Simultaneous determination
of arbutin, kojic acid and
hydroquinone in skin
lightening cosmetics by MEKC
Yi-Hui Lin1, Shou-Mei Wu1,2,3*
1Graduate
Institute of Pharmaceutical Science, College of Pharmacy, Kaohsiung
Medical University, Kaohsiung, Taiwan
2Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical
University, Kaohsiung, Taiwan
3Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
Abstract
A simple method of micellar electrokinetic capillary electrophoresis
(MEKC) was established for simultaneous analysis of arbutin, kojic
acid and hydroquinone. Untreated fused-silica capillary was operated
using phosphate buffer (20 mM, pH 6.5) under 20 kV and detection at
200 nm. Baseline separation was attained within 9 minutes. The
quantitative ranges were 20-200 μg/mL for arbutin, 20 -100μg/mL for
kojic acid and 8-80μg/mL for hydroquinone with correlation
coefficients ≥ 0.9994. The R.S.D. and R.E. were all less than 3.0 % for
the intra-day and inter-day analysis, and all recoveries were greater
than 99 %. The limits of detection was arbutin 5.4 μg/mL, kojic acid
7.1μg/mL and hydroquinone 2.2 μg/mL (S/N = 3, hydrodynamic
injection 5 sec). Our method was applied to determine the quality of
commercial cosmetic. Assay results were all within the labeled amount
of 99.6-102.5%.
2
Introduction
Arbutin (AR)
HO
O
O
OH
OH
OH
OH
 4-hydroxyphenyl-β-D-glucopyranoside
 extracted from leaves of common bearberry
 It can compete with L-dopa for receptor site on
tyrosinase and hinders the oxidation of L-dopa to
L-dopaquinone, thus, can inhibit the formation of
eumelanin and phaeomelanin
 often used in skin care products as a lightening
agent
3
Introduction
Hydroquinone (HQ)
OH
HO
 1,4-benzenediol
 It can produce reversible depigmentation of the
skin by suppression of melanocyte metabolic
processes
4
Introduction
Kojic acid (KA)
O
OH
HO
O
 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one
 a natural substance produced by fungi or bacteria,
such as Aspergillus, Penicillium or Acetobacter spp
 It is able to inhibit the creolase and catecholase
activities of tyrosinase.
 can be used in cosmetics to prevent serious sunburning caused by an accumulation of melanin
5
Introduction
 According to Department of Health, Taiwan, R.O.C
Ingredients
Limit
1、Kojic Acid (5-Hydroxy-2-Hydroxymethyl-4HPyran-4-one)
2%
2、Arbutin (4-hydroxyphenyl-β-D-glucopyranoside)
7%
 It also recommended that HQ should be used under
prescription because long term used of high
concentration hydroquinone (over 5 %) may cause
severe side effects including dermatitis, erythema,
leukoderma, burning and hyperpigmentation.
6
Aim of this study
 To develop a rapid, sensitive and quantitative assay
for the simultaneous determination of AR, KA and
HQ by capillary electrophoresis.
 Optimization, validation and application of this
method were investigated.
7
Materials
•
•
•
•
•
•
AR (98%)
KA
HQ
Urea (I.S.)
Sodium dodecyl sulfate
NeoStrata® pigment lightening gel (Princeton, NJ,
USA)
• Shiseido® white lucent (Tokyo, Japan)
• Grape® quincare ointment (Chung-Li, Taiwan)
8
CE System
• A Beckman P/ACE System 2200 equipped with a
filter UV detector
• The fused-silica capillaries:
I.D.= 50 µm; Lt= 57.0 cm; Ld= 50.0 cm
• Phosphate buffer containing SDS
• Applied voltage: 20 kV
• Temperature: 25 ˚C
• Wavelength for detection: 200 nm
• Hydrodynamic injection pressure: 50 mbar, 5 sec
9
Results and discussion
10
A
0.006
C
B
D
AR+HQ
HQ
AR
HQ
KA+HQ
KA
absorbance
AR
0.004
AR
KA
KA
0.002
0.000
5
6
7 5
6
7 5
6
7 5
6
7
time (min)
Figure 1. Effects of SDS concentrations on the migration of AR, KA
and HQ each at 200 μM. Electropherograms: (A) overlapping peaks
from separation in the absence of SDS, (B) with 60 mM SDS, (C) with
80 mM SDS, (D) with 100 mM SDS.
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Table 1. Regression analysis for the determination of AR, KA and HQ.
Analysis
intra-daya
AR
KA
HQ
inter-dayb
AR
KA
HQ
Regression equation
Coefficient of
correlation (r)
Y=(5.2316±0.0166)X+(0.0149±0.0020)
Y=(6.0875±0.0336)X+(0.0023±0.0046)
Y=(11.469±0.0249)X+(0.0116±0.0032)
0.9998
0.9994
0.9998
Y=(5.2295±0.0516)X+(0.0109±0.0053)
Y=(6.0724±0.1173)X+(0.0018±0.0053)
Y=(11.3938±0.1129)X+(0.0009±0.007)
0.9997
0.9996
0.9998
* Concentration ranges for the intra- and inter-day analysis:
AR: 0.02-0.2 mg/ml
KA: 0.02-0.1 mg/ml
HQ: 0.008-0.08 mg/ml
a The regression equations of intra-day analysis were calculated from the assay values of prepared
standards on a single day (n=3).
b The regression equations of inter-day analysis were calculated from the assay values of prepared
standards on a single day (n=5).
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Table 2. Precision and accuracy for the determination of AR, KA and HQ.
Concentration
Concentration
RSD
RE
know
found
(mg/ml)
(mg/ml)
(%)
(%)
0.05
0.0504±0.0006
1.20
0.86
0.09
0.0904±0.0004
0.48
0.41
0.16
0.1609±0.0007
0.43
0.57
0.03
0.0299±0.0006
2.03
-0.34
0.07
0.0697±0.0008
1.13
-0.41
0.09
0.0891±0.0007
0.77
-0.95
0.015
0.0150±0.0001
0.57
0.01
0.03
0.0292±0.0005
1.75
-2.63
0.07
0.0698±0.0007
1.05
-0.23
0.05
0.0503±0.0010
2.05
0.70
0.09
0.0902±0.0009
1.04
0.28
0.16
0.1606±0.0010
0.61
0.35
0.03
0.0296±0.0008
2.59
-1.21
0.07
0.0693±0.0009
1.28
-0.96
0.09
0.0893±0.0009
1.05
-0.75
0.015
0.0150±0.0001
0.87
-0.14
0.03
0.0292±0.0006
2.01
-2.80
0.07
0.0701±0.0008
1.17
0.08
Intra-day analysis (n=3)
AR
KA
HQ
Inter-day analysis (n=5)
AR
KA
HQ
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Table 3. Recovery of AR, KA and HQ added to the commercial formulation
Concentration
Concentration
Recovery
spiked
found
(mg/ml)
(mg/ml)
(%)
–
0.0303±0.0002
–
0.02
0.0512±0.0008
104.55
0.07
0.1017±0.0015
102.00
0.16
0.1928±0.0012
101.55
–
0.0222±0.0009
–
0.02
0.0421±0.0010
99.44
0.04
0.0631±0.0002
102.16
0.07
0.0924±0.0005
100.30
–
0.0109±0.0002
–
0.02
0.0313±0.0002
101.9497
0.04
0.0511±0.0002
100.3522
0.06
0.0720±0.0005
101.6940
AR
KA
HQ
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Table 4. Quantification of AR, KA and HQ in marketing cosmetics
Samples
(Labeled amount)
AR
(3%)
KA
(2%)
HQ
(20mg/g)
Amount found
1
3.00
2
2.99
3
3.01
Mean
3.00
SD
0.0068
1
2.01
2
2.05
3
2.04
Mean
2.03
SD
0.0152
1
19.94
2
20.27
3
20.35
Mean
20.19
SD
0.1778
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Conclusions
 This method can be used for the simultaneous
determination of arbutin, kojic acid and
hydroquinone and was successfully applied to
the content assays of skin whiting cosmetics.
16