Guide to Method Format

(Method shown is incomplete to allow space for description.)

Locator number identifies Method number is a

50.2.10
a method by chapter, permanent codification that
AOAC Official Method 2016.05
subchapter, and sequence identifies a method by year
Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders,
within the subchapter for ease and sequence of adoption.
Infant Formulas, and Adult/Pediatric Nutritional Formulas
of location. 50 = Chapter 50; 2016 = First Action 2016; .05 =
Liquid Chromatography–Tandem Mass Spectrometry
2 = subchapter 2; 10 = 10th 5th method adopted in 2016.
First Action 2016
method found in chapter 50, Final Action 2017
subchapter 2. The locator
Title typically includes analyte
number is not a permanent AOAC–ISO Method*
and matrix, technique, and
number and is included only (Applicable to the determination of total vitamin D2 and vitamin D3 in fortified official status (First Action,
for convenient accessibility. milk powders, infant formulas, and adult/pediatric nutritional formulas.) Final Action, Revised First
Caution: Refer to the Material Safety Data Sheets for all chemicals prior to use. Use all Action, Repealed). May
Applicability statement
specifies utility and limitations
appropriate personal protective equipment and follow good laboratory practices. include adoption by other
on scope of method and other organizations.
A. Principle
pertinent information.
Samples are saponified at high temperature, and then lipid-soluble components
are extracted into isooctane. A portion of the isooctane layer is transferred and
Cautionary statement washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is added to
specifies precautions and derivatize vitamin D to form a high-molecular-mass, easily ionizable adduct.
possible hazards in carrying B. Apparatus
out method, including safety Methods are divided into
information on equipment,
(a)  Ultra-high-performance LC (UHPLC) system.—Nexera (Shimadzu, Kyoto, several descriptive sections,
techniques and practices,
Japan) or equivalent LC system, consisting of a dual pump system, sample injector including specifications
and safe handling of
unit, degasser unit, and column oven. for necessary laboratory
chemicals, acids, alkalies, C. Reagents apparatus and reagent
microorganisms, and solvents. (a)  Vitamin D2 (ergocalciferol).—CAS No. 50-14-6, purity: ≥99%. preparations. See also
D. Reagent Preparation Definition of Terms and
Explanatory Notes.
(a)  PTAD solution (10 mg/mL).—To a 5 mL volumetric flask, add 50 mg PTAD,
then add 4 mL acetone, and dissolve; dilute to volume with acetone. Expiry: 1 day.
E. Standard Preparation
Vitamin D is sensitive to light. Perform all steps under UV-shielded lighting. If
vitamin D3 is exclusively required for analysis, then standards pertaining to vitamin
D2 need not be used and vice versa.
J. Calculations
(a)  Concentration of SIL vitamin D2 in SILD2SS.—
Calculation symbols are
defined and show correct SILD2SSabs(λmax)
units. SILD2SSD2concn = × 10000
E1%
1 cm

where SILD2SSD2concn = concentration of d6-vitamin D2 in the stock standard

(μg/mL); SILD2SSabs(λ max) = UV absorbance of the stock standard at 265 nm (cm−1);
1 cm = extinction coefficient for vitamin D2 in ethanol (461 dL/g·cm); and 10 000
E1% References direct the user
= concentration conversion factor (g/dL to μg/mL). to published validation
studies (single- and/or
References: J. AOAC Int. 99, 1321(2016)
DOI: 10.5740/jaoacint.16-0160 (First Action) multilaboratory), subsequent
revisions, and relevant
J. AOAC Int. 101, 256(2018)
SMPRs®. Other informative
DOI: 10.5740/jaoacint.17-0149 (Final Action)
references may be included.
Method may include Chemical J. AOAC Int. 95, 292(2012) (AOAC SMPR 2011.004)
DOI: 10.5740/jaoac.int.11-0440 Revision date at the end
Abstracts Service (CAS)
registry numbers, unique of method signifies that
CAS-50-14-6 (ergocalciferol) the method received minor
identifiers used to search
a number of data-retrieval editorial revisions (that do not
Revised: January 2019
systems. affect method procedures).

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