Determination of trans and Total Vitamin K 1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Post Column Reduction and Fluorescence Detection: Multilaboratory Testing Study, AOAC Final Action 2015.09.

A multilaboratory study was completed with AOAC INTERNATIONAL First Action Official Method ^SM 2015.09, "Determination of trans and Total ( cis+trans ) Vitamin K ₁ in Infant, Pediatric, and Adult Nutritionals by HPLC with Post Column Reduction and Fluorescence Detection." Eight laboratories from six countries participated in the multilaboratory study. Each laboratory analyzed 19 infant, pediatric, and adult nutritionals in duplicate. The product matrixes analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders; milk-based infant formula ready-to-feed liquids; pediatric powders; adult low- and high-fat powders; and high-protein ready-to-drink nutritionals. Vitamin K ₁ was extracted from product matrixes with isooctane after precipitation of proteins and the release of lipids with methanol. Prepared samples were injected onto a silica HPLC column in which cis and trans vitamin K ₁ were separated with an isooctane-isopropanol mobile phase. The column eluent was mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and cis and trans vitamin K ₁ were reduced to fluorescent derivatives in a zinc reactor column. Overall, trans vitamin K ₁ repeatability averaged 3.06% relative standard deviation (RSD) with a range of 0.99-7.16% RSD and reproducibility averaged 6.36% RSD with a range of 3.15-16.1% RSD. Repeatability Standard Method Performance Requirements (SMPR ^® ) were met for all 19 matrixes, and reproducibility SMPRs were met for 18 of the 19 product matrixes analyzed. Repeatability and reproducibility for total ( cis + trans ) vitamin K ₁ averaged 3.15% RSD with a range of 1.06-6.87% RSD and 6.11% RSD with a range of 2.94-16.7% RSD, respectively.