or other award.
Grace Kwamboka Abere
I56/CE/22232/10
Signature……………………………… Date………………………….
This thesis has been submitted with our approval as the University supervisors
Prof. Hudson Nyambaka
Department of Chemistry
Kenyatta University
Signature…………………………….Date……………………………..
Dr. Ruth Wanjau
Department of Chemistry
Kenyatta University
Signature……………………………Date…………………………….
iii
DEDICATION
This work is dedicated to my parents Handson Abere and Victoria Moige who initiated my
education at a tender age.
iv
ACKNOWLEDGEMENTS
I am indebted to God with whom all things are made possible. I am grateful to Kenyatta
University (KU) for the abundant support accorded to me. KU provided the necessary facilities
that created a conducive environment for learning and facilitating the learning sessions and
research. I also owe a debt of gratitude to my supervisors Prof. Hudson Nyambaka and Dr. Ruth
Wanjau for their patience in guiding me throughout the study period. The two were always there
for me whenever I called on them.
I would like to acknowledge the technical support I received from the staff of both KU and
JKUAT, in particular Mr. Dennis Osoro, Mr Kevin Odhiambo, Mr Elias Maina and Mr Samwel
Kangethe of Chemistry Department KU, Mr Amos Karanja and Mr Richard Votha of the
Department of Food Science and Technology Jomo Kenyatta University of Agriculture and
Technology (JKUAT) for their assistance during the laboratory work.
Special gratitude goes to the National Council Science Technology and Innovation (NACOSTI)
for the financial aid and the Grants Office of the Kenyatta University for their tireless efforts in
ensuring that the funds given by the NACOSTI were well managed. I thank my friends and
colleagues, Erick Salano, Shylock Onduso, Linet Ondogo and Zipporah Onyambu of Kenyatta
University for their supportive ideas.
I will also not forget the family of Prof. Vincent Omwoyo of Masai Mara University for their
moral support during my studies in KU. May God bless everyone who contributed in one way or
another towards the success of this work. Finally, I would like to express my gratitude to my
husband Tom Mong’are Nyauma, our children Spencer, Sophine and Annet for their patience
and prayers more especially when I was away from them. I also thank my parents, brothers and
sisters for their encouragement, patience and moral support during my studies.
v
ABSTRACT
Cosmetics are generally used to improve the appearance by the removal or correction of
blemishes and to treat diseases of the human skin and hair. Skin-lighteners which include
hydroquinone, mercury, arbutin, kojic acid, ascorbyl glucoside, magnesium ascorbyl phosphate
have been frequently used in skin lightening creams and soaps. However chronic exposure to
mercury compounds in skin lightening creams and soaps may lead to various disorders such as
nephritic, peripheral neuropathy, anxiety, depression and damage the developing foetus. Mercury
may also cause skin rashes, skin discoloration and scarring, as well as a reduction in the skin’s
resistance to bacterial and fungal infections. Similarly when hydroquinone is applied at levels
higher than the allowed, it can result in ochronosis, dermatitis, exonogenous ochronosis, macular
hyperchromia, fingernail discolouration, hydroquinone neuropathy and damage of the nervous
system. On the other hand if arbutin, kojic acid, ascorbyl glucoside, or magnesium ascorbyl
phosphate are used above the permissible levels they cause pigmentation increasing to the joints
at the finger, toes, buttocks and ears. Although there are allowed levels of the skin lighteners,
some producers use levels above the set limits. There is scanty information reported on the levels
of skin lighteners in creams and soaps that contain them. The labels on the market products do
not indicate levels of active compounds among the ingredient list. Some of the active compounds
have been banned but yet they are in continue use. The aim of this study therefore was to assess
the levels of these compounds in some skin lightening facial creams and soaps in the Kenyan
market. Samples of cosmetics were obtained from small outlets in Nairobi and Kisii. Cold
Vapour Atomic Absorption Spectrometry (CV-AAS) was used to determine mercury and High
Performance Liquid Chromatography (HPLC) was used to determine hydroquinone, arbutin,
ascorbyl glucoside, kojic acid and magnesium ascorbyl phosphate. The mean levels in of skin
lighteners in creams ranged as follows; mercury 47.87 ± 0.00 to 513.06 ± 26.74 ppm,
hydroquinone 0.002 ± 0.00 % to 0.05 ± 0.00 %, arbutin 0.95 ± 0.02 to 107 ± 0.06 %, kojic acid
0.06 ± 0.00 to 15 ± 0.65 %, ascorbyl glucoside 5.83 ± 0.00 to 61. 47 ± 0.00 % and magnesium
ascorbyl phosphate 1.68 ± 0.01 to 40.48 ± 4.5 %. In soaps the levels were as follows; ascorbyl
glucoside 5.83 ± 0.00 to 5.84 ± 0.00 %, kojic acid 1.12 ± 0.086 to 3.50 ± 0.44 %, and mercury
578.25 ± 77.63 to 1108.754 ± 1.32 ppm. In most cases, there were significant differences
(p<0.05) in the levels of skin lighteners among creams and between the creams and soaps. The
mean levels of most skin lighteners were found to be above the maximum permissible limits set
by WHO except for hydroquinone. These findings indicate that the use of skin lightening creams
and soaps may potentially not be safe as far as toxicity is concerned. Although hydroquinone was
not above the set limit, continuous use of cosmetics containing it may lead to bioaccumulation
hence the need to have it monitored.
vi
TABLE OF CONTENT
DECLARATION....................................................................................................................................... ii
DEDICATION.......................................................................................................................................... iii
ACKNOWLEDGEMENTS .................................................................................................................... iv
ABSTRACT ................................................................................................................................................ v
TABLE OF CONTENT ............................................................................................................................. vi
List of Figures ............................................................................................................................................. x
List of Tables ............................................................................................................................................. xi
ABBREVIATIONS AND ACRONYMS ............................................................................................... xii
CHAPTER ONE ......................................................................................................................................... 1
1. INTRODUCTION .................................................................................................................................. 1
1.1 Background information ................................................................................................................... 1
1.2 Problem statement and justification .................................................................................................. 6
1.3 Hypothesis ........................................................................................................................................ 8
1.4 Objectives ......................................................................................................................................... 8
1.4.1 General objective ....................................................................................................................... 8
1.4.2 Specific objectives ..................................................................................................................... 8
1.5 Significance of study ........................................................................................................................ 9
1.6 Limitations and scope of study ......................................................................................................... 9
CHAPTER TWO ...................................................................................................................................... 10
2. LITERATURE REVIEW ..................................................................................................................... 10
2.1 Motivation for use of skin lighteners .............................................................................................. 10
vii
2.2 Cosmetics and their classification ................................................................................................... 11
2.3 Active compounds in skin lightening cosmetics ............................................................................. 12
2.3.1 Mercury .................................................................................................................................... 13
2.3.2 Hydroquinone .......................................................................................................................... 16
2.3.3 Arbutin ..................................................................................................................................... 19
2.3.4 Kojic acid ................................................................................................................................. 20
2.3.5 Magnesium ascorbyl phosphate (MAP)................................................................................... 21
2.3.6 Ascorbyl glucoside (AG) ......................................................................................................... 23
2.4 Methods of analysis ........................................................................................................................ 24
2.4.1 Method of analyzing mercury .................................................................................................. 24
2.4.1.1 Introduction ........................................................................................................................... 24
2.4.1.2 Theory of atomic absorption spectroscopy ....................................................................... 25
2.4.1.3 The cold vapour atomic absorption spectroscopy (CV-AAS) .......................................... 27
2.4.2 Analytical methods for organic lighteners ............................................................................... 28
CHAPTER THREE .................................................................................................................................. 33
3. MATERIALS AND METHODS .......................................................................................................... 33
3.1 Research design .............................................................................................................................. 33
3.2 Sampling ......................................................................................................................................... 33
3.3 Chemical, reagents and solvents ..................................................................................................... 33
3.4 Cleaning of apparatus ..................................................................................................................... 34
3.5 Instrumental conditions of operation .............................................................................................. 34
3.6 Laboratory procedures .................................................................................................................... 34
viii
3.6.1 Preparation of mercury standards ............................................................................................ 34
3.6.2 Preparation of other standards ................................................................................................. 35
3.6.3 Method validation .................................................................................................................... 35
3.6.4 Sample preparation .................................................................................................................. 37
3.7 Data analysis ................................................................................................................................... 38
CHAPTER FOUR ..................................................................................................................................... 39
4. RESULTS AND DISCUSSION ........................................................................................................... 39
4.1 Introduction ..................................................................................................................................... 39
4.2 Method validation ........................................................................................................................... 39
4.3 Mean levels of skin lighteners in facial creams .............................................................................. 41
4.3.1 Mercury (Hg) ........................................................................................................................... 42
4.3.2 Hydroquinone (HQ) ................................................................................................................. 44
4.3.3 Arbutin (ART) ......................................................................................................................... 45
4.3.4 Kojic acid (KA)........................................................................................................................ 46
4.3.5 Magnesium ascorbyl phosphate (MAP)................................................................................... 47
4.3.6 Ascorbyl glucoside) (AG) ........................................................................................................ 48
4.4 Skin lightening compounds in soaps .............................................................................................. 49
CHAPTER FIVE ...................................................................................................................................... 53
5. CONCLUSIONS AND RECOMMENDATIONS ............................................................................... 53
5.1 Conclusions ..................................................................................................................................... 53
5.2 Recommendations ........................................................................................................................... 54
ix
5.2.1 Recommendations from the study ........................................................................................... 54
5.2.2 Recommendations for further work ......................................................................................... 55
REFERENCES ......................................................................................................................................... 56
APPENDICES
Appendix 1: Calibration curve for kojic acid ........................................................................................... 57
Appendix 2: Calibration curve for magnesium ascorbyl phosphate ......................................................... 57
Appendix 3: Calibration curve for arbutin ............................................................................................... 58
Appendix 4: Calibration curve for hydroquinoe ...................................................................................... 58
Appendix 5: Calibration curve for ascorbyl glucoside ............................................................................ 59
Appendix 6: Calibration curve for mercury ............................................................................................. 60
Appendix 2: Limit of detection (ppm) for parameter analyzed ............................................................... 65
x
LIST OF FIGURES
Figure 2:1 Structure of hydroquinoe ......................................................................................................... 16
Figure 2:4 Structure of arbutin .................................................................................................................. 19
Figure 2:3 Structure of kojic acid ............................................................................................................. 20
Figure 2:4 Structure of magnesium ascorbyl phosphate ........................................................................... 21
Figure 2:5 Structure of ascorbyl glucoside ............................................................................................... 23
Figure 2:6 Diagram to illustrate instrumentation of AAS ........................................................................ 27
Figure 2:7 Flow scheme for HPLC ........................................................................................................... 30
Figure 4:1 Calibration curve for arbutin ................................................................................................... 40
Figure 4:2Mean levels of hydroquinone in creams ................................................................................... 44
Figure 4:3 Mean levels of arbutin in creams ............................................................................................ 45
Figure 4.4 Mean levels of kojic acid in creams ........................................................................................ 46
Figure 4:5 Mean levels of magnesium ascorbyl phosphate in creams ...................................................... 47
Figure 4:6 Mean levels of ascorbyl phosphate in creams ......................................................................... 48
xi
LIST OF TABLES
Table 3:1 Concentration of spiked, unspiked and standards added .......................................................... 34
Table 4:1 Method validation result ........................................................................................................... 40
Table 4:2 Mean levels of skin lighteners in creams .................................................................................. 39
Table 4:3 Mean levels of mercury, ascorbyl glucoside and kojic acid in lightening soaps ...................... 45
Table 4:4 Comparision of the levels of Hg, AG and KA in facial creams and soaps ............................... 49
Table 4:5 Mean levels of Hg,AG and KA in facial creams and soaps with same brand names ............... 49
xii
ABBREVIATIONS AND ACRONYMS
ANOVA Analysis of Variance
CV-AAS Cold Vapour Atomic Absorption Spectrophotometry
WHO World Health Organization
MAP Magnesium Ascorbyl Glucoside
KA Kojic Acid
HQ Hydroquinone
ART Arbutin
AG Ascorbyl Glucoside
HPLC High Performance Liquid Chromatography
FDA Food and Drug Administration
ZMWG Zero Mercury Working Group
KEBS Kenya Bureau of standards
DHEY Department of Health Exercutive
USFDA United States of America Food and Drug Association
1
CHAPTER ONE
1 INTRODUCTION
1.1 Background information
Skin is an organ that covers the entire outside of the body that varies in colour, texture and
thickness throughout the body. It has several functions including keeping the body together,
regulating body temperature, protects the body against injuries by providing tactile sensation and
helping in excreting waste products from the body and synthesizes vitamin D (Liesl, 2006).
Structurally there are three layers of the skin, namely epidermis, dermis and subcutis (Liesl,
2006). The epidermis is the thin outer of the skin which is made up of stratum cornea (honey
layer). This layer contains shedding dead keratinocytes. The next layer of epidermis is
keratinocytes (squamous cells) which contains living squamous cells which protect the rest of the
body. The innermost layer of the epidermis is the basal layer that contains basal cells. Basal cells
continually divide, forming new keratinocytes and replacing the old ones that shed from the skin
surface. Melanocytes are also contained in the epidermis. Melanocytes produce melanin (Liesl,
2006).
Melanin is the pigment that is located in a very small granule called melasome found in the
melanocyte cells. It is produced by a chemical reaction via the enzyme tyrosinase. Its main factor
is to determine the skin colour (Walters and Roberts, 2008). It has two forms that give varying
skin tones. The two forms are eumelanin which produce a range of brown skin and hair colour,
while pheomelanin produce a yellow to reddish blue colour. Pigmentation is the colour of
2
melanin which is produced by melanocytes cells in the dermis, the superficial layer of the skin.
Uneven pigmentation affects most people, regardless of ethnic background or skin colour
(Walters and Roberts, 2008).
Uneven skin pigmentation occurs because the body produces either too much or too little
melanin. Increased production of melanin is called hyper-pigmentation usually known as
melasma, chloasma or solar lentigen (Dahl, 2004). Melasma generally describe darkening of the
skin. Chloasma describe skin discolouration caused by hormonal changes due to pregnancy, birth
control pills or oestrogens replacement therapy. While solar lentigen is a term for darkened spots
caused by sun, common in adults with a long history of unprotected sun exposure. This hyper-
pigmentation disorder can be treated using skin lighteners which inhibit biosynthesis of melanin
via different mechanism (Oyedeji et al., 2010). Hypo-pigmentation is another type of skin
pigmentation where the skin develops white patches as a result of little production of melanin.
This condition is also called vitiligo or eczema or cytotoxicity (Walters and Roberts, 2008).
Cosmetics, including facial creams and soaps are defined as any preparation intended to be
rubbed, poured, sprinkled or sprayed on, introduced into or otherwise applied to the human body
for purpose of cleansing, beautifying, promoting attractiveness or altering the appearance
without affecting the body’s structure and function (Liesl, 2006; Oyedeji et al., 2010). These
cosmetic products include; skin creams, lotions, perfumes, lipsticks, fingernail polishes, eye and
facial make-up preparations, shampoos, permanent weaves, hair colours, toothpastes, deodorants,
and any material intended for use as a component of a cosmetic product (Liesl, 2006; Reed,
2007).
3
Currently, skin-spots represent an aesthetic concern in humans. This skin disorder can be due to
a variety of reasons, including overexposure to solar radiation, ageing and hormonal dysfunction
during pregnancy or taking certain medicines (Claudia et al., 2011). This disorder can be reduced
with cosmetic treatment through the use of so-called skin-whitening cosmetic products. The
practice of using these chemical substances in an attempt to lighten skin tone or provide an even
skin complexion by lessening the concentration of melanin is called skin lightening.
Skin lightening is currently one of the most common forms of potentially harmful body
modification practices in the world (Lewis, 2011). African women are among the most widely
represented users of skin-lightening products because of a believe that fair skin leads to beauty
than dark skin (WHO, 2007). A large fraction of the population primarily women and young
girls in Asia, Africa and Latin America use skin lighteners. Survey reports these figures of users
in specific countries; Senegal 27 %, Mali 25 %, Togo 59 %, South Africa 35 %, Nigeria 77 %,
Hong Kong 45 %, Republic of Korea 28 %, Malaysia 41 %, Philippines 50 % and Taiwan 37 %
. Reason given by 61 % of the respondents to the survey is that they felt younger with a fair
complexion (Sin and Tsang, 2003).
Skin listeners contain compounds for lightening the skin which include mercury, hydroquinone,
kojic acid, azleic acid, arbutin, vitamin C, magnesium ascorbyl phosphate, calcium ascorbate,
ascorbyl glucoside, which produces a whitening effect on the skin, based on the inhibition of
melanin biosynthesis via different mechanisms (Gallarate et al., 1999; Kuo et al., 2005; Amanda,
2011). Mercury is a heavy metal that can exist in three forms; elemental, inorganic and organic.
4
Inorganic compound such as ammoniated mercury and mercuric iodide are used as lighteners in
cosmetics. It is used as a preservative in eye make-ups, skin lightening creams and soaps
(Oyedeji et al., 2010). Its use in skin lightening products is because it inactivates the enzyme that
lead to production of melanin (Doreen, 2010). Mercury is toxic to the nervous system. Users of
mercury containing soaps in Kenya have had symptoms of nervous system toxicity which
include tremor, lassitude, vertigo, loss of memory. These are all classic signs of inorganic
mercury poisoning (Harada et al., 2001).
United Nations Environment programme has developed a mercury awareness raising toolkit
which include information about mercury in skin lightening products and mercury uses in
cosmetic products are prohibited by law in the European Union and United States (ZMWG,
2010). Several national government including Kenya and Indonesia have mounted public
education campaigns and banned long list of specific products (FDA, 2009; ZMWG, 2010).
Arbutin, a hydroquinone glucoside containing cream is used as a lightener (Kuo et al., 2005) and
its active compound is hydroquinone. Both arbutin and hydroquinone inhibit the conversion of
tyrosinase to melanin by inhibiting tyrosinase activity (Gallarate et al., 1999). It is considered to
be a topical ingredient for inhibiting melanin production thus preventing the skin from making
substances responsible for the skin colour. Hydroquinone is potentially carcinogenic and is
known to be a skin and respiratory irritant (Kooyer and Westerhof, 2005; Doreen, 2010).
Because of its carcinogenic properties, it has been banned in some countries like Kenya and
Indonesia because of the fear of a cancer risk and only allowed to be used as a drug (WHO,
2007; ZMWG, 2010).
5
Major effects of hydroquinone are common in Africa where adulterated skin lightening products
are common. Among the chronic problems associated with long term exposure to hydroquinone
are diseases such as thyroid disorder, leukemia, liver damage among others (Oyiedeji, 2010). It
can also cause neurological effects which include; headache, dizziness, tinnitus, delirium, muscle
twitching, tremor, nausea, vomiting, and the production of green to brown-green urine may occur
(FDA, 2009).
The use of hydroquinone in cosmetics was banned in the European Union in 2001 and products
intended for treatment of abnormal conditions containing this compound, henceforth were not
generally classified as cosmetics but as drugs (WHO, 2007). Clinical preparations containing 2-4
% hydroquinone are prescribed for the treatment of hyper pigmentation such as melasma,
freckles, and senile lentigines as well as chloasma (WHO, 2007). The Kenya Bureau of
Standards through gazette legal notices number 4310 of 14th
August 1998 and 7169 of November
2000 prohibited use of hydroquinone in cosmetics and issued a public notice in the media to
inform and educate the consumers on the harmful effects of hydroquinone.
The products containing hydroquinone continue to be inappropriately used for skin lightening
purposes. Despite the ban of such hydroquinone containing cosmetics, over-the-counter products
containing hydroquinone in concentrations exceeding 2% in skin-lightening creams continue to
be freely sold in the Kenyan market. Curiously, most of these products are not appropriately
labeled.
6
Kojic acid is a tyrosinase inhibitor produced by various fungal species such as Aspergillus,
acetobactor and penicillium (Shou-chieh et al., 2004). It acts as a skin lightener by inhibiting
melanin production where it works by chelating the copper ions in tyrosinase (Engasser and
Maibach, 2003). In large dosage kojic acid may be carcinogenic and can cause allergic contact,
dermatitis and skin irritation (Engasser and Maibach, 2003).
Ascobyl glucoside is a derivative of ascorbic acid like magnesium ascorbyl phosphate (MAP)
and works by inhibiting melanin production. Magnesium ascorbyl phosphate (MAP), kojic acid,
arbutin and ascorbyl glucoside (AG) were admitted as lightening ingredients in cosmetic by the
Department of Health Executive Yuan in Taiwan in 2006 (Shou-chieh et al., 2004). Both
magnesium ascorbyl phosphate and ascorbyl glucoside have been recognized in reducing the
aging of facial skin. They also provide a range of benefits such as inhibition of biosynthesis of
melanogenesis, promotion of collagen synthesis and prevention of free radical formation (Shou-
chieh et al., 2004).
1.2 Problem statement and justification
Skin cosmetics (creams and soaps) have gained increased use both in the herbal and synthetic
forms. Skin lighteners are used by a large fraction of the population. Survey reports these figures
of users in specific countries; Senegal 27 %, Mali 25 %, Togo 59 %, South Africa 35 %, Nigeria
77 %, Hong Kong 45 %, Republic of Korea 28 %, Malaysia 41 %, Philippines 50 % and Taiwan
37 % (Sin and Tsang, 2003). Skin lighteners such as hydroquinone and mercury have many side
effects. Arbutin, ascorbyl glucoside, magnesium ascorbyl phosphate and kojic acid on the other
hand will have side effects when concentrations above the allowable levels are used. These
7
cosmetic products available in pharmacies and beauty shops do not indicate the presence, levels
neither the side effects of the active compounds in the ingredient list, so the consumer does not
have any choice for selecting suitable products (WHO, 2007).
In the Kenyan market production and supply of cosmetics undergo a quality control check by the
Kenya Bureau of Standards (KEBS). Through this body KEBS; the government has limits of the
levels of skin lighteners in cosmetics. The allowable levels of skin lighteners in creams and soaps
are 2 % for hydroquinone, 1 ppm for mercury, 2 % for ascorbyl glucoside, 2 % for kojic acid, 3
% for magnesium ascorbyl phosphate and 7 % for arbutin in skin care products (DHEY, 2000;
WHO, 2007; FDA, 2009).
Levels above maximum permissible limits would be harmful to both skin and other body organs.
However as much as KEBS make efforts of controlling the production and supply of the
products, some creams in the market are produced and marketed without KEBS approval and
some come into the country illegally.
Despite the efforts to ban products containing mercury and hydroquinone and public health
campaigns discouraging their use because of the side effects of the lightening agents, the desire
for light skin has accelerated over the last few decades and the market for skin-lightening
products has boomed in many parts of the world (Oyiedeji, 2010 ; Claudia et al., 2011). Studies
indicate that their use is growing fastest among young, urban and educated women in the global
south where light skin operates as a form of symbolic capital (Lewis, 2011).
8
Considering the toxic effects of these lightening compounds, it is necessary therefore to control
their exposure to human by quantifying their levels in skin lightening creams. Little work has
been reported on the levels of mercury, hydroquinone and other active lightening compounds in
cosmetics sold in the Kenyan market. In view of the above situation, the purpose of the research
work was to determine the levels of hydroquinone, mercury, arbutin, kojic acid, ascorbyl
glucoside and magnesium ascorbyl phosphate in facial lightening creams available and soaps in
the Kenyan market.
1.3 Hypothesis
The levels of active skin lightening compounds in facial creams and soaps sold in Kenyan
market are within the required limits.
1.4 Objectives
1.4.1 General objective
To determine the levels of active skin lightening compounds in facial creams and soaps in
Kenyan market.
1.4.2 Specific objectives
1. To determine the levels of mercury, hydroquinone, arbutin, kojic acid, magnesium
ascoryl phosphate and ascorbyl glucoside, in selected lightening facial creams.
2. To determine the levels of mercury, hydroquinone, arbutin, kojic acid, magnesium
ascoryl phosphate and ascorbyl glucoside, in selected skin lightening soaps.
9
1.5 Significance of study
The results of this study is aimed at providing information on the levels of mercury,
hydroquinone, mercury ascorbyl phosphate, ascorbyl glucoside, kojic acid, arbutin in facial
creams and soaps. It is anticipated that the findings will give the relevant authorities information
on the levels of skin lightening compounds in soaps and creams in the Kenyan market in order to
take necessary measures and sensitize the public consumers on the hazards. The result will also
form a data base of these compounds in cosmetic products.
1.6 Limitations and scope of study
Though there are many cosmetic creams and soaps in the Kenyan market, the study focused only
on skin lightening facial creams and soaps available in the market. Six active compounds in these
creams and soaps were investigated since they are the commonly used compounds and can be
analyzed by the same technique of HPLC. Variations such as batch difference, difference in
manufacturing companies that may occur in the products were not considered
10
CHAPTER TWO
2 LITERATURE REVIEW
2.1 Motivation for use of skin lighteners
The use of bleaching creams cuts across all sociodemographic characteristics with people of all
classes using the products. Studies indicate that a desire to lighten and to have a more uniform
complexion is the main reason why people use these products (Al-Sahel et al., 2004). Other
reasons include: believe that lighter and brighter skin equals to younger healthier loving face
(Lewis, 2011), the desire to be beautiful, to look like Arabians or Europeans, to be attractive to
people especially men, due to peer pressure, to go with the existing fashion trend, to treat skin
blemishes like acnes, dark spot, fade scars and melasma, to satisfy the taste of one’s spouse. Men
on the other hand claim to use lighteners because their wives use (Lewis, 2011).
Some marketers use skin lighteners in order to advertise their wares. Commercial sex workers
use skin lighteners for reasons of looking attractive in order to maintain their business.
Surprisingly even people with naturally fair complexion use lighteners to maintain the light skin
and to prevent tanning or blotches from sunlight (Yetunde et al., 2008). Besides the above
reasons for use of lighteners, there is a myth that lighter paler complexion portrays beauty, riches
and success (Claudia et al., 2011). The reasons have resulted to the huge market of skin
lightening products.
The racist remnants left in the wake of colonization unequivocally contribute to the preference
for lighter skin tone. Establishing a racial hierarchy in which dark-skinned native Africans were
considered primitive and inferior to light-skinned, Europeans was a key method of control during
11
the colonial period (Al-Sahel et al., 2005). Some scholars argue that pre-colonial conceptions of
female beauty favored lighter skin tones. If racial preferences existed before, they were
intensified by colonial racial hierarchies attaching privilege to light skin (Gleen, 2008). Media
images would portray lighter skin as beautiful and preferable over darker skin (DeSouza, 2008).
Even media, such as billboards, originating in Africa portrayed white-skinned people as icons of
beauty. Studies suggest that the use of skin-lightening products is increasing in places where
modernization and the influence of western culture and capitalism are most prominent (Gleen,
2008).
2.2 Cosmetics and their classification
Cosmetic product can be defined as any substance or preparation intended to be placed in contact
with the various external parts of the human body (epidermis, hair system, nails, lips and external
genital organs) or applied to the teeth and the mucous membranes of the oral cavity with a view
exclusively or mainly for the purpose of cleaning, perfuming, protection, changing their
appearance, correcting body odours and keeping the surfaces in good condition (Reed, 2007;
Oyedeji et al., 2010).
Dermatologist have classified cosmetics according to different uses, such as; skin care cosmetics
(cleansing agent, moistening agent and others), hair care cosmetics (shampoo, hair colorants
styling agent etc), face care cosmetics (facial creams including skin lighteners, powders, eye
shadow, mascara used to enhance eye lashes, eye liners, eye shadows (colour eye lids), lipstick
and lip glow to colour the lips, nail care cosmetics (paint removers, nail varnishes used to colour
fingernails and toenails, fragrance products (deodorants, aftershaves perfumes among others),
12
ultraviolet (UV) light screening preparations (Liesl, 2006; Reed, 2007), products for internal
intimate hygiene, sunbathing products, skin-whitening products and anti-wrinkle products
(Anton, 2005).
2.3 Active compounds in skin lightening cosmetics
Skin lightening refers to the practice of using chemical substances which lighten skin tone or
provide an even skin complexion by lessening the concentration of melanin. Most skin lightening
products contain one of the two active products; mercury and hydroquinone. In the past, the
quickest way to lighten skin was with hydroquinone. But this active ingredient has toxic
ramifications and has been banned in many countries across Africa and Europe (FDA, 2009;
ZMWG, 2010).
Other lightening products may have niacinamide (vitamin B), licorice extract, chromabright,
azelaic acid aleossin from aloevera plant, kojic acid, alpha arbutin, beta arbutin, ascorbyl
glucoside or magnesium ascorbyl phosphate, mulberry extract, glycoric acid, licorine extract,
niacinamidel lactic acid, lemon juice extract, emlica, potato and turmeric. All the compounds
work by inhibiting the production of melanin (Yetunde et al., 2008). A review of literature
reveals that kojic acid, magnesium ascorbyl phosphate (MAP), arbutin, and ascorbyl glucoside
are at investigational stages (Shou-chieh et al., 2004).
Both magnesium ascorbyl phosphate and ascorbyl glucoside are forms of vitamin C. The MAP is
a water soluble form of vitamin C while ascorbyl glucoside is a fat soluble form of vitamin C
(Al-Sahel et al., 2005). The two have been recognized in reducing the ageing of facial skin.
13
Kojic acid is able to chelate the copper containing enzyme tyrosinase in the formation of melanin
hence it has skin lightening property since it inactivates tyrosinase (Shou-chieh et al., 2004).
Arbutin is similar to hydroquinone and both inhibit the conversion of tyrosinase to melanin
(Shou-chieh et al., 2004). It is used to prevent the skin against damage caused by free radicals. It
does not have side effects that hydroquinone seems to have. It also has ant-cancer activity on
melanoma cells (Shou-chieh et al., 2004).
The four lighteners are (arbutin, kojic acid, ascorbyl glucoside and magnesium ascorbyl
phosphate) commonly used as alternatives lighteners to mercury and hydroquinone. They have
not shown adverse effects when used in the right concentration. However long term use of these
skin lighteners can lead to pigmentation increasing to the joints of the finger toes, buttocks and
ears (Oluminde, 2010). Lightener levels above maximum permissible limits would be harmful to
both skin and other body organs. The allowable levels are 2 % by weight for hydroquinone, 1
ppm for mercury, 3 % by weight for magnesium ascorbyl phosphate, 2 % by weight for ascorbyl
glucoside, 2 % by weight for kojic acid and 7 % by weight for arbutin in skin care products
(DHEY, 2000; WHO,2007). The skin lightening compounds are discussed in the following
subsections.
2.3.1 Mercury
Mercury is a heavy metal. Heavy metals are those that have higher than 3 g/cm3 densities, have
biological effects at low concentrations and are toxic when present in cells at higher than the
tolerable physiological levels (Banfalvi, 2011; Rai et al., 2011). They are persistent
environmental contaminants since they cannot be degraded or destroyed. Mercury exists in three
14
forms; elemental, inorganic and organic mercury and in cosmetics it exists in any of the forms
(Ladizinski et al., 2011). Inorganic mercury such as ammoniated mercury and mercuric iodide is
used in skin lightening soaps and creams and organic mercury such ethyl mercury is used as
cosmetic preservatives in eye makeup cleansing products and mascara ((Ladizinski et al., 2011).
The principal organ systems affected by mercury poisoning are the central nervous system and
kidneys. Poisoning may lead to tremor, sensitivity disturbance, reduced memory and
intelligence, sleeping disorders and even death in severe cases (FDA, 2009). Chronic exposure to
either inorganic or organic mercury may damage brain, kidneys, nervous system and developing
foetus (Barrat et al., 2006). By inhibiting the production of melanin, the skin is more susceptible
to skin cancer. Nephro- toxic effects have been attributed to application of inorganic mercury
salts. Exposure of mercury to placental cells causes damage to the developing foetus (Kinabo,
2003; Bingol and Ackay, 2005).
Other complications of mercurial toxicity are exogenous ochronosis, dermatitis, facial acnes,
facial hypertricosis, hyper and hypo-pigmentation, reduction in the skin resistance to bacterial
and fungal infections, anxiety, depression, psychosis, impaired wound healing, the fish odour
syndrome, nephropaty, steroid addition syndrome and predispotion to infections (Doreen, 2010;
Oyelakin, 2010). A high concentration of mercury in pregnant women is known to considerably
increase the risk of permanent brain damage and may lead to acrodymia (pink baby syndrome).
Studies done on 128 professionals of using mercury soaps in disinfecting their hands showed that
the group that used mercury soap had urine concentration of mercury being three times more
15
than the controlled group (Peter, 2008). It is quite unfortunate as the users are often young fertile
women and mercury is toxic and can cause permanent brain damage.
It has also been reported that women using creams and soaps containing mercury attain
concentration of 0.03 to 0.15 ppm in urine (Glahder et al., 1999). This poses major risks of
negative effects on their central nervous system and kidney (Glahder et al., 1999). A study of
119 Latino women from California and Arizona that were using lighteners showed that 87 % of
them had elevated mercury levels of 20 ppm in urine (Weldon et al., 2000). Mercury
concentrations in urine greater than 20 ppm are associated with symptoms of mercury poisoning
(Oyedeji et al., 2010).
Analysis carried out on facial creams produced in Thailand, Lebanon and England showed
highest levels of mercury ranging from 1281 ppm to 5650 ppm (Al-Sahel et al., 2004). These
levels are above the United States of America Food and Drugs Administration (FDA)
permissible levels of 1 ppm mercury in creams (FDA, 2009). In Tanzania a study of two skin
lightening soaps namely jaribu and rico recorded mercury levels of 6200 ppm and 6900 ppm
respectively (Peter, 2008).
A study by California medical officials on some facial lightening creams contained mercury
levels of 20,000 to 56,000 ppm (Melisa and Jay, 2009). Claudia (2011) recorded levels of
mercury between 878 and 36,000 ppm in six lightening creams studied in Mexican. In Saudia
Arabia a study by Al-Ashban (2006) on skin lightening creams recorded levels of mercury
between 2.46 to 23222 ppm while Voegborio et al., (2008) in the same country recorded mercury
levels between 1.18 to 5650 ppm in 17 skin lightening cream samples. In Kenya a study by
16
Maina (2013) on mercury in seven different brands of skin lightening creams showed the
following levels of mercury in ppm: 20,867-33,508, 14,330-22,167, 3,742-9,949, 5,444-32,270,
8,837-16,187,1,182-1,969 and 3,743- 5,444. All the levels of mercury from the above four
studies are far much above the set limits of 1 ppm (WHO, 2007; FDA, 2009).
Reports from above studies including Al-Ashban (2006) and Maina (2013) recommend that the
manufacturers should be informed about the hazards of high concentrations of mercury.
Furthermore, keeping in view the potential health risk of such creams, they suggest that mercury
levels of skin-lightening creams and soaps be monitored officially before marketing such creams.
Maina (2013) recommended specifically Kenya Bureau of standards to find ways of preventing
products with mercury levels above the set limits from entering the market.
2.3.2 Hydroquinone
Hydroquinone occur naturally as a conjugate with beta-D-glucopyranoside in leaves, barks and
fruits of a number of plants such as cranberry, cowberry, bearberry and blueberry (Yetunde et
al., 2008). Figure 1 is the structure of hydroquinone.
Figure 2.1 Structure of hydroquinone
Hydroquinone is an important phenol compound used as an intermediate in the manufacturing of
antioxidants for rubber, dyestuffs and foodstuffs. It is used as a reducing agent in photographic
17
developing solution to reduce silver halides to elemental silver in black and white photography
and lithography. It is used as a stabilizer in paints, varnishes, motor fuels and oils (Weldon et al.,
2000). Hydroquinone is also used as medicine up to a concentration of 5 % to treat dyschromias
such as melasma, which is acquired hypermelanosis. It is used in cosmetics up to 2 % as a
depigmenting agent in a number of skin creams. It is also found in other cosmetics such as hair
dyes and products for coating fingernails (Weldon et al., 2000).
Besides its importance, it is toxic and has hazardous health effects (Doreen, 2010). It is a strong
inhibitor of melanin production that has long been established as the most effective ingredients
for reducing and potentially eliminating melasma (Yoshimura, 2001) thus it prevents the skin
from making the melanin responsible for skin colour. Over the counter hydroquinone products
can contain 0.5 % to 2 %. Sometimes higher concentrations of 4 % and above are available from
dermatologist in some countries for the gradual lightening of hyper-pigmented skin in conditions
such as melasma, freckles and senile lentigenines as well as chloasma (Odumuso and Ekwe,
2010).
The WHO (2007) report a raised awareness of the side effects of skin bleaching due to
hydroquinone which range from skin irritation to cancer, skin rashes, swelling of the skin,
seizures, numbness, pain tremor and memory loss. Exposure to high level of hydroquinone
affects the central nervous system, can result in ochronosis in which skin area exposed to
hydroquinone becomes dark and thick, exonogenous ochronosis which a serious ochronosis,
dermatitis a condition in which the skin area exposed to hydroquinone get inflamed, macular
hyperchromia a condition where there is increased pigmentation of the area surrounding the eyes
18
(Decarporio, 1999; Claudia et al., 2011). Fingernail discolouration and hydroquinone neuropathy
can also be caused by high levels of hydroquinone (Claudia et al., 2011). Neurological effects of
hydroquinone include; headache, dizziness, tinnitus, delirium, muscle twitching, tremor, nausea,
vomiting, and the production of green to brown green urine may occur (Melisa and Jay, 2009).
Studies done on rodents showed some evidence that hydroquinone may act as a carcinogen
although its cancer causing properties have not been proven in humans (FDA, 2009). The FDA
(2009) reported abnormal function of the adrenal glands in people who used hydroquinone
containing cosmetics. Chronic occupational exposure to hydroquinone dust has resulted in eye
injuries varying from mild irritation and staining of conjunctivae and cornea to changes in the
thickening and curvature of the cornea, loss of corneal lustre and impaired vision (Doreen,
2010).
Brown discolouration of nails has been reported occasionally when application of 2 %
hydroquinone is used on the back of the hand (Oyedeji et al., 2010). Deaths have been reported
after ingestion of photographic developing agent containing hydroquinone (Doreen, 2010).
Hydroquinone also causes body weakness, a burning sensation, loss of deep tendon reflexes and
impaired sensation along with a very low pressure which are all symptoms of a peripheral
neuropathy (Karamagi et al., 2001). Exposure to airborne hydroquinone usually during
production and packaging cause noticeable eye irritation. Concentration of hydroquinone dust of
over 0.1 ppm may result in irreversible eye damage. Hydroquinone can cause vitiligo or
leukodemia, a skin disease characterized by the death or dysfunction of melanocytes (Karamagi
et al., 2001).
19
A research on levels of hydroquinone in some skin lightening creams in Nigeria reported levels
of below 0.001 to 3.45 % (Doreen, 2010). Other studies in Taiwan recorded levels of
hydroquinone to be 3.96 % in facial creams (Shou-chieh et al., 2004). While Terer et al (2013)
reported the levels of hydroquinone in body creams and lotions of between 0.00025 % 0.03457
% in their study on levels of hydroquinone in body creams and lotions sold in retail outlets in
Baraton Kenya. These levels of latter study were within the permissible level for hydroquinone
of 2 %. (WHO, 2007)
2.3.3 Arbutin
Arbutin is the most popular and safest skin lightening agent (Lewis, 2011). This skin de-
pigmentation and whitening agent is derived from wheat, pears and the bearberry plant. Figure 2
is the structure of arbutin.
Figure 2.2 Structure of arbutin
It has been used to prevent pigmentation and whiten skin beautifully in Japan and across Asia.
Arbutin treats skin discoloration conditions by blocking the production of melanin by the body.
Arbutin also contains anti-aging properties to make skin look younger. Through continued use,
20
skin is moisturized and left softer. This is because arbutin is an antioxidant with antibacterial
qualities (Lewis, 2011).
There are two types of arbutins. These are beta arbutin and alpha arbutin. These compounds have
inhibiting function against tyrosinase enzyme. Alpha arbutin is a powerful yet gentle lightening
agent derived from bearberry trees. Its inhibition is effective than that of its beta version. Like
hydroquinone it brings all the benefits of all the strong melanin inhibitors without strong odour
and potential side effects. Alpha arbutin is quickly becoming the alternative to harsh skin
lightening chemicals like hydroquinone Many dermatologists recommend alpha arbutin because
of its relatively quick results and have fewer side effects (Terer et al., 2013). Excessive
concentration could be potentially harmful. Hence it is safe so long as it is used within the
permissible levels of 7 % (WHO, 2007; FDA, 2009). Studies done on facial cosmetics in Taiwan
recorded levels of arbutin to be 2.07 % (Shou-chieh et al., 2004). Achieng et al. (2011) reported
the levels of arbutin in some cream and lotions to be 2.26 % in Taiwan.
2.3.4 Kojic acid
Kojic acid is a byproduct in the fermentation process of malting rice for use in manufacturing of
Japenese rice wine. Kojic acid can also be derived from mushroom. Figure 3 is the structure is
the kojic acid.
Figure 2.3 Structure of kojic acid
21
It is an inhibitor in the formation of melanin hence common in skin lighteners, facial and body
moisturizers, anti-aging creams, lotions and other skin care products. Its major purpose in the
skin care products is to treat hyper pigmentation (Terer et al., 2013).
Although effective in skin lightening gel, it has been reported to have high sensitizing issues and
may cause irritant contact dermatitis characterized by red rashes, itching pain and dry skin
(Lewis, 2011). Kojic acid can cause cell mutation in mammals (Yingshing et al., 2007). A study
done on animals showed that it can cause liver, kidney, reproductive cardiovascular and
respiratory side effects (Melisa and Jay, 2009). Studies done on facial cosmetics in Taiwan
reported the levels of kojic acid to be 1% (Shou-chieh et al., 2004). Yingshing et al. (2007)
reported levels in the range of 5.04 % to 10.30 % of kojic acid in cosmetic skin lightening
creams in Taiwaan. While the permissible levels of kojic acid in cosmetics are 2 % (DHEY,
2000)
2.3.5 Magnesium ascorbyl phosphate (MAP)
Magnesium ascorbyl phosphate (MAP) is a water soluble and stable form of vitamin C. It can be
obtained from citrus fruits, grape fruits, tropical fruits and vegetables. Figure 4 is the structure of
magnesium ascorbyl phosphate.
Figure 2:4 Structure of magnesium ascorbyl phosphate
22
It is found in skin products such as face masks, sunscreen and body lotion (Segnall et al., 2008).
MAP in skin care products is used for UV protection and repair, collagen production, skin
lightening and brightening and as an antinflammatory. It helps in anti-aging by removing
wrinkles and fine lines on the skin. It is a potent antioxidant and is considered an excellent non-
irritating skin lightening agent that inhibit skin cells to produce melanin and lightens age spots
and it is a great alternative of hydroquinone (Segnall et al., 2008).
The MAP has the same prospective function as L-ascorbic acid that enhances collagen
production. But unlike L-ascorbic acid, MAP is efficient even on lower concentrations of 10 %
to suppress melanin build up and at this concentration it can reduce aging. Studies show that
MAP is a better alternative of L-ascorbic acid because of its efficiency even on lower
concentration of 10 % (Lewis, 2011). This lessens the exfoliating effects and irritation caused by
L-ascorbic acid which is effective on a concentration of 20 % (Achieng et al. 2011). Another
reason why MAP is better than L-ascorbic acid is that L-ascorbic is not stable when exposed to
air which reduces its efficiency whereas MAP is very stable than L-ascorbic acid. Magnesium
ascorbyl phosphate is therefore for people who want to look lighter, young and age defying
appeal that has sensitive skin. Studies show that MAP has no side effects although those with
sensitive skin need to be aware of vitamin C’s acidic and exfoliating effect. Thus MAP is
considered to be more gentle than the traditional vitamin C and therefore safer on sensitive skin
(Segnall et al., 2008). Shou-chieh et al. (2004) reported MAP levels in marketing facial
cosmetics of 0.93%. Achieng et al. (2011) reported MAP levels in some cream and lotions
23
ranging from 1.35-1.47 percent. The allowable level of MAP in cosmetic products is 3 %
(DHEY, 2000).
2.3.6 Ascorbyl glucoside (AG)
Ascorbyl glucoside is a type of vitamin C that is fat soluble. Figure 5 shows the structure of
ascorbyl glucoside.
Figure 2:5 Structure of ascorbyl glucoside
It has a structure in which the C2- hydroxyl group of L-ascorbic acid is masked with glucose.
Once AG is permeated into the skin, AG is broken down into L-ascorbic acid and glucose by an
enzyme alpha- glucosidase. Hence AG has same functions as L-ascorbic acid. The functions are
reducing melanin products by inhibiting tyrosinase activity, reducing existence of black melanin
and promoting collagen synthesis (Segnall et al., 2008). Collagen is a product that plays an
important role in the structure and firmness of the skin. Therefore AG is very beneficial to
improve wrinkles and unevenness of the skin no wonder it is used in antaging cosmetics (Shou-
chieh et al., 2004). The benefits of using products with AG as opposed to usual vitamin C in skin
care products are that AG has greater stability than L-ascorbic acid. L-ascorbic acid tends to
break down in heat, light and in the presence of oxygen and certain pH levels hence vitamin C
24
will turn yellow or brown after a few uses (Segnall et al., 2008). The AG has excellent stability
in heat, light, in the presence of oxygen and in metal ions when compared to other vitamin C’s
(Segnall et al., 2008). The AG therefore benefits the skin since it provide the skin with a stable
form of vitamin hence one need not to worry about the product breaking down in air, heat and
light.
The AG however has one foreseeable detriment whereby it will add glucose in the skin in the
process of breaking into L-ascorbic acid and glucose. This may lead to formation of advanced
glycation endpoint that can age or harden collagen. Studies by Shou-chieh et al. (2004) reported
AG levels in marketing facial cosmetics ranging from 1.89-1.98 %. The permissible limit is 2%
(DHEY, 2000).
2.4 Methods of analysis
2.4.1 Method of analyzing mercury
2.4.1.1 Introduction
Mercury levels in biological and environmental samples are usually determined by cold vapour
atomic absorption spectrometry (CV-AAS) (Mester and Sturgeon, 2003), cold vapour atomic
fluorescence spectrometry (CV-AFS) (Mester and Sturgeon, 2003), inductively coupled plasma
mass spectrometry (ICP-MS) (Horwitz, 2001) and direct analysis by thermal decomposition
(Bingol and Akcay, 2005). Cold vapour atomic absorption spectroscopy (CV-AAS) was used
because all others exhibit poor sensitivity while ICP-MS involve special sample handling
including the addition of small amount of gold to pre-concentrate the mercury and the instrument
is not readily available. The Cold vapour atomic absorption spectroscopy (CV-AAS) instruments
25
are more sensitive, more automated, smaller, faster, less expensive, provide improved detection
limit of a few parts per trillion because the entire mercury sample is introduced into the
absorption cell within a few seconds. The detection limit for mercury by this cold vapor
technique is approximately 0.02 ppm (Doreen, 2010).
2.4.1.2 Theory of atomic absorption spectroscopy
Atomic absorption spectroscopy (AAS) is a technique for determining the concentration of a
particular metal element in a sample and analyze over 62 different metals in a solution.
Typically, the technique makes use of a flame to atomize the sample, but other atomizers such as
a graphite furnace and cold vapour devices can also be used in flame atomization. Three steps
are involved in turning a liquid sample into an atomic gas. The steps are; desolvation; where the
liquid solvent is evaporated and the dry sample remains, vaporization; this is where the solid
sample is vaporized to a gas and finally volatilization; where the compounds making up the
sample are broken into free atoms (Skoog and Leary, 1992).
The flame is arranged such that it is laterally long (usually 10cm). The height of the flame must
also be controlled by controlling the flow of the fuel mixture. A beam of light from a hollow
cathode lamp is focused through the flame at its longest axis (the lateral axis) onto a detector
placed a head of the flame. The lamp consists of cylindrical metal cathode containing the metal
for excitation, and an anode. When a high voltage is applied across the anode and cathode, the
metal atoms in the cathode are excited into producing light with a certain emission spectrum
(Skoog and Leary, 1992).
26
The type of hollow cathode tube depends on the metal being analyzed. For analyzing the
concentration of copper, a copper cathode tube would be used, and likewise for any other metal
being analyzed. The cathode produces specific radiations that are absorbed by atoms in the flame
thereby exciting electrons to higher orbitals for an instant by absorbing a set quantity of energy
(a quantum). This amount of energy is specific to a particular electron transition in a particular
element (Skoog and Leary, 1992). As the quantity of energy put into the flame is known, and the
quantity remaining at the other side (at the detector) can be measured, it is possible to calculate
how many of these transitions took place, and thus get a signal that is proportional to the
concentration of the element being measured using Beer-Lambert’s law ((Khopkar, 2004). Beer-
Lambert’s law relates absorbance, a to the concentration of metallic atoms in the atom cell, c as
follows;
LogT-1
= a b c………………………………………………………………………………..Eq 2.1
Where:
a is the absorptive in grams per litre-centimetre
b is the atom width in centimeters
c is the concentration of atoms
The AAS involves the measurement of the drop in light intensity of initial radiation Io to final
radiation I depending on the concentration of the metal. Modern instruments automatically
convert logarithmic values into absorbance (Nollet, 2011). Figure 6 below illustrates AAS
instrumentation.
27
Figure 2:6 Diagram to illustrate instrumentation of AAS (Skoog and Leary, 1992).
2.4.1.3 The cold vapour atomic absorption spectroscopy (CV-AAS)
Most elements do not exist in free states in environment or biological samples, and therefore
require heat to free atoms. The only notable exception to this is mercury. Free mercury atoms can
exist at room temperature and, therefore, mercury can be measured by atomic absorption without
a heated sample cell, hence cold vapour atomic absorption spectroscopy technique (Skoog and
Leary, 1992). In the cold vapor mercury technique, mercury is chemically reduced to the free
atomic state by reacting the sample with a strong reducing agent like stannous chloride or
sodium borohydride in a closed reaction system. The volatile free mercury is then driven from
the reaction flask by bubbling air or argon through the solution. Mercury atoms are carried in the
gas stream through tubing connected to an absorption cell, which is placed in the light path of the
atomic absorption spectrometer. Sometimes the cell is heated slightly to avoid water
condensation but otherwise the cell is completely unheated.
28
As the mercury atoms pass into the sampling cell, measured absorbance rises indicating the
increasing concentration of mercury atoms in the light path. Some systems allow the mercury
vapor to pass from the absorption tube to waste, in which case the absorbance peaks and then
falls as the mercury is depleted. The highest absorbance observed during the measurement will
be taken as the analytical signal. The absorbance will rise until an equilibrium concentration of
mercury is attained in the system. The absorbance will then level off, and the equilibrium
absorbance is used for quantization. The amount of mercury is determined by measuring the
absorption at the mercury resonance wavelength of 253.7 nm (Khopkar, 2004).
2.4.2 Analytical methods for organic lighteners
Organic based active skin lightening compounds can be determined by several analytical
techniques such as flow injection analysis, kinetic spectrophotomertry, gas chromatography mass
spectrometry (GC-MS), differential pulse voltametry, and capillary electrochromatography
(Doreen, 2010). Although gas chromatography is widely used and is a powerful chromatographic
method, it is limited to compounds that have a significant vapour pressure at temperatures up to
about 200 atmospheres. Thus compounds with high molecular weight and high polarity cannot
be separated by gas chromatography (Doreen, 2010).
High-performance liquid chromatography (HPLC) is a chromatographic technique used to split a
mixture of compounds in the fields of analytical chemistry, biochemistry and industrial. The
main purposes for using HPLC are for identifying, quantifying and purifying the individual
components of the mixture (Bassam and Rasool, 2012). The HPLC play an important and critical
29
role in the field of analysis since it can be used to test the products and to detect the raw
ingredient used to make them that is it can do both qualitative and quantitative analysis (Bassam
and Rasool, 2012). Moreover it has the advantages of using relatively small amounts of the
solvent, it is rapid and can accomplish difficult separation (Harada et al., 2001).
HPLC, a powerful tool in analysis uses the same principles as in thin layer chromatography and
column chromatography. Chromatography has a stationery phase (a solid or a liquid supported
on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationery
phase and carriers the components of the mixture with it. Different components flow at different
rates based on their polarity. In thin layer chromatography, the stationary phase is a thin layer of
silica gel or alumina on a glass, metal or plastic plate. Column chromatography works on a much
larger scale by packing the same materials into a vertical glass column.
High performance liquid chromatography is therefore a highly improved form of column
chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is
forced through under high pressures of up to 400 atmospheres making it faster and allows one to
use small particle size for the column packing material which gives a much greater surface area
for interactions between the stationary phase and the molecules flowing past it. This allows a
much better separation of the components of the mixture. The other major improvement over
column chromatography concerns the detection methods used, making it sensitive and automated
(Fifield and Kealey, 1995).
There are two types of HPLC depending on the polarity of the solvent and the stationery phase;
normal HPLC and reversed HPLC. Normal is essentially the same as column chromatography.
30
Although it is described as "normal", it isn't the most commonly used form of HPLC. In the
normal HPLC, the column is filled with tiny silica particles, and the solvent is non-polar. Polar
compounds in the mixture being passed through the column will stick longer to the polar silica
than non-polar compounds will. The non-polar ones will therefore pass more quickly through the
column (Fifield and Kealey, 1995).
In reversed HPLC, the column size is the same as in normal HPLC, but the silica is modified to
make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or
18 carbon atoms in them. A polar solvent is used where there will be a strong attraction between
the polar solvent and polar molecules in the mixture being passed through the column hence
polar molecules that will travel through the column more quickly. Reversed phase HPLC is the
most commonly used form of HPLC and figure 7 shows the major steps followed in HPLC
(Doreen, 2010).
Figure 2:7 Flow scheme for HPLC (Skoog and Leary, 1992).
31
After injection of the sample the time taken for a particular compound to travel through the
column to the detector is called retention time. This time is measured from the time at which the
sample is injected to the point at which the display shows a maximum peak height for that
compound. Different compounds have different retention times. For a particular compound, the
retention time will vary depending on the pressure used (because that affects the flow rate of the
solvent), the nature of the stationary phase (not only what material it is made of, but also particle
size), exact composition of the solvent and the temperature of the column. This means that
conditions have to be carefully controlled if retention times are used as a way of identifying
compounds.
There are several ways of detecting when a substance has passed through the column. Common
methods use ultra-violet absorption since many organic compounds absorb UV light of various
wavelengths. The amount of light absorbed will depend on the amount of a particular compound
that is passing through the beam at the time. The output will be recorded as a series of peaks each
one representing a compound in the mixture passing through the detector and absorbing UV
light. As long as the conditions on the column are carefully controlled, retention times can be
used to identify the compounds present provided, retention times for pure samples of the various
compounds are measured under same identical conditions as for the mixture. Peaks can also be
used as a way of measuring the quantities of the compounds present since the peak area is
directly proportional to the concentration of compound of that peak (Doreen, 2010).
Achieng et al (2011) analysed using HPLC with Intertsil ODS -3v 250 mm×4.6 mm (5µm
particle) column and a mobile phase of acetonitrile: buffer mixture dector was set at a(1:99 v/v) .
The detector was set at a wavelength of 270 nm. With this analysis, MAP recorded a level of 1.5
32
% while arbutin recorded a level of 2 %. Shou-chieh et al. (2004). In this study using HPLC, the
column used was Cosmosil 5 CI8-AR-II, the mobile phase of a mixture of 0.05 M KH2PO4
buffer solution (pH 2.5) and methanol (99.1v/v) and UV detector set at 280 nm to analyze
arbutin, kojic acid, ascorbyl glucoside, magnesium ascorbyl phosphate and hydroquione.
33
CHAPTER THREE
3 MATERIALS AND METHODS
3.1 Research design
Purposive non probability sampling design was used in this study where the cases best
contributing to the information needs of the study were selected (Khopkar, 2004). In this design
samples were purchased from the various outlets and levels of mercury, hydroquinone, arbutin,
ascobyl glucoside, kojic acid and magnesium ascorbyl phosphate determined.
3.2 Sampling
Forty six skin lightening facial creams and fourteen skin lightening soaps were purchased from
small outlets in Nairobi and Kisii based on their availability. There were twenty six skin
lightening creams and eight skin lightening soaps that were sourced from Kisii and the rest from
Nairobi.
3.3 Chemical, reagents and solvents
Chemicals, reagents and solvents used were of analytical grade. Analar standards of mercury
chloride solution, pyridoxine solution, hydroquinone, arbutin, magnesium ascorbyl phosphate,
kojic acid and ascorbyl glucoside were sourced from Sigma-Aldrich. Solvents for separation
were of HPLC grade. Concentrated nitric acid, concentrated hydrochloric acid, sulphuric acid,
stannous chloride and potassium permanganate were sourced from Thomas Baker Chemicals
Ltd, Mumbai, India. Distilled water was used throughout.
34
3.4 Cleaning of apparatus
All apparatus were soaked in detergent solution overnight, rinsed and then soaked in 10 %
analytical grade nitric acid overnight and then rinsed with distilled water. The glassware were
dried in an oven at 105 0C.
3.5 Instrumental conditions of operation
Atomic absorption spectrophotometer with mercury vaporizer unit (model MVU-IA) was used to
analyze mercury. Analysis was done at a wavelength of 253.6 nm, slit width of 0.7 nm, detection
limit of 0.0002 ppm, and an optimum working range of 80-200 ppm. The HPLC system
(Shimadzu) consisted of reversed-phase column (Cosmosil 5 C18- AR-II) and photodiode array
detector (LC-10AS- Shimadzu) was used to analyze arbutin, kojic acid, ascorbyl glucoside,
magnesium ascorbyl phosphate and hydroquione. Mobile phase consisted of the mixtures of 0.05
M KH2PO4 buffer with methanol in the ratio of 99:1. The flow rate was 0.9 mL/min, pressure
ranged between 68-75 bars and the detecting wavelength was set at 280 nm. The volume for each
injection was 20 μL.
3.6 Laboratory procedures
3.6.1 Preparation of mercury standards
Mercury standard stock (1000 mgL-1
) solution was used to prepare serial standard solutions; 10
ppm, 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm. Standard solutions of stannous chloride
solution was prepared by dissolving 20 g of the stannous chloride (SnCl2.2H20) salt in 40 mL of
1 M hydrochloric acid and then the solution diluted to 200 mL total volume. This solution was
used as a reducing agent.
35
3.6.2 Preparation of other standards
Magnesium ascorbyl phosphate (20.0 mg/mL) was prepared by dissolving 2 g of magnesium
ascorbyl phosphate in 100 millilitre of water, ascorbyl glucoside (10.0 mg/mL) by dissolving
0.5 g of ascorbyl glucoside in 50 millilitre of water, arbutin (10.0 mg/mL) 1 g in 100 millilitre
of water, hydroquinone (5.0 mg/mL) 0.5 g of hydroquinone in 100 millilitre of water, and kojic
acid (2.0 mg/mL) dissolving 0.2 gin 100 millilitre of water, All these were prepared as the
standard stock solutions. The stock solutions were diluted using de-ionized water to prepare a
series of standard solutions; magnesium ascorbyl phosphate (200, 400, 600, 800 and 1000),
ascorbyl glucoside (100, 200, 300, 400, and 500), kojic acid (20, 40, 60, 80 and 100), arbutin
(100, 200, 300, 400 and 500) while series standard solutions of hydroquinone were (50,100, 150,
200 and 250 (Shou-chieh et al., 2004). Internal standard stock solution of pyridoxine with a
concentration of 1.0 mg/mL was prepared by dissolving 1 g of pyridoxine solid in 1000 millilitre
of water.
3.6.3 Method validation
The accuracy of CV-AAS was investigated by using the spiking method. Spiking is an addition
method where the standard is directly added to the aliquots of the sample to be analyzed. This
method is used in situations where sample matrix also contributes to the analytical signal (matrix
effect) thus making it impossible to compare the analytical signal between sample and standard
in the calibration curve approach. In this study samples were spiked with a known amount of
standards. Table 1 shows the concentration of unspiked samples, the known concentration of the
standard added to each sample and the concentration of the spiked samples. Results were used to
calculate the percentage recovery (Equation 3.1).
36
R%= (Cf – Cu /Ca) 100.....................................................................................................Eq 3.1
Where:
R- % recovery
Cf- Concentration of the sample after spiking
Cu- Concentration of the sample before spiking
Ca-Concentration of standard used for spiking
Table 3:1 Concentration of spiked, unspiked and standards added
Skin lightener
Concentration of
unspiked sample
Mean ± SE(ppm)
Concentration of
Standard added
to sample(ppm)
Concentration of
spiked sample.
Mean±SE.(ppm)
MAP
Ag
1.65±0.01
0.95±0.01
300
150
301.000± 0.01
149.112±0.02
HQ 0.51±0.00 75 75.100±0.01
KA 0.96±0.05 30 31.202±0.01
AR 1.33±0.01 150 148.122±0.02
Hg 47.87±0.01 10 58.099±0.00
Recovered amount after digestion of the spiked samples was used to calculate percentage
recovery (Borosova et al, 2002). The mean recovery of the matrix was evaluated at 95%
confidence level (Miller and Miller, 1988). Limit of detection (LOD) was calculated using
equation (3.2) (EURACHEM guide,1998) using the determined absorbance values for 10
replicates of the blank solution, then transformed into concentration values in order to be
compared with the data obtained from the calibration curve.
LOD x blank + 3sblank..................................................................................................................(3.2)
x blank –mean absorbance obtained with the blank solutions: sblank-standard deviations of the
blank:xi- values of the blank solutions. Results of LOD are shown in table 4.1.
37
High performance liquid chromatography (HPLC) was validated using calibration and precision.
Calibration is a general method for determining the concentration of a substance in an unknown
sample by comparing the unknown to a set of standards samples of known concentration.
Precision is the degree to which repeated measurement under unchanged conditions show same
results. In calibration, five different concentrations of standard solutions were prepared from the
stock solutions and 40 μg/mL of an internal standard was added and analyzed, respectively.
Linear regression equations and correlation coefficients were obtained from the plots of
concentration versus peak area ratio of standard to internal standard solutions. In precision, the
standard stock solution and the internal standard stock solution were quantified precisely and
diluted with distilled water to three different concentrations in µg/ML within the standard
calibration range, and then 40 μg/mL of internal standard was added to each standard solution.
The samples were analyzed in triplicates by HPLC. The standard deviation and relative standard
deviation were then calculated.
3.6.4 Sample preparation
For analysis of mercury, 1.000 g of each cream and each soap was weighed accurately into a
conical flask. A 20 mL acid mixture of concentrated nitric and hydrochloric acid in the ratio 3:1
was added to the sample. The conical flask was covered and mixture heated at 200 oC until there
were no more brown fumes produced. The solution was cooled, filtered through Whatman paper
(Number 1) into a 50 mL volumetric flask and then made up to the mark using distilled water
and used for analysis.
For analysis hydroquine, arbutin, kojic acid, magnesium ascorbyl phosphate and ascorbyl
glucoside, 1.000 g of each cream and each soap was weighed precisely, mixed with an
38
appropriate amount of the internal standard stock solution and diluted with twenty-fold of 0.05
M KH2PO4 buffer (pH 2.5). A homogeneous suspension was obtained after 30 min of
sonification. The suspension was filtered and the filtrate further diluted with 0.05 M KH2PO4
buffer (pH 2.5) until the final concentrations of the whitening ingredients was within the
standard calibration range and the internal standard 40 μg/mL before HPLC analysis. The ratio of
the peak area of the sample to the internal standard was compared to determine the concentration
of each sample.
The mean levels of skin lighteners analysed by HPLC, were transformed from mg/g into
percentage since their results are always recorded in percentage (Shou-chieh et al., 2002). The
transformation was done using the following formula Xmg/1000mg×100. This was done for
ascobyl glucoside, arbutin, magnesium ascorbyl phosphate, hydroquinone and kojic acid in skin
lightening soaps. To determine the final concentration of mercury in the samples, the
concentration values from the calibration curve in appendix 6 was multiplied by the dilution
factor which was 50.
3.7 Data analysis
Mean values obtained for mercury, hydroquinone, arbutin, ascorbyl glucoside, kojic acid and
magnesium ascorbyl phosphate in the two types of skin cosmetics were compared by using a
one-way ANOVA with post hoc multiple comparisons in each column at significant differences
(p<0.05). The standard t-test was used to compare the means in levels of the lighteners between
the two types. Whenever a significance difference exists, the means were compared at P<0.05
significance level (Salvador and Chisvert, 2007).
39
CHAPTER FOUR
4 RESULTS AND DISCUSSION
4.1 Introduction
The levels of mercury, hydroquinone, arbutin, kojic acid, ascorbyl glucoside and magnesium
ascorbyl phosphate in creams and soaps were determined and the results obtained are presented
and discussed in the following sections.
4.2 Method validation
The analytical methods used were validated using recovery tests and regression analysis.
Recovery is a method of validation that is used to test the reliability of the results. It is usually
calculated in percentage. Accepted values range from 95 % to 110 % (Miller and Miller, 1988).
The percentage recovery as shown in table 4:1 lied within the range 97.86-103 %. This shows
that the method is accurate and fit for analysis of the above parameters (Miller and Miller, 1988).
Calibration curves were drawn for all compounds analyzed and their curves presented in the
appendices I to 6, while the calibration curve for arbutin as an example is shown in figure 4:1.
40
Figure 4:1 Calibration curve for arbutin
Regression equations were then determined for every calibration curve. In all the cases
regression was found to be above 0.978. The regression and regression equations are shown in
the table 4:1. The detection limits were also calculated as shown in table 4:1.The result of the
validation parameters are given in table 4:1.
Table 4:1 Method validation result
Analyte R2 Regression equation LOD (ppm) % Recovery
Hg 0.978 y=219x - 0.028 15.74 102.29 MAP 0.998 y=456.8x -7496 1.00 99.78
AG 0.999 y=991.7x -57836 5.57 98.70
HQ 0.997 y=16802x -72553 1.2×10-4
99.45
ART 0.987 y=8951x -84600 0.92 97.87 KA 0.981 y=56973x-32685 0.05 100.81 R
2 - Correlation coefficient LOD-Limit of detection
41
4.3 Mean levels of skin lighteners in facial creams
The mean levels (± standard deviation) of mercury, hydroquinone, arbutin, kojic acid,
magnesium ascobyl phosphate and ascobyl glucoside in different creams were determined and
results shown in table 4:2.
Table 4:2 Mean levels various skin lighteners in skin lightening creams
Sample name
(n=3)
MAP
(Mean±SD)
AG %
(Mean±SD)
KA %
(Mean±SD)
ART %
(Mean±SD)
HQ %
(Mean±SD)
Hg (ppm)
(Mean±SD)
Gold touch/oil skin <DL 5.83±0.00a <DL 11.37±0.06c <DL 47.87±0.00
Neovate <DL <DL <DL <DL 0.002±0.00a <DL
Movate 1.65±0.00a <DL <DL 1.09±0.00a <DL <DL
Gold touch/dry skin 1.64±0.00a <DL <DL 11.09±0.29c <DL 469.41±18.27
Biocarote 1.65±0.01a 5.83±0.00a <DL 49.19±0.67e <DL 63.67±11.85
Tentclair <DL 7.31±0.06a <DL <DL 0.05±0.00b 161.38±4.05
Miss caro 1.65±0.00a 5.85±0.00a 0.06±0.00a <DL <DL 82.26±22.76
pure skin <DL 38.66±1.66b 15.00±0.65 <DL <DL 99.59±2.65
skin succe <DL <DL <DL 4.51±0.07ab <DL 132.05±6.61
F/L/Ayuve 4.19±0.12a <DL <DL <DL <DL 147.84±1.54
white moon 1.67±0.00a 5.84±0.00a <DL 107.62±0.6g <DL 271.79±72.81
Cocoderm 1.65±0.00a 5.84±0.00a <DL 40.37±5.36d <DL 277.85±35.49
Rico <DL 6.64±0.03a <DL <DL 0.02±0.00a 271.25±22.31
caro 7 15.61±0.1c <DL 0.06±0.00a <DL <DL 233.83±29.21
G&G <DL 5.83±0.00a <DL <DL <DL 253.64±17.20
Naturally fair <DL 5.83±0.00a <DL 11.33±0.74c <DL 299.30±27.45
Bio 26 <DL <DL <DL <DL <DL 261.35±13.36
maxi light <DL <DL <DL 1.52±0.11a <DL 333.69±36.32
F/L/MULT <DL 61.47±12.82d <DL <DL <DL 327.36±3.13
idole medical 1.65±0.00a <DL <DL <DL <DL 357.63±99.58
Epicliar <DL <DL 0.06±0.00a <DL <DL 293.25±22.41
Neucliar 1.65±0.00a <DL 0.96±0.05b <DL <DL 287.75±19.64
Carolight 1.65±0.00a 5.84±0.00a <DL 51.00±4.09e <DL 297.38±28.88
F/L/Fairness 1.64±0.00a 5.83±0.00a <DL 0.95±0.00a <DL 339.20±18.22
Idole gold 40.07±1.8d <DL <DL <DL <DL 315.54±21.48
Betasol 1.65±0.00a 5.84±0.00a 7.09±1.25b <DL <DL 325.45±9.79
Elagance/rico 40.48±4.5d 5.84±0.00a 0.58±0.00a <DL <DL 326.82±33.29
42
MAP-Magnesium ascobyl phosphate, AG-Ascorbyl glucoside, KA-Kojic acid, ART-Arbutin, HQ-Hydroquinone,
Hg-Mercury, DL-Detected Limit Values with different letters (superscript) indicates significant differences
(p<0.05).
4.3.1 Mercury (Hg)
From the results, mercury was detected in all the forty six skin lightening creams except two.
The levels of mercury differed significantly among the different skin lightening creams studied.
Levels ranged from 47.87 ± 0.00 ppm to 513.06 ± 26.74 ppm.
These levels are far above the maximum recommended limit of 1 ppm set by WHO (2007) and
therefore the skin lightening creams are not safe for use. Analysis carried out on facial creams
produced in Thailand, Lebanon and England showed highest levels of mercury ranging from
1281 ppm to 5650 ppm (Al-Saleh et al., 2004). California Medical Officials carried a study on
vul/herbal 1.65±0.00a <DL <DL <DL <DL 345.91±47.53
Miki 1.65±0.00a 5.83±0.00a 10.92±0.19d <DL <DL 311.96±23.59
Faemark <DL <DL 1.61±0.12c <DL <DL 318.84±25.77
Sivoclair <DL 6.19±0.00a <DL <DL <DL 318.56±29.76
Princess <DL <DL <DL <DL <DL 319.66±12.90
Diproson <DL 5.84±0.00a <DL 6.59±0.80b <DL 340.02±16.11
Siri 1.67±0.00a <DL <DL <DL <DL 341.40±11.08
G/T/Normal <DL 50.90±3.24c <DL <DL 0.004±0.00a 330.39±15.31
Mediven <DL <DL <DL <DL <DL 549.37±151.78
Fairness <DL <DL 1.66±0.15c <DL <DL 409.90±44.55
Alovera <DL 5.84±0.00a 0.06±0.00a <DL 0.02±0.01a 393.39±41.44
Peuclair <DL <DL <DL <DL <DL 429.70±20.45
Fair&handsome <DL <DL <DL <DL <DL 409.62±16.70
Epiderm cream <DL 58.34±0.00a <DL 6.16±0.06b <DL 419.80±30.95
No mark <DL <DL 0.90±0.02b <DL <DL 398.34±21.43
Extra clair 1.64±0.00a 58.35±0.00a <DL 87.23±1.13f <DL 483.35±25.12
Max/clear/lemon <DL 58.33±0.00a 0.91±0.01b <DL <DL 462.71±18.40
Fashion fair <DL <DL <DL 9.54±0.44c 0.004±0.00a 472.06±7.87
Idole/lemon <DL 5.84±0.00a <DL <DL 0.030±0.00a 513.06±26.74
Fairever 1.65±0.00a 5.84±0.00a <DL <DL <DL 434.60±9.61
p-value <0.001 <0.001 <0.001 <0.001 0.001 <0.001
43
some facial lightening creams which recorded mercury levels of 20,000 to 56,000 parts per
million (Melisa and Jay, 2009). Claudia (2011) recorded levels of mercury between 878 and
36,000 ppm in six lightening creams studied in Mexican. In Saudia Arabia a study by Al-Ashban
(2006) on skin lightening creams recorded levels of mercury between 2.46 to 23222 ppm while
Voegborio et al. (2008) in the same country recorded mercury levels between 1.18 to 5650 ppm
in 17 skin lightening cream samples. In Kenya a study by Maina (2013) on mercury in seven
different brands of skin lightening creams showed the following levels of mercury in ppm:
20,867-33,508, 14,330-22,167, 3,742-9,949, 5,444-32,270, 8,837-16,187,1,182-1,969 and 3,743-
5,444. United States of America Food and Drugs Association (USFDA) and World Health
Organization (WHO) allows levels of less than 1 ppm (WHO, 2007; FDA, 2009).The levels
recorded in above studies are higher than the levels recorded in this study. This is probably due
to the banning of use of mercury in cosmetic (FDA, 2009; ZMWG, 2010) hence manufacturers
have started reducing the levels of mercury being added to cosmetics. It is possible therefore that
the high levels of mercury in creams that originate from certain parts of the world can be
attributed to the unavailability of regulations and laws against the manufacture, export, import
and sale of mercury containing creams.
44
4.3.2 Hydroquinone (HQ)
Hydroquinone was detected in only eight out of the forty six creams studied, with the mean
levels ranging from 0.002 ± 0.00 % to 0.05 ± 0.00 %. Figure 4.2 below show mean levels of
hydroquinone.
Figure 4:2 Mean levels of hydroquinone in creams
The levels differed significantly among the creams studied (p<0.05). The levels are much lower
than the maximum allowable levels of 2 % by WHO (2007). This means that most manufacturers
are not adding hydroquinone to the cosmetic since it has been banned (WHO, 2007; ZMWG,
2010). Study on levels of hydroquinone and mercury in some skin lighteners in Nigeria recorded
the levels of hydroquinone from 0.001 to 3.45 % (Doreen, 2010). These levels are higher than
the ones recorded in this study. Terer et al. (2013) reported the levels of hydroquinone in body
creams and lotions of between 0.00025 % - 0.03457 % in their study on levels of hydroquinone
45
in body creams and lotions sold in retail outlets in Baraton Kenya. The levels recorded by Terer
et al are comparable with those levels recorded in this study.
4.3.3 Arbutin (ART)
Arbutin was detected in thirteen out of the forty six facial creams analyzed and the levels ranged
from 0.95 ± .0.02 % to 107.00 ± 0.06 %. The levels of arbutin are shown in figure 4.3 below
Figure 4:3 Mean levels of arbutin in creams
The levels differed significantly among the various types of lightening creams (p<0.05). Nine out
of the sixteen lighteners with arbutin had levels below the maximum permissible limit with the
rest having levels above the recommended limit of 7 % (DHEY, 2000; WHO, 2007). A study
done in Taiwan recorded levels of between 2.00 % - 2.07% of arbutin in the marketed skin
46
lighteners (Shou-chieng et al, 2002). These levels are not comparable to the levels obtained in
this study.
4.3.4 Kojic acid (KA)
Kojic acid was detected in thirteen of the forty six creams analyzed. There was a significant
difference in the mean levels of kojic acid which ranged from 0.06 ± 0.00 % to 15.00 ± 0.65 %.
Only three out of the fourteen creams had levels above the permissive limits of 2 % (DHEY,
2000; WHO, 2007). Levels of kojic acid are shown in figure 4.4 below.
Figure 4:4 Mean levels of kojic acid in creams
In Taiwan a study done to investigate the levels of some lightening facial creams recorded levels
of kojic acid ranging from 0.93- 1.00 % (Shou-chieh et al., 2002).These levels are comparable to
the majority of the results obtained for kojic acid in this study.
47
4.3.5 Magnesium ascorbyl phosphate (MAP)
Magnesium ascorbyl phosphate (MAP) was detected in twenty one of the forty six creams
analyzed. The mean levels ranged from 1.64 % ± 0.01 to 40.48 ± 0 .5 %. Levels of magnesium
ascorbyl phosphate are shown in figure 4.5
Figure 4:5 Mean levels of magnesium ascorbyl phosphate in creams
There was significant difference among the levels of magnesium ascorbyl phosphate in
different skin lightening creams analyzed(p<0.05). Four of the creams had levels above the
maximum recommended limit of 3 % in skin lighteners (DHEY, 2000; WHO, 2007). Shou-chieh
et al. (2002) reported levels of MAP in creams ranging from 0.93- 1 % in Nigeria. Similarly
Achieng et al. (2011) from the same country reported levels of MAP in some creams and lotions
48
to be between 1.35-1.47 % which was slightly higher than that of Shou-chieh et al. (2002). The
levels of Shou-chieh et al .(2002) are lower than the recorded levels in this study, while those of
Achieng et al. (2011) are comparable with the levels of most creams that were analyzed in this
study.
4.3.6 Ascorbyl glucoside) (AG)
Ascorbyl glucoside (AG) was found to be contained in twenty six out of the forty six creams
analyzed. The mean levels ranged from 5.83 ± 0.00 % to 61. 47 ± 0.00 %. Figure 4.6 show the
mean levels of ascorbyl glucoside in creams.
Figure 4:6 Mean levels of ascorbyl phosphate in creams
The levels of ascorby glucoside differed significantly among the skin lightening creams analyzed
(p<0.05). All creams had levels of AG above the set limits of 2 % (Shou-chieh et al., 2002). The
49
levels of AG recorded in facial skin lightening creams marketed in Taiwan ranged from 0.93-
1.00 % (Shou-chieh et al., 2002). These levels were much less than the levels obtained in this
study.
4.4 Skin lightening compounds in soaps
Three out of six active compounds were detected in skin lightening soaps (Table 4.3). The results
for ascorbyl glucoside and kojic acid were transformed from mg/g to percentage using formula
Xmg/1000mg×100 since from the literature review, these compounds are reported in percentage.
Table 4:3 Mean levels of mercury (ppm) ascorbyl glucoside(%) and kojic acid (%) in skin
lightening soaps
Sample name AG %(Mean±SD)
KA%(Mean±SD) Hg ppm (Mean±SD)
Miss caro 5.84±0.00bc
3.31±0.19ab
700.73±30.95a
Carolight <DL 1.49±0.09ab
578.25±77.63a
Carambolla <DL <DL 582.93±16.96a
Rico 5.83±0.00ab
1.36±0.10ab
587.33±4.76a
Mekako 5.84±0.00d 3.50±0.44
c 11048.75±1.32
f
Jaribu 5.84±0.00d 1.37±0.03
ab 10919.70±152.81
f
Vitamin C 5.83±0.00a 1.19±0.38
a 10709.56±63.40
e
Sivoclair 5.84±0.02e 1.56±0.0800
ab 10476.24±83.13
d
Magic mix <DL 1.74±0.15b 10584.08±74.93
d
Topshirly 5.83±0.00a 1.39±0.05
ab 10613.24±55.67
de
Dark spot remover <DL 1.25±0.04ab
10449.83±87.26cd
Super baby face <DL <DL 10310.36±192.13c
Acne soap 5.84±0.00c 1.12±0.086
a 10315.31±87.20
c
Peuclair <DL 1.50±0.01ab
9174.20±221.09b
p-value 2.05 x 10-11
4.49 x 10-14
1.36 x 10-44
AG-Ascorbyl glucoside, KA-Kojic acid, Hg-Mercury, DL-Detected Limit. Mean±SD
followed by different letters (superscript) indicates significant differences (p<0.05).
Mercury was detected in all the soap analyzed and the levels ranged from 582.93 ± 16.96 ppm to
11048.75 ± 1.32 ppm. All the samples had concentration of mercury levels higher than the
maximum allowable levels in cosmetics of 1 ppm (WHO, 2007), making the skin lightening
soaps not safe. In Tanzania a study of two skin lightening soaps namely jaribu and rico recorded
50
mercury levels of 6200 ppm and 6900 ppm respectively (Peter, 2008). These levels are above the
set limit of 1 ppm in cosmetics products (WHO, 2007). From this study four skin lightening
soaps had levels of mercury below the levels Peter (2008) recorded while eight soaps from this
study had levels of mercury above what Peter recorded.
Ascorbyl glucoside was detected in eight out of the fourteen soaps analysed. The levels ranged
from 5.83 ± 0.00 % to 5.84 ± 0.00 % .These levels were almost constant in all the soaps that
were detected to have ascorbyl glucoside. The levels are above the set limit of 2 % in cosmetic
products (DHEY, 2000; WHO, 2007). Results from this study are also much higher than those
reported from cosmetics in Taiwan. Ascorbyl glucoside does not have adverse effects if used in
the right concentrations. However, it causes exfoliation for sensitive skin because of the acidity
effects of vitamin C.
Kojic acid was detected in twelve soaps out of the fourteen soaps analyzed. The levels ranged
from 1.12 ±0.086 % to 3.50 ± 0.04 %. Out of the twelve soaps containing kojic acid, only two
had levels above the set limits of 2 % (DHEY, 2000; WHO, 2007). Most of the levels obtained in
this study compare well with those reported for cosmetics in Taiwan (Shou-chieh et al., 2002).
In this study, six parameters were looked, at these are: hydroquinone, arbutin, kojic acid,
magnesium ascorbyl phosphate, ascorbyl glucoside and mercury. Out of the six, kojic acid,
ascorbyl glucoside and mercury were detected in both skin lightening facial creams and soaps.
The result comparing their levels in soaps and creams are as shown in table 4.4.
51
Table 4:4 Comparison of the levels of Hg, AG and KA in facial creams and soaps
Independent t-test showed a significant difference between facial cream and Soaps for
Ascorbyl glucoside and Mercury (Tcalculated>t critical, 95% confidence level).
Table 4.5 shows a comparison of mean levels of mercury, ascorbyl glucoside and kojic acid in
facial creams and soaps with the same brand names for example peuclair cream and peuclair
soap. The letters in bracket after the levels indicate the name of the sample in table 4.2 for
creams and table 4.3 for soaps.
Table 4:5 Mean levels of Hg, AG and KA in facial creams and soaps with same brand
names
Mean levels of AG Mean level of KA Mean levels of Hg
Cream Soap Cream Soap Cream Soap
5.85±0.00a (ms) 5.84±0.00bc(ms) 0.06±0.00a(ms) 3.31±0.19ab(ms) 82.26±22.76(ms) 700.73±30.95a(ms)
6.64±0.03a(rc) 5.83±0.00ab(rc) <DL(rc) 1.36±0.10ab(rc) 271.25±22.31(rc) 587.33±4.76a(rc)
5.84±0.00a(sc) 5.84±0.02a(sc) <DL(sc) 1.56±0.0800ab(sc) <DL(sc) 10476.24±83.13dsc)
5.84±0.00a(cl) <DL(cl) <DL(cl) 1.49±0.09ab(cl) 297.38±28.88(cl) 578.25±77.63a(cl)
AG-Ascorbyl glucoside, KA-Kojic acid, Hg-Mercury. Values with different letters (superscript) indicates significant
differences (p<0.05). Ms-miss caro,rc-rico, sc-sivoclair and cl- carolight.
From the study as shown in table 4.5, only kojic acid, ascorbyl glucoside and mercury are
comparable between facial creams and soaps. There is a significant difference in the levels of
ascorbyl glucoside and mercury between facial creams and soaps for (p < 0.05). There was no
Lightener Creams Soaps P-value
AG (mg/mL) 110.73±16.74 58.36±0.01 0.002
KA (mg/mL) 23.56±6.92 17.33±1.32 0.381
Hg (µg/g) 341.17±14.89 7646.47±697.87 <0.001
52
significant difference between facial creams and soaps for kojic acid (p > 0.05). Although levels
of kojic acid are below the maximum set limits, there is fear that continues use of soaps
containing kojic acid may cause irritant contact dermatitis (Lewis, 2012).
The results obtained could be different from those of other authors due to the weather conditions,
geographical locations and human activities such as industrial and agricultural activities;
(Abulude et al., 2010; Sukender et al., 2012). The information from the package indicates that
most products are from outside Kenya with only three produced in Kenya. Most products were
from West African countries, others from Italy, Europe and United States of America. In view of
this therefore if our country cannot produce these products, KEBS should ensure the once that
are imported are analyzed for the levels of the lighteners to ensure that they are safe for use.
53
CHAPTER FIVE
5 CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
Results from the study showed that Magnesium ascorbyl phosphate was detected in twenty four
facial creams but was not detected in soaps; arbutin was detected in sixteen facial creams and
was not detected in soaps while hydroquinone was detected in eight facial creams but was not
detected in soaps. Mercury, ascobyl gucoside and kojic acid were detected in both facial creams
and soaps. Mercury was detected in all creams and in all soaps; ascorbyl glucoside was detected
in twenty seven creams and in eight soaps while kojic acid was detected in fourteen creams and
in twelve soaps.
Other conclusions from the study include those samples that had levels above the set limits.
Magnesium ascorbyl phosphate was above 3 % in five creams, ascorbyl glucoside was above 2
% in all creams, kojic acid was above 2 % in three creams, arbutin was above 7 % in seven
creams while mercury was above 1 ppm in all creams analysed except two. In soaps, ascorbyl
glucoside was above 2 % in all soaps, kojic acid was above 2% in two soaps while mercury was
above 1 ppm in all soaps analysed. Mercury levels were far much above the set limits in all soaps
and creams where it was detected. Hydroquinone was below the limits in all the creams
analysed. Only two creams had the levels below the limits of all the parameters measured hence
can be considered safe, however the LOD is above the set limit for mercury and ascorbyl
glucoside as shown in appendix 7. Actually most creams analysed had their source outside
Kenya and some of them might have been smuggled into the country and are illegally sold and
54
used since they did not have KEBS rubber stamp. This necessitates KEBS to be on watch to
establish which creams and of which quality are sold in our country.
Based on the objectives of the study and the result obtained from the study, most facial creams
contain high levels of skin lighteners with respect to WHO. Skin lightening soaps contain kojic
acid, ascorbyl glucoside and mercury. Skin lightening soaps contain higher levels of mercury
than in skin lightening creams. There were variations between facial creams and soaps in the
levels of all the skin lighteners detected.
5.2 Recommendations
5.2.1 Recommendations from the study
The study noted that most skin lighteners in both creams and soaps have high levels of lightening
compounds with respect to the permissible levels of cosmetic products and the following may be
recommended from this research.
1. Caution on the use of facial creams and soaps containing skin lighteners since it would
result in the increase of the levels of the lightening compounds in the human body has the
study indicate that some creams and soaps contain these compounds in levels above the
maximum permissible limits by WHO.
2. Members of the public should be sensitized on the effects of skin lightening creams and
soaps.
3. Levels of these lighteners should be monitored regularly by KEBS in skin lightening
products.
55
5.2.2 Recommendations for further work
1. Hair care cosmetics, other facial care cosmetics such as powders, eye shadows and eye
liners, lipsticks and lip glow, nail care cosmetics, fragrance products such as deodorants
and perfumes should be studied to determine the levels of lighteners in them.
2. Blood and urine of consumers of skin lightening products should also be analyzed for
skin lighteners.
56
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APPENDICES
Appendix 1: Calibration curve for kojic acid
Appendix 2: Calibration curve for magnesium ascorbyl phosphate
62
Appendix 3: Calibration curve for arbutin
Appendix 4: Calibration curve for hydroquinone
63
Appendix 5: Calibration curve for ascorbyl glucoside
Appendix 6: Calibration curve for mercury
64
Appendix 7: Limit of detection (ppm) for the parameters analyzed
ANALYTE Hg MAP AG HQ ART KA
LOD 15.74 1.0007 5.56522 0.00012 0.91537 0.05244