Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection.
A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.
Ahmed
Sameh
S
Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Kishikawa
Naoya
N
Nakashima
Kenichiro
K
Kuroda
Naotaka
N
D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D049449:Luminescence; D008297:Male; D010070:Oxalates; D015203:Reproducibility of Results; D014466:Ultraviolet Rays; D010837:Vitamin K 1 / Q000097:blood* / Q000528:radiation effects; D024482:Vitamin K 2 / Q000031:analogs & derivatives* / Q000097:blood / Q000528:radiation effects; D014815:Vitamins / Q000097:blood* / Q000528:radiation effects
D010070:Oxalates; D014815:Vitamins; D024482:Vitamin K 2; C030814:menatetrenone; C044622:peroxyoxalate; D010837:Vitamin K 1; C062629:menaquinone 7