RESUMO
A rapid and sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously quantitate vitamin K1 (PK) and vitamin K2 (MK-4) concentrations in human plasma to evaluate nutritional interventions. Charcoal-stripped human plasma was used for standard and calibration preparation with vitamin E-d6 as the internal standard. Patient plasma samples were extracted using n-hexane and analytes separated using an ACE -PFP C18 column (50â¯×â¯2.1â¯mm, 3 µ) equipped with standard C18 guard. The mobile phase consisted of 0.1 % formic acid in water and 0.1 % formic acid in methanol at a flow rate of 0.5â¯mL/min. Analyte quantification was achieved by using MS/MS with a positive atmospheric pressure chemical ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.01â¯ng/mL for both PK and MK-4. The assay was linear over the concentration range from 0.01-50â¯ng/mL for all analytes with a determination coefficient (r2) of 0.998 or better. The intra-and inter-day accuracy and precision were within the acceptable limits per FDA guidance. The validated method was successfully applied to a clinical study for quantification of PK and MK-4 in human plasma to assess nutritional status and dietary interventions in patients with neurodegenerative diseases. Baseline plasma concentrations of PK and MK-4 ranged from 0.23 to 1.81â¯ng/mL and 0.33-0.57â¯ng/mL, respectively.
