|Sample Preparation Procedure:|
A 500 µL aliquot of fortified sample (SeraFlx BIOMATRIX) was mixed with 5 µL of internal standard solution (K1-d7, MK4-d7, and MK7-d7 at 100 ng/mL in methanol) and 1.5 mL of acetonitrile followed by vortexing for 20 seconds at 3000 rpm. After centrifugation at 4300 rpm for 10 minutes, the supernatant was loaded onto a Biotage ISOLUTE PLD+ 96-well plate (50 mg) and vacuum was applied to collect the eluate. The eluate was then evaporated to dryness at 50 °C under a gentle stream of nitrogen. The dried extract was reconstituted with 100 μL of diluent and 5 μL of sample was injected for analysis.