Abstract: In this study, an ultrasonic-assisted cloud point extraction combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole (QqQ) mass detector method was developed and optimized for determining the natural vitamin K homologues phylloquinone (PK) and menaquinone-4 (MK) in vegetables. The analytes were isolated from vegetable samples by ultrasound-assisted extraction using acetonitrile as the extraction solvent followed by microwave-assisted cloud point extraction with Triton X-45, chromatographed by gradient elution using ammonium formate solution-methanol as the mobile phase, detected using an electrospray ionization source in the positive ion mode (ESI+) with multiple reaction monitoring (MRM), and quantified by the external standard method. The results showed that the optimized conditions for ultrasonic-assisted cloud point extraction were as follows: 1.5 g of the sample was ultrasonically extracted with 2 mL of acetonitrile, and the extract was re-extracted with 50 μL of Triton X-45 after adding 0.15 g of sodium chloride to it. Good linearity was achieved for the two vitamin K homologues in the range of 1.0–500.0 ng/mL with correlation coefficients of greater than 0.997 0. The limits of detection (LODs) were 1.0 ng/g and 0.8 ng/g for PK and MK, respectively. The recoveries for samples spiked at three concentration levels of 40, 80 and 120 ng/g ranged from 94.5% to 106.2%, and the relative standard deviations (RSDs) of inter-day and intra-day precision were less than 10%. No natural vitamin K was detected in the root and stem vegetables. Only PK was detected in the leafy vegetable samples analyzed at levels in the range of 96.2–1 704.5 ng/g, with the highest value being observed in spinach.

Key words: cloud point extraction; liquid chromatography-tandem mass spectrometry; vegetables; vitamins K