AOAC Method 2011.11: UHPLC/MS/MS Analysis of Vitamin D₂ and Vitamin D₃ on Titan C18

column

Titan C18, 10 cm x 2.1 mm I.D., 1.9 μm particles (577124-U)

mobile phase

[A] 0.1% formic in methanol:water (20:80); [B] 0.1% formic acid in methanol

gradient

60 to 90% B in 0.4 min; to 100% B in 0.3 min; held at 100% B for 7.8 min; to 60% B in 0.1 min; held at 60% B for 1.5 min

flow rate

0.25 mL/min for 5.55 min; to 0.50 mL/min in 0.1 min; held at 0.5 mL/min for 3.84 min; to 0.25 mL/min in 0.1 min; held at 0.25 mL/min for 0.7 min

pressure

5859 psi (404 bar)

column temp.

25 °C

detector

MS, APCI(+), MRM 397/125 (ergocalciferol, D₂), MRM 385/259 (cholecalciferol, D₃)

injection

5 μL

sample

1 IU/mL in methanol