Assessment of levels of some active skin lightening compounds in selected facial creams and soaps in the Kenyan market

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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

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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

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DEDICATION

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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.

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ABSTRACT

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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

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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

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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

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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

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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

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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

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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

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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).

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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).

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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).

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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).

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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.

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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

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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.

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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.

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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

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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.

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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).

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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).

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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

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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).

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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).

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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

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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).

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(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

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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

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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.

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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.

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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).

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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

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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

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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).

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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 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

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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)

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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).

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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).

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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).

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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).

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% 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

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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±0.44c 11048.75±1.32f

Jaribu 5.84±0.00d 1.37±0.03ab 10919.70±152.81f

Vitamin C 5.83±0.00a 1.19±0.38a 10709.56±63.40e

Sivoclair 5.84±0.02e 1.56±0.0800ab 10476.24±83.13d

Magic mix <DL 1.74±0.15b 10584.08±74.93d

Topshirly 5.83±0.00a 1.39±0.05ab 10613.24±55.67de

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±0.086a 10315.31±87.20c

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).

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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).

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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

(64)

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).

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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.

Figure

Figure 2.1 Structure of hydroquinone

Figure 2.1

Structure of hydroquinone p.28
Figure 2.3 Structure of kojic acid

Figure 2.3

Structure of kojic acid p.32
Figure 2:6 Diagram to illustrate instrumentation of AAS (Skoog and Leary, 1992).

Figure 2:6

Diagram to illustrate instrumentation of AAS (Skoog and Leary, 1992). p.39
Figure 2:7 Flow scheme for HPLC (Skoog and Leary, 1992).

Figure 2:7

Flow scheme for HPLC (Skoog and Leary, 1992). p.42
Table 4:1 Method validation result

Table 4:1

Method validation result p.52
Figure 4:1 Calibration curve for arbutin

Figure 4:1

Calibration curve for arbutin p.52
Table 4:2 Mean levels various skin lighteners in skin lightening creams

Table 4:2

Mean levels various skin lighteners in skin lightening creams p.53
Figure 4:2 Mean levels of hydroquinone in creams

Figure 4:2

Mean levels of hydroquinone in creams p.56
Figure 4:3 Mean levels of arbutin in creams

Figure 4:3

Mean levels of arbutin in creams p.57
Figure 4:4 Mean levels of kojic acid in creams

Figure 4:4

Mean levels of kojic acid in creams p.58
Figure 4:5 Mean levels of magnesium ascorbyl phosphate in creams

Figure 4:5

Mean levels of magnesium ascorbyl phosphate in creams p.59
Figure 4:6 Mean levels of ascorbyl phosphate in creams

Figure 4:6

Mean levels of ascorbyl phosphate in creams p.60
Table 4:3 Mean levels of mercury (ppm) ascorbyl glucoside(%) and kojic acid (%) in skin lightening soaps

Table 4:3

Mean levels of mercury (ppm) ascorbyl glucoside(%) and kojic acid (%) in skin lightening soaps p.61
Table 4:5 Mean levels of Hg, AG and KA in facial creams and soaps with same brand names

Table 4:5

Mean levels of Hg, AG and KA in facial creams and soaps with same brand names p.63
Table 4:4 Comparison of the levels of Hg, AG and KA in facial creams and soaps

Table 4:4

Comparison of the levels of Hg, AG and KA in facial creams and soaps p.63

References

Related subjects : Skin Lightening