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ChromatographiaAn International Journal for RapidCommunication in Chromatography,Electrophoresis and AssociatedTechniques ISSN 0009-5893Volume 79Combined 13-14 Chromatographia (2016) 79:851-860DOI 10.1007/s10337-016-3104-2

Simultaneous Detection of Glabridin,(−)-α-Bisabolol, and AscorbylTetraisopalmitate in Whitening CosmeticCreams Using HPLC-PAD

Jong-Sup Jeon, Han-Taek Kim, Myeong-Gil Kim, Moon-Seog Oh, Se-Ra Hong,Mi-Hye Yoon, Ho-Chul Shin, Jae-HanShim, Nehal Aly Afifi, et al.

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DOI 10.1007/s10337-016-3104-2Chromatographia (2016) 79:851–860

ORIGINAL

Simultaneous Detection of Glabridin, (−)‑α‑Bisabolol, and Ascorbyl Tetraisopalmitate in Whitening Cosmetic Creams Using HPLC‑PAD

Jong‑Sup Jeon1 · Han‑Taek Kim1 · Myeong‑Gil Kim1 · Moon‑Seog Oh1 · Se‑Ra Hong1 · Mi‑Hye Yoon1 · Ho‑Chul Shin2 · Jae‑Han Shim3 · Nehal Aly Afifi4 · Ahmet Hacımüftüoglu5 · A. M. Abd El‑Aty2,4

Received: 12 February 2016 / Revised: 14 April 2016 / Accepted: 9 May 2016 / Published online: 19 May 2016 © Springer-Verlag Berlin Heidelberg 2016

(−)-α-bisabolol, and ascorbyl tetraisopalmitate, respec-tively. The calibration curves showed good linearity with determination coefficients (R2) ≥ 0.999. The mean recov-eries were ranged between 89.8 and 103.9 % with relative standard deviations (RSDs) <5 %. The intra- and inter-day precision was <2 %. The limits of detection (LODs) were 0.03, 0.4, and 4.02 μg mL−1 for glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate, respectively. The method was successfully applied for monitoring 11 market sam-ples, in which glabridin was quantified in the range of 17.5–25 mg 100 g−1, (−)-α-bisabolol in the range of 25.1–677 mg 100 g−1, and 140.6–291.5 mg 100 g−1 for ascorbyl tetraisopalmitate. The proposed analytical method is sim-ple, sensitive, and versatile and can be used for the quan-tification of lipophilic compounds in cosmetics in a single chromatographic run.

Keywords High-performance liquid chromatography · Glabridin · (−)-α-Bisabolol · Ascorbyl tetraisopalmitate · Functional cosmetic

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. Cosmeceu-ticals are intended to carry out their functions as whiten-ing, 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,

Abstract A simultaneous analytical method was devel-oped and validated to quantify three lipophilic com-pounds; namely glabridin (an isoflavonoid isolated from crude licorice), (−)-α-bisabolol (a sesquiterpene alcohol obtained from plant extracts), and ascorbyl tetraisopal-mitate (a fat-soluble molecule derived from vitamin C) in functional cosmetic cream using high-performance liquid chromatography (HPLC) coupled with photodiode array detection (PAD). Cosmetic cream samples were extracted with a mixture of acetonitrile and isopropyl alcohol (45:55, v/v) and the target compounds were separated on a C18 column with a gradient mobile phase consisting of deion-ized water, acetonitrile, and isopropyl alcohol. The detec-tor wavelengths were 228, 202, and 221 nm, for glabridin,

* Jong-Sup Jeon vet089@gg.go.kr

* A. M. Abd El-Aty abdelaty44@hotmail.com; amabdelaty@konkuk.ac.kr

1 Public Health Research Division, Gyeonggi Province Institute of Health and Environment, 95, Pajang cheon-ro, Jangan-gu, Suwon-Si, Gyeonggi province 440-290, Republic of Korea

2 Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Nuengdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea

3 Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, Yongbong-ro 77, Buk-gu, Gwangju 500-757, Republic of Korea

4 Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt

5 Department of Medical Pharmacology, Medical Faculty, Ataturk University, Erzurum, Turkey

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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 deriva-tives, (−)-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 tetraiso-palmitate, 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-sol-uble compounds. In our previous research studies, we have developed methodologies to determine hydrophilic sub-stances 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, antibacte-rial, 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. Addition-ally, it alters the gene expression of microphthalmia-asso-ciated transcription factor (MITF) and tyrosinase; imply-ing 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]; exhib-iting a good percutaneous absorption and a strong anti-oxidant 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 mili-spheres 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 nei-ther for simultaneous determination of the three lipophilic compounds (glabridin, (−)-α-bisabolol, and ascorbyl tetrai-sopalmitate) nor in whitening cosmetic creams.

Thus, the aim of this study was to develop and vali-date a simple and sensitive method to quantify glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmitate in whiten-ing 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 tetraisopalmi-tate (purity: 88.8 %) were obtained from Wako Chemi-cals (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 (Advan-tec, 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 puri-fied deionized water.

Standard Preparation

Stock standard solutions of glabridin (5.0 μg mL−1), (−)-α-bisabolol (60.0 μg mL−1), and ascorbyl tetraisopal-mitate (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 con-centration 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.

Sample Preparation

Commercial creams (0.1 g) was accurately weighed (XSE 205DU Analytical Balance, Mettler Toledo, Greifensee,

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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 (Vor-tex-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 super-natant was filtered through a 0.20 μm polytetrafluorethyl-ene (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 detec-tion. 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 ana-lytes. Capcell Pak C18 MG (250 × 4.6 mm, 5 μm, Shiseido Co, Ltd., Tokyo, Japan) was used to evaluate the chro-matographic robustness test. The target compounds were

Table 1 Detection wavelength (nm), concentration range (μg mL−1), calibration curve, linearity (R2), and limits of detection (LOD), and quan-tification (LOQ) of the tested compounds

a n = 5b Standard deviation

Compounds Detection wavelength (nm)

Concentration range (μg mL−1)

Calibration curvea R2 LOD (μg mL−1)

LOQ (μg mL−1)

Glabridin 228 0.25–5.0 y = 0.9151 (±0.008)b X − 0.0355 (±0.009)

1.000 0.03 0.10

(−)-α-Bisabolol 202 3.0–60.0 y = 0.296 (±0.003) 1X − 0.6659 (±0.047)

0.999 0.40 1.10

Ascorbyl tetraisopal-mitate

221 10.0–200.0 y = 0.2961 (±0.001) X − 0.6659 (± 0.106)

0.999 4.02 12.18

Table 2 Variables and their levels in chromatographic separation in the Youden’s robustness test experimental design

The uppercase (A–G) and lowercase (a–g) letters denote the assigned reasonable high and low values of the selected parameters with a balance between capital and small letters

The observed results (S–Z) are the values of the standard deviation of peak area of sample in each experi-ment

Selected variable Experiment #

1 2 3 4 5 6 7 8

Slope of mobile phase gradient A A A A a a a a

Column temperature B B b b B B b b

Flow rate C c C c C c C c

Wavelength D D d d d d D D

Injection volume E e E e e E e E

Column supplier F f f F F f f F

Initial mobile phase composition (D.W. %) G g g G g G G g

Observed results (R) S T U V W X Y Z

Variable Units Abbreviation High level Low level

Slope of mobile phase gradient – A, a 6 4

Column temperature °C B, b 27 23

Flow rate mL/min C, c 1.1 0.9

Wavelength nm D, d 204 200

Injection volume μL E, e 11 9

Column supplier – F, f Agilent Shiseido

Initial mobile phase composition (D.W. %) % G, g 32 28

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separated using a stepwise gradient mobile phase consist-ing 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 determi-nation coefficient (R2) was calculated and evaluated as lin-earity. 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 devel-oped 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 separa-tion 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 gra-dient, column temperature, flow rate, detection wavelength, injection volume, column manufacturer, and the initial mobile phase composition were chosen as the seven vari-ables 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 Di. The standard deviation of the differ-ences, SDi

, was calculated by the formula:

When SDi is significantly higher than the standard devia-

tion of the method, it means that all the chosen factors together have an effect on the result [35].

SDi=

√

√

√

√

2×∑

(

D2

i

7

)

.

Fig. 1 HPLC-PAD chromatograms of standard solution using vari-ous 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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Additionally, with t test, it is possible to evaluate the influence of each investigated factor. The experimental t value is given by the equation:

where n is the number of experiments carried out at each level for each parameter (n = 4) The standard devia-tion 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 (tcrit) at n − 1 degree of freedom, where n is the number of determinations used in the esti-mation of S.D. at 95 % confidence level. If t value is greater than tcrit, the investigated variable shows a significant influ-ence, 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

t =√n× |Di|√2× S.D.

of them as following: (A) 100 % acetonitrile, (B) 100 % isopropyl alcohol, (C) acetonitrile and isopropyl alco-hol (30:70 %, v/v), (D) acetonitrile and isopropyl alco-hol (40:60 %, v/v), (E) acetonitrile and isopropyl alco-hol (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 ace-tonitrile and isopropyl alcohol (45:55 %, v/v) efficiently extracted the three analytes compared to others. From previous studies, for instance, Almeida et al. used isopro-pyl 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 prep-arations 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 %

Fig. 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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acetonitrile) was tested as a mobile phase (data not shown); however, the ascorbyl tetraisopalmitate peak was not detected. Afterwards, isopropyl alcohol was added to ace-tonitrile 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 glabri-din were 38.30, 12.19, 6.55, and 7.80, in mobile phase A, B, C, and D, respectively (Fig. 1).

The enlarged chromatograms of dotted zone in Fig. 1 are presented in Fig. 2 at 202 nm. The mobile phase composi-tion 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.

Method Performance

The linearity, expressed as determination coefficient (R2), was calculated by external standard calibration curves as shown in Table 1. The R2 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 satis-factory 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 abovemen-tioned chromatographic parameters were deemed accept-able [38], indicating that the validated method is accurate for analysis. SDi

value in robustness test (=0.07) was lower than the estimated method precision value (=0.21, stand-ard 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: tcrit = 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 experi-ments (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

Table 3 Conditions of mobile phase illustrated in Fig. 1

Time (min) Composition of mobile phase (%)

A (Deionized water)

B (Acetonitrile) C (Isopropyl alcohol)

(A)

0 30 70 0

5 30 70 0

12 0 100 0

15 0 0 100

25 0 0 100

32 0 100 0

37 0 100 0

45 30 70 0

(B)

0 0 100 0

12 0 100 0

15 0 0 100

25 0 0 100

32 0 100 0

45 0 100 0

(C)

0 0 70 30

12 0 70 30

15 0 0 100

25 0 0 100

32 0 70 30

45 0 70 30

(D)

0 0 80 20

12 0 80 20

15 0 20 80

25 0 20 80

32 0 80 20

45 0 80 20

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that can occur during the routine application of the method are significantly affecting its performance characteristics.

Analysis of Commercial Products

The proposed method was applied for the quantitation of glabridin, (−)-α-bisabolol, and ascorbyl tetraisopalmi-tate in 11 functional cosmetic products. The three com-pounds 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.

Conclusions

A simple and sensitive analytical method using an HPLC-PAD was developed for simultaneous quantification of

Fig. 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

Table 4 Accuracy and precision of the tested compounds in spiked cosmetic formulations

* Three independent samples individually extracted

Compounds Concentration (μg mL−1)

Recovery (%) (n = 3)*

RSD (%) Intra-day vari-ability (RSD %) (n = 3)

Inter-day vari-ability (RSD %) (n = 9)

Glabridin 0.5 95.0 0.95 0.62 0.45

2.5 99.4 0.54 0.07 0.41

5.0 100.6 0.71 0.77 0.54

(−)-α-Bisabolol 6.0 89.8 1.83 1.34 0.83

30.0 99.7 1.18 0.90 0.83

60.0 100.8 0.34 0.89 0.58

Ascorbyl tetraiso-palmitate

20.0 103.9 4.88 0.55 0.79

100.0 91.9 1.00 0.82 0.76

200.0 93.7 2.44 0.41 0.56

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Table 6 Robustness test results for (−)-α-bisabolol sample

a t =√n×|Di |√2×S.D.

where n = 4 and S.D. = 0.21; tcrit = 4.30, degree of freedom = 3−1, 95 % confidence level

Variable Difference (Di) in standard deviation value t valuea

Slope of mobile phase gradient 0.05 0.33

Column temperature 0.03 0.23

Flow rate 0.02 0.11

Wavelength −0.06 0.42

Injection volume 0.02 0.14

Column supplier −0.09 0.63

Initial mobile phase composition (D.W. %) −0.04 0.27

Table 7 Effect of variation of the column supplier (bold results in Table 6)

a Mean value (n = 4)b In this study, Df = ∑(Fi)/4−∑(fi)/4 = (0.08 + 0.08 + 0.12 + 0.02)/4−(0.18 + 0.25 + 0.17 + 0.07)/4 = −0.09

Column brand Experimental results Standard deviation valuea

Agilent (F) S 0.08

V 0.08

W 0.12

Z 0.02

Average 0.08

Shiseido (f) T 0.18

U 0.25

X 0.17

Y 0.07

Average 0.17

Df (difference f)b −0.09 (=0.08−0.17)

Selected variable Experiment

1 2 3 4 5 6 7 8

Column supplier F f f F F f f F

Observed results S T U V W X Y Z

Numerical value 0.08 0.18 0.25 0.08 0.12 0.17 0.07 0.02

Table 5 Chromatographic parameters

a Instrument precision indicates the performance of the HPLC, including plumbing, column, environmental conditions, and the time at which the samples were analyzedb Capacity factor is a measure of the location of the peak of interest with respect to the void volumec Resolution is a measure of how well two peaks are separatedd Accuracy of quantitation decreases with increase in peak tailing because of the difficulties encountered by the integrator in determining where or when the peak ends, and hence the calculation of the area under the peake Theoretical plate number is a measure of column efficiency; that is, how many peaks can be located per unit run time of the chromatogram

Compounds Acceptance criteria

Retention time (min)

Instrument precision, RSDa (%), ≤1.0

Capacity factorb, ≥2.0

Resolu-tionc, ≥2.0

Tailing fac-tord, ≤2.0

Plate counte, ≥2000

Glabridin 4.98 0.37 1.51 – 1.15 14,194

(−)-α-Bisabolol 11.90 0.26 5.10 34.59 1.13 63,710

Ascorbyl tetraisopalmitate 20.09 0.79 9.32 113.31 1.09 82,781

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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 knowl-edge, this is the first report for simultaneous detection of the three lipophilic compounds in functional cosmetics.

Acknowledgments The authors gratefully acknowledge the financial support from the Gyeonggi Province Institute of Health and Environ-ment (Suwon city, Republic of Korea).

Compliance with standards

Conflict of interest The authors have declared no conflict of interest.

References

1. Korea Ministry of drug and food safety (2013) Cosmetic act, Seoul, Republic of Korea

2. Gao XH, Zhang L, Wei H, Chen HD (2008) Efficacy and safety of innovative cosmeceuticals. Clin Dermatol 26:367–374

3. Amer M, Maged M (2009) Cosmeceuticals versus pharmaceuti-cals. Clin Dermatol 27:428–430

4. Jeon JS, Kim HT, Kim MG, Oh MS, Hong SR, Yoon MH, Cho SM, Shin HC, Shim JH, Ramadan A, Abd El-Aty AM (2016) Simultaneous determination of water-soluble whitening ingredi-ents and adenosine in different cosmetic formulations by high-performance liquid chromatography coupled with photodiode array detection. Int J Cosmet Sci 38:286–293

5. Chang ML, Chang CM (2003) Simultaneous HPLC determi-nation of hydrophilic whitening agents in cosmetic products. J Pharm Biomed Anal 33:617–626

6. Jain P, Sonti S, Garruto J, Mehta R, Banga AK (2012) Formula-tion optimization, skin irritation, and efficacy characterization of a novel skin-lightening agent. J Cosmet Dermatol 11:101–110

7. Rendon M, Berneburg M, Arellano I, Picardo M (2006) Treat-ment of melasma. J Am Acad Dermatol 54:272–281

8. Huang SC, Lin CC, Huang MC, Wen KC (2004) Simultaneous determination of magnesium ascorbyl phosphate, ascorbyl glu-coside, kojic acid, arbutin and hydroquinone in skin whitening cosmetics by HPLC. J Food Drug Anal 12:13–18

9. Jin W, Wang WY, Zhang YL, Yang YJ, Chu QC, Ye JN (2013) Determination of phenolic whitening agents in cosmetics by micellar electrokinetic capillary chromatography with ampero-metric detection. Chin Chem Lett 24:636–638

10. Jeon JS, Kim BH, Lee SH, Kwon HJ, Bae HJ, Kim SK, Park JA, Shim JH, Abd El-Aty AM, Shin HC (2015) Simultaneous deter-mination of arbutin and its decomposed product hydroquinone in whitening creams using high-performance liquid chromatogra-phy with photodiode array detection: effect of temperature and pH on decomposition. Int J Cosmet Sci 37:567–573

11. Korea Ministry of drug and food safety (2013) Korean Func-tional Cosmetics Codex, Seoul, Republic of Korea

12. Jeon JS, Lee MJ, Yoon MH, Park JA, Yi H, Cho HJ, Shin HC (2014) Determination of arbutin, niacinamide, and adenosine in functional cosmetic products by high-performance liquid chro-matography. Anal Lett 47:1650–1660

13. Simmler C, Pauli GF, Chen S-N (2013) Phytochemistry and bio-logical properties of glabridin. Fitoterapia 90:160–184

14. Yokota T, Nishio H, Kubota Y, Mizoguchi M (1998) The inhibi-tory effect of glabridin from licorice extracts on melanogenesis and inflammation. Pigment Cell Res 11:355–361

15. Kamal YT, Singh M, Tamboli ET, Parveen R, Zaidi SM, Ahmad S (2012) Rapid RP-HPLC method for the quantification of glabridin in crude drug and in polyherbal formulation. J Chroma-togr Sci 50:779–784

16. Rosenblat M, Belinky P, Vaya J, Levy R, Hayek T, Coleman R, Merchav S, Aviram M (1999) Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell-medi-ated oxidation of low density lipoprotein a possible role for pro-tein kinase c. J Biol Chem 274:13790–13799

17. Waleczek KJ, Marques HMC, Hempel B, Schmidt PC (2003) Phase solubility studies of pure (−)-α-bisabolol and camomile essential oil with β-cyclodextrin. Eur J Pharm Biopharm 55:247–251

18. Murugan R, Mallavarapu GR (2013) α-Bisabolol, the main con-stituent of the essential oil of Pogostemon speciosus. Ind Crops Prod 49:237–239

19. Bhatia SP, McGinty D, Letizia CS, Api AM (2008) Fragrance material review on α-bisabolol. Food Chem Toxicol 46:72–76

20. Vila R, Santana AI, Pérez-Rosés R, Valderrama A, Castelli MV, Mendonca S, Zacchino S, Gupta MP, Cañigueral S (2010) Com-position and biological activity of the essential oil from leaves of Plinia cerrocampanensis, a new source of α-bisabolol. Bioresour Technol 101:2510–2514

21. Kamatou GP, Viljoen A (2010) A review of the application and pharmacological properties of α-bisabolol and α-bisabolol-rich oils. J Am Oil Chem Soc 87:1–7

22. Kim S, Lee J, Jung E, Huh S, Park J-O, J-w Lee, Byun SY, Park D (2008) Mechanisms of depigmentation by α-bisabolol. J Der-matol Sci 52:219–222

23. Hakozaki T, Takiwaki H, Miyamoto K, Sato Y, Arase S (2006) Ultrasound enhanced skin-lightening effect of vitamin C and niacinamide. Skin Res Technol 12:105–113

24. Manela-Azulay M, Bagatin E (2009) Cosmeceuticals vitamins. Clin Dermatol 27:469–474

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

a Sample no. 9 contained two whitening ingredientsb n = 3

Samples Whitening ingredient Amount detected (mg 100 g−1)b

1 (−)-α-Bisabolol 25.1

2 Glabridin 17.5

3 Glabridin 19.3

4 (−)-α-Bisabolol 677.0

5 (−)-α-Bisabolol 624.6

6 Glabridin 25.0

7 Ascorbyl tetraisopalmitate 291.5

8 (−)-α-Bisabolol 499.0

9a Ascorbyl tetraisopalmitate 140.6

(−)-α-Bisabolol 634.6

10 (−)-α-Bisabolol 647.1

11 (−)-α-Bisabolol 670.2

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25. Almeida MM, Alves JMP, Patto DCS, Lima CRRC, Quenca-Guillen JS, Santoro MIRM, Kedor-Hackmann ERM (2009) Determination of tocopheryl acetate and ascorbyl tetraisopal-mitate in cosmetic formulations by HPLC. Int J Cosmet Sci 31:445–450

26. Maia Campos PMBG, Gonçalves GMS, Gaspar LR (2008) In vitro antioxidant activity and in vivo efficacy of topical formu-lations containing vitamin C and its derivatives studied by non-invasive methods. Skin Res Technol 14:376–380

27. Gaspar LR, Campos PMBGM (2007) Photostability and efficacy studies of topical formulations containing UV-filters combina-tion and vitamins A, C and E. Int J Pharm 343:181–189

28. Pedro AS, Detoni C, Ferreira D, Cabral-Albuquerque E, Sar-mento B (2009) Validation of a high-performance liquid chroma-tography method for the determination of (−)-α-bisabolol from particulate systems. Biomed Chromatogr 23:966–972

29. Perbellini L, Gottardo R, Caprini A, Bortolotti F, Mariotto S, Tagliaro F (2004) Determination of alpha-bisabolol in human blood by micro-HPLC–ion trap MS and head space-GC–MS methods. J Chromatogr B 812:373–377

30. Scalia S, Giuffreda L, Pallado P (1999) Analytical and prepara-tive supercritical fluid extraction of chamomile flowers and its comparison with conventional methods. J Pharm Biomed Anal 21:549–558

31. Weber B, Herrmann M, Hartmann B, Joppe H, Schmidt CO, Ber-tram H-J (2008) HPLC/MS and HPLC/NMR as hyphenated tech-niques for accelerated characterization of the main constituents

in Chamomile (Chamomilla recutita [L.] Rauschert). Eur Food Res Technol 226:755–760

32. Aoki F, Nakagawa K, Tanaka A, Matsuzaki K, Arai N, Mae T (2005) Determination of glabridin in human plasma by solid-phase extraction and LC-MS/MS. J Chromatogr B Analyt Tech-nol Biomed Life Sci 828:70–74

33. US Pharmacopeia (2009) General Chapters <1225> Validation of compendial methods. United States Pharmacopeal Conven-tion, Inc., Rockville, Maryland, USA

34. Karageorgou E, Samanidou V (2014) Youden test application in robustness assays during method validation. J Chromatogr A 1353:131–139

35. Commission Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Commun L221 8

36. Scortichini G, Annunziata L, Haouet MN, Benedetti F, Krusteva I, Galarini R (2005) ELISA qualitative screening of chloram-phenicol in muscle, eggs, honey and milk: method validation according to the Commission Decision 2002/657/EC criteria. Anal Chim Acta 535:43–48

37. Furman WB, Dorsey JG, Snyder LR (1998) System suitability tests in regulatory liquid and gas chromatographic methods: adjustments versus modifications. Pharm Technol 22:58–65

38. US Food and Drug Administration (1994) Reviewer Guidance, validation of chromatographic methods. Centre for Drug Evalua-tion and Research, Washington

Author's personal copy