6 8 Vol. 6 No. 8 2015 8 Journal of Food Safety and Quality Aug., 2015 蔡 伟 江 *, 邓 鹏 飞, 刘 光 兰, 李 丹 (, 519040) 摘 要 : 目 的, 方 法 (4.6 mm 250 mm, 5.0 μm) ; : : : =70: 16: 14(V:V:V),, : 1.0 ml/min; : 40 ; : 10 µl; : 205 nm;, 结 果, 0.1~0.8 mg/ml(r=0.9999), 0.04 μg/ml, 20 μg/g, 66.666 μg/g, (RSD) 0.6%, 99.37%(n=9) 结 论,,, 关 键 词 : ; ; Determination of phosphatidylcholine from soybean phospholipids by normal phase chromatography CAI Wei-Jiang *, DENG Peng-Fei, LIU Guang-Lan, LI Dan (By-Health Co., Ltd., Zhuhai 519040, China) ABSTRACT: Objective To establish a method for the determination and quality control of phosphatidylcholine from soybean phospholipids in health food by normal-phase chromatography. Methods Chromatographic conditions for determination phosphatidylcholine were silica gel column (4.6 mm 250 mm, 5 μm), isocratic elution with isopropanol: n-hexane: water =70:16:14 (V:V:V) as mobile phase, the flow rate of 1.0 ml/min, the column temperature of 40, the sample volume of 10 μl and the detection wavelength of 205 nm. The methodology validation was used to investigate method detection limit, precision, recovery, etc. Results The results showed that phosphatidylcholine had a good linear relationship at the range of 0.1~0.8 mg/ml between peak area and concentration (r=0.9999). The detection concentration was 0.04 µg/ml, the LOQ was 20 µg/g, the DOQ was 66.666 µg/g, the RSD was 0.6% and the average recovery rate was 99.37 % (n=9). Conclusion The method is simple, accurate, sensitive and with good reproducibility by the method validation. It can be used for the determination of phosphatidylcholine from soybean phospholipids. KEY WORDS: soybean phospholipids; normal phase chromatography; phosphatidyl choline 1 引 言,,,,,,, ; * 通 讯 作 者 :,, E-mail: 714524253@qq.com *Corresponding author: CAI Wei-Jiang, By-Health Co., Ltd., Zhuhai 519040, China. E-mail: 714524253@qq.com
8, : 3099,,,, [1-5] [6] [7] [8-12] [13] [14],, 2 材 料 与 方 法 2.1 材 料 与 仪 器 ( Sigma ; : SLBB9069V; : 99.0%); (, ); (, ); ; ( ) (EQ-500, ); (15C, ( ) ); (, 4.6 mm 250 mm, 5.0 μm, ( ) ) 2.2 实 验 方 法 2.2.1 色 谱 条 件 (, 4.6 mm 250 mm, 5.0 μm, ); : : : =70:16:14(V:V:V); : 1.0 ml/min; : 40 ; : 10 µl; : 205 nm; 20 min 2.2.2 标 准 品 溶 液 的 配 制 100.8 mg 50 ml,,, 1.996 mg/ml, 2.50 5.00 10.00 15.00 20.00 ml 50 ml,,, 0.0100 0.200 0.399 0.599 0.798 mg/ml 2.2.3 供 试 品 溶 液 的 配 制 0.05 g, 25 ml,, 20 min,,,,, 0.45 μm, 2.2.4 标 准 曲 线 的 绘 制 0.0100 0.200 0.399 0.599 0.798 mg/ml HPLC,,,,, 2.2.5 检 出 限 实 验 0.0100 mg/ml 1 ml 50 ml,, 0.002 mg/ml, 1 ml 50 ml,, 0.040 μg/ml, HPLC 2.2.6 精 密 度 实 验 6, 2.2.3,, RSD(%) 2.2.7 回 收 率 实 验 0.05 g 9, 25 ml, 3, 3, ( 2.00 mg/ml)4 5 6 ml,, 20 min,, 0.45 μm, 3 结 果 与 讨 论 3.1 标 准 曲 线 确 证 结 果 1, A=6234647.64026C-54020.42073(r=0.9999), 0.1~0.8 mg/ml 1 Fig. 1 Standard curve of phosphatidyl choline
3100 6 3.2 检 出 限 和 定 量 限 DL QL (S/N) DL S/N=3, QL S/N=10 3.2.1 检 出 限 0.010 mg/ml 1 ml 50 ml,, 0.002 mg/ml, 1 ml 50 ml,, 0.040 μg/ml, 3.57, (3:1) : DL( )=0.040 μg/ml,, : 0.040 μg/ml 25 ml/0.05 g =20 μg/g 3.2.2 定 量 限 S/N=10 : QL 0.040 μg/ml/3 10=0.133 μg/ml,, : 20 μg/g 10/3=66.666 μg/g 3.3 精 密 度 1, 6 176.87~179.49 mg/g, 178.1 mg/g, (RSD) 0.6%, 3.4 回 收 率 3 2, 3 98.08%~100.72%, : 99.37%, (RSD) 0.9% Table 1 表 1 精 密 度 的 实 验 结 果 (n=6) The precision of the experiment (n=6) (g) (mg/ml) (mg/g) (mg/g) RSD(%) 1 0.06311 0.43694 176.865 2 0.06194 0.43247 178.277 3 0.06208 0.43556 179.014 4 0.06301 0.43772 177.277 178.1 0.6% 5 0.06181 0.43504 179.489 6 0.06330 0.43986 177.673 Table 2 表 2 加 标 回 收 率 实 验 (n=9) Standard addition recovery of the experiment (n=9) (mg) (mg) (%) (%) RSD(%) 1 7.98336 8.041059 100.722 2 7.98336 7.861175 98.469 3 7.98336 7.936347 99.411 4 9.97920 9.787373 98.077 5 9.97920 9.898489 99.191 99.37 0.9 6 9.97920 9.932468 99.531 7 11.97504 11.936214 99.675 8 11.97504 11.859555 99.035 9 11.97504 11.996478 100.179
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