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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