6. AOACSPIFANMethods-2018Awards

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Bidlack et al.: J ournal of AOAC I nternational V ol. 98, N o. 5, 2015  1385

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 1 in infant, pediatric, and adult nutritionals. Vitamin K 1 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 1 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 1 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 .

Figure 1. Vitamin K 1

standard curve calibration errors; L = level.

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 r ) and intermediate precision (RSD IP ) ranged from 0.6 to 3.5% and 1.1 to 6.0%, respectively. Pooled across all samples, the RSD r was 2.0% and the RSD IP 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 1 with r 2 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 1 in Infant, Pediatric, and Adult Nutritionals HPLC with Fluorescence Detection First Action 2015

(Applicable to the determination of trans vitamin K 1 in infant, pediatric, and adult nutritional formulas.) Caution : Refer to Material Safely Data Sheets (MSDS)

C. Reagents (a) Acetic acid .—Glacial, ACS. (b) Helium .—Zero grade or equivalent .

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.

(c) Iso-octane (2,2,4-trimethylpentane) .—HPLC grade. (d) Isopropanol (isopropyl alcohol) .—HPLC grade. (e) Methanol .—HPLC grade.