Methods to be Reviewed by OMB in 2016

Bidlack et al.:

ournal of

AOAC I

nternational

ol.

98, N

5, 2015

1385

naturally fluoresce or form fluorescent derivatives when

exposed to zinc.

Acceptance Criteria

Previously established method performance requirements for

vitamin K (SMPR 2014.001) are summarized in Table 1.

Results

The SRM repeatability and recovery requirements were met

for all 12 matrixes. Repeatability data are summarized in Table

2. Repeatability precision (RSD

) and intermediate precision

(RSD

) ranged from 0.6 to 3.5% and 1.1 to 6.0%, respectively.

Pooled across all samples, the RSD

was 2.0% and the RSD

was 2.8%. Mean spike recovery data are summarized in Table 3

and ranged from 91.9 to 106%, averaging 97.8% across all

samples. The method demonstrated good linearity over a

standard range of approximately 2–90 µg/L

trans

vitamin K

with r

averaging 0.99995. These data are summarized in

Table 4. Average calibration errors were <1% and are illustrated

graphically in Figure 1.

The LOQ requirement wasmet. LODand LOQwere estimated

to be 0.1 and 0.4 µg/L, respectively, with standards and 0.03

and 0.09 µg/100 g ready-to-feed (RTF) liquids, respectively, for

products assuming a 4 g sample diluted to 10 mL.

AOAC Official Method 2015.09

Trans

Vitamin K

in Infant, Pediatric, and Adult Nutritionals

HPLC with Fluorescence Detection

First Action 2015

(Applicable to the determination of

trans

vitamin K

infant, pediatric, and adult nutritional formulas.)

Caution

: Refer to Material Safely Data Sheets (MSDS)

of chemicals prior to use and use the suggested

personal protective equipment. Zinc powder should

be handled in a fume hood; it is self-heating and

may catch fire. Zinc powder should not be allowed

to come into contact with water, which may release

flammable gases that may spontaneously ignite.

See

Tables 2 and 3 for infant, pediatric, and nutritional

matrixes for which the method has been validated.

A. Principle

This normal-phase (NP) HPLC method with postcolumn

reduction and fluorescence detection allows for the quantitative

determination of

trans

vitamin K

in infant, pediatric, and

adult nutritionals. Vitamin K

is extracted from products with

iso-octane after precipitation of proteins and release of lipids

with methanol. Prepared samples are injected onto a silica

HPLC column where

cis

and

trans

vitamin K

are separated

with an iso-octane–isopropanol mobile phase. The column

eluent is mixed with a dilute ethanolic solution of zinc chloride,

sodium acetate, and acetic acid, and vitamin K

is reduced to a

fluorescent derivative in a zinc reactor column. The resulting

fluorescent compound is then detected by fluorescence at an

excitation wavelength of 245 nm and an emission wavelength

of 440 nm.

B. Apparatus and Materials

(a)

HPLC system

.—Two isocratic pumps; autosampler

capable of injecting 20 µL; fluorescence detector; high-

pressure mixing tee; and postcolumn reactor column 20 × 4 mm

stainless steel (Waters, Milford, MA; Part No. WAT084550

or equivalent). The system should be configured as shown in

Figure

2015.09A

(b)

Analytical column

.—Silica 150 × 3.0 mm, 3 µm, 60 Å,

or equivalent.

(c)

Analytical balance

.—Capable of weighing to the nearest

0.00001 g.

(d)

Beakers

.—Glass, assorted sizes.

(e)

Centrifuge

(f)

Centrifuge tubes and caps

.—50 mL glass tubes with

Teflon-lined caps.

(g)

Cylinders

.—Graduated, glass, assorted sizes.

(h)

Gas regulator

.—Helium.

(i)

Helium sparge

.—Tubing and filtering assembly.

(j)

Magnetic stirrer.—

Variomag Telesystem (Daytona

Beach, FL) HP 60 S (Part No. HP 46040U) with tube rack

(Part No. HP 86029) and egg-shaped Teflon stirring bars (Part

No. PI20110) or equivalent.

(k)

Pipet.—

Disposable glass, Pasteur.

(l)

Pipet.—

Mechanical, variable volume, 0.5–5 mL and

10–100 µL.

(m)

Pipet

.—Repeating 5 and 25 mL or equivalent.

(n)

Volumetric flasks.—

Glass, Class A, assorted sizes.

(o)

Volumetric pipets.—

Glass, Class A, assorted sizes.

(p)

Vortex mixers

(q)

Yellow lights or yellow shields with cutoff of at least

440 nm

C. Reagents

(a)

Acetic acid

.—Glacial, ACS.

(b)

Helium

.—Zero grade or equivalent

(c)

Iso-octane (2,2,4-trimethylpentane)

.—HPLC grade.

(d)

Isopropanol (isopropyl alcohol)

.—HPLC grade.

(e)

Methanol

.—HPLC grade.

Figure 1. Vitamin K

standard curve calibration errors; L = level.

Candidates for 2016 Method of the Year

112