Craig Aurand, David Bell, Anders Fridstrom, Klaus Buckendahl
Figure 1.Vitamin D Metabolites on Ascentis® Express C18
Retention Time:
25-hydroxyvitamin D3 1.09 min.
epi-25-hydroxyvitamin D3 1.09 min.
25-hydroxyvitamin D2 1.29 min.
column: Ascentis Express C18, 5 cm x 2.1 mm, 2.7 μm (53822-U)
mobile phase A: 15%, 5 mM ammonium formate water
mobile phase B: 85%, 5 mM ammonium formate (95:5 acetonitrile:water)
flow rate: 0.4 mL/min.
temp.: 35 °C
det.: UV 265 nm, MS ESI+ 100-1000m/z
injection: 1 μL
Figure 2. Vitamin D Metabolites on Ascentis Express F5
Retention Time:
25-hydroxyvitamin D3 2.57 min.
epi-25-hydroxyvitamin D3 2.76 min.
25-hydroxyvitamin D2 2.77 min.
column: Ascentis Express F5, 10 cm x 2.1 mm, 2.7 μm (53569-U)
mobile phase A: 25%, 5 mM ammonium formate water
mobile phase B: 75%, 5 mM ammonium formate (methanol)
flow rate: 0.4 mL/min.
temp.: 40 °C
det.: UV 265 nm, MS ESI+ 100-1000m/z
injection: 1 μL
Wellplate: HybridSPE-Phospholipid 50 mg 96-well plate (575656U)
Rat serum was spiked at 300 ng/mL with 25-hydroxyvitamin D2, 25- hydroxyvitamin D3 and epi-25-hydroxyvitamin D3. Protein precipitation was performed offline by adding 100 μL of spiked serum into a 500 μL 96-well collection plate followed by 300 μL of 1% formic acid acetonitrile. Samples were mixed by performing five 300 μL draw/aspiration cycles using a digital pipetter, then allow to set for 5 minutes before transferring 200 μL of precipitate into the HybridSPE-Phospholipid 96-well plate. Samples were passed through the HybridSPE-Phospholipid plate by applying 10” Hg vacuum for 4 minutes, the resulting filtrate was analyzed directly.
As a comparison, spiked rat serum was also processed using standard protein precipitation by adding 100 μL of serum into 2 mL centrifuge vials followed by 300 μL of 1% formic acid in acetonitrile. Samples were vortexed and centrifuged, and the resulting supernatant was collected and analyzed directly.
Figure 3. Sample process for HybridSPEPhospholipid 96-well Plate
Figure 4. Sample Preparation Comparison: Phospholipid Monitoring
Phospholipid Monitoring (m/z):
Lysophosphatidylcholines: 496.3, 524.3 m/z
Glycerophosphocholines: 758.5, 786.5, 806.5, 810.5 m/z
instrument: Agilent 1200 SL Rapid Resolution, 6210 TOF
column: Ascentis Express F5, 10 cm x 2.1 mm, 2.7 μm (53569-U)
mobile phase A: 25%, 5mM ammonium formate water
mobile phase B: 75%, 5mM ammonium formate methanol
flow rate: 0.4 mL/min
temp.: 40 °C
MS det.: 100-1000m/z
Cap V: 3000V, Fragmenter: 200V, Dry Gas: 10L/min, Gas Temp: 350 °C
Table 2. Sample Prep Analyte Recovery Comparison from Spiked Rat Serum
Sample spike level of 300 ng/mL, n=48 replicates, external calibration
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