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column	Titan C18, 10 cm x 2.1 mm I.D., 1.9 µm particles (577124-U)
column temp.	25 °C
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)
sample	1 IU/mL in methanol
injection	5 µL
detector	MS, APCI(+), MRM 397/125 (ergocalciferol, D2), MRM 385/259 (cholecalciferol, D3)

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Analysis Note	AOAC method 2011.11 describes the procedure the analysis of vitamin D in infant formula and adult/pediatric nutritional formula using UHPLC/MS/MS. Two forms of vitamin D are recognized, vitamin D₂ or ergocalciferol, and vitamin D₃ or cholecalciferol. Separation of vitamin D₂ and D₃ following the AOAC method is shown here using a Titan C18 UHPLC column.
Featured Industry	Food and Beverages
suitability	application for UHPLC-MS

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