International Journal of Cosmetic Science, 2016, 38, 286–293
doi: 10.1111/ics.12292
Simultaneous determination of water-soluble whitening ingredients and adenosine in different cosmetic formulations by high-performance liquid chromatography coupled with photodiode array detection J.-S. Jeon*, H.-T. Kim*, M.-G. Kim*, M.-S. Oh*, S.-R. Hong*, M.-H. Yoon*, S.-M. Cho†, H.-C. Shin†, J.-H. Shim‡, A. Ramadan§ and A. M. Abd El-Aty†,§ *Public Health Research Division, Gyeonggi Province Institute of Health and Environment, 95, Pajang cheon-ro, Jangan-gu, Suwon-Si 440-290, Republic of Korea, †Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Nuengdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea, ‡Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea and §Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt
Received 17 September 2015, Accepted 7 November 2015
Keywords: adenosine, arbutin, ascorbyl glucoside, ethyl ascorbyl ether, high-performance liquid chromatography, niacinamide
Abstract OBJECTIVE: The Korean Cosmetic Act regulates the use of functional cosmetics) by the law. Four functional cosmetic groups, whitening, anti-wrinkle, UV protection and combination of whitening and anti-wrinkle, were categorized according to the Korean Cosmetic Act and Functional Cosmetics Codex. In this study, highperformance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) was employed for the simultaneous detection of arbutin (and its decomposition product, hydroquinone), niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine in functional cosmetic products such as creams, emulsions and lotions. METHODS: Separation by HPLC-DAD was conducted using a C18 column with a gradient elution of 5 mM KH2PO4 buffer (containing 0.1% phosphoric acid) and methanol (containing 0.1% phosphoric acid). The wavelengths for the detection of arbutin, hydroquinone, niacinamide, adenosine, ascorbyl glucoside and ethyl ascorbyl ether were 283, 289, 261, 257, 238 and 245 nm, respectively. RESULTS: This method exhibited good linearity (R2 ≥ 0.999), precision (expressed as relative standard deviation (RSD) < 2%) and mean recoveries (89.42–104.89%). The results obtained by monitoring 100 market samples showed that the detected levels of the tested materials are within the acceptable authorized concentration. CONCLUSION: The method developed herein is simple and can be used for market survey and quality control of functional cosmetics. sume Re Correspondence: J.-S. Jeon, Public Health Research Division, Gyeonggi Province Institute of Health and Environment, 95, Pajang cheon-ro, Jangan-gu, Suwon-Si 440-290, Republic of Korea. Tel.: +82 31 250 2582; fax: +82-31-250-2588; e-mail: [email protected] and A. M. Abd El-Aty, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Nuengdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea. Tel.: +82 10 5934 0701; fax: +82 2 444 4396. e-mail: [email protected]
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OBJECTIF: La Loi Cosmetique coreenne reglemente l’utilisation de cosmetiques ou de produits fonctionnels). Quatre groupes cosmeti savoir les blanchissants ment, les anti-rides, les ques fonctionnels, a anti-UV, et les combinaisons de blanchiment et anti-rides, ont ete classes selon la reglementation cosmetique coreenne et le Codex des Functional Cosmetics. Dans cette etude, la Chromatographie haute performance (HPLC) couplee a une detection a liquide a reseau de photodiodes (DAD) a ete utilisee pour la detection simultanee de l’arbutine (et son produit de degradation, l’hydroquinone), du niacinamide, du glucoside d’ascorbyle, de l’ether d’ascorbyle d’ethyle, et de l’adenosine dans les produits cosmetiques fonctionnels tels que les cremes, emulsions, lotions etc. METHODES: La separation par HPLC-DAD a ete effectuee en utilisant une colonne C18 avec un gradient d’elution d’un tampon 5 mM KH2PO4 (contenant 0.1% d’acide phosphorique) et de methanol (contenant 0.1% d’acide phosphorique). Les longueurs d’onde pour la detection de l’arbutine, de l’hydroquinone, du niacinamide, de l’adenosine, du glucoside d’ascorbyle, de l’ether l’ascorbyl d’ethyle sont 283, 289, 261, 257, 238 et 245 nm, respectivement. RESULTATS: Cette methode presente une bonne linearite 2% (exprimee en ecart(R2 ≥ 0.999), une precision inferieure a type relatif (RSD), et des recouvrements moyens (89.42 de 104.89%). Les resultats obtenus en analysant les 100 echantillons du marche ont montre que les taux des substances testees se situent dans les limites des concentrations autorisees. CONCLUSION: La methode developpee ici est simple et peut ^etre utilisee pour l’etude du marche et le contr^ole de qualite des produits cosmetiques fonctionnels.
Introduction According to the Korean Cosmetic Act, cosmetics are defined as goods that are used by various applications for the purpose of increasing attractiveness by cleaning and beautifying the human body, increasing brightening appearance and maintaining or improving the health of skin and hair [1]. In addition, the Korean
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
Determination of whitening ingredients and adenosine in functional cosmetics using HPLC
Cosmetic Act regulates the use of functional cosmetics (cosmeceuticals) by the law. The word ‘cosmeceuticals’ was popularized by Albert Kligman in the late 1970s. Before the late 1970s, the term ‘cosmeceuticals’ was first used by Raymond Reed, who used it for the purpose of explaining active and science-based cosmetics [2, 3]. Cosmeceuticals may also be considered as hybrids between cosmetics and pharmaceuticals, which are intended to enhance the health and beauty of skin [4]. Thus far, only the Republic of Korea and Japan have legislations that distinguish cosmetic products and functional (quasi-drug) cosmetics. However, the U.S. Food and Drug Administration does not recognize any such categorization into cosmeceuticals, and the use of the term cosmeceutical has no meaning [5]. Tanned skin is considered healthy and beautiful by Westerners, whereas light skin is considered beautiful in oriental countries. Hence, whitening cosmetics are very popular in the oriental countries [6]. In particular, in the Republic of Korea, there is rapid growth towards the development of functional cosmetics as different cosmetics by the simple correction of the colour to an active creation of beauty [7]. The Korean Cosmetic Act and the Korean Functional Cosmetics Codex (KFCC) have categorized four functional cosmetic groups: whitening, anti-wrinkle, UV protection and the combination of whitening and anti-wrinkle [1, 8]. Arbutin, niacinamide, ascorbyl glucoside, ascorbyl tetraisopalmitate, abisabolol, ethyl ascorbyl ether, Broussonetia extract, and glabridin, which are active ingredients of oil-soluble licorice (Glycyrrhiza) extract, are registered as whitening agents. Arbutin, niacinamide, ascorbyl glucoside and ethyl ascorbyl ether are water-soluble substances, whereas the remaining are fat-soluble substances. Hydroquinone has been recognized as one of the pharmaceuticals used for the treatment of hyperpigmentation. However, the prolonged usage of hydroquinone may cause disorders such as ochronosis, dermatitis, cataract and pigmented colloid milium. Because of these reasons, hydroquinone is no longer being authorized for use in formulations for cosmetic skin lightening in several countries, including the Republic of Korea. This limitation leads to the development of new ingredients such as arbutin, kojic acid, niacinamide and vitamin C derivatives [9–13]. Arbutin, a derivative of hydroquinone, is a safe skin-whitening topical agent used for depigmentation without cytotoxicity and side effects [12, 14]. Niacinamide, also known as nicotinamide and nicotinic acid amide, is a water-soluble vitamin [3]. It acts as a powerful antioxidant and an effective skin lightener by the inhibition of the transfer of melanosome from melanocytes to keratinocytes, subsequently decreasing the appearance of hyperpigmented spots [15–18]. The topical application of niacinamidecontaining cosmetics has been reported to exhibit beneficial antiwrinkle effects such as improving barrier function and decreasing the appearance of signs of chronological and facial photoaging [19, 20]. Because vitamin C exhibits antioxidant activity and effect for the inhibition of tyrosinase [18], it has been used as a skin lightener. However, a major limitation of vitamin C and its derivatives is degradation, which can be accelerated in the presence of oxygen, water, light, heat and metals. To prevent rapid degradation, different derivatives have been introduced, such as ascorbyl phosphate salts, ascorbyl palmitate, ascorbyl glucoside and ethyl ascorbyl ether. These derivatives are susceptible to enzymatic hydrolysis by tissue esterase and a-glucosidase, thereby yielding ascorbic acid [21–23]. Adenosine exhibits anti-wrinkle properties [24], and it is mostly used as an active ingredient in anti-wrinkle and combination of whitening and anti-wrinkle cosmetics in the Republic of Korea.
J.-S. Jeon et al.
High-performance liquid chromatography (HPLC) is a general technique for the quantitative estimation of arbutin in whitening cosmetics [10, 14, 25–30] and hydroquinone in cosmetic creams, topical dermatologic drugs and other matrices [27, 30–35]. Previously, arbutin, niacinamide and adenosine have been simultaneously determined in cosmetic products by HPLC [29]. Methods for determining ascorbyl glucoside or ethyl ascorbyl ether have been previously reported [27, 36]. This study aims to develop a simple method for the simultaneous detection of arbutin (and its decomposed product, hydroquinone), niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine in different cosmetic formulations for the quality control of functional cosmetics. To the best of our knowledge, this is the first report for the detection of the tested compounds in one chromatographic run. Materials and methods Chemicals and reagents Various brands of commercial functional cosmetics containing active ingredients (arbutin, niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine) were purchased from wholesale markets and cosmetic shops located in Suwon City, Republic of Korea. Niacinamide, adenosine, hydroquinone and potassium dihydrogen phosphate (KH2PO4, reagent grade) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Arbutin was supplied by Fluka (Buchs, Switzerland). Ascorbyl glucoside and ethyl ascorbyl ether were purchased from Tokyo Chemical Industry Co., LTD. (Tokyo, Japan). HPLC-grade methanol was provided by J.T. Baker (Griesheim, Germany). Phosphoric acid (85%, reagent grade) was obtained from Wako Chemicals (Tokyo, Japan). A 0.4-lm nylon membrane filter (Advantec, Tokyo, Japan) was used to filter the mobile phase. Sample solutions were filtered using 0.20-lm polytetrafluorethylene (PTFE) syringe filters (Advantec, Tokyo, Japan). Deionized water was purified using a Barnstead Nano pure Diamond (Dubuque, IA, U.S.A.). Standard preparation Stock standard solutions of arbutin (401 lg mL 1), hydroquinone (100.5 lg mL 1), niacinamide (401.6 lg mL 1), adenosine (40.2 lg mL 1), ascorbyl glucoside (400.2 lg mL 1) and ethyl ascorbyl ether (201.0 lg mL 1) were separately prepared by dissolving in deionized water. Fresh working solutions were prepared daily by the further dilution of the stock solutions with 5 mM KH2PO4 buffer (containing 0.1% phosphoric acid). Table I shows the concentration ranges used for the calibration of each standard. Sample preparation The samples were prepared according to our previous study [30]. First, 0.1 g each of cream, lotion and emulsion was accurately weighed in 15-mL polypropylene centrifuge tubes, and methanol (3 mL) was added to each of the tubes and mixed by vortex for 2 min. Second, the solution was sonicated (Branson 8510 sonicator, Danbury, CT, U.S.A.) for 30 min, and the total volume was adjusted to 10 mL using deionized water. Subsequently, the solution was sonicated for 30 min and then centrifuged (Sigma 2–6 centrifuge, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at 3500 rpm for 20 min. Finally, the solutions were diluted with the mobile phase A [5 mM KH2PO4 buffer (containing 0.1% phosphoric acid)] and then filtered using PTFE syringe filters.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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Determination of whitening ingredients and adenosine in functional cosmetics using HPLC Table I Detection wavelength (nm), concentration range (lg mL the tested compounds
1
), calibration curve, linearity (R2), and limits of detection (LOD) and quantification (LOQ) of
Compounds
Detection wavelength (nm)
Concentration range (lg mL 1)
Calibration curve
Arbutin Hydroquinone Niacinamide Adenosine Ascorbyl glucoside Ethyl ascorbyl ether
283 289 261 257 238 245
10–200.5 2–40.6 10–200.8 1–20.1 10–200.1 5–100.5
y y y y y y
= = = = = =
1.2610x 0.7504x 3.2662x 0.2620x 4.0854x 2.8608x
+ + + + + +
0.0090 0.0251 0.1194 0.0053 0.1316 0.1217
R2
LOD (lg mL 1)
LOQ (lg mL 1)
0.999 1.000 1.000 1.000 1.000 1.000
0.75 0.45 0.10 0.08 0.62 0.24
2.48 1.50 0.34 0.25 2.04 0.78
Table II Extraction efficiency (%) of the tested compounds using various solvents
Niacinamide
Ascorbyl glucoside
Arbutin
Ethyl ascorbyl ether
Adenosine
Solvents
Meana
SDb
Mean
SD
Mean
SD
Mean
SD
Mean
SD
A B C D E F G
Sc 85.21 102.04 89.80 99.23 101.00 98.01
– 0.13 5.36 4.69 0.72 6.55 1.14
Sc Md 102.25 104.52 107.74 Sc 108.14
– – 5.61 2.48 0.84 – 0.56
87.67 Sc 104.98 105.35 102.28 109.77 101.45
6.88 – 0.75 1.46 0.95 1.10 1.76
58.18 9.86 91.26 84.72 96.08 99.18 94.64
3.65 0.31 4.75 4.43 0.77 5.36 1.09
67.19 Sc 91.44 81.25 90.64 93.38 89.54
0.86 – 4.81 4.05 0.65 5.42 0.88
n = 3. SD, standard deviation. c Peak splitting. d Chromatographic peak moved forward. (A) methanol, (B) acetonitrile, (C) 5 mM KH2PO4, (D) 5 mM KH2PO4 buffer containing 0.1% phosphoric acid, (E) methanol: deionized water (3 : 7, v/v), (F) acetonitrile: deionized water (3 : 7, v/v) and (G) methanol: 5 mM KH2PO4 buffer containing 0.1% phosphoric acid (3 : 7, v/v). Hydroquinone is not an active ingredient. It is a decomposed product of arbutin. a
Retention time (min)
b
KH2PO4 conc (mM) Figure 1 Various concentrations of KH2PO4 in the mobile phase A. Solvents with the asterisk * contained 0.1% phosphoric acid.
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© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
J.-S. Jeon et al.
Retention time (min)
Determination of whitening ingredients and adenosine in functional cosmetics using HPLC
Phosphoric acid Figure 2 Various concentrations of phosphoric acid in the mobile phase A.
Analytical method Analysis was conducted using a Dionex UltiMate 3000 HPLC system (Sunnyvale, CA, U.S.A.) equipped with an UltiMate 3000 pump, an UltiMate 3000 autosampler column compartment and an UltiMate 3000 photodiode array detector. Data acquisition was performed with CHROMELEON software (Sunnyvale, CA, U.S.A.). A CAPCELL PAK C18 MG column (250 mm 9 4.6 mm, 5 lm, Shiseido, Japan) maintained at room temperature was used to achieve selective chromatographic conditions. The target compounds were separated using a stepwise gradient mobile phase consisting of 5 mM KH2PO4 buffer (containing 0.1% phosphoric acid) (A) and methanol (containing 0.1% phosphoric acid) (B), which was run as follows: 0–5 min, 0% B (v/v); 5–12 min, 10% B (v/v); 12–18 min, 20% B (v/v); and 18–23 min, 30% B (v/v), 23–26 min, 50% B (v/ v); 26–33 min, 0% B (v/v); and 33–35 min, 0% B (v/v). The flow rate and injection volume were 1 mL min 1 and 10 lL, respectively. Table I shows the wavelengths for the detection of each compound. Method validation The linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy and precision [37] of the method were validated for the quantification of the target compounds. The standard of each compound (at three concentrations) was spiked into blank samples (cream, lotion and emulsion) for conducting recovery tests, the results of which are evaluated as accuracy. To determine intraand interday sample variations, the developed method was repeated three times a day on three consecutive days.
pounds from commercially available cream samples was investigated using one of the following solvents: (A) methanol, (B) acetonitrile, (C) 5 mM KH2PO4, (D) 5 mM KH2PO4 buffer containing 0.1% phosphoric acid, (E) methanol: deionized water (3 : 7, v/v), (F) acetonitrile: deionized water (3 : 7, v/v) and (G) methanol: 5 mM KH2PO4 buffer containing 0.1% phosphoric acid (3 : 7, v/v). Sonication times were 30 min for A, B, C and D, and the remaining solvents were first sonicated (30 min), followed by another sonication with deionized water or buffer. The extraction efficiency (%) was calculated by dividing the concentration of each compound in sample solutions by the concentration of standard solutions, except
Table III Intraday and interday precision using standard solutions
Compounds
Arbutin
Hydroquinone
Niacinamide
Adenosine
Ascorbyl glucoside
Results and discussion Extraction procedure In previous studies, various extraction solvents such as water, acetonitrile and 0.05 M KH2PO4 buffer (pH 2.5) [6, 26, 27] have been used for the determination of the hydrophilic functional ingredients in cream. In this study, the extraction efficiency of the tested com-
Ethyl ascorbyl ether
Concentration (lg mL 1)
Intraday variabilitya, RSD (%)
Interday variabilityb, RSD (%)
20.1 100.3 200.5 4.1 20.3 40.6 20.1 100.4 200.8 0.4 2.1 4.2 20.0 100.1 200.1 10.1 50.3 100.5
0.42 0.19 0.53 0.29 0.59 0.83 0.29 0.10 0.51 0.46 0.07 0.14 0.28 0.14 0.45 0.25 0.3 0.48
0.74 0.84 0.81 0.78 0.95 0.95 0.71 0.82 0.64 0.45 0.96 0.89 0.76 0.77 0.90 0.95 1.03 0.86
n = 3. n = 9, triplicate injection each day for three consecutive days.
a b
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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as the extraction solvent-exhibited recovery satisfactory for the determination of adenosine in royal jelly. Our previous study has reported that using methanol: deionized water (1 : 9, v/v) as the extraction solvent-exhibited acceptable recovery [29]. Clearly, the standard deviation of the extraction efficiency of samples extracted with methanol: deionized water (3 : 7, v/v) was steady and less than one. Overall, the extraction efficiency using methanol (A) and acetonitrile (B) was not good. In sum, the use of methanol:
hydroquinone (not an original compound). As shown in Table II, the chromatograms of niacinamide and ascorbyl glucoside in methanol, arbutin and ethyl ascorbyl ether in acetonitrile, and ascorbyl glucoside in acetonitrile: deionized water (3 : 7, v/v) exhibited peak splitting. Moreover, the retention time of ascorbyl glucoside in acetonitrile moved forward. Adenosine in acetonitrile exhibited the lowest extraction efficiency (9.86%), caused by variance in solubility. Xue et al. [38] have reported that 80% ethanol
Table IV Recovery of the tested compounds in various spiked cosmetic formulations
Cream
Compounds
Arbutin
Hydroquinone
Niacinamide
Adenosine
Ascorbyl glucoside
Ethyl ascorbyl ether
Lotion
Emulsion
Concentration (lg mL 1)
Recoverya (%)
SDb (%)
Recovery (%)
SD (%)
Recovery (%)
SD (%)
20.1 100.3 200.5 4.1 20.3 40.6 20.1 100.4 200.8 0.4 2.1 4.2 20 100.1 200.1 10.1 50.3 100.5
102.35 102.45 101.42 102.99 100.63 98.75 94.54 96.39 103.46 101.90 103.96 98.03 99.64 99.19 95.64 104.89 103.50 97.38
2.82 1.69 1.72 3.58 0.30 3.99 2.72 0.86 2.75 0.91 2.47 3.69 0.55 1.51 1.63 0.51 3.40 1.20
93.30 99.74 95.21 96.88 102.28 96.04 89.42 91.18 99.74 94.76 99.58 98.06 97.46 96.79 98.40 103.92 95.75 96.47
2.14 2.46 0.53 3.65 4.87 2.28 0.90 0.83 0.66 0.52 2.87 1.02 2.36 3.96 2.69 0.94 0.40 0.89
93.95 102.24 95.13 92.98 104.71 99.00 93.82 90.60 103.19 96.71 98.22 100.37 93.78 94.75 98.65 96.54 103.92 99.64
1.18 2.84 0.47 1.39 0.64 0.47 4.77 0.92 0.39 5.02 2.99 1.18 3.10 4.21 0.92 1.39 0.52 2.49
n = 3. SD, standard deviation.
a b
Table V Chromatographic parameters
Acceptance criteria
Compounds
Retention time (min)
Instrument precisiona, RSD (%) ≤ 1.0
Capacity factorb ≥ 2
Niacinamide Ascorbyl glucoside Arbutin Adenosine Hydroquinone Ethyl ascorbyl ether
4.7 5.6 11.8 12.2 12.8 18.8
0.25 0.52 0.19 0.17 0.03 0.04
1.0 1.4 4.1 4.3 4.6 7.2
Resolutionc ≥ 2.0
– 5.91 32.55 2.60 3.17 22.25
Tailing factord ≤ 2.0
Theoretical plate numbere ≥ 2000
0.97 1.01 0.94 0.98 0.94 0.94
20 016 13 208 66 338 100 240 60 572 133 311
a Instrument precision indicates the performance of the HPLC, inclusive of the plumbing, column, environmental conditions and the time at which the samples were analysed. b Capacity factor is a measure of the location of the peak of interest with respect to the void volume. c Resolution is a measure of how well two peaks are separated. d 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 peak. e Theoretical plate number is a measure of column efficiency; that is, how many peaks can be located per unit run time of the chromatogram.
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© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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Determination of whitening ingredients and adenosine in functional cosmetics using HPLC Table VI Detection of the target compounds in commercial functional cosmetics
Category
Number of samples
Group
Whitening
Niacinamide Arbutin Ethyl ascorbyl ether Ascorbyl glucoside Adenosine Niacinamide Adenosine Arbutin Adenosine Niacinamide Ethyl ascorbyl etherb Adenosine Ethyl ascorbyl ether Adenosine
Anti-wrinkle Combined whitening/ anti-wrinkle
17 8 4 2 36 22
Range (%)
93.1–158.4 93.6–116.2 105.1–111.7 100.5–105.2 91.2–120.0 91.3–156.8 91.2–138.3 100.5–107.0 90.1–137.2 102.4 110.3 106.9 96.9 90.1
9 1
1
SDa
17.4 7.0 2.9 3.3 7.4 15.1 11.5 2.5 15.7 – – – – –
a
SD, standard deviation. Not declared on the product label.
b
450
deionized water (3 : 7, v/v) as an extraction solvent was the optimum condition to extract the tested compounds. Optimization of chromatographic conditions First, a mobile phase consisting of deionized water and methanol was tested [30]. However, the peaks of some analytes such as hydroquinone, niacinamide, ascorbyl glucoside and ethyl ascorbyl ether overlapped, and peak symmetry was poor. Afterwards, the KH2PO4 concentrations and acidity of phosphoric acid in mobile phase A were investigated. At the start, the tested KH2PO4 concentrations were 2.5, 5, 10, 20 and 50 mM at 0.1% phosphoric acid and 25 mM of KH2PO4 at 0.05% phosphoric acid. As can be seen in Fig. 1, the best resolution was achieved at 50 mM of KH2PO4 containing 0.1% phosphoric acid and 25 mM of KH2PO4 containing 0.05% phosphoric acid. Meanwhile, at 25 mM of KH2PO4 containing 0.05% phosphoric acid (data not shown), ascorbyl glucoside exhibited poor peak symmetry, and the precipitation of KH2PO4 possibly occurred in the HPLC column for 50 mM of KH2PO4 with 0.10% phosphoric acid; as a result, the backpressure of HPLC possibly increases. Consequently, we chose the KH2PO4 concentration of 5 mM. In the next step, the phosphoric acid concentrations (0.05,
1 mAU
Niacinamide
(A)
Adenosine
Ascorbyl glucoside
200
Arbutin
Ethyl ascorbyl ether
Hydroquinone
-50 700
mAU
500
(B)
250
-100 60.0
mAU
(C)
40.0
20.0
Hydroquinone
-10.0 450
mAU
Adenosine
Arbutin (D)
200
-50 140
mAU
(E)
100
50
min
-20 0.0
1.3
2.5
3.8
5.0
6.3
7.5
8.8
10.0
11.3
12.5
13.8
15.0
16.3
17.5
18.8
20.0
21.3
22.5
23.8
25.0
Figure 3 High-performance liquid chromatography chromatograms of standard solution and cosmetic products. (A) Standard solutions: niacinamide (200.8 lg mL 1, 261 nm), ascorbyl glucoside (200.1 lg mL 1, 238 nm), arbutin (200.5 lg mL 1, 283 nm), adenosine (4.2 lg mL 1, 257 nm) hydroquinone (20.1 lg mL 1, 289 nm), and ethyl ascorbyl ether (100.5 lg mL 1, 245 nm), (B and C) cream sample solution, (D) emulsion sample solution and (E) lotion sample solution.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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0.1, 0.2, 0.4, 0.6 and 0.8%) were tested, whereas the KH2PO4 concentration remained constant at 5 mM. As shown in Fig. 2, the best resolution was achieved at 0.1% phosphoric acid. On the other hand, the pH of the solvents containing 0.2% phosphoric acid was 90% of the labelled contents and does not allow for the existence of hydroquinone in whitening cosmetics [8]. As compiled in Table VI, the content of the active ingredients of all samples was
doi: 10.1111/ics.12292
Simultaneous determination of water-soluble whitening ingredients and adenosine in different cosmetic formulations by high-performance liquid chromatography coupled with photodiode array detection J.-S. Jeon*, H.-T. Kim*, M.-G. Kim*, M.-S. Oh*, S.-R. Hong*, M.-H. Yoon*, S.-M. Cho†, H.-C. Shin†, J.-H. Shim‡, A. Ramadan§ and A. M. Abd El-Aty†,§ *Public Health Research Division, Gyeonggi Province Institute of Health and Environment, 95, Pajang cheon-ro, Jangan-gu, Suwon-Si 440-290, Republic of Korea, †Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Nuengdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea, ‡Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea and §Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt
Received 17 September 2015, Accepted 7 November 2015
Keywords: adenosine, arbutin, ascorbyl glucoside, ethyl ascorbyl ether, high-performance liquid chromatography, niacinamide
Abstract OBJECTIVE: The Korean Cosmetic Act regulates the use of functional cosmetics) by the law. Four functional cosmetic groups, whitening, anti-wrinkle, UV protection and combination of whitening and anti-wrinkle, were categorized according to the Korean Cosmetic Act and Functional Cosmetics Codex. In this study, highperformance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) was employed for the simultaneous detection of arbutin (and its decomposition product, hydroquinone), niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine in functional cosmetic products such as creams, emulsions and lotions. METHODS: Separation by HPLC-DAD was conducted using a C18 column with a gradient elution of 5 mM KH2PO4 buffer (containing 0.1% phosphoric acid) and methanol (containing 0.1% phosphoric acid). The wavelengths for the detection of arbutin, hydroquinone, niacinamide, adenosine, ascorbyl glucoside and ethyl ascorbyl ether were 283, 289, 261, 257, 238 and 245 nm, respectively. RESULTS: This method exhibited good linearity (R2 ≥ 0.999), precision (expressed as relative standard deviation (RSD) < 2%) and mean recoveries (89.42–104.89%). The results obtained by monitoring 100 market samples showed that the detected levels of the tested materials are within the acceptable authorized concentration. CONCLUSION: The method developed herein is simple and can be used for market survey and quality control of functional cosmetics. sume Re Correspondence: J.-S. Jeon, Public Health Research Division, Gyeonggi Province Institute of Health and Environment, 95, Pajang cheon-ro, Jangan-gu, Suwon-Si 440-290, Republic of Korea. Tel.: +82 31 250 2582; fax: +82-31-250-2588; e-mail: [email protected] and A. M. Abd El-Aty, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Nuengdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea. Tel.: +82 10 5934 0701; fax: +82 2 444 4396. e-mail: [email protected]
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OBJECTIF: La Loi Cosmetique coreenne reglemente l’utilisation de cosmetiques ou de produits fonctionnels). Quatre groupes cosmeti savoir les blanchissants ment, les anti-rides, les ques fonctionnels, a anti-UV, et les combinaisons de blanchiment et anti-rides, ont ete classes selon la reglementation cosmetique coreenne et le Codex des Functional Cosmetics. Dans cette etude, la Chromatographie haute performance (HPLC) couplee a une detection a liquide a reseau de photodiodes (DAD) a ete utilisee pour la detection simultanee de l’arbutine (et son produit de degradation, l’hydroquinone), du niacinamide, du glucoside d’ascorbyle, de l’ether d’ascorbyle d’ethyle, et de l’adenosine dans les produits cosmetiques fonctionnels tels que les cremes, emulsions, lotions etc. METHODES: La separation par HPLC-DAD a ete effectuee en utilisant une colonne C18 avec un gradient d’elution d’un tampon 5 mM KH2PO4 (contenant 0.1% d’acide phosphorique) et de methanol (contenant 0.1% d’acide phosphorique). Les longueurs d’onde pour la detection de l’arbutine, de l’hydroquinone, du niacinamide, de l’adenosine, du glucoside d’ascorbyle, de l’ether l’ascorbyl d’ethyle sont 283, 289, 261, 257, 238 et 245 nm, respectivement. RESULTATS: Cette methode presente une bonne linearite 2% (exprimee en ecart(R2 ≥ 0.999), une precision inferieure a type relatif (RSD), et des recouvrements moyens (89.42 de 104.89%). Les resultats obtenus en analysant les 100 echantillons du marche ont montre que les taux des substances testees se situent dans les limites des concentrations autorisees. CONCLUSION: La methode developpee ici est simple et peut ^etre utilisee pour l’etude du marche et le contr^ole de qualite des produits cosmetiques fonctionnels.
Introduction According to the Korean Cosmetic Act, cosmetics are defined as goods that are used by various applications for the purpose of increasing attractiveness by cleaning and beautifying the human body, increasing brightening appearance and maintaining or improving the health of skin and hair [1]. In addition, the Korean
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
Determination of whitening ingredients and adenosine in functional cosmetics using HPLC
Cosmetic Act regulates the use of functional cosmetics (cosmeceuticals) by the law. The word ‘cosmeceuticals’ was popularized by Albert Kligman in the late 1970s. Before the late 1970s, the term ‘cosmeceuticals’ was first used by Raymond Reed, who used it for the purpose of explaining active and science-based cosmetics [2, 3]. Cosmeceuticals may also be considered as hybrids between cosmetics and pharmaceuticals, which are intended to enhance the health and beauty of skin [4]. Thus far, only the Republic of Korea and Japan have legislations that distinguish cosmetic products and functional (quasi-drug) cosmetics. However, the U.S. Food and Drug Administration does not recognize any such categorization into cosmeceuticals, and the use of the term cosmeceutical has no meaning [5]. Tanned skin is considered healthy and beautiful by Westerners, whereas light skin is considered beautiful in oriental countries. Hence, whitening cosmetics are very popular in the oriental countries [6]. In particular, in the Republic of Korea, there is rapid growth towards the development of functional cosmetics as different cosmetics by the simple correction of the colour to an active creation of beauty [7]. The Korean Cosmetic Act and the Korean Functional Cosmetics Codex (KFCC) have categorized four functional cosmetic groups: whitening, anti-wrinkle, UV protection and the combination of whitening and anti-wrinkle [1, 8]. Arbutin, niacinamide, ascorbyl glucoside, ascorbyl tetraisopalmitate, abisabolol, ethyl ascorbyl ether, Broussonetia extract, and glabridin, which are active ingredients of oil-soluble licorice (Glycyrrhiza) extract, are registered as whitening agents. Arbutin, niacinamide, ascorbyl glucoside and ethyl ascorbyl ether are water-soluble substances, whereas the remaining are fat-soluble substances. Hydroquinone has been recognized as one of the pharmaceuticals used for the treatment of hyperpigmentation. However, the prolonged usage of hydroquinone may cause disorders such as ochronosis, dermatitis, cataract and pigmented colloid milium. Because of these reasons, hydroquinone is no longer being authorized for use in formulations for cosmetic skin lightening in several countries, including the Republic of Korea. This limitation leads to the development of new ingredients such as arbutin, kojic acid, niacinamide and vitamin C derivatives [9–13]. Arbutin, a derivative of hydroquinone, is a safe skin-whitening topical agent used for depigmentation without cytotoxicity and side effects [12, 14]. Niacinamide, also known as nicotinamide and nicotinic acid amide, is a water-soluble vitamin [3]. It acts as a powerful antioxidant and an effective skin lightener by the inhibition of the transfer of melanosome from melanocytes to keratinocytes, subsequently decreasing the appearance of hyperpigmented spots [15–18]. The topical application of niacinamidecontaining cosmetics has been reported to exhibit beneficial antiwrinkle effects such as improving barrier function and decreasing the appearance of signs of chronological and facial photoaging [19, 20]. Because vitamin C exhibits antioxidant activity and effect for the inhibition of tyrosinase [18], it has been used as a skin lightener. However, a major limitation of vitamin C and its derivatives is degradation, which can be accelerated in the presence of oxygen, water, light, heat and metals. To prevent rapid degradation, different derivatives have been introduced, such as ascorbyl phosphate salts, ascorbyl palmitate, ascorbyl glucoside and ethyl ascorbyl ether. These derivatives are susceptible to enzymatic hydrolysis by tissue esterase and a-glucosidase, thereby yielding ascorbic acid [21–23]. Adenosine exhibits anti-wrinkle properties [24], and it is mostly used as an active ingredient in anti-wrinkle and combination of whitening and anti-wrinkle cosmetics in the Republic of Korea.
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High-performance liquid chromatography (HPLC) is a general technique for the quantitative estimation of arbutin in whitening cosmetics [10, 14, 25–30] and hydroquinone in cosmetic creams, topical dermatologic drugs and other matrices [27, 30–35]. Previously, arbutin, niacinamide and adenosine have been simultaneously determined in cosmetic products by HPLC [29]. Methods for determining ascorbyl glucoside or ethyl ascorbyl ether have been previously reported [27, 36]. This study aims to develop a simple method for the simultaneous detection of arbutin (and its decomposed product, hydroquinone), niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine in different cosmetic formulations for the quality control of functional cosmetics. To the best of our knowledge, this is the first report for the detection of the tested compounds in one chromatographic run. Materials and methods Chemicals and reagents Various brands of commercial functional cosmetics containing active ingredients (arbutin, niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine) were purchased from wholesale markets and cosmetic shops located in Suwon City, Republic of Korea. Niacinamide, adenosine, hydroquinone and potassium dihydrogen phosphate (KH2PO4, reagent grade) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Arbutin was supplied by Fluka (Buchs, Switzerland). Ascorbyl glucoside and ethyl ascorbyl ether were purchased from Tokyo Chemical Industry Co., LTD. (Tokyo, Japan). HPLC-grade methanol was provided by J.T. Baker (Griesheim, Germany). Phosphoric acid (85%, reagent grade) was obtained from Wako Chemicals (Tokyo, Japan). A 0.4-lm nylon membrane filter (Advantec, Tokyo, Japan) was used to filter the mobile phase. Sample solutions were filtered using 0.20-lm polytetrafluorethylene (PTFE) syringe filters (Advantec, Tokyo, Japan). Deionized water was purified using a Barnstead Nano pure Diamond (Dubuque, IA, U.S.A.). Standard preparation Stock standard solutions of arbutin (401 lg mL 1), hydroquinone (100.5 lg mL 1), niacinamide (401.6 lg mL 1), adenosine (40.2 lg mL 1), ascorbyl glucoside (400.2 lg mL 1) and ethyl ascorbyl ether (201.0 lg mL 1) were separately prepared by dissolving in deionized water. Fresh working solutions were prepared daily by the further dilution of the stock solutions with 5 mM KH2PO4 buffer (containing 0.1% phosphoric acid). Table I shows the concentration ranges used for the calibration of each standard. Sample preparation The samples were prepared according to our previous study [30]. First, 0.1 g each of cream, lotion and emulsion was accurately weighed in 15-mL polypropylene centrifuge tubes, and methanol (3 mL) was added to each of the tubes and mixed by vortex for 2 min. Second, the solution was sonicated (Branson 8510 sonicator, Danbury, CT, U.S.A.) for 30 min, and the total volume was adjusted to 10 mL using deionized water. Subsequently, the solution was sonicated for 30 min and then centrifuged (Sigma 2–6 centrifuge, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) at 3500 rpm for 20 min. Finally, the solutions were diluted with the mobile phase A [5 mM KH2PO4 buffer (containing 0.1% phosphoric acid)] and then filtered using PTFE syringe filters.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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Determination of whitening ingredients and adenosine in functional cosmetics using HPLC Table I Detection wavelength (nm), concentration range (lg mL the tested compounds
1
), calibration curve, linearity (R2), and limits of detection (LOD) and quantification (LOQ) of
Compounds
Detection wavelength (nm)
Concentration range (lg mL 1)
Calibration curve
Arbutin Hydroquinone Niacinamide Adenosine Ascorbyl glucoside Ethyl ascorbyl ether
283 289 261 257 238 245
10–200.5 2–40.6 10–200.8 1–20.1 10–200.1 5–100.5
y y y y y y
= = = = = =
1.2610x 0.7504x 3.2662x 0.2620x 4.0854x 2.8608x
+ + + + + +
0.0090 0.0251 0.1194 0.0053 0.1316 0.1217
R2
LOD (lg mL 1)
LOQ (lg mL 1)
0.999 1.000 1.000 1.000 1.000 1.000
0.75 0.45 0.10 0.08 0.62 0.24
2.48 1.50 0.34 0.25 2.04 0.78
Table II Extraction efficiency (%) of the tested compounds using various solvents
Niacinamide
Ascorbyl glucoside
Arbutin
Ethyl ascorbyl ether
Adenosine
Solvents
Meana
SDb
Mean
SD
Mean
SD
Mean
SD
Mean
SD
A B C D E F G
Sc 85.21 102.04 89.80 99.23 101.00 98.01
– 0.13 5.36 4.69 0.72 6.55 1.14
Sc Md 102.25 104.52 107.74 Sc 108.14
– – 5.61 2.48 0.84 – 0.56
87.67 Sc 104.98 105.35 102.28 109.77 101.45
6.88 – 0.75 1.46 0.95 1.10 1.76
58.18 9.86 91.26 84.72 96.08 99.18 94.64
3.65 0.31 4.75 4.43 0.77 5.36 1.09
67.19 Sc 91.44 81.25 90.64 93.38 89.54
0.86 – 4.81 4.05 0.65 5.42 0.88
n = 3. SD, standard deviation. c Peak splitting. d Chromatographic peak moved forward. (A) methanol, (B) acetonitrile, (C) 5 mM KH2PO4, (D) 5 mM KH2PO4 buffer containing 0.1% phosphoric acid, (E) methanol: deionized water (3 : 7, v/v), (F) acetonitrile: deionized water (3 : 7, v/v) and (G) methanol: 5 mM KH2PO4 buffer containing 0.1% phosphoric acid (3 : 7, v/v). Hydroquinone is not an active ingredient. It is a decomposed product of arbutin. a
Retention time (min)
b
KH2PO4 conc (mM) Figure 1 Various concentrations of KH2PO4 in the mobile phase A. Solvents with the asterisk * contained 0.1% phosphoric acid.
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Retention time (min)
Determination of whitening ingredients and adenosine in functional cosmetics using HPLC
Phosphoric acid Figure 2 Various concentrations of phosphoric acid in the mobile phase A.
Analytical method Analysis was conducted using a Dionex UltiMate 3000 HPLC system (Sunnyvale, CA, U.S.A.) equipped with an UltiMate 3000 pump, an UltiMate 3000 autosampler column compartment and an UltiMate 3000 photodiode array detector. Data acquisition was performed with CHROMELEON software (Sunnyvale, CA, U.S.A.). A CAPCELL PAK C18 MG column (250 mm 9 4.6 mm, 5 lm, Shiseido, Japan) maintained at room temperature was used to achieve selective chromatographic conditions. The target compounds were separated using a stepwise gradient mobile phase consisting of 5 mM KH2PO4 buffer (containing 0.1% phosphoric acid) (A) and methanol (containing 0.1% phosphoric acid) (B), which was run as follows: 0–5 min, 0% B (v/v); 5–12 min, 10% B (v/v); 12–18 min, 20% B (v/v); and 18–23 min, 30% B (v/v), 23–26 min, 50% B (v/ v); 26–33 min, 0% B (v/v); and 33–35 min, 0% B (v/v). The flow rate and injection volume were 1 mL min 1 and 10 lL, respectively. Table I shows the wavelengths for the detection of each compound. Method validation The linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy and precision [37] of the method were validated for the quantification of the target compounds. The standard of each compound (at three concentrations) was spiked into blank samples (cream, lotion and emulsion) for conducting recovery tests, the results of which are evaluated as accuracy. To determine intraand interday sample variations, the developed method was repeated three times a day on three consecutive days.
pounds from commercially available cream samples was investigated using one of the following solvents: (A) methanol, (B) acetonitrile, (C) 5 mM KH2PO4, (D) 5 mM KH2PO4 buffer containing 0.1% phosphoric acid, (E) methanol: deionized water (3 : 7, v/v), (F) acetonitrile: deionized water (3 : 7, v/v) and (G) methanol: 5 mM KH2PO4 buffer containing 0.1% phosphoric acid (3 : 7, v/v). Sonication times were 30 min for A, B, C and D, and the remaining solvents were first sonicated (30 min), followed by another sonication with deionized water or buffer. The extraction efficiency (%) was calculated by dividing the concentration of each compound in sample solutions by the concentration of standard solutions, except
Table III Intraday and interday precision using standard solutions
Compounds
Arbutin
Hydroquinone
Niacinamide
Adenosine
Ascorbyl glucoside
Results and discussion Extraction procedure In previous studies, various extraction solvents such as water, acetonitrile and 0.05 M KH2PO4 buffer (pH 2.5) [6, 26, 27] have been used for the determination of the hydrophilic functional ingredients in cream. In this study, the extraction efficiency of the tested com-
Ethyl ascorbyl ether
Concentration (lg mL 1)
Intraday variabilitya, RSD (%)
Interday variabilityb, RSD (%)
20.1 100.3 200.5 4.1 20.3 40.6 20.1 100.4 200.8 0.4 2.1 4.2 20.0 100.1 200.1 10.1 50.3 100.5
0.42 0.19 0.53 0.29 0.59 0.83 0.29 0.10 0.51 0.46 0.07 0.14 0.28 0.14 0.45 0.25 0.3 0.48
0.74 0.84 0.81 0.78 0.95 0.95 0.71 0.82 0.64 0.45 0.96 0.89 0.76 0.77 0.90 0.95 1.03 0.86
n = 3. n = 9, triplicate injection each day for three consecutive days.
a b
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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as the extraction solvent-exhibited recovery satisfactory for the determination of adenosine in royal jelly. Our previous study has reported that using methanol: deionized water (1 : 9, v/v) as the extraction solvent-exhibited acceptable recovery [29]. Clearly, the standard deviation of the extraction efficiency of samples extracted with methanol: deionized water (3 : 7, v/v) was steady and less than one. Overall, the extraction efficiency using methanol (A) and acetonitrile (B) was not good. In sum, the use of methanol:
hydroquinone (not an original compound). As shown in Table II, the chromatograms of niacinamide and ascorbyl glucoside in methanol, arbutin and ethyl ascorbyl ether in acetonitrile, and ascorbyl glucoside in acetonitrile: deionized water (3 : 7, v/v) exhibited peak splitting. Moreover, the retention time of ascorbyl glucoside in acetonitrile moved forward. Adenosine in acetonitrile exhibited the lowest extraction efficiency (9.86%), caused by variance in solubility. Xue et al. [38] have reported that 80% ethanol
Table IV Recovery of the tested compounds in various spiked cosmetic formulations
Cream
Compounds
Arbutin
Hydroquinone
Niacinamide
Adenosine
Ascorbyl glucoside
Ethyl ascorbyl ether
Lotion
Emulsion
Concentration (lg mL 1)
Recoverya (%)
SDb (%)
Recovery (%)
SD (%)
Recovery (%)
SD (%)
20.1 100.3 200.5 4.1 20.3 40.6 20.1 100.4 200.8 0.4 2.1 4.2 20 100.1 200.1 10.1 50.3 100.5
102.35 102.45 101.42 102.99 100.63 98.75 94.54 96.39 103.46 101.90 103.96 98.03 99.64 99.19 95.64 104.89 103.50 97.38
2.82 1.69 1.72 3.58 0.30 3.99 2.72 0.86 2.75 0.91 2.47 3.69 0.55 1.51 1.63 0.51 3.40 1.20
93.30 99.74 95.21 96.88 102.28 96.04 89.42 91.18 99.74 94.76 99.58 98.06 97.46 96.79 98.40 103.92 95.75 96.47
2.14 2.46 0.53 3.65 4.87 2.28 0.90 0.83 0.66 0.52 2.87 1.02 2.36 3.96 2.69 0.94 0.40 0.89
93.95 102.24 95.13 92.98 104.71 99.00 93.82 90.60 103.19 96.71 98.22 100.37 93.78 94.75 98.65 96.54 103.92 99.64
1.18 2.84 0.47 1.39 0.64 0.47 4.77 0.92 0.39 5.02 2.99 1.18 3.10 4.21 0.92 1.39 0.52 2.49
n = 3. SD, standard deviation.
a b
Table V Chromatographic parameters
Acceptance criteria
Compounds
Retention time (min)
Instrument precisiona, RSD (%) ≤ 1.0
Capacity factorb ≥ 2
Niacinamide Ascorbyl glucoside Arbutin Adenosine Hydroquinone Ethyl ascorbyl ether
4.7 5.6 11.8 12.2 12.8 18.8
0.25 0.52 0.19 0.17 0.03 0.04
1.0 1.4 4.1 4.3 4.6 7.2
Resolutionc ≥ 2.0
– 5.91 32.55 2.60 3.17 22.25
Tailing factord ≤ 2.0
Theoretical plate numbere ≥ 2000
0.97 1.01 0.94 0.98 0.94 0.94
20 016 13 208 66 338 100 240 60 572 133 311
a Instrument precision indicates the performance of the HPLC, inclusive of the plumbing, column, environmental conditions and the time at which the samples were analysed. b Capacity factor is a measure of the location of the peak of interest with respect to the void volume. c Resolution is a measure of how well two peaks are separated. d 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 peak. e Theoretical plate number is a measure of column efficiency; that is, how many peaks can be located per unit run time of the chromatogram.
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Determination of whitening ingredients and adenosine in functional cosmetics using HPLC Table VI Detection of the target compounds in commercial functional cosmetics
Category
Number of samples
Group
Whitening
Niacinamide Arbutin Ethyl ascorbyl ether Ascorbyl glucoside Adenosine Niacinamide Adenosine Arbutin Adenosine Niacinamide Ethyl ascorbyl etherb Adenosine Ethyl ascorbyl ether Adenosine
Anti-wrinkle Combined whitening/ anti-wrinkle
17 8 4 2 36 22
Range (%)
93.1–158.4 93.6–116.2 105.1–111.7 100.5–105.2 91.2–120.0 91.3–156.8 91.2–138.3 100.5–107.0 90.1–137.2 102.4 110.3 106.9 96.9 90.1
9 1
1
SDa
17.4 7.0 2.9 3.3 7.4 15.1 11.5 2.5 15.7 – – – – –
a
SD, standard deviation. Not declared on the product label.
b
450
deionized water (3 : 7, v/v) as an extraction solvent was the optimum condition to extract the tested compounds. Optimization of chromatographic conditions First, a mobile phase consisting of deionized water and methanol was tested [30]. However, the peaks of some analytes such as hydroquinone, niacinamide, ascorbyl glucoside and ethyl ascorbyl ether overlapped, and peak symmetry was poor. Afterwards, the KH2PO4 concentrations and acidity of phosphoric acid in mobile phase A were investigated. At the start, the tested KH2PO4 concentrations were 2.5, 5, 10, 20 and 50 mM at 0.1% phosphoric acid and 25 mM of KH2PO4 at 0.05% phosphoric acid. As can be seen in Fig. 1, the best resolution was achieved at 50 mM of KH2PO4 containing 0.1% phosphoric acid and 25 mM of KH2PO4 containing 0.05% phosphoric acid. Meanwhile, at 25 mM of KH2PO4 containing 0.05% phosphoric acid (data not shown), ascorbyl glucoside exhibited poor peak symmetry, and the precipitation of KH2PO4 possibly occurred in the HPLC column for 50 mM of KH2PO4 with 0.10% phosphoric acid; as a result, the backpressure of HPLC possibly increases. Consequently, we chose the KH2PO4 concentration of 5 mM. In the next step, the phosphoric acid concentrations (0.05,
1 mAU
Niacinamide
(A)
Adenosine
Ascorbyl glucoside
200
Arbutin
Ethyl ascorbyl ether
Hydroquinone
-50 700
mAU
500
(B)
250
-100 60.0
mAU
(C)
40.0
20.0
Hydroquinone
-10.0 450
mAU
Adenosine
Arbutin (D)
200
-50 140
mAU
(E)
100
50
min
-20 0.0
1.3
2.5
3.8
5.0
6.3
7.5
8.8
10.0
11.3
12.5
13.8
15.0
16.3
17.5
18.8
20.0
21.3
22.5
23.8
25.0
Figure 3 High-performance liquid chromatography chromatograms of standard solution and cosmetic products. (A) Standard solutions: niacinamide (200.8 lg mL 1, 261 nm), ascorbyl glucoside (200.1 lg mL 1, 238 nm), arbutin (200.5 lg mL 1, 283 nm), adenosine (4.2 lg mL 1, 257 nm) hydroquinone (20.1 lg mL 1, 289 nm), and ethyl ascorbyl ether (100.5 lg mL 1, 245 nm), (B and C) cream sample solution, (D) emulsion sample solution and (E) lotion sample solution.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 38, 286–293
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0.1, 0.2, 0.4, 0.6 and 0.8%) were tested, whereas the KH2PO4 concentration remained constant at 5 mM. As shown in Fig. 2, the best resolution was achieved at 0.1% phosphoric acid. On the other hand, the pH of the solvents containing 0.2% phosphoric acid was 90% of the labelled contents and does not allow for the existence of hydroquinone in whitening cosmetics [8]. As compiled in Table VI, the content of the active ingredients of all samples was
