Method development and validation for dieckol in the standardization of phlorotannin preparations
© Kim et al. 2016
Received: 2 February 2016
Accepted: 16 February 2016
Published: 11 March 2016
Phlorotannins are reported to have diverse biological properties. However, no analytical methods for the standardization of phlorotannin preparations have been reported. Herein, we developed and validated an analytical method for the determination of dieckol in phlorotannin extracts (PRT) using high-performance liquid chromatography (HPLC). The optimum HPLC conditions consisted of a Supelco Discovery C18 column stationary phase, a mobile phase (A: 15 % HPLC grade methanol in deionized water, B: methanol), UV detection at 230 nm, and a flow rate of 0.7 mL/min. The optimized chromatographic conditions were validated and exhibited good specificity and linearity (R 2 > 0.9994–1.0000). The recoveries were in the range of 100.9–102.3 %. The method had good intermediate (%RSD 1.2) and intra-day (%RSD 0.4–1.7) assay precisions. This HPLC method had good accuracy and quality in the determination of dieckol in PRT.
KeywordsDieckol HPLC measurement Validation Phlorotannins Standardization
Marine polyphenol phlorotannins are produced from secondary metabolites via the acetate-malonate pathway in brown seaweeds (Shibata et al., 2004; Isaza Martínez & Torres Castañeda 2013). They have fundamentally different structures than the polyphenols of terrestrial plants (Isaza Martínez & Torres Castañeda 2013; Shibata et al., 2002). Terrestrial plant polyphenols are based on gallic acids or flavones, whereas phlorotannins are only derived from oligomers and polymers of phloroglucinol (1,3,5-trihydroxybenzene) (Koivikko et al., 2007). Thus far, many phlorotannins, such as dieckol, eckol, triphlorethol A, bieckol, fucol, fucophlorethol, have been identified (Isaza Martínez and Torres Castañeda 2013; Kim et al., 2014; Cho et al., 2012).
Over the past 10 years, studies on the biological activities of phlorotannins have increased exponentially (Isaza Martínez & Torres Castañeda 2013). They have a wide range of biological properties, such as antioxidant (Zou et al., 2008), anti-inflammatory (Kim et al., 2009), anti-allergic (Li et al., 2008), and neuroprotective effects (Ahn et al., 2012). Recently, Cho et al. (2014) reported that a phlorotannin preparation and its constituent, eckstolonol, promoted non-rapid eye movement sleep via the benzodiazepine site of gamma-aminobutyric acid type A receptors. Therefore, phlorotannins are considered a promising material for the development of functional foods and supplements.
The standardization of phlorotannin preparations is required for the development of functional foods (Hwang et al., 2009). However, a validation for the standardization of phlorotannin products has not been reported. Dieckol is generally the most abundant compound in phlorotannin preparations, and is used as an indicator compound (Shibata et al., 2004; Cho et al., 2012; Goo et al., 2010). Therefore, we developed and validated an HPLC method for the determination of dieckol for the commercialization of phlorotannin preparations.
Reagents and materials
Phlorotannin preparations (PRT) were obtained from S&D Co., Ltd. (Cheongwon-gun, Korea). The PRT were prepared from the brown seaweed, Ecklonia cava, using a macroporous adsorption resin, as described in our previous work (Kim et al., 2014). The total phlorotannin content (TPC) of the PRT was assessed by the Folin-Ciocalteu method (Slinkard and Singleton, 1977), which determined that there were 905 mg phloroglucinol equivalents/g. Dieckol, a standard compound, was isolated using silica gel and Sephadex LH-20 column chromatography. All of the reagents used were of HPLC grade and purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Preparation of dieckol and PRT samples
A dieckol stock solution was prepared by dissolving 5 mg in 2 mL of dimethyl sulfoxide (DMSO) and 3 mL of methanol. Analytical working solutions were prepared by diluting the stock solution with methanol to seven concentrations, i.e., 12.5, 25, 50, 100, 200, 300, and 400 μg/mL. The phlorotannin extract (50 mg) was added to a volumetric flask, and was then dissolved in 10 mL of DMSO and 40 mL of methanol using ultrasonication at ambient temperature. The PRT sample solution was filtered through a 0.45 μm PTFE syringe filter paper (Whatman, Maidstone, UK), and was used as a sample solution for the HPLC analysis.
Method development for dieckol
Chromatographic conditions for dieckol in phlorotannin preparations
HPLC measurement conditions
A: 15 vol% MeOH in deionized water, B: methanol
Shideido capcellpak C18
Supelco Discovery C18
Mobile phase (A:B)
Flow rate (mL/min)
Oven temperature (°C)
Injection volume (μL)
Elution time (min)
The method was validated in terms of its specificity, linearity, accuracy, recovery, and precision according to the guidelines of the International Conference on the Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) (Jeong et al., 2013; International Conference on Harmonization ICH 1997).
Linearity was tested at seven concentrations, i.e., 12.5, 25, 50, 100, 200, 300, 400 μg/mL. The linear regression equation was calculated from the calibration curve as Y = Ax + B, where A and B are the slope and intercept of the calibration curve, respectively, x is the concentration of dieckol, and Y is the peak area (Goo et al., 2010; Kim et al., 2013). The correlation coefficient (R 2) values were determined for the calibration curve.
Accuracy and recovery
Results and Discussion
Development of HPLC conditions for dieckol
Validation of the proposed analytical method
The validation provides reliable documentation for standardization through regulated experiments (Shabir, 2003; Epshtein 2004). According to the United States Food and Drug Administration (FDA) and Pharmacopeia, validation is a critical factor in the development of the functional food industry (Epshtein 2004; Kazakevich and Lobrutto 2007). Therefore, the validation of the analytical HPLC conditions was conducted in terms of specificity, linearity, accuracy, recovery, and precision (Satinder and Henrik 2011; Meyer, 2010).
Linear regression equations for dieckol in phlorotannin preparationsa
Correlation coefficient (R 2 )
Accuracy validation for dieckol in phlorotannin preparations
Phlorotannin Sample (mg)
Add dieckol (μg/mL)
Total amount found (μg/mL)
Average recovery (%)a
Precision (Intermediate assay and Intra-day) validation for dieckol in phlorotannin preparations
Dieckol content (mg/g)
Average content (mg/g)
Phlorotannin sample (mg)
Dieckol content (mg/g)
Average content (mg/g)
Intra-day: Agilent 1260 Infinitya
We successfully developed an HPLC method for the determination of dieckol in PRT. In addition, we systematically investigated the validation of this analysis. Because information regarding the standardization of phlorotannin preparations or brown seaweed extracts is limited, our study could be useful for the development and commercialization of phlorotannins.
This study was supported by grants from the Korea Food Research Institute (E0191402-07) and the Small and Medium Business Administration (G01981).
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- Ahn BR, Moon HE, Kim HR, Jung HA, Choi JS. Neuroprotective Effect of Edible Brown Algae Eisenia bicyclis on Amyloid Beta Peptide-induce Toxicity in PC12 Cells. Arch Pharm Res. 2012;11:1989–98.View ArticleGoogle Scholar
- Ariffin AA, Ghazali HM, Kavousi P. Validation of a HPLC method for determination of hydroxymethylfufural in crude palm oil. Food Chem. 2014;154:102–7.View ArticlePubMedGoogle Scholar
- César IDC, Braga FC, Soares CDV, Nunan EDA, Pianetti GA, Condessa FA, et al. Development and validation of a RP-HPLC method for quantification of isoflavone aglycones in hydrolyzed soy dry extracts. J Chromatogr B. 2006;836:74–8.View ArticleGoogle Scholar
- Cho S, Yang H, Jeon YJ, Lee CJ, Jin YH, Baek NI, et al. Phlorotannins of the edible brown seaweed Ecklonia cava Kjellman induce sleep via positive allosteric modulation of gamma-aminobutyric acid type A-benzodiazepine receptor: A novel neurological activity of seaweed polyphenols. Food Chem. 2012;132:1133–11424.View ArticleGoogle Scholar
- Cho S, Yoon M, Pae AN, Jin YH, Cho NC, Takata Y, et al. Marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the GABAA receptor. Psychopharmacology. 2014;231:2825–37.View ArticlePubMedGoogle Scholar
- Epshtein NA. Structure of Chemical Compounds, Methods of Analysis and Process Control: Validation of HPLC Techniques for Pharmaceutical Analysis. Pharm Chem J. 2004;38:2120–228.Google Scholar
- Goo HR, Choi JS, Na DH. Quantitative Determination of Major Phlorotannins in Ecklonia stolonifera. Arch Pharm Res. 2010;33:539–44.View ArticlePubMedGoogle Scholar
- Henry BE, Alstyne KL. Effects of UV Radiation on Growth and Phlorotannins in Fucus gardneri (Phaeophyceae) Juveniles and Embryos. J Phycol. 2004;40:527–33.View ArticleGoogle Scholar
- Hwang JS, Kim SJ, Kim HB. Development and Industry of Health Functional Food in Korea. Food Sci Technol Res. 2009;15:1–4.View ArticleGoogle Scholar
- International Conference on Harmonization (ICH). Q2A: Text on Validation of Analytical Procedures. US FDA Federal Register. 1995;60:11260.Google Scholar
- International Conference on Harmonization (ICH). Q2b: Validation of Analytical Procedures: Methodology. US FDA Federal Register. 1997;62:27463.Google Scholar
- Isaza Martínez JH, Torres Castañeda HG. Preparation and chromatographic analysis of phlorotannins. J Chromatogr Sci. 2013;51:825–38.View ArticlePubMedGoogle Scholar
- Jeong HC, Shim YS, Rhee YK, Choi SY, Hong HD, Chung J, et al. Quantification of Marker Compounds in Cirsium setidens Nakai by HPLC-DAD. Food Sci Biotechnol. 2013;22:1481–6.View ArticleGoogle Scholar
- Kazakevich Y, Lobrutto R. HPLC for Pharmaceutical Scientists. Hoboken, New Jersey, USA: John Wiley & Sons, Ltd; 2007.View ArticleGoogle Scholar
- Kim AR, Shin TS, Lee MS, Park JY, Park KE, Yoon NY, et al. Isolation and Identification of Phlorotannins from Ecklonia stolonifera with Antioxidant and Anti-inflammatory Properties. J Agr Food Chem. 2009;57:3483–9.View ArticleGoogle Scholar
- Kim SM, Kang SW, Jeon JS, Jung YJ, Kim WR, Kim CY, et al. Determination of major phlorotannins in Eisenia bicyclis using hydrophilic interaction chromatography: Seasonal variation and extraction characteristics. Food Chem. 2013;138:2399–406.View ArticlePubMedGoogle Scholar
- Kim J, Yoon M, Yang H, Jo J, Han D, Jeon Y, et al. Enrichment and purification of marine polyphenol phlorotannins using macroporous adsorption resins. Food Chem. 2014;162:135–42.View ArticlePubMedGoogle Scholar
- Koivikko R, Loponen J, Pihlaja K, Jormalainen V. High-performance Liquid Chromatographic Analysis of Phlorotannins from the Brown Alga Fucus Vesiculosus. Phytochem Analysis. 2007;18:326–32.View ArticleGoogle Scholar
- Li Y, Lee SH, Le QT, Kim MM, Kim SK. Anti-allergic Effect of Phlorotannins on Histamine Release via Binding Inhibition between lgE and FcεRI. J Agr Food Chem. 2008;56:12073–80.View ArticleGoogle Scholar
- Meyer VR. Practical High-Performance Liquid Chromatogaphy. 5th ed. Chichester, UK: John Wiley & Sons, Ltd; 2010.View ArticleGoogle Scholar
- Mittal A, Parmar S. Development and Validation of Rapid HPLC Method for Determination of Doxofylline in Bulk Drug and Pharmaceutical Dosage Forms. J Anal Chem. 2010;65:293–7.View ArticleGoogle Scholar
- Moussata J, Wang Z, Wang J. Development and validation of an HPLC method for the simultaneous quantification of indole-3-carbinol acetate, indole-3-carbinol, and 3,3’-diindolylmethane in mouse plasma, liver, and kidney tissues. J Chromatogr B. 2014;958:1–9.View ArticleGoogle Scholar
- Sarkar M, Khandavilli S, Panchagnula R. Development and validation of RP-HPLC and ultraviolet spectrophotometric methods of analysis for the quantitative estimation of antiretroviral drugs in pharmaceutical dosage forms. J Chromatogr B. 2006;830:349–54.View ArticleGoogle Scholar
- Satinder A, Henrik R. HPLC Method Development for Pharmaceuticals. London, UK: Academic; 2011.Google Scholar
- Shabir GA. Validation of high-performance liquid chromatography method for pharmaceutical analysis understanding the differences similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J Chromatogr A. 2003;987:57–66.View ArticlePubMedGoogle Scholar
- Shah SR, Dey S, Pradhan P, Jain HK, Upadhyay UM. Method development and validation for simulataneous estimation of albendazole and praziquantel in bulk and in a synthetic mixture. J Taibah Univ Sci. 2014;8:54–63.View ArticleGoogle Scholar
- Shibata T, Fujimoto K, Nagayama K, Yamaguchi K, Nakayama T. Inhibitory activity of brown algal phlorotannins against hyaluronidase. Int J Food Sci Tech. 2002;37:703–9.View ArticleGoogle Scholar
- Shibata T, Kawaguchi S, Hama Y, Inagaki M, Yamaguchi K, Nakamura T. Local and chemical distribution of phlorotannins in brown algae. J Appl Phycol. 2004;16:291–6.View ArticleGoogle Scholar
- Slinkard K, Singleton VL. Total phenol analysis: Automation and comparison with manual methods. Am J Enol Viticult. 1977;28:49–55.Google Scholar
- Zou Y, Qian ZJ, Li Y, Kim MM, Lee SH, Kim SK. Antioxidant effects of phlorotannins isolated from Ishige okamurae in free radical mediated oxidative systems. J Agr Food Chem. 2008;56:7001–9.View ArticleGoogle Scholar