AOAC Final Action Methods in 2018

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 or T-junction; post-column reactor column 20 × 4 mm stainless steel or similar dimensions (old HPLC column with packing removed or 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 .—Compatible with helium or nitrogen. (i)  Gas sparge .—Tubing and filtering assembly (not required if can sufficiently degas mobile phase and post-column solution with instrument vacuum degasser). (j)  Magnetic stirrer.— Variomag Telesystem (Daytona Beach, FL, USA) 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)  Spectrophotometer .—Capable of measuring absorbance at 248 nm. (o)  Volumetric flasks.— Glass, Class A, assorted sizes. (p)  Volumetric pipets.— Glass, Class A, assorted sizes.

AOAC Official Method 2015.09 Trans and Total ( cis + trans ) Vitamin K 1 in Infant, Pediatric, and Adult Nutritionals HPLC with Fluorescence Detection

First Action 2015 Final Action 2018

Codex-Adopted AOAC-ISO Method [Applicable to the determination of trans and total ( cis + trans ) vitamin K 1 in infant, pediatric, and adult nutritional formulas.] Caution : Refer toMaterial Safely Data Sheets (MSDS) of chemicals prior to use and follow 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 come into contact with water, which may release flammable gases that may spontaneously ignite. See Tables 2015.09A – D for infant, pediatric, and nutritional matrixes for which the method has been validated and for study results supporting acceptance of the method. A. Principle This normal-phase (NP) HPLC method with post-column reduction and fluorescence detection allows for the quantitative determination of trans and total ( cis + 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 cis and trans vitamin K 1 are reduced to fluorescent derivatives in a zinc reactor column. The resulting fluorescent compounds are then detected by fluorescence at an

Table 2015.09A.  Trans vitamin K 1 SLV data—precision

No. of replicates (duplicates on multiple days)

Mean, µg/100 g RTF

Sample type

SD r

RSD r , % SD IP

RSD IP , %

Child formula powder, placebo Infant elemental powder, placebo

6 6 6 6 6

2.03 2.03 3.47 3.07 2.16 1.11 a 3.26 7.69 8.99 2.92 2.66 7.57 6.09 6.26 9.01 9.10 10.7

0.040 0.020 0.021 0.034 0.038 0.022 0.097 0.123 0.141 0.102 0.049 0.129 0.105 0.105 0.168 0.283 0.120

2.0 1.0 0.6 1.1 1.8 2.0 3.0 1.6 1.6 3.5 1.8 1.7 1.7 1.7 1.9 3.1 1.1

0.045 0.025 0.040 0.034 0.038 0.025 0.142 0.170 0.226 0.102 0.050 0.451 0.131 0.211 0.189 0.299 0.179

2.2 1.2 1.2 1.1 1.8 2.3 4.4 2.2 2.5 3.5 1.9 6.0 2.2 3.4 2.1 3.3 1.7

Adult nutritional RTF high protein, placebo Adult nutritional RTF high fat, placebo Infant formula RTF milk based, placebo

SRM 1849a

12 12 12 12 12 12 12 12 12 12 16 12

Adult nutritional powder milk protein based

Infant formula powder partially hydrolyzed milk based Infant formula powder partially hydrolyzed soy based

Adult nutritional powder low fat

Child formula powder Infant elemental powder

Infant formula powder milk based Infant formula powder soy based Infant formula RTF milk based Adult nutritional RTF high protein Adult nutritional RTF high fat a  Results reported as mg/kg powder.

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