Vitamin K is essential for blood clotting, bone metabolism, and other diverse physiological functions. It is thought to play a role in reducing osteoporosis and coronary heart disease. Vitamin K is the generic term for a family of fat-soluble compounds with a common chemical structure comprising 2-methyl-1,4-naphthouinone. The naturally occurring forms are vitamin K1 (phylloquinone) and a series of vitamin K2 homologues (menaquinones). Menaquinones are referred to as MK4 through MK13, based on the length of their unsaturated isoprenyl side chains. Two of the most well-known menaquinones are vitamin K2-MK4 and -MK7. Vitamin K is fat-soluble and stored in fatty tissue. It is rapidly metabolized and excreted, so is present at relatively low concentrations in the blood and tissue compared with other fat-soluble vitamins such as retinol (vitamin A) and tocopherol (vitamin E).
Vitamin K Compounds Raise Multiple Analytical Challenges
The ability to precisely analyze—that is, detect, differentiate, quantify, and characterize—these vitamin K compounds is an important capability for clinical researchers. However, the low concentration of vitamin K compounds in biological samples and their complex chemical structures and properties make this challenging. The analytical measurement range for vitamin K compounds is as low as 50 pg/mL and at least as high as 2,000 pg/mL.
The hydrophobicity of vitamin K compounds, demonstrated by their LogP values (see Figure 1), means that they are prone to adhering to glass and plastic. These compounds are also very difficult to ionize for detection by mass spectrometry.
Figure 1: The chemical structure of vitamin K compounds and their LogP values demonstrating high hydrophobicity. Source: Column Selectivity Screening for Vitamin K Compounds (webinar). Credit: Phenomenex.
Therefore, for LC-MS/MS analysis, it is important to select the right high performance liquid chromatography (HPLC) column to sufficiently resolve the vitamin K analytes and separate them from endogenous matrix interferences.
Vitamin K analysis can be further complicated when laboratories desire to quantify all fat soluble vitamins (K, A, and E) in a single panel because the typical concentration ranges for vitamins A and E are an order of magnitude higher than those for vitamin K. Therefore, it can be better to have separate panels: one for vitamin K compounds and one for vitamins A and E. This can reserve a more vigorous and laborious sample extraction for vitamin K analysis and allow a less complex sample cleanup protocol for vitamins A and E.
Several Factors Influence Resolution
The key variables affecting HPLC resolution are efficiency, retention, and selectivity, with selectivity being the most powerful. Selectivity is, in turn, influenced by six key parameters: hydrophobicity, steric interaction, hydrogen donation, hydrogen acceptance, and cationic selectivity. Hydrophobicity is the strongest determining factor. This is especially true of hydrophobic analytes such as vitamin K compounds. Understanding the column attributes with regard to these factors will help guide the selection of the optimal HPLC column for the desired application.
Traditionally, HPLC columns contain media comprised of fully porous spherical particles of silica. However, about a decade ago, the particle technology evolved to include a new kind of particle consisting of a solid, impermeable core of silica surrounded by layers of fully porous silica grafted on top (see Figure 2). These particles, based on core shell technology, are referred to as superficially porous.
Figure 2: Transmission electron micrograph of fully porous particles, a core-shell particle, and schematic representation of a core-shell particle (left to right). Source: What is Core-shell and Superficially Porous Technology? Science Unfiltered (Phenomenex). July 25, 2017. Credit: Phenomenex.
Core Shell Columns Performed Better Than Fully Porous Columns for Vitamin K Analysis
We compared the performance of three 5 µm 50 x 2.1 mm HPLC columns—one core shell (the Kinetex EVO C18 column), and two fully porous columns (a traditional C18 column called the Gemini C18 and one with a C18 ligand optimized for improved peak shape called the Luna C18(2))—for the separation and analysis of vitamin K1, K2-MK4, and K2-MK7 using LC-MS/MS. The comparison data revealed the fully porous traditional column showed some higher selectivity and resolution values, whereas both the fully porous C18 modified for optimized peak shape and the core-shell C18 columns were somewhat comparable. However, the core shell C18 column demonstrated the highest efficiency. This was seen chromatographically as narrower peaks with more peak height that displayed the highest signal to noise ratios with the shortest run time (see Figure 3).
Figure 3: Comparison of two fully porous (A and B) and one core shell (C) HPLC columns for the separation of vitamin K compounds for LC-MS/MS based analysis. Source: Column Selectivity Screening for Vitamin K Compounds (webinar). August 17, 2023. Credit: Phenomenex.
The particular core shell column tested was one that is unique for its organo-silica chemistry—with less silanol and more carbons in aliphatic chains, which resists repulsion of the aliphatic bond. This attribute, along with the higher efficiency, contributed to the achievement of increased peak heights and higher signal to noise ratios with a shorter run time (see Figure 3C).
While LC-MS/MS with fully porous columns may be more appropriate depending on the demands and instrumentation of a particular application and laboratory, the highest quality separation in the shortest amount of time was achieved using a core shell HPLC column over fully porous columns.
