HPLC-荧光衍生法同时测定人血浆中维生素K1和K2的浓度

Determination of Vitamin K1 and K2 in Human Plasma by HPLC-FLU and Its Clinical Application

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-C_(18)柱(4.6 mm×150 mm,5μm),流动相甲醇-水(98∶2,V/V),流速1m L·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Y_a=2.944ρ_a+0.023(r=0.999 8),定量范围1.000~50.036 ng·m L^(-1),内源性维生素K2(VK2)的标准曲线方程为Y_b=1.195ρ_b+2.653(r=0.999 7),定量范围5.006~100.124 ng·m L^(-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。

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 SnC12 in acidic medium. An AQ-C18 columm 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. 944pa ＋ 0. 023 （ r = 0. 999 8,1. 000 - 50. 036 ng · mL- 1 ）, Yh = 1. 195pb ＋ 2. 653 （ r = 0.999 7,5. 006 - 100. 124 ng· mL-1 ） , respectively. Their precision RSD, stability RSD, repeatability RSD and freezingthawing 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.