Journal of Cosmetics, Dermatological Sciences and Applications, 2012, 2, 224-228 Published Online September 2012 (
Qualitative and Quantitative Estimation of Hydroquinone
in Skin Whitening Cosmetics
Saima Siddique*, Zahida Parveen, Zeeshan Ali, Muhammad Zaheer
PCSIR Labs Complex, Lahore, Pakistan.
Email: *
Received February 1st, 2012; revised March 5th, 2012; accepted March 19th, 2012
Hydroquinone has been used for decades as a skin lightening agent. Its use in cosmetics has been banned as a result of
skin problems including contact dermatitis and ochronosis. A total of 22 samples of different skin whitening cosmetics
were collected from local market. They were analyzed by using thin layer chromatography and HPLC for qualitative
and quantitative determination of their hydroquinone contents. The hydroquinone was extracted from samples by using
96% ethanol and was subjected to TLC analysis. Eleven out of 22 samples were found to contain hydroquinone. The
HPLC analysis showed the concentration of hydroquinone ranged from 0.002% to 0.092% in the cosmetic samples.
Keywords: Hydroquinone; HPLC; Whitening Cosmetics; Thin Layer Chromatography
1. Introduction
Visible pigmentation in mammals results from the syn-
thesis and distribution of melanin pigment in the skin and
hair bulbs [1,2]. Melanin also plays a crucial role in the
absorption of free radicals generated within cytoplasm
and in shielding of the host from various types of ioniz-
ing radiations including UV [3]. However, the dark skin
caused by melanin accumulation is not considered cos-
metically pleasing to many people [4-6]. The increased
levels of melanin are also characteristic of a great num-
ber of skin diseases including melasma, solar lentigines,
and post-inflammatory hyperpigmentation. Thus, there is
an increasing desire for the development of skin whiten-
ing agents for both beauty and therapeutic purposes [7,8].
Tyrosine is the precursor for the synthesis of melanin.
Tyrosinase is the key and rate-limiting enzyme responsi-
ble for the conversion of tyrosine into melanin by me-
lanocytes in human skin [9,10]. Inhibition of the enzy-
matic activity of tyrosinase by competitive inhibitors
results in decreased or absent melanin synthesis by the
melanocytes in human skin [11,12].
Many compounds that bind to the tyrosinase active site
and inhibit melanin synthesis have been developed as
agents to lighten skin color, including hydroquinone [13].
Hydroquinone is the most conventional skin whitening
agent. However, it has numerous unfavorable effects
with long-term application, including irritative dermatitis,
melanocyte destruction, contact dermatitis and ochrono-
sis [14,15]. Its use has been recommended to ban in cos-
metics [16-18]; however, it is still being used in devel-
oping countries in skin lightening cosmetics. The aim of
the present study is to analyze different skin whitening
cosmetics present in the market for the determination of
their hydroquinone contents.
2. Experimental
Different samples of skin whitening creams were col-
lected randomly from the local market of Lahore District,
Punjab, Pakistan.
3. TLC Analysis
3.1. Sample Preparation
Two grams of sample was weighed in a 25 ml beaker and
15 ml of 96% (V/V) ethanol was added. The mixture was
homogenized on water bath at 60˚C for 10 min and then
cooled in an ice bath till the separation of fats occurred.
Finally it was filtered and filtrate was used for TLC analy-
sis. The same procedure was repeated for all samples.
3.2. Preparation of Reference Solution
It was prepared by dissolving 0.05 g of hydroquinone in
small amount of ethanol 96% (V/V) in 25 ml volumetric
flask and finally making up the volume up to the mark.
Note: The standard solution should be freshly prepared
as it is stable for less than one day at room temperature.
*Corresponding author.
Copyright © 2012 SciRes. JCDSA
Qualitative and Quantitative Estimation of Hydroquinone in Skin Whitening Cosmetics 225
3.3. TLC Procedure
TLC plates (20 × 20 cm) of 0.25 mm thickness were
prepared by using silica gel (90 g) and water (180 ml)
[16]. The plates were air dried and activated by heating
in an oven at 105˚C for an hour. The solvent system; n-
Hexane/Acetone, 3:2 was used [19]. Twenty micro litres
of each sample and standard solution were deposited on
plates and they were developed at room temperature in a
vertical separating chamber to the height of approxi-
mately 16 cm from the start. The chamber was previ-
ously saturated with the appropriate mobile phase (satu-
ration time was 1 hour). After drying, visualization was
performed in two ways:
1) In short UV light (254 nm);
2) Spraying with 0.2% ethanolic dichlorofluorescein;
chromatograms were interpreted in long wave UV light
(366 nm).
The Rf values were calculated for each spot and their
results are given in Table 1.
4. HPLC Analysis
4.1. Sample Preparation
2 ± 0.1 g of a sample was weighed accurately into a
beaker and 25 ml of mobile phase (Water: Methanol mix-
ture 45:55) was added and mixed. It was homogenized in
a water bath at 60˚C for 10 min and then cooled in an ice
bath until the separation of fats and waxes occurred. The
mixture was filtered and transferred to 100 ml volumetric
flask. The volume was made up to the mark by mobile
4.2. Preparation of Reference Solution
0.05 g of hydroquinone was weighed and transferred to a
50 ml volumetric flask. It was dissolved in small amount
of mobile phase and volume was made upto the mark. 5
ml of this solution was pipetted into a 50 ml volumetric
flask. It was diluted and volume was made up to the
mark by mobile phase.
Table 1. Rf Values of analyzed samples.
Sample No. Sample Name Manufacturers Rf value
1 WT Whitens Cream Anonymous 0.61
2 Face Fresh Beauty Cream Shaheen Cosnetics Company (Pvt.) Ltd., Pakistan 0.61
3 Barbie Whitening Cream 0.50
4 Faiza Beauty Cream Poonia Brothers, Pakistan 0.50
5 Blesso Cream Blesso Cosmetics 0.91
6 Care Cream Coslab (Pvt.) Ltd. 0.86
7 English Fairness Cream English Laboratories (Pvt.) Ltd., Pakistan 0.86
8 Virgo Acne Cream Virgo Cosmetics, Pakistan 0.50
9 White Gold Cream Trade Masters, Pakistan 0.50
10 19-Herbs Cream Allied Medical & Chemical Research Co., Pakistan 0.50
11 Pearl Whitening Cream Cosmo Care, Pakistan 0.50
12 Chun Chehra 0.81
13 Sanober Beauty Cream Future Sky Marketing Ltd., London 0.50
14 Kamal Face Cream Anonymous 0.86
15 X-Cream Anonymous 0.81
16 Farzana Beauty Cream Farzana Cosmetics, Pakistan 0.50
17 Fair & Lovely Unilever (Pvt.) Ltd., Pakistan 0.88
18 Bio Nikhar Forvil Cosmetics, Pakistan 0.50
19 Roop Nikhar Singh Herbals, India 0.50
20 Gipsy Cream Gipsy Cosmetics, Pakistan 0.88
21 Dabar Cream H. & Sons Enterprises, Pakistan 0.88
22 Ever Green Cream Anonymous 0.50
f values of Reference Hydroquinone Solution = 0.50.
Copyright © 2012 SciRes. JCDSA
Qualitative and Quantitative Estimation of Hydroquinone in Skin Whitening Cosmetics
5. HPLC Procedure
A Schimadzu LC-9 was used with UV detector set at 295
nm having ODS column (25 cm × 4.6 mm). Twenty was
adjusted at 35˚C ± 1˚C. The mobile phase was a mixture
of water and ethanol (45:55) with a flow rate of 1.5 ml/
20 μl of each sample solution was injected and chro-
matogram was recoded. Peak area for each sample was
measured and comparison was made between reference
and sample solutions peaks. The amount of hydro-
quinone was calculated as percentage by mass using the
formula A
ref spl
%age ofHydroquinonebipiwwd100=× ××
bi = Peak area of hydroquinone in sample solution;
pi = Peak area of hydroquinone in reference solution;
d (dilution factor) = 0.1;
wref = Weight of hydroqinone in reference solution;
wspl = Weight of hydroqinone in sample solution.
6. Discussion
The concept of skin whitening is very old. Variations in
the skin colour are caused by different levels of melanin
pigment in the skin. Melanin is synthesized in organelles
called melanosomes, in melanocytes cell, by the action of
an enzyme called tyrosinase. Most skin lightening prod-
ucts aim at tyrosinase production inhibition as it is the
one of the first steps in the pigment formation and can
therefore block all pigment producing pathways.
Hydroquinone was considered as one of the most ef-
fective skin lightening agent as it decreases tyrosinase
activity by 90% [20]. However its side effects include
skin irritation or contact dermatitis, development of ex-
ogenous ochronosis; an adverse effect that is character-
ized by darkening of the skin area where hydroquinone
containing cream is applied [21]. The present study fo-
cuses on the determination of hydroquinone in various
skin lightening cosmetics flooding the Pakistani market.
A review of literature showed use of different techniques
for the determination of hydroquinone in cosmetics mainly
GC-MS and HPLC [22-24].
In the present study a combination of TLC and HPLC
was used for the qualitative and quantitative determina-
tion of hydroquinone from skin whitening cosmetics re-
spectively. On spraying developed plates with 0.2% e-
thanolic dichlorofluorescein, they showed several spots
some of which coincided with that of the reference solu-
tion. The presence of more than one spot in a sample
revealed the presence of more than one ingredient in the
sample. A further confirmation was made by comparing
the Rf value of the reference spot (0.50) with the Rf val-
ues of samples spots. Based on TLC results 11 out of 22
samples were found to contain hydroquinone. The Rf
values for different samples are given in Table 1. HPLC
was used for the quantitative estimation of hydroquinone
concentration. The sample and reference solutions show-
ed UV absorption at λmax 295 nm. The retention time
(5.480) of reference and sample also coincided (Figures
1 and 2). The peak areas for both reference and sample
solutions were also measured and the quantity of hydro-
quinone was also calculated by using formula A. The re-
sults are given in Table 2. The concentration of hydro-
quinone in the samples ranged from 0.002% to 0.092%.
Figure 1. Chromatogram of reference solution of hydroquinone.
Copyright © 2012 SciRes. JCDSA
Qualitative and Quantitative Estimation of Hydroquinone in Skin Whitening Cosmetics 227
Figure 2. Chromatogram of representative sample (3) solution of hydroquinone.
Table 2. Percentage concentration of hydroquinone.
Sample No. Sample Name Sample Concentration (Y) Percentage Concentration of Hydroquinone
ref spl
ipiwwd 100=× ××
3 Barbie Whitening Cream 35.071 0.005
4 Faiza Beauty Cream 40.817 0.006
8 Virgo Acne Cream 10.00 0.002
9 White Gold Cream 220.855 0.034
10 19-Herbs Cream 607.071 0.092
11 Pearl Whitening Cream 25.318 0.004
13 Sanober Beauty Cream 549.325 0.08
18 Bio Nikhar 30.574 0.005
19 Roop Nikhar 21.652 0.003
16 Farzana Beauty Cream 230.76 0.035
22 Ever Green Cream 25.512 0.004
Peak area of reference solution of hydroquinone(pi) = 1643.
7. Recommendations
Based on the above findings and keeping in view the harm-
ful effects caused by hydroquinone as cited in this publi-
cation it is highly recommended that there should be a
regulatory body appointed by government to check the
quality of cosmetics available at market.
[1] M. Seiberg, C. Paine, E. Sharlow, et al., “Inhibition of
Mela-Nosomes Transfer Results in Skin Lightening,” Jour-
nal of Investigative Dermatology, Vol. 115, 2000, pp.
162-167. doi:10.1046/j.1523-1747.2000.00035.x
[2] J. V. Schaffer and J. L. Bolongia, “The Melanocortin-1
Receptor: Red Hair and Beyond,” Archives of Dermatol-
ogy, Vol. 137, No. 11, 2001, pp. 1477-1485.
[3] H. S. Raper, “The Anaerobic Oxidases,” Physiological
Reviews, Vol. 8, No. 2, 1928, pp. 245-282.
[4] S. S. Tai, C. G. Lin, M. H. Wu and T. S. Chang, “Evalua-
tion of Depigmenting Activity by 8-Hydroxydaidzein in
Mouse B16 Melanoma Cells and Human Volunteers,”
International Journal of Molecular Sciences, Vol. 10, No.
10, 2009, pp. 4257-4266. doi:10.3390/ijms10104257
[5] H. Ando, M. Ichihashi and V. J. Hearing, “Role of the
Ubiquitin Proteasome System in Regulating Skin Pig-
mentation,” International Journal of Molecular Sciences,
Vol. 10, No. 10, 2009, pp. 4428-4434.
[6] G. E. Costin and V. J. Hearing, “Human Skin Pigmenta-
tion: Melanocytes Modulate Skin Color in Response to
Stress,” FASEB Journal, Vol. 21, No. 4, 2007, pp. 976-
994. doi:10.1096/fj.06-6649rev
[7] J. P. Ebanks, R. R. Wickett and R. E. Boissy, “Mecha-
Copyright © 2012 SciRes. JCDSA
Qualitative and Quantitative Estimation of Hydroquinone in Skin Whitening Cosmetics
nisms Regulating Skin Pigmentation: The Rise and Fall
of Complexion Coloration,” International Journal of
Molecular Sciences, Vol. 10, No. 9, 2009, pp. 4066-4087.
[8] J. Lee, K. Jung, Y. S. Kim and D. Park, “Diosgenin Inhi-
bits Melanogenesis through the Activation of Phospha-
tidylinositol-3-Kinase Pathway (PI3K) Signaling,” Life
Sciences, Vol. 81, No. 3, 2007, pp. 249-254.
[9] T. S. Chang, “An Updated Review of Tyrosinase Inhibi-
tors,” International Journal of Molecular Sciences, Vol.
10, No. 6, 2009, pp. 2440-2475.
[10] M. Mastore, L. Kohler and A. J. Nappi, “Production and
Utilization of Hydrogen Peroxide Associated with Mela-
nogenesis and Tyrosinase-Mediated Oxidations of DOPA
and Dopamine,” FEBS Journal, Vol. 272, No. 10, 2005,
pp. 2407-2415. doi:10.1111/j.1742-4658.2005.04661.x
[11] N. Smit, J. Vicanova and S. Pavel, “The Hunt for Natural
Skin Whitening Agents,” International Journal of Mo-
lecular Sciences, Vol. 10, No. 12, 2009, pp. 5326-5349.
[12] R. Halaban, R. S. Patton, E. Cheng, S. Svedine, E. S. Trom-
betta, M. L. Wahl, S. Ariyan and D. N. Hebert, “Abnor-
mal Acidification of Melanoma Cells Induces Tyrosinase
Retention in the Early Secretory Pathway,” The Journal
of Biological Chemistry, Vol. 277, No. 17, 2002, pp.
[13] S. H. Hamed, P. Sriwiiyanont, R. R. Wickett and R.
Boissy, “Effect of Deoxyarbutin on Melanogenesis: In
Vivo Comparison with Other Melanogenesis Inhibitor,”
Journal of Cosmetic Science, Vol. 55, No. 1, 2004, pp.
[14] S. B. Adebajo, “An Epidermiological Survey of the Use
of Cosmetic Skin Lightening Cosmetics among Traders in
Lagos, Nigeria,” West African Journal of Medicine, Vol.
21, No. 1, 2002, pp. 51-55.
[15] N. Hardwick, et al., “Exogenous Ochronosis: An Epider-
miological Study,” British Journal of Dermatology, Vol.
120, No. 2, 1989, pp. 229-238.
[16] Twenty Fourth Directive 2000/6/EG Publication nr L056.
European Union, 2000.
[17] Food and Drug Administration, “Skin Bleaching Drug
Products for Over-the-Counter Human Use; Proposed
Rule,” US Department of Health and Human Services, 29
August 2006.
[18] Federal Register, “Proposed Rules,” Vol. 71, No. 167,
2006, pp. 51146-51155.
[19] E. Stahal, “Thin Layer Chromatography,” George Allen
and Unwin Limited, London, 1969.
[20] M. W. Akhtar, N. Kausar, M. N. Nawazish and Z. Hus-
sain,” Pakistan Journal of Biological Sciences, Vol. 16,
No. 2, 1981, p. 71.
[21] V. M. Veralla Rowell, V. Verallo, K. Graupe, L. Lopez-
Villafuerte and M. Garcia-Lopez, “Double-Blind Com-
parison of Azelaic Acid and Hydroquinone in the Treat-
ment of Melasma,” Acta Dermato-Venereological Sup-
plementum (Stocckh), Vol. 143, 1981, pp. 58-61.
[22] K. B. Penny, C. J. Smith and J. C. Allen, “Depigmenting
Action of Hydroquinone Depends on Disruption of Fun-
damental Cell Processes,” Journal of Investigative Der-
matology, Vol. 82, 1984, pp. 308-310.
[23] H. Jiang and T. Yan, “Determination of Hydroquinone in
Cosmetics by GC-MS,” Weishing Yanjiu, Vol. 30, No. 1,
2001, p. 22.
[24] L. Cheng-Hui, et al., “Determination of Hydroquinone in
Cosmetic Emulsion Using Microdialysis Sampling Cou-
pled with HPLC,” Journal of Pharmaceutical & Bio-
medical Analysis, Vol. 38, No. 3, 2005, pp. 414-419.
Copyright © 2012 SciRes. JCDSA