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HomeSmall Molecule HPLCAnalysis of Arbutin and Hydroquinone in whitening serum

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


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.

Two images- on the left bond line chemical structure of arbutin and on the right bond line chemical structure of hydroquinone

EXPERIMENTAL CONDITIONS

Results

Calibration and Repeatability

A Chromatogram obtained for the blank run in the HPLC-UV analysis of arbutin and hydroquinone with intensity on the y-axis and retention time measured in minutes on the x-axis showing a straight line running parallel to the x-axis

Figure 1.Chromatogram blank.

A Chromatogram obtained for a standard solution of arbutin and hydroquinone with 10 µg/mL each in an HPLC-UV analysis with intensity on the y-axis and retention time measured in minutes on the x-axis showing three distinct peaks labeled 1,2, and 3.

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

Chromatographic Data - Standard Solution (10 µg/mL )

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)
Table 2.Repeatability of sample solution (serum with arbutin)
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).
Calibration curve with mean area on y-axis and concentration in µg/mL on the x-axis, obtained for arbutin measured at different concentrations in the range of 0.10-15.5 µg/mL, showing a straight line starting from 0, following the equation for straight line y=mx+c with m value of 0.0791 and c value of 0.0141, along with R2 value of 0.9992

Figure 3.Calibration curve arbutin.

Calibration curve with mean area on y-axis and concentration in µg/mL on the x-axis, obtained for hydroquinone measured at different concentrations in the range of 0.10-15.3 µg/mL, showing a straight line starting from 0, following the equation for straight line y=mx+c with m value of 0.1490 and c value of 0.0132, along with R2 value of 0.9994

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

A Chromatogram obtained for a serum sample not containing arbutin in an HPLC-UV analysis with intensity on the y-axis and retention time measured in minutes on the x-axis showing only time of the unretained peak (t0) nad no peak for arbutin

Figure 5.Chromatogram serum without arbutin.

A Chromatogram obtained for a serum sample containing arbutin in an HPLC-UV analysis with intensity on the y-axis and retention time measured in minutes on the x-axis showing two peaks labeled, 1 and 2. Peak 1 corresponds to time of the unretained peak (t0) and peak 2 for arbutin

Figure 6.Chromatogram serum containing arbutin

Chromatographic Data - Serum with Arbutin

Calculation

ωSample  =         cst/cPr

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

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

where,

ωSample  = Concentration of Arbutin in the sample 

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

cPr     = 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.

See more chromatograms for cosmetic applications


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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
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