Simultaneous Determination of Arbutin and Hydroquinone in Different Herbal Slimming Products Using Gas Chromatography-mass Spectrometry/Arbutin ve Hidrokinonun Farkli Bitkisel Zayiflama Urunlerinde Gaz Kromatografisi-Kutle Spektrometresi ile Eszamanli Belirlenmesi.
Arbutin (4-hydroxyphenyl-[beta]-D-glucopyranoside) is a phenolic glucoside that is found mainly in the Ericaceae and Saxifragaceae families. (1) It is an inhibitor of tyrosinase, which is the essential enzyme for melanin formation. Melanin is important for the protection of skin from harmful ultraviolet (UV)-A and UVB radiation. Arbutin is disinfectant in genitourinary diseases and has anti-inflammatory, antioxidant, and antitumor effects. It is used in urinary therapeutics, skin-whitening, and depigmenting cosmetics. (1-3)
Hydroquinone is 1,4-dihydroxybenzene and a metabolite of arbutin. (4) It has antibacterial, astringent, disinfectant, and antioxidant effects. It is used for the treatment of hyperpigmentation and is a component of topical pharmaceutical agents. (1,5)
Calluna vulgaris (C. vulgaris) (L.) Hull (heather) is a perennial shrub that is a member of the Ericaceae family. It is distributed throughout most of Europe, Russia, Asia Minor, and the Atlantic coast of North America. (6,7) Secondary metabolites of C. vulgaris are flavonoids, tannins, proanthocyanidins, caffeic acid derivatives, phenols, triterpenes, steroids and hydroquinone glycosides (arbutin). The infusion of aerial parts of C. vulgaris is traditionally used in Turkey as a urinary tract disenfectant, diuretic, and antidiaretic. (8) C. vulgaris has diuretic, antimicrobial, antirheumatic, antioxidant, antibacterial, and monoamine oxidase-A inhibitory effects (7,9-11) and is presented in herbal slimming teas due to its diuretic and digestive effects.
Obesity is a serious disease that can be due to genetic and environmental reasons, defined as abnormal or excessive fat accumulation that may impair health by the World Health Organization. In addition to the various synthetic medicines used in obesity treatment, there is an increasing trend towards herbal products in this field. (12) The effectiveness of herbal slimming products and their adherence to standards is a matter of debate. Thus, the quality control analysis of these products is important for public health. Arbutin and hydroquinone are among the effective components in herbal slimming products containing C. vulgaris.
Several analytical methods have been reported for the determination of arbutin and/or hydroquinone including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), capillary zone electrophoresis, and densitometry. (5,13-22)
However, no method has been used to quantify the presence of arbutin and hydroquinone simultaneously from different herbal slimming products. Therefore, analytical methods for their separation and simultaneous quantification are required for quality control purpose. Hence, in the present research, a simple, rapid, precise and accurate GC-MS method was developed and validated using International Conference on Harmonization Guidelines for the simultaneous determination and quantification of arbutin and hydroquinone form different herbal slimming products. (23)
MATERIALS AND METHODS
Methanol (France), arbutin (England) and hydroquinone (Switzerland) were purchased from Sigma-Aldrich. Acetonitrile and N-methyl-N-(trimethylsilyl) (TMS) trifluoroacetamide (MSTFA) were obtained from Sigma (St. Louis, MO, USA).
Plant material and pharmaceutics
Nine herbal slimming products containing C. vulgaris were acquired from the internet and different pharmacies in Turkey. The herbal slimming products from 1 to 7 were tea mixtures, product 8 was a slimming tea capsule, product 9 was C. vulgaris tea.
Extraction of the herbal slimming products
Ten grams of each product were extracted with distilled water and methanol (100 mLx2) separately at 40[degrees]C for 30 min. The extracts were filtered. Then, the aqueous extracts were cooled at -80[degrees]C and lyophilized. Methanol was evaporated to dryness and the methanol extracts were obtained.
Arbutin, hydroquinone, and contained extracts were derivatized using MSTFA to increase the performance of the gas chromatographic separation. The hydroxy (-OH) groups were converted to the corresponding silyl (-O-TMS) groups. After establishing the optimum reaction conditions, the compounds were analyzed.
Chromatographic analysis was performed on an Agilent 7820A GC system equipped with a 5977 series mass selective detector, 7673 series autosampler, and Agilent chemstation (Agilent Technologies, Palo Alto, CA). An HP-5 MS column with 0.25-[micro]m film thickness (30 m x 0.25 mm I.D., USA) was used for separation. Splitless injection was used and the carrier gas was helium at a flow rate of 1.0 mL/min. The injector volume was 1 mL. The MS detector parameters were transfer line temperature 230[degrees]C, solvent delay 3 min and electron energy 70 eV. The GC temperature gradient program was as follows: the initial temperature was 100[degrees]C, held for 2 min, which was increased to 220[degrees]C at a rate of 30[degrees]C/min, held for 1 min, and finally to 300[degrees]C at a rate of 20[degrees]C/min and held for 2.0 min. The MS detector parameters were: transfer line temperature 280[degrees]C; solvent delay 3 min; electron energy 70 eV; the MS was run in electron impact mode with selected ion monitoring (SIM) for quantitative analysis (m/z 254 for arbutin, m/z 239 for hydroquinone).
Standards and quality control samples
Stock solutions of arbutin and hydroquinone were prepared by dissolving the accurately weighed reference compounds in acetonitrile to give a final concentration of 100 [micro]g/mL of both. The solution was then serially diluted with chloroform to achieve standard working solutions at concentrations of 5, 10, 25, 50, 100, 250 and 500 ng/mL for arbutin and hydroquinone, respectively. Structural formula and derivatization of arbutin and hydroquinone are shown in Figure 1.
All solutions were stored at 4[degrees]C and were brought to room temperature before use. Quality control solutions were prepared by adding aliquots of standard working solution of final concentrations of 7.5, 75 and 375 ng/mL for arbutin and hydroquinone, respectively.
There were no experimental animal or clinical studies in our study. Thus, we did not need ethics committee approval for the study.
Method development and optimization
Arbutin and hydroquinone are polar molecules so a capillary column coated with 5% phenyl and 95% dimethylpolysiloxane were used for separation. During GC-MS method development, the injection port and detector temperatures were set to 250[degrees]C and 290[degrees]C, respectively. Different temperature programs were investigated to give an optimum temperature program as follows; initial temperature was 100[degrees]C, held for 2 min, increased to 220[degrees]C at 30[degrees]C/min, held for 1 min, and finally to 300[degrees]C at 20[degrees]C/min with a final hold of 2.0 min. The injector volume was 1 mL in splitless mode.
MSTFA is an effective TMS donor. MSTFA reacts to replace labile hydrogens on a wide range of polar compounds with a TMS group and is used to prepare volatile and thermally stable derivatives for GC-MS. (24)
The effects of time and temperature on the reaction were investigated. Therefore, arbutin and hydroquinone were dissolved in acetonitrile. 50 [micro]L of MSTFA solution was added to 50 [micro]L of 200 ng/mL arbutin and hydroquinone solution and reacted at room temperature, 50 and 75[degrees]C for 5, 15, 30 and 45 min. The resulting samples were quantitated using the GC-MS system. The optimized conditions for derivatization were 50[degrees]C and 30 min.
Then, dry residue of the herbal slimming products was dissolved in 100 [micro]L of a mixture of acetonitrile and MSTFA (50:50, v/v). The mixture was vigorously shaken and then delayed at 50[degrees]C for 30 min. A 1-[micro]L sample was injected into the GC-MS system.
To evaluate the validation of the present method, parameters such as selectivity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) were investigated according to ICH validation guidelines.
The selectivity of the GC-MS method was investigated by observing interferences between arbutin and hydroquinone. For GC-MS, the electron impact mode with SIM was used for quantitative analysis (m/z 254 for arbutin and m/z 239 for hydroquinone). The mass spectra of the arbutin and hydroquinone are shown in Figure 2.
The retention times of arbutin and hydroquinone in GC-MS method were approximately 11.32 and 5.44 min with good peak shape (Figure 3).
Linearity was determined for arbutin and hydroquinone in the range of 5-500 ng/mL. The calibration curves constructed were evaluated using their correlation coefficients. The calibration equations from three replicate experiments demonstrated the linearity of the method. Standard deviations of the slope and intercept for the calibration curves are given in Table 1.
Precision and accuracy
The precision of the GC-MS method was determined through repeatability (intra-day) and intermediate precision (inter-day). Repeatability was evaluated by analyzing quality control samples six times per day, at three different concentrations, which were quality control samples. The intermediate precision was evaluated by analyzing the same samples once daily for three days. The relative standard deviation (RSD) of the predicted concentrations from the regression equation was taken as precision. The accuracy of this analytic method was assessed as the percentage relative error. For all the concentrations studied, intra- and inter-day RSD values were [less than or equal to]2.73% and for all concentrations of arbutin and hydroquinone the relative errors were [less than or equal to]2.56%.
LOD and LOQ
The LOD is the lowest amount of arbutin and hydroquinone in a sample that can be detected but not necessarily quantitated as an exact value. The LOQ is the lowest amount of arbutin and hydroquinone that can be quantitatively determined with suitable precision. The LOD and LOQ of the developed method were determined by injecting progressively lower concentrations of the standard solution under the chromatographic conditions. The lowest concentrations assayed where the signal/noise ratio was at least 10:1 was regarded as the LOQ. The LOD was defined as a signal/noise ratio of 3:1. The results are shown in Table 1.
Application of method
The developed GC-MS method was used for the simultaneous determination of arbutin and hydroquinone from different herbal slimming products. The sample working solutions (1 [micro]L) were injected and the height of both arbutin and hydroquinone peaks were measured. From the calibration curve, the amounts of arbutin and hydroquinone in different herbal slimming products were calculated. The retention time of arbutin and hydroquinone in sample solutions were 11.32 and 5.44 min, respectively (Figure 4). The mean amounts and percent values of arbutin and hydroquinone found in different herbal slimming products are presented in Table 2.
Comparison of methods
Today, GC-MS is a powerful technique for highly specific and quantitative measurements of low levels of analytes in samples. As compared with HPLC, high-resolution capillary GC has been less frequently used. (25)
During the development of the method, it became evident that arbutin and hydroquinone were very sensitive to matrix effects during the derivatization process in different herbal slimming products. Sample preparation techniques such as extraction and derivatization were used in order to minimize matrix suppression effects.
GC-MS sensitivity is not enough for the determination of arbutin and hydroquinone in different herbal slimming products. For this reason, MSTFA was chosen as a chromagenic derivatization reagent. In this study, the purpose of the derivatization reaction is to raise the sensitivity, thus the possibility of working at low concentrations was able to be realized.
A literature survey revealed that some of the related methods have been reviewed. A GC-MS method was reported for separating and determining arbutin and hydroquinone from strawberry tree leaf extracts. (4) In a reported method, the calibration curve of GC-MS method was linear for arbutin and hydroquinone in the range 0.5-200 [micro]g/mL. Intra- and inter-day precision, expressed as the RSD were less than 5.0%, and accuracy (relative error) was better than 3.80%. In statistical comparison (p<0.05) with the other method in the literature, the proposed method indicated high accuracy, precision, and sensitivity. (4) The minimum determinable concentration wss 9 ng/mL. The present method has the following advantage over the reported method. The LOQ of the reported method was 29 ng/mL, whereas the the LOQ of the present method was 0.093 ng/mL.
When this method is applied to different herbal slimming product samples, its sensitivity was found to be adequate for analysis studies. The present method has the following advantages over the reported method. The sensitivity was evaluated using the LOQ, which was determined as 0.093 ng/mL. This method is as good as or superior to that reported in the other papers. (4,16,19,20)
Calibration curves of arbutin and hydroquinone were linear over the concentration range of 5-500 ng/mL for the study, which is as good as or superior to that reported in other papers. (15-20)
In the present work, a new, simple and sensitive GC-MS method was developed for the simultaneous quantitation of arbutin and hydroquinone in whole plant powder of different herbal slimming products. The method was validated to track the active principles in the complex mixture of herbal ingredients. The method can be extended for the marker-based standardization of other herbal products containing arbutin and hydroquinone. The method was found to be simple, precise, accurate, specific and sensitive and can be used for routine quality control of herbal raw materials and for the quantification of these compounds in plant materials.
Benan Dursunoglu would like to acknowledge the scholarship during her postgraduate program provided by the Turkish Scientific and Technical Research Council (TUBITAK).
Conflict of Interest: No conflict of interest was declared by the authors.
(1.) Migas P, Baranowska MK. The significance of arbutin and its derivatives in therapy and cosmetics. Phytochem Lett. 2015;13:35-40.
(2.) Thongchai W, Liawruangrath B, Liawruangrath S. Arbutin determination in medicinal plants and creams. Int J Cosmet Sci. 2009;31:87-96.
(3.) Pop C, Vlase L, Tamas M. Natural resources containing arbutin. Determination of arbutin in the leaves of Bergenia crassifolia (L.) Fritsch. acclimated in Romania. Not Bot Hort Agrobot Cluj. 2009;37:129-132.
(4.) Jurica K, Karaconji IB, Segan S, Opsenica DM, Kremer D. Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry. Arh Hig Rada Toksikol. 2015;66:197-202.
(5.) Jeon JS, Kim BH, Lee SH, Kwon HJ, Bae HJ, Kim SK, Park JA, Shim JH, Abd El-Aty AM, Shin HC. Simultaneous determination of arbutin and its decomposed product hydroquinone in whitening creams using high-performance liquid chromatography with photodiode array detection: Effect of temperature and pH on decomposition. Int J Cosmet Sci. 2015;37:567-573.
(6.) Monschein M, Neira JI, Kunert O, Bucar F. Phytochemistry of heather (Calluna vulgaris (L.) Hull) and its altitudinal alteration. Phytochem Rev. 2010;9:205-215.
(7.) Gruenwald J, Brendler T, Jaenicke C. PDR for Herbal Medicines (2nd ed). New Jersey; Medical Economics Company; 2007:383-384.
(8.) Baytop T. Turkiye'de Bitkiler ile Tedavi: Gecmiste ve Bugun (2nd ed). Istanbul; Nobel Tip Kitabevleri; 1999:208.
(9.) Drozdz P, Sentkowska A, Pyrzynska K. Biophenols and antioxidant activity in wild and cultivated heather. Nat Prod Res. 2017;31:1181-1184.
(10.) Vucic DM, Petkovic MR, Rodic-Grabovac BB, Stefanovic OD, Vasic SM, Comic LR. In vitro activity of heather [Calluna vulgaris (L.) Hull] extracts on selected urinary tract pathogens. Bosn J Basic Med Sci. 2014;14:234-238.
(11.) Saaby L, Rasmussen HB, Jager AK. MAO-A inhibitory activity of quercetin from Calluna vulgaris (L.) Hull J Ethnopharmacol. 2009;121:178-181.
(12.) Cayir A, Atak N, Kose SK. Beslenme ve diyet klinigine basvuranlarda obezite durumu ve etkili faktorlerin belirlenmesi. Ank Uni Tip Fak Mecm. 2011;64:13-19.
(13.) Gimeno P, Maggio AF, Bancilhon M, Lassu N, Gornes H, Brenier C, Lempereur L. HPLC-UV method for the identification and screening of hydroquinone, ethers of hydroquinone and corticosteroids possibly used as skin-whitening agents in illicit cosmetic products. J Chromatogr Sci. 2016;54:343-352.
(14.) Wittig J, Wittemer S, Veit M. Validated method for the determination of hydroquinone in human urine by high-performance liquid chromatography-coulometric-array detection. J Chromatogr B Biomed Sci Appl. 2001;761:125-132.
(15.) Chang ML, Chang CM. Simultaneous HPLC determination of hydrophilic withening agents in cosmetic products. J Pharm Biomed Anal. 2003;33:617-626.
(16.) Thongchai W, Liawruangrath B, Liawruangrath S. High-performance liquid chromatographic determination of arbutin in skin-whitening creams and medicinal plant extracts. J Cosmet Sci. 2007;58:35-44.
(17.) Kittipongpatana N, Chaiwan A, Pusod U, Kittipongpatana OS. High-performance liquid chromatographic method for separation and quantitative analysis of arbutin in plant tissue cultures. CMU J Nat Sci. 2007;6:65-74.
(18.) Parejo I, Viladomat F, Bastida J, Codina C. A single extraction step in the quantitative analysis of arbutin in bearberry (Arctostaphylos uva-ursi) leaves by high-performance liquid chromatography. Phytochem Anal. 2001;12:336-339.
(19.) Rychlinska I, Nowak S. Quantitative determination of arbutin and hydroquinone in different plant materials by HPLC. Not Bot Horti Agrobo. 2012;40:109-113.
(20.) Lamien-Meda A, Lukas B, Schmiderer C, Franz CH, Novak J. Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva-ursi extracts. Phytochem Anal. 2009;20:416-420.
(21.) Glockl I, Blaschke G, Veit M. Validated methods for direct determination of hydroquinone glucuronide and sulfate in human urine after oral intake of bearberry leaf extract by capillary zone electrophoresis. J Chromatogr B Biomed Sci Appl. 2001;761:261-266.
(22.) Pyka A, Bober K, Stolarczyk A. Densitometric determination of arbutin in cowberry leaves (Vaccinium vitis idaea). Acta Pol Pharm. 2007;64:395-400.
(23.) ICH Steering Committee. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use: Harmonised Tripartite Guideline on Validation of Analytical Procedures: Methodology, Recommended for Adoption at Step 4 of the ICH. Switzerland; IFPMA; 1996:1-17.
(24.) Donike M. N-Methyl-N-trimethylsilyl-trifluoracetamid, ein neues Silylierungsmittel aus der Reihe der silylierten Amide. J Chromatogr. 1969;42:103-104.
(25.) Yilmaz B, Arslan S, Akba V. Gas chromatography-mass spectrometry method for determination of metoprolol in the patients with hypertension. Talanta. 2009;80:346-351.
[iD] Benan DURSUNOGLU (1), [iD] Hafize YUCA (1), [iD] Zuhal GUVENALP (1*), [iD] Sefa GOZCU (2), [iD] Bilal YILMAZ (3)
(1) Ataturk University, Faculty of Pharmacy, Department of Pharmacognosy, Erzurum, Turkey
(2) Erzincan University, Faculty of Pharmacy, Department of Pharmacognosy, Erzincan, Turkey
(3) Ataturk University, Faculty of Pharmacy, Department of Analytical Chemistry, Erzurum, Turkey
(*) Correspondence: E-mail: email@example.com, Phone: +90 505 521 20 43 ORCID-ID: orcid.org/0000-0002-8803-8147
Received: 09.08.2017, Accepted: 02.11.2017
Table 1. Features of the calibration curves of arbutin and hydroquinone Parameters Arbutin Hydroquinone Linearity (ng/mL) 5-500 5-500 Regression equation (a) y=1215.7x+810.48 y=1195.4x+4902 Correlation coefficent 0.9933 0.9915 Standard deviation of 5.0x[10.sup.-3] 2.9x[10.sup.-3] correlation coefficent Limit of detection 0.555 0.031 (ng/mL) Limit of quantification 1.665 0.093 (ng/mL) (a) Based on three calibration curves, y: peak-height, x: arbutin and hydroquinone concentration Table 2. Application of arbutin and hydroquinone in different herbal slimming products (1.0 mg/mL) Sample name (1 mg/mL) Arbutin (ng/mL) % Concentration Aqueous extract n.d. - 1 Methanol extract 20.0256 0.0020 Aqueous extract n.d. - 2 Methanol extract 33.9755 0.0034 Aqueous extract n.d. - 3 Methanol extract 63.4331 0.0063 Aqueous extract n.d. - 4 Methanol extract 2.7145 0.0002 Aqueous extract n.d. - 5 Methanol extract 34.4716 0.0034 Aqueous extract n.d. - 6 Methanol extract 36.2843 0.0036 Aqueous extract n.d. - 7 Methanol extract 18.8898 0.0019 Aqueous extract n.d. - 8 Methanol extract n.d. - Aqueous extract 60.6236 0.0060 9 Methanol extract 35.2968 0.0035 Sample name (1 mg/mL) Hydroquinone (ng/mL) % Concentration 8.4867 0.0008 1 15.4885 0.0015 8.6724 0.0008 2 26.2841 0.0026 7.2009 0.0007 3 18.0860 0.0018 9.2036 0.0009 4 23.9543 0.0024 7. 6376 0.0007 5 41.1235 0.0041 7. 7472 0.0007 6 16.9257 0.0017 8.1354 0.0008 7 16.3477 0.0016 7.4452 0.0007 8 25.7495 0.0026 15.7178 0.0016 9 11.6379 0.0012 n.d.: not determined
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Dursunoglu, Benan; Yuca, Hafize; Guvenalp, Zuhal; Gozcu, Sefa; Yilmaz, Bilal|
|Publication:||Turkish Journal of Pharmaceutical Sciences|
|Date:||Dec 1, 2018|
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