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Vitamin K1 and K2 in Fortified Artificial Serum on Raptor Biphenyl by LC-MS/MS

PeakstR (min)Conc.
Precursor IonProduct Ion
1.Vitamin MK41.440.5445.5187.2
2.Vitamin K11.470.5451.5187.2
3.Vitamin MK72.200.5649.7187.2
ColumnRaptor Biphenyl (cat.# 9309A52)
Dimensions:50 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Temp.:40 °C
Diluent:15:85 Water:methanol
Conc.:0.5 ng/mL Fortified SeraFlx BIOMATRIX sample
Inj. Vol.:5 µL
Mobile Phase
A:Water, 0.1% formic acid, 5 mM ammonium formate
B:Methanol, 0.1% formic acid
Time (min)Flow (mL/min)%A%B
Ion Mode:ESI+
Sample PreparationA 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.