A new, simple, and sensitive spectrometric method was developed for hydroquinone (HQ) determination in the presence of other depigmenting agents (kojic acid (KA), glycolic acid (GA), and ascorbic acid (AA)), commonly introduced in skin lightening products. The method is based on the oxidation of the depigmenting agents by potassium dichromate in sulfuric acid medium and subsequent measurement of the amplitude of the first-order derivative absorption spectrum at 268 nm. By applying the zero-crossing method, at this wavelength, the oxidation products of KA, AA, and GA do not interfere in the indirect determination of HQ. Beer’s law was obeyed in the range of 0.22–22
Human skin contains melanocytes (cells located at the base of the epidermis), which produce melanin (a dark macromolecular vital pigment) by a combination of enzymatically catalyzed chemical reactions. This process is named melanogenesis and it intensifies after exposure to UVB radiation, causing the skin to visibly
Hydroquinone (HQ) is considered to be one of the strongest inhibitors of melanin production and for more than 25 years it has been established as the most effective ingredient for treating melasma [
Taking into consideration both benefits and risks of using HQ-containing cosmetics, the quantitative determination of the HQ level in bleaching creams is imperative. For this purpose, many studies on HQ determination in different cosmetics are reported. The employed analytical methods are based on the specific properties of HQ exploited by chromatographic (HPLC [
In the present work a simple, accurate, and precise first-order derivative spectrometric method was proposed for the first time to quantify HQ in the presence of other depigmenting agents, namely, KA, GA, and AA, commonly present in cosmetic products. The method is based on the oxidation of HQ by K2Cr2O7 in sulfuric acid medium and subsequent absorbance measurement of the first-order derivative spectrum of the oxidation product (BQ) at 268 nm.
All chemicals were of analytical reagent grade and were purchased from Sigma-Aldrich. Deionized-distilled water was used throughout the experiments.
Aqueous stock solutions of HQ, KA, GA, and AA (10−2 mol·L−1) were freshly prepared and used to obtain the working standard solutions. When not used, the solutions were stored in the refrigerator. A 5 mol·L−1 H2SO4 solution was obtained by diluting concentrated sulfuric acid (98%, 1.84 g·mL−1). Working solutions of 10−3 and 5 × 10−3 mol·L−1 K2Cr2O7 resulted after dilution of a 10−1 mol·L−1 K2Cr2O7 stock solution. Eppendorf vary-pipettes (10–100; 100–1000; and 500–2500
An aliquot (1 mL) of 10−3 mol·L−1 solution of HQ, AA, KA, or GA was transferred into a 5 mL volumetric flask. After adding 5 mol·L−1 H2SO4 (1 mL), the mixture was brought to the mark with distilled water and homogenized. Then, the absorbance spectrum was recorded against water as reference. The derivative spectra were plotted with a 2 nm interval from the zero-order spectra of the individual analyzed solutions.
An aliquot (1 mL) of 10−3 mol·L−1 solution of HQ, KA, GA, or AA, 1 mL of 5 mol·L−1 H2SO4, and a known volume of 10−3 or 5 × 10-3 mol·L−1 K2Cr2O7 (added in small excess, to obtain a weak yellow colored solution) were transferred into a 5 mL volumetric flask and diluted to the mark. For each solution the absorption spectrum was recorded in the range 215–400 nm, against water as reference. This was necessary due to the fact that between 325 and 400 nm only K2Cr2O7 is absorbed, thus being mandatory the removal of the K2Cr2O7 excess influence. For each mixture, a corresponding blank solution was prepared by transferring into a 5 mL calibrated flask 1 mL of 5 mol·L−1 H2SO4 and a known volume of 10−3 or 5 × 10−3 mol·L−1 K2Cr2O7 to obtain a final aqueous solution having the concentration equal to that of the unreacted K2Cr2O7 (deduced from its absorbance at 350 nm; see the details given in Section
Binary, ternary, and quaternary mixtures of HQ and other dermatological active agents (KA, GA, and AA) in acidic medium and in the presence of K2Cr2O7 were prepared in the same manner as the individual depigmenting agents. The differences between absorbencies of the mixtures and the corresponding blank solutions were also obtained.
Absorbance measurements were performed in the 215–400 nm wavelength range on a UV-VIS spectrometer (V-530 Jasco-Japan), with fully integrated PC running Spectra Manager software, equipped with quartz cells of 1.00 cm. Suitable settings were slit width 1 cm and scan speed 100 nm·min−1.
HQ, KA, GA, and AA absorb UV radiation having characteristic absorption spectra (Figure
UV absorption spectra of HQ, KA, AA, and GA; every compound has the same concentration (2 × 10−4 mol·L−1).
By applying the zero-crossing method, the first- and second-order derivative spectra of HQ, KA, and AA do not permit the determination of HQ in ternary mixtures due to the fact that there is no wavelength where only HQ presents a measurable signal (Figures
(a) First-order, (b) second-order, and (c) third-order derivative spectra of HQ, KA, AA, and GA; every compound has the same concentration (2 × 10−4 mol·L−1).
It was observed that HQ presents an analytical signal at 303 nm in its third-order derivative spectrum, while the same order derivative spectra of the other tested compounds intersect the abscissa (Figure
Under these circumstances, a new methodology was established. This one considers the capacity to be oxidized of the above-mentioned compounds when K2Cr2O7 in acidic medium is used as oxidizing agent. Thus, another series of UV spectra were recorded for the individual active compounds in the presence of K2Cr2O7 in sulfuric acid medium. As depicted in Figure
UV spectra of (1) 2 × 10−4 mol·L−1 HQ in the presence of 2 × 10−4 mol·L−1 K2Cr2O7 (in excess), in 1 mol·L−1 sulfuric acid; (2) K2Cr2O7 at a level of concentration corresponding to those unreacted in solution (1);
The concentration of the unreacted K2Cr2O7 was deduced from the absorbance of the mixture at 350 nm, where only dichromate ion is absorbed. To achieve this aim a calibration curve was accomplished using K2Cr2O7 solutions with different concentrations (
The UV spectra of the investigated compounds in the absence and in the presence of K2Cr2O7 differ significantly in the case of HQ and AA (Figure
Overlaid spectra of the studied compounds in the absence (solid lines) and in the presence (dashed line) of K2Cr2O7 in 1 mol·L−1 sulfuric acid; every compound has the same concentration (2 × 10−4 mol·L−1).
The redox reactions between HQ, GA [
By overlaying the spectra of the reaction products of HQ, KA, GA, AA, and K2Cr2O7, the first one cannot be determined in presence of the others (Figure
Overlaid (a) zero-order and (b) first-order derivative spectra of HQ, KA, GA, and AA in the presence of K2Cr2O7 in 1 mol·L−1 sulfuric acid; every compound has the same concentration (2 × 10−4 mol·L−1).
Applying the first-order derivative, the analytical signal attributed to the oxidation product of HQ (benzoquinone (BQ)) could be used for the indirect determination of HQ in the presence of the other ingredients, at the zero-crossing of KA oxidation product (268 nm), where the amplitudes of the first-order derivative spectra of AA and GA oxidation products are also zero (Figure
In order to optimize the working conditions of the proposed method, the influence of the sulfuric acid concentration at constant HQ and dichromate contents was studied. It was observed that the analytical signal increases with increasing the concentration of H2SO4 up to 1 mol·L−1; then it remains almost constant. Further experiments were made on samples prepared in 1 mol·L−1 H2SO4. The stability of the reaction product between HQ and dichromate was monitored by spectrometry in the time range 0–1800 sec. The measurements made at 240 nm (the wavelength corresponding to the maximum absorbance of the HQ oxidation product in the presence of dichromate) indicated that the absorbance was stable within the tested period.
Using the above optimized spectrophotometric method developed for the indirect determination of HQ (via its oxidation in the presence of dichromate) a linear relationship was obtained between d
Analytical parameters of the first-order derivative spectrometric method for the indirect HQ determination.
Parameter | Value |
---|---|
Linear range, | 0.22–22 |
Intercept, | 0.0018 |
Intercept standard deviation, | 0.00032 |
Slope, | 0.0031 |
Slope standard deviation, | 0.00003 |
Determination coefficient, | 0.9994 |
LOD, | 0.07 |
LOQ, | 0.22 |
A comparison with other reported methods shows that the proposed spectrometric method is more sensitive, with a larger linear range of 0.22–22
Comparison of the proposed method with the reported spectrometric methods for HQ determination.
Analytical signal/working conditions | Linear range, | Application | Ref. |
---|---|---|---|
| 2–12 | Body lotions | [ |
| 10–50 | Cosmetic creams | [ |
| 0.025–2 | Cosmetic creams | [ |
| 10–26 | Cosmetic creams | [ |
| 10–100 | Skin whitening formulations | [ |
| 1–26 | Pharmaceuticals | [ |
| 0.22–22 | Synthetic mixtures | Present work |
The precision and the accuracy of the proposed spectrometric method were obtained using solutions containing HQ at three different concentration levels, within the established linear range. The results presented in Table
The precision and accuracy of the results obtained by the proposed spectrometric method;
HQ, | RSD, % | | |
---|---|---|---|
Considered | Found ± SD | ||
2.2 | 2.19 ± 0.05 | 2.31 | 99.39 |
5.5 | 5.51 ± 0.07 | 1.34 | 100.15 |
11 | 10.99 ± 0.29 | 2.60 | 99.88 |
The obtained percent recovery values lie within the accepted limits for these concentration levels [
The literature data report various combinations of different topical agents for melasma treatment [
As it can be seen, the four ingredients are found in the dermatological formulations in variable mixtures and concentrations (HQ ≤ 4%; AA ≤ 10%; KA ≤ 2%; GA ≤ 10%). In order to determine how the possible interfering compounds affect the HQ quantitation, binary mixtures were prepared, the concentration of the other active compounds being smaller than, equal to, and higher than the HQ concentration.
The recipes of the dermatological formulations that contain all the four active ingredients do not contain information about the KA, GA, and AA concentrations. Therefore, in the present study, the ternary mixtures contain all the four components at the lowest concentration level (percent ratio, HQ : KA : GA : AA = 1 : 1 : 1 : 1) and at the highest accepted concentration level (percent ratio, HQ : KA : GA : AA = 2 : 1 : 5 : 5).
The determination of HQ in combination with the mentioned active ingredients was studied by applying the proposed indirect derivative spectrometric method. Different combinations of HQ and the other depigmenting compounds and the analytical results for HQ determination are given in Tables
Results of the HQ determination in synthetic binary mixtures by applying the proposed spectrometric method;
Mixtures | Percent ratio, HQ : KA/AA/GA | HQ concentration, | | |
---|---|---|---|---|
Considered | Found ± SD | |||
HQ + KA | 4 : 1 | 4 | 4.04 ± 0.08 | 101.06 |
2 : 1 | 4 | 4.04 ± 0.05 | 101.06 | |
1 : 1 | 4 | 4.06 ± 0.07 | 101.33 | |
1 : 1.5 | 4 | 3.96 ± 0.05 | 99.47 | |
1 : 2 | 4 | 3.97 ± 0.04 | 99.20 | |
| ||||
HQ + AA | 2 : 1 | 4 | 3.99 ± 0.06 | 99.73 |
1 : 1 | 4 | 3.98 ± 0.05 | 99.47 | |
1 : 2 | 4 | 4.02 ± 0.04 | 100.53 | |
1 : 2.5 | 4 | 4.00 ± 0.06 | 100.00 | |
1 : 5 | 4 | 4.04 ± 0.07 | 101.06 | |
| ||||
HQ + GA | 1 : 1 | 4 | 4.04 ± 0.05 | 101.06 |
1 : 2 | 4 | 4.05 ± 0.06 | 101.17 | |
1 : 3 | 4 | 4.03 ± 0.07 | 100.80 | |
1 : 4 | 4 | 4.02 ± 0.06 | 100.53 | |
1 : 5 | 4 | 4.06 ± 0.08 | 101.60 |
Results of the HQ determination in ternary and quaternary synthetic mixtures by applying the proposed spectrometric method;
Percent ratio, HQ : GA : KA : AA | HQ concentration, | | |
---|---|---|---|
Considered | Found ± SD | ||
1 : 5 : 1 : 0 | 2 | 1.85 ± 0.08 | 92.55 |
1 : 5 : 2 : 0 | 2 | 1.87 ± 0.06 | 93.62 |
4 : 0.75 : 0 : 2.5 | 8 | 8.34 ± 0.35 | 104.26 |
1 : 1 : 1 : 1 | 2 | 1.83 ± 0.05 | 91.49 |
2 : 5 : 1 : 5 | 4 | 4.19 ± 0.05 | 104.79 |
In the case of ternary and quaternary mixtures, HQ can be determined by the proposed spectrometric method with good recovery values, the standard deviations supporting also the fact that the results are reliable and comparable.
All the experimental data obtained during this study led to the results presented throughout this paper and these revealed that the developed spectrometric method is a useful analytical tool for HQ quantitation in the presence of GA, KA, and AA from real samples containing varied mixtures of the mentioned compounds.
The new proposed method for the hydroquinone determination in the presence of other dermatologic active ingredients (kojic acid, ascorbic acid, and glycolic acid) is cheap and simple, and it is not time consuming. The used reaction system contains an oxidant (K2Cr2O7) that does not necessitate any additional steps. Moreover, the method does not contain variables which influence the reliability of the results. The accuracy, the precision, and the results obtained analyzing the synthetic mixtures recommend the spectrometric procedure for skin depigmenting products analysis and control by means of hydroquinone level monitoring, in binary, ternary, or quaternary mixtures with kojic acid, glycolic acid, and ascorbic acid. The application of the spectrometric method on synthetic mixtures represents the first step for further researches on real samples.
The authors declare that they have no competing interests.