动物性食物不同形式维生素K检测方法的建立
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摘要
目的建立不同形式维生素K在线分析技术,探讨动物性食物中维生素K的含量分布。方法动物性食物样品在pH为8、脂肪酶(0.2 g)、蛋白酶(0.1 g)条件下水解4 h,再用异辛烷提取,经旋转蒸发、复溶后测定。使用Atlantis T3(4.6 mm×250 mm,5μm)色谱柱分离,以900 mL甲醇∶100 mL四氢呋喃(包含5 mmol冰乙酸、11 mmol氯化锌、6 mmol无水乙酸钠)为流动相,激发波长为320 nm,发射波长为410.3 nm,高效液相色谱-荧光检测法分离维生素K_1、四烯甲萘醌(menaquinone-4, MK-4)和七烯甲萘醌(menaquinone-7,MK-7)。结果方法线性范围为0.01～0.40μg/mL(决定系数>0.999),加标回收率为84.4%～124.2%,相对标准偏差<6%(n=6)。MK-4是猪、鸡肉中维生素K的主要形式,牛肉中以维生素K_1含量最高,白贝和鱿鱼中可检测到MK-7。结论建立的高效液相色谱柱后衍生法适用于动物性食物维生素K的测定,不同形式维生素K在不同动物体内的分布特征不同。
OBJECTIVE To establish a high performance liquid chromatography method with fluorescence detection for a variety of vitamin K and to assess the content of vitamin K in animal foods. METHODS Animal foods were hydrolyzed by 0.2 g lipase and 0.1 g protease in pH 8 for 4 hours, extracted with isooctane followed by rotary evaporation and reconstitution. The mobile phase was 900 mL methanol and 100 mL tetrahydrofuran which contained 5 mmol glacial acetic acid, 11 mmol zinc chloride and 6 mmol anhydrous sodium acetate. The content of vitamin K_1, menaquinone-4(MK-4), and menaquinone-7(MK-7)were separated on Atlantis T3 column(4.6 mm×250 mm,5 μm)by high-performance liquid chromatography. The fluorescence detector was set at a wavelength of 320 nm for excitation and 410.3 nm for emission. RESULTS The linear range of the method was 0.01-0.40 μg/mL, and coefficient of determination was > 0.999. The spiked recoveries were 84.4%-124.2% with relative standard deviation was <6%(n=6). MK-4 was the main form of vitamin K in pork and chicken. The highest content of vitamin K_1 was found in beef, and MK-7 could be detected in aquatic products. CONCLUSION The proposed method is successfully applied for the determination of vitamin K in animal foods. A variety of vitamin K are distributed differently in distinct animals.
OBJECTIVE To establish a high performance liquid chromatography method with fluorescence detection for a variety of vitamin K and to assess the content of vitamin K in animal foods. METHODS Animal foods were hydrolyzed by 0.2 g lipase and 0.1 g protease in pH 8 for 4 hours, extracted with isooctane followed by rotary evaporation and reconstitution. The mobile phase was 900 mL methanol and 100 mL tetrahydrofuran which contained 5 mmol glacial acetic acid, 11 mmol zinc chloride and 6 mmol anhydrous sodium acetate. The content of vitamin K_1, menaquinone-4(MK-4), and menaquinone-7(MK-7)were separated on Atlantis T3 column(4.6 mm×250 mm,5 μm)by high-performance liquid chromatography. The fluorescence detector was set at a wavelength of 320 nm for excitation and 410.3 nm for emission. RESULTS The linear range of the method was 0.01-0.40 μg/mL, and coefficient of determination was > 0.999. The spiked recoveries were 84.4%-124.2% with relative standard deviation was <6%(n=6). MK-4 was the main form of vitamin K in pork and chicken. The highest content of vitamin K_1 was found in beef, and MK-7 could be detected in aquatic products. CONCLUSION The proposed method is successfully applied for the determination of vitamin K in animal foods. A variety of vitamin K are distributed differently in distinct animals.
引文
[1] 张萌萌.维生素K2调节骨代谢的生物学研究回顾[J].中国骨质疏松杂志,2016,22(12):1597-1600.
[2] 张红梅,赖建强.维生素K的膳食摄入及其与成人健康关系的研究进展[J].卫生研究,2012,41(1):126-131.
[3] 范加金,彭延杰,严为留.维生素K2的生理作用及其在纳豆中的研究进展[J].黑龙江医药,2016,29(3):395-399.
[4] 国家食品药品监督管理总局,国家卫生和计划生育委员会.食品安全国家标准食品中维生素K1的测定:GB 5009.158—2016 [S].北京:中国标准出版社,2016.
[5] KOIVUTIKKANEN T J,VELIMATTI OLLILAINEN A,PIIRONEN V I.Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc[J].J Agr Food Chem,2000,48(12):6325.
[6] AOAC.AOAC official method 999.15 vitamin K in milk and infant formulas liquid chromatographic method first action 1999 [S].Rockville:AOAC,1999.
[7] 卫生部.食品安全国家标准食品营养强化剂使用标准:GB 14880—2012 [S].北京:中国标准出版社,2012.
[8] MANOURY E,JOURDON K,BOYAVAL P,et al.Quantitative measurement of vitamin K-2 (menaquinones) in various fermented dairy products using a reliable high-performance liquid chromatography method[J].J Dairy Sci,2013,96(3):1335-1346.
[2] 张红梅,赖建强.维生素K的膳食摄入及其与成人健康关系的研究进展[J].卫生研究,2012,41(1):126-131.
[3] 范加金,彭延杰,严为留.维生素K2的生理作用及其在纳豆中的研究进展[J].黑龙江医药,2016,29(3):395-399.
[4] 国家食品药品监督管理总局,国家卫生和计划生育委员会.食品安全国家标准食品中维生素K1的测定:GB 5009.158—2016 [S].北京:中国标准出版社,2016.
[5] KOIVUTIKKANEN T J,VELIMATTI OLLILAINEN A,PIIRONEN V I.Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc[J].J Agr Food Chem,2000,48(12):6325.
[6] AOAC.AOAC official method 999.15 vitamin K in milk and infant formulas liquid chromatographic method first action 1999 [S].Rockville:AOAC,1999.
[7] 卫生部.食品安全国家标准食品营养强化剂使用标准:GB 14880—2012 [S].北京:中国标准出版社,2012.
[8] MANOURY E,JOURDON K,BOYAVAL P,et al.Quantitative measurement of vitamin K-2 (menaquinones) in various fermented dairy products using a reliable high-performance liquid chromatography method[J].J Dairy Sci,2013,96(3):1335-1346.