柱后还原-高效液相色谱法同时测定调制乳粉中维生素K1和维生素K2

doi:10.3724/SP.J.1123.2019.11020

色谱 ›› 2020, Vol. 38 ›› Issue (7): 847-852.DOI: 10.3724/SP.J.1123.2019.11020

柱后还原-高效液相色谱法同时测定调制乳粉中维生素K₁和维生素K₂

孙珊珊¹, 公丕学¹, 张艳侠¹, 王明栋¹, 卢兰香¹, 李新玲¹, 薛霞¹, 刘艳明¹^,^*(), 张峰²^,^*()

¹ 山东省食品药品检验研究院, 山东济南 250101
² 中国检验检疫科学研究院, 北京 100176

收稿日期:2019-11-22 出版日期:2020-07-08 发布日期:2020-12-10
通讯作者: 刘艳明,张峰
作者简介:张峰.Tel:53898008, E-mail:fengzhang@126.com
刘艳明.Tel:81216719, E-mail:msymliu@163.com;
基金资助:
山东省重点研发计划（医用食品）(2019YYSP020);国家重点研发计划(2017YFC1601600)

Determination of vitamin K₁ and vitamin K₂ in modulation milk powder by post-column reduction-high performance liquid chromatography

SUN Shanshan¹, GONG Pixue¹, ZHANG Yanxia¹, WANG Mingdong¹, LU Lanxiang¹, LI Xinling¹, XUE Xia¹, LIU Yanming¹^,^*(), ZHANG Feng²^,^*()

¹ Shandong Institute for Food and Drug Control, Ji'nan 250101, China
² Chinese Academy of Inspection and Quarantine, Beijing 100176, China

Received:2019-11-22 Online:2020-07-08 Published:2020-12-10
Contact: LIU Yanming,ZHANG Feng
Supported by:
Shandong Keypoint Research and Invention Program (Food for Special Medical Purpose, FSMP)(2019YYSP020);the National Key Research and Development Program of China(2017YFC1601600)

摘要/Abstract

摘要：

建立了一种同时检测调制乳粉中维生素K₁和维生素K₂的柱后还原-高效液相色谱-荧光检测方法。样品用水溶解，经脂肪酶酶解，2.5 mol/L氢氧化钠溶液和乙醇溶液皂化，正己烷萃取，氮吹浓缩后，用甲醇复溶。通过Xbridge C₁₈色谱柱分离，锌粉还原柱柱后还原，荧光检测器检测，激发波长为326 nm，发射波长为432 nm，外标法定量。结果表明，维生素K₁在0.0025~2.0 μg/mL、维生素K₂在0.01~2.0 μg/mL范围内线性关系良好，相关系数均大于0.999，维生素K₁和维生素K₂的检出限分别为0.07 μg/100 g和0.24 μg/100 g，定量限分别为0.2 μg/100 g和0.8 μg/100 g；方法的加标回收率为80.39%~94.39%，精密度为0.85%~3.98%。该方法灵敏度高，重复性好，结果准确，适用于调制乳粉中维生素K₁和维生素K₂的分析检测。

关键词: 高效液相色谱, 荧光检测器, 柱后还原, 维生素K₁, 维生素K₂, 调制乳粉

Abstract:

A method for the determination of vitamin K₁ and vitamin K₂ in modulation milk powder was developed by high performance liquid chromatography (HPLC) coupled with post-column reduction. The samples were dissolved in water, lipase hydrolyzed, saponified with 2.5 mol/L sodium hydroxide solution and ethanol solution, extracted with n-hexane, and dissolved in methanol after concentration. The vitamin K were first separated on an Xbridge C₁₈ column and then on a zinc powder reduction column, and detected using a fluorescence detector. The excitation and emission wavelengths were 326 nm and 432 nm, respectively. An external standard method was used for quantification. The results showed that the linearities of vitamin K₁ and vitamin K₂ was in the ranges of 0.0025-2.0 μg/mL and 0.01-2.0 μg/mL, respectively, with correlation coefficients both greater than 0.999. The spiked recoveries were 80.39%-94.39% and the precisions were 0.85%-3.98%. The limits of detection of vitamin K₁ and vitamin K₂ were 0.07 μg/100 g and 0.2 μg/100 g, respectively. The limits of quantification of vitamin K₁ and vitamin K₂ were 0.2 μg/100 g and 0.8 μg/100 g, respectively. The method has high sensitivity and good repeatability, and gives accurate results. It is suitable for the analysis and determination of the vitamin K₁ and vitamin K₂ in formula milk powder.

Key words: high performance liquid chromatography (HPLC), fluorescence detector, post-column reduction, vitamin K₁, vitamin K₂, modulation milk powder

孙珊珊, 公丕学, 张艳侠, 王明栋, 卢兰香, 李新玲, 薛霞, 刘艳明, 张峰. 柱后还原-高效液相色谱法同时测定调制乳粉中维生素K₁和维生素K₂[J]. 色谱, 2020, 38(7): 847-852.

SUN Shanshan, GONG Pixue, ZHANG Yanxia, WANG Mingdong, LU Lanxiang, LI Xinling, XUE Xia, LIU Yanming, ZHANG Feng. Determination of vitamin K₁ and vitamin K₂ in modulation milk powder by post-column reduction-high performance liquid chromatography[J]. Chinese Journal of Chromatography, 2020, 38(7): 847-852.

图/表 6

图1 还原后维生素K1和维生素K2的(a)激发光谱和(b)荧光光谱图

Fig. 1 (a) Excitation and (b) fluorescence spectra of the vitamin K1 (VK1) and vitamin K2 (VK2) after reduction a. emission wavelength (λem)=430 nm; b. excitation wavelength (λex)=326 nm.

图2 维生素K1和维生素K2混合标准溶液(1.0 μg/mL)的色谱图

Fig. 2 Chromatogram of the VK1 and VK2 in the mixed standard solution (1.0 μg/mL)

图3 (a) 脂肪酶用量和(b)酶解时间对维生素K1和维生素K2回收率的影响(n=3)

Fig. 3 Effect of (a) dosage of lipase and (b) enzymolysis time on the recoveries of the VK1 and VK2 (n=3)

图4 氢氧化钠溶液(2.5 mol/L)的加入量对维生素K1和维生素K2回收率的影响(n=3)

Fig. 4 Effect of volumes of sodium hydroxide solution (2.5 mol/L) on the recoveries of the VK1 and VK2 (n=3)

表1 萃取条件对维生素K1和维生素K2提取回收率的影响(n=3)

Table 1 Effect of extraction conditions on the extraction recoveries of the VK1 and VK2 (n=3)

No.	Conditions	VK₁		VK₂
No.	Conditions	Recovery/%	RSD/%	Recovery/%	RSD/%
1	10 mL hexane, extracted once	78.37	1.06	75.82	0.76
2	10 mL hexane, extracted twice	93.01	2.67	92.65	1.29
3	15 mL hexane, extracted once	85.30	2.79	84.82	2.70
4	15 mL hexane, extracted twice	92.03	1.38	93.60	1.32

表2 方法的回收率和精密度(n=6)

Table 2 Recoveries and RSDs for the method (n=6)

Spiked/ (μg/100 g)	VK₁		VK₂
Spiked/ (μg/100 g)	Recovery/%	RSD/%	Recovery/%	RSD/%
5	80.83	2.44	80.39	3.98
50	94.39	1.16	92.50	0.85
100	92.59	1.30	91.23	2.96

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柱后还原-高效液相色谱法同时测定调制乳粉中维生素K₁和维生素K₂

Determination of vitamin K₁ and vitamin K₂ in modulation milk powder by post-column reduction-high performance liquid chromatography

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