AOAC SPIFAN Final Review Forms (March 16, 2016)

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Infant Formula and Adult Nutrition - Review Form

Evaluation of Method VitD-17

Title:

Determination of Vitamin D in Milk Products by UPLC-MS-MS with 4-Phenyl-1,2,4-triazoline-3,5-

dione (PTAD) Derivatization

Author:

CDC-SH

Summary of Method:

Method Principle

Samples are prepared using a procedure based on AOAC Method 2011.11. Powder samples are

reconstituted per SMPR. Liquid samples are delivered to a 100-mL flask to which pyrogallol is added as an

antioxidant and tri-deuterated vitamin D3 is added as an internal standard. Potassium hydroxide (50%, aq)

is added to the sample which is then flushed with nitrogen, tightly sealed. Saponification is carried out at

room temperature overnight.

The reaction solution is quantitatively transferred to a 250-mL separatory funnel to which Hexane (w/BHT)

is added. The sample is shaken and the aqueous layer removed. The hexane extraction solution is then

washed 4x20mL with water and cooled at -20C for 60 mins.

A six-mL aliquot of the Hexane solution is transferred to a small vial and dried under nitrogen. PTAD

solution (60 mcL) is added to the dried sample, mixed by vortex and sonication. Derivatization is complete

after 30 mins at RT. Water (400 mcL) is added to the sample and the sample filtered prior to analysis by

UPLC-MS/MS.

Instrument:

Instrument analysis for this work was completed using a 1290 Infinity UPLC (Agilent) with a 50 x 2.1 mm,

1.7micron BEH C18 column (Waters) interfaced to a 6490 Triple Quadrupole Mass Spectrometer (Agilent).

Ionization is by ESI (+). Two mass transitions, one for quantitation and a second for qualitative verification,

are used for each analyte, vitamin D

, D

and tri-deuterated D3 IS.

Method Scope/Applicability:

The method as submitted includes validation data for SRM 1849a, overspike data for vitamins D2 and D3 in

four SPIFAN matrices (Amino Acid Based Powder, Milk-Based IF Powder, Soy-Based IF Powder, and High Fat

Adult Nutritional RTF).

Applicability is consistent with SMPR definition for Infant Formula and Adult Nutritional products; Vitamins

and D

General comments about the method:

VitD-17 Review Forms