Determination of phylloquinone (vitamin K1) in plasma and serum by HPLC with fluorescence detection

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 K-1) in plasma or serum samples. Compensation for procedural losses of vitamin K, 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 K, 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, R-2 = 0.99. Validation also included linearity of response, absence of interference and confirmation of vitamin K, peak purity. (C)2004 Elsevier B.V. All rights reserved.