Food Sci. Biotechnol. 24(5): 1603-1607 (2015) DOI 10.1007/s10068-015-0207-8
Comparison of Extraction Methods for the Determination of Phylloquinone Content in Legumes and Nuts Hyeongi Kim, Youngmin Choi1, Jeehye Sung, Younghwa Kim2, Heon Sang Jeong, and Junsoo Lee* Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju, Chungbuk 28644, Korea 1 National Academy of Agricultural Science, Rural Development Administration, Jeonju, Jeonbuk 55365, Korea 2 School of Food Biotechnology & Nutrition, Kyungsung University, Busan 48434, Korea Received January 22 2015 Revised May 21 2015 Accepted May 21 2015 Published online October 31 2015 *Corresponding Author Tel: +82-43-261-2566 Fax: +82-43-271-4412 E-mail:
[email protected] pISSN 1226-7708 eISSN 2092-6456 © KoSFoST and Springer 2015
Abstract The objective of this study was to compare extraction methods for the determination of phylloquinone content in legumes and nuts. Phylloquinone was quantified using high-performance liquid chromatography by post-column derivatization and fluorescence detection. The higher analytical values in legumes were obtained using direct solvent extraction. However, the higher analytical values in nuts were acquired using enzyme extraction. Phylloquinone contents of legumes and nuts ranged from 15.26 to 37.77 and 0.00 to 22.31 µg/100 g, respectively. In addition, analytical method validation parameters, including accuracy and precision, were determined to ensure the validity of the extraction procedures for phylloquinone analysis. Our study provides reliable data on phylloquinone contents in commonly consumed legumes and nuts in Korea. Reliable data regarding vitamin content in food are needed to further understand the nutritional role of vitamin K. Keywords: phylloquinone, HPLC, method validation, legumes, nuts
Introduction The natural forms of vitamin K are phylloquinone (vitamin K1) and menaquinones (vitamin K2). Phylloquinone is synthesized by plants, whereas menaquinones are of microbiological origin. Menadione (vitamin K3) is a synthetic form of vitamin K that is generally used in commercial animal feed. Phylloquinone is a fat-soluble vitamin that functions as a coenzyme, and it is involved in the synthesis of several proteins that participate in blood clotting and bone metabolism (1). The role vitamin K plays as a cofactor in the synthesis of gammacarboxyglutamic acid, which is associated with both blood coagulation factors and osteocalcin, is generally accepted (2,3). Many analytical methods such as colorimetry, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography (HPLC) have been used in vitamin K analyses (1-9). At the present time, the method of choice for vitamin K quantification is HPLC because it is simpler, more precise, and more specific than other methods. Prior to the implementation of HPLC assays, the most critical and time-consuming step is the extraction of vitamin K from the food matrix. Depending on the nature of the sample, various extraction methods have been used to release vitamin K from various food matrices, including liquid-liquid (solvent) extraction (5,6,10,11), solid-phase extraction (1,12,13), acid hydrolysis method (10,11), and
enzyme extraction (9,11). Many studies have reported extraction methods for the analysis of vitamin K in foods, however there was no comparison data for the extraction methods. Therefore, it is necessary to compare the extraction methods to ensure the complete extraction of phylloquinone from the sample matrix. Many studies have reported the vitamin K content in food. However, the Korean nutritional database lacks vitamin K information as compared to database of other countries. In this study, we evaluated the effectiveness of various extraction methods, including enzyme extraction, two different direct solvent extractions, and Soxhlet extraction for the determination of phylloquinone content from soybeans and pine nuts. In addition, the analytical method validation parameters, including accuracy and precision, were determined to ensure the validity of extraction procedures for phylloquinone analysis
Materials and Methods Chemicals All chemicals were of analytical reagent grade. Phylloquinone (vitamin K1) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Lipase (from Candida rugosa, 1000 units/mg, type VII) was purchased from Sigma-Aldrich (St. Louis, MO,
1604 Kim et al.
USA). Sodium acetate, acetic acid (Merck, Darmstadt, Germany), monobasic potassium phosphate, potassium hydroxide, potassium carbonate (Sigma-Aldrich) and zinc powder (particle size
