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Determination of Vitamin D in Milk Products by UPLC-MS-MS with
4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) Derivatization
1.
Material and Apparatus
1.1 Materials
Infant formula/nutritional products reference (NIST SRM 1849a);
Vitamin D2 and Vitamin D3: Dr. Ehrenstorfer GmbH, Germany;
Vitamin D3-[
2
H
3
] isotope internal standard: King of Prussia, USA.
1.2
Chemicals and Reagents
2,6-Di-
tert
-butyl-4-methylphenol (BHT): CP grade, Shanghai Lingfeng Chemical Reagent Co.,
Ltd., Shanghai, China;
Formic acid: LC-MS grade (98%), ANPEL Laboratory Technologies (Shanghai) Inc., Shanghai,
China;
4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD): TCI, Shanghai, China;
Acetonitrile, methanol, and n-hexane: HPLC grade, Thermo Fisher, USA;
KOH, pyrogallic acid, and anhydrous ethanol: AR grade;
PVDF filter membrane: 0.22
μ
m
1.3
Instrument and Apparatus
Agilent 1290 Infinity UPLC equipped with Agilent 6490 Triple Quad MS-MS;
Nitrogen evaporator.
2.
Method
2.1 Preparation of Solutions
2.1.1 VD2 and VD3 stock standard solution (500
μ
g/mL, 20000 IU/mL): Accurately weigh 25 mg
(sensitive to 0.1 mg) of VD2 or VD3 into a 50 mL volumetric flask, and dissolve and bring to
volume with acetonitrile.
2.1.2 ID3 stock internal standard solution (20
μ
g/mL, 800 IU/mL): Accurately pipet 1 mL of 1
mg/mL ID3 standard into a 50 mL volumetric flask, and dissolve and bring to volume with
ethanol.
2.1.3 VD2/VD3 standard mixture 2 (10
μ
g/mL, 400 IU/mL): Accurately pipet 0.2 mL of VD2 and
VD3 stock standard solutions (2.1.1) into a 10 mL volumetric flask, bring to volume with
acetonitrile.
2.1.4 VD2/VD3 standard mixture 1 (1.0
μ
g/mL, 40 IU/mL): Accurately pipet 1.0 mL of VD2/VD3
standard mixture 2 (2.1.3) into a 10 mL volumetric flask, bring to volume with acetonitrile.
2.1.5 ID3 working internal standard solution (0.1
μ
g/mL, 4 IU/mL): Accurately pipet 0.5 mL of
ID3 stock internal standard solution (2.1.2) into a 100 mL volumetric flask, bring to volume with
VitD-17 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE