HPLC DETERMINATION OF ARBUTIN 39
Mobile phase flow rate. The optimum flow rate of the mobile phase should provide good
separation, high sensitivity, and short analysis time. In this work, after the optimal
wavelength was selected, optimization of the flow rate was carried out by injecting the
same concentration of mixed standard solutions at varying flow rates from 0. 5 ml/min -l
to 1.0 ml/min- 1
.
Recommended RP-HPLC procedure. A sample and/or standard solution containing arbutin
was separated on a reverse-phase ODS Hypersil® C
18
column (125 mm x 4 mm, 5.0 µm)
and detected at 222 nm. An aliquot of 100 µl of a series of arbutin standard solutions
and 100 µl of sample extract was injected into the LC system and eluted with the mobile
phase, water:methanol:0.1 M hydrochloric acid (89: 10: 1, v/v/v) (flow rate =1.0 ml/
min- 1 ).The area of the arbutin peak was measured. Arbutin concentration in the plant
extract was determined by reference to the calibration curve prepared under identical
experimental conditions.
RESULTS AND DISCUSSION
A high-performance liquid chromatographic method for the determination of arbutin in
skin-whitening creams and medicinal plant extracts containing arbutin was developed.
The experimental conditions were investigated and the proposed method was also vali-
dated.
OPTIMIZATION OF RP-HPLC CONDITIONS
The optimal conditions of HPLC for determining arbutin were carried out under iso-
cratic conditions. Various mobile phase systems with different compositions were in-
vestigated. First, the optimal wavelength for the detection of arbutin and other com-
pounds, as mentioned earlier, was investigated, and the UV spectrum of each standard
compound showed the absorption maxima at 222 nm. A wavelength of 222 nm showed
the highest sensitivity for arbutin. Second, among the mobile phases studied, a mixture
consisting of water:methanol:0.1 M hydrochloric acid (89: 10: 1, v/v/v) was used as the
mobile phase, and it was found that this mobile phase was the most suitable because it
resulted in good retention times, resolution, and satisfactory peak profiles (Figure 2).
Finally, the optimum flow rate was 1.0 ml/min-1, as it gave a good resolution, high
sensitivity, a short analysis time, etc. In the RP-HPLC analysis, arbutin and resorcinol
(internal standard) showed single symmetrical peaks (retention time 5. 7 min and 10. 7
min), respectively.
METHOD VALIDATION
The proposed method was validated according to U.S. Pharmacopoeia USP (19).
Sensitivity. The sensitivity of the assay was determined in terms of the detection limit
(LOD) and the quantitation limit (LOQ). Detection limits and quantitation limits were
estimated for each of the examined compounds. The values were calculated from the
standard deviation (S.D.) of response and the slope of the curve (S) by means of the
equations: LOD =3.3 (S.D./S) and LOQ =10 (S.D./S). Low LOD and LOQ values were

40 JOURNAL OF COSMETIC SCIENCE
mAU
l l
j
120:i r
801
l
40
0
2 4
Resorcinol
Arbutin
6 8 10 12 14
mm
Figure 2. HPLC chromatogram of the arbutin standard (5 µg/ml- 1 )and the resorcinol internal standard (5
µg/ml- 1 ).
obtained as shown in Table I, which indicates the good sensitivity of the proposed LC
method.
Linearity. To determine linearity, five different concentrations of the arbutin standard
were used in a working range of 0. 5-3 0. 0 µg/ ml -1
• The linear regression equations and
the correlation coefficient (r2) values for arbutin and the internal standard are given in
Table I. The r2 values show good linearity in the examined concentration range.
PRECISION AND ACCURACY
The intraday reproducibility study was performed during a period of three days. The
results obtained showed that the arbutin peak area variabilities for standard solutions
were within 0.00-0.02% R.S.D. For interday reproducibility, five replicate injections of
various concentrations of arbutin were made within a day. The results obtained showed
that the arbutin peak area variabilities for the standard solutions were within 0.00-
0.02% R.S.D. The results are shown in Table II. The R.S.D. values demonstrated good
precision.
Table I
Linear Regression Analysis (n =3) and Limits of Detection (S/N =3) and Quantitation (SIN =10)
Linearity range Equation r 2 1OD LOQ
Standard (�1g/ml- 1
)Y =SX +C Mean (±S.D.) (µg/ml- ')(µg/ml- 1
)
Arbutin 0.5-30 Y =132.84 (±0.36)X 0.9999 5.07
X 10-5 1.01
X 10-2
-9.32 (± 1.23) (±5.77
X 10-S)
Resorcinol *0.5-30 Y =257.03 (±0.9l)X 0.9999 5.04
X 10-5 1.00
X 10-2
+7 .34 (±0.04) (± 1.49
X 10- 8
)*Internal standard.