Wang Laura Y, Bates Chris J, Yan Liya, Harrington Dominic J, Shearer Martin J, Prentice Ann
Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK.
Clin Chim Acta. 2004 Sep;347(1-2):199-207. doi: 10.1016/j.cccn.2004.04.030.
A modified high-performance liquid chromatography (HPLC) method, based on Davidson and Sadowski [Meth. Enzymol. 282 (1997) 408], with fluorescence detection after zinc postcolumn reduction was developed and validated for the analysis of phylloquinone (vitamin K1) in plasma or serum samples. Compensation for procedural losses of vitamin K1 was made by the method of internal standardization using a proprietary vitamin K derivative. Increased sensitivity of detection by the use of a high-sensitivity Waters 440 fluorescence detector and optimized chromatography conditions increased the sensitivity to 4 fmol vitamin K1. The response was linear and free from interfering peaks and from baseline drift. It is therefore adequately sensitive for 0.25 ml or less of plasma sample. Long-term reproducibility of quality assurance (QA) samples was verified over a period of 4 months. The intraassay precision estimates of the QA samples within-run with mean vitamin K1 concentrations of 0.4, 1.4 and 3.4 nmol/l were 5.2% (n=6), 8.2% (n=6) and 3.0% (n=12), respectively, while interassay precision estimates between runs were 16% (n=22), 12% (n=21) and 8.1% (n=15), respectively. The assay accuracy was validated by comparing the results we obtained for 14 samples from the Vitamin K External Quality Assessment Scheme (KEQAS) with the consensus of the results from the other participating laboratories. Good agreement was obtained, with y=1.06x-0.09, R2=0.99. Validation also included linearity of response, absence of interference and confirmation of vitamin K1 peak purity.
基于戴维森和萨多夫斯基[《酶学方法》282(1997)408]，开发了一种改良的高效液相色谱(HPLC)方法，该方法在锌柱后还原后进行荧光检测，并经过验证可用于分析血浆或血清样品中的叶绿醌(维生素K1)。使用一种专有的维生素K衍生物通过内标法对维生素K1的程序损失进行补偿。通过使用高灵敏度的沃特世440荧光检测器和优化的色谱条件提高了检测灵敏度，使维生素K1的检测灵敏度提高到4飞摩尔。响应呈线性，无干扰峰和基线漂移。因此，对于0.25 ml或更少的血浆样品具有足够的灵敏度。在4个月的时间内验证了质量保证(QA)样品的长期重现性。QA样品在运行内的批内精密度估计值，维生素K1平均浓度为0.4、1.4和3.4 nmol/l时，分别为5.2%(n = 6)、8.2%(n = 6)和3.0%(n = 12)，而批间精密度估计值分别为16%(n = 22)、12%(n = 21)和8.1%(n = 15)。通过将我们从维生素K外部质量评估计划(KEQAS)的14个样品中获得的结果与其他参与实验室的结果共识进行比较，验证了该分析方法的准确性。得到了良好的一致性，y = 1.06x - 0.09，R2 = 0.99。验证还包括响应线性、无干扰以及维生素K1峰纯度的确认。
