Journal of Chromatography A, 1338 (2014) 102–110 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma Rapid, high performance method for the determination of vitamin K 1 , menaquinone-4 and vitamin K 1 2,3-epoxide in human serum and plasma using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry Alessandra Gentili a,∗ , Arturo Cafolla b , Tecla Gasperi c , Simona Bellante a , Fulvia Caretti a , Roberta Curini a , Virginia Pérez Fernández a a Department of Chemistry, Faculty of Mathematical, Physical and Natural Science, “Sapienza” University of Rome, Piazzale Aldo Moro 5, P.O. Box 34, Posta 62, 00185 Rome, Italy b Division of Haematology, Department of Cellular Biotechnologies and Haematology, “Sapienza” University of Rome, via Benevento 6, 00161 Rome, Italy c Department of Sciences, Faculty of Mathematics, Physics and Natural Sciences, Roma Tre University, Via della Vasca Navale 79, 00146 Rome, Italy a r t i c l e i n f o Article history: Received 27 June 2013 Received in revised form 19 February 2014 Accepted 20 February 2014 Available online 28 February 2014 Keywords: Phylloquinone Phylloquinone-epoxide Menaquinone-4 Human serum and plasma HPLC–MS a b s t r a c t Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phyl- loquinone (K 1 ) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K 2 family and metabolite of K 1 in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in bio- logical fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K 1 , K 1 2,3-epoxide (K 1 O), and MK-4 in human serum and plasma at concentrations in the ng/L–g/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sen- sitivity was achieved by removing signal “endogenous suppressors” and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K 1 but levels comparable with those of healthy people and an accumulation of K 1 O up to 3.760 g/L. MK-4 was not detected in either of the two groups. © 2014 Elsevier B.V. All rights reserved. ∗ Corresponding author. Tel.: +39 06 49693230; fax: +39 06 490631. E-mail addresses: alessandra.gentili@uniroma1.it, alessandragentili1965@gmail.com (A. Gentili). 1. Introduction Vitamin K is a generic descriptor for compounds having a com- mon 2-methyl-1,4-naphthoquinone nucleus and a variable alkyl substituent attached at the 3 position [1]. Vitamin K 1 or phylloqui- none (K 1 ) has a phytyl side chain and occurs in green plants [2]. Vitamin K 2 has a bacterial origin and it is represented by a group of homologs known as menaquinones-n (MK-n), where n (from 4 to http://dx.doi.org/10.1016/j.chroma.2014.02.065 0021-9673/© 2014 Elsevier B.V. All rights reserved.