LC/MS/MS of 25-Dihydroxyvitamin D2, 25-Hydroxyvitamin D3, and 3-epi-25-Hydroxyvitamin D3 in Serum or Plasma on Ascentis® Express F5 after SPE with HybridSPE®-Phospholipid, Minimization of Matrix Effects

LC/MS/MS of 25-Dihydroxyvitamin D2, 25-Hydroxyvitamin D3, and 3-epi-25-Hydroxyvitamin D3 in Serum or Plasma on Ascentis® Express F5 after SPE with HybridSPE®-Phospholipid, Minimization of Matrix Effects

Conditions

sample preparation SPE (Solid Phase Extraction)
sample/matrix plasma or serum spiked at 25 ng/mL with each vitamin D metabolite
SPE well plate 575656-U (HybridSPE-Phospholipid 96-well plate, 50 mg/2 mL)
sample addition 100 µL spiked rat plasma followed by 300 µL 1% formic acid in acetonitrile
elution apply vacuum at 10 in Hg for 4 min
eluate post-treatment collect filtrate and analyze directly
column Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 µm particles (53569-U)
column temp. 40 °C
mobile phase (A) 5 mM ammonium formate in 75:25 (v/v) methanol:water
flow rate 0.4 mL/min
injection 1 µL
detector MS, ESI(+), m/z 100-1000; phospholipids monitored at 496.3, 524.3, 758.5, 786.5, 806.5, and 810.5 m/z

Description

Analysis Note Chromatographic resolution of the various homologs of vitamin D2 is necessary for accurate quantification, especially considering several key metabolites are isobaric and not distinguishable by MS alone. A pentafluorophenyl HPLC phase (Ascentis Express F5) was chosen because of its ability to rapidly resolve the vitamin D homologs tested, especially the 25-hydroxyvitamin D3 and the 3-epi-25-hydroxyvitamin D3 that coelute on C18 stationary phases. HybridSPE-Phospholipid selectively depleted the phospholipid matrix and precipitated proteins, providing no interference from the serum matrix. Cerilliant CRMs provided reliable quantification.
Categories Analytical Chromatography, Clinical, SPE Applications, vitamins
Featured Industry Clinical
Legal Information Ascentis is a registered trademark of Sigma-Aldrich Co. LLC, HybridSPE is a registered trademark of Sigma-Aldrich Co. LLC
suitability application for LC-MS, application for SPE

Materials

     
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