Department of Clinical Biochemistry and Immunology, Lillebaelt Hospital, Vejle, Denmark; Department of Regional Health Research, University of Southern Denmark, Odense, Denmark; Odense Patient Data Explorative Network (OPEN), Department of Clinical Research, University of Southern Denmark and Odense University Hospital, Odense, Denmark.
Department of Clinical Biochemistry and Immunology, Lillebaelt Hospital, Vejle, Denmark; Department of Regional Health Research, University of Southern Denmark, Odense, Denmark; Odense Patient Data Explorative Network (OPEN), Department of Clinical Research, University of Southern Denmark and Odense University Hospital, Odense, Denmark.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jun 1;1117:41-48. doi: 10.1016/j.jchromb.2019.04.010. Epub 2019 Apr 7.
Vitamin K1 (phylloquinone) is one of the vitamin Ks. Several studies have previously investigated the role of vitamin K1 status in respect to disease, but without consistent results. Since vitamin K deficiency has been associated with different disease states it is important to develop a biochemical analysis with sufficient sensitivity and a low limit of quantitation (LOQ). The vitamin Ks are very fat-soluble. This non-polar nature has given rise to several challenges during the method development, because vitamin K1 sticks to materials used during the process and is lost during evaporation. We found that reducing the sample preparation as much as possible offline, instead using online SPE-LC-MS/MS improves recovery and gives satisfactory chromatograms. An Protein BEH C4 column, 300 Å (50 × 2.1 mm, 1.7 μm particle size) was used as trap column and a Phenyl-Hexyl-LC-column, 100 Å (100 × 2.1 mm, 2.6 μm particle size) was used as analytical column. The mobile phases consisted of 30 μmol/L NHF in water and 30 μmol/L NHF in MeOH. A triple quadrupole tandem mass spectrometer with atmospheric pressure chemical ionization (APCI) ion source, positive ion mode, was used to perform the mass spectrometric measurements. The method is simple, highly sensitive and fast. The method was validated for vitamin K1 with good analytical performance. With a LOQ of 0.05 nmol/L it is to our knowledge the vitamin K1 method with lowest LOQ reported to date in the literature. It can easily be automated and applied in a routine diagnostic laboratory. Blood collection tubes with different additives were tested and showed no difference. Stability of vitamin K1 in serum was tested at different temperatures (-20 °C, 4 °C and in light and dark at 20 °C over a period of 30 days) and showed that vitamin K1 is light sensitive in serum even after only one day.
维生素 K1（叶绿醌）是维生素 K 族之一。先前已有多项研究调查了维生素 K1 状态与疾病的关系，但结果并不一致。由于维生素 K 缺乏与多种疾病状态有关，因此开发一种具有足够灵敏度和低定量下限 (LOQ) 的生化分析方法非常重要。维生素 K 非常脂溶性。这种非极性性质在方法开发过程中带来了一些挑战，因为维生素 K1 会粘在处理过程中使用的材料上，并在蒸发过程中丢失。我们发现，尽可能减少离线样品制备，而使用在线 SPE-LC-MS/MS 可以提高回收率并获得满意的色谱图。我们使用 300Å（50×2.1mm，1.7μm 粒径）的 Protein BEH C4 柱作为捕集柱和 100Å（100×2.1mm，2.6μm 粒径）的 Phenyl-Hexyl-LC 柱作为分析柱。流动相由水中的 30μmol/L NHF 和 MeOH 中的 30μmol/L NHF 组成。采用大气压化学电离（APCI）离子源的三重四极杆串联质谱仪，正离子模式，进行质谱测量。该方法简单、灵敏、快速。该方法已针对维生素 K1 进行了验证，具有良好的分析性能。LOQ 为 0.05nmol/L，据我们所知，这是迄今为止文献中报道的维生素 K1 方法中 LOQ 最低的方法。它可以轻松实现自动化，并应用于常规诊断实验室。测试了不同添加剂的采血管，结果没有差异。在不同温度（-20°C、4°C 和 20°C 光照和黑暗下）下测试血清中维生素 K1 的稳定性，结果表明即使在一天后，血清中的维生素 K1 对光也很敏感。
