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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	5860 psi (404 bar)
Temp.	25 °C
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. Although both forms, as well as the hydroxy-metabolites, can be present in the formula, vitamin D3 is more commonly used for food fortification. The AOAC method was followed in the work presented here to measure vitamin D in infant formula using a Titan C18 UHPLC column. Vitamin D3 was found to be present, but D2 and the hydroxy metabolites were absent.
Featured Industry	Food and Beverages
Legal Information	Titan is a trademark of Sigma-Aldrich Co. LLC
Other Notes	Sample prep other: Saponification followed by liquid-liquid extraction per AOAC method 2011.11
Suitability	Application for LC-MS

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