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.
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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