Introduction

The Korean Cosmetic Act defines cosmetics as safe goods used via rubbing, spraying or in similar ways for cleaning and beautifying the human body, brightening appearance, maintaining or improving the health of skin and hair [1]. To be used, the cosmeceuticals (functional cosmetics in Republic of Korea) are regulated by The Korean Cosmetic Act and Korean Functional Cosmetics Codex. Cosmeceuticals are intended to carry out their functions as whitening, tanning, anti-wrinkle, antiaging, and nail and hair care [2]. Pharmaceuticals are agents intended to alter, change, or protect skin from abnormal or pathological conditions, whereas cosmeceuticals represent a category of products placed between non-prescribed and prescribed [3].

There were several cosmeceutical ingredients that have been shown to be effective in skin whitening, such as hydroxyacetic acid, kojic acid, azelaic acid, hydroquinone, resorcinol, arbutin, niacinamide, vitamin C and its derivatives, (−)-α-bisabolol, and glabridin [2, 4–8]. However, for safety reason, some of them have been banned for uses in cosmetics [9, 10]. In Republic of Korea, ascorbyl tetraisopalmitate, glabridin, (−)-α-bisabolol, arbutin, niacinamide, ascorbyl glucoside, and ethyl ascorbyl ether are the main ingredients used in whitening functional cosmetics [11]. Glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate are lipophilic substances, whereas the arbutin, niacinamide, ascorbyl glucoside, and ethyl ascorbyl ether are water-soluble compounds. In our previous research studies, we have developed methodologies to determine hydrophilic substances in whitening functional cosmetics [4, 12].

Glabridin is an isoflavonoid originally isolated from crude licorice (Glycyrrhiza glabra L.). Glabridin has been associated with numerous biological properties such as anticancer, antioxidant, anti-inflammatory, antibacterial, and skin-whitening activities [13–15]. Because it is a potent tyrosinase inhibitor, the skin-whitening effect was due to the inhibition of melanin [16]. (−)-α-Bisabolol is sesquiterpene alcohol obtained from several plant extracts, such as Chamomilla recutita, Plinia cerrocampanensis Barrie, Pogostemon speciosus Benth and others. It has been used in cosmetic products, fine fragrances, toilet soaps and other toiletries as well as in non-cosmetic products [17–21]. (−)-α-Bisabolol inhibits the cAMP response element (CRE) induced by α-melanocyte-stimulating hormone (α-MSH), thereby reducing the melanin content. Additionally, it alters the gene expression of microphthalmia-associated transcription factor (MITF) and tyrosinase; implying that it inhibits the melanogenesis by reducing the intra cellular cAMP levels [22]. Vitamin C, or L-ascorbic acid, is the most plentiful antioxidant in human skin and has an ability to inhibit the activity of tyrosinase [23, 24]. Due to oxidation, vitamin C is easily degraded and unstable when exposed to air, light, etc. To overcome this defect, ascorbyl tetraisopalmitate and other vitamin C’s derivatives have been introduced [24]. The ascorbyl tetraisopalmitate is a fat-soluble substance derived from vitamin C [25]; exhibiting a good percutaneous absorption and a strong antioxidant activity in vitro in a lipid system [26]. Analytical methods for determination of ascorbyl tetraisopalmitate in cosmetic formulations using HPLC were reported in the literatures [25, 27]. Pedro et al. validated a HPLC method for the determination of (−)-α-bisabolol in chitosan milispheres and liposomes [28]. Meanwhile, there were a very few literatures for determination of (−)-α-bisabolol in human blood and chamomile flowers, but not cosmetics [29–31]. The HPLC method for quantitation of glabridin in polyherbal preparations and crude extracts was evaluated by Kamal et al. [15]. Similar to (−)-α-bisabolol, there was no report for detection glabridin in cosmetics, however, it was detected in crude drugs and human plasma [15, 32]. So far, no such method is described in the literature neither for simultaneous determination of the three lipophilic compounds (glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate) nor in whitening cosmetic creams.

Thus, the aim of this study was to develop and validate a simple and sensitive method to quantify glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate in whitening creams using a single HPLC-PAD chromatographic run.

Experimental

Chemicals and Reagents

Commercial functional cosmetics containing glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate were bought from internet markets and cosmetic shops located in Suwon City, Republic of Korea. An analytical standard of glabridin (purity: 97.0 %) and ascorbyl tetraisopalmitate (purity: 88.8 %) were obtained from Wako Chemicals (Tokyo, Japan). (−)-α-Bisabolol (purity: 95.0 %) was supplied by Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol (purity: 99.99 %) and isopropyl alcohol (purity: 99.99 %) were provided by J. T. Baker (Griesheim, Germany). A 0.4 μm nylon membrane filter and 0.20 μm polytetrafluorethylene syringe filter (Advantec, Tokyo, Japan) was used to filter the mobile phase and sample solutions, before using. A Barnstead Nano pure Diamond (Dubuque, IA, USA) was used to produce purified deionized water.

Standard Preparation

Stock standard solutions of glabridin (5.0 μg mL−1), (−)-α-bisabolol (60.0 μg mL−1), and ascorbyl tetraisopalmitate (200.0 μg mL−1) were prepared by dissolving in a mixture of acetonitrile and isopropyl alcohol (45:55, v/v). Working standard solutions were obtained by diluting the stock solutions with the same mixture solution. The concentration ranges of each standard calibration curve are presented in Table 1. The standard solutions were stable for 1 month when stored at 4 °C in refrigerator.

Table 1 Detection wavelength (nm), concentration range (μg mL−1), calibration curve, linearity (R 2 ), and limits of detection (LOD), and quantification (LOQ) of the tested compounds
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Sample Preparation

Commercial creams (0.1 g) was accurately weighed (XSE 205DU Analytical Balance, Mettler Toledo, Greifensee, Switzerland) in 15 mL polypropylene centrifuge tubes (SPL Life Sciences, Gyeonggi-do, Republic of Korea) to which a mixture of acetonitrile and isopropyl alcohol (45:55, v/v, 10 mL) was added and vortex-mixed (Vortex-Genie 2, Scientific Industries, NY, USA) for 2 min. Subsequently, the solution was sonicated (Branson 8510 sonicator, CT, USA) for 30 min, then centrifuged (Sigma 2–6 centrifuge, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at 3500 rpm for 20 min. The supernatant was filtered through a 0.20 μm polytetrafluorethylene (PTFE) syringe filter before injection onto the HPLC system.

Analytical Method

HPLC-PAD system (Dionex, UltiMate 3000, Sunnyvale, CA, USA) consisting of a degasser, a quaternary pump, an auto sampler, and column compartment was used for detection. The Chromeleon software (Dionex, Sunnyvale, CA, USA) was used for data acquisition. An Eclipse Plus C18 column (250 × 4.6 mm, 5 μm, Agilent Technologies, CA, USA) maintained at 25 °C was used to separate the analytes. Capcell Pak C18 MG (250 × 4.6 mm, 5 μm, Shiseido Co, Ltd., Tokyo, Japan) was used to evaluate the chromatographic robustness test. The target compounds were separated using a stepwise gradient mobile phase consisting of deionized water (A), acetonitrile (B), and isopropyl alcohol (C), as following: 0–5 min, 30:70:0, v/v/v (A:B:C); 5–12 min, 0:100:0, v/v/v (A:B:C); 12–15 min, 0:0:100, v/v/v (A:B:C); and 15–25 min, 0:0:100, v/v/v (A:B:C), 25–32 min, 0:100:0, v/v/v (A:B:C); 32–37 min, 0:100:0, v/v/v (A:B:C); and 37–45 min, 30:70:0, v/v/v (A:B:C). The flow rate was 1.0 mL min−1 and the injection volume was 10 μL. The detection wavelengths were 228 nm for glabridin, 202 nm for (−)-α-bisabolol, and 221 nm for ascorbyl tetraisopalmitate.

Method Validation

For the quantification of the target compounds, the determination coefficient (R 2) was calculated and evaluated as linearity. The limit of detection and limit of quantitation were determined using the signal-to-noise (S/N) ratios of 3.3 and 10, respectively. The recovery and precision of the developed method were validated according to US Pharmacopeia [33]. Recovery (expressed as accuracy) was determined via spiking three different concentration levels of each compound into blank cream samples (n = 3). The method precision (expressed as intra- and inter-day variations) was evaluated by the repeated analysis of cream samples (n = 3) spiked with various compounds during 1 day and was repeated for another couple of days, respectively. The robustness evaluation of the chromatographic method was tested by the introduction of minor changes in the separation techniques of sample containing (−)-α-bisabolol (the compound with low detection wavelength value = 202 nm) by means of Youden’s test. The slope of mobile phase gradient, column temperature, flow rate, detection wavelength, injection volume, column manufacturer, and the initial mobile phase composition were chosen as the seven variables for Youden’s robustness test. As shown in Table 2, eight experiments were conducted to evaluate the selected factors [34]. For each variable, the calculated difference was indicated as D i . The standard deviation of the differences, \( S_{{D_{i} }} \), was calculated by the formula:

Table 2 Variables and their levels in chromatographic separation in the Youden’s robustness test experimental design
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$$ S_{{D_{i} }} = \sqrt {2 \times \mathop \sum \nolimits \left( {\frac{{ D_{i}^{2} }}{7}} \right)}. $$

When \( S_{{D_{i} }} \) is significantly higher than the standard deviation of the method, it means that all the chosen factors together have an effect on the result [35].

Additionally, with t test, it is possible to evaluate the influence of each investigated factor. The experimental t value is given by the equation:

$$ t = \frac{{\sqrt n \times \left| {D_{i} } \right|}}{{\sqrt 2 \times {\text{S}}.{\text{D}}.}} $$

where n is the number of experiments carried out at each level for each parameter (n = 4) The standard deviation was obtained from the analysis of (−)-α-bisabolol at 50 μg mL−1 during the inter-day precision test. For all seven variables, the obtained t value was compared with the 2-tailed t critical value (t crit) at n − 1 degree of freedom, where n is the number of determinations used in the estimation of S.D. at 95 % confidence level. If t value is greater than t crit, the investigated variable shows a significant influence, and the method is not sufficiently robust against the chosen modification [36].

Results and Discussion

Extraction Procedure

The extraction efficiency of the tested compounds from commercially available cream samples was investigated using acetonitrile, isopropyl alcohol, or a combination of them as following: (A) 100 % acetonitrile, (B) 100 % isopropyl alcohol, (C) acetonitrile and isopropyl alcohol (30:70 %, v/v), (D) acetonitrile and isopropyl alcohol (40:60 %, v/v), (E) acetonitrile and isopropyl alcohol (45:55 %, v/v), (F) acetonitrile and isopropyl alcohol (50:50 %, v/v), (G) acetonitrile and isopropyl alcohol (55:45 %, v/v), (H) acetonitrile and isopropyl alcohol (60:40 %, v/v), and (I) acetonitrile and isopropyl alcohol (70:30 %, v/v). It has to be noted that a mixture of acetonitrile and isopropyl alcohol (45:55 %, v/v) efficiently extracted the three analytes compared to others. From previous studies, for instance, Almeida et al. used isopropyl alcohol as an extraction solvent to determine ascorbyl tetraisopalmitate in cosmetic cream [25] and n-hexane was used as an extraction solvent for determination of (−)-α-bisabolol in particulate systems by Pedro et al. [28]. Kamal et al. [15] extracted glabridin from polyherbal preparations using 30 % aqueous ethanol.

Optimization of Chromatographic Conditions

Because, the λ max value of (−)-α-bisabolol was 192 nm under the chromatographic conditions, therefore, methanol cannot be used as a mobile phase, as its UV cutoff value = 205 nm. First, acetonitrile and deionized water (gradient condition: 10 % acetonitrile → 90 % acetonitrile → 10 % acetonitrile) was tested as a mobile phase (data not shown); however, the ascorbyl tetraisopalmitate peak was not detected. Afterwards, isopropyl alcohol was added to acetonitrile and deionized water. In this context, Almeida et al. used isocratic elution with methanol and isopropyl alcohol (25:75, v/v) to separate tocopheryl acetate and ascorbyl tetraisopalmitate in cosmetic formulations using HPLC [25]. Kamal et al. used acetonitrile and deionized water in gradient elution method [15]. In this study, the mobile phase gradient condition described in “Analytical method” was selected to separate the three compounds, as shown in Fig. 1A. The chromatograms of (B), (C), and (D) were built with mobile phases without deionized water; resulting in poor resolution of glabridin and (−)-α-bisabolol compared to (A). The resolution values of (−)-α-bisabolol and glabridin were 38.30, 12.19, 6.55, and 7.80, in mobile phase A, B, C, and D, respectively (Fig. 1).

Fig. 1
figure 1

HPLC-PAD chromatograms of standard solution using various mobile phase composition. Initial mobile phase compositions; A deionized water:acetonitrile (30:70, v:v), B 100 % acetonitrile, C acetonitrile:isopropyl alcohol (70:30, v:v), D acetonitrile:isopropyl alcohol (80:20, v:v). Entire mobile phase compositions are stated in Table 3. Standard solutions; glabridin (5.0 μg mL−1), (−)-α-bisabolol (60.0 μg mL−1), and ascorbyl tetraisopalmitate (200.0 μg mL−1)

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The enlarged chromatograms of dotted zone in Fig. 1 are presented in Fig. 2 at 202 nm. The mobile phase composition is shown in Table 3. Finally, mobile phase containing deionized water, acetonitrile, and isopropyl alcohol was used for separation and detection of the tested compounds.

Fig. 2
figure 2

Enlarged HPLC-PAD chromatograms of dotted zone from Fig. 1 at UV 202 nm. A–D is the same condition used in Fig. 1

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Table 3 Conditions of mobile phase illustrated in Fig. 1
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Method Performance

The linearity, expressed as determination coefficient (R 2), was calculated by external standard calibration curves as shown in Table 1. The R 2 values of the three compounds were ≥0.999. The method was specific since there is no overlap or interference peak around the retention time of the tested compounds (Figs. 1, 2, 3). The LOD values of 0.03 μg mL−1 (glabridin), 0.4 μg mL−1 ((−)-α-bisabolol), and 4.02 μg mL−1 (ascorbyl tetraisopalmitate) were satisfactory for analysis. The current LODs were considerably lower than those reported for ascorbyl tetraisopalmitate in cosmetic products (15.05 μg mL−1 [15]), glabridin in crude drug (0.35 μg mL−1 [25]), and (−)-α-bisabolol in particulate systems (0.5 μg mL−1 [28]). Recoveries at three fortification levels were ranged from 89.8 to 103.9 % with RSD < 5 % (Table 4). The precision values using standard solutions shown in Table 4 were <2 % for both inter- and intra-day variation. System suitability testing was investigated using mixed standard solutions [37] (Table 5). As shown in Fig. 3, although the capacity factor of glabridin was lower than 2.0, the instrumental analysis was not affected. The abovementioned chromatographic parameters were deemed acceptable [38], indicating that the validated method is accurate for analysis. \( S_{{D_{i} }} \) value in robustness test (=0.07) was lower than the estimated method precision value (=0.21, standard deviation value of (−)-α-bisabolol at 50 μg mL−1). All selected variables for Youden’s robustness test have no effect on the results. The experimental t values are lower than that of the 2-tailed t critical value for all seven factors: t crit = 4.30 for 2 degrees of freedom at 95 % confidence level (Table 6). Regarding the variable “column supplier” all standard deviation values obtained in the eight robustness experiments (Table 2) are reported in Table 7. This factor was confirmed as the most critical (t = 0.63) with a change of column supplier. The alteration of column supplier did not present significant variations in the standard deviation value of (−)-α-bisabolol (n = 3) The tested procedure proved to be robust, since minor fluctuations in the operative parameters that can occur during the routine application of the method are significantly affecting its performance characteristics.

Fig. 3
figure 3

HPLC-PAD chromatograms of analyzed cosmetic products. A Standard solutions, B sample solution no. 6, C Sample solution no. 5, and D sample solution no. 7

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Table 4 Accuracy and precision of the tested compounds in spiked cosmetic formulations
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Table 5 Chromatographic parameters
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Table 6 Robustness test results for (−)-α-bisabolol sample
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Table 7 Effect of variation of the column supplier (bold results in Table 6)
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Analysis of Commercial Products

The proposed method was applied for the quantitation of glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate in 11 functional cosmetic products. The three compounds were not labeled as major functional ingredients, just general components. As shown in Table 8, the detected amounts were in the range of 25.1–677.0 mg 100 g−1 for (−)-α-bisabolol, 17.5–25 mg 100 g−1 for glabridin, and 140.6–291.5 mg 100 g−1 for ascorbyl tetraisopalmitate. According to the Korean Ministry of Food and Drug safety, the content of (−)-α-bisabolol should be higher than 0.5 % (w/w) to enhance its functional effect. From the above reported data, (−)-α-bisabolol was considered as a major functional ingredient (except for sample no. 1) and the other couples were considered as minor ingredients. Figure 3 shows the typical HPLC chromatograms of market samples.

Table 8 Detection of the target compounds in commercial whitening cosmetic creams
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Conclusions

A simple and sensitive analytical method using an HPLC-PAD was developed for simultaneous quantification of glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate in functional cosmetic products. The validated method exhibited good linearity, sensitivity, recovery, precision, and robustness and can be used for detection of lipophilic compounds in cosmetic creams. Up to the author knowledge, this is the first report for simultaneous detection of the three lipophilic compounds in functional cosmetics.