目的 建立高效液相色谱荧光检测法,测定人血浆中内源性维生素K同系物的浓度。方法 应用氯化亚锡使维生素K同系物发生荧光衍生,盐析液液萃取后采用色谱柱AQ-C18柱(4.6 mm×150 mm,5 μm),流动相甲醇-水(98∶2,V/V),流速1 mL·min-1,激发波长248 nm,发射波长418 nm,柱温30 ℃进行分析。结果 内源性维生素K1(VK1)的标准曲线方程为Ya=2.944ρa+0.023(r=0.999 8),定量范围1.000~50.036 ng·mL-1,内源性维生素K2(VK2)的标准曲线方程为Yb=1.195ρb+2.653(r=0.999 7),定量范围5.006~100.124 ng·mL-1。VK1和VK2的精密度、稳定性、重复性及冻融试验的相对标准偏差(RSD)均小于10%,符合生物样品的测定需要。结论 该法操作简便、灵敏、准确,适用于的内源性维生素K同系物测定。
Abstract
OBJECTIVE To establish a rapid and sensitive method for the determination of vitamin K homologue in human plasma using HPLC-fluorescence detection. METHODS With salting-out assisted liquid/liquid extraction, the fluorescence derivatization reaction of vitamin K homologues was proposed by ethanolic SnCl2 in acidic medium. An AQ-C18 column was used as the chromatographic column with a mobile phase of a mixture of methanol-water(98∶2) at a flow rate of 1 mL·min-1,the fluorescence detector was set at λex/λem=248 nm/418 nm, and the column temperature was maintained at 30 ℃. RESULTS The calibration curves of vitamin K1(VK1) and vitamin K2 (VK2) were as followed:Ya=2.944ρa+0.023(r=0.999 8,1.000-50.036 ng·mL-1),Yb=1.195ρb+2.653(r=0.999 7,5.006-100.124 ng·mL-1), respectively. Their precision RSD, stability RSD, repeatability RSD and freezing-thawing test RSD were all less than 10%.CONCLUSION The method is simple, sensitive and accurate, which can be used for the assay of endogenous vitamin K homologue in human plasma.
关键词
维生素K同系物 /
荧光衍生法 /
高效液相色谱法
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Key words
vitamin K homologue /
fluorescence derivatization /
HPLC
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中图分类号：
R969
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参考文献
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脚注
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基金
国家自然科学基金资助项目资助(81503140);江苏省医学重点人才项目资助(ZDRCA2016048)
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