title: Simultaneous determination of vitamin K analogs in human serum by sensitive and selective high-performance liquid chromatography with electrochemical detection.
abstract: OBJECTIVE
  We report a sensitive and selective method for the simultaneous determination of vitamin K1 and K2 analogs (VKs) with high-performance liquid chromatography in which a platinum catalyst reduction column and an electrochemical detector operated in the oxidation mode are incorporated into the detection system. We also applied this trace analysis method to the simultaneous determination of VKs in human serum to investigate the physiologic and pathophysiologic roles of VKs in the bone metabolism.
  
  
  METHODS
  Our high-performance liquid chromatographic method with postcolumn catalyst reduction and electrochemical detection was applied for the simultaneous determination of VKs in human serum samples. After separation of VKs on a reversed-phase separation column by using a mixture of ethanol and methanol (1:1, v/v), containing 0.025 M of sodium perchlorate as the mobile phase, the VKs were reduced once in a platinum catalyst reduction column connected online and then monitored quantitatively by an electrochemical detector with a glassy carbon working electrode operated in the oxidation mode (+0.6 V versus Ag/AgCl).
  
  
  RESULTS
  VKs were clearly separated from each other within 80 min. The detection limits at a signal-to-noise ratio of 3 were 2 to 10 pg for VKs. Quantitative recovery from serum was obtained in the range of 86% to 91% for VKs. The average coefficients of variation of within-day and between-day assays were 1.6% to 2.1% and 2.4% to 3.6%, respectively, for all VKs.
  
  
  CONCLUSIONS
  This method was sensitive and selective for detection of VKs and was satisfactory in the simultaneous determination of VKs in small volumes of human serum.
authors.affiliation: Department of Clinical Pharmacotherapy, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan. waka@niigata-pharm.ac.jp
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Kenji
authors.initials: K
authors.lastname: Onodera
authors.forename: Susumu
authors.initials: S
authors.lastname: Yamato
authors.forename: Kenji
authors.initials: K
authors.lastname: Shimada
mesh_terms: D001842:Bone and Bones / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D005260:Female; D006801:Humans; D008297:Male; D010024:Osteoporosis / Q000378:metabolism; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D045730:Soy Foods; D013997:Time Factors; D014812:Vitamin K / Q000031:analogs & derivatives / Q000097:blood / Q000302:isolation & purification; D010837:Vitamin K 1 / Q000097:blood / Q000302:isolation & purification*; D024482:Vitamin K 2 / Q000097:blood / Q000302:isolation & purification*
chemical_list: D024482:Vitamin K 2; D014812:Vitamin K; D010837:Vitamin K 1