Determination of plasma Vitamin K by high-performance liquid chromatography with fluorescence detection using Vitamin K analogs as internal standards

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2005-02, Vol.816 (1), p.41-48
• Main Authors: Kamao, Maya, Suhara, Yoshitomo, Tsugawa, Naoko, Okano, Toshio
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Fluorescence; Fluorescence detection; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Internal standards; Medical sciences; Osteoporosis - blood; Osteoporosis - drug therapy; Pharmacology. Drug treatments; Reference Standards; Spectrometry, Mass, Electrospray Ionization; Vitamin K; Vitamin K - analogs & derivatives; Vitamin K - blood; Vitamin K 2 - analogs & derivatives; Vitamin K 2 - blood; Vitamin K 2 - therapeutic use
• ISSN: 1570-0232; 1873-376X
• 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.