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, Taiwan2Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan3Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan

2

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%.

3

Introduction

Arbutin (AR)

4-hydroxyphenyl-β-D-glucopyranoside extracted from leaves of common bearberry It can compete with L-dopa for receptor site on tyr

osinase 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

O

OH

O

OH

OH

HO

OH

4

Hydroquinone (HQ)

1,4-benzenediol It can produce reversible depigmentation of the sk

in by suppression of melanocyte metabolic processes

OH

HO

Introduction

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Kojic acid (KA)

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 ac

tivities of tyrosinase. can be used in cosmetics to prevent serious sun-b

urning caused by an accumulation of melanin

O

O

OH

HO

Introduction

6

According to Department of Health, Taiwan, R.O.C

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.

Ingredients Limit

1、 Kojic Acid (5-Hydroxy-2-Hydroxymethyl-4H-Pyran-4-one)

2%

2、 Arbutin (4-hydroxyphenyl-β-D-glucopyranoside) 7%

Introduction

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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.

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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)

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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

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Results and discussion

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time (min)

5 6 7

abso

rban

ce

0.000

0.002

0.004

0.006

KA

AR+HQ

A

5 6 7

AR

KA+HQ

B

5 6 7

AR

KA

HQ

C

5 6 7

AR

KA

HQ

D

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 Regression equation Coefficient of correlation (r)

intra-daya AR KA HQ

inter-dayb AR KA HQ

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)

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)

0.99980.99940.9998

0.99970.99960.9998

* 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/mla 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) (%) (%)

Intra-day analysis (n=3)

AR 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

KA 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

HQ 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

Inter-day analysis (n=5)

AR 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

KA 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

HQ 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

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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) (%)

AR

– 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

KA

– 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

HQ

– 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

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Table 4. Quantification of AR, KA and HQ in marketing cosmetics

Samples (Labeled amount)

Amount found

AR(3%)

1 3.00

2 2.99

3 3.01

Mean 3.00

SD 0.0068

KA(2%)

1 2.01

2 2.05

3 2.04

Mean 2.03

SD 0.0152

HQ(20mg/g)

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.