Kamao Maya, Suhara Yoshitomo, Tsugawa Naoko, Okano Toshio
Department of Hygienic Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Feb 25;816(1-2):41-8. doi: 10.1016/j.jchromb.2004.11.003.
A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K(1), phylloquinone, PK and K(2), menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7-9.2% for MK-4, 4.9-9.6% for PK and 6.3-19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R(2)=0.988), PK (R(2)=0.979) and MK-7 (R(2)=0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.
建立了一种采用柱后还原和内标法的高效液相色谱荧光检测法，用于测定维生素K衍生物（维生素K(1)、叶绿醌、PK和K(2)、甲萘醌、MK - 4和MK - 7）。通过优化色谱条件提高了选择性和重现性，使用合成内标获得了令人满意的精密度和准确度。向血浆样品中加入内标后，用乙醇和己烷提取脂质。在C18柱上通过等度反相分离进行色谱分析。维生素K衍生物在430 nm处检测，MK - 4的激发波长为320 nm，PK和MK - 7的激发波长为240 nm。MK - 4、PK和MK - 7的检测限分别为4、2和4 pg。MK - 4、PK和MK - 7的回收率大于92%，批间和批内相对标准偏差值，MK - 4为5.7 - 9.2%，PK为4.9 - 9.6%，MK - 7为6.3 - 19.3%。数据表明，该方法与LC - APCI/MS法对MK - 4（R(2)=0.988）、PK（R(2)=0.979）和MK - 7（R(2)=0.986）具有良好的相关性。该方法可用于测定维生素K以评估其临床和营养状况。
