Analysis of Arbutin and Hydroquinone in whitening serum using a Chromolith^® HighResolution RP-18e 2 mm I.D. HPLC column

Anita Piper, R&D Chemist, Lara Celia Bergner

Section Overview

• Introduction
• Experimental Conditions
• Results - Calibration and Repeatability
• Serum Sample Results
• Conclusion
• Related Products

Introduction

Bleaching or skin whitening serums are widely used to reduce melanin content in the skin, and for such products, analytical quality control is required. Hydrochinone is a decomposition product of arbutin which can cause severe contact dermatitis in humans.

This report focuses on the testing of arbutin and hydroquinone in formulated serum products. A Chromolith^® HighResolution RP-18 endcapped column 100x2mm was used on an HPLC-UV instrument, and can improve existing methods (e.g. Int. J. Appl. Sci. Eng., 2011.9.4)¹.

EXPERIMENTAL CONDITIONS
Column:	Chromolith® HighResolution RP-18e, 100x2mm (1.52322)
Detection:	Dionex Ultimate 3000 VWD-3400, UV @ 220 nm, micro flow cell (1.4 µL/7 mm)
Mobile phase:	[A] Water, [B] methanol; isocratoc at A/B 90/10 (v/v)
Flow Rate:	91 µL/min
Pressure Drop:	22 bar (319 psi)
Column temp.:	25 °C
Injection volume:	0.5 µL
Samples
Diluent	Water/Methanol 30/70 (v/v)
Standard solution (10 mg/mL)	Accurately weigh 5 mg of each standard into a 50 mL volumetric flask and add 40 mL diluent. Sonicate for 5 min and fill up to mark with diluent. Pipette 5 mL of this solution into a 50 ml volumetric flask and fill up to mark with diluent. Pass through a 0.45 µm syringe filter before injection.
Sample solution	Place 1 g of homogenized serum in a 50 mL volumetric flask, add 30 mL of diluent and sonicate for 5 minutes. Fill up to volume with diluent. Transfer 3 mL of this suspension into a 25 mL volumetric flask and fill up to volume with diluent. The resulting cream sample concentration in the final dilution is 2.4 mg/mL. Pass through a 0.45 µm syringe filter before injection.

Results - Calibration and Repeatability

 

Figure 1. Chromatogram blank.

Figure 2. Chromatogram standard solution arbutin and hydroquinone each at 10 µg/mL.

Chromatographic Data - Standard Solution (10 µg/mL )
No.	Compound	Retention Time (min)	S/N	Area (mAU*min)	Tailing Factor
1	t0 void volume	2.9
2	Arbutin	4.1	94.3	0.8407	1.4
3	Hydroquinone	5.1	368.4	1.6358	1.8

Specificity Test: The standard solution of arbutin and hydroquinone (each at 10 µg/mL) was injected and the retention time and content of desired analyte determined (Table 1). Repeatability was determined by 5 injections of a serum sample solution (Table 2). Calibration and sensitivity results for arbutin and hydroquinone (calibration range 0.10-15.5 µg/mL for arbutin and 0.10-15.3 µg/mL for hydroquinone with 9 calibrators) are shown in Table 3.

Table 1. Specificity test results (standard solution of arbutin and hydroquinone at 10 µg/mL each)

	Compound	Retention Time (min)	Area (mAU*min)	Tailing Factor	S/N
1	Arbutin	4.1	0.8407	1.4	141.7
2	Hydroquinone	5.1	1.6358	1.8	488.9

Table 2.Repeatability of sample solution (serum with arbutin)

Sample	Area (mAU*min)
STD 1	5.2146
STD 2	5.2615
STD 3	5.2657
STD 4	5.2224
STD 5	5.3311
Mean	5.2702
Standard Deviation	0.0451
(%) RSD	0.9

Table 3.Calibration and sensitivity results for arbutin and hydroquinone (calibration range 0.10-15.5 µg/mL for arbutin and 0.10-15.3 µg/mL for hydroquinone with 9 calibrators).

Compound	LOD (µg/mL)	LOQ (µg/mL)	R2
Arbutin	2.7	8.2	0.9992
Hydoquinone	0.5	1.4	0.9994

Figure 3. Calibration curve arbutin.

Figure 4. Calibration curve hydroquinone.

The LOD & LOQ in the injected standard solution represent limits for the serum samples of 1.13 mg/g (LOD) and 3.42 mg/g (LOQ) for arbutin 0.21 mg/g (LOD) and 0.58 mg/g (LOQ) for hydroquinone.

Serum Sample Results

Figure 5. Chromatogram serum without arbutin.

Figure 6. Chromatogram serum containing arbutin

Chromatographic Data - Serum with Arbutin
No.	Compound	Retention Time (min)	S/N	Area (mAU*min)	Tailing Factor
1	t0 void volume	2.9
2	Arbutin	4.1	563.5	4.9694	1.7
3	Hydroquinone	n.d.

Calculation

ω_Sample  =         c_st/c_Pr

ω_Sample  =   (63.00 μg/mL) / (2.4 mg/mL)

ω_Sample  =    26.25 µg/mg  or 2.63% Arbutin in Serum

where,

ω_Sample  = Concentration of Arbutin in the sample 

c_st    = Concentration calculated from calibration curve (µg/mL)

c_Pr     = Concentration of serum sample in final dilution (mg/mL)

Conclusion

It was shown, that a Chromolith^® HighResolution RP-18e 100x2mm column coupled to UV detection can be utilized for the determination of arbutin and hydrochinone in serum. For arbutin, the resulting limit of detection (LOD) was 2.7 µg/mL, and the limit of quantitation (LOQ) was 8.2 µg/mL in the final dilution sample, representing limits for the serum samples of 1.13 mg/g (LOD) and 3.42 mg/g (LOQ); for hydroquinone, the results were 0.5 µg/mL (LOD) and 1.4 µg/mL (LOQ) in the final dilution sample, representing limits for the serum samples of 0.21 mg/g (LOD) and 0.58 mg/g (LOQ). Due to the excellent permeability and low backpressure of this column type, an analytical HPLC-System with a micro cell could be used.

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References

1. Wang A, Cheng S, Kwan C. 2011. Simultaneous determination of five whitening agents by ion-pair reversed-phase high performance liquid chromatography. [Internet]. Int. J. Appl. Sci. Eng. Available from: https://gigvvy.com/journals/ijase/articles/ijase-201112-9-4-287